benzofurans and fura-2-am

Treatment of Madin-Darby canine kidney (MDCK) cells with the peptide hormone angiotensin II (Ang II) results in an increase in the concentrations of cytosolic free calcium ([Ca(2+)](i)) and sodium ([Na(+)](i)) with a concomitant decrease in cytosolic free Mg(2+) concentration ([Mg(2+)](i)). In the present study we demonstrate that this hormone-induced decrease in [Mg(2+)](i) is independent of [Ca(2+)](i) but dependent on extracellular Na(+). [Mg(2+)](i), [Ca(2+)](i), and [Na(+)](i) were measured in Ang II-stimulated MDCK cells by fluorescence digital imaging using the selective fluoroprobes mag-fura-2AM, fura-2AM, and sodium-binding benzofuran isophthalate (acetoxymethyl ester), respectively. Ang II decreased [Mg(2+)](i) and increased [Na(+)](i) in a dose-dependent manner. These effects were inhibited by irbesartan (selective AT(1) receptor blocker) but not by PD123319 (selective AT(2) receptor blocker). Imipramine and quinidine (putative inhibitors of the Na(+)/Mg(2+) exchanger) and removal of extracellular Na(+) abrogated Ang II-mediated [Mg(2+)](i) effects. In cells pretreated with thapsigargin (reticular Ca(2+)-ATPase inhibitor), Ang II-stimulated [Ca(2+)](i) transients were attenuated (p < 0.01), whereas agonist-induced [Mg(2+)](i) responses were unchanged. Clamping the [Ca(2+)](i) near 50 nmol/liter with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis(acetoxymethyl ester) inhibited Ang II-induced [Ca(2+)](i) increases but failed to alter Ang II-induced [Mg(2+)](i) responses. Benzamil, a selective blocker of the Na(+)/Ca(2+) exchanger, inhibited [Na(+)](i) but not [Mg(2+)](i) responses. Our data demonstrate that in MDCK cells, AT(1) receptors modulate [Mg(2+)](i) via a Na(+)-dependent Mg(2+) transporter that is not directly related to [Ca(2+)](i). These data support the notion that rapid modulation of [Mg(2+)](i) is not simply a result of Mg(2+) redistribution from intracellular buffering sites by Ca(2+) and provide evidence for the existence of a Na(+)-dependent, hormonally regulated transporter for Mg(2+) in renally derived cells.

Topics: 3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester; Adrenergic alpha-Antagonists; Adrenergic Uptake Inhibitors; Amiloride; Angiotensin II; Animals; Antihypertensive Agents; Benzofurans; Biphenyl Compounds; Calcium; Calcium Channel Agonists; Cell Line; Cells, Cultured; Chelating Agents; Cytosol; Dogs; Dose-Response Relationship, Drug; Egtazic Acid; Ethers, Cyclic; Fluorescent Dyes; Fura-2; Imidazoles; Imipramine; Irbesartan; Kidney; Kinetics; Magnesium; Microscopy, Fluorescence; Peptides; Pyridines; Quinidine; Receptor, Angiotensin, Type 1; Receptor, Angiotensin, Type 2; Receptors, Angiotensin; Sodium; Tetrazoles; Time Factors

We investigated the mechanism by which H2O2 mediates an increase in [Na+]i in L929 cells and the relevance of this Na+ load for H2O2-induced cell injury. [Na+]i increased early after exposure to H2O2 as monitored by fluorescence spectrophotometry of cells loaded with SBFI. The omission of Na+ from the incubation buffer significantly reduced H2O2-cytotoxicity. This protection could not be mimicked by inhibition of either the Na+/H+-antiporter, the Na+/HCO3- -cotransporter, or the Na+/K+/2Cl- -cotransporter by using Hoechst 694 (0.02 mM) or 4-acetamido-4'-isothio-cyanatostilbene-2,2'-disulfonic acid (SITS) (0.02 mM) or furosemide (1 mM) and bumetanide (0.5 mM). Only the blocker of the Na+/Ca2+-exchanger bepridil (0.2 mM) significantly reduced H2O2-cytotoxicity but without interfering with the increase in [Na+]i. H2O2 caused a rapid and sustained increase in [Ca2+]i, which was significantly reduced in bepridil pretreated cells and after replacing extracellular Na+ by choline. H2O2 was found to initiate a cellular uptake of unphysiological Ni2+ by using Newport Green diacetate as fluorescent dye. Our data suggest that H2O2 mediates Na+-influx across the plasma membrane rather unspecifically than through specific transporters. The protective effect of bepridil against H2O2-cytotoxicity occurs as a consequence of a reduced cellular Ca2+-uptake. We conclude that H2O2-mediated unspecific accumulation of Na+ seems to favor a Ca2+-influx into the cells, which takes place on the Na+/Ca2+-exchanger operating in reverse mode in exchange for Na+-efflux. Therefore, H2O2-induced cellular Na+ accumulation appears to play a permissive rather than a triggering role in H2O2-mediated cell injury.

Topics: Animals; Benzofurans; Bepridil; Calcium; Cell Line; Cell Membrane Permeability; Fluorescent Dyes; Fura-2; Hydrogen Peroxide; Mice; Nickel; Ouabain; Phthalic Acids; Sodium; Sodium Channel Blockers; Spectrometry, Fluorescence

Endothelin is a novel peptide secreted by endothelial cells, the vasoconstrictor effects of which appear dependent on the activation of phospholipase C. We examined in tissue culture its potential as a growth factor for vascular smooth muscle. In quiescent cultures of rat aortic smooth muscle cells, endothelin rapidly elevated levels of c-fos and c-myc mRNA. Peak effects on c-fos mRNA occurred between 15 and 30 min and were completely gone after 2 h. The elevation in c-fos mRNA was, in part, dependent on protein kinase C, since phorbol myristate acetate (PMA) also elevated c-fos mRNA and further increased c-fos mRNA expression by endothelin, but the effects were not additive. Furthermore, the endothelin-induced elevation in c-fos mRNA was attenuated but not abolished in protein kinase C-depleted cells. Maximum levels of c-myc mRNA occurred between 15 and 30 min after exposing the cells to endothelin and persisted for at least 6 h. The effects of simultaneous addition of endothelin and PMA on c-myc mRNA levels were essentially similar to those observed with c-fos mRNA. [3H]thymidine incorporation into DNA occurred 8 h after exposing the cells to endothelin. The mitogenic effect of endothelin was smaller than that observed with either fetal calf serum or epidermal growth factor and was dependent on both pertussis toxin-insensitive and -sensitive pathways. Sensitivity to the latter pathway did not appear dependent on attenuation of phospholipase C activity, since neither peak intracellular calcium concentrations nor c-fos mRNA levels were reduced in pertussis toxin-treated cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Aorta; Benzofurans; Calcium; Cell Division; Cells, Cultured; DNA Replication; Endothelins; Endothelium, Vascular; Epidermal Growth Factor; Fluorescent Dyes; Fura-2; Kinetics; Muscle, Smooth, Vascular; Peptides; Pertussis Toxin; Rats; Rats, Inbred SHR; RNA; Tetradecanoylphorbol Acetate; Virulence Factors, Bordetella

The effect of TRH on cytosolic free calcium concentrations, [Ca2+]i, was evaluated on cell suspensions obtained from 6 human PRL secreting pituitary adenomas. In these cells resting [Ca2+]i levels were variable (mean +/- SE; 103.8 +/- 6.5, n = 25); the addition of 100 nM TRH caused a marked [Ca2+]i rise within 20 sec., the peak values ranging from 200 to 437 nM (285 +/- 10.8 nM, n = 10). The transients induced by TRH were composed by a rapid increase, due to mobilization of calcium from intracellular stores, followed within a few seconds by a lower plateau which was due to stimulated influx from the extracellular space. In fact, when EGTA and verapamil were applied after TRH they caused the Ca2+ plateau to dissipate rapidly. The addition of 1 microM dopamine (DA) caused a substantial decrease of resting [Ca2+]i (about 10-30%) as well as an inhibition of the plateau phase induced by TRH. The effect of DA completely depended on extracellular Ca2+. The TRH-induced transients observed in adenomatous cells were quite similar in size and time course to those recorded in normal rat lactotrophs. As previously observed in rat lactotrophs, in adenomatous cells treatment with pertussis toxin (PTx, 1 microgram/ml for 4 h) was unable to affect the [Ca2+]i transients induced by TRH while completely abolished the effect of DA. The effects of TRH on in vivo and in vitro PRL secretion were also evaluated. Before surgery, no patient showed a positive response to the iv administration of 200 micrograms TRH (serum PRL levels: 95 +/- 62 ng/ml in basal conditions vs 124 +/- 92 after TRH, P = NS).(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Benzofurans; Calcium; Cytosol; Dopamine; Egtazic Acid; Female; Fura-2; Humans; Pertussis Toxin; Pituitary Gland, Anterior; Pituitary Neoplasms; Prolactin; Prolactinoma; Rats; Rats, Inbred Strains; Thyrotropin-Releasing Hormone; Virulence Factors, Bordetella

Cytosolic free calcium ([Ca2+]i) and fusion of secondary granules with the phagosomal membrane (phagosome-lysosome fusion, P-L fusion) were assessed in single adherent human neutrophils during phagocytosis of C3bi-opsonized yeast particles. Neutrophils were loaded with the fluorescent dye fura2/AM and [Ca2+]i was assessed by dual excitation microfluorimetry. Discharge of lactoferrin, a secondary granule marker into the phagosome was verified by immunostaining using standard epifluorescence, confocal laser scanning and electron microscopy. In Ca2(+)-containing medium, upon contact with a yeast particle, a rapid rise in [Ca2+]i was observed, followed by one or more Ca2+ peaks (maximal value 1,586 nM and median duration 145 s): P-L fusion was detected in 80% of the cells after 5-10 min. In Ca2(+)-free medium the amplitude, frequency and duration of the [Ca2+]i transients were decreased (maximal value 368 nM, mostly one single Ca2+ peak and median duration 75 s): P-L fusion was decreased to 52%. Increasing the cytosolic Ca2+ buffering capacity by loading the cells with MAPT/AM led to a dose-dependent inhibition both of [Ca2+]i elevations and P-L fusion. Under conditions where basal [Ca2+]i was reduced to less than 20 nM and intracellular Ca2+ stores were depleted, P-L fusion was drastically inhibited while the cells ingested yeast particles normally. P-L fusion could be restored in Ca2(+)-buffered cells containing ingested particles by elevating [Ca2+]i with the Ca2(+)-ionophore ionomycin. The present findings directly indicate that although the ingestion step of phagocytosis is a Ca2(+)-independent event, [Ca2+]i transients triggered upon contact with opsonized particles are necessary to control the subsequent fusion of secondary granules with the phagosomal membrane.

Topics: Benzofurans; Calcium; Cytosol; Egtazic Acid; Fluorescent Antibody Technique; Fluorescent Dyes; Fura-2; Humans; Immunohistochemistry; Lactoferrin; Lysosomes; Membrane Fusion; Microscopy, Electron; Neutrophils; Phagocytosis; Phagosomes

The aim of this study was to elucidate the cellular mechanisms of action of epidermal growth factor (EGF) inhibition of parietal cell secretion. EGF effects on histamine- and carbachol-stimulated [14C]aminopyrine (AP) uptake and intrinsic factor (IF) secretion were evaluated in isolated rabbit parietal cells. EGF inhibited histamine-stimulated [14C]AP uptake and IF secretion through a reduction in stimulated adenosine 3',5'-cyclic monophosphate (cAMP) levels. EGF decreased the phosphorylation of a cytosolic 30-kDa, histamine-stimulated, cAMP-dependent protein kinase substrate. These effects on histamine-stimulated activation were reversed by pertussis toxin preincubation. EGF inhibited carbachol-stimulated [14C]AP uptake and IF secretion, but did not alter the carbachol-stimulated Ca2+ transient. These results indicate that EGF inhibits histamine-stimulated secretion through the inhibitory Gi guanosine 5'-triphosphate-binding protein and carbachol-stimulated secretion through a mechanism independent of the activation of an increase in intracellular Ca2+.

Topics: Aminopyrine; Animals; Benzofurans; Buffers; Calcium; Carbachol; Colforsin; Cyclic AMP; Cytosol; Epidermal Growth Factor; Fluorescent Dyes; Fura-2; Gastric Acid; Histamine; In Vitro Techniques; Intrinsic Factor; Kinetics; Parietal Cells, Gastric; Phosphorylation; Proteins; Rabbits; Signal Transduction

Among three endothelin (ET) isopeptides, ET-3 shows the most potent initial depressor response through the endothelium-dependent mechanism. We studied the presence of specific binding sites for ET-3 in cultured bovine endothelial cells (EC) and its cellular mechanism of action. Binding studies revealed the presence of two distinct subclasses of ET-3 receptors with high and low affinities. ET-3 dose-dependently (10(-10)-10(-7) M) increased both intracellular Ca2+ levels ([Ca2+]i) and inositol trisphosphate (IP3) formation. The ET-3-induced increase in [Ca2+]i was not affected by either removal of extracellular Ca2+ or Ca2(+)-channel blockers. These data suggest that ET-3 induces phosphoinositide breakdown and increase in [Ca2+]i in ECs, possibly resulting from intracellular Ca2+ mobilization, thereby leading to vasodilatation.

Topics: Animals; Benzofurans; Binding, Competitive; Calcium; Cattle; Cells, Cultured; Endothelins; Endothelium, Vascular; Fluorescent Dyes; Fura-2; Inositol 1,4,5-Trisphosphate; Peptides; Receptors, Cell Surface; Receptors, Endothelin

Olfactory neurons from the channel catfish, Ictalurus punctatus, were isolated by a brief (15 min) treatment with papain. After incubation with fura-2 acetoxymethyl ester (fura-2/AM) for 1 h, the isolated olfactory receptor cells are found to hydrolyze fura-2/AM to fura-2 free acid without detectable traces of intermediate products of hydrolysis. Intracellular calcium measured with fura-2 in single cells covers a wide range (from less than 2 to 100 nM with a median of 17.6 nM, n = 140 cells). Twenty-one percent of the cells respond to potassium-induced depolarization with an increase in intracellular calcium mediated by influx of extracellular calcium. The L-type calcium channel antagonist nimodipine inhibits the increase in intracellular calcium triggered by membrane depolarization and blocks small unitary barium currents displaying the characteristics of L-type calcium currents (unitary conductance of 29 +/- 5 pS in 55 mM BaCl2 and high selectivity for Ba2+ over Na+ and K+) recorded from azolectin bilayers at the tip of patch pipettes into which isolated olfactory cilia membrane vesicles had been incorporated. Olfactory neurons are found to be functionally heterogeneous in their response to membrane depolarization and can be separated into three groups: one in which the increase in intracellular calcium is rapid and transient, another in which calcium increases slowly, and a third group of cells in which depolarization causes no change in intracellular calcium.

Topics: Animals; Barium; Benzofurans; Calcium; Calcium Channels; Central Nervous System; Egtazic Acid; Fluorescent Dyes; Fura-2; Ictaluridae; In Vitro Techniques; Kinetics; Lipid Bilayers; Neurons; Nimodipine; Olfactory Pathways; Osmolar Concentration; Potassium; Sensory Receptor Cells; Spectrometry, Fluorescence

Topics: Benzofurans; Calcium; Cytosol; Fluorescent Dyes; Fura-2; Spectrometry, Fluorescence

The fluorescent probe, fura 2, is widely used to measure agonist-induced changes in intracellular calcium concentration ([Ca2+]i) in cultured cells. However, in many instances, the results obtained in the same cell type have differed from one study to the next. The possibility that such differences might be due to experimental conditions was examined by using fura 2 in four different cell types responding to appropriate agonists when the cells were incubated in either CO2/HCO3-- or HEPES-buffered media. Examined were: 1) the response of rat glomerular mesangial cells to arginine vasopressin, 2) the response of vascular smooth muscle cells to angiotensin II, 3) the response of adrenal glomerulosa cells to angiotensin II, and 4) the response of hypothalamic cells to insulin-like growth factor-1. In each cell type there was a significant difference in the pattern of agonist-induced change in [Ca2+]i when HEPES vs. CO2/HCO3- was used as the buffer system: in HEPES buffer, agonist addition led to a transient rise in [Ca2+]i followed by a fall to a sustained plateau 27 to 34 nM higher than the original basal value, whereas in CO2/HCO3- buffer, agonist addition led to an identical transient increase in [Ca2+]i followed by a fall to a value within 10 nM or less of the preagonist level. The plateau value of [Ca2+]i in the different buffers was examined in relationship to known differences in intracellular pH (pHi). It was found that measurements of [Ca2+]i with fura 2 were influenced by shifts in pHi that occur when cells are incubated in either HEPES-buffered or CO2/HCO3- media of differing pHo values. However, at any given value of pHi, the apparent [Ca2+]i measured in cells incubated in HEPES-buffered media was slightly higher than in cells incubated in CO2/HCO3- buffered media.

Topics: Adrenal Medulla; Animals; Benzofurans; Bicarbonates; Buffers; Calcium; Fluorescent Dyes; Fura-2; Glomerular Mesangium; HEPES; Hydrogen-Ion Concentration; Hypothalamus; Muscle, Smooth, Vascular; Rats

We examined the effect of fluoride (F) on intracellular ionic calcium [Ca2+]i in normal human osteoblasts maintained in culture. Cells were grown on glass coverslips to near-confluency and loaded with the Ca-sensitive dye, fura-2AM. Fluorescence changes were monitored in single cells using an inverted microscope coupled by fiberoptic to a microspectrofluorometer. The addition of F (100 ng/mL) to the medium promoted a rapid and significant increase in free [Ca2+]i from a resting level of 245 +/- 36 SE nM to a peak concentration of 440 +/- 51 nM (p less than 0.04). This increase in [Ca2+]i began at 10-20 s after addition of F and was maximal by 30 s. Intracellular [Ca2+]i levels then returned to near resting values by 60-80 s after F addition. This response was evident with as little as 25 ng/ml of fluoride and was dose dependent up to 500 ng/ml. At concentrations greater than 500 ng/ml, there appeared to be an attenuation of the rise in [Ca2+]i. The observed rise in [Ca2+]i was dependent on extracellular calcium since lowering extracellular calcium concentration or incubation with calcium channel blockers abolished the response. This observation supports a role of increased [Ca2+]i as one of the initial events of fluoride on action osteoblasts.

Topics: Benzofurans; Calcium; Cells, Cultured; Cytosol; Diltiazem; Fluorescent Dyes; Fluorides; Fura-2; Humans; Nifedipine; Osteoblasts; Parathyroid Hormone; Time Factors; Verapamil

Gastric parietal cells in primary culture have been tested to determine their utility as a model for the study of the role of intracellular calcium ([Ca2+]i) in the control of HCl secretion. Changes in [Ca2+]i were measured in single cells on a microscope stage using the cell-permeant form of the fluorescent calcium probe, fura-2. Simultaneous images of cell fluorescence and morphology were acquired using a digitized video image analysis system and two video cameras, one for low-light level fluorescence detection and one for high resolution DIC/transmitted light images. Both histamine and carbachol, which are known stimulants of HCl secretion, increased [Ca2+]i and stimulated dramatic changes in morphology in these cultured cells. Changes in morphology were accompanied by an increased uptake of the weak base, [14C]-aminopyrine (AP), and a shift from green to red fluorescence of another weak base, acridine orange. These results indicate that cultured parietal cells, maintained under controlled conditions on a microscope stage, retain viability and secretagogue responsiveness. Thus, this cellular model appears to be suitable for correlation of changes in [Ca2+]i and activation of HCl secretion.

Topics: Animals; Benzofurans; Calcium; Cells, Cultured; Fluorescent Dyes; Fura-2; Gastric Acid; Histamine; Image Processing, Computer-Assisted; Male; Omeprazole; Parietal Cells, Gastric; Rabbits; Second Messenger Systems; Video Recording

Procedures for loading fura 2 acetoxymethyl ester (fura 2/AM) into smooth muscle cells isolated from guinea pig taenia coli have been investigated. It was difficult to load these cells with fura 2 in the absence of diisopropylfluorophosphate (DFP). The presence of DFP, a potent cholinesterase (ChE) inhibitor during the loading, significantly enhanced the incorporation of the fura 2 into the cells. More than 80% of the single cells treated with DFP and fura 2/AM were viable. DFP did not affect the ability of the cell to shorten in response to either Ca2+ or carbachol (CCh). The single cells contracted transiently with caffeine and the intracellular Ca2+ concentration increased simultaneously. The results indicate that the amount of fura 2/AM incorporated into the single smooth muscle cells depends on the activity of ChE or various serine proteases located outside the cells and suppression of these enzymes induces more efficient incorporation, which permits shorter incorporation periods. Since the presence of DFP may shorten the incubation time significantly, the viability of these cells is improved. The procedure might be applicable for measuring simultaneously the contraction of cells and the behavior of intracellular Ca2+ in the same cells.

Topics: Animals; Benzofurans; Biological Transport; Caffeine; Calcium; Carbachol; Cholinesterases; Colon; Egtazic Acid; Fluorescent Dyes; Fura-2; Guinea Pigs; Histocytochemistry; In Vitro Techniques; Isoflurophate; Kinetics; Muscle Contraction; Muscle, Smooth; Spectrometry, Fluorescence

The effect of Pluronic F-127 (PF-127), a surfactant polyol, on the loading of fura 2/AM into smooth muscle cells isolated from guinea pig taenia coli was investigated. The presence of PF-127 during the loading of fura 2 acetoxymethyl ester (fura 2/AM), an intracellular Ca indicator, occasionally increased the incorporation of the dye into the cells. However, long time loading (over approximately 60 min) in the presence of PF-127 decreased the incorporation of fura 2. When the extracellular medium obtained from cells treated with DFP, then incubated with fura 2/AM in the presence of PF-127 was analyzed with excitation spectra, the fluorescence peaks shifted to longer wave lengths by addition of EGTA. However, the peak of the extracellular medium obtained from cells treated with DFP, then incubated without PF-127, did not shift. These results show that PF-127 affects the membranous permeability of the dye in single smooth muscle cells, permitting fura 2 hydrolyzed in the cytoplasm to leak out through the membrane.

Topics: Animals; Benzofurans; Carbachol; Colon; Egtazic Acid; Fluorescent Dyes; Fura-2; Guinea Pigs; In Vitro Techniques; Isoflurophate; Muscle Contraction; Muscle, Smooth; Poloxalene; Polyethylene Glycols; Spectrometry, Fluorescence

1. Changes in cytoplasmic Ca2+ concentration ([Ca2+]1) were measured simultaneously with force by a microfluorometric method using a calcium indicator, fura-2, in canine coronary arterial smooth muscle cells. 2. Depolarization by high (30-90 mM) KCl-physiological salt solution (PSS) produced concentration-dependent increases in force and [Ca2+]i. 3. The KCl-induced increase in [Ca2+]i abolished by Ca2+-free conditions and almost abolished by verapamil 10-5 M, suggesting that it was entirely due to the increased Ca2+ influx through voltage-dependent Ca2+ channels. 4. The [Ca2+]i force relationship in the presence of verapamil was not distinguishable from that of control. 5. Nitroglycerin produced a concentration-dependent, reversible contraction of the coronary artery that had been contracted by high KCl-PSS, without reduction of the increased [Ca2+]i. 6. The KCl-induced increase in [Ca2+]i was not affected by nitroglycerin and in a presence of nitroglycerin it was abolished by 10-5 M verapamil suggesting that it was caused by the influx of extracellular Ca2+. 7. The [Ca2+]-force curve was shifted to the right by nitroglycerin. 8. It is likely that nitroglycerin relaxes the coronary arterial smooth muscle b reducing the amount of myosin light chain phosphorylation even in the presence of raised [Ca2+]i produced by increased Ca2+ influx following depolarization.

Topics: Animals; Benzofurans; Calcium; Coronary Vessels; Dogs; Female; Fura-2; In Vitro Techniques; Male; Muscle Relaxation; Muscle, Smooth, Vascular; Nitroglycerin; Potassium Chloride; Spectrometry, Fluorescence; Verapamil

The ionic composition of the mitochondrial matrix, under both physiological and pathophysiological conditions, remains controversial. Although fura-2 and 2',7'-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF), fluorescent probes for [Ca2+] and [H+] respectively, have successfully been loaded into mitochondria [Lukács & Kapus (1987) Biochem. J. 248, 609-613; Davis, Altschuld, Jung & Brierley (1987) Biochem. Biophys. Res. Commun. 49, 40-45], the adaptation of fluorescence-ratio spectroscopy to the study of the matrix ion content poses unique problems. In this report, we describe a method for successfully attaching viable rat cardiac mitochondria to glass coverslips, allowing continuous superfusion of isolated organelles during fluorescence microscopy. This technique obviated the need to correct for the accumulation of ion-sensitive and -insensitive fluorescent species of dye both within the matrix and outside of mitochondria in suspension in a cuvette, a particular problem with fura-2. By using this technique for superfusion of immobilized mitochondria, we found the pKa of BCECF for H+ at 25 degrees C shifted from 6.8 in buffer to 7.2 in rat cardiac mitochondria, with a marked hysteresis effect noted for intramitochondrial BCECF calibration curves. At higher pH, photobleaching of BCECF was enhanced. The dissociation constant (Kd) of fura-2 for Ca2+ was found to be 315 nM at 25 degrees C, pH 8.0, but only at [Ca2+] below 1 microM. At matrix [Ca2+] greater than 1 microM, the Kd shifted into the micromolar range, an effect that appeared to be pH-dependent. Importantly, the matrix [Ca2+] was determined to be between 10 and 100 nM at perfusion buffer [Ca2+] below 500 nM, but rose rapidly at the higher extramitochondrial [Ca2+] reported to occur in ischaemic cardiac myocytes. Importantly, mitochondrial transmembrane H+ and Ca2+ gradients both appeared to be maximal at perfusion buffer [H+] and [Ca2+] that approximate those of the cytosol of many resting cells.

Topics: Animals; Benzofurans; Calcium; Fluoresceins; Fluorescent Dyes; Fura-2; Hydrogen-Ion Concentration; Hydrolysis; Microscopy, Fluorescence; Mitochondria, Heart; Photochemistry; Protons; Rats; Rats, Inbred Strains

Effects of the monoclonal antiparathyroid antibodies G11 and E11 on Mn2+ interaction with individual normal human parathyroid cells were studied. At 0.5mM Ca2+, 3mM Mn2+ induced a rapid transient increase in cytoplasmic Ca2+ [Ca2+i] followed by quenching of the fluorescence from the Ca2+ indicator fura-2 as Mn2+ entered into the cells. Whereas the antibody E11 had no effects, treatment with G11 abolished the Ca2+i transient and considerably delayed the entry of Mn2+. The results support the presence of a cation-sensitive receptor mechanism on parathyroid cells and indicate that the antibody G11 not only blocks the interaction between Ca2+ and this receptor mechanism but also that of Mn2+.

Topics: Antibodies, Monoclonal; Benzofurans; Calcium; Calcium Channel Blockers; Calcium Channels; Cell Membrane; Fluorescent Antibody Technique; Fura-2; Humans; Manganese; Parathyroid Glands; Receptors, Nicotinic

The effects of ATP on the concentration of cytosolic calcium [( Ca2+]i) were examined with respect to early events associated with activation of the superoxide (O2-)-generating system in human neutrophils. Addition of ATP to cytochalasin B-treated neutrophils resulted in two sequential increase in [Ca2+]i: an initial phase presumably related to the mobilization of Ca2+ from intracellular stores and a second phase dependent upon the presence of extracellular Ca2+. The second phase was associated with an increase in the rate of O2- production, which also required the presence of extracellular Ca2+. The results suggest that increased Ca2+ influx may act to trigger a cascade of Ca2+-sensitive events, leading to stimulated O2-production.

Topics: Adenosine Triphosphate; Benzofurans; Calcium; Cytochalasin B; Extracellular Space; Fluorescent Dyes; Fura-2; Humans; Kinetics; Neutrophils; Superoxides

Single cell [Ca2+], studies were performed in chicken and rat osteoclasts loaded with fura-2 and exposed to a variety of treatments. Under resting conditions, basal [Ca2+]i, was 79.2 +/- 47.3 and 84.3 +/- 65.7 nM (averages +/- S.D.; n = 141 and 126) in the osteoclasts of the two species, respectively. Basal [Ca2+]i was stable in all rat and in approximately 80% of chicken osteoclasts. In the remaining 20%, spontaneous, irregular [Ca2+], fluctuations were observed (amplitude range: 50-200 nm over basal values). Increase of [Ca2+]o over the concentration of the Krebs-Ringer incubation medium (2 mM) induced rises of [Ca2+] in almost all cells investigated. [Ca2+] rises were already appreciable with 0.5 mM [Ca2+]o additions and reached high values with 4 mM additions: 390 +/- 113 and 364 +/- 214 nM [Ca2+], in rat and chicken osteoclasts, respectively (n = 122 and 101). Qualitatively, the responses to [Ca2+]o additions consisted of discrete [Ca2+]i transients, biphasic (an initial spike followed by a plateau), or monophasic (either the spike or the plateau). In a few chicken osteoclasts, the [Ca2+]i increase occurring after [Ca2+]o addition consisted of multiple, irregular fluctuations, similar to those observed in 20% of these cells under resting conditions. In individual osteoclasts subsequently exposed to multiple [Ca2+]o increase pulses, the type of the [Ca2+]i transient (mono- or biphasic) was maintained, and the size was dependent on the magnitude of the [Ca2+]o additions. Effects similar to those of [Ca2+]o were induced by the addition of Cd2+ or Ba2+ (but not La3+ or Mg2+) into the medium. The Cd2+ effect was maintained in part even in a Ca2+-free medium. Of various hormones and factors, parathormone, 1,25-dihydroxyvitamin D3, and prostaglandin E2 were inactive. In contrast, calcitonin was active in rat osteoclasts (which express numerous receptors). [Ca2+]i increases were small (19 +/- 17.9 nM; n = 21) when the hormone was administered alone; they were synergistic (severalfold potentiation) when the hormone was administered before or after [Ca2+]o. The [Ca2+]i effects of calcitonin were mimicked by 8Br-cAMP (31 +/- 26 nM; n = 12) when the nucleotide was administered alone; marked synergism when it was administered in combination with [Ca2+]o. This paper demonstrates for the first time that changes of [Ca2+]i are induced in osteoclasts by treatments with [Ca2+]o and calcitonin and can therefore be involved in intracellular mediation of the physiologica

Topics: Animals; Benzofurans; Cadmium; Calcitonin; Calcium; Chickens; Cyclic GMP; Cytosol; Drug Synergism; Extracellular Space; Female; Fura-2; Hormones; Lanthanum; Magnesium; Osteoclasts; Rats; Rats, Inbred Strains

Because metabolic acids stimulate bone resorption in vitro and in vivo, we focused on the cellular events produced by acidosis that might be associated with stimulation of bone remodeling. To this end, we exposed isolated chicken osteoclasts to a metabolic (butyric) acid and observed a fall in both intracellular pH and cytosolic calcium [( Ca2+]i). These phenomena were recapitulated when bone resorptive cells, alkalinized by HCO3 loading, were transferred to a bicarbonate-free environment. The acid-induced decline in osteoclast [Ca2+]i was blocked by either NaCN or Na3VO4, in a Na+-independent fashion, despite the failure of each inhibitor to alter stimulated intracellular acidification. Moreover, K+-induced membrane depolarization also reduced cytosolic calcium in a manner additive to the effect of protons. These findings suggest that osteoclasts adherent to bone lack functional voltage-operated Ca2+ channels, and they reduced [Ca2+]i in response to protons via a membrane residing Ca-ATPase. Most importantly, acidosis enhances formation of podosomes, the contact areas of the osteoclast clear zone, indicating increased adhesion to substrate, an early step in bone resorption. Thus, extracellular acidification of osteoclasts leads to decrements in intracellular pH and calcium, and appears to promote cell-matrix attachment.

Topics: Animals; Benzofurans; Bicarbonates; Buffers; Calcium; Cell Adhesion; Chickens; Cytosol; Ethers; Extracellular Matrix; Female; Fluorescent Dyes; Fura-2; Hydrogen-Ion Concentration; Ionomycin; Isoelectric Point; Membrane Potentials; Osteoclasts; Protons; Sodium; Sodium Cyanide; Vanadates

The relationship between stimulation of single C-cells (rMTC-6-23 cell line) with extracellular calcium, glucagon or 8-bromo-cAMP and fluctuations of intracellular free calcium concentration was studied. After pretreatment of rMTC cells with either 1 microM glucagon (30-60 min) or 1 mM 8-bromo-cAMP (5 min) [Ca2+]i started to oscillate when extracellular calcium was raised to 3 mM. These fluctuations in [Ca2+]i could be stopped by chelating the external calcium with EGTA or by adding calcium channel blockers. The voltage-dependent calcium channels in the plasma membrane seem to play a major role in maintaining the oscillations of [Ca2+]i.

Topics: 8-Bromo Cyclic Adenosine Monophosphate; Animals; Benzofurans; Calcium; Carcinoma; Cyclic AMP; Cytosol; Fluorescent Dyes; Fura-2; Glucagon; Rats; Thyroid Neoplasms; Tumor Cells, Cultured

Topics: Acetylcholine; Aminoquinolines; Animals; Benzofurans; Calcium Channels; Fluorescent Dyes; Fura-2; In Vitro Techniques; Male; Muscle Contraction; Muscle, Smooth, Vascular; Myosins; Phosphorylation; Rabbits; Tetradecanoylphorbol Acetate

In the present study, we have investigated the role of Ca2+ in the coupling of membrane depolarization to neurotransmitter secretion. We have measured (a) intracellular free Ca2+ concentration ([Ca2+]i) changes, (b) rapid 45Ca2+ uptake, and (c) Ca2+-dependent and -independent release of endogenous glutamate (Glu) and gamma-aminobutyric acid (GABA) as a function of stimulus intensity by elevating the extracellular [K+] to different levels in purified nerve terminals (synaptosomes) from rat hippocampus. During stimulation, Percoll-purified synaptosomes show an increased 45Ca2+ uptake, an elevated [Ca2+]i, and a Ca2+-dependent as well as a Ca2+-independent release of both Glu and GABA. With respect to both amino acids, synaptosomes respond on stimulation essentially in the same way, with maximally a fourfold increase in Ca2+-dependent (exocytotic) release. Ca2+-dependent transmitter release as well as [Ca2+]i elevations show maximal stimulation at moderate depolarizations (30 mM K+). A correlation exists between Ca2+-dependent release of both Glu and GABA and elevation of [Ca2+]i. Ca2+-dependent release is maximally stimulated with an elevation of [Ca2+]i of 60% above steady-state levels, corresponding with an intracellular concentration of approximately 400 nM, whereas elevations to 350 nM are ineffective in stimulating Ca2+-dependent release of both Glu and GABA. In contrast, Ca2+-independent release of both Glu and GABA shows roughly a linear rise with stimulus intensity up to 50 mM K+. 45Ca2+ uptake on stimulation also shows a continuous increase with stimulus intensity, although the relationship appears to be biphasic, with a plateau between 20 and 40 mM K+.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Benzofurans; Calcium; Calcium Radioisotopes; Ethers; Fluorescent Dyes; Fura-2; gamma-Aminobutyric Acid; Glutamates; Glutamic Acid; Hippocampus; Ionomycin; Kinetics; Male; Perfusion; Potassium; Rats; Rats, Inbred Strains; Synaptosomes

In the embryonic and in the adult heart muscarinic stimulation reduces the heart rate. Here we demonstrate that in the embryonic heart an additional muscarinic system is present, which is characterized by Ca++ mobilization and corresponds to the embryonic muscarinic system in other organ anlagen. In suspensions of embryonic chick heart cells we measured release of Ca++ from intracellular stores and influx of extracellular Ca++ with fura-2 and chlorotetracycline after muscarinic stimulation and determined the [3H]quinuclidinylbenzilate binding sites. We observed intense Ca++ mobilization at day 4, weaker reactions between day 4.5 and 11, and no Ca++ response at day 13. The pharmacological profile was identical to the profile of Ca++ mobilization in cells from other embryonic tissues in which the general embryonic muscarinic system is expressed. In parallel, we studied the effect of muscarinic agonists and antagonists on the heart rate of isolated embryonic hearts at day 4 and 5 in a perfusion chamber. Oxotremorine and bethanechol being antagonists or weak partial agonists in Ca++ mobilization, behaved as full agonists in frequency regulation. Thus, the pharmacological profile of the transient embryonic muscarinic system was different from that of the definitive adult form.

Topics: Animals; Benzofurans; Bethanechol; Bethanechol Compounds; Calcium; Carbachol; Chick Embryo; Chlortetracycline; Female; Fura-2; Heart; Heart Rate, Fetal; In Vitro Techniques; Methacholine Chloride; Methacholine Compounds; Oxotremorine; Parasympathomimetics; Pilocarpine; Pregnancy; Receptors, Muscarinic

The molecular mechanisms associated with the effects of various pathological stimuli on myocardial tissue, as well as the mechanisms by which Ca2+ antagonists exert their protective effect, are poorly understood. With the use of digital image processing of Fura-2 fluorescence, we have shown that the mean intracellular free Ca2+ concentration of single isolated rat cardiomyocytes is increased upon exposure to various pathological stimuli (high extracellular Ca2+, veratrine). This increased Ca2+ content coincided with an increased number of hypercontracted cells. Pretreatment with flunarizine under these experimental conditions lowered the free intracellular Ca2+ concentration, thereby reducing the number of hypercontracted cells. Verapamil had no effect. The kinetics of changes in intracellular Ca2+ in electrically paced cardiomyocytes were not affected by flunarizine, but were significantly altered by the beta agonist isoprenaline. In addition, isoprenaline increased the mean diastolic intracellular free Ca2+ concentration of paced cardiomyocytes, whereas it remained unchanged in flunarizine treated cells. We conclude that flunarizine reduces intracellular free Ca2+ levels in isolated cardiomyocytes under pathological conditions, but does not affect physiological processes mediated by Ca2+. The report also illustrates the possibilities of digital imaging microscopy in the study of ion distributions in living cells.

Topics: Animals; Benzofurans; Calcium; Depression, Chemical; Flunarizine; Fluorescent Dyes; Fura-2; Heart; Image Processing, Computer-Assisted; Isoproterenol; Kinetics; Myocardium; Rats; Verapamil; Veratrine

Changes in the cytosolic free calcium ion concentrations ([Ca2+]i) induced by LHRH were studied in individual rat granulosa cells using fura-2 microspectrofluorimetry. The resting [Ca2+]i concentration was 96.7 +/- 2.9 nM (n = 115). The majority of the granulosa cells responded to LHRH at doses between 10(-7) and 10(-5) M; the latter dose was the highest concentration required to initiate alterations in [Ca2+]i in these cells. The alterations in [Ca2+]i induced by LHRH were transient and returned to resting levels within 84 +/- 3 sec (n = 64). A potent LHRH antagonist completely blocked the effect of LHRH on [Ca2+]i. Within a single granulosa cell, three consecutive injections of the same dose of LHRH, delivered 5 min apart, induced three discrete peaks of [Ca2+]i of similar amplitudes. Sustained perfusions of LHRH, however, resulted in a desensitization of the [Ca2+]i response to LHRH, but not to the calcium ionophore Br-A23187. These results, which were obtained from individual cells, provide strong support for the hypothesis that acute changes in [Ca2+]i are involved in the early cellular transduction of the LHRH signal in the ovary.

Topics: Animals; Benzofurans; Calcium; Cells, Cultured; Cytosol; Dose-Response Relationship, Drug; Female; Fura-2; Gonadotropin-Releasing Hormone; Gonadotropins; Granulosa Cells; Rats; Rats, Inbred Strains; Spectrometry, Fluorescence

A rise in cytosolic free Ca2+ is the immediate trigger for contraction in mammalian vascular smooth muscle. We used the fluorescent calcium indicator fura-2 and digital imaging microscopy to study the spatial distribution of intracellular Ca2+ in arterial myocytes and the changes elicited by activation with norepinephrine (NE). Viable arterial myocytes were obtained from bovine tail arteries by enzymatic digestion. In modified Krebs' solution containing 1.8 mM Ca2+, these myocytes were relaxed and spindle-shaped. The cells contracted rapidly when exposed to NE or high-K+ solution ejected from a micropipette; they relaxed slowly when the activator was washed away. NE evoked a rise in Ca2+ concentration ([Ca2+]) in the cells within 100 msec, at a time when the cells had not yet begun to contract. Maximal [Ca2+] levels were attained within 600 msec, at which time the cells were substantially contracted. Digital analysis of images of cellular fura-2 fluorescence revealed that the intracellular [Ca2+] was relatively uniformly distributed prior to activation, with an average resting level of 111 +/- 14 nM (n = 6). During NE-evoked contractions, intracellular [Ca2+] increased, and the distribution of [Ca2+] became much more heterogeneous. On recovery from activation, the cells relaxed, usually attaining less than 90% of their original resting length. In contrast to the relatively uniform Ca2+ distribution observed prior to NE activation, discrete regions of elevated [Ca2+] were observed throughout the recovered cells. The large spatial variation of [Ca2+] after cell activation implies that Ca2+ was sequestered at localized sites in the cell during relaxation.

Topics: Animals; Arteries; Benzofurans; Calcium; Cattle; Fura-2; Microscopy, Fluorescence; Models, Biological; Muscle Contraction; Muscle, Smooth, Vascular; Norepinephrine; Rats; Rats, Inbred Strains

There are a number of reports of the plasma membrane transport of Ca2+ in biological systems being enhanced by low frequency electromagnetic fields (EMF), including reports that the enhancement involves a resonance-type response at the cyclotron frequency for Ca2+ ions for geomagnetic values of the magnetic field. Using the fluorescent probe fura2, we find no evidence for changes in cytosolic calcium concentration in BALB/c3T3, L929, V-79, and ROS, a rat osteosarcoma cell line, at the application of both resonant and nonresonant EMF.

Topics: Animals; Benzofurans; Calcium; Cell Membrane; Cells, Cultured; Cricetinae; Electromagnetic Fields; Electromagnetic Phenomena; Fura-2; Particle Accelerators; Rats

We examined the role of intracellular and extracellular calcium on the ability of human polymorphonuclear leukocytes to migrate chemotactically and reexpress (or recycle) formyl peptide receptors when challenged with the synthetic chemotactic peptide, N-formyl-methionyl-leucyl-phenylalanine (FMLP). Extracellular calcium was not required for either optimal chemotactic responses or receptor reexpression. Depletion and chelation of intracellular calcium resulted in significant diminution in the ability of polymorphonuclear leukocytes to release the specific granule constituents lactoferrin and vitamin B12-binding protein during the process of chemotaxis, but had no effect on the capability of these cells to respond chemotactically. Similarly, chelation of intracellular calcium did not affect the ability of these cells to reexpress a population of formyl peptide receptors. Inhibition of receptor reexpression, by a nonagglutinating derivative of wheat-germ agglutinin, was associated with inhibition of chemotactic responses to FMLP. Thus, it appears that large changes in cytosolic free calcium are not necessary for formyl peptide-induced polymorphonuclear leukocyte chemotaxis. In contrast, continuous reexpression (or recycling) of formyl peptide receptors is required for polymorphonuclear leukocyte chemotactic responses to FMLP, a process that appears to be independent from specific granule fusion with plasma membrane.

Topics: Adult; Benzofurans; Calcium; Chemotaxis, Leukocyte; Cytoplasmic Granules; Cytosol; Fura-2; Humans; N-Formylmethionine Leucyl-Phenylalanine; Neutrophils; Receptors, Formyl Peptide; Receptors, Immunologic; Scattering, Radiation; Temperature; Wheat Germ Agglutinins

Cytosolic free calcium concentration, [Ca2+]i, in human peripheral blood mononuclear leukocytes was increased by platelet-activating factor (PAF) dose-dependently. The response was transient and was attenuated by EGTA (2 mM) in the medium. Furthermore, pretreatment of the cells with pertussis toxin did not alter significantly the rise in [Ca2+]i after stimulation by PAF. The data suggest that elevation of [Ca2+]i in human mononuclear leukocytes induced by PAF depends on: (1) calcium influx through calcium channels in the plasma membranes and (2) calcium mobilization from internal stores mediated via pathway(s) that is insensitive to the action of pertussis toxin.

Topics: Benzofurans; Calcium; Cytosol; Fura-2; Humans; In Vitro Techniques; Neutrophils; Pertussis Toxin; Phytohemagglutinins; Platelet Activating Factor; Virulence Factors, Bordetella

Changes in cytosolic calcium concentration (Cai2+) have been implicated in the regulation of intracellular pH (pHi) in several cell types. In the present study we investigated the mechanism by which an increase in Cai2+ stimulates H+ secretion and a rise in pHi in cultured rat inner medullary collecting duct (IMCD) cells. Confluent monolayers were made quiescent by incubation for 24 h in 0.1% serum before study. Changes in pHi and Cai2+ were measured with the fluorescent probes, 2,7-biscarboxyethyl-5(6)-carboxyfluorescein (BCECF) and fura-2. In nominally bicarbonate-free media containing 110 mM Na+ and 1 mM Ca2+, addition of the Ca2+ inophore, ionomycin (10 microM), produced a biphasic response in pHi, a transient acidification from 7.29 +/- 0.07 to 7.12 +/- 0.05 at 2 min followed by a sustained alkalinization to a steady-state value of 7.51 +/- 0.10 at 10 min. The rate of alkalinization was dose dependent. The alkalinization was not affected by 1 mM amiloride, removal of extracellular Na+, or by the proton pump inhibitor, N-ethyl maleimide (NEM). Metabolic energy was not required, but removal of extracellular Ca2+ prevented the alkalinization. By use of the fluorescent probe bisoxonol to assess membrane potential, ionomycin was shown to cause depolarization under the same experimental conditions as those for alkalinization. The Ca2+-induced alkalinization was mimicked by cell depolarization (induced by raising extracellular K+ in the presence of valinomycin 1 microM). We conclude that changes in Cai2+ are important in the regulation of pHi in the IMCD. Ca2+-induced cell alkalinization may be mediated by changes in membrane ionic conductance.

Topics: Amiloride; Animals; Benzofurans; Calcium; Cells, Cultured; Cytosol; Ethers; Fluorescent Dyes; Fura-2; Hydrogen-Ion Concentration; Ionomycin; Kidney Medulla; Kidney Tubules; Kidney Tubules, Collecting; Kinetics; Membrane Potentials; Rats; Spectrometry, Fluorescence

Rat heart mitochondria were incubated for 5 min at 30 degrees C and at approx. 40 mg protein.ml-1 and in the presence of 10 microM fura-2/AM. This allowed the entrapment of free fura-2 within the mitochondrial matrix and its use as a probe for Ca2+, but without affecting the apparent viability of the mitochondria. Parallel measurements of the activities of the intramitochondrial Ca2+-sensitive enzymes, pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase, allowed an assessment of their sensitivity to measured free Ca2+ within intact mitochondria incubated under different conditions; the enzymes responded to matrix Ca2+ over the approximate range 0.02-2 microM with half-maximal effects at about 0.3-0.6 microM Ca2+. Effectors of Ca2+-transport across the inner membrane (e.g., Na+, Mg2+, Ruthenium red, spermine) did not appear to affect these ranges, but did bring about expected changes in Ca2+ distribution across this membrane. Significantly, when mitochondria were incubated in the presence of physiological concentrations of both Na+ and Mg2+, and at low extramitochondrial Ca2+ (less than 400 nM), there was a gradient of Ca2+ (in:out) of less than unity; at higher extramitochondrial [Ca2+] (but still within the physiological range) the gradient was greater than unity indicating a highly cooperative nature of transmission of the Ca2+ signal into the matrix under such conditions.

Topics: Animals; Benzofurans; Biological Transport; Calcium; Enzyme Activation; Fluorescent Dyes; Fura-2; Intracellular Membranes; Ketoglutarate Dehydrogenase Complex; Male; Mitochondria, Heart; Pyruvate Dehydrogenase Complex; Rats

Maitotoxin, a potent marine toxin isolated from toxic tropical dinoflagellates and poisonous fishes induces contraction of different smooth muscle preparations. Actions of maitotoxin on phosphoinositides and calcium metabolism were studied using a primary culture of aortic smooth muscle cells. Maitotoxin induced a very large increase of cytosolic calcium concentration as evaluated by fura-2 acetoxymethyl ester fluorescence. This increase was concomitant with stimulation of inositol-phosphate accumulation and loss of viability of aortic smooth muscle cells. These responses to maitotoxin were abolished in Ca2+-free medium, and were mimicked by saponin. Calcium ionophores or K+ depolarisation did not induce inositol-phosphate formation. These results suggest that maitotoxin acts by altering smooth muscle cells permeability allowing a sustained calcium influx which is able to activate inositol-phosphate formation and which is lethal for the cells.

Topics: Angiotensin II; Animals; Aorta, Thoracic; Benzofurans; Calcium; Cell Survival; Cells, Cultured; Fluorescent Dyes; Fura-2; Male; Marine Toxins; Muscle, Smooth, Vascular; Oxocins; Phosphatidylinositols; Potassium; Rats; Rats, Inbred Strains; Saponins; Spectrometry, Fluorescence

Topics: Aminoquinolines; Animals; Benzofurans; Calcium; Cells, Cultured; Cytosol; Fura-2; Guinea Pigs; Histamine; Parietal Cells, Gastric; Spectrometry, Fluorescence

We report here the use of the fluorescent Ca2+-chelator fura-2 to directly measure free Ca2+ concentration within intact human erythrocytes and the influence of viscosity on the fluorescence of this probe. The bright fluorescence of fura-2 has permitted the use of low concentrations of indicator and cells, thus minimizing the screening effect and the intrinsic fluorescence of haemoglobin. Erythrocytes (10(8) cells/ml) were loaded with 0.5 microM fura-2AM then diluted at 10(7) cells per ml for measurements. The extracellular signal was suppressed by addition of manganese ions just before recording spectra. Under these conditions, a blood sample of 100 microliter was sufficient for analysis. To study the influence of viscosity on fura-2 fluorescence, gelatin and polyvinylpyrrolidone at various concentrations were added to a physiological buffer to perform fura-2-Ca fluorescence standard curves. Fluorescence intensities and the apparent affinity constant for Ca2+ were modified by viscosity. When intra-erythrocytic viscosity was simulated with 21 g/l polyvinylpyrrolidone to obtain a mean viscosity of 14 mPa.s similar to that observed in human erythrocytes, the mean value of free Ca2+ concentration measured in erythrocytes from healthy subjects was 78 +/- 16 nM (mean +/- S.D., n = 29).

Topics: Benzofurans; Calcium; Erythrocytes; Fluorescent Dyes; Fura-2; Humans; Spectrometry, Fluorescence

1. Intracellular free calcium levels were recorded in strips of longitudinal smooth muscle from guinea-pig ileum, by the use of the fluorescent calcium indicator Fura-2. 2. The resting intracellular free calcium concentration was estimated to be 210 nM. Many muscle strips showed spontaneous bursts of contractions, accompanied by bursts of calcium transients. Following these the calcium level often fell transiently below the resting level. The spontaneous transients were unaffected by tetrodotoxin (TTX) and atropine. 3. Field electrical stimulation of muscle strips evoked a series of calcium transients comprising: (i) an initial rise in free calcium, reaching a peak within 20-30 ms of stimulation, (ii) a second rise in calcium, beginning after a few hundred milliseconds, and finally (iii) a decline in calcium to below the resting level, persisting for a few seconds. The mean peak increase in free calcium above the resting level during components (i) and (ii) was, respectively, 130 and 200 nM. The mean decrease in free calcium during the third component was to 20 nM below the resting level. 4. The short-latency calcium transient required relatively long stimuli for activation, and was not blocked by TTX and atropine. The long-latency transient was selectively activated by brief stimuli, and was abolished by TTX and atropine. Thus, the short-latency component probably arose because of direct electrical stimulation of muscle fibres, while the long-latency component was due to stimulation of muscarinic nerves. 5. The first detectable increase in tension began about 100 ms after the peak of the initial calcium transient. Contractions associated with the long-latency calcium transient were much larger than those associated with the short-latency transient, even in muscle strips where the calcium levels were similar for both transients. 6. Removal of calcium in the bathing solution caused the resting intracellular calcium level to fall, following an initial rise accompanied by increased spontaneous transients. Electrically evoked contractions and calcium transients were abolished in calcium-free solution, and by the addition of verapamil or diltiazem to normal Krebs solution.

Topics: Action Potentials; Animals; Atropine; Benzofurans; Calcium; Fura-2; Guinea Pigs; Ileum; In Vitro Techniques; Muscle Contraction; Muscle, Smooth; Tetrodotoxin; Time Factors; Verapamil

Fura-2, loaded into J774.2 macrophages as the acetoxymethyl ester, is sequestered into intracellular vacuoles within 90 min after the beginning of the loading at 37 degrees C. The dye is also efficiently secreted from the cells. Sequestration and secretion of fura-2 reduce the accuracy of measurements of cytosolic free Ca2+ concentration in this cell line. Fura-2 is also sequestered and secreted by J774.2 when the dye is loaded into the cytoplasm as the pentapotassium salt by reversible permeabilization of the plasma membrane. Regardless of the mechanism by which fura-2 is loaded into the cytoplasm, both sequestration and secretion are prevented by 2.5 mM probenecid, a blocker of organic anion transport. Probenecid has no effect on resting or stimulated cytosolic free Ca2+ levels or on FcR-mediated phagocytosis. These findings suggest that macrophages express a transport mechanism for the anionic form of fura-2. This transport system is responsible for the clearance of fura-2 from the cytoplasm of this cell type. Furthermore we suggest that use of probenecid to block secretion and intracellular sequestration of fura-2 may overcome problems arising in the application of this Ca2+ indicator to macrophages and perhaps to other cell types.

Topics: Animals; Benzofurans; Biological Transport; Calcium; Cell Compartmentation; Cell Line; Cell Membrane Permeability; Extracellular Space; Fluorescent Dyes; Fura-2; Intracellular Fluid; Macrophages; Mice; Probenecid

Intracellular free Ca2+ was monitored in suspensions of 1321N1 astrocytoma cells by using the Ca2+ indicator fura-2. The cytoplasmic Ca2+ concentration increased from 237 +/- 6 nM to 1580 +/- 170 nM within 3-5 s of addition of 300 microM-carbachol. After the peak in response, the Ca2+ concentration diminished, establishing a new steady state in about 1 min that was approx. 150 nM above the previous baseline. Histamine increased cytoplasmic Ca2+ to about 40% of the maximal value seen with carbachol. In Ca2+-free buffer each agonist elicited a normal initial increase in cytoplasmic Ca2+, but the sustained portion of the response was abolished. The increase in Ca2+ in response to either carbachol or histamine could be completely inhibited by pretreating the cells with carbachol; the response to carbachol could be partially inhibited by pretreating the cells with histamine. The Ca2+ responses did not recover in the continued presence of carbachol. However, if the carbachol was washed out or if atropine was added after carbachol, the responses to agonist recovered in a time-dependent manner (half-time 3-4 min), and recovery depended on the presence of extracellular calcium. The results indicate that carbachol and histamine stimulate release of Ca2+ from the same intracellular Ca2+ store, that depletion of this store is responsible for heterologous desensitization between these two agonists, and that repletion of the agonist-sensitive Ca2+ pool does not occur in the continued presence of agonist or in the absence of extracellular Ca2+.

Topics: Astrocytoma; Benzofurans; Calcium; Carbachol; Cytoplasm; Dose-Response Relationship, Drug; Fluorescent Dyes; Fura-2; Histamine; Intracellular Fluid; Tumor Cells, Cultured

The polycation-coated latex bead is a potent stimulus for the induction of postsynaptic-type differentiation in cultured Xenopus myotomal muscle cells. Specializations characteristic of the neuromuscular junction, such as clusters of acetylcholine receptors and other postsynaptic-specific proteins, develop at the bead-muscle contact. Previous studies have shown that a deprivation of extracellular calcium inhibits the effect of the beads in causing the development of these specializations. This suggests that an increase in intracellular Ca2+ is a necessary condition for the development of this specialization. In this study, we tested whether an increase in intracellular calcium is observable upon the bead-muscle contact. The measurement was carried out on cells loaded with the fluorescent calcium indicator fura-2 AM by digitized video microscopy. When polycation-coated beads were added to cells, an increase in intracellular calcium concentration in the range of 5 to 57% of the resting level was observed within 10 sec after bead-muscle contact. Afterward, the calcium level gradually returned to the resting level with a time course of about 3 min. Uncoated beads, which do not induce the formation of acetylcholine receptor clustering, failed to elicit this calcium transient. Removal of extracellular calcium as well as blocking calcium channels with 50 microM verapamil also suppressed this transient induced by the polycation-coated beads. Both treatments are known to suppress the formation of receptor clusters by these beads. These results suggest that the polycation-coated beads cause an influx of calcium by increasing the membrane conductance to this ion. This process may underlie the signaling of the postsynaptic differentiation.

Topics: Animals; Anti-Bacterial Agents; Benzofurans; Caffeine; Calcium; Cell Differentiation; Cells, Cultured; Ethers; Fluorescent Dyes; Fura-2; Ionomycin; Ionophores; Microscopy, Fluorescence; Microspheres; Muscles; Neuromuscular Junction; Peptides; Verapamil; Xenopus laevis

Localization of myoplasmic free calcium was measured in fura2-loaded single rat myocardial cells to determine whether the mechanism of norepinephrine desensitization includes redistribution of calcium. Fluorescence intensities at each pixel were quantitated by use of a photon-counting, microchannel plate camera. From these images, values of calcium-dependent fluorescence intensity averages in whole cells, areas of calcium release (as zones of high intracellular calcium concentrations), and ratios of fluorescence intensity in central vs. peripheral sites were determined. Stimulation by 1 nM norepinephrine caused an increase in total free intracellular calcium and an activation of intracellular calcium release sites from subsarcolemmal pools initially and later from centrally located calcium pools. Subsequent addition of 100 nM norepinephrine failed to cause significant intracellular calcium release from centrally located pools. In contrast, forskolin exposure still released high concentrations of calcium from these central pools. These results indicate that pretreatment with even a relatively small concentration of norepinephrine causes markedly decreased subsequent intracellular calcium release from centrally located sarcoplasmic reticulum because of a refractoriness of the link between receptor activation and calcium release.

Topics: Animals; Animals, Newborn; Benzofurans; Calcium; Cells, Cultured; Colforsin; Computers; Fluorescent Dyes; Fura-2; Microscopy, Fluorescence; Myocardium; Norepinephrine; Rats; Rats, Inbred WKY; Videotape Recording

To determine whether increases in the cytosolic free Ca2+ concentration ([Ca2+]i) accompany agonist-stimulated surfactant secretion by cultured alveolar type II cells, we measured the [Ca2+]i of quin2-loaded cells isolated from adult rats before and after cells were stimulated with ionomycin, terbutaline or tetradecanoylphorbol acetate (TPA). To determine whether increases in [Ca2+]i are necessary for stimulated surfactant secretion to occur, we measured secretion in cells after [Ca2+]i had been reduced by loading cells with quin2 in medium containing low [Ca2+]. Ionomycin increased [Ca2+]i and stimulated surfactant secretion in a dose-dependent manner. Reductions in [Ca2+]i correlated with reductions in secretion stimulated by ionomycin, terbutaline or TPA. Ionomycin-stimulated secretion was most sensitive to reductions in [Ca2+]i; terbutaline-stimulated secretion was more sensitive than TPA-stimulated secretion. When [Ca2+]i was less than 65 nM, all stimulated secretion was blocked. Restoration of [Ca2+]i to greater than 100 nM restored ionomycin-stimulated secretion. We conclude that ionomycin increases [Ca2+]i and stimulates surfactant secretion in cultured alveolar type II cells, and that increased [Ca2+]i appears to be necessary for ionomycin-stimulated secretion to occur. Terbutaline-stimulated surfactant secretion seems to be more easily inhibited by a reduction in [Ca2+]i than does TPA-stimulated secretion.

Topics: Aminoquinolines; Animals; Benzofurans; Calcium; Cells, Cultured; Cytosol; Ethers; Fura-2; Ionomycin; Male; Phosphatidylcholines; Pulmonary Alveoli; Pulmonary Surfactants; Rats; Rats, Inbred Strains; Spectrometry, Fluorescence; Terbutaline; Tetradecanoylphorbol Acetate

A wave front of increased free calcium traversing the egg at fertilization is demonstrated in the sea urchin Lytechinus pictus. The use of the fluorescent calcium chelator fura-2 in combination with low-light-level TV microscopy and image processing allows the visualization of the Ca2+ wave front with high spatial and temporal resolution. Such a wave is demonstrated as increased fluorescence after an excitation of 340-nm wavelength and as the reciprocal image in form of a reduced fluorescence when excited at 380 nm. The band-like appearance of the wave resembles the Ca2+ wave described for larger eggs of other species. In a dispermic egg the high resolution of the system used allows us to recognize two waves of Ca2+ originating from the respective points of sperm entry.

Topics: Animals; Benzofurans; Calcium; Female; Fertilization; Fluorescent Dyes; Fura-2; Ovum; Sea Urchins

Fura-2 AM is an esterified cell-permeant form of the Ca2+ indicator fura-2 (1-[2-(5-carboxyoxal-2-yl)-6-aminobenzofuran-5-oxyl]-2-(2'-a mino-5'- methylphenoxy)-ethane-N,N,N',N'-tetraacetic acid). Fura-2 AM has been reported to be completely cleaved by cellular esterases to fura-2 which is trapped within cells and is used to measure intracellular free Ca2+ concentration by a fluorescence ratio method. Successful application of the method requires that fura-2 be the major cellular fluorescent metabolite of fura-2 AM. We have used high-performance liquid chromatography to study fura-2 AM hydrolysis by cells. Murine N1E-115 neuroblastoma cells incubated with 10 microM fura-2 AM formed fura-2 at a rate of 9.7 pmol/min/10(6) cells. The concentration of fura-2 in the cells after 60 min, assuming uniform distribution, was 137 microM. Smaller amounts of at least four other metabolites were present, as well as large amounts of unhydrolyzed fura-2 AM. Washing the cells with medium containing 2% bovine serum albumin decreased the concentration of fura-2 to 40 microM and that of fura-2 AM to 90 microM. The half-time for loss of fura-2 from neuroblastoma cells after washing was 34 min. Human pulmonary artery endothelial (HPAE) cells formed fura-2 at a rate of 2.6 nmol/min/10(6) cells and the concentration of fura-2 after 60 min of incubation and washing with albumin containing medium was 130 microM, and the concentration of fura-2 AM was 58 microM. The half-time for loss of fura-2 from washed HPAE cells was 74 min.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Benzofurans; Calcium; Cells, Cultured; Chromatography, High Pressure Liquid; Fluorescence; Fluorescent Dyes; Fura-2; Humans; Hydrolysis; In Vitro Techniques; Inactivation, Metabolic; Liver; Male; Rats; Rats, Inbred Strains; Tumor Cells, Cultured

[Ca2+]i transients, elicited by voltage-clamp depolarization of single guinea pig cardiac ventricular cells, were observed through use of the fluorescent Ca2+ indicator, fura-2. Individual cells, loaded with fura-2 either by internal perfusion or by exposure to fura-2/AM, were studied with the use of an inverted microscope that was equipped with ultraviolet epifluorescence illumination, an intensified silicon intensifier target camera, and a photomultiplier tube. Variation of membrane voltage and exposure of cells to verapamil (a Ca2+ channel blocker) and ryanodine (which was assumed to abolish selectively the release of Ca2+ from the sarcoplasmic reticulum) were used to investigate the cellular processes that determine the [Ca2+]i transient. The principal results of the study are: When appropriate methods are used, the properties of cytosolic fura-2 inside guinea pig cells are similar to those of fura-2 in solution, irrespective of the method of loading. The amplitude (at 100 msec) of verapamil-sensitive fluorescence transients elicited by pulse depolarization (range -30 to 80 mV) has a bell-shaped dependence on membrane voltage (maximum at 10 mV). Rapid, ryanodine-sensitive and verapamil-sensitive "tail transients" are elicited on repolarization from membrane potentials greater than 30 mV; their amplitude increases as the amplitude of the preceding pulse increases. The amplitude of slow fluorescence transients that are insensitive to verapamil and ryanodine increases continuously with membrane potential throughout the range -20 to 80 mV. The voltage dependence and pharmacology of the rapid transients elicited by pulse depolarization or by repolarization are consistent with their having arisen from Ca2+ released from the sarcoplasmic reticulum, via Ca2+-induced Ca2+ release.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Benzofurans; Calcium; Carrier Proteins; Cricetinae; Fluorescence; Fura-2; Intracellular Fluid; Ion Channels; Membrane Potentials; Myocardial Contraction; Myocardium; Purkinje Fibers; Ryanodine; Sodium-Calcium Exchanger; Verapamil

Intracellular free calcium concentration [( Ca2+]i) was measured with the fluorescent Ca2+ indicator Fura 2 within individual human neutrophils during the phagocytosis of several types of particles, including serum-treated zymosan (STZ), immunoglobulin G (IgG)-coated zymosan (IGZ), C3b-coated zymosan (C3Z), nontreated zymosan (Z), and serum-treated similarly sized latex particles (STL). STZ was coated with both IgG and C3b. IGZ was coated with only IgG, and C3Z was coated with only C3b. STL was coated with only C3b but to a lesser extent than C3Z. The ingestion of particles was greatest for STZ and somewhat lower for C3Z. Ingestion of IGZ and STL was much less than ingestion of C3Z. The relative efficiencies of the particles for inducing superoxide production were as follows: STZ greater than IGZ = C3Z greater than Z = STL. [Ca2+]i significantly increased from the resting level of approximately 70 to greater than 240 nM (P less than 0.01) during phagocytosis of the particles. The increment in [Ca2+]i was greater in the paraphagosomal region than in the cell body after the ingestion of STZ or IGZ. The mean peak [Ca2+]i values in the paraphagosomal cytoplasm of neutrophils ingesting one particle of STZ, IGZ, C3Z, Z and STL were 536.1 +/- 57.6, 424.7 +/- 55.8, 373.8 +/- 62.7, 272.3 +/- 31.5, and 270.8 +/- 38.0 nM, respectively, which showed good correlation (r = 0.97) with the efficiency of the particles for inducing superoxide production. Depletion of extracellular Ca2+ by EGTA [ethylene glycol-bis(beta-aminoethyl ether)-N,N,N'N'-tetraacetic acid] attenuated both [Ca2+]i increase and superoxide production induced by particles. Thus [Ca2+]i increased after ingestion of several types of particles, and the subcellular pattern of [Ca2+]i was different depending on the type of particle ingested. Greater increases in paraphagosomal [Ca2+]i were closely associated with greater increases in superoxide production by neutrophils.

Topics: Benzofurans; Calcium; Cell Compartmentation; Cytoplasm; Egtazic Acid; Fura-2; Humans; Neutrophils; Opsonin Proteins; Phagocytosis; Phagosomes; Superoxides; Time Factors

A method was developed to monitor continuously the matrix free Ca2+ concentration ([Ca2+]m) of heart mitochondria by use of the fluorescent Ca2+ indicators, fura-2 and quin2. The acetoxymethyl esters of fura-2 and quin2 were accumulated in and hydrolysed by isolated mitochondria. An increase of the mitochondrial Ca content from 0.3 nmol/mg of protein to 6 nmol/mg corresponded to a rise of [Ca2+]m from 30 to 1000 nM. The results indicate that physiological fluctuations of the mitochondrial Ca content elicit changes of [Ca2+]m in that range which regulates the matrix dehydrogenases.

Topics: Aminoquinolines; Animals; Benzofurans; Calcium; Fluorescent Dyes; Fura-2; Manganese; Mitochondria, Heart; Rats

The new, fluorescent Ca2+ indicator, fura-2, promises to expand our understanding of the role of subcellular changes in Ca2+ underlying cell function. During an investigation of the role of Ca2+ in the polarization response of human polymorphonuclear leukocytes to formyl-methionyl-leucyl-phenylalanine, we found that fura-2 trapped by cells incubated with the acetoxy-methyl ester of fura-2, F2-AM, yielded measurements of Ca2+ that were depressed at rest and during the response to formyl-methionyl-leucyl-phenylalanine. Fura-2, trapped by the cells, exhibited a spectrum in the presence of saturating Ca2+ that differed from that of fura-2 free acid. We have shown that the cellular fluorescence can be spectrally decomposed into two components: one with Ca2+ sensitivity identical to fully deesterified fura-2, and another which is Ca2+-insensitive. The Ca2+-insensitive component appears to be more fluorescent than F2-AM as well as spectrally different from F2-AM. The insensitive form probably results from incomplete deesterification of F2-AM by the cells. In order to accurately measure Ca2+ in polymorphonuclear leukocytes, it is imperative to check for the presence of Ca2+-insensitive fluorescence. The contribution of Ca2+-insensitive fura-2 fluorescence can be assessed routinely from spectral data obtained by calibration of intracellular fura-2 with known [Ca2+] using ionomycin. The end-of-experiment calibration step not only ensures accurate [Ca2+] measurements in polymorphonuclear leukocytes and in other cell types that display Ca2+-insensitive, contaminating fluorescence but also yields the spectral characteristics of the insensitive species.

Topics: Benzofurans; Calcium; Fura-2; Humans; Mathematics; N-Formylmethionine Leucyl-Phenylalanine; Neutrophils

The effect of diisopropyl fluorophosphate (DFP), a potent cholinesterase (ChE) inhibitor, on loading quin 2 acetoxymethyl ester (quin 2/AM) and fura 2/AM into smooth muscle cells isolated from guinea pig taenia coli was investigated spectrofluorometrically. The presence of DFP during the loading permitted the incorporation of quin 2 into the cells, so that it became possible to measure intracellular Ca2+ concentrations using the ester of this dye. Also, DFP significantly enhanced the incorporation of fura 2 into the cells. These results indicate that loading of quin 2/AM and fura 2/AM into the smooth muscle cells may depend on the suppression of ChE or various serine protease activities outside cells.

Topics: Aminoquinolines; Animals; Benzofurans; Calcium; Fluorescent Dyes; Fura-2; Guinea Pigs; Hydrolysis; In Vitro Techniques; Isoflurophate; Muscle, Smooth

In order to analyze the factors regulating agonist-stimulated Ca2+ mobilization, cytosolic free [Ca2+] ([Ca2+]i) was measured directly in fura-2-loaded rat parotid acinar cells. Stimulation of muscarinic receptors by carbachol produced a dose-dependent rise in [Ca2+]i. In the presence of external Ca2+, the initial transient rise was followed by a maintained elevation. The maintained elevation is dependent on the presence of external Ca2+. Removal of Ca2+ by addition of EGTA caused a rapid decline in [Ca2+]i back to base line. In the absence of external Ca2+, only an initial transient peak in [Ca2+]i was seen which then declined to base line; the maintained elevation in [Ca2+]i could then be evoked by addition of Ca2+ in the continued presence of carbachol. Muscarinic receptor occupation by carbachol is required to maintain the elevated level of [Ca2+]i; addition of the muscarinic antagonist, atropine, caused [Ca2+]i to decline back to the basal level. The maintained elevation in [Ca2+]i, but not the initial transient peak, can also be blocked by Ni2+ but was unaffected by the organic Ca2+ antagonists. Total substitution of external Na+ with the impermeant cation, N-methyl-D-glucamine, had no effect on either the initial or the maintained response to carbachol; however, total substitution of Na+ with K+ attenuated the maintained response while not affecting the initial peak. Refilling of the intracellular Ca2+ store was also studied and found to take place in the absence of agonist and with no substantial elevation in [Ca2+]i. These experiments also showed that not all of the intracellular vesicular Ca2+ stores can be released by agonists. From these results, we propose a model for the regulation of [Ca2+]i.

Topics: Aluminum; Aluminum Compounds; Animals; Atropine; Benzofurans; Calcium; Carbachol; Cytosol; Fluorescent Dyes; Fluorides; Fura-2; Male; Nickel; Parotid Gland; Rats; Rats, Inbred Strains

Fura-2-am, the pentaester precursor of the fluorescent Ca(2+) indicator fura-2, is modified when it is exposed to isolated skeletal muscle sarcoplasmic reticulum vesicles. The modified fura-2-am has enhanced fluorescence, is not sensitive to Ca(2+), and is partially bound to the SR membrane. The isolated product is further converted into fura-2 by esterase. It is suggested that the SR-induced modification is a selective enzymatic hydrolysis of only some of the five ester moieties on fura-2-am. A structure is proposed to account for the results. The potential for this effect of SR on fura-2-am to cause complications in measurements of in vivo intracellular free [Ca(2+)] is noted.

Topics: Animals; Benzofurans; Calcium; Fluorescent Dyes; Fura-2; In Vitro Techniques; Rabbits; Sarcoplasmic Reticulum; Solubility; Spectrometry, Fluorescence