carbocyanines and 3-3--dipropyl-2-2--thiadicarbocyanine

Both innate and adaptive immune cells are critical players in autoimmune destruction of insulin-producing β cells in type 1 diabetes. However, the early pathogenic events triggering the recruitment and activation of innate immune cells in islets remain obscure. Here we show that circulating fatty acid binding protein 4 (FABP4) level was significantly elevated in patients with type 1 diabetes and their first-degree relatives and positively correlated with the titers of several islet autoantibodies. In nonobese diabetic (NOD) mice, increased FABP4 expression in islet macrophages started from the neonatal period, well before the occurrence of overt diabetes. Furthermore, the spontaneous development of autoimmune diabetes in NOD mice was markedly reduced by pharmacological inhibition or genetic ablation of FABP4 or adoptive transfer of FABP4-deficient bone marrow cells. Mechanistically, FABP4 activated innate immune responses in islets by enhancing the infiltration and polarization of macrophages to proinflammatory M1 subtype, thus creating an inflammatory milieu required for activation of diabetogenic CD8+ T cells and shift of CD4+ helper T cells toward Th1 subtypes. These findings demonstrate FABP4 as a possible early mediator for β cell autoimmunity by facilitating crosstalk between innate and adaptive immune cells, suggesting that pharmacological inhibition of FABP4 may represent a promising therapeutic strategy for autoimmune diabetes.

Topics: Adult; Animals; Autoantibodies; Benzothiazoles; Bone Marrow Transplantation; Carbocyanines; Diabetes Mellitus, Experimental; Diabetes Mellitus, Type 1; Fatty Acid-Binding Proteins; Female; Humans; Islets of Langerhans; Macrophages; Male; Mice, Inbred NOD; Mice, Mutant Strains; Middle Aged; T-Lymphocytes

An amphipathic α-helical peptide, Hp1404, was isolated from the venomous gland of the scorpion

Topics: 1-Naphthylamine; Acinetobacter baumannii; Animals; Anti-Bacterial Agents; Antimicrobial Cationic Peptides; Benzothiazoles; Biofilms; Carbocyanines; Cell Survival; Circular Dichroism; Drug Resistance, Multiple, Bacterial; Escherichia coli; Gram-Negative Bacteria; Gram-Positive Bacteria; Hemolysis; Listeria monocytogenes; Methicillin-Resistant Staphylococcus aureus; Microbial Sensitivity Tests; Protein Conformation, alpha-Helical; Pseudomonas aeruginosa; Salmonella; Scorpions

It is widely believed that most xenobiotics cross biomembranes by diffusing through the phospholipid bilayer, and that the use of protein transporters is an occasional adjunct. According to an alternative view, phospholipid bilayer transport is negligible, and several different transporters may be involved in the uptake of an individual molecular type. We recognise here that the availability of gene knockout collections allows one to assess the contributions of all potential transporters, and flow cytometry based on fluorescence provides a convenient high-throughput assay for xenobiotic uptake in individual cells.. We used high-throughput flow cytometry to assess the ability of individual gene knockout strains of E coli to take up two membrane-permeable, cationic fluorescent dyes, namely the carbocyanine diS-C3(5) and the DNA dye SYBR Green. Individual strains showed a large range of distributions of uptake. The range of modal steady-state uptakes for the carbocyanine between the different strains was 36-fold. Knockouts of the ATP synthase α- and β-subunits greatly inhibited uptake, implying that most uptake was ATP-driven rather than being driven by a membrane potential. Dozens of transporters changed the steady-state uptake of the dye by more than 50% with respect to that of the wild type, in either direction (increased or decreased); knockouts of known influx and efflux transporters behaved as expected, giving credence to the general strategy. Many of the knockouts with the most reduced uptake were transporter genes of unknown function ('y-genes'). Similarly, several overexpression variants in the 'ASKA' collection had the anticipated, opposite effects. Similar results were obtained with SYBR Green (the range being approximately 69-fold). Although it too contains a benzothiazole motif there was negligible correlation between its uptake and that of the carbocyanine when compared across the various strains (although the membrane potential is presumably the same in each case).. Overall, we conclude that the uptake of these dyes may be catalysed by a great many transporters of putatively broad and presently unknown specificity, and that the very large range between the 'lowest' and the 'highest' levels of uptake, even in knockouts of just single genes, implies strongly that phospholipid bilayer transport is indeed negligible. This work also casts serious doubt upon the use of such dyes as quantitative stains for representing either bioenergetic parameters or the amount of cellular DNA in unfixed cells (in vivo). By contrast, it opens up their potential use as transporter assay substrates in high-throughput screening.

Topics: Benzothiazoles; Biological Transport; Carbocyanines; Diamines; Escherichia coli; Escherichia coli Proteins; Flow Cytometry; Fluorescent Dyes; Membrane Transport Proteins; Organic Chemicals; Quinolines

Naturally occurring antimicrobial peptides important for innate immunity are widely studied for their antimicrobial and anticancer activity. The primary target of these AMPs is believed to be the bacterial cytoplasmic membrane. However, the interaction between cytoplasmic membrane and the antimicrobial peptides remains poorly understood. Therefore to focus on the target membrane composition that is required by AMPs to interact with membranes, we have examined the interaction of the antimicrobial and anticancer active 11-residue GA-K4 (FLKWLFKWAKK) peptide with model and intact cell membranes. Effect on the structural conformational properties of GA-K4 peptide was investigated by means of far-UV CD and fluorescence spectroscopic methods. The different conformation of GA-K4 peptide in large unilamellar vesicles (LUV) bilayer and micelle environment suggest that the curvature has an influence on the secondary structure acquired by the peptide. Furthermore, the leakage experiment result confirmed that GA-K4 induced the leakage of cytoplasmic membrane in Staphylococcus аureus bacterial cells. Fluorescence data revealed the interfacial location of GA-K4 peptide in the model membranes. The blue-shift in emission wavelength by tryptophan residues in fluorescence data indicated the penetration of GA-K4 peptide in micelles and phospholipid bilayers. These results showed that the GA-K4 peptide is a membrane-active peptide and its activity depends on membrane curvature and lipid composition. Although further studies are required to confirm the mechanism of action, the data suggest mechanism of toroidal pore formation for the interaction of GA-K4 peptide with membranes. Our studies will be helpful in better understanding of the membrane requirment of peptides to express their therapeutic effects.

Topics: Amino Acid Sequence; Anti-Bacterial Agents; Antimicrobial Cationic Peptides; Antineoplastic Agents; Benzothiazoles; Carbocyanines; Cell Membrane; Cell Membrane Permeability; Fluorescent Dyes; Kinetics; Lipid Bilayers; Lysophosphatidylcholines; Micelles; Phosphatidylcholines; Phosphatidylethanolamines; Phosphatidylglycerols; Protein Structure, Secondary; Spectrometry, Fluorescence; Staphylococcus aureus; Unilamellar Liposomes

Bac8c (RIWVIWRR-NH2) is an analogue peptide derived through complete substitution analysis of the linear bovine host defense peptide variant Bac2A. In the present study, the antifungal mechanism of Bac8c against pathogenic fungi was investigated, with a particular focus on the effects of Bac8c on the cytoplasmic membrane. We used bis-(1,3-dibutylbarbituric acid) trimethine oxonol [DiBAC4(3)] staining and 3,3'-dipropylthiacarbocyanine iodide [DiSC3(5)] assays to show that Bac8c induced disturbances in the membrane potential of Candida albicans. An increase in membrane permeability and suppression of cell wall regeneration were also observed in Bac8c-treated C. albicans. We studied the effects of Bac8c treatment on model membranes to elucidate its antifungal mechanism. Using calcein and FITC-labeled dextran leakage assays from Bac8c-treated large unilamellar vesicles (LUVs) and giant unilamellar vesicles (GUVs), we found that Bac8c has a pore-forming action on fungal membranes, with an estimated pore radius of between 2.3 and 3.3 nm. A membrane-targeted mechanism of action was also supported by the observation of potassium release from the cytosol of Bac8c-treated C. albicans. These results indicate that Bac8c is considered as a potential candidate to develop a novel antimicrobial agent because of its low-cost production characteristics and high antimicrobial activity via its ability to induce membrane perturbations in fungi.

Topics: Animals; Antifungal Agents; Antimicrobial Cationic Peptides; Barbiturates; Benzothiazoles; Biological Transport; Candida albicans; Carbocyanines; Cattle; Cell Membrane; Cell Membrane Permeability; Cell Wall; Dextrans; Fluorescein-5-isothiocyanate; Fluoresceins; Fluorescent Dyes; Isoxazoles; Membrane Potentials; Microbial Sensitivity Tests; Potassium; Spectrometry, Fluorescence; Unilamellar Liposomes

Isoquercitrin is a flavonoid isolated from Aster yomena, which has been used as a traditional medicinal herb. In the present study, we investigated the antifungal activity and the underlying mechanism of isoquercitrin. Isoquercitrin had a potent effect in the susceptibility test against pathogenic fungi and almost no hemolysis. Propidium iodide and potassium release assays were conducted in Candida albicans, and these studies confirmed that isoquercitrin induced membrane damage, thereby, increasing permeability. Membrane potential was analyzed using 3,3'-dipropylthiacarbocyanine iodide [DiSC3(5)], and the transition of membrane potential was indicated by an increased fluorescence intensity. To further analyze these results using model membranes, giant unilamellar vesicles and large unilamellar vesicles that encapsulated calcein were prepared and the detection of calcein leakage from liposomes indicated that membrane was disturbed. We further verified membrane disturbance by observing the disordered status of the lipid bilayer with 1,6-diphenyl-1,3,5-hexatriene fluorescence. Moreover, changes in size and granularity of the cell were revealed in flow cytometric analysis. All these results suggested the membrane disturbance and the degree of disturbance was estimated to be within a range of 2.3 nm to 3.3 nm by fluorescein isothiocyanate-dextran analysis. Taken together, isoquercitrin exerts its fungicidal effect by disturbing the membrane of cells.

Topics: Antifungal Agents; Aster Plant; Benzothiazoles; Candida albicans; Carbocyanines; Cell Membrane; Cell Membrane Permeability; Dextrans; Diphenylhexatriene; Flow Cytometry; Fluorescein-5-isothiocyanate; Fluoresceins; Fluorescent Dyes; Membrane Potentials; Microbial Sensitivity Tests; Plants, Medicinal; Quercetin; Republic of Korea; Unilamellar Liposomes

Scolopendin 2 is a 16-mer peptide (AGLQFPVGRIGRLLRK) derived from the centipede Scolopendra subspinipes mutilans. We observed that this peptide exhibited antimicrobial activity in a salt-dependent manner against various fungal and bacterial pathogens and showed no hemolytic effect in the range of 1.6 μM to 100 μM. Circular dichroism analysis showed that the peptide has an α-helical properties. Furthermore, we determined the mechanism(s) of action using flow cytometry and by investigating the release of intracellular potassium. The results showed that the peptide permeabilized the membranes of Escherichia coli O157 and Candida albicans, resulting in loss of intracellular potassium ions. Additionally, bis-(1,3-dibutylbarbituric acid) trimethine oxonol and 3,3'-dipropylthiacarbocyanine iodide assays showed that the peptide caused membrane depolarization. Using giant unilamellar vesicles encapsulating calcein and large unilamellar vesicles containing fluorescein isothiocyanate-dextran, which were similar in composition to typical E. coli O157 and C. albicans membranes, we demonstrated that scolopendin 2 disrupts membranes, resulting in a pore size between 4.8 nm and 5.0 nm. Thus, we have demonstrated that a cationic antimicrobial peptide, scolopendin 2, exerts its broad-spectrum antimicrobial effects by forming pores in the cell membrane.

Topics: Amino Acid Motifs; Animals; Anti-Infective Agents; Antimicrobial Cationic Peptides; Arthropod Proteins; Arthropods; Barbiturates; Benzothiazoles; Candida albicans; Carbocyanines; Cell Membrane; Dextrans; Erythrocytes; Escherichia coli O157; Fluorescein-5-isothiocyanate; Fluoresceins; Fluorescent Dyes; Humans; Isoxazoles; Microbial Sensitivity Tests; Molecular Sequence Data; Spectrometry, Fluorescence; Unilamellar Liposomes

The antimicrobial activity of analogs obtained by substituting arginine and lysine in CL(14-25), a cationic α-helical dodecapeptide, with alanine against Porphyromonas gingivalis, a periodontal pathogen, varied significantly depending on the number and position of cationic amino acids. The alanine-substituted analogs had no hemolytic activity, even at a concentration of 1 mM. The antimicrobial activities of CL(K20A) and CL(K20A, K25A) were 3.8-fold and 9.1-fold higher, respectively, than that of CL(14-25). The antimicrobial activity of CL(R15A) was slightly lower than that of CL(14-25), suggesting that arginine at position 15 is not essential but is important for the antimicrobial activity. The experiments in which the alanine-substituted analogs bearing the replacement of arginine at position 24 and/or lysine at position 25 were used showed that arginine at position 24 was crucial for the antimicrobial activity whenever lysine at position 25 was substituted with alanine. Helical wheel projections of the alanine-substituted analogs indicate that the hydrophobicity in the vicinity of leucine at position 16 and alanines at positions 18 and/or 21 increased by substituting lysine at positions 20 and 25 with alanine, respectively. The degrees of diSC3 -5 release from P. gingivalis cells and disruption of GUVs induced by the alanine-substituted analogs with different positive charges were not closely related to their antimicrobial activities. The enhanced antimicrobial activities of the alanine-substituted analogs appear to be mainly attributable to the changes in properties such as hydrophobicity and amphipathic propensity due to alanine substitution and not to their extents of positive charge (cationicity).

Topics: Alanine; Amino Acid Sequence; Amino Acid Substitution; Animals; Anti-Infective Agents; Arginine; Benzothiazoles; Carbocyanines; Carbon-Nitrogen Lyases; Cations; Hemolysis; Inhibitory Concentration 50; Lysine; Microbial Sensitivity Tests; Molecular Sequence Data; Oligopeptides; Oryza; Peptide Fragments; Porphyromonas gingivalis; Sheep; Time Factors; Unilamellar Liposomes

In this study, the antifungal activity and mode of action(s) of hibicuslide C derived from Abutilon theophrasti were investigated. Antifungal susceptibility testing showed that hibicuslide C possessed potent activities toward various fungal strains and less hemolytic activity than amphotericin B. To understand the antifungal mechanism(s) of hibicuslide C in Candida albicans, flow cytometric analysis with propidium iodide was done. The results showed that hibicuslide C perturbed the plasma membrane of the C. albicans. The analysis of the transmembrane electrical potential with 3,3'-dipropylthiacarbocyanine iodide [DiSC3(5)] indicated that hibicuslide C induced membrane depolarization. Furthermore, model membrane studies were performed with calcein encapsulating large unilamellar vesicles (LUVs) and FITC-dextran (FD) loaded LUVs. These results demonstrated that the antifungal effects of hibicuslide C on the fungal plasma membrane were through the formation of pores with radii between 2.3 nm and 3.3 nm. Finally, in three dimensional flow cytometric contour plots, a reduced cell sizes by the pore-forming action of hibicuslide C were observed. Therefore, the present study suggests that hibicuslide C exerts its antifungal effect by membrane-active mechanism.

Topics: Amphotericin B; Antifungal Agents; Benzothiazoles; Candida albicans; Carbocyanines; Cell Membrane; Cell Membrane Permeability; Dextrans; Erythrocytes; Flow Cytometry; Fluorescein-5-isothiocyanate; Fluoresceins; Fluorescent Dyes; Hemolysis; Humans; Malvaceae; Membrane Potentials; Phenylpropionates; Plant Extracts; Unilamellar Liposomes

Piracetam is a cyclic derivative of γ-aminobutyric acid (GABA). It was the first nootropic drug approved for clinical use. However, mechanism of its action is still not clear. In present paper, I investigated effects of piracetam on neurotransmitter release, plasma membrane potential monitored by fluorescent dye DiSC3(5) and chloride transport monitored by fluorescent dye SPQ in rat brain synaptosomes. It was shown that piracetam (1 mM) induces slow weak plasma membrane depolarization. This effect was decreased on 43% and 58% by both AMPA/kainate receptor blockers NBQX (10 μM) and CNQX (100 μM), respectively, on 84% by GABA ionotropic receptor blocker picrotoxin (50 μM) and on 91% upon withdrawal of HCO(3-) ions from incubation medium. GABA (1 mM) and kainate (100 μM) were found not to produce changes of plasma membrane potential. Also, it was found that piracetam induces chloride efflux which seems to be the reason of depolarization. Thereby, piracetam induces depolarization of plasma membrane of isolated neuronal presynaptic endings by picrotoxin-sensitive way.

Topics: Animals; Benzothiazoles; Biological Transport; Brain; Carbocyanines; Cell Membrane; Cell Polarity; Chlorides; Fluorescent Dyes; GABA Antagonists; Male; Membrane Potentials; Nootropic Agents; Quinolinium Compounds; Rats, Wistar; Receptors, AMPA; Receptors, Kainic Acid; Synaptosomes

Pleurocidin, a 25-mer antimicrobial peptide, has been known to exhibit potent antibacterial activity. To investigate the functional roles in N- and C-terminal regions of pleurocidin on the antibacterial activity, we designed four truncated analogs. The antibacterial susceptibility testing showed that pleurocidin and its analogs exerted antibacterial effect against various bacterial strains and further possessed specific activity patterns corresponding with their hydrophobic scale [pleurocidin > Anal 3 (1-22) > Anal 1 (4-25) > Anal 4 (1-19) > Anal 2 (7-25)]. Fluorescence experiments using 1,6-diphenyl-1,3,5-hexatriene (DPH) and 3,3'-dipropylthiadicarbocyanine iodide [diSC3(5)] indicated that the differences in antibacterial activity of the peptides were caused by its membrane-active mechanisms including membrane disruption and depolarization. Blue shift in tryptophan fluorescence demonstrated that the decrease in net hydrophobicity attenuates the binding affinity of pleurocidin to interact with plasma membrane. Therefore, the present study suggests that hydrophobicity in the N- and C-terminal regions of pleurocidin plays a key role in its antibacterial activity.

Topics: Amino Acid Sequence; Anti-Bacterial Agents; Antimicrobial Cationic Peptides; Benzothiazoles; Carbocyanines; Cell Membrane; Diphenylhexatriene; Drug Resistance, Bacterial; Enterococcus faecium; Escherichia coli O157; Fish Proteins; Fluorescence; Hydrophobic and Hydrophilic Interactions; Microbial Sensitivity Tests; Molecular Sequence Data; Pseudomonas aeruginosa; Staphylococcus aureus; Tryptophan

Lariciresinol is an enterolignan precursor isolated from the herb Sambucus williamsii, a folk medicinal plant used for its therapeutic properties. In this study, the antifungal properties and mode of action of lariciresinol were investigated. Lariciresinol displays potent antifungal properties against several human pathogenic fungal strains without hemolytic effects on human erythrocytes. To understand the antifungal mechanism of action of lariciresinol, the membrane interactions of lariciresinol were examined. Fluorescence analysis using the membrane probe 3,3'-diethylthio-dicarbocyanine iodide (DiSC(3)-5) and 1,6-diphenyl-1,3,5-hexatriene (DPH), as well as a flow cytometric analysis with propidium iodide (PI), a membrane-impermeable dye, indicated that lariciresinol was associated with lipid bilayers and induced membrane permeabilization. Therefore, the present study suggests that lariciresinol possesses fungicidal activities by disrupting the fungal plasma membrane and therapeutic potential as a novel antifungal agent for the treatment of fungal infectious diseases in humans.

Topics: Antifungal Agents; Benzothiazoles; Candida albicans; Carbocyanines; Cell Membrane; Cells, Cultured; Diphenylhexatriene; Erythrocytes; Flow Cytometry; Fluorescent Dyes; Furans; Hemolysis; Hemolytic Agents; Humans; Lignans; Propidium; Sambucus

Here, we communicate that the design of the anti-oxidant consisting of amphiphilic manganese porphyrin with one dimethylimidazolium group and three phenyl groups for mitochondrion targeting. The resulting Mn-porphyrin MnMImP(3)P exhibited high partition coefficient (logP(ow) = +4.78) as well as significant superoxide dismutase and peroxynitrite decomposition activities. Accordingly, the MnMImP(3)P exhibited a little increase in fluorescence intensity attributed to 3,3'-dipropyl-2,2'-thiadicarbocyanine iodide [diS-C(3)-(5)], a tracer dye to assess the mitochondrial membrane potential, which suggested the interaction of the MnMImP(3)P, leading to the release of the fluorescence dye from the mitochondrial membrane, with the mitochondria. As a result, the MnMImP(3)P rescued the cell death under oxidative stress concerned with mitochondrial damage.

Topics: Antioxidants; Benzothiazoles; Carbocyanines; Cell Line, Tumor; Cell Survival; Humans; Manganese; Mitochondria; Molecular Structure; Peroxynitrous Acid; Porphyrins; Superoxide Dismutase

The effects of hydrophobic thickness and the molar phosphatidylglycerol (PG) content of lipid bilayers on the structure and membrane interaction of three cationic antimicrobial peptides were examined: aurein 2.2, aurein 2.3 (almost identical to aurein 2.2, except for a point mutation at residue 13), and a carboxy C-terminal analog of aurein 2.3. Circular dichroism results indicated that all three peptides adopt an alpha-helical structure in the presence of a 3:1 molar mixture of 1,2-dimyristoyl-sn-glycero-3-phosphocholine/1,2-dimyristoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (DMPC/DMPG), and 1:1 and 3:1 molar mixtures of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine/1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (POPC/POPG). Oriented circular dichroism data for three different lipid compositions showed that all three peptides were surface-adsorbed at low peptide concentrations, but were inserted into the membrane at higher peptide concentrations. The (31)P solid-state NMR data of the three peptides in the DMPC/DMPG and POPC/POPG bilayers showed that all three peptides significantly perturbed lipid headgroups, in a peptide or lipid composition-dependent manner. Differential scanning calorimetry results demonstrated that both amidated aurein peptides perturbed the overall phase structure of DMPC/DMPG bilayers, but perturbed the POPC/POPG chains less. The nature of the perturbation of DMPC/DMPG bilayers was most likely micellization, and for the POPC/POPG bilayers, distorted toroidal pores or localized membrane aggregate formation. Calcein release assay results showed that aurein peptide-induced membrane leakage was more severe in DMPC/DMPG liposomes than in POPC/POPG liposomes, and that aurein 2.2 induced higher calcein release than aurein 2.3 and aurein 2.3-COOH from 1:1 and 3:1 POPC/POPG liposomes. Finally, DiSC(3)5 assay data further delineated aurein 2.2 from the others by showing that it perturbed the lipid membranes of intact S. aureus C622 most efficiently, whereas aurein 2.3 had the same efficiency as gramicidin S, and aurein 2.3-COOH was the least efficient. Taken together, these data show that the membrane interactions of aurein peptides are affected by the hydrophobic thickness of the lipid bilayers and the PG content.

Topics: Animals; Anti-Infective Agents; Antimicrobial Cationic Peptides; Anura; Benzothiazoles; Carbocyanines; Cell Membrane; Cell Membrane Permeability; Dimyristoylphosphatidylcholine; Fluoresceins; Gramicidin; Lipid Bilayers; Membrane Potentials; Phosphatidylcholines; Phosphatidylglycerols; Protein Structure, Secondary; Staphylococcus aureus

Lovastatin, an inhibitor of cellular cholesterol synthesis, has an apparent anti-cancer property, but the detailed mechanisms of its anti-cancer effects remain poorly understood. We investigated the molecular mechanism of Lovastatin anti-tumor function through the study of its effect on membrane ion flow, gap junctional intercellular communication (GJIC), and the pathways of related signals in MCF-7 mammary cancer cells. After treatment for 24-72 h with 4, 8 or 16 micromol/L Lovastatin, cellular proliferation was examined via the MTT assay, and changes in membrane potential and cellular [Ca(2+)](i) were monitored using confocal laser microscopy. In addition, the expression of plasma membrane calcium ATPase isoform 1 (PMCA1) mRNA was analyzed via RT-PCR, the GJIC function was examined using the scrape-loading dye transfer (SLDT) technique, and MAPK phosphorylation levels were tested with the kinase activity assay. The results showed that Lovastatin treatment significantly inhibited the growth of MCF-7 breast cancer cells. It also increased the negative value of the membrane potential, leading to the hyperpolarization of cells. Moreover, Lovastatin treatment continuously enhanced [Ca(2+)](i), although the levels of PMCA1 mRNA were unchanged. GJIC was also upregulated in MCF-7 cells, with transfer of LY Fluorescence reaching 4 to 5 rows of cells from the scraped line after treatment with 16 micromol/L Lovastatin for 72 h. Finally, downregulation of ERK1 and p38(MAPK) phosphorylation were found in Lovastatin-treated MCF-7 cells. It could be deduced that Lovastatin can induce changes in cellular hyperpolarization and intracellular Ca(2+) distributions, and increase GJIC function. These effects may result in changes in the downstream signal cascade, inhibiting the growth of MCF-7 cells.

Topics: Antineoplastic Agents; Benzothiazoles; Breast Neoplasms; Calcium; Carbocyanines; Cell Line, Tumor; Cell Proliferation; Enzyme Activation; Female; Fluorescence; Gap Junctions; Gene Expression Regulation, Neoplastic; Humans; Ion Transport; Lovastatin; Membrane Potentials; Microscopy, Confocal; Mitogen-Activated Protein Kinase 3; p38 Mitogen-Activated Protein Kinases; Plasma Membrane Calcium-Transporting ATPases; RNA, Messenger; Signal Transduction

The relationship between antibacterial activity of tetra-substituted tetrahydrofuran lignans (1-4) and their absolute configurations was tested. Only compound 4 among two virgatusins and two related compounds exhibited growth inhibitory activity against the Gram-positive bacteria Bacillus subtilis, Staphylococcus aureus and Listeria denitrificans. Compound 4 affected the growth of B. subtilis in a bactericidic manner, and its ability to dissipate the cytoplasmic membrane potential was investigated using the fluorescence probe 3,3'-dipropylthiadicarbocyanine iodide. These results suggested that compound 4 damages cells by causing the loss of the proton motive force and disruption of the cellular integrity of the membrane, leading to cell death. In addition, it was shown that the antibacterial activity of a lignan was closely related to its absolute configuration and functional groups.

Topics: Anti-Bacterial Agents; Benzothiazoles; Carbocyanines; Cell Membrane; Fluorescent Dyes; Furans; Gram-Positive Bacteria; Membrane Potentials; Microbial Viability

Morphine, a mu-opioid agonist, suppressed the Ca(2+)-dependent release of glutamate that was evoked by exposing cerebrocortical synaptosomes to the potassium channel blocker 4-aminopyridine. The presynaptic inhibition produced by morphine was concentration-dependent and blocked by the nonselective opioid receptor antagonist naloxone. As determined by examining the mechanism of mu-opioid receptor-mediated inhibition of glutamate release, morphine caused a significant reduction in 4-aminopyridine-evoked increase in the cytoplasmic free Ca(2+) concentration ([Ca(2+)](c)), but failed to alter both 4-aminopyridine-evoked depolarization of the synaptosomal plasma membrane potential and Ca(2+) ionophore (ionomycin)-induced glutamate release. In addition, morphine was not capable of producing further inhibition on 4AP-evoked glutamate release in synaptosomes pretreated with the cannabinoid CB(1) receptor agonist WIN 55212-2, which has been shown to depress glutamate release through a suppression of presynaptic voltage-dependent Ca(2+) channel activity. These data suggest that morphine exerts its inhibitory effect presynaptically, likely through the reduction of Ca(2+) influx into nerve terminals, and thereby inhibits the release of glutamate in the cerebral cortex. This may therefore indicate that mu-opioid receptor agonists have neuroprotective properties, especially in the excessive glutamate release that occurs under certain pathological conditions.

Topics: 4-Aminopyridine; Analgesics, Opioid; Animals; Benzothiazoles; Benzoxazines; Calcium; Calcium Channel Blockers; Carbocyanines; Cerebral Cortex; Dose-Response Relationship, Drug; Exocytosis; Fluorometry; Fura-2; Glutamic Acid; Ionomycin; Ionophores; Male; Membrane Potentials; Morphine; Morpholines; Naloxone; Naphthalenes; Narcotic Antagonists; Potassium Channel Blockers; Rats; Rats, Sprague-Dawley; Synaptosomes

Gramicidin S (GS) is a 10-residue cyclic beta-sheet peptide with lytic activity against the membranes of both microbial and human cells, i.e. it possesses little to no biologic specificity for either cell type. Structure-activity studies of de novo-designed 14-residue cyclic peptides based on GS have previously shown that higher specificity against microbial membranes, i.e. a high therapeutic index (TI), can be achieved by the replacement of a single L-amino acid with its corresponding D-enantiomer [Kondejewski, L.H. et al. (1999) J. Biol. Chem. 274, 13181]. The diastereomer with a D-Lys substituted at position 4 caused the greatest improvement in specificity vs. other L to D substitutions within the cyclic 14-residue peptide GS14, through a combination of decreased peptide amphipathicity and disrupted beta-sheet structure in aqueous conditions [McInnes, C. et al. (2000) J. Biol. Chem. 275, 14287]. Based on this information, we have created a series of peptide diastereomers substituted only at position 4 by a D- or L-amino acid (Leu, Phe, Tyr, Asn, Lys, and achiral Gly). The amino acids chosen in this study represent a range of hydrophobicities/hydrophilicities as a subset of the 20 naturally occurring amino acids. While the D- and L-substitutions of Leu, Phe, and Tyr all resulted in strong hemolytic activity, the substitutions of hydrophilic D-amino acids D-Lys and D-Asn in GS14 at position 4 resulted in weaker hemolytic activity than in the L-diastereomers, which demonstrated strong hemolysis. All of the L-substitutions also resulted in poor antimicrobial activity and an extremely low TI, while the antimicrobial activity of the D-substituted peptides tended to improve based on the hydrophilicity of the residue. D-Lys was the most polar and most efficacious substitution, resulting in the highest TI. Interestingly, the hydrophobic D-amino acid substitutions had superior antimicrobial activity vs. the L-enantiomers although substitution of a hydrophobic D-amino acid increases the nonpolar face hydrophobicity. These results further support the role of hydrophobicity of the nonpolar face as a major influence on microbial specificity, but also highlights the importance of a disrupted beta-sheet structure on antimicrobial activity.

Topics: 1-Naphthylamine; Amino Acid Substitution; Anti-Infective Agents; Benzothiazoles; Candida albicans; Carbocyanines; Cell Membrane Permeability; Circular Dichroism; Glycine; Gram-Negative Bacteria; Gram-Positive Bacteria; Gramicidin; Hemolysis; Humans; Hydrophobic and Hydrophilic Interactions; Membrane Potentials; Microbial Sensitivity Tests; Peptides, Cyclic; Protein Structure, Secondary; Structure-Activity Relationship; Yeasts

3,3'-Dipropyl-2,2'-thiadicarbocyanine iodide [DiS-C(3)(5)], often used as a tracer dye to assess the mitochondrial membrane potential, was investigated in detail regarding its effects on the structure and function of isolated mitochondria. As reported previously, DiS-C(3)(5) had an inhibitory effect on NADH-driven mitochondrial electron transfer. On the contrary, in the presence of inorganic phosphate, DiS-C(3)(5) showed dose-dependent biphasic effects on mitochondria energized by succinate. At higher concentrations, such as 50 micro m, DiS-C(3)(5) accelerated mitochondrial oxygen consumption. Measurements of the permeability of DiS-C(3)(5)-treated mitochondrial membranes to poly(ethylene glycol) and analysis of mitochondrial configuration by transmission electron microscopy revealed that the accelerating effect of DiS-C(3)(5) on mitochondrial oxygen consumption reflects the induction of the mitochondrial permeability transition (PT). When the mitochondrial PT was induced by DiS-C(3)(5), release of mitochondrial cytochrome c was observed, as in the case of the PT induced by Ca(2+). On the contrary, at a low concentration such as 5 micro m, DiS-C(3)(5) showed an inhibitory effect on the latent oxygen consumption by mitochondria. This effect was shown to reflect inhibition of the PT induced by a low concentration of Ca(2+). Furthermore, in the absence of inorganic phosphate, DiS-C(3)(5) caused mitochondrial swelling. Under this condition, DiS-C(3)(5) caused changes in the membrane status of the mitochondria, but did not induce a release of mitochondrial cytochrome c.

Topics: Animals; Benzothiazoles; Calcium; Carbocyanines; Cytochromes c; Dose-Response Relationship, Drug; Electron Transport; Fluorescent Dyes; Intracellular Membranes; Male; Mitochondria; Molecular Structure; NAD; Oxygen; Permeability; Phosphates; Rats; Rats, Wistar

Fluorescent dyes have been widely employed as optical indicators of the membrane potential difference in cells, isolated organelles and lipid vesicles that are too small to make microelectrode measurements feasible. We describe here the application of a carbocyanine dye, 3,3'-dipropylthiodicarbocyanine iodide [DiS-C3-(5)], to monitor the transmembrane potential changes induced by a variation of the K+ concentration for the cells of Escherichia (E.) coli and photosynthetic bacterium Rhodospirillum (R.) rubrum. The cells were first incubated in buffers containing DiS-C3-(5) and K+ ions of various concentrations until the fluorescence intensity reached a constant value. Valinomycin was then added to the solution, which caused changes in the fluorescence intensity, depending on the K+ concentrations. The membrane potential is shown to have a linear relationship with the fluorescence intensity of DiS-C3-(5). The results demonstrate that the K+ concentrations inside intact cells are 4.6 mM and 5.3 mM for E. coli and R. rubrum, respectively. The diffusion potentials of K+ ions were determined using the Nernst equation over the range of -1.3 mV to 44 mV, corresponding to K+ concentrations of 5 mM -25 mM outside of the cells.

Topics: Benzothiazoles; Biological Transport, Active; Carbocyanines; Cell Membrane; Escherichia coli; Fluorescent Dyes; Membrane Potentials; Potassium; Rhodospirillum rubrum; Valinomycin

We have previously shown that anacardic acid has an uncoupling effect on oxidative phosphorylation in rat liver mitochondria using succinate as a substrate (Life Sci. 66 (2000) 229-234). In the present study, for clarification of the physicochemical characteristics of anacardic acid, we used a cyanine dye (DiS-C3(5)) and 9-aminoacridine (9-AA) to determine changes of membrane potential (DeltaPsi) and pH difference (DeltapH), respectively, in a liposome suspension in response to the addition of anacardic acid to the suspension. The anacardic acid quenched DiS-C3(5) fluorescence at concentrations higher than 300 nM, with the degree of quenching being dependent on the log concentration of the acid. Furthermore, the K(+) diffusion potential generated by the addition of valinomycin to the suspension decreased for each increase in anacardic acid concentration used over 300 nM, but the sum of the anacardic acid- and valinomycin-mediated quenching was additively increasing. This indicates that the anacardic acid-mediated quenching was not due simply to increments in the K(+) permeability of the membrane. Addition of anacardic acid in the micromolar range to the liposomes with DeltaPsi formed by valinomycin-K(+) did not significantly alter 9-AA fluorescence, but unexpectedly dissipated DeltaPsi. The DeltaPsi preformed by valinomycin-K(+) decreased gradually following the addition of increasing concentrations of anacardic acid. The DeltaPsi dissipation rate was dependent on the pre-existing magnitude of DeltaPsi, and was correlated with the logarithmic concentration of anacardic acid. Furthermore, the initial rate of DeltapH dissipation increased with logarithmic increases in anacardic acid concentration. These results provide the evidence for a unique function of anacardic acid, dissimilar to carbonylcyanide p-trifluoromethoxyphenylhydrazone or valinomycin, in that anacardic acid behaves as both an electrogenic (negative) charge carrier driven by DeltaPsi, and a 'proton carrier' that dissipates the transmembrane proton gradient formed.

Topics: Aminacrine; Anacardic Acids; Benzothiazoles; Carbocyanines; Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; Dose-Response Relationship, Drug; Fluorescent Dyes; Gramicidin; Hydrogen-Ion Concentration; Liposomes; Membrane Potentials; Models, Chemical; Molecular Structure; Salicylates; Spectrometry, Fluorescence; Valinomycin

Erythrocytes can reduce extracellular ascorbate free radicals by a plasma membrane redox system using intracellular ascorbate as an electron donor. In order to test whether the redox system has electrogenic properties, we studied the effect of ascorbate free radical reduction on the membrane potential of the cells using the fluorescent dye 3,3'-dipropylthiadicarbocyanine iodide. It was found that the erythrocyte membrane depolarized when ascorbate free radicals were reduced. Also, the activity of the redox system proved to be susceptible to changes in the membrane potential. Hyperpolarized cells could reduce ascorbate free radical at a higher rate than depolarized cells. These results show that the ascorbate-driven reduction of extracellular ascorbate free radicals is an electrogenic process, indicating that vectorial electron transport is involved in the reduction of extracellular ascorbate free radical.

Topics: Ascorbic Acid; Benzothiazoles; Carbocyanines; Cell Membrane; Erythrocytes; Fluorescent Dyes; Free Radicals; Humans; In Vitro Techniques; Membrane Potentials; Oxidation-Reduction

A fluorometric assay for mitochondrial membrane potential in permeabilized yeast cells has been developed. This method involves permeabilizing the plasma membrane and measuring the distribution of a mitochondrial membrane potential sensitive probe 3,3'-dipropylthiadicarbocyanine iodide (DiSC(3)(5); DiSC(3)). In permeabilized cells, DiSC(3) fluorescence decreased when introduced into energized mitochondria and increased three- to sixfold when the mitochondrial membrane potential was dissipated by the chemical uncoupler carbonylcyanide m-chlorophenyl hydrazone. Plasma membrane potential was abolished by permeabilization, as shown by a lack of polarization of the plasma membrane induced by K(+) and glucose. Uncoupling protein 1 (UCP1), a mitochondrial H(+) transporter, was used as a model for method validation. The fluorescence intensity responded vigorously to specific modulators in UCP1-expressing cells. This method has been adapted as a high-throughput assay to screen for modulators of mitochondrial membrane potential.

Topics: Benzothiazoles; Biological Assay; Carbocyanines; Carbonyl Cyanide m-Chlorophenyl Hydrazone; Carrier Proteins; Cell Membrane Permeability; Dinitrophenols; Glucosephosphate Dehydrogenase; Iodides; Ion Channels; Ionophores; Membrane Potentials; Membrane Proteins; Mitochondria; Mitochondrial Proteins; Spectrometry, Fluorescence; Uncoupling Agents; Uncoupling Protein 1; Valinomycin; Yeasts

The DNA binding behavior of a tricationic cyanine dye (DiSC3+(5)) was studied using the [Poly(dA-dT)]2, [Poly(dI-dC)]2 and Poly(dA) x Poly(dT) duplex sequences and the Poly(dA) x 2Poly(dT) triplex. Optical spectroscopy and viscometry results indicate that the dye binds to the triplex structure by intercalation, to the nonalternating Poly(dA) x Poly(dT) duplex through minor groove binding and to the alternating [Poly(dA-dT)]2 duplex by a combination of two binding modes: intercalation at low concentration and dimerization within the minor groove at higher concentration. Dimerization occurs at lower dye concentrations for the [Poly(dI-dC)]2 sequence, consistent with our previous investigations on an analogous monocationic cyanine dye. [Seifert, J.L., et al. (1999) J. Am. Chem. Soc. 121, 2987-2995] These studies illustrate the diversity of DNA binding modes that are available to a given ligand structure.

Topics: Base Pairing; Base Sequence; Benzothiazoles; Carbocyanines; Cations; Circular Dichroism; Coloring Agents; Dimerization; Intercalating Agents; Molecular Structure; Nucleic Acid Conformation; Poly A; Poly dA-dT; Poly T; Polydeoxyribonucleotides; Spectrometry, Fluorescence; Temperature; Viscosity

The influence of cholesterol on the sphingomyelin (SM)/dioleoylphosphatidylcholine (DOPC) binary system was investigated in various respects. Electron spin resonance (ESR) measurements reveal that the order parameter of 5DS (5-doxyl stearic acid) in SM/DOPC bilayers increases notably when the concentration of cholesterol is over 30 mol%. Membrane potential measurements indicate that the K+ permeability of the SM/DOPC bilayer decreases steeply at 40 mol% cholesterol concentration. Both these experiments suggest that cholesterol reduces the motion amplitude of hydrocarbon chains abruptly above 30 mol%. In contrast to the ordering effects on the hydrocarbon chains, (31)P-NMR results indicate that cholesterol slightly increases the motion of phosphate groups of the lipids. (31)P-NMR also raises the possibility of domain formation in the presence of cholesterol. Fluorescence-quenching experiments verified that solid domains appear in the binary system when cholesterol is present, and percolation threshold occurs at 50 mol% cholesterol concentration. The solid domains bear the properties of liquid ordered phase, which is the basic structure of caveolae and functional rafts. So this work provides an artificial model for the study of rafts and caveolae on biological membranes.

Topics: Animals; Benzothiazoles; Biophysical Phenomena; Biophysics; Carbocyanines; Cholesterol; Egg Yolk; Electron Spin Resonance Spectroscopy; Fluorescence; Lipid Bilayers; Magnetic Resonance Spectroscopy; Membrane Lipids; Membrane Potentials; Membranes, Artificial; Phosphatidylcholines; Sphingomyelins; Spin Labels; Temperature; Time Factors

The effects of phase transition from normal to interdigitated lipid bilayer on the function and structure of membrane proteins were studied using linear gramicidin (gramicidin A) as a model. Interdigitated bilayer structure of dipalmitoylphosphatidylglycerol (DPPG) liposomes that was induced by atropine could not be changed notably by intercalating of gramicidin. The K+ transportation of gramicidin in both normal and interdigitated bilayer was assayed by measuring the membrane potential. Results showed that gramicidin in interdigitated bilayer exhibited lower transport capability. Intrinsic fluorescence spectrum of gramicidin in interdigitated bilayer blue-shifted 2.8 nm from the spectrum in normal bilayer, which means that interdigitation provides a more hydrophobic environment for gramicidin. Circular dichroism measurement results indicated that the conformation of gramicidin in interdigitated bilayer is not the typical beta6.3 helix as in the normal bilayer. The results suggested that the interdigitated lipid bilayer might largely affect the structure and function of membrane proteins.

Topics: 1,2-Dipalmitoylphosphatidylcholine; Benzothiazoles; Carbocyanines; Circular Dichroism; Fluorescent Dyes; Gramicidin; Intercalating Agents; Kinetics; Lipid Bilayers; Membrane Potentials; Protein Conformation; Spectrometry, Fluorescence; Thermodynamics

When human sperm was incubated in medium deprived of glucose, glucose restoration caused a transient hyperpolarization of the plasma membrane. This hyperpolarization was also induced by fructose but not by 2-deoxyglucose, a substrate that cannot be metabolized. The hyperpolarization was inhibited by NaF, a glycolysis inhibitor, but not by mitochondrial inhibitors (cyanide, rotenone and antimycin), suggesting that it depended on glycolysis. Furthermore, the hyperpolarization was still induced in medium containing a high concentration of KCl and was insensitive to the K(+) channel blocker TEA and the Cl(-) channel blocker niflumic acid, but it was blocked by ouabain. This suggested that upon glucose addition, there was an increase in the concentration of ATP, that in turns increased the Na(+),K(+)-ATPase activity. Since this pump is electrogenic (2K(+)/3Na(+)) the plasma membrane hyperpolarized. On the other hand, CCCP, a proton ionophore, inhibited the hyperpolarization induced by glucose. When CCCP was added to glucose-treated hyperpolarized sperm, it caused a depolarization that triggered a Ca(2+) influx sensitive to nickel, an inhibitor of voltage-dependent calcium channels. Moreover, CCCP caused hyperpolarization in sperm incubated in medium without calcium, a known condition that depolarizes sperm. This indicated that CCCP induced proton permeability in the plasma membrane that was able to change the membrane potential to a value corresponding to the E(H) and that was also able to clamp it, so that it prevented the hyperpolarization induced by glucose.

Topics: Benzothiazoles; Carbocyanines; Carbonyl Cyanide m-Chlorophenyl Hydrazone; Cell Membrane; Fluorescent Dyes; Fura-2; Glucose; Humans; Ionophores; Male; Membrane Potentials; Mitochondria; Sodium-Potassium-Exchanging ATPase; Spermatozoa

In micromolar concentrations both antagonists suppressed CA2+ entry and simultaneously elevate the agonist-induced plasma membrane depolarization due to Na+ inward current via these channels. Potentiation by nicardipine of the Na+ current induced by the platelet-activating factor, was revealed. Both antagonists caused plasma membrane depolarization suppressed by Na+ and Ca2+ ions. The depolarization disappeared after substitution of NaCl by an isotonic solution of choline chloride. The antagonists nicardipine and verapamil seem to modulate the platelet receptor-operated channels suppressing Ca2+ entry and elevating Na+ current via these channels.

Topics: Benzofurans; Benzothiazoles; Blood Platelets; Calcium; Calcium Channel Blockers; Carbocyanines; Ethers, Cyclic; Fluorescent Dyes; Humans; In Vitro Techniques; Nicardipine; Sodium; Sodium Channels; Verapamil

Erythrocytes of diabetic patients have abnormal membrane properties. We examined in vitro transmembrane potential and the possible effect of resorcylidene aminoguanidine (RAG) on its modulation in erythrocytes of diabetic subjects. The transmembrane potential was assessed in RAG-treated and untreated erythrocytes, respectively, using a fluorescent dye (3,3'-dipropylthiadicarbocyanine iodide [DiSC3(5)]). We confirmed earlier findings that the transmembrane potential of diabetic erythrocytes is significantly increased compared with control (P < 0.01). The membrane hyperpolarization found in diabetic cells seems to be a result of oxidative stress present in diabetes mellitus. On one hand, the RAG treatment induced decrease in abnormal transmembrane potential values in diabetic erythrocytes (P < 0.01), presumably via its antioxidant and antiglycation activity. On the other hand, RAG moderately hyperpolarized the control erythrocytes (P < 0.05). We suggest that the drug-induced transient membrane expansion leads to an intracellular potassium loss and a subsequent change of the transmembrane potential. However, if controlled by an appropriate dosage, RAG can eliminate certain types of erythrocyte membrane damage induced by diabetes mellitus.

Topics: Benzothiazoles; Carbocyanines; Diabetes Mellitus, Type 1; Erythrocyte Membrane; Fluorescent Dyes; Guanidines; Humans; Membrane Potentials

Antimicrobial cationic peptides are prevalent throughout nature as part of the intrinsic defenses of most organisms, and have been proposed as a blueprint for the design of novel antimicrobial agents. They are known to interact with membranes, and it has been frequently proposed that this represents their antibacterial target. To see if this was a general mechanism of action, we studied the interaction, with model membranes and the cytoplasmic membrane of Escherichia coli, of 12 peptides representing all 4 structural classes of antimicrobial peptides. Planar lipid bilayer studies indicated that there was considerable variance in the interactions of the peptides with model phospholipid membranes, but generally both high concentrations of peptide and high transmembrane voltages (usually -180 mV) were required to observe conductance events (channels). The channels observed for most peptides varied widely in magnitude and duration. An assay was developed to measure the interaction with the Escherichia coli cytoplasmic membrane employing the membrane potential sensitive dye 3,5-dipropylthiacarbocyanine in the outer membrane barrier-defective E. coli strain DC2. It was demonstrated that individual peptides varied widely in their ability to depolarize the cytoplasmic membrane potential of E. coli, with certain peptides such as the loop peptide bactenecin and the alpha-helical peptide CP26 being unable to cause depolarization at the minimal inhibitory concentration (MIC), and others like gramicidin S causing maximal depolarization below the MIC. We discuss the mechanism of interaction with the cytoplasmic membrane in terms of the model of Matsuzaki et al. [(1998) Biochemistry 37, 15144-15153] and the possibility that the cytoplasmic membrane is not the target for some or even most cationic antimicrobial peptides.

Topics: Amino Acid Sequence; Anti-Bacterial Agents; Antimicrobial Cationic Peptides; Benzothiazoles; Carbocyanines; Cell Membrane; Cell Membrane Permeability; Escherichia coli; Fluorescent Dyes; Lipid Bilayers; Membrane Potentials; Models, Chemical; Molecular Sequence Data; Peptides, Cyclic; Potassium Chloride; Protein Structure, Secondary; Proteins

The aim of our investigation was to study the red blood cell (RBC) membrane effects of NaNO(2)-induced oxidative stress. Hyperpolarization of erythrocyte membranes and an increase in membrane rigidity have been shown as a result of RBC oxidation by sodium nitrite. These membrane changes preceded reduced glutathione depletion and were observed simultaneously with methemoglobin (metHb) formation. Changes of the glutathione pool (total and reduced glutathione, and mixed protein-glutathione disulfides) during nitrite-induced erythrocyte oxidation have been demonstrated. The rates of intracellular oxyhemoglobin and GSH oxidation highly increased as pH decreased in the range of 7.5-6.5. The activation energy of intracellular metHb formation obtained from the temperature dependence of the rate of HbO(2) oxidation in RBC was equal to 16.7+/-1.6 kJ/mol in comparison with 12.8+/-1.5 kJ/mol calculated for metHb formation in hemolysates. It was found that anion exchange protein (band 3 protein) of the erythrocyte membrane does not participate significantly in the transport of nitrite ions into the erythrocytes as band 3 inhibitors (DIDS, SITS) did not decrease the intracellular HbO(2) oxidation by extracellular nitrite.

Topics: Benzothiazoles; Biological Transport; Carbocyanines; Erythrocyte Membrane; Erythrocytes; Glutathione; Humans; Hydrogen-Ion Concentration; Membrane Potentials; Methemoglobin; Oxidation-Reduction; Sodium Nitrite; tert-Butylhydroperoxide; Thiobarbituric Acid Reactive Substances

This study aimed to investigate the membrane potential and fluidity changes of human red blood cells subjected to isotonic glucose solutions. For the control erythrocytes a membrane potential value of -10.1 +/- 1.8 mV was obtained, whereas erythrocyte membranes in diabetic cells were hyperpolarized, with a potential of -13.9 +/- 2.3 mV. Incubation of both types of red blood cells with increasing glucose concentrations resulted in a substantial hyperpolarization of the cell membranes. Glucose solutions in water had a much stronger effect on membrane potential than glucose dissolved in phosphate-buffered saline. Red blood cell membrane fluidity measurements using the fluorescent label TMADPH did not reveal any significant changes upon incubation with glucose.

Topics: Benzothiazoles; Carbocyanines; Diabetes Mellitus; Diphenylhexatriene; Erythrocyte Membrane; Fluorescent Dyes; Glucose; Humans; Isotonic Solutions; Membrane Fluidity; Membrane Potentials

Pentobarbital sodium is a widely used anesthetic for animal experiments. We have already reported that a high dose of pentobarbital sodium (100 mg/kg i.p., twice the usual anesthetic dose) depresses myocardial mechanoenergetics in the canine heart. Furthermore, it has been reported that a high concentration of pentobarbital sodium inhibits myocardial contractility and mitochondrial electron transport and energy transfer. Therefore, we were afraid that the mitochondrial respiratory function would be impaired by pentobarbital anesthesia and that pentobarbital sodium would be an inappropriate anesthetic for cardiac mechanoenergetic studies. In this study, we investigated the respiratory function of mitochondria isolated from the hearts of anesthetized rats with high-dose pentobarbital sodium. We examined the mitochondrial respiratory function by the ADP/O ratio, respiratory control index (RCI), oxygen consumption rate in state III (State III O2) by oximetry, and membrane potential using a fluorescent dye, 3,3'-dipropylthiodicarbocyanine iodide (diS-C3-(5). The ADP/O ratio, RCI, and State III O2 values and changes in membrane potential induced by various respiratory substances were not significantly different between the rats with and without anesthesia. These results indicate that the respiratory function is not suppressed in the isolated myocardial mitochondria of rat hearts after high-dose pentobarbital anesthesia, although the pentobarbital sodium blood concentration was of the same order as that which exerts mitochondrial uncoupling in rat isolated mitochondrial preparation. Therefore, pentobarbital sodium anesthesia up to 100 mg/kg i.p. is applicable for mechanoenergetic studies of excised rat hearts, at least from the energetic aspect.

Topics: Adjuvants, Anesthesia; Anesthesia, General; Animals; Benzothiazoles; Carbocyanines; Dose-Response Relationship, Drug; Female; Fluorescent Dyes; Male; Membrane Potentials; Mitochondria, Heart; Oximetry; Oxygen Consumption; Pentobarbital; Rats; Rats, Wistar

Topics: Animals; Apamin; Benzothiazoles; Binding Sites; Calcium; Calcium Channels; Carbocyanines; Enzyme Inhibitors; Erythrocytes; Fluorescent Dyes; Membrane Potentials; Potassium Channel Blockers; Potassium Channels; Rats; Second Messenger Systems; Staurosporine; Tetraethylammonium; Tetraethylammonium Compounds

A photokinetic method of detection of fluorescence resonance energy transfer (FRET) between special fluorescent labels is applied to study time-averaged spatial distribution of labeled proteins in protein assemblies. Prolonged irradiation of a sample at the absorption maximum of the energy donor label initiates FRET-sensitized fluorescence photobleaching of the energy acceptor label, which was monitored by steady-state fluorimetric measurements. Kinetics of the acceptor photobleaching and kinetics of decreasing the efficiency of FRET from donors to unbleached acceptors were determined. The FRET efficiency was found from measuring sensitization of acceptor fluorescence. Analysis of the photokinetic data permits to estimate the time-averaged distribution of acceptors on donor-acceptor distances in the range of characteristic distances of FRET. Dynamic processes influencing donor-acceptor distances can be also investigated by the method. Application of the method is demonstrated by the studies of a complex of biotinylated IgM with streptavidin and aggregates composed of concanavalin A and sodium dodecyl sulphate. A new thiadicarbocyanine dye was used as the acceptor label, R-phycoerythrin and tetramethylrhodamine isothio-cyanate were the donor labels. In the IgM-streptavidin complex, 16% of acceptors most contributed to FRET provided 90% of FRET efficiency, whereas acceptors made about the same time-averaged contribution to FRET in the concanavalin A aggregates.

Topics: Animals; Benzothiazoles; Biotin; Biotinylation; Carbocyanines; Concanavalin A; Energy Transfer; Fluorescence; Fluorescent Dyes; Humans; Immunoglobulin M; Mice; Molecular Structure; Phycoerythrin; Rhodamines; Sodium Dodecyl Sulfate; Streptavidin

Topics: Aerobiosis; Anaerobiosis; Benzothiazoles; Carbocyanines; Fluorescent Dyes; Glucose; Membrane Potentials; Potassium; Saccharomyces cerevisiae; Spectrometry, Fluorescence; Uncoupling Agents

Charge translocation by the Kdp-ATPase of Escherichia coli was measured by adsorption of proteoliposomes to a planar lipid membrane. The proteoliposomes were prepared by reconstitution of purified Kdp-ATPase into liposomes prepared from E. coli lipids. The protein was activated by a ATP concentration jump produced by photolysis of a protected derivative of ATP, caged ATP. Charge translocation was measured with a time resolution of 15-40 ms. Stationary currents demonstrated the continuous pumping activity of the enzyme. Control measurements with the potential-sensitive dye DiSC3(5) showed a negative potential inside the proteoliposomes after activation with ATP. The measured electrical signals as well as the dye measurements correspond to the transport of positive charge to the intracellular face of the protein. The electrical signal was increased when K+ was inside the proteoliposomes (K0.5 approximately 50 microM) and was inhibited by vanadate. These experiments demonstrate the electrogeneity of the Kdp-ATPase in a purified reconstituted system.

Topics: Adenosine Triphosphatases; Adenosine Triphosphate; Benzothiazoles; Carbocyanines; Carrier Proteins; Cation Transport Proteins; Enzyme Activation; Escherichia coli; Escherichia coli Proteins; Fluorescent Dyes; Kinetics; Liposomes; Osmolar Concentration; Potassium; Proteolipids; Spectrometry, Fluorescence

A synthetic peptide with the sequence of the first 20 residues of melittin and terminating with an additional cysteine amide was found to have cytolytic activity similar to that of melittin. It was apparent from MS data that the cysteine-terminating peptides had formed disulphide dimers. A peptide in which the thiol was blocked by iodoacetate showed no activity, whereas the same peptide blocked by acetamidomethyl showed activity marginally less haemolytic than that of melittin. Cytolytic activity of melittin analogues comprising the full 26 residues could be obtained with wide sequence permutations providing that a general amphipathic helical structure was preserved. In contrast, the activity of the dimers was dependent not only on retention of an amphipathic helix but also on certain individual residues and a free positive charge. A free N-terminus was essential for haemolytic activity. In addition, a lysine or arginine residue at position 7 and a proline at position 14 were found to be necessary for activity, although it was apparent that additional residues are important for retention of the full lytic potential.

Topics: Amino Acid Sequence; Benzothiazoles; Carbocyanines; Cell Survival; Cysteine; Disulfides; Flow Cytometry; Fluorescent Dyes; Hemolysis; Humans; Melitten; Membrane Potentials; Molecular Sequence Data; Peptides; Protein Conformation; Tumor Cells, Cultured

Three fluorescent probes, tetramethyl rhodamine ethyl ester (TMRE), 3,3'-dipropylthiacarbocyanine iodide (diS-C3(3)) and 3,3'-dipropyloxacarbocyanine iodide (diO-C3(3)), were tested for their suitability as fluorescent indicators of membrane potential in Saccharomyces cerevisiae in studies performed by flow cytometry. For all these dyes the intensity of fluorescence of stained cells increased with probe concentration in the range of 60-3000 nmol/L. The optimum staining period was 15-20 min for diS-C3(3). Depolarization of cells by increased extracellular potassium level and by valinomycin elicited with all probes a drop in fluorescence intensity. In some yeast batches this depolarization was accompanied by a separation of subpopulations with different fluorescence properties.

Topics: Artifacts; Benzothiazoles; Carbocyanines; Flow Cytometry; Fluorescence; Fluorescent Dyes; Fluorometry; Indicators and Reagents; Membrane Potentials; Organometallic Compounds; Pyridinium Compounds; Saccharomyces cerevisiae; Scattering, Radiation; Time Factors

We demonstrate for the first time in this study that triadic vesicles derived from skeletal muscle display a voltage dependence of depolarization-induced calcium release similar to that found in intact muscle. We confirm previous studies by Dunn (1989) which demonstrated that changes in extravesicular potassium induced membrane potential changes in isolated transverse tubules with the voltage sensitive dye DiSC(3)-5. Depolarization-induced calcium release was studied in isolated triadic vesicles through similar changes in extravesicular [K] while clamping extravesicular Ca++ to submicromolar concentrations. The amplitude of fast phase of calcium release, identified as depolarization-induced calcium release, varied with the percentage of transverse tubules in the preparation (determined through 3H-PN200-110 specific activity) and different levels of depolarization. Threshold activation of calcium release was obtained with a 40.5 mV potential change; maximal calcium release was obtained with a 75 to 81 mV potential change. Boltzmann fits to the normalized depolarization induced calcium release plotted against the membrane potential change yielded a voltage dependence (k = 4.5 mV per e-fold change) very similar to that found in intact muscle (k = 3-4 mV per e-fold change; Baylor, Chandler & Marshall 1978, 1983; Miledi et al., 1981). Substitution of methanesulfonate for propionate as the impermeant ion or addition of valinomycin in the depolarizing solutions had little effect on the voltage dependence of calcium release.

Topics: Adenosine Triphosphate; Animals; Benzothiazoles; Calcium; Carbocyanines; Fluorescent Dyes; In Vitro Techniques; Isradipine; Kinetics; Membrane Potentials; Muscle, Skeletal; Nitrendipine; Potassium; Rabbits; Ryanodine; Signal Transduction; Spectrometry, Fluorescence; Tritium

In many cell types depletion of the intracellular Ca2+ stores promotes divalent cation entry across the plasma membrane, although the mechanism of such store-regulated Ca2+ entry remains unclear. It has been suggested that cytochrome P-450 plays a role in the communication from intracellular stores to plasma membrane in human platelets and other cells. These studies involved the use of the imidazole antimycotics, econazole and miconazole, which are inhibitors of cytochrome P-450. Here we report additional effects of the imadazole antimycotics, which we show to inhibit agonist-evoked protein-tyrosine phosphorylation and to evoke plasma membrane depolarisation in human platelets. Both of these effects might be expected to influence agonist-evoked Ca2+ entry in these and other cells. These data suggest that great caution is required in interpreting the results from studies using imadazole antimycotics and in particular imply that the effects of these inhibitors cannot be taken as evidence for a role for cytochrome P-450 in the Ca2+ entry pathway.

Topics: Adenosine Diphosphate; Benzothiazoles; Blood Platelets; Calcium; Carbocyanines; Cell Membrane; Cytochrome P-450 Enzyme Inhibitors; Econazole; Fluorescent Dyes; Humans; Membrane Potentials; Miconazole; Phosphorylation; Phosphotyrosine; Signal Transduction; Tyrosine

The search for improved photosensitizers for laser phototherapy of malignancies has led to the examination of a new group of carbocyanine dyes as effective fluorochromes. In this study, four carbocyanine dyes with different absorption maxima of 483 nm [DiOC6(3)], 545.5 nm (DiIC5(3)], 556.6 nm [DiSC5(3)], and 651.0 nm [DiSC3(5)] were tested in vitro. The kinetics of uptake and toxicity of these four dyes were assessed for P3 human squamous cell carcinoma, HT29 colon carcinoma, M26 melanoma, and TE671 fibrosarcoma cell lines at 15, 30, 45, 60, and 180 minutes after exposure with each dye. After sensitization with DiOC6(3), the P3 and M26 cell lines were also tested for phototherapy by treatment with 488-nm light from an argon laser. The results showed that these four carbocyanine dyes had rapid and significant uptake by the carcinoma cell lines with no toxicity at concentrations < 0.1 micrograms/mL. Nontoxic DiOC6(3) levels in sensitized tumor cells after laser phototherapy resulted in approximately 85% inhibition of P3 and approximately 95% inhibition of M26 cell lines by MTT assays. The results suggest that these carbocyanine dyes can be used for tumor photosensitization and wavelength-matched laser photodynamic therapy. Further in vivo studies will be necessary to define the clinical potential of carbocyanine dyes as tumor-targeting agents for phototherapy of cancer.

Topics: Adenocarcinoma; Argon; Benzothiazoles; Carbocyanines; Carcinoma, Squamous Cell; Cell Survival; Colonic Neoplasms; Fibrosarcoma; Fluorescent Dyes; Humans; Laser Therapy; Lung Neoplasms; Medulloblastoma; Melanoma; Neoplasms; Photochemotherapy; Tetrazolium Salts; Tumor Cells, Cultured

Upon exposure of human red blood cells to hypertonic sucrose, the fluorescence of the potentiometric indicator 3,3'-dipropylthiadicarbocyanine iodide, denoted diS-C3(5), displays a biphasic time course indicating the rapid development of an inside-positive transmembrane voltage, followed by a slow DIDS (4,4'-diisothiocyano-2,2'-disulfonic acid stilbene)-sensitive decline of the voltage. In addition to monitoring membrane potential, proton (or hydroxide) fluxes were measured by a pH stat method, cell volume was monitored by light scattering, and cell electrolytes were measured directly when red cells were shrunken either with hypertonic NaCl or sucrose. Shrinkage by sucrose induced an initial proton efflux (or OH- influx) of 5.5 mu eq/g Hb.min and a Cl shift of 21-31 mu eq/g Hb in 15 min. Upon shrinkage with hypertonic NaCl, the cells are initially close to Donnan equilibrium and exhibit no detectable shift of Cl or protons. Experiments with the carbonic anhydrase inhibitor ethoxzolamide demonstrate that for red cell suspensions exposed to air and shrunken with sucrose, proton fluxes mediated by the Jacobs-Stewart cycle contribute to dissipation of the increased outward Cl concentration gradient. With maximally inhibitory concentrations of ethoxzolamide, a residual proton efflux of 2 mu eq/g Hb.min is insensitive to manipulation of the membrane potential with valinomycin, but is completely inhibited by DIDS. The ethoxzolamide-insensitive apparent proton efflux may be driven against the electrochemical gradient, and is thus consistent with HCl cotransport (or Cl/OH exchange). The data are consistent with predictions of equations describing nonideal osmotic and ionic equilibria of human red blood cells. Thus osmotic equilibration after shrinkage of human red blood cells by hypertonic sucrose occurs in two time-resolved steps: rapid equilibration of water followed by slower equilibration of chloride and protons (or hydroxide). Under our experimental conditions, about two-thirds of the osmotically induced apparent proton efflux is mediated by the Jacobs-Stewart cycle, with the remainder being consistent with mediation via DIDS-sensitive HCl cotransport (or Cl/OH exchange).

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Benzothiazoles; Body Water; Carbocyanines; Chlorides; Erythrocytes; Ethoxzolamide; Hemoglobins; Humans; Hydroxides; In Vitro Techniques; Indicators and Reagents; Light; Membrane Potentials; Osmolar Concentration; Potassium; Protons; Saline Solution, Hypertonic; Scattering, Radiation; Sodium; Sucrose

We examined whether swelling-activated K-Cl cotransport is electrogenic in human erythrocytes. Baseline membrane potential, measured by the change in fluorescence of the carbocyanine dye diS-C3-5, was not different in hypotonically swollen (-7.6 mV) or isosmotically swollen cells (-9.5 mV). We used hemisodium, a new highly selective Na ionophore, in varying concentrations, in the presence of a fixed outwardly directed Na gradient (intracellular Na, 75 mM; external Na, 1 mM) to vary membrane potential over a wide range despite identical K and Cl concentrations. The membrane potential varied between -8 and -90 mV. K influx increased slightly with hyperpolarization in swollen and nonswollen cells. However, the difference between the two fluxes, swelling-activated K influx, a measure of K-Cl cotransport, was unaffected by voltage changes, as was swelling-activated K efflux. We conclude that K-Cl cotransport in human erythrocytes is electroneutral and by inference has a 1:1 stoichiometry.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Benzothiazoles; Biological Transport; Calibration; Carbocyanines; Erythrocytes; Fluorescent Dyes; Hematocrit; Homeostasis; Humans; Ionophores; Membrane Potentials; Potassium; Potassium Chloride; Sodium

The rabbit intestinal brush-border membrane Na(+)-phosphate cotransporter was purified from sodium dodecyl sulfate (SDS)-brush-border membrane vesicles (BBMV) protein (SDS-treated Ca(2+)-precipitated BBMV) by a three-column chromatography protocol. The purification included a preparative scale chromatofocusing chromatography column over the pH range from 7.4 to 4 after solubilization in 3-[(3-cholamidopropyl)-diamethylammonia]-1-propanesulfonate (CHAPS), a chromatofocusing column over the pH range from 5.6 to 4 after solubilization in n-octyl glucoside, and gel filtration chromatography on a Sephacryl S-200 column. Verification of Na(+)-phosphate cotransporter purification involved substrate affinities, substrate stoichiometry, and inhibitor sensitivity after proteoliposome reconstitution and SDS-polyacrylamide gel electrophoresis (PAGE). After gel filtration Na(+)-dependent phosphate uptake was 3,300-fold enriched compared with the cell homogenate. A single 130-kDa polypeptide was visualized by SDS-PAGE under reducing conditions using silver stain. The coenrichment of this 130-kDa polypeptide and proteoliposome reconstituted Na(+)-dependent phosphate uptake suggest that the intestinal brush-border membrane Na(+)-phosphate cotransporter has been purified and proteoliposome reconstituted.

Topics: Animals; Benzothiazoles; Carbocyanines; Carrier Proteins; Cholic Acids; Electrophoresis, Polyacrylamide Gel; Fluorescent Dyes; Intestinal Mucosa; Kinetics; Liposomes; Membrane Potentials; Microvilli; Molecular Weight; Phosphates; Proteolipids; Rats; Sodium-Phosphate Cotransporter Proteins; Symporters

The membrane potential responsiveness of human myeloid leukemia cells (ML-1 line) was studied with the voltage sensitive fluorescent dye diS-C3-(5). The experimental procedure used in this study enabled us to assess the magnitude of the membrane potential change in cells treated with ouabain, 12-0-tetradecanoylphorbol-13-acetate (TPA) and N-formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP), relative to the membrane potential in the untreated control. Inhibition of the Na, K-ATPase by ouabain was followed by a (20 +/- 4) mV depolarization. In undifferentiated homogeneous cell population TPA caused a (19.4 +/- 4.4) mV depolarization while FMLP had virtually no effect. Cells in which granulocytic or monocytic differentiation was induced by retinoic acid or 1,25-dihydroxyvitamin D3 exhibited under the effect of TPA a (57.8 +/- 7.1) mV and (34.8 +/- 10.9) mV depolarization, respectively. A very small transient depolarization was also observed up on treating of the cells with FMLP. The changes in the membrane potential responsiveness in the induced cells are obviously connected with the cell differentiation.

Topics: Benzothiazoles; Carbocyanines; Cell Differentiation; Fluorescence; Granulocytes; Humans; Kinetics; Leukemia, Myeloid; Membrane Potentials; Monocytes; N-Formylmethionine Leucyl-Phenylalanine; Ouabain; Sodium-Potassium-Exchanging ATPase; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured; Valinomycin

To clarify the inhibitory mechanisms by triphenyltin chloride (TPTCl) on superoxide anion formation in neutrophils, the effects of phenyltin compounds [TPTCl, diphenyltin dichloride (DPTCl2) and phenyltin trichloride (MPTCl3) on the increase of cytosolic free calcium and the changes in membrane potential in neutrophils stimulated by n-formyl-methionyl-leucyl-phenylalanine (FMLP) were examined. TPTCl and DPTCl2 concentration dependently inhibited the increase of fluorescence intensity of the dye 3,3'-dipropyl-thiodicarbocyanine iodide [diS-C3-(5)] (membrane potential probe) in neutrophils induced by 0.1 microM FMLP in the presence or absence of extracellular calcium (1.26 mM). TPTCl had a greater inhibitory effect on FMLP-mediated membrane potential change than that of DPTCl2. In the presence of extracellular calcium, TPTCl and DPTCl2 increased intracellular free calcium [( Ca2+]i) of unstimulated fura-2-loaded neutrophils at concentrations from 1.0 to 10 microM TPTCl and from 2.5 to 10 microM DPTCl2. TPTCl and DPTCl2 also increased slightly, in the absence of extracellular calcium, [Ca2+]i without stimulation of FMLP in neutrophils. However, TPTCl and DPTCl2 significantly inhibited the rise of [Ca2+]i in neutrophils stimulated by FMLP at concentrations from 2.5 microM to 10 microM TPTCl and at a concentration of 10 microM DPTCl2 in the absence of extracellular calcium. TPTCl and DPTCl2 significantly inhibited the superoxide anion production by FMLP at concentrations over 2.5 microM in the presence of extracellular calcium. In the absence of extracellular calcium, TPTCl and DPTCl2 also inhibited the superoxide anion production by FMLP at concentrations over 1.5 microM TPTCl and over 5.0 microM DPTCl2.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Benzothiazoles; Calcium; Carbocyanines; Cell Membrane; Cell Survival; Cytosol; Fluorescent Dyes; Fura-2; Humans; Membrane Potentials; N-Formylmethionine Leucyl-Phenylalanine; Neutrophils; Organotin Compounds; Superoxides

In addition to previous observations indicating that membrane potential changes generated by various Donnan- and Nernst-potentials lead to erythrocyte shape transformations, we show in this paper that diffusion potential change, induced by valinomycin, governs erythrocyte shape transformations. In low KCl-medium valinomycin, transferring the positive Nernst-potential into a negative diffusion potential, transforms stomatocytes into echinocytes. Using modified erythrocytes with a reversed K/Na ratio, even positive diffusion potentials can be induced by valinomycin. In these cases, stomatocytes can be generated by valinomycin. It is shown that, additionally, valinomycin in large concentrations is itself stomatocytogenic, and that the fluorescent dye diS-C3-(5) also induces stomatocytes. This, however, is a side effect which does not contradict the potential dependence of shape transformation. Using non washed erythrocytes, resuspended in plasma, valinomycin, inducing negative diffusion potential, transforms most erythrocytes to echinocytes despite the stomatocytogenic effect of albumin.

Topics: Albumins; Benzothiazoles; Carbocyanines; Erythrocytes; Humans; Hydrogen-Ion Concentration; Membrane Potentials; Potassium; Sodium; Valinomycin

Phosphate ion (Pi) in sufficient concentrations is crucial for bone mineralization. The osteoblast (OB) may be responsible for the transport of Pi into the bone interstitium, where mineralization occurs. We previously characterized a Na(+)-dependent Pi transporter (NaPi) in the osteoblastic UMR-106-01 cell line. In the present study, the alteration of Na(+)-dependent Pi transport by changes in membrane potential was investigated. Depolarizing the cells with increasing concentrations of ambient K+ and valinomycin resulted in a progressive decline in Na(+)-dependent Pi uptake to a maximum of 28% at a membrane potential of -18 mV compared to control Na(+)-dependent Pi uptake at a membrane potential of approximately -60 mV. Hyperpolarizing the cells with SCN- increased Na(+)-dependent Pi uptake over control by 50% at an SCN- concentration of 70 mM. Determination of membrane potential by using the fluorescent probe, DiSC3(5), showed that the addition of Pi to cells in Na(+)-containing medium resulted in a small depolarization. These data show that NaPi activity can be altered by membrane potential changes and that the initiation of Na(+)-dependent Pi uptake is associated with depolarization of the plasma membrane of UMR-106-01 cells. Taken together, the cotransport of Na+ and Pi results in the movement of a net positive charge into the cell.

Topics: Benzothiazoles; Biological Transport; Carbocyanines; Carrier Proteins; Cell Line; Hydrogen-Ion Concentration; Kinetics; Membrane Potentials; Membrane Proteins; Osteoblasts; Phosphate-Binding Proteins; Phosphates; Potassium; Sodium; Thiocyanates; Valinomycin

We have used the membrane-permeant charged fluorescent dye, 3,3'-dipropylthiadicarbocyanine iodide (diS-C3[5]), to monitor electrical potentials across the membranes of isolated bovine disks. Calibration curves obtained from experiments where a potential was created across the disk membrane by a potassium concentration gradient and valinomycin showed an approximately linear relation between dye fluorescence and calculated membrane potential from 0 to -120 mV. Light exposure in the presence of the permeant buffer, imidazole, caused a rapid decay of the membrane potential to a new stable level. Addition of CCCP, a proton ionophore, in the dark produced the same effect as illumination. When the permeant buffer, imidazole, was replaced by the impermeant buffer, Hepes, neither light nor CCCP discharged the gradient. We interpret the changes in membrane potential measured upon illumination to be the result of a light-induced increase in the permeability of the disk membrane to protons. A permeant buffer is required to prevent the build-up of a pH gradient which would inhibit the sustained proton flow needed for an observable change in membrane potential.

Topics: Animals; Benzothiazoles; Carbocyanines; Cattle; Cell Membrane Permeability; Fluorescent Dyes; Kinetics; Light; Mathematics; Membrane Potentials; Models, Biological; Photoreceptor Cells; Potassium Channels; Protons; Valinomycin

The conditions to increase the sensitivities of liposomes to odorants were examined. The results obtained are as follows. (1) The minimum concentration of amyl acetate to induce the membrane potential changes (threshold) in phosphatidylcholine (PC) liposomes was about 10(-4) M and addition of 10 or 20% phosphatidylserine (PS) lowered the threshold to about 10(-9) M, which was lower than the thresholds for amyl acetate in the turtle and frog olfactory systems. (2) Similar to amyl acetate, addition of PS to PC greatly lowered the threshold for beta-ionone. On the other hand, addition of PS to PC in certain ratio increased the threshold for citral, suggesting that addition of PS to PC does not always increase the responses to all odorants. (3) The membrane fluidity change of the liposomes in response to odorants occurred at similar concentration region where the membrane potential changes occurred. The presence of CaCl2 in external solution much greatly increased both the magnitude of the membrane potential changes and the membrane fluidity changes of the PC-PS liposomes in response to amyl acetate than the presence of NaCl and MgCl2. These results suggest that the membrane fluidity change is related to generation of the membrane potential change. (4) It was estimated that adsorption of less than a few molecules of amyl acetate on single liposome elicits detectable changes in the membrane potential and the membrane fluidity.

Topics: Benzothiazoles; Calcium Chloride; Carbocyanines; Fluorescence Polarization; Fluorescent Dyes; Kinetics; Liposomes; Membrane Potentials; Odorants; Pentanols; Phosphatidylcholines; Phosphatidylserines

The fluorescence of the voltage sensitive dye, diS-C3-(5), has been analyzed by means of synchronous excitation spectroscopy. Using this rather rare fluorescence technique we have been able to distinguish between the slightly shifted spectra of diS-C3-(5) fluorescence from cells and from the supernatant. It has been found that diS-C3-(5) fluorescence in the supernatant can be selectively monitored at lambda exc = 630 nm and lambda em = 650 nm, while the cell associated fluorescence can be observed at lambda exc = 690 nm and lambda em = 710 nm. A modified theory for the diS-C3-(5) fluorescence response to the membrane potential is presented, according to which a linear relationship exists between the logarithmic increment of the dye fluorescence intensity in the supernatant, 1n I/I degrees, and the underlying change in the plasma membrane potential, delta psi p = psi p - psi p degrees. The theory has been tested on human myeloid leukemia cells (line ML-1) in which membrane potential changes were induced by valinomycin clamping in various K+ gradients. It has been demonstrated that the membrane potential change, delta psi p, can be measured on an absolute scale.

Topics: Benzothiazoles; Carbocyanines; Fluorescent Dyes; Humans; Kinetics; Leukemia, Myeloid; Mathematics; Membrane Potentials; Potassium; Spectrometry, Fluorescence; Tumor Cells, Cultured; Valinomycin

Sperm from trout, like other sperm, are immotile in the seminal tract and initiate motility upon dilution into an appropriate fertilizing environment. Trout sperm motility is inhibited by high extracellular [K+] and can be activated by dilution of extracellular [K+]. Activation of trout sperm by the dilution of extracellular [K+] suggests regulation by membrane potential. Using the membrane potential-sensitive fluorescent dye 3,3'-dipropylthiocarbocyanine iodide (diS-C3-(5)) we directly measured the K+ contribution to the membrane potential. Manipulating the membrane potential with Cs+ and the ionophore valinomycin can override K+ regulation. We show that trout sperm can also be activated in the presence of inhibitory [K+] by the addition of divalent cations. Activation by divalent cations is explained by the cations' ability to mask membrane surface potential and thus alter the potential sensed by membrane voltage sensors. Using the surface potential-sensitive dye, 1-anilino-8-naphthosulfonate (ANS), we directly measure the divalent cations' ability to mask surface potential. We propose a model where membrane hyperpolarization is the trigger that initiates the cascade of events leading to trout sperm activation. An increase in intracellular pH has been suggested to be a conserved step in the activation of sperm motility. We show that increasing intracellular pH by procedures that activate sea urchin and mammalian sperm does not activate trout sperm. In contrast, there is a decrease in intracellular pH upon activation of trout sperm motility. Artificially decreasing intracellular pH is not sufficient for activation of motility in trout sperm in an inhibitory [K+]. Thus, unlike some other sperm, changes in intracellular pH do not regulate trout sperm motility.

Topics: Anilino Naphthalenesulfonates; Animals; Benzothiazoles; Carbocyanines; Cesium; Fluoresceins; Hydrogen-Ion Concentration; Magnesium; Male; Membrane Potentials; Models, Biological; Potassium; Sperm Motility; Spermatozoa; Trout; Valinomycin

In human T (Jurkat) lymphoblasts we have studied the calcium signals induced by monoclonal antibodies reacting with the T-cell antigen receptor complex (TCR and CD3). Jurkat cells were preloaded with the fluorescent calcium indicator Indo-1 and the stimulus-induced rise in cytoplasmic free calcium concn was followed in the absence or in the presence of external calcium. The technique allowed the separate investigation of the intracellular calcium release and the external calcium influx processes. The changes in the membrane potential of Jurkat cells were followed simultaneously by using fluorescent indicators. We found that the activation of protein kinase C by phorbol ester (PMA) or by the permeable diacyl glycerol, DiC8, rapidly eliminated the calcium signal, independently of the presence or absence of external calcium, while these treatments did not appreciably change the membrane potential. In contrast, cell membrane depolarization achieved by various treatments selectively blocked the stimulus-induced calcium influx, while did not affect stimulus-induced calcium release from internal stores. The magnitude of the stimulus-induced calcium influx was found to be largely independent of the external calcium concns between about 2-2500 microM. It is demonstrated that the inhibitory effect of membrane depolarization on calcium influx is not simply due to the reduction of the inward calcium gradient under these conditions. These observations indicate a significant down-regulation of the stimulus-induced calcium signal by protein kinase C activation and a selective inhibition of the receptor-operated calcium channels by membrane depolarization.

Topics: 4-Chloromercuribenzenesulfonate; Antibodies, Monoclonal; Benzothiazoles; Biological Transport; Calcium; Carbocyanines; Cell Line; Diglycerides; Gramicidin; Humans; Membrane Potentials; Muromonab-CD3; Protein Kinase C; T-Lymphocytes; Tetradecanoylphorbol Acetate

The mechanism of carbocyanine dye diS-C3-(5) fluorescence intensity variations with transmembrane potential changes has been studied using time-resolved fluorescence spectroscopy. Clear evidence is given of the transmembrane-potential-dependent partition of the dye among various sites with different fluorescence lifetimes. It was found that fluorescence decay profiles reflect the transmembrane potential changes.

Topics: Benzothiazoles; Calibration; Carbocyanines; Diffusion; Fluorescent Dyes; Humans; Membrane Potentials; Potassium; Quinolines; Spectrometry, Fluorescence; Time Factors; Tumor Cells, Cultured

Topics: Animals; Benzothiazoles; Biological Transport, Active; Ca(2+) Mg(2+)-ATPase; Calcium; Calcium Channels; Carbocyanines; Fluorescent Dyes; In Vitro Techniques; Membrane Potentials; Muscle, Smooth; Sarcolemma; Swine; Valinomycin

This work comments on the location and orientation of 3,3'-dipropylthiodicarbocyanine (diS-C3-(5)) in renal brush-border membrane vesicles (RBBMV) (Cabrini, G. and Verkman, A.S. (1986) Biochim. Biophys. Acta 862, 285-293) evaluated from collisional quenching of n-(9-anthroyloxy)stearic acid (n-AS) fluorescence. At variance with these authors, it is concluded that the quenching is due to resonance energy transfer. It is also shown that the fluorescence data are not clear evidence for the reported monomer and dimer locations.

Topics: Benzothiazoles; Carbocyanines; Chemical Phenomena; Chemistry, Physical; Energy Transfer; Fluorescence; Fluorescent Dyes; Kidney; Liposomes; Microvilli; Quinolines; Stearic Acids

The basolateral membrane of the thick ascending loop of Henle (TALH) of the mammalian kidney is highly enriched in Na+/K+ ATPase and has been shown by electrophysiological methods to be highly conductive to Cl-. In order to study the Cl- conductive pathways, membrane vesicles were isolated from the TALH-containing region of the porcine kidney, the red outer medulla, and Cl- channel activity was determined by a 36Cl uptake assay where the uptake of the radioactive tracer is driven by the membrane potential (positive inside) generated by an outward Cl- gradient. The accumulation of 36Cl- inside the vesicles was found to be dependent on the intravesicular Cl- concentration and was abolished by clamping the membrane potential with valinomycin. The latter finding indicated the involvement of conductive pathways. Cl- channel activity was also observed using a fluorescent potential-sensitive carbocyanine dye, which detected a diffusion potential induced by an imposed inward Cl- gradient. The anion selectivity of the channels was Cl- greater than NO3- = I- much greater than gluconate. Among the Cl- transport inhibitors tested, 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPAB), 4,4'-diisothiocyano-stilbene-2,2'-disulfonate (DIDS), and diphenylamine-2-carboxylate (DPC) showed IC50 of 110, 200 and 550 microM, respectively. Inhibition of 36Cl uptake by NPPAB and two other structural analogues was fully reversible, whereas that by DIDS was not. The nonreactive analogue of DIDS, 4,4'-dinitrostilbene-2,2'-disulfonate (DNDS), was considerably less inhibitory than DIDS (25% inhibition at 200 microM). The irreversible inhibition by DIDS was prevented by NPPAB, whereas DPC was ineffective, consistent with its low inhibitory potency.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Animals; Anions; Benzothiazoles; Carbocyanines; Chlorides; Fluorescent Dyes; Ion Channels; Kidney Medulla; Membrane Potentials; Membranes; ortho-Aminobenzoates; Swine; Valinomycin

Transverse tubule membranes isolated from rabbit skeletal muscle consist mainly of sealed vesicles that are oriented primarily inside out. These membranes contain a high density of binding sites for 1,4-dihydropyridine calcium channel antagonists. The presence of functional voltage-dependent calcium channels in these membranes has been demonstrated by their ability to mediate 45Ca2+ efflux in response to changes in membrane potential. Fluorescence changes of the voltage-sensitive dye, 3,3'-dipropyl-2,2'-thiadicarbocyanine, have shown that transverse tubule vesicles may generate and maintain membrane potentials in response to establishing potassium gradients across the membrane in the presence of valinomycin. A two-step procedure has been developed to measure voltage-dependent calcium fluxes. Vesicles loaded with 45Ca2+ are first diluted into a buffer designed to generate a membrane potential mimicking the resting state of the cell and to reduce the extravesicular Ca2+ to sub-micromolar levels. 45Ca2+ efflux is then measured upon subsequent depolarization. Flux responses are modulated with appropriate pharmacological specificity by 1,4-dihydropyridines and are inhibited by other calcium channel antagonists such as lanthanum and verapamil.

Topics: 3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester; Animals; Benzothiazoles; Calcium Channel Blockers; Calcium Channels; Calcium Radioisotopes; Carbocyanines; Cell Membrane; Dihydropyridines; Edetic Acid; Fluorescent Dyes; Isradipine; Lanthanum; Membrane Potentials; Muscles; Nitrendipine; Oxadiazoles; Potassium; Rabbits; Verapamil

Topics: Antineoplastic Agents; Benzothiazoles; Carbocyanines; Cell Line; Colonic Neoplasms; Fluorescent Dyes; Humans; Membrane Potentials; Mitomycin; Mitomycins

The cationic fluorescent probe, DiSC3(5) was used to measure the membrane potential in human platelets. Hyperpolarization was induced by the addition of Ca2+ to the medium and also by the addition of the Ca2+ ionophore, A23187. In the absence of extracellular Ca2+ ([Ca2+]o) there was no response to A23187. The threshold concentration for [Ca2+]o was 20 microM and for A23187 was 12 nM. The increase polarity induced by [Ca2+]o was not affected by various K+ channel blockers. However, the effect of A23187 was inhibited by quinine and charybdotoxin, while apamin, tetraethylammonium, and the calmodulin inhibitors trifluoperazine and compound R24571 were ineffective. The resting membrane potential was -66 +/- 0.9 mV and was decreased by quinine. There are three conclusions from this study: (i) Ca2+-activated K+ channels exist in human platelets; (ii) they are the type that are apamin insensitive, charybdotoxin sensitive; and (iii) they may contribute to the resting membrane potential.

Topics: Adult; Apamin; Benzothiazoles; Blood Platelets; Calcimycin; Calcium; Carbocyanines; Female; Fluorescent Dyes; Humans; Kinetics; Male; Membrane Potentials; Middle Aged; Potassium Channels; Quinine; Tetraethylammonium; Tetraethylammonium Compounds

The applicability of the potential-sensitive dye diS-C3-(5) for the study of A23187 + Ca2+ induced plasma membrane hyperpolarization was tested in rat brain synaptosomes. An appropriate dye synaptosome ratio was chosen for the fluorescence titration dye in Ca-free Krebs-Ringer solution. The fluorescence intensity of the probe was increased upon the addition of Ca2+ (1 microM) to the synaptosomes in the presence of A23187 (1 microM). The effect of Ca2+ + A23187 persisted in a Na+-free medium or when Na+ channels were inhibited by tetrodotoxin as well as in high K+-depolarized synaptosomes (75 microM KCl). In the presence of oligomycin or a protonophore (1 microM) the effect of Ca2+ + A23187 was suppressed. This suggests that the A23187-induced fluorescence increase is due to a depolarization of intrasynaptosomal mitochondria. Therefore, the use of the dye diS-C3-(5) for the study of Ca-induced hyperpolarization does not seem to be feasible unless a quantitative model of changes in fluorescence related to the plasma and mitochondrial membrane potentials is elaborated.

Topics: Animals; Benzothiazoles; Brain; Calcimycin; Calcium; Calcium Channels; Carbocyanines; Cell Membrane; Dose-Response Relationship, Drug; Fluorescent Dyes; In Vitro Techniques; Intracellular Membranes; Membrane Potentials; Mitochondria; Rats; Spectrometry, Fluorescence; Synaptosomes

The membrane potential in murine epididymal sperm was determined with a voltage-sensitive, fluorescent probe. In freshly collected sperm, the potential was inside-negative, viz., -13 mV, and was associated with an intracellular K+ concentration of about 122 mM. Following incubation of sperm in a medium capable of sustaining capacitation and fertilization efficacy, the potential became gradually positive. An inside-positive potential, +24 mV, was obtained after 40 min of incubation, concomitant with an intracellular K+ concentration of approximately 30 mM. At this time, about 70 percent of sperm had capacitated. An inside-positive membrane potential may play a role in facilitating the acrosome reaction.

Topics: Animals; Benzothiazoles; Carbocyanines; Cell Membrane; Epididymis; Fluorescent Dyes; Kinetics; Luminescent Measurements; Male; Membrane Potentials; Mice; Mice, Inbred Strains; Spectrometry, Fluorescence; Sperm Capacitation; Spermatozoa

Using a new methodological approach based on the binding of 125I-labeled troponin C to troponins I and T immobilized on polyvinylchloride, the Ca2+-dependent interaction of troponin components was investigated. In the absence of Ca2+, two types of sites of troponin C--troponin T interaction were revealed (Kd = 3.6.10(-8) M and 5.10(-7) M). It was found that Ca2+ induced the formation of a troponin I--troponin C complex which was resistant to 5 M urea (Kd = 4.10(-8) M). In the absence of Ca2+, the binary troponin T--troponin C complex also revealed two types of interaction sites (Kd = 7.1.10(-8) M and 2.10(-7) M); however, in the presence of Ca2+ only high affinity sites whose number increased almost 2-fold were revealed. The events that may take place in the whole troponin complex during Ca2+ binding by troponin C are discussed.

Topics: Animals; Benzothiazoles; Binding, Competitive; Calcium; Carbocyanines; Fluorescent Dyes; In Vitro Techniques; Muscles; Polyvinyl Chloride; Rabbits; Troponin; Troponin C; Troponin I; Troponin T; Urea

The membrane potential of primitive red cells from 4- and 6-day old chick embryos has been determined using the fluorescent dye Dis-C3-(5). At day 4 the membrane potential Em was -44 mV for pH 7.4 and 20 degrees C and -36 mV at day 6. Both values are far removed from the equilibrium potential for chloride, which is about -14 mV at day 6. Changes in the external potassium, sodium or chloride concentration were without effect on the membrane potential, except at very high potassium concentrations, where a small but significant depolarization was observed at day 6. The measurements gave the same results in the absence or presence of the anion exchange blocking agent DIDS. Three pieces of evidence indicate that the membrane potential of primitive red cells is primarily caused by an electrogenic H+ conductance: 1) The measured membrane potential of -36 mV at day 6 is close to the previously determined proton equilibrium potential (Baumann and Haller, 1983) EH + of -36 mV. 2) Addition of the electrosilent Cl-/OH- exchanger tributyltin causes a significant depolarization of about 20 mV at day 4 and about 14 mV at day 6. 3) Measurement of hydrogen ion fluxes demonstrate a potential dependent proton conductance, which increases with depolarization. These results indicate that large qualitative differences exist with regard to the mechanisms involved in the generation of membrane potential and hydrogen distribution between red cell and plasma of embryonic and adult chicken.

Topics: Animals; Benzothiazoles; Carbocyanines; Chick Embryo; Chlorides; Erythrocyte Membrane; Fluorescent Dyes; Membrane Potentials; Potassium; Protons; Trialkyltin Compounds; Valinomycin

The membrane potential of platelets suspended in physiological medium and membrane potential changes induced by high potassium concentrations, ouabain and cooling have been measured using a cyanine fluorescent dye (3,3'-dipropylthiodicarbocyanine) [corrected]. The membrane potential of platelets suspended in physiological medium was -63.8 mV. High potassium concentrations, ouabain and cooling induced depolarization of platelet membrane. Depolarization using the above procedures enhanced platelet aggregation induced by ADP, adrenaline and collagen. These results suggest that the membrane potential could modulate platelet activity.

Topics: Adult; Benzothiazoles; Carbocyanines; Female; Fluorescent Dyes; Humans; Male; Membrane Potentials; Ouabain; Platelet Aggregation; Sodium-Potassium-Exchanging ATPase

42K+ transport properties of isolated rat parotid acini were characterized concomitant with measurements of membrane potentials (Em) by means of the fluorescent dye diSC3-(5). In unstimulated acini suspended in a 5 mM K+ buffer, Em was governed by the K+ and Cl- gradients and amounted to about -59 mV, a value that remained unaffected on cholinergic stimulation. In unstimulated acini, 42K+ influx was largely mediated by the Na+-K+ pump, and the residual influxes were mediated by a bumetanide-sensitive component (cotransport system) and by K+ channels. Efflux of 42K+ was largely mediated by a bumetanide-sensitive component and by K+ channels. In the unstimulated state, the cotransport system was mediating K+-K+ exchange without contributing to the net uptake of K+. Within 10 s after stimulation, a approximately 10-fold increase in the acinar K+ conductance (gK) occurred, resulting in a rapid net efflux of K+ that amounted to approximately 3.8 mmol.l cells-1.s-1. Measurements of 42K+ fluxes as a function of the external K+ concentration revealed that in the stimulated state gK increases when external K+ is raised from 0.7 to 10 mM, consistent with an activation of acinar gK by the binding of external K+ to the channel. 42K+ flux ratios as well as the effect of the K+ channel inhibitor from scorpion venom (LQV) suggest that approximately 90% of K+ transport in the stimulated state is mediated by "maxi" K+ channels.

Topics: Animals; Benzothiazoles; Bumetanide; Carbachol; Carbocyanines; Cells, Cultured; Fluorescent Dyes; Intracellular Fluid; Kinetics; Membrane Potentials; Ouabain; Parotid Gland; Potassium; Potassium Channels; Potassium Radioisotopes; Rats; Spectrometry, Fluorescence; Valinomycin

Transmembrane potential (TMP) of synaptic vesicles (SV) from the rat brain was registered by means of potential-sensitive fluorescent probe 3,3' dipropylthiocarbocyanine iodide (dis-C3-(5)). It was found that Mg-ATP generates TMP on the SV membrane suspended in sucrose-buffer medium. TMP did not appear if 0.25 M sucrose medium was replaced by 0.125 M KCl medium or 0.125 NaCl medium, or if the SV membrane was disrupted by triton X-100 or alamethicin, or if there was protonophore m-chlorophenylhydrazone or N-ethylmaleimide influence on SV. The data obtained indicate that functioning of SV proton pump is electrogenic and the level of TMP depends on the presence of anion Cl- in the incubation medium.

Topics: Adenosine Triphosphate; Animals; Benzothiazoles; Brain; Carbocyanines; Fluorescent Dyes; In Vitro Techniques; Membrane Potentials; Rats; Synaptic Vesicles

A preparation of viable epithelial cells, suitable for transport studies, was prepared from rabbit distal colon. Enzymatic digestion of scraped mucosa liberated a population of intact colonic glands that were dissociated into cells by gentle agitation in culture medium. Metabolism of [14C]glucose was constant over 2 h and was inhibited 70% by 1 mM ouabain. Cells were loaded with the trapped, pH-sensitive fluorescent dye 2'-7'-bis(carboxyethyl)-5(6)-carboxyfluorescein acetoxy methyl ester and leaked 0.1% of the dye per minute. Cell pH was 7.21 +/- 0.03 (SE) (n = 15) in pH 7.4 Ringer medium. Intracellular potassium concentration, as measured by a nigericin null-point technique, was 128 +/- 8 mM. Plasma membrane potential measured with the potential-sensitive fluorescent dye 3,3-dipropylthiadicarbocyanine iodide was -52 +/- 2 mV. Recovery of intracellular pH in acid-loaded cells occurred after exposure to sodium-containing medium and was inhibited by 5 x 10(-4) M amiloride. It is concluded that viable epithelial cells can be prepared from rabbit distal colon with relative simplicity and in high yield. These cells are suitable for measurement of intracellular pH and membrane potential and are thus a convenient model system for study of colonic cell physiology.

Topics: Animals; Benzothiazoles; Carbocyanines; Cell Separation; Cell Survival; Colon; Epithelial Cells; Fluoresceins; Fluorescent Dyes; Glucose; Glycolysis; Kinetics; Male; Membrane Potentials; Microscopy, Electron; Potassium; Rabbits

Various bitter substances were found to depolarize liposomes. The results obtained are as follows: (1) Changes in the membrane potential of azolectin liposomes in response to various bitter substances were monitored by measuring changes in the fluorescence intensity of 3,3'-dipropylthiocarbocyanine iodide [diS-C3(5)]. All the bitter substances examined increased the fluorescence intensity of the liposome-dye suspension, which indicates that the substances depolarize the liposomes. There existed a good correlation between the minimum concentrations of the bitter substances to depolarize the liposomes and the taste thresholds in humans. (2) The effects of changed lipid composition of liposomes on the responses to various bitter substances vary greatly among bitter substances, suggesting that the receptor sites for bitter substances are multiple. The responses to N-C=S substances and sucrose octaacetate especially greatly depended on the lipid composition; these compounds depolarized only liposomes having certain lipid composition, while no or hyperpolarizing responses to these compounds were observed in other liposomes examined. This suggested that the difference in "taster" and "nontaster" for these substances can be explained in terms of difference in the lipid composition of taste receptor membranes. (3) It was confirmed that the membrane potential of the planar lipid bilayer is changed in response to bitter substances. The membrane potential changes in the planar lipid bilayer as well as in liposomes in response to the bitter substances occurred under the condition that there is no ion gradient across the membranes. These results suggested that the membrane potential changes in response to bitter substances stem from the phase boundary potential changes induced by adsorption of the substances on the hydrophobic region of the membranes.

Topics: Benzothiazoles; Carbocyanines; Chemical Phenomena; Chemistry; Fluorescent Dyes; Liposomes; Membrane Potentials; Models, Biological; Quinolines; Spectrometry, Fluorescence; Structure-Activity Relationship; Taste; Taste Buds

We re-examined the electrical and stoichiometric properties of the Na+-L-lactate cotransporter using highly purified brush-border membrane vesicles prepared from the whole cortex of rabbit kidney. A valinomycin-induced K+ diffusion potential (interior-negative) stimulated Na+ gradient-dependent L-lactate uptake. However, this stimulation reflected catalytic rather than energetic activation as an inside-negative membrane potential did not induce net uphill lactate accumulation in the presence of Na+ but in the absence of a Na+ concentration gradient. Additional evidence for electroneutrality of the cotransporter was the finding that, under voltage-clamped conditions, L-lactate flux was a hyperbolic function of extravesicular Na+ concentration with a Hill coefficient (n) of 1.0. Moreover, the plot of V/[Na+]n versus V was linear for n = 1, indicating that one Na+ ion is co-transported with an anionic lactate1- molecule. Finally, addition of L-lactate to vesicles under Na+ equilibrium conditions failed to generate an inside-positive membrane potential as monitored by 3,3'-dipropylthiodicarbocyanine iodide fluorescence quenching, arguing against Na+-L-lactate cotransport by an electrogenic process. Taken together, these data indicate that the luminal Na+-L-lactate co-transporter is electroneutral with a stoichiometry of 1.

Topics: Animals; Benzothiazoles; Biological Transport; Carbocyanines; Carrier Proteins; Electrochemistry; Fluorescent Dyes; Glucose; Kidney Cortex; Lactates; Lactic Acid; Membrane Potentials; Microvilli; Monocarboxylic Acid Transporters; Potassium; Rabbits; Sodium; Spectrometry, Fluorescence; Symporters; Valinomycin

The pH sensitive fluorescent probe acridine orange and membrane potential-sensitive fluorescent probe acridine orange and membrane potential-sensitive fluorescent probe 3,3'-dipropylthiadicarbocyanine iodide were used to evaluate the Cl-/HCO-3 antiporter and proton and potassium conductances, respectively, in human red blood cell ghosts. Acidic, chloride-loaded ghosts alkalinized rapidly in pH 8.5 chloride-free media. Alkalinization could not be ascribed to conductive proton efflux with either depolarizing potassium influx or chloride efflux. Alkalinization was consequent to flux on the Cl-/HCO-3 antiporter: this process displayed saturation kinetics, competitive inhibition by external chloride, and inhibition by 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid. The mean Kms for internal chloride, external bicarbonate, and external chloride were 2.19, 0.24, and 0.44 mM, respectively. These studies confirm both the asymmetry of this carrier and the high affinity for external HCO-3; however, the affinities for internal and external chloride are significantly greater than prior estimates. The Km for internal chloride (2.19 mM) was considerably lower than levels previously reported (20-65 mM) unless external (trans) chloride was raised above 2 mM. The present studies thus demonstrate and emphasize the critical importance of trans substrate concentration in assessing the kinetics of a carrier whose mobility is faster in the loaded than in the unloaded state.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; Acridine Orange; Alkalies; Benzothiazoles; Bicarbonates; Buffers; Calibration; Carbocyanines; Carrier Proteins; Chloride-Bicarbonate Antiporters; Chlorides; Erythrocyte Membrane; Humans; Hydrogen-Ion Concentration; Kinetics; Protons; Quinolines

We isolated myeloid precursors from human marrow and studied the effects of phorbol myristate acetate (PMA) and N-formyl-methionyl-leucyl-phenylalanine (fMLP) upon transmembrane potentials and cytosolic calcium ([Ca2+]i) as the cells matured. Using a panel of fluorescent probes, we found that membrane depolarization induced by PMA and fMLP in granulocytes, and elevation in [Ca2+]i stimulated by fMLP, were absent in myeloblasts. When we induced differentiation with granulocyte-macrophage colony-stimulating factors, we found that both ionic responses appeared at approximately the promyelocyte stage. By using di-O-C5(3), we detected an initial phase of fMLP-induced hyperpolarization which appeared ontogenetically earlier than depolarization and which could be evoked in mature granulocytes with lower concentrations of the ligand. Hyperpolarization was partially dependent on extracellular Na+, was abrogated by increasing the external K+ concentration, and was accompanied by mild acidification of the cytoplasm. Bordetella pertussis toxin abolished both hyperpolarization and depolarization. Our findings indicate that shifts in [Ca2+]i and membrane potential changes in response to PMA and fMLP evolve as granulocytes mature. In addition, transmembrane ionic fluxes induced by fMLP appear to be more complex than previously considered, involving at least two separable phases of membrane potential change.

Topics: Aminoquinolines; Benzothiazoles; Bone Marrow Cells; Calcium; Carbocyanines; Cell Differentiation; Cell Membrane; Cytosol; Fluorescent Dyes; Granulocytes; Hematopoietic Stem Cells; Humans; Hydrogen-Ion Concentration; Interleukin-3; Membrane Potentials; N-Formylmethionine Leucyl-Phenylalanine; Pertussis Toxin; Potassium; Sodium; Tetradecanoylphorbol Acetate; Virulence Factors, Bordetella

It is generally assumed that electrolytes equilibrate readily across renal cortical brush-border membrane vesicles (BBMV). This assumption was tested by use of two new methods in rat BBMV prepared with free-flow electrophoresis (FFE), Mg aggregation, or Ca aggregation. Intravesicular KCl and RbCl concentrations, as well as the conductance of Cl relative to K (GCl/GK) and GNa/GK were determined with the fluorescent, potential-sensitive probe 3,3'-dipropylthiadicarbocyanine iodide [diS-C3-(5)]; intravesicular KCl concentration was also approximated utilizing the response of Na-dependent [3H]glucose uptake to variations in the membrane potential. These studies demonstrated that KCl fails to attain electrochemical equilibrium in BBMV prepared by the three methods, despite prolonged incubation at 22 degrees C; a significant, inwardly directed electrolyte gradient was sustained in all cases. The intravesicular electrolyte concentration was lower in BBMV prepared with FFE than in those prepared with Mg or Ca. GCl/GK was lowest in BBMV prepared with FFE and highest in those prepared with Ca; GNa/GK was comparable in all preparations. The apparent impermeance of BBMV may impact significantly in interpreting data from studies that require knowledge of the precise concentration of intravesicular electrolytes.

Topics: Animals; Benzothiazoles; Carbocyanines; Cell Fractionation; Cell Membrane Permeability; Electric Conductivity; Glucose; In Vitro Techniques; Kidney Cortex; Membrane Potentials; Microvilli; Potassium; Rats; Rubidium; Sodium

The interaction of the probe diS-C3-(5) with dipalmitoylphosphatidylcholine (DPPC) liposomes has been studied using fluorescence and differential scanning calorimetry (DSC). The partition coefficients (K) of the probe for the lipid and the aqueous phase (in terms of molar part units) were (1.20 +/- 0.4) X 10(6) at 45 degrees C and (0.50 +/- 0.07) X 10(6) at 23 and 36 degrees C. In terms of volume concentration units, these values correspond to Kp = (2.88 +/- 0.10) X 10(4) and Kp = (1.20 +/- 0.17) X 10(4), respectively. DSC thermograms were practically identical both for large unilamellar and multilamellar liposomes. The main transition peak remained practically unchanged over the entire range of the probe concentrations used. The pretransition could be observed up to maximal probe concentrations applied and it widened and shifted from 35.4 degrees C in pure DPPC to approximately 32 degrees C at a probe/lipid ratio of 0.027. These results suggest that in both quasicrystalline and liquid crystalline lipid bilayers the probe molecules are included in "defects" between structurally ordered microregions (microdomains or clusters). The dependence of the fluorescence response on the transmembrane potential in a suspension of unilamellar DPPC vesicles suggest that the equilibrium thermodynamic model is valid for liquid crystalline bilayers.

Topics: 1,2-Dipalmitoylphosphatidylcholine; Benzothiazoles; Carbocyanines; Fluorescent Dyes; Kinetics; Liposomes; Membrane Potentials; Models, Biological; Quinolines; Spectrometry, Fluorescence

The effect of a number of commonly employed potential-sensitive molecular probes on the 31P-NMR properties of dimyristoylphosphatidylcholine vesicles at two field strengths has been investigated in order to obtain information on the location and effect of these probes on the membrane bilayer. In comparison to the control dye-free vesicle spectrum, the probes diS-C3-(5) and diS-C4-(5), when added to a vesicle suspension, cause a substantial broadening of the 31P resonance with no detectable chemical shift within an uncertainty of +/- 0.05 ppm at 24 MHz. The spin-lattice and spin-spin relaxation times are also reduced when the cyanines are present by well over 20% relative to those of the control vesicle preparation. The addition of anionic probes, including several oxonol derivatives and merocyanine 540, causes no chemical shift, line broadening, or changes in the relaxation times. Possible explanations for the failure of the anionic probes to alter the vesicle 31P-NMR properties include charge repulsion between these dyes and the phosphate group that prevents the probes from penetrating the bilayer to a depth sufficient to alter the local motion of the phosphate moiety. The 31P resonance broadening and reduction in the relaxation times caused by the two cyanines is at least in part due to an increase in vesicle size as judged by electron microscopy measurements, although an inhibition of the local phosphate motion as well cannot be completely eliminated. The cyanine-mediated increase in vesicle size appears to be due to an irreversible vesicle-fusion process possibly initiated by the screening of surface charge by these probes. The implications of these observations in relation to functional energy-transducing preparations is discussed.

Topics: Benzothiazoles; Carbocyanines; Dimyristoylphosphatidylcholine; Indicators and Reagents; Isoxazoles; Kinetics; Lipid Bilayers; Magnetic Resonance Spectroscopy; Membrane Potentials; Microscopy, Electron; Oxazoles; Pyrimidinones; Quinolines

Quinine inhibits mitogenesis at the same concentration (10(-4) M) as that which blocks Ca++-dependent potassium transport in lymphocytes. Lower quinine concentrations (10(-8)-10(-6) M) induce a comitogenic effect which is most pronounced when Ca++-ionophore A23187 is used as a mitogen. Thus, activation of Ca++-dependent K+-channels is not necessary to trigger mitogenesis, but is important for further stages of the process.

Topics: Benzothiazoles; Calcimycin; Calcium; Carbocyanines; Cell Membrane Permeability; Cells, Cultured; Concanavalin A; DNA; Fluorescent Dyes; Humans; Ion Channels; Lymphocyte Activation; Membrane Potentials; Mitogens; Phytohemagglutinins; Potassium; Quinine

We have incorporated the myosin fragment heavy meromyosin (HMM), which is known to interact mechanochemically and enzymatically with actin filaments, into intact chromaffin cells of the bovine adrenal medulla, in order to study the possible involvement of actin and myosin in stimulus-secretion coupling. HMM was found to stimulate secretion of catecholamines, to cause depolarization of the plasma membrane, and to enhance 22Na+ uptake. HMM-stimulated catecholamine secretion was dependent on the presence of extracellular Na+. The Na+ uptake caused by HMM was inhibited by 10 microM amiloride. Acetylcholine-stimulated catecholamine secretion and 22Na+ uptake were both enhanced by HMM incorporation. A Na+/H+ antiporter, activated by the interaction of HMM with the cells' microfilaments, seems to be involved in HMM action and could possibly also be a component of stimulus-secretion coupling in chromaffin cells, induced by regular agonists.

Topics: 3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester; Actin Cytoskeleton; Actins; Adrenal Medulla; Animals; Benzothiazoles; Carbocyanines; Catecholamines; Cattle; Cell Membrane; Chromaffin System; Cytoskeleton; Fluorescent Dyes; Liposomes; Membrane Potentials; Myosin Subfragments; Myosins; Nifedipine; Peptide Fragments; Phencyclidine; Tetraethylammonium; Tetraethylammonium Compounds; Tetrodotoxin

Some aspects of the interaction of the extrinsic, potential-sensitive, molecular probe diS-C3-(5) with pigeon heart mitochondria are reported in this paper. Binding studies based on fluorimetry indicate that the ratio of the dissociation constant to the maximum number of binding sites, KD/n, is larger for succinate-containing mitochondria than that for cyanide-inhibited preparations. These observations suggest that the basis of the energy-dependent diS-C3-(5) optical signals is the ejection of the probe from the mitochondrial membrane. A more detailed analysis indicated that the major change in the binding parameters is a reduction in the maximum number of binding sites, n, when a charge gradient is formed at the expense of substrate. Using rapid mixing techniques, the time course of the passive association of diS-C3-(5) with mitochondria, that of the glutamate- and ATP-dependent optical signals, and the effect of this probe on the rate at which the energy-dependent cytochrome c oxidase Soret band shift signal develops have been monitored. Retardation the ATP-dependent cytochrome c oxidase Soret band shift signal suggests that the probe readily permeates the mitochondrial membrane. The first-order rate law that the glutamate-dependent signal obeys suggests that the rate-limiting step in the development of this signal is the dissociation of the dye from the mitochondrial membrane or the permeation of this membrane by the probe. The faster phase of the ATP-induced signal likely reflects the initial transfer of dye from the bulk aqueous phase followed by a slower probe permeation process that obeys a first-order rate law. This probe appears to distribute across the mitochondrial membrane in accordance with the transmembrane potential as judged by its effect on the ATP-dependent cytochrome c oxidase Soret band shift signal. DiS-C3-(5) also appears to inhibit the NADH dehydrogenase.

Topics: Adenosine Triphosphate; Animals; Benzothiazoles; Binding Sites; Biological Transport; Carbocyanines; Coloring Agents; Columbidae; Electron Transport Complex IV; Glutamates; In Vitro Techniques; Kinetics; Mathematics; Membrane Potentials; Mitochondria, Heart; Quinolines; Spectrophotometry; Time Factors

Fluorescence quenching of the pH gradient sensitive dye acridine orange and that of the membrane potential sensitive dye Di-S-C3(5) have been studied in purified basolateral membrane vesicles obtained from rabbit small intestine. Basolateral membranes contain an electroneutral, carrier mediated, Na+/H+ exchange activity. They also appear to contain an electrogenic pathway for H+ movement. Based on the comparison of acridine orange fluorescence quenching in the presence of an outwardly directed Na+ gradient and in the presence of known K+ diffusion gradients it can be estimated that at least 50% of the observed proton fluxes are due to the activity of the exchanger. Acridine orange fluorescence recovery measurements have been used to assess the kinetic properties of the exchanger.

Topics: Acridine Orange; Animals; Benzothiazoles; Carbocyanines; Carrier Proteins; Cell Compartmentation; Cell Membrane; Cell-Free System; Chlorides; Hydrogen; Hydroxides; Intestine, Small; Membrane Potentials; Rabbits; Sodium; Sodium-Hydrogen Exchangers

Polymorphonuclear leukocytes undergo a series of morphological and biochemical changes in response to various chemical stimuli. Transmembrane potential change is an early event that follows stimulation and membrane depolarization may act as a trigger for superoxide generation. To determine if there is a correlation between membrane depolarization and superoxide generation, we investigated the effects of different membrane modulators on stimulus-dependent depolarization. The membrane modulators mepacrine, chlorpromazine and cepharanthine inhibited the superoxide generation produced by chemotactic peptide, FMLP, and/or digitonin in neutrophils. Inhibitory profiles of the activation parameters, however, demonstrate that membrane depolarization is not associated with superoxide generation: FMLP-induced depolarization was inhibited by the modulators tested and was accompanied by the suppression of superoxide generation, but the depolarization produced by digitonin was stimulated somewhat by these drugs. Our results indicate that receptor-mediated membrane depolarization is not a necessary event for the activation of superoxide generation by digitonin.

Topics: Animals; Benzothiazoles; Calcimycin; Carbocyanines; Cell Membrane; Cell Membrane Permeability; Digitonin; Fluorescent Dyes; Guinea Pigs; Kinetics; Membrane Potentials; N-Formylmethionine Leucyl-Phenylalanine; Neutrophils; Oxygen Consumption; Potassium; Spectrometry, Fluorescence; Superoxides

The equilibrium binding mechanism and kinetics of binding of diS-C3-(5) (3,3'-dipropylthiodicarbocyanine iodide) to rabbit renal brush-border membrane vesicles (BBMV) were examined using steady-state and time-resolved fluorescence, and fluorescence stopped-flow methods. In aqueous solution, diS-C3-(5) exists as a monomer at concentrations less than 5 microM with fluorescence emission peak at 670 nm (excitation 622 nm), anisotropy r = 0.102, and lifetime tau = 1.2 nsec (23 degrees C). Upon addition of increasing BBMV (voltage clamped to 0 mV using K+/valinomycin), the 670 nm emission peak decreases, corresponding to formation of a nonfluorescent membrane dimer, and subsequently a new emission peak at 695 nm increases, corresponding to membrane monomer. Dynamic depolarization studies show that aqueous diS-C3-(5) rotation is unhindered with a rotational rate R = 0.57 nsec-1 while membrane monomer is hindered with steady-state anisotropy r = 0.190, lifetime tau = 2.1 nsec, R = 0.58 nsec-1 and limiting anisotropy r infinity = 0.11. Based on equilibrium fluorescence titrations, the membrane monomer-dimer (M-D) dissociation constant, Kd = [M]2/[D][BBMV], is 0.0013, where BBMV is expressed as membrane phospholipid concentration. Three distinct kinetic processes are identified by stopped-flow experiments in which BBMV are mixed with diS-C3-(5). There is rapid binding of diS-C3-(5) to the membrane to form bound monomer with a 6-msec exponential time constant. The membrane monomer at the membrane outer surface then aggregates to form bound dimer at the outer surface with a concentration independent time constant of 30 msec. The overall dimerization reaction probably consists of a rate-limiting reorientation process (30 msec) followed by a rapid dimerization which occurs on a nanosecond time scale. Finally, there is a 0.8 to 1 sec translocation of membrane dimer between symmetric sites at the inner and outer membrane surfaces. The translocation reaction is the step which is probably sensitive to changes in transmembrane electrical potential.

Topics: Animals; Benzothiazoles; Carbocyanines; Fluorescent Dyes; Kidney; Kinetics; Lipid Bilayers; Mathematics; Membrane Lipids; Membrane Potentials; Microvilli; Models, Biological; Rabbits; Solvents; Spectrometry, Fluorescence

The effects of uncouplers (FCCP, DNF), oligomycin, and rotenone on the fluorescence of potential-sensitive dyes, rhodamine 123 and diS-C3-(5), in lymphocyte suspensions were compared. The fluorescence of these optical probes gradually increased at higher FCCP concentrations. The dependences of fluorescence intensities and FCCP concentrations were similar for both dyes, and only diS-C3-(5) fluorescence started increasing at lower FCCP concentrations. Rotenone (1 microM) significantly increased rhodamine 123 fluorescence. TMPD-induced and uncoupler-induced diS-C3-(5) fluorescence changes increased 1.5- to 2-fold if the incubation mixture was supplemented with oligomycin (0.1-0.2 microgram/ml). The fluorescence responses of the dyes in the lymphocyte suspension correlate with the effects of mitochondrial energetics inhibitors on delta psi m in isolated mitochondria. The results suggest the possibility of using these dyes for estimating the direction of the delta psi m changes in the lymphocyte suspension.

Topics: Animals; Benzothiazoles; Carbocyanines; Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; Energy Metabolism; Female; Fluorescence; Fluorescent Dyes; In Vitro Techniques; Kinetics; Lymphocytes; Membrane Potentials; Mitochondria; Oligomycins; Rats; Rhodamine 123; Rhodamines; Rotenone; Uncoupling Agents

The potential-sensitive response mechanism of 3,3'-dipropylthiodicarbocyanine iodide (diS-C3-(5] was examined based on our previous model of diS-C3-(5) interaction with brush border membrane vesicles (BBMV) in the absence of a membrane potential. The model contained binding (6 msec), reorientation (30 msec), dimerization (less than 10 nsec), and translocation (1 sec) reaction steps (Cabrini & Verkman, 1986. J. Membrane Biol. 90:163-175). Transmembrane potentials (psi) were induced in BBMV by K+ gradients and valinomycin. Steady-state diS-C3-(5) fluorescence (excitation 622 nm, emission 670 nm) increased linearly with psi. The reorientation and translocation reaction steps were resolved by the stopped-flow technique as a biexponential decrease in fluorescence following mixture of diS-C3-(5) with BBMV at varying psi. The fractional amplitude of the faster exponential increased from 0.36 to 0.73 with increasing psi (-17 to 87 mV); the time constant for the faster exponential (30-35 msec) was independent of psi. There were single exponential kinetics (0.5-1.5 sec) for diS-C3-(5) fluorescence response to a rapid (less than 2 msec) change in psi in the absence of a diS-C3-(5) concentration gradient. These results, and similar findings in placental brush border vesicles, red cell vesicles, and phosphatidylcholine vesicles, support a translocation mechanism for diS-C3-(5) response, where induced membrane potentials drive diS-C3-(5) redistribution between sites at the inner and outer membrane leaflets, with secondary effects on diS-C3-(5) dimerization and solution/membrane partitioning. Fluorescence lifetime and dynamic depolarization measurements showed no significant change in diS-C3-(5) rotational characteristics or in the polarity of the diS-C3-(5) environment with changes in psi. Based on the experimental results, a mathematical model is developed to explain the quantitative changes in diS-C3-(5) fluorescence which accompany changes in psi at arbitrary dye/lipid ratios.

Topics: Animals; Benzothiazoles; Carbocyanines; Female; Humans; Kidney Cortex; Kinetics; Liposomes; Mathematics; Membrane Potentials; Microvilli; Models, Biological; Phosphatidylcholines; Placenta; Pregnancy; Quinolines; Rabbits; Spectrometry, Fluorescence

The location and orientation of 3,3'-dipropylthiodicarbocyanine (diS-C3-(5)) binding sites in renal brush-border membrane vesicles was examined from the quenching of n-(9-anthroyloxy) fatty acid (n-AS) fluorescence. Based on previous kinetic studies (Cabrini, G. and Verkman, A.S. (1986) J. Membrane Biol. 90, 163-175) monomeric aqueous diS-C3-(5) binds to brush-border membrane vesicles (BBMV) by an initial 6 ms association to form bound monomer, a 30-40 ms equilibrium between bound monomer (M) and bound dimer (D), and a 1-1.3 s translocation of D from the outer to inner membrane leaflet. Based on Stern-Volmer and lifetime analyses, M and D quench the fluorescence of the n-AS probes by a collisional mechanism. At low [diS-C3-(5)]/[BBMV] (R), where M predominates, the n-AS quenching efficiencies (Q) are similar (n = 2-16); at high R, where D predominates, Q increases with n (16 greater than 12 much much greater than 6 greater than 2), suggesting that M is oriented parallel, and D perpendicular, to the phospholipid chains deep within the membrane. Mixture of diS-C3-(5) with brush-border membrane vesicles containing n-AS in a stopped-flow apparatus gave a biexponential fluorescence decrease (excitation 390 nm, emission above 450 nm) with time constants 30-40 ms and 1-1.5 s; there was no 6 ms quenching process. These findings are incorporated into a model in which diS-C3-(5) adheres loosely to the outer membrane surface in 6 ms, binds parallel to the membrane phospholipid in 30-40 ms, dimerizes and rotates by 90 degrees in much less than 30 ms, and translocates to the opposite half of the bilayer in 1-15 s.

Topics: Animals; Benzothiazoles; Biological Transport; Carbocyanines; Female; Fluorescent Dyes; Kinetics; Liposomes; Membrane Potentials; Microvilli; Models, Biological; Quinolines; Rabbits; Spectrometry, Fluorescence

The influence of transmembrane ion fluxes on mycoplasma membrane potentials was studied. Electric membrane potential was calibrated vs fluorescence intensity of a potential-sensitive carbocyanine dye according to delta psi = (RT/F) X log([aIN(1 - IN) - b]/Kint), where IN = I/I0, I0 = maximal fluorescence intensity (obtained for delta psi----infinity), and a and b are constants. Fluorescence intensity was calibrated vs membrane potential by inducing a K+ diffusion potential. The calibration procedure was based on the assumption that in the presence of valinomycin the membrane potential was determined entirely by K+ diffusion. Then the dependence of fluorescence intensity on the external K+ concentration, Kext, could be described by Ival = I0[1 + a/(Kext + b)]-1. For Mycoplasma mycoides subsp. capri and enterococci, the constants were determined from experimental data using nonlinear least-squares computer-assisted methods. The validity of our assumption was proved using the "null-point" method. Here the Ca2+ ionophore A23187 and varying external Ca2+ concentrations were used to change the membrane potential experimentally. K+ and Na+ diffusion potentials significantly contributed to mycoplasma membrane potential whereas Cl- had no influence. Under growth conditions the mycoplasma membrane potential was estimated to be delta psi = -68 mV.

Topics: Benzothiazoles; Calcium; Carbocyanines; Chlorides; Diffusion; Fluorescence; Membrane Potentials; Mycoplasma; Potassium; Quinolines; Sodium

The cytochrome d complex from Escherichia coli has been reconstituted in proteoliposomes. Previous studies have shown that the enzyme rapidly oxidizes ubiquinol-8 within the bilayer as well as the soluble homologue, ubiquinol-1, and that quinol oxidase activity is accompanied by the formation of a transmembrane potential across the vesicle bilayer. In this work, the proton pumping activity of the cytochrome in the reconstituted vesicles is examined. Ubiquinol-1 oxidase activity is shown to be accompanied by the net alkalinization of the interior space of the reconstituted vesicles and by the release of protons in the external volume. H+/O ratios varying from 0.6 to 1.2 were measured in different preparations, by the oxygen pulse technique. Antibodies which bind specifically to subunit I (cytochrome b558) of the 2-subunit oxidase were used to estimate the topology of the reconstituted oxidase in the vesicles. It was concluded that 70-85% of the molecules were oriented with subunit I facing the outside and that this population of molecules is responsible for the observed proton release. Correction for the fraction of the oxidase which pumps protons into the vesicle interior yields an estimate of H+/O = 1.7 +/- 0.2. It is proposed that the enzyme does not function as an actual proton pump, but that the enzyme oxidizes ubiquinol and reduces oxygen (to water) on opposite faces of the membrane. Hence, scalar chemistry would yield H+/O = 2 and an electrogenic reaction by virtue of the transmembrane electron transfer between the proposed active sites.

Topics: Aerobiosis; Antibodies, Monoclonal; Benzothiazoles; Carbocyanines; Carbonyl Cyanide m-Chlorophenyl Hydrazone; Cell Membrane; Cytochrome d Group; Cytochromes; Electron Transport; Escherichia coli; Fluorescent Dyes; Hydrogen-Ion Concentration; Immunologic Techniques; Ion Channels; Liposomes; Membrane Potentials; Oxygen Consumption; Protons; Quinone Reductases; Spectrometry, Fluorescence

The role of membrane potential in the activation of human platelets by thrombin, ADP and PAF was assessed, using the fluorescent probe diSC3(5). Thrombin, ADP and PAF transiently depolarised the platelet membrane by 6-8 mV from its resting level (-70 mV). This depolarisation had a similar time course to that of shape change. The ionophores valinomycin and gramicidin hyperpolarised and depolarised the platelets respectively but did not activate them. In contrast, exposure of platelets to high K+ media both depolarised and caused them to change shape. Removal of Na+ from the suspension media abolished the depolarisation induced by thrombin, ADP and PAF but the platelets under these conditions were still capable of changing shape and aggregating. This result indicates that the observed depolarisation depends on Na+ fluxes. Amiloride or tetrodotoxin did not mimic the effect of Na+ removal suggesting that any Na+ movement involved does not go through the classic "Na+ channel". Thrombin, ADP and PAF still depolarised the platelet membrane in the absence of added Ca++. Under these conditions, however, the membrane did not repolarise. It is evident that all three agents, thrombin, ADP and PAF, change the membrane potential of human washed platelets through a similar mechanism and this change seems to be a consequence of stimulus-receptor interaction (and platelet activation?). A causal relationship however between these events cannot be clearly shown.

Topics: Adenosine Diphosphate; Amiloride; Benzothiazoles; Blood Platelets; Carbocyanines; Cell Membrane; Fluorescent Dyes; Gramicidin; Humans; Membrane Potentials; Platelet Activating Factor; Platelet Aggregation; Potassium Chloride; Quinolines; Tetrodotoxin; Thrombin; Valinomycin; Verapamil

The interference of cytochrome c with absorption and fluorescence changes of the cyanine dye, diS-C3-(5), was investigated in the presence of liposomes and cytochrome c-oxidase reconstituted proteoliposomes. The apparent cytochrome c-dependent quenching of diS-C3-(5) fluorescence, and the associated absorbance losses in the presence of liposomes and proteoliposomes in low ionic strength media, are due to destruction of the dye caused by cytochrome c-mediated lipid peroxidation. The rate of this reaction was further enhanced in the presence of hydrogen peroxide. Even in the absence of liposomes or proteoliposomes, a cytochrome c-induced breakdown of dye was observed in the presence of hydrogen peroxide. The cytochrome c mediated absorbance and fluorescence losses of diS-C3-(5) in liposomal or proteoliposomal systems are prevented by Ca2+ and La3+ ions, by ascorbate, by high ionic strength and by the antioxidant BHT. Under these conditions, the process of lipid peroxidation mediated by cytochrome c is inhibited either directly (e.g. by BHT) or indirectly, by preventing the binding of cytochrome c to lipid vesicles. The impact of these findings upon the experimental estimation of membrane potential in aa3-reconstituted proteoliposomes is discussed.

Topics: Animals; Ascorbic Acid; Benzothiazoles; Butylated Hydroxytoluene; Carbocyanines; Cations, Divalent; Cytochrome c Group; Electron Transport Complex IV; Fluorescent Dyes; Horses; Kinetics; Liposomes; Myocardium; Osmolar Concentration; Proteolipids; Quinolines

Safranine and the cyanine dye, 3',3'-dipropylthiadicarbocyanine (diSC3-5), were examined as membrane potential probes in cytochrome c oxidase vesicles. The spectra of the vesicle-associated dyes resemble those of the same dyes in organic solvents, indicating that safranine and diSC3-5 probably dissolve in a hydrophobic region of the proteoliposomal membrane. This binding of safranine to proteoliposomes occurs with a dye-membrane dissociation constant in the micromolar range. The binding of safranine and of diSC3-5 to liposomes or proteoliposomes is accompanied by fluorescence enhancement. This enhanced fluorescence is quenched by respiration or by the establishment of a K+ diffusion potential by valinomycin (negative interior). An optimal dye/lipid ratio was required to secure maximum fluorescence quenching of the dyes, whether that quenching was active or passive. Calibrations of both the safranine and the diSC3-5 responses with K+ diffusion potentials were also affected by the dye/lipid ratio. At lower dye/lipid ratios, the calibration curve was linear at higher potentials but deviated from linearity at lower potentials. The converse was true at higher dye/lipid ratios. The non-linearity of the calibration curve at higher potential was attributed to a 'saturation' effect; it may also involve increased permeability of proteoliposomal membrane to protons. Destacking of dye at the lower dye/lipid ratio was probably responsible for the non-linearity of the calibration curves at lower potentials. When all these effects are taken into account, the steady-state value of delta psi generated during maximal proteoliposomal respiration was calculated to be between 140 and 160 mV (interior negative) when measured with either safranine or diSC3-5. We conclude that quantitative estimates of delta psi values can be made using these probes in cytochrome c oxidase reconstituted proteoliposomes provided that appropriate precautions are taken.

Topics: Animals; Benzothiazoles; Carbocyanines; Cattle; Coloring Agents; Electron Transport Complex IV; Kinetics; Liposomes; Mathematics; Membrane Potentials; Methods; Phenazines; Potassium; Proteolipids; Quinolines; Spectrometry, Fluorescence; Valinomycin

Strain N.C.Y.C. 193 of Candida utilis was grown aerobically at 30 degrees C with nitrate as limiting nutrient in a chemostat. The washed yeast cells depleted of ATP absorbed up to 5 nmol of nitrate/mg dry wt. of yeast. At pH 4-6, extra protons and nitrate entered the yeast cells together, in a ratio of about 2:1. Charge balance was maintained by an outflow of about 1 equiv. of K+. Nitrate stimulated the uptake of about 1 proton equivalent during glycolysis or aerobic energy metabolism. Studies with 3,3'-dipropylthiadicarbocyanine indicated that the proton-linked absorption of nitrate, amino acids or glucose depolarized the yeast cells. Proton uptake along with lactate led neither to net expulsion of K+ nor to membrane depolarization.

Topics: Absorption; Benzothiazoles; Biological Transport, Active; Candida; Carbocyanines; Fluorescent Dyes; Glucose; Lactates; Lactic Acid; Maltose; Membrane Potentials; Nitrates; Protons; Spectrometry, Fluorescence

Peptide transport in purified rabbit intestinal brush-border membrane vesicles has been studied using a potential-sensitive fluorescent dye, di-S-C3(5). Transport of dipeptides is accompanied by an increase in the fluorescence of the dye in the presence and absence of Na+, indicating electrogenic, Na+-independent peptide transport. Dipeptides containing D-amino acids also increase the fluorescence, showing that these peptides too possess significant affinity for the peptide transport system. beta-Alanylglycylglycine and prolylglycylglycine, very much like the dipeptides, increase the fluorescence even in the absence of Na+ which demonstrates the Na+-independent, electrogenic transport of tripeptides. However, concentrations needed for half-maximal fluorescence changes are higher for tripeptides than for dipeptides suggesting different affinities for the carriers. The studies, in addition, provide evidence for the existence of more than one carrier system for translocation of small peptides in rabbit intestinal brush-border membrane.

Topics: Animals; Benzothiazoles; Biological Transport; Carbocyanines; Dipeptides; Fluorescent Dyes; Intestine, Small; Kinetics; Microvilli; Oligopeptides; Peptides; Quinolines; Rabbits; Sodium; Spectrometry, Fluorescence; Stereoisomerism; Substrate Specificity

We have studied Microciona prolifera cells as a model for inflammation and secretion. Dissociated in Ca-, Mg-free seawater with 2.5 mM EDTA, the cells aggregate when exposed to Ca (greater than 5 mM) and Ca ionophores. Extracellular Ca is not required over the course of aggregation; brief pulses of Ca suffice. Aggregation was induced by A23187 in excess EDTA after cells were prepared by pulse Ca. It appeared that Ca ionophore stimulated the secretion of Microciona aggregation factor (MAF) to a locus or in a form inaccessible to external EDTA. Pulse-induced aggregation depended on MAF because it was inhibited by MAF fragments, which are ligands for MAF-binding sites. Sponge cells were preloaded with three fluorescent dyes that monitor aspects of stimulus-secretion coupling: 1) 3,3'-dipropylthiadicarbocyanine iodide (dis-C3-(5)), a carbocyanine dye presumed to report changes in membrane potential; 2) 9-aminoacridine (9AA), which presumably reports secretion from acid vesicles; and 3) chlortetracycline (CTC), presumed to report mobilization of membrane-associated Ca. Exposure of cells either to constant Ca or to pulse Ca stimuli caused prompt decreases in the fluorescence of cells with diS-C3-(5) and increases in fluorescence of cells with 9AA. In contrast, although constant Ca provoked decreases in fluorescence of cells with CTC, a pulse Ca was without effect. Moreover, inhibitors of stimulus-response coupling (e.g., aspirin, sodium salicylate, 5 mM; diclofenac, 100 microM) inhibited sponge aggregation induced by either constant or pulse stimuli. In contrast, like the endogenous mediator of inflammation, leukotriene B4, trienoic alkyl catechols (urushiol) from poison ivy provoked aggregation. These studies suggest the utility of this marine model for analysis of stimulus-response coupling in cells of higher species that also respond to secretagogues in the absence of external Ca.

Topics: Aminacrine; Animals; Anti-Inflammatory Agents; Benzothiazoles; Calcimycin; Calcium; Carbocyanines; Catechols; Cell Adhesion Molecules; Cell Aggregation; Chlortetracycline; Fluorescence; Fluorescent Dyes; Inflammation; Porifera; Potassium; Proteins

Optical indicators of the cationic, cyanine and anionic oxonol classes were used to evaluate the plasma membrane potential of animal cells in suspension and in monolayer culture. The optical signals were calibrated by using diffusion potentials either of K+ (in the presence of valinomycin) or of H+ (in the presence of carbonyl cyanide p-trifluoromethoxyphenylhydrazone; FCCP); both classes of dye gave similar values of plasma membrane potential, in the range -40 to -90 mV for different cell types. Addition of haemolytic Sendai virus or Staphylococcus aureus alpha-toxin depolarizes cells and causes them to leak monovalent cations; these effects are antagonized by extracellular Ca2+. Cells infected with vesicular stomatitis or Semliki Forest virus become depolarized during an infectious cycle; infection with other viruses was without affect on plasma membrane potential.

Topics: Animals; Benzothiazoles; Carbocyanines; Cell Membrane; Isoxazoles; Membrane Potentials; Mice; Mice, Inbred Strains; Oxazoles; Toxins, Biological; Virus Diseases

Investigations in this laboratory have demonstrated that thrombin induces dose-dependent changes in the transmembrane electrical potential of gel-filtered human platelets. This change is monitored with the fluorescent lipophilic cation, 3,3'-dipropylthiodicarbocyanine (diS-C3-(5], whose rapid release from the platelet (maximal within 30 s) correlates with a rapid, dose-dependent influx of sodium, a depolarization, and an increase in the intracellular pH. There is also a later release of this probe, detectable only 60 s after activation by thrombin. It is shown that this latter probe release is also thrombin dose dependent, and correlates in time course and extent with the secretion of beta-glucuronidase from the platelet's lysosomal granules, implying that it corresponds to probe sequestered in these granules in the resting platelet. Such a conclusion is corroborated by the fact that both the thrombin-induced secondary release of diS-C3-(5) and the secretion of the lysosomal enzyme, beta-glucuronidase, are inhibitable to the same extent by pretreatment of the probe-equilibrated platelets with valinomycin, a K+ ionophore, are partially inhibited to a comparable extent when thrombin is removed from the platelet membrane by an excess of hirudin within 15 s of activation, and are unaffected by amiloride, a Na+ blocking agent. We suggest therefore that some of the membrane potential probe diS-C3-(5) is accumulated by the platelet lysosomal granules and is secreted when the platelets are stimulated by the high doses of thrombin which induce lysosomal enzyme secretion. This secondary dye release is linearly proportional to, and can be used as a continuous and quantitative indicator of, the thrombin-induced lysosomal enzyme secretion by human platelets.

Topics: Benzothiazoles; Blood Platelets; Carbocyanines; Chromatography, Gel; Fluorescent Dyes; Glucuronidase; Hirudins; Humans; Lysosomes; Membrane Potentials; Platelet Aggregation; Quinolines; Spectrometry, Fluorescence; Thrombin

The ability of rabbit jejunal brush borders to transport inhibitors of the imino carrier was investigated in membrane vesicles by measuring their ability to depolarize the membrane potential. Membrane potentials were monitored using a voltage-sensitive cyanine dye. Piperidine and pyrrolidine carboxylic acids, which are potent inhibitors of Na+-dependent proline transport (Ki less than 0.5 mM) depolarize the potential in a Na+-dependent, saturable manner indicating transport. On the other hand, N-methylated amino acids, which are fair inhibitors (Ki 2-10 mM), do not depolarize the membrane to any significant extent, but they competitively inhibit the L-proline transport signal. This indicates that these analogs are nontransported inhibitors of the imino carrier. The poor inhibitors niacin and pipolinic acid (Ki greater than 60 mM) depolarize the membrane about twice as much as proline and with low Kf values. This suggests separate carriers for these substrates.

Topics: Amino Acids; Animals; Benzothiazoles; Biological Transport; Carbocyanines; Fluorescent Dyes; In Vitro Techniques; Intestinal Mucosa; Jejunum; Kinetics; Membrane Potentials; Microvilli; Proline; Quinolines; Rabbits

A lipophilic fluorescent cation diS-C3-(5) and rotenone suppress the oxygen consumption rate of thymocytes in similar concentrations. Seventy percent inhibition corresponds to an inhibitor:cytochrome a molar ratio of about 1:1. Addition of uncouplers decreases the inhibition of respiration by diS-C3-(5) (but not rotenone). FCCP in similar concentrations increases O2 consumption in the absence of diS-C3-(5) and the diS-C3-(5) fluorescence intensity in the presence of TMPD in thymocyte suspensions. In most thymocyte preparations, oligomycin (0.05-0.1 microgram/mL) increases the fluorescence of diS-C3-(5) and further addition of TMPD (50-100 microM) decreases the fluorescence. Addition of NaCN (400 microM) after oligomycin leads to a fluorescence increase that is hardly affected by subsequent addition of 0.2 microM FCCP. Nigericin (10-50 nM) decreases the diS-C3-(5) fluorescence. The data indicate that the diS-C3-(5) fluorescence associated with mitochondrial transmembrane potential (delta psi m) may be an essential part of the diS-C3-(5) fluorescence in lymphocyte suspensions. The changes of the diS-C3-(5) fluorescence intensity in the presence of TMPD after FCCP addition reflect delta psi m.

Topics: Animals; Benzothiazoles; Carbocyanines; Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; Ethylenediamines; Fluorescent Dyes; Lymphocytes; Membrane Potentials; Mitochondria; Oligomycins; Oxygen Consumption; Quinolines; Rats; Rotenone; Spectrometry, Fluorescence; Thymus Gland

Effects of three probes for measuring membrane potential, tetraphenylphosphonium (TPP+), rhodamine 6G and 3,3'-dipropylthiocarbocyanine (diS-C3-(5)) on energy metabolism in synaptosomes were investigated. None of the three probes had any effect on lactate production in synaptosomes. TPP+ and rhodamine 6G did not inhibit the respiration of synaptosomes with pyruvate and succinate as exogenous substrate and were only weakly inhibitory with endogenous substrates. In contrast, diS-C3-(5) markedly inhibited the respiration of synaptosomes with glucose, pyruvate and endogenous substrates. All three probes reduced ATP content in synaptosomes and depolarized the membrane potential in synaptosomes with increasing concentrations of the probes. It is, therefore, preferable to estimate membrane potential with TPP+ or rhodamine 6G at their low concentrations where their effect on metabolism is negligible.

Topics: Adenosine Triphosphate; Animals; Benzothiazoles; Brain Chemistry; Carbocyanines; Coloring Agents; Glucose; In Vitro Techniques; Lactates; Membrane Potentials; Onium Compounds; Organophosphorus Compounds; Oxygen Consumption; Pyruvates; Pyruvic Acid; Quinolines; Rats; Rhodamines; Succinates; Succinic Acid; Synaptosomes; Xanthenes

It was shown that 3,3'-dipropylthiocarbocyanine iodide diS-C3-(5) can be used as a fluorescent probe for registration of conformational changes in calmodulin and troponin C, as well as for determination of concentrations of these Ca-binding proteins in experimental samples. The sensitivity of the method (10(-7) M) is only 5 times less than that of determination of calmodulin by phosphodiesterase and by 1 or 2 orders of magnitude more than that of other techniques based on conformational changes of the protein. The spectral parameters of the fluorescent probe allow to conduct the measurements in turbid media and in the presence of many other optically active substances. Evidence is given testifying the promiscuity of the use of this approach to the study of conformational changes in calmodulin under the action of Ca2+, Mg2+, monovalent cations, temperature and target proteins (troponin I, phosphodiesterase). using phosphodiesterase and Ca-ATPase, it was shown that under certain conditions diS-C3-(5) as well as other amphypathic compounds can modify the activity of calmodulin-dependent enzymes.

Topics: Animals; Benzothiazoles; Brain Chemistry; Calcium; Calcium-Binding Proteins; Calmodulin; Carbocyanines; Cattle; Fluorescent Dyes; Magnesium; Phosphoric Diester Hydrolases; Protein Conformation; Quinolines; Temperature; Troponin; Troponin C

An attempt was made to use 3,3'-dipropylthiacarbocyanine as a membrane potential probe in yeast by following both its fluorescence changes and its uptake by the cells under different conditions. It was found that the uptake of the dye into the cytoplasmic compartment was translated into an increased fluorescence, and the uptake by the mitochondria produced a quenching of the fluorescence. The experiments to measure uptake showed that a large amount of the dye was taken up by the cells under "deenergized" conditions. The uptake of the cyanine, however, was significantly reduced by the omission of the substrate, by deenergization of the mitochondria, or by the addition of K+, but not by Na+. This cyanine seems to be a good, qualitative indicator of the potential of the plasma membrane and of the mitochondria of the cells, with a faster response than those probes used before in yeast.

Topics: Benzothiazoles; Carbocyanines; Fluorescent Dyes; Membrane Potentials; Mitochondria; Quinolines; Saccharomyces cerevisiae; Spectrometry, Fluorescence

The dependence of both the magnitude and the sign of fluorescent responses of the probe diS-C3-(5) in egg lecithin vesicle suspensions on the magnitude of the inside-negative transmembrane potential and on the total probe concentration in the sample volume has been studied. Results were compared with theoretical calculations made on the basis of the equilibrium thermodynamic model suggested earlier as well as on the observed concentration dependence of the probe fluorescence in aqueous media and membranes. It was shown that transmembrane potential results in a redistribution of the probe between the aqueous and the membrane phases and in the membrane interior. The model calculations showed that the probe concentrations in the external aqueous medium and in the outer lipid monolayer of a vesicle significantly decrease. At the same time, the dye concentration in the inner membrane monolayer increases significantly, which should lead to a marked quenching of the dye fluorescence. The changes in the fluorescence and absorption spectra are well explained in terms of the proposed mechanism. The highest responses of diS-C3-(5) to changes of the transmembrane potential were observed in a shorter wavelength region of the fluorescence spectrum at the probe to lipid ratios in membrane of 15-20 moles of probe per 1000 moles of lipid. In the longwave region, the increase in fluorescence is not an obligatory indication of a decrease in the transmembrane potential and, under certain conditions, this process can take place when the transmembrane potential increases. The generation of a quasi-equilibrium diffusion transmembrane potential results in an increase in the average probe concentration in membranes if the signs of the probe charge and the potential inside the vesicles are opposite. Thus, the "on-off" mechanism, working under conditions of steady-state processes, is not valid under equilibrium conditions.

Topics: Benzothiazoles; Carbocyanines; Fluorescent Dyes; Liposomes; Membrane Potentials; Phosphatidylcholines; Quinolines; Spectrometry, Fluorescence

Egg yolk phosphatidyl choline liposomes containing variable amounts of phosphatidyl ethanolamine, phosphatidyl inositol or phosphatidyl serine demonstrated important variations in the fluorescence of 3.3' dipropylthiodicarbocyanine. When the membrane contained no cholesterol, fluorescence was not correlated with membrane fluidity as measured by diphenyl hexatriene polarization. Increasing cholesterol concentration in valinomycin containing liposome membranes decreased the potassium induced apparent membrane potential and prevented sorption of dye to the membrane. Discontinuity in the apparent potential occurred at 30 mol% cholesterol but could not be correlated with changes in microviscosity. These results indicate that great care should be taken when correlating rapid variations of fluorescence to changes in membrane potential. We propose that changes in phospholipid metabolism could well explain fluorescent changes when monitoring the fluorescence of cyanine dye molecules sorbed to biological membranes.

Topics: Benzothiazoles; Carbocyanines; Cholesterol; Egg Yolk; Female; Fluorescent Dyes; Liposomes; Phosphatidylcholines; Phosphatidylethanolamines; Phosphatidylinositols; Phosphatidylserines; Spectrometry, Fluorescence; Structure-Activity Relationship

Studies were carried out on the electrogenicity of the lysosomal proton pump using dipropylthiadicarbocyanine iodide (diS-C3-(5] as a membrane potential probe. Pure lysosome preparations (tritosomes) quenched the fluorescence of diS-C3-(5). The quenching correlated well with the potassium ion diffusion potential (inside negative) generated by K+ with or without valinomycin. The quenching caused by lysosomes was reversed by lipophilic cations, tetraphenylarsonium (TPA) or triphenylmethylphosphonium (TPMP). Mg-ATP also reversed the quenching, which was inhibited by a protonophore, 3,5-di-tert-butyl-4-hydroxybenzylidene-malononitrile (SF-6847). The properties of the ATP-induced recovery of the quenching were exactly the same as those of ATP-induced acidification, as measured with fluorescein isothiocyanate-dextran (FD) (Ohkuma, S., et al. (1982) Proc. Natl. Acad. Sci. U.S. 79, 2758-2762) and acridine orange (Moriyama, Y., et al. (1982) J. Biochem. 92, 1333-1336), except replacement of the anion by an impermeable one enhanced ATP-induced recovery of quenching, but reduced ATP-induced acidification. Amines which dissipate delta pH across the lysosomal membrane also enhanced the Mg-ATP-induced fluorescence recovery. These results suggest that isolated lysosomes exhibit an inside negative membrane potential, especially in low K+ medium, mostly due to the K+-diffusion potential, and that the Mg-ATP-driven proton pump causes membrane depolarization (in the direction of inside positive). These possibilities were supported by results on the uptake of the radioactive membrane-permeant ions [3H]TPMP and [14C]SCN. The present results provide evidence for the electrogenic nature of the lysosomal proton pump.

Topics: Adenosine Triphosphate; Animals; Benzothiazoles; Biological Transport, Active; Carbocyanines; Fluorescent Dyes; Hydrogen-Ion Concentration; In Vitro Techniques; Kinetics; Liver; Lysosomes; Male; Potassium; Quinolines; Rats; Rats, Inbred Strains

The interaction of thymocyte mitochondria with two types of dyes - potential indicators commonly used in lymphocyte studies, has been investigated. Ethylrhodamine at concentrations up to 16 microM does not influence the systems of oxidation and energy coupling in lymphocyte mitochondria. Carbocyanines-diS-C3-(5) and diO-C3-(5) inhibit oxygen uptake by the lymphocytes in the presence of glucose and pyruvate at the same low concentrations as does rotenone (40% inhibition occurs at 10 nM). DNP reduces the inhibition of respiration by carbocyanines but not by rotenone. The increase in the fluorescence of diS-C3-(5) and in the rate of oxygen uptake in the absence of diS-C3-(5) occurs at close concentrations of the uncoupler. This indicates that the changes in the fluorescence caused by FCCP reflect the membrane potential of lymphocyte mitochondria. The maintenance of the membrane potential in lymphocyte mitochondria in the presence of diS-C3-(5) provides evidence for the absence of the corresponding changes in mitochondrial ultrastructure after addition of 0.6 microM diS-C3-(5) which completely inhibits oxygen uptake.

Topics: Animals; Benzothiazoles; Carbocyanines; Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; Fluorescent Dyes; Intracellular Membranes; Lymphocytes; Membrane Potentials; Mitochondria; Oxygen Consumption; Quinolines; Rats; Rhodamines; Thymus Gland; Xanthenes

Topics: Animals; Benzothiazoles; Carbocyanines; Cattle; Cell Membrane Permeability; Erythrocytes; In Vitro Techniques; Membrane Potentials; Potassium; Spectrometry, Fluorescence

Based on an analysis of back fluorometric titration data a partition coefficient, Kp = (5.70 +/- 0.95) x 10(4), and partition constant, K = (2.37 +/- 0.43) x 10(6), were found for the probe diS-C3-(5) in egg lecithin vesicle suspension. The relative probe quantity in an aqueous medium and in liposomes was calculated using these parameters. The number of chromophore states in this system was computer-analysed and it was shown that the probe fluorescence could be described by two fluorescing dye forms, aqueous and membrane monomers. The dependence of fluorescence intensity on the probe concentration was studied in various salt media, and a dimerization (association) constant Ka = 5 x 10(4) mol -1 . l in the buffer, and Ka = (8.1 +/- 1.5) x 10(4) mol-1 . l in 0.1 or 0.2 mol/l salt medium (KCl or NaCl) was found. From the fluorescence and absorption data critical concentrations of the onset of large probe aggregate formation were calculated for various aqueous media. The concentration dependence of the probe fluorescence in the membrane phase was calculated. The critical concentration of interaction characterizing the efficiency of the fluorescence concentration quenching processes (CCI) was found to be approx. 5-6 mol probe per 1000 mol lipid. The top probe concentration in a membrane (the "saturation" concentration) was estimated from the slope of the initial linear parts of the back fluorometric titration curves, and was found to be equal to (59 +/- 13) mol probe per 1000 mol lipid.

Topics: Benzothiazoles; Carbocyanines; Chemical Phenomena; Chemistry, Physical; Fluorescent Dyes; Liposomes; Membranes, Artificial; Phosphatidylcholines; Quinolines; Spectrometry, Fluorescence; Water

The membrane potential of the human platelet was investigated using the membrane potential probes 3,3'-dipropyl-2,2'-thiadicarbocyanine iodide and tritiated triphenylmethylphosphonium bromide. The membrane potential in physiologic buffer was estimated to be 52-60 mV inside negative. The membrane was depolarized when extracellular potassium or hydrogen ion concentrations were increased. Changes in extracellular sodium, chloride, or calcium ion concentration had no measurable effect on membrane potential. Elevated extracellular potassium has been shown to increase platelet sensitivity to the aggregating agent, adenosine diphosphate. Our results show that changes in extracellular ion concentrations that depolarize platelets increase platelet sensitivity to aggregating agents. These results suggest that membrane potential changes may play a role in modulating the response of platelets to aggregating agents.

Topics: Benzothiazoles; Blood Platelets; Carbocyanines; Electrolytes; Humans; Inulin; Membrane Potentials; Onium Compounds; Platelet Aggregation; Spectrometry, Fluorescence; Trityl Compounds; Valinomycin

The accumulation of [3H]triphenylmethylphosphonium cation in neuroblastoma N1E 115 cells in the presence of tetraphenylboron is reduced by 3,3'-diethylthiadicarbocyanine iodide and by 3,3'-dipropylthiadicarbocyanine iodide. This reduction in uptake of the lipophilic cation is not due to the carbocyanine dyes depolarizing the plasma membrane of these cells but due to an interaction between the carbocyanine dyes and tetraphenylboron leaving less of the lipophilic anion free in solution to assist uptake of the lipophilic cation. This interaction is shown to have a 1:1 stoicheiometry.

Topics: Animals; Benzothiazoles; Biological Transport; Carbocyanines; Cell Line; Fluorescent Dyes; Indicators and Reagents; Kinetics; Membrane Potentials; Mice; Neoplasms, Experimental; Neuroblastoma; Onium Compounds; Quinolines; Thiazoles; Tritium; Trityl Compounds

The kinetics of Na-coupled solute transport by renal and jejunal brush-border vesicles in the rabbit were examined using the potential-sensitive fluorescent dye diS-C3-(5). All organic solutes known to be transported across these membranes by Na-coupled mechanisms increase the fluorescence of the dye in the presence of Na, but not K. An increase in fluorescence (delta F) corresponds to a depolarization of the electrical potential difference (5-60 mV) across the brush-border membrane in the intact cell. delta F was independent of the valency of the transported solute. The fluorescence response was saturable, and for twelve solutes the Kf, i.e. the concentration of the substrate generating 50% of the maximal response, agreed quite closely with the Kt values reported from tracer studies. For six solutes increasing the Na concentration decreased Kf, and this agrees with the effect of Na on the kinetics of succinate transport in renal vesicles. We conclude that D-glucose, neutral amino acids and imino acids are co-transported with Na across both renal and jejunal brush-border membranes, and that carboxylic acids, beta-amino acids, and dibasic amino acids are co-transported with Na across the renal, but not jejunal, membranes.

Topics: Amino Acids; Animals; Benzothiazoles; Biological Transport, Active; Carbocyanines; Carboxylic Acids; Fluorescent Dyes; Glucose; In Vitro Techniques; Jejunum; Kidney; Kinetics; Membrane Potentials; Microvilli; Rabbits; Sodium

The membrane potential of human platelets, and the role of this potential in platelet aggregation, was assessed using the noncovalent, fluorescent probe DiS-C3-5. High K+ and Gramicidin depolarised the cells, whereas valinomycin in standard (4 mMK+) solution produced a hyperpolarisation. Very small changes in potential were observed when choline Cl replaced NaCl. These findings indicate that platelets possess a relatively K+-perm-selective membrane. The resting potential calculated from the "valinomycin null point" (the K+ concentration gradient at which valinomycin did not change the potential) was approximately -60 mV. Other factors that contribute to the platelet membrane potential include a significant Cl- permeability, demonstrated by replacing Cl- with methylsulphate, and an electrogenic Na+ pump, demonstrated using strophanthidin. Little or no change in potential was observed upon addition of ADP, collagen, U44069 or thrombin. Neither strong depolarisation with high K+ or gramicidin nor hyperpolarisation with valinomycin induced platelet aggregation or altered platelet responses to agonists. It is concluded that the information transduction mechanisms involved in platelet activation do not include changes in platelet membrane potential.

Topics: Benzothiazoles; Blood Platelets; Carbocyanines; Cell Membrane Permeability; Egtazic Acid; Fluorescent Dyes; Gramicidin; Humans; Ion Channels; Membrane Potentials; Platelet Aggregation; Potassium; Valinomycin

Methods for the measurement of membrane potentials in cells not easily penetrated by microelectrodes were assessed for use in epithelia. The lipophilic cation triphenylmethylphosphonium appeared to distribute in a Nernstian fashion in the epithelial cells of Necturus gallbladder as judged by parallel microelectrode measurements. In the toad urinary bladder, the distribution of this cation gave a value for the membrane potential of epithelial cells under short-circuit conditions of -62 mV in normal Ringer's solution and -51 mV after 2 h treatment with ouabain. In our laboratory the dye 3,3'-dipropylthiadicarbocyanine iodide, when used with cell suspensions, yielded results comparable to those of other workers, but we were unable to record a redistributional signal from epithelial sheets or scraped cells. The dye appeared to enter cells and become irreversibly bound. The membrane-bound merocyanine dyes have not been used on epithelial cells. They appear to hold the greatest promise for dynamic experiments on epithelial membrane potentials.

Topics: Animals; Anions; Anura; Benzothiazoles; Carbocyanines; Cations; Epithelium; Membrane Potentials; Necturus; Onium Compounds; Trityl Compounds; Urinary Bladder; Urodela

It is shown that during deamination of AMP with membrane-bound AMP-aminohydrolase of sarcoplasmic reticulum, which is accompanied by the pH increase, there occurs a fluorescence quenching of 1-anilino-8-naphthalinsulphonate and 3-3(1)-dipropylthio-dicarbocyanine bound to membranes. Such a direction in the fluorescence changes is observed when pH is artificially increased from 6.5 to 8.5. No changes are marked in fluorescence of the applied probes in experiments with the use of the sarcoplasmic reticulum fragment vesicles with AMP-aminohydrolase only or in deamination of AMP in a strongly buffered medium as well as under the effect of IMP or NH4Cl in concentrations up to 10 mM. The studies conducted show that the charge of sarcoplasmic reticulum membranes may change in the process of amp deamination.

Topics: Adenosine Monophosphate; AMP Deaminase; Anilino Naphthalenesulfonates; Animals; Benzothiazoles; Carbocyanines; Fluorescent Dyes; Hydrogen-Ion Concentration; Intracellular Membranes; Kinetics; Nucleotide Deaminases; Quinolines; Rabbits; Sarcoplasmic Reticulum; Spectrometry, Fluorescence

Changes in fluorescence intensity of thiodicarbocyanine, DiS-C3(5), were correlated with direct microelectrode potential measurements in red blood cells from Amphiuma means and applied qualitatively to evaluate the effects of extracellular Ca2+, K+ and pH on the membrane potential of human red cells. Increasing extracellular [Ca2+] from 1.8 to 15 mM causes a K+-dependent hyperpolarization and decrease in fluorescence intensity in Amphiuma red cells. Both the hyperpolarization and fluorescence change disappear when the temperature is raised from 17 to 37 degrees C. No change in fluorescence intensity is observed in human red cells with comparable increase in extracellular Ca2+ in the temperature range 5-37 degrees C. Increasing the extracellular pH, however, causes human red cells to respond to an increase in extracellular Ca2+ with a significant but temporary loss in fluorescence intensity. This effect is blocked by EGTA, quinine or by increasing extracellular [K+], indicating that at elevated extracellular pH, human erythrocytes respond to an increase in extracellular Ca2+ with an opening of K+ channels and associated hyperpolarization of the plasma membrane.

Topics: Animals; Benzothiazoles; Calcium; Carbocyanines; Erythrocyte Membrane; Erythrocytes; Fluorescent Dyes; Hydroxides; Kinetics; Membrane Potentials; Potassium; Urodela

The membrane potential of guinea pig polymorphonuclear leukocytes has been assessed with two indirect probes, tetraphenylphosphonium (TPP+) and 3,3'-dipropylthiadicarbocyanine (disS-C3-(5)). The change in TPP+ concentration in the medium was measured with a TPP+-selective electrode. By monitoring differences in accumulation of TPP+ in media containing low and high potassium concentrations, a resting potential of -58.3 mV was calculated. This potential is composed of a diffusion potential due to the gradient of potassium, established by the Na+, K+ pump, and an electrogenic potential. The chemotactic peptide fMet-Leu-Phe elicits a rapid efflux of accumulated TPP+ (indicative of depolarization) followed by its reaccumulation (indicative of repolarization). In contrast, stimulation with concanavalin A results in a rapid and sustained depolarization without a subsequent repolarization. The results obtained with TPP+ and diS-C3-(5) were comparable. Such changes in membrane potential were observed in the absence of extracellular sodium, indicating that an inward movement of sodium is not responsible for the depolarization. Increasing potassium levels, which lead to membrane depolarization, had no effect on the oxidative metabolism in nonstimulated or in fMet-Leu-Phe-stimulated cells. Therefore, it seems unlikely that membrane depolarization per se is the immediate stimulus for the respiratory burst.

Topics: Animals; Benzothiazoles; Carbocyanines; Cell Membrane; Electric Stimulation; Fluorescent Dyes; Guinea Pigs; Hydrogen-Ion Concentration; Indicators and Reagents; Kinetics; Membrane Potentials; Neutrophils; Onium Compounds; Organophosphorus Compounds; Potassium

The protonmotive force in Saccharomyces fragilis has been estimated under various experimental conditions. The transmembrane potential has been monitored with tetraphenylphosphonium and 3,3'-dipropylthiadicarbocyanine. The distribution ratio of these cations between intracellular and extracellular water appeared to be governed by the electrical potential difference across the membrane of this yeast strain. The transmembrane pH difference was deduced from dimethyloxazolidinedione uptake experiments and from direct measurements of intracellular pH after freezing and boiling of the cells. Both methods yielded similar results. D-Fucose is transported by S. fragilis via H+ symport, with a H+/fucose stoichiometry of approximately 1. Accumulation of this sugar appeared to be closely correlated with the protonmotive force.

Topics: Benzothiazoles; Biological Transport, Active; Carbocyanines; Cell Membrane; Coloring Agents; Fucose; Hydrogen-Ion Concentration; Indicators and Reagents; Membrane Potentials; Onium Compounds; Organophosphorus Compounds; Saccharomyces

Activation of normal or myeloperoxidase-deficient human granulocytes by phorbol myristate acetate resulted in an initial membrane depolarization as indicated by an increase in fluorescence of the lipophilic cation probe of membrane potential, 3,3'-dipropylthiodicarbocyanine. A subsequent apparent hyperpolarization (decrease in fluorescence) was observed in normal but not myeloperoxidase-deficient cells. Addition of purified myeloperoxidase restored a normal pattern of fluorescence changes to the enzyme-deficient granulocytes. The secondary decrease in fluorescence in normal cells was markedly blunted by addition of azide, cyanide, or catalase. In a cell-free system, the fluorescence of 3,3'-dipropylthiodicarbocyanine, but not that of 3,3'-dipentyloxadicarbocyanine, was rapidly eliminated by myeloperoxidase in the presence of hydrogen peroxide and a halide; this loss of fluorescence was inhibited by azide, cyanide, or catalase. These findings indicate that secretion of myeloperoxidase and hydrogen peroxide by activated granulocytes results in decreased fluorescence of 3,3'-dipropylthiodicarbocyanine, probably by thioether oxidation. While the determination of initial rates of depolarization using this probe is unaffected by the myeloperoxidase system, measurement of extent of depolarization and any subsequent membrane potential changes requires the addition of inhibitors. In the absence of inhibitors, the secondary decrease in fluorescence can be used as an indicator of secretion of myeloperoxidase and hydrogen peroxide.

Topics: Benzothiazoles; Carbocyanines; Fluorescent Dyes; Granulocytes; Humans; Kinetics; Membrane Potentials; Peroxidase; Peroxidases; Quinolines; Tetradecanoylphorbol Acetate

Phagocytic cells generate superoxide in response to stimulation by opsonized particles. A continuous assay for opsonized zymosan-stimulated granulocyte superoxide production shows that there is a lag time between the addition of particles and the onset of detectable superoxide production. Superoxide production is preceded by membrane potential depolarization. Neither superoxide production nor membrane depolarization occurs in granulocytes from patients with chronic granulomatous disease. The extent of activation by opsonized zymosan is affected by the dose of zymosan from 0.5 to 4.5 mg/ml, but the time necessary for activation (lag time) is not. Similarly, the extent of depolarization but not the time necessary for attaining maximum depolarization is concentration-dependent. Effects of temperature, divalent cations, 2-deoxyglucose, cyanide, and N-ethyl maleimide on superoxide production are similar for granulocytes treated with soluble stimuli and with opsonized zymosan. Thus, zymosan stimulates granulocytes to generate superoxide and undergo membrane depolarization in a manner similar to that elected by soluble stimuli.

Topics: Animals; Benzothiazoles; Calcium; Carbocyanines; Cyanides; Cytochrome c Group; Deoxyglucose; Ethylmaleimide; Granulocytes; Granulomatous Disease, Chronic; Guinea Pigs; Humans; Membrane Potentials; Neutrophils; Opsonin Proteins; Oxygen; Superoxides; Temperature; Zymosan

Topics: Bacillus subtilis; Benzothiazoles; Biological Transport, Active; Carbocyanines; Carbonyl Cyanide m-Chlorophenyl Hydrazone; Cell Membrane; Fluorescent Dyes; Kinetics; Magnesium; Membrane Potentials; Rubidium; Valinomycin

Changes in fluorescence of 3,3'-dipropylthiodicarbocyanine iodide which had been equilibrated with suspensions of the wild-type yeast Saccharomyces cerevisiae and of respiration-deficient mutants were followed. The changes have been attributed to changes of yeast membrane potentials, since the fluorescence with wild-type yeast could be affected in a predictable manner by uncouplers and the pore-forming agent nystatin. As in other systems, a rise of steady-state fluorescence was ascribed to depolarization and a drop of the fluorescence to hyperpolarization. (1) A considerable rise in steady-state fluorescence was brought about by addition of antimycin A or some other mitochondrial inhibitors to respiring cells. A major part of the composite membrane potential monitored in intact yeast cells appeared to be represented by the membrane potential of mitochondria. (2) Addition of D-glucose and of other substrates of hexokinase, including non-metabolizable 2-deoxy-D-glucose, induced a two-phase response of fluorescence, indicating transient depolarization followed by repolarization. Such a response was not elicited by other sugars which had been reported to be transported into the cells by a glucose carrier or by D-galactose in galactose-adapted cells. The depolarization was explained by electrogenic ATP exit from mitochondria to replenish the ATP consumed in the Hexokinase reaction and the repolarization by subsequent activation of respiration. (3) In non-respiring cells only a drop in fluorescence was induced by glucose and this was ascribed to an ATP-dependent polarization of the plasma membrane. (4) Steady-state fluorescence in suspensions of respiration-deficient mutants, lacking cytochrome a, cytochrome b, or both, was high an remained unaffected by uncouplers and nystatin. This indicates that membranes of the mutants may have been entirely depolarized. A partial polarization, apparently restricted to the plasma membrane, could be achieved by glucose addition.

Topics: Antimycin A; Benzothiazoles; Carbocyanines; Deoxyglucose; Energy Metabolism; Fluorescent Dyes; Glucose; Membrane Potentials; Mutation; Nystatin; Quinolines; Saccharomyces cerevisiae; Spectrometry, Fluorescence

The effect of the transport of tricarboxylic acid cycle intermediates on the membrane potential of renal brush border vesicles was studied using fluorescence of the cyanine dye, 3,3'-dipropylthiadicarbocyanine iodide. The behavior of the dye in the preparation was established with valinomycin-induced K+-diffusion potentials; increases in fluorescence were associated with depolarizing conditions. Addition of 1 mm succinate or citrate to membrane/dye suspensions produced transient increases in fluorescence, indicative of a depolarizing event(s) associated with the transport of these substrates. The transient response in fluorescence was Na+ dependent, of greater magnitude under Na+-gradient as compared to Na+-equilibrium conditions, and was a saturable function of substrate concentration. The specificity of the fluorescence response was identical to that obtained from studies of the competitive inhibition of succinate transport by tricarboxylic acid cycle intermediates and analogs We conclude that the major tricarboxylic acid cycle intermediates are transported via a common Na+-dependent transport system in renal brush border membranes.

Topics: Animals; Benzothiazoles; Biological Transport; Carbocyanines; Carboxylic Acids; Cell Membrane; Citrates; Citric Acid Cycle; Kidney; Kinetics; Membrane Potentials; Microvilli; Rabbits; Sodium; Spectrometry, Fluorescence; Succinates

Topics: Benzothiazoles; Carbocyanines; Cations; Cell Line; Fluorescent Dyes; Glioma; Hybrid Cells; Membrane Potentials; Neoplasms, Experimental; Neuroblastoma; Prostaglandins; Prostaglandins D; Serotonin

The experiments presented below compare the interaction of diO-C3-(5) and diS-C3-(5) with erythrocytes, erythrocyte ghosts and phospholipid vesicles derived from erythrocyte membranes. The results confirm earlier reports of diS-C3-(5) dimerization in the presence of hemoglobin and of dye aggregate formation in erythrocyte suspensions. DiO-C3-(5), on the other hand, binds to vesicles and ghosts freed of hemoglobin in a potential-dependent manner but without forming dye aggregates. The two dyes bind to the different preparations in similar proportions, but diS-C3-(5) is bound in amounts 3-40 times greater depending on the degree of polarization. The results show that mechanism other than binding to hemoglobin must occur in order to explain the potential-dependent binding of both dyes to ghosts and vesicles. A primary interaction must exist between the dye molecule and the lipid bilayer in a biological membrane, and this would be expected to occur in the presence of hemoglobin or other cytosolic components. DiO-C3-(5) is a better dye to use than diS-C3-(5) for mechanistic studies, in order to avoid problems associated with formation of complex aggregates of the latter dye, especially in hyperpolarized membrane suspensions.

Topics: Benzothiazoles; Carbocyanines; Erythrocyte Membrane; Erythrocytes; Fluorescence; Fluorescent Dyes; Humans; Lipid Bilayers; Membrane Potentials; Potassium; Quinolines; Spectrophotometry

The characterization of the anion transport system in sarcoplasmic reticulum (SR) vesicles was performed by kinetic analysis of the reversible inhibition of gluconate efflux by a disulfonic stilbene derivative; 4-acetamido-4'-isothiocyano-stilbene-2,2'-disulfonate (SITS). The gluconate efflux in SR vesicles was measured by following the change in Cl- ion-diffusion potential due to the mutual diffusion of Cl- and gluconate- using a potential probe; 3,3'-dipropylthiadicarbocyanine iodide (diS-C3-(5)). The main results are as follows. (1) Gluconate efflux was increased by intravesicular gluconate- and was decreased by both extravesicular gluconate- and extravesicular Cl-. (2) Gluconate efflux was depressed when membrane potential became inside-negative. (3) SITS reacted with the site of the anion transport system from the outside of the vesicles and one molecule of SITS inhibited each transport unit. (4) Extravesicular Cl- competed with SITS at the common site of the anion transport system. The dissociation constants for Cl- and SITS are 70 mM and 0.048 microM, respectively. (5) The inhibition of gluconate efflux by SITS was noncompetitive with both intravesicular gluconate and extravesicular gluconate. These results suggest that the anion transport system in SR vesicles can be described by a carrier-mediated transport model in which anions are transported by a mobile transport site accompanied by a net negative charge.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; Animals; Anions; Benzothiazoles; Biological Transport; Carbocyanines; Chlorides; Diffusion; Dose-Response Relationship, Drug; Fluorescent Dyes; Gluconates; Kinetics; Membrane Potentials; Quinolines; Rabbits; Sarcoplasmic Reticulum; Stilbenes

The fluorescent dye, 3,3'-dipropylthiadicarbocyanine iodide, is frequently used to estimate the cell membrane potential of small cells. We have tested the effects of this dye on glycolytic rate, O2 consumption, and cellular ATP content in Ehrlich ascites tumor cells. Addition of the dye to steady state cells in the absence of glucose induces a rapid depletion (half-time approximately equal to 5 min) of cellular ATP, which is secondary to a 76% inhibition of O2 consumption. Aerobic glycolysis is stimulated by 76%. Valinomycin produces an additional stimulation of acid production. Our findings indicate that this fluorescent dye alters energy metabolism of Ehrlich ascites tumor cells. The response is consistent with primary inhibition of oxidative phosphorylation.

Topics: Adenosine Triphosphate; Animals; Benzothiazoles; Carbocyanines; Carcinoma, Ehrlich Tumor; Energy Metabolism; Glycolysis; Kinetics; Mice; Oxidative Phosphorylation; Oxygen Consumption; Quinolines; Valinomycin

Topics: Amino Acids; Animals; Benzothiazoles; Biological Transport, Active; Carbocyanines; Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; Cell Membrane; Diffusion; Fluorescent Dyes; Glucose; Glutamates; Gramicidin; Hydrogen-Ion Concentration; In Vitro Techniques; Kidney; Microvilli; Rabbits; Sodium; Valinomycin

The fluorescent dye 3,3'-dipropylthiodicarbocyanin iodide [diS-C3-(5)] is used as a probe of fast membrane potential variations in the study of rod-outer-segment membranes. The formation of metarhodopsin II is found to be associated with the generation of a transmembrane potential, which is dissipitated by ionic movements across the membrane; its risetime is very similar to that of the protonation of the protein, which is also associated with the formation of metarhodopsin II.

Topics: Animals; Benzothiazoles; Carbocyanines; Cattle; Cell Membrane; Hydrogen-Ion Concentration; Kinetics; Light; Membrane Potentials; Photoreceptor Cells; Quinolines; Retinal Pigments; Rhodopsin; Spectrometry, Fluorescence

Topics: Animals; Benzothiazoles; Biological Transport, Active; Carbocyanines; Carcinoma, Ehrlich Tumor; Cell Membrane; Fluorescent Dyes; Kinetics; Membrane Potentials; Mice; Ouabain; Potassium; Quinolines; Sodium

Rabbit neutrophil leucocytes take up the cationic, fluorescent dye 3,3'-dipropylthiadicarbocyanine iodide (DiS-C3-(5)). Treatment with valinomycin and K+ then produces characteristic changes in suspension fluorescence that indicate that the dye enters the cells in a potential-dependent fashion and that the resting membrane potential lies between -66 and -86 mV. The peptide, N-fMet-Leu-Phe, a potent chemoattractant for neutrophils, added to stained cell suspensions, induces fluorescence intensity changes. These occur over an 8-10 min period. The time course of this response is profoundly affected by the omission of Ca2+ from the medium. When this ion is present (1.26 mM) a small, transient increase in intensity is observed, superimposed on a sustained decrease. On the other hand, in the absence of added Ca2+ a large, transient increase is observed. The ED50 for this is 1.1 x 10(-10) M. These changes are not elicited by N-fMet-Phe (10(-9) M) and are inhibited by the antagonist Boc-Leu-Phe-Leu-Phe. However, a component of zymosan-activated rabbit plasma, which is complement-derived, induces identical fluorescence changes that are not inhibited by the antagonist, confirming that neutrophil activation by complement operates through an independent receptor. The fluorescence responses to the chemotactic peptide and the activated-plasma component may be interpreted in terms of changes in neutrophil membrane potential brought about by alterations in cell ionic permeability at an early stage during activation. The transient increase corresponds to a depolarisation that may be associated with a change in Na+ permeability, while the sustained decrease corresponds to a membrane hyperpolarisation.

Topics: Anaphylatoxins; Animals; Benzothiazoles; Calcium; Carbocyanines; Cell Membrane Permeability; Chemotactic Factors; Complement C5; Complement C5a; Fluorescent Dyes; Membrane Potentials; Methionine; N-Formylmethionine; N-Formylmethionine Leucyl-Phenylalanine; Neutrophils; Oligopeptides; Quinolines; Rabbits; Spectrometry, Fluorescence

The Donnan potential in sarcoplasmic reticulum vesicles prepared from rabbit skeletal muscle was measured by using a fluorescent cyanine dye, diS-C3-(5) (3,3'-dipropylthiadicarbocyanine iodide). The Donnan potential is caused by the existence of negative fixed charges inside the vesicles. On average, sarcoplasmic reticulum vesicles have internal negative fixed charges of 420 nmol/mg protein at pH 6.8. The fixed charges are mainly due to low affinity Ca2+ binding proteins. The Donnan potential in sarcoplasmic reticulum vesicles was about -72 mV in 5 mM K-MES buffer (pH 6.8). When Ca2+ was accumulated into sarcoplasmic reticulum vesicles with ATP, the concentration of negative fixed charges decreased from 140 mM to 95 mM due to Ca2+ binding to the negative fixed charges, and sarcoplasmic reticulum vesicles were depolarized by about 10 mV. It was found that there are two types of sarcoplasmic reticulum vesicles, one type (60%) having many negative fixed charges inside the vesicles and the other (40%) having few fixed charges.

Topics: Animals; Benzothiazoles; Calcium; Carbocyanines; Carrier Proteins; Fluorescent Dyes; Membrane Potentials; Muscles; Potassium; Quinolines; Rabbits; Sarcoplasmic Reticulum

The cyanine dye 3,3'-dipropyl-2,2'-thiadicarbocyanine iodide [diS-C3(5)] was found to be a potent inhibitor of endogenous respiration of Ehrlich ascites tumor cells. This effect is believed to involve both rotenone and uncoupler type actions of the dye on the electron transport system of the mitochondria.

Topics: Adenosine Triphosphatases; Animals; Benzothiazoles; Carbocyanines; Carcinoma, Ehrlich Tumor; Dinitrophenols; Electron Transport; Fluorides; Kinetics; Mice; Mitochondria; Oligomycins; Oxygen Consumption; Quinolines; Rotenone