sq-23377 and indo-1

Gonadotropin releasing hormone (GnRH) stimulates both transcription and secretion of the alpha subunit of the gonadotropins in a Ca2+-dependent fashion. In this study, we examined the role of Ca2+ as the signal coupling agonist occupancy of GnRH receptors to hormone secretion using the gonadotropic cell line alphaT3-1. Treatment of alphaT3-1 cells for 60 min with GnRH (0.1-100 nM), veratridine (50 microM) or high K+ (56 mM) was completely ineffective in stimulating secretion. The lack of effect occurred in spite of a robust, specific, and dose-dependent biphasic [Ca2+]i response consisting of a rapid peak sensitive to thapsigargin (200 nM) followed by a smaller plateau sensitive to the extracellular application of EGTA (5 mM). On the other hand, treatment of alphaT3-1 cells with the Ca2+ ionophore ionomycin resulted in a significant dose-dependent stimulation of secretion and [Ca2+]i responses comparable to those elicited by GnRH. Binding assays revealed the presence of Ins(1,4,5)P3 receptors (Kd = 3.2 nM, Bmax = 50.5 fmol/mg protein) but not ryanodine receptors in alphaT3-1 cell membranes. Together, these results show a functional uncoupling between the [Ca2+]i response and secretion in this cell line, suggesting that the increase in [Ca2+]i triggered by GnRH and depolarization may be necessary but not sufficient to stimulate exocytosis.

Topics: Calcium; Calcium Channel Blockers; Calcium Signaling; Cell Line; Cell Survival; Chelating Agents; Egtazic Acid; Enzyme Inhibitors; Exocytosis; Fluorescent Dyes; Gadolinium; Glycoprotein Hormones, alpha Subunit; Glycoproteins; Gonadotropin-Releasing Hormone; Indoles; Inositol 1,4,5-Trisphosphate; Ionomycin; Ionophores; L-Lactate Dehydrogenase; Membrane Potentials; Nifedipine; Pituitary Gland; Receptors, LHRH; Ryanodine; Ryanodine Receptor Calcium Release Channel; Sulfur Radioisotopes; Thapsigargin; Tritium

Ca2+ is an essential activator of motility in the obligate intracellular parasite Toxoplasma gondii. Ca2+ ionophore A23187 and intracellular microinjection of Ca2+ initiate motility of parasites residing in parasitophorous vacuoles (PV). The source of Ca2+ and the mechanism by which it activates motility in vivo remain uncertain. Exposure of the parasites to dithiothreitol (DTT) can activate egress of previously nonmotile intravacuolar parasites within 60 sec. DTT is also known to activate both isoforms of the highly concentrated nucleoside triphosphate hydrolase (NTPase) produced by T. gondii. Using an adherent cell analysis system (ACAS) for Ca2+ imaging, a brief 15-50% increase in intra-PV fluorescence ratio was observed after exposure of infected fibroblasts to 5 mM DTT. Chelation of intracellular Ca2+ with BAPTA-AM and extracellular Ca2+ with EGTA blocked the DTT effect; however, this chelation did not prevent the activation of parasites nor the Ca2+ response to the Ca2+ ionophore ionomycin, suggesting that the Ca2+ that activates motility may reside near or within the parasite itself. This result demonstrates that an increase in Ca2+ within the vacuole precedes the onset of motility and the correlation of the DTT effect on motility and tachyzoite NTPase suggests that NTPase activation may be involved in the Ca2+ flux.

Topics: Animals; Calcium; Cells, Cultured; Chelating Agents; Dithiothreitol; Drug Interactions; Egtazic Acid; Fibroblasts; Fluorescent Dyes; Humans; Image Processing, Computer-Assisted; Indoles; Ionomycin; Ionophores; Microscopy, Confocal; Reproducibility of Results; Ryanodine; Sulfhydryl Reagents; Thapsigargin; Toxoplasma; Vacuoles

Improved time resolution of kinetic cellular events in flow cytometry is demonstrated by using a coaxial flow-mixing device integrated within a flow-injection (FI) system. The instrument is used in combination with a Becton Dickinson FACS Analyzer for on-line reagent addition, rapid sample mixing, and temperature control of cell suspensions. The coaxial flow device can instantaneously (< 60 ms) mix reagent and sample streams, allowing cytometric analysis of subsecond events to be performed. Kinetic measurements can be performed on the FACS analyzer in a variable time range of from 100 ms to 3 min. The system also allows the collection of unlimited cellular events at a specific incubation time point. Because the system operates continuously and no boost in core flow is required, disturbances of flow conditions are avoided. The capabilities of the flow injection cytometer have been demonstrated by the determination of internal [Ca2+]i mobilization in Jurkat T lymphocytes perfused internally with INDO-1 and stimulated by ionomycin.

Topics: Flow Cytometry; Fluorescent Dyes; Humans; Indoles; Ionomycin; Ionophores; Jurkat Cells; Kinetics; Lymphocyte Activation; Statistics as Topic

CD3 mAb induced calcium movements are unaffected by hyperpolarization of the membrane in Jurkat T cells treated with valinomycin. By contrast, the CD3 induced Ca2+ influx was impaired by depolarization of the membrane with either gramicidin or by equimolar substitution of KCl for NaCl in the medium. In depolarized cells, the synthesis of phosphatidylserine was strongly diminished as a result of impaired transport of the [3H]serine substrate. In depolarized cells, the CD3-induced release of Ca2+ from intracellular stores (endoplasmic reticulum) was unaffected. Emptying of the Ca2+ stores by CD3 was shown by the lack of effect of additional treatment of the cells with the Ca2+ ionophore, ionomycin. The empty status of the calcium stores was also confirmed by measurements of phosphatyidylserine synthesis through the Ca2+ -dependent base exchange enzyme system that was found to be significantly decreased despite the low amount synthesized in the presence of a defective [3H]serine transport in depolarized cells.

Topics: Antibodies, Monoclonal; Barbiturates; Calcium; CD3 Complex; Fluorescent Dyes; Gramicidin; Humans; Indoles; Ionomycin; Ionophores; Isoxazoles; Jurkat Cells; Membrane Potentials; Phosphatidylserines; T-Lymphocytes; Valinomycin

The acrosome reaction in many animals is a coupled reaction involving an exocytotic step and a dramatic change in cell shape. It has been proposed that these morphological changes are regulated by intracellular ions such as Ca2+ and H+. We report here simultaneous visualization, under a multiview microscope, of intracellular free Ca2+ concentration ([Ca2+]i), intracellular pH (pHi), and morphological changes in a single starfish sperm (Asterina pectinifera). [Ca2+]i and pHi were monitored with the fluorescent probes indo-1 and SNARF-1, respectively. The acrosome reaction was induced with ionomycin. After the introduction of ionomycin in the medium, [Ca2+]i increased gradually and reached a plateau in approximately 30 s. The fusion of the acrosomal vacuole took place abruptly before the plateau, during the rising phase. Although the speed of the [Ca2+]i increase varied among the many sperm tested, exocytosis in all cases occurred at the same [Ca2+]i of approximately 2 microM (estimated using the dissociation constant of indo-1 for Ca2+ of 1.1 microM). This result suggests that the exocytotic mechanism in starfish sperm responds to [Ca2+]i rapidly, with a reaction time of the order of one second or less. Unlike the change in [Ca2+]i, an abrupt increase in pHi was observed immediately after exocytosis, suggesting the presence of a proton mobilizing system that is triggered by exocytosis. The rapid increase in pHi coincided with the formation of the acrosomal rod and the beginning of vigorous movement of the flagellum, both of which have been proposed to be pHi dependent. The exocytotic event itself was visualized with the fluorescent membrane probe RH292. The membrane of the acrosomal vacuole, concealed from the external medium in an unreacted sperm, was seen to fuse with the plasma membrane.

Topics: Acrosome; Animals; Benzopyrans; Calcium; Cell Size; Exocytosis; Fluorescent Dyes; Hydrogen-Ion Concentration; Image Processing, Computer-Assisted; Indoles; Intracellular Membranes; Ionomycin; Ionophores; Kinetics; Male; Microscopy, Fluorescence; Naphthols; Rhodamines; Spermatozoa; Starfish; Vacuoles

Large amounts of Ca2+ (almost 20 nmol/10(8) cells) are released from platelets by exocytosis. This secretory-granule-associated Ca2+ does not contribute to the cytosolic free Ca2+ ([Ca2+]i), which is controlled by the much smaller agonist-sensitive Ca2+ pool, unless high (1 microM), but not low (0.04 microM) concentrations of ionomycin are present. Low concentrations of ionomycin release Ca2+ almost exclusively from the agonist-sensitive stores. In aspirinated platelets incubated in the presence of 0.5 mM EGTA the extensive depletion of the agonist-sensitive stores is obtained by the combined action of low ionomycin and the endomembrane Ca(2+)-ATPase inhibitor thapsigargin (which individually promote only a partial depletion). The subsequent decay of [Ca2+]i is increased by phorbol-myristate acetate, confirming that Ca2+ efflux from platelets is potentiated by the activation of protein kinase C [Pollock, W. K., Sage, S. O. & Rink, T. J. (1987) FEBS Lett. 210, 132-140]. A novel type of control of Ca2+ efflux appears to be exerted by the filling state of the stores. Treatment with low ionomycin or thapsigargin determines the release of a fraction of the stores-associated Ca2+; the subsequent decay of [Ca2+]i is slow. The decay rate of [Ca2+]i accelerates after extensive depletion of the stores following the addition of thapsigargin or ionomycin. If the depletion of the stores is induced by thrombin, added alone or in combination with thapsigargin, the increases of [Ca2+]i are the same and the subsequent decay rates are largely superimposable; however a large fraction of [Ca2+]i is reaccumulated into the stores in the absence, but not in the presence of thapsigargin, indicating that Ca2+ efflux is activated when the stores are empty. Ca2+ efflux can proceed against a concentration gradient. In 45Ca-loaded platelets, the thrombin-promoted 45Ca efflux is potentiated by thapsigargin. The protein-kinase-C-dependent and store-depletion-dependent stimulations of 45Ca efflux are additive. These observations indicate that, in addition to being activated by protein kinase C, Ca2+ efflux from platelets is activated by the depletion of the stores. The two activations appear to be additive.

Topics: Alkaloids; Blood Platelets; Calcium; Calcium Radioisotopes; Calcium-Transporting ATPases; Cytoplasmic Granules; Cytosol; Dose-Response Relationship, Drug; Egtazic Acid; Exocytosis; Fluorescent Dyes; Humans; In Vitro Techniques; Indoles; Ionomycin; Kinetics; Protein Kinase C; Staurosporine; Terpenes; Tetradecanoylphorbol Acetate; Thapsigargin; Thrombin

We have developed a perifusion system that can measure both changes of cytosolic free calcium concentration [Ca2+]i and prolactin release simultaneously from cultured lactotrophs. This model incorporated a commonly-used perifusion system to a spectrofluorometer. Indo-1 loaded cells were injected into Sephadex G-150 matrix in the cuvette at a site where the emitting light of the fluorometer projects. During perifusion periods, the perifusate was collected in a fraction collector, while optical density of the emitting light at 405 nm was recorded. The [Ca2+]i was calculated based on an ionomycin and Mn2+ quenching technique. As expected, TRH (1 mumol/l) stimulated prolactin release from cultured lactotrophs in this system. We further observed that prolactin releases as induced by TRH and ionomycin were not proportional with changes of the [Ca2+]i, suggesting that changes of [Ca2+]i is not the sole final pathway of intracellular transduction systems for prolactin release.

Topics: Animals; Calcium; Cells, Cultured; Culture Media; Cytosol; Estradiol; Extracellular Space; Hydrogen-Ion Concentration; Indoles; Ionomycin; Male; Manganese; Phenolsulfonphthalein; Pituitary Gland, Anterior; Prolactin; Rats; Rats, Sprague-Dawley; Signal Transduction; Thyrotropin-Releasing Hormone

Activation of lymphocytes with 10 microM ionomycin leads to a rapid increase in the concentration of free cytoplasmic calcium ([Ca2+]i) and, at a slower rate, also to an increase in the cytoplasmic free magnesium concentration ([Mg2+]i). The ionomycin-induced Mg(2+)-mobilization response is dependent on the influx of extracellular Ca2+. After receptor-mediated lymphocyte activation, induced by mitogens or anti-receptor antibodies, a Mg(2+)-mobilization response does occur in a small fraction of the cells. Simultaneous measurement of [Ca2+]i and [Mg2+]i in individual cells showed that the receptor-triggered Mg(2+)-mobilization response is restricted to cells that have a high [Ca2+]i. It can therefore be concluded that a high [Ca2+]i induces the release into the cytoplasm of Mg2+ from intracellular stores.

Topics: Burkitt Lymphoma; Calcium; Cells, Cultured; Cytoplasm; Flow Cytometry; Fluorescent Dyes; Humans; Indoles; Ionomycin; Kinetics; Lymphocyte Activation; Magnesium; T-Lymphocytes; Time Factors; Tumor Cells, Cultured

ATP has been demonstrated to act as a co-transmitter or neuromodulator in various physiological processes. There is recent evidence that ATP receptors, characterized as P2 purinergic receptors, are expressed in the sensory hair cells of the auditory organ. The aim of the present study was to know whether other cell types of the organ of Corti, the supporting cells, were also sensitive to external ATP. In both types of supporting cells considered in this study, Deiters' cells (DCs) and Hensen's cells (HEs), extracellular ATP at sub-micromolar concentrations evoked a transient increase in [Ca2+]i as monitored with fluorescence microscopy using the calcium probe Indo-1. An apparent Kd of 0.5 and 0.9 microM was determined for DCs and HEs, respectively. Furthermore, we demonstrated that ATP stimulated Ca2+ release from internal stores in DCs, but not in HEs. Dynamic calcium imaging by confocal laser scanning microscopy of ATP induced Ca2+ mobilization demonstrated a calcium wave propagation in the cell body of DCs which originated in the phalangeal processes, suggesting a functional organization of Ca2+ sequestering stores in DCs.

Topics: Adenosine Triphosphate; Aniline Compounds; Animals; Calcium; Dose-Response Relationship, Drug; Guinea Pigs; Indoles; Ionomycin; Microspectrophotometry; Organ of Corti; Receptors, Purinergic; Thionucleotides; Xanthenes

Under whole-cell recording, bradykinin (BK) produced an initial outward membrane current followed by an inward current in voltage-clamped NG108-15 cells. The initial outward current was associated with a rise in intracellular Ca2+ and was accompanied by the opening of Ca(2+)-dependent K(+)-channels recorded with a cell-attached patch electrode. This current was inhibited by intracellular Mg2+. The inward current was associated with inhibition of the voltage-dependent K(+)-current IK(M). These effects accord with those previously observed in microelectrode-impaled cells, with the difference that BK produced much more pronounced and long-lasting desensitization in the patch-clamped cells.

Topics: Acetylcholine; Animals; Apamin; Bradykinin; Calcium; Calcium Channels; Charybdotoxin; Fluorescent Dyes; Glioma; Hybrid Cells; Indoles; Inositol 1,4,5-Trisphosphate; Ionomycin; Membrane Potentials; Mice; Neuroblastoma; Neurotoxins; Norepinephrine; Potassium Channels; Rats; Scorpion Venoms; Virulence Factors, Bordetella

1. Ca2+ homeostasis in freshly dissociated neurons from embryonic rat hypothalamus, cortex, and brain stem was investigated with flow cytometry. Cells were dissociated from embryonic brain by enzymatic and mechanical means and were incubated with the acetoxymethylester derivative of the Ca(2+)-sensitive dye indo-1. Neurons hydrolyzed and retained the dye as determined by the intensity of fluorescence emission, whereas similarly treated cultured astrocytes gave very low-level fluorescence. 2. The fluorescence of the indo-1 dye was measured at two wavelengths (405 and 485 nm) for each cell. Data were collected only from those cells (presumptive neurons) with high levels of fluorescence. Methods were developed to calibrate the level of intracellular free calcium ([Ca2+]i) as the ratio of fluorescence at 410 and 485 nm. The level of intracellular free Ca2+ was then calculated for each neuron. 3. A wide distribution of resting [Ca2+]i was found, with a median of approximately 90 nM. After addition of ionomycin to cells in Ca(2+)-free medium, there was a transient increase in [Ca2+]i, suggesting that all embryonic neurons had internal Ca2+ stores. The presence of active calcium extrusion mechanisms was demonstrated with the use of ionomycin in Ca(2+)-containing medium and with metabolic inhibitors. Furthermore, incubation in sodium-free medium resulted in a transient increase in [Ca2+]i and a reduced ability to eliminate elevated [Ca2+]i from the cytoplasm, suggesting that calcium homeostasis was dependent on the activity of the Na(+)-Ca2+ exchange mechanism. 4. Depolarization with K+ or veratrine increased [Ca2+]i in approximately 20% of the cells. This increase was blocked by eliminating extracellular free Ca2+ or adding Co2+, nifedipine, or verapamil, suggesting mediation by voltage-sensitive calcium channels. 5. Neurons were sorted on the basis of high [Ca2+]i and placed into dissociated culture. After 24 h, neurons in culture retained indo-1 fluorescence, suggesting that populations of neurons can be collected on the basis of their levels of [Ca2+]i. 6. These results demonstrate that flow cytometric analysis allows the characterization of a variety of Ca(2+)-regulatory mechanisms in populations of freshly dissociated embryonic neurons. Although only a proportion of embryonic day 17 neurons exhibit voltage-sensitive calcium channels, all neurons have developed the ability to sequester and extrude Ca2+.

Topics: Animals; Brain; Calcium; Calcium Channel Blockers; Calcium Channels; Cells, Cultured; Egtazic Acid; Female; Flow Cytometry; Homeostasis; Indoles; Ionomycin; Neurons; Potassium; Pregnancy; Rats; Rats, Inbred Strains; Veratrine

We have measured the distribution of cytoplasmic calcium in lily pollen tubes by microinjecting them with indo-1 and performing fluorescence ratio image analysis on them. All of the 16 tubes that were growing at the time of the calcium measurements showed a gradient of [Ca2+]i in the tip region, with Ca2+ being 1.25 to 3.32 times higher at the distal end in 15 cases and more than 5 times higher in one case. The extent of the gradient ranged from 22 to 65 microns. Most of the 15 nongrowing tubes either had no gradient or had lower Ca2+ in the tip region. While we have confirmed a previous report that lily pollen tubes can be loaded with the membrane-permeable acetoxymethyl ester forms of calcium indicators, the dyes loaded in this way are visibly partitioned into organelles and this method of loading is, therefore, not useful for the measurement of [Ca2+]i. Iontophoresis of the dye free acids into tubes produces a more uniform and diffuse fluorescence which does not appear to partition into organelles. Indo-1 remains in the pollen tubes longer than fura-2. The correlation between growth and the [Ca2+]i gradient in the apical portion of the pollen tube is discussed in relation to previous reports that have suggested that such a gradient should exist during polarized growth.

Topics: Calcium; Cell Polarity; Cytoplasm; Fertilization; Indoles; Ionomycin; Plant Cells; Plant Physiological Phenomena; Pollen

The aim of the present study was to analyse whether an increase in the intracellular free Ca2+ concentration ([Ca2+]i) plays a role as a signal mediating synthesis of nitric oxide (NO) in bone-marrow-derived macrophages, either by stimulating induction of NO synthase or by regulating the activity of the enzyme. Therefore we compared the effects of various synthetic analogues of bacterial lipopeptide and of lipopolysaccharide (LPS) on NO production (assessed as nitrite formation during an incubation for 24 h) and on [Ca2+]i [measured with the fluorescent probe indo-1 (1-[2-amino-5-(6-carboxyindol-2-yl)phenoxy]-2- 2-(2'-amino-5'-methylphenoxy)ethane-NNN'N'-tetra-acetic acid)]. Strongly dissociating effects were evoked on nitrite formation and on [Ca2+]i by the stimuli. LPS was preferentially effective on nitrite formation, whereas the Ca2+ ionophore ionomycin and AlF3 induced increases only in [Ca2+]i. The lipopeptides N-palmitoyl-(S)-[2,3-bis(palmitoyloxy)-(2RS)- propyl]-(R)-cysteinylalanylglycine, N-palmitoyl-(S)-[2,3-bis(palmitoyloxy)- (2RS)-propyl]-(R)-cysteinylseryl-lysyl-lysyl-lysine and (S)-(1,2- dicarboxyhexadecyl)ethyl-N-palmitoylcysteinylseryl-lysyl-lys yl-lysine stimulated both parameters, but the maximal effects on nitrite formation and the shape of the dose-response curves did not parallel the effects on [Ca2+]i. Reduction of extracellular Ca2+ with EGTA significantly inhibited increases in [Ca2+]i, but did not change nitrite formation. Furthermore, NO synthesis in the cytosolic fraction of stimulated macrophages was not affected by Ca2+ over the concentration range 10 nM-2 microM. We conclude that increases in [Ca2+]i are not required for NO production in bone-marrow-derived macrophages. Thus the cellular regulation of NO production strikingly differs from that in the vascular endothelium, brain and adrenal gland.

Topics: Aluminum; Aluminum Compounds; Amino Acid Oxidoreductases; Amino Acid Sequence; Animals; Bone Marrow Cells; Calcium; Cytosol; Egtazic Acid; Enzyme Induction; Fluorescent Dyes; Fluorides; Indoles; Ionomycin; Lipopolysaccharides; Lipoproteins; Macrophages; Mice; Mice, Inbred BALB C; Molecular Sequence Data; Nitric Oxide Synthase; Nitrites; Sodium Fluoride

Changes in intracellular calcium levels induced in mast cells by either antigen or ionomycin were monitored using flow cytometry and the calcium binding dye indo-1. Both stimuli increased calcium levels in responsive cells by a similar amount, but not all cells examined were responsive to antigen. Antigen-induced increases in intracellular calcium levels could be completely blocked by the calcium antagonist TMB-8. Flow cytometry appears to be a useful method for monitoring mast cell calcium concentrations, as it provides the capability to study large numbers of individual cells.

Topics: Animals; Antigens; Calcium; Calcium Channel Blockers; Cells, Cultured; Flow Cytometry; Fluorescent Dyes; Gallic Acid; Indoles; Ionomycin; Kinetics; Mast Cells; Mice

Recent studies have demonstrated that murine lymphocytes express specific cell-surface receptors for a range of sulfated polysaccharides. In order to determine whether polysaccharide binding induces transmembrane signaling, the effects of sulfated polysaccharides on the free intracellular calcium ion concentration [( Ca2+]i) of mouse thymocytes and spleen cells were determined. Cells were loaded with Indo-I, a fluorescent indicator of calcium ion concentration. The validity and limitations in the use of this indicator in the determination of [Ca2+]i are documented. Dextran sulfate (Mn = 500,000), iota-carrageenan, lambda-carrageenan and kappa-carrageenan all cause relatively large changes in the [Ca2+]i of thymocytes (change in [Ca2+]i greater than 50 nM). Of these, dextran sulfate (Mn = 500,000) always had the greatest effect on [Ca2+]i. Smaller responses were obtained with heparin and dextran sulfate (Mn = 5000), while no response was obtained with chondroitin 4-sulfate, chondroitin 6-sulfate, pentosan sulfate or fucoidin. This response pattern (with the exception of fucoidin and pentosan sulfate) corresponds with the expression of thymocyte receptors for these polysaccharides. The increase in [Ca2+]i caused by the sulfated polysaccharides requires extracellular Ca2+ ions however, it is unlikely that voltage-dependent ion channels are involved in these responses. In contrast to thymocytes, although spleen cells express receptors for sulfated polysaccharides, they were unresponsive to all of the sulfated polysaccharides tested, suggesting a basic difference between thymocytes and peripheral T and B lymphocytes in their response to the binding of sulfated polysaccharides.

Topics: Animals; Calcium; Carrageenan; Concanavalin A; Dextran Sulfate; Dextrans; Ethers; Female; Fluorescence Polarization; Fluorescent Dyes; Indoles; Ionomycin; Kinetics; Lymphocytes; Male; Mice; Mice, Inbred BALB C; Polysaccharides; Spectrometry, Fluorescence; Spleen; Thymus Gland