Coupling membranes as energy-transmitting cables. II. Cyanobacterial trichomes

Power transmission along trichomes of filamentous cyanobacteria Phormidium uncinatum has been studied with the use of ethylrhodamine fluorescence as a probe for the transmembrane electric potential difference (delta psi). It is found that agents preventing the light-induced delta psi formation (photosynthetic redox chain inhibitor dibromothymoquinone) or dissipating delta psi (uncoupler tetrachlorotrifluoromethylbenzimidazole) strongly decrease the fluorescence of the ethyl-rhodamine-stained trichomes. K+-H+ antiporter nigericin converting delta pH to delta psi increases the fluorescence. These relationships are in agreement with the assumption that ethylrhodamine electrophoretically accumulates inside the cyanobacterial cells. Illumination of a single cell in the P. uncinatum trichome gives rise to quenching of the fluorescence in this cell and usually in one or two neighbor cells, whereas the rest of trichome remains fluorescing. A small light spot (5% of the trichome length) causes an increase in the ethylrhodamine fluorescence not only in the illuminated but also in the nonilluminated parts of the trichome up to the laser-treated cell or its neighbor(s). It is concluded ethylrhodamine can be used to monitor the power transmission which was previously demonstrated by microelectrode studies of the cyanobacterial trichomes. In certain trichomes, several "dark" cells appear during the storage of the trichomes without energy sources. Illumination for several minutes results in dark cells becoming fluorescing. Thus some cells or cell clusters can be reversibly excluded from the lateral delta psi-transmitting system of the trichome, the rest being still electrically connected. This means that filamentous cyanobacteria possess mechanisms to transmit power along the trichome and to switch off this transmission.     

Effect of the Ca ionophore A-23187 on the plasmatic and mitochondrial potentials of the brain synaptosomes in rats: fluorescence measurements

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

Regulation of Cyclic AMP Levels by Calcium in Bovine Adrenal Medullary Cells

Both nicotine and histamine have been reported to increase cyclic AMP levels in chromaffin cells by Ca(2+)-dependent mechanisms. The present study investigated whether Ca2+ was an adequate and sufficient signal for increasing cyclic AMP in cultured bovine adrenal medullary cells. Depolarization with 50 mM K+ caused a two- to three-fold increase in cellular cyclic AMP levels over 5 min, with no change in extracellular cyclic AMP. This response was abolished by omission of extracellular Ca2+ and by 100 microM methoxyverapamil, and was unaffected by 1 microM tetrodotoxin and by 1 mM isobutylmethylxanthine. Veratridine (40 microM) also increased cellular cyclic AMP levels by two- to fourfold. This response was abolished by either methoxyverapamil or tetrodotoxin. The Ca2+ ionophore A23187 (10-50 microM) had little or no effect on cellular cyclic AMP levels. When the concentration of K+ used to depolarize the cells was reduced to 12-15 mM, the catecholamine release was similar to that induced by 50 microM A23187, and the cyclic AMP response was almost abolished. The results suggest that Ca2+ entry into chromaffin cells is a sufficient stimulus for increasing cellular cyclic AMP production. The possible involvement of a Ca2+/calmodulin-dependent isozyme of adenylate cyclase is discussed.     

Calcium control of ciliary reversal in ionophore-treated and ATP- reactivated comb plates of ctenophores

Previous work showed that ctenophore larvae swim backwards in high-KCl seawater, due to a 180 degrees reversal in the direction of effective stroke of their ciliary comb plates (Tamm, S. L., and S. Tamm, 1981, J. Cell Biol., 89: 495-509). Ion substitution and blocking experiments indicated that this response is Ca2+ dependent, but comb plate cells are innervated and presumably under nervous control. To determine whether Ca2+ is directly involved in activating the ciliary reversal mechanism and/or is required for synaptic triggering of the response, we (a) determined the effects of ionophore {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187 and Ca2+ on the beat direction of isolated nerve-free comb plates dissociated from larvae by hypotonic, divalent cation-free medium, and (b) used permeabilized ATP- reactivated models of comb plates to test motile responses to known concentrations of free Ca2+. We found that 5 microM {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187 and 10 mM Ca2+ induced dissociated comb plate cells to beat in the reverse direction and to swim counterclockwise in circular paths instead of in the normal clockwise direction. Detergent/glycerol-extracted comb plates beat actively in the presence of ATP, and reactivation was reversibly inhibited by micromolar concentrations of vanadate. Free Ca2+ concentrations greater than 10(-6)M caused reversal in direction of the effective stroke but no significant increase in beat frequency. These results show that ciliary reversal in ctenophores, like that in protozoa, is activated by an increase in intracellular free Ca2+ ions. This allows the unique experimental advantages of ctenophore comb plate cilia to be used for future studies on the site and mechanism of action of Ca2+ in the regulation of ciliary motion.     

Commitment to differentiation of murine erythroleukemia cells involves a modulated plasma membrane depolarization through Ca2+-activated K+ channels

The role of the plasma membrane potential (delta psi p) in the commitment to differentiation of murine erythroleukemia (MEL) cells has been studied by analyzing the ionic basis and the time course of this potential in the absence or the presence of different types of inducers. delta psi p was determined by measuring the distribution of tetraphenylphosphonium (TPP+) across the plasma membrane and displayed a 22-hour depolarization phase (from -28 to +5 mV) triggered by factors contained in foetal calf serum (FCS) and followed by a nearly symmetrical repolarization phase. After measuring the electrochemical equilibrium potential of Na+, K+, and Cl-, the relative contribution of these ions to delta psi p was evaluated by means of ion substitution experiments and by the addition of ion flux inhibitors (tetrodotoxin [TTX], 4-acetoamide-4'-isothiocyanostilbene-2,2'-disulfonate [SITS]) and ionophores (Valinomycin, A23187). The Na+ contribution to delta psi p appeared negligible, the potential being essentially generated by K+ and Cl- fluxes. When evaluated by a new mathematical approach, the effects of Valinomycin and A23187 at different times of incubation provided evidence that both the depolarization and the repolarization phase were due to variations of the K+ permeability across the plasma membrane (PK) mediated by Ca2+-activated K+ channels. All the inducers tested (dimethylsulfoxide [DMSO], hexamethylen-bis-acetamide [HMBA], diazepam), although they did not modify the ionic basis of delta psi p, strongly attenuated the depolarization rate of this potential. This attenuation was not brought about when the inducers were added to noninducible MEL cell clonal sublines. Cell commitment occurred only during the depolarization phase and increased proportionally to the attenuation of this phase up to a threshold beyond which the further increase of the attenuation was associated with the inhibition of commitment. The major role of the inducers apparently consisted of the stabilization of the Ca2+-activated K+ channels, suggesting that a properly modulated delta psi p depolarization through these channels is primarily involved in the signal generation for MEL cell commitment to differentiation.     

Regulation of goby intestinal ion absorption by the calcium messenger system

The role of the calcium messenger system in the regulation of ion absorption across the teleost intestine was studied using pharmacological intervention. Radiochloride transport was independent of external Ca2+ over the range 10 microM to 2.5 mM. Treatment with the Ca2+ ionophore A23187 (to hyperpolarization of the apical membrane potential of intestinal epithelial cells. The Ca2+-calmodulin antagonists trifluoperazine (TFP) and calmidazolium (R24571) produced opposite effects, i.e., stimulation of Cl- absorption and cellular depolarization. Treatment with TFP or R24571 will block or override the inhibitory action of A23187. These data suggest a regulatory role for Ca2+ in the control of intestinal NaCl absorption and mediation via calmodulin.     

A one-instant mechanism of phototaxis in the cyanobacterium Phormidium uncinatum

Abstract The front portion ('head') of a Phormidium uncinatum trichome responds to a step-down in light intensity [10], whereas the rear end ('tail') responds to step-up stimuli [11]. We studied this phenomenon further and found that: (i) illumination of the head caused a reversal within 1 min in only 6% of the trichomes, whereas illumination of the tail produced a reversal in 56% of trichomes; (ii) if a light spot trained on the head of a trichome was moved together with the trichome, there were no reversals for > 20 min, while the normal rate of spontaneous reversals was once per 3–5 min. Shifting the light spot from the head to the tail caused a reversal within 1–2 min; (iii) both the step-up response of the tail and phototaxis were suppressed by an inhibitor of methylation, ethionine, but not by inhibitors of photosynthesis (DCMU, DBMIB); phototaxis and the step-up response of the tail were absent in red light ( λ > 670 nm). It was concluded that trichomes of P. uncinatum possess a one-instant mechanism of phototaxis, which involves a simultaneous comparison of light intensities between two parts of the organism.     

Ca++-ionophore-induced dissipation of intracellular proton gradients in rat thymocytes

Rat thymocytes incorporate large amounts of acridine orange at concentrations of approximately 10 microM in about 10 minutes at 37 degrees C. The addition of (NH4)2SO4 (at a final concentration of several mMolars) releases about 30% of the incorporated dye into the medium. The NH4+-releasable dye uptake is almost completely abolished by 20 minutes' incubation with 4 microM of the Ca++-ionophore A23187. Dye uptake is associated with an absorbance change at 492 mm and thus may be followed spectrophotometrically. However, NH4+ at the above concentrations or nigericin (0.5 mg/ml) completely annul this change in absorbance, indicating that it reflects only the accumulation of the dye within the acidic cellular compartments. In a Ca++-containing physiological saline, the addition of A23187 (at a final concentration of 8 microM) at the end of the dye uptake phase initiated a reversal in the absorbance change; in the absence of Ca++, reversal occurred at a much lower rate. Incubation of cells for 30 minutes with 2 microM A23187 in Ca++-containing saline completely abolished NH4+-sensitive dye uptake; less A23187 (0.5 microM) and like or a longer incubation period brought about a striking decrease in NH4+-sensitive dye uptake. Similar results were obtained with cells suspended in RPLM 1640 medium. In the absence of external Ca++, A23187 impaired cell capacity to incorporate dye in a delta pH-dependent manner, but a longer incubation time or higher concentrations of the ionophore were required to obtain a comparable effect. It is thus concluded that the ionophore dissipates intracellular pH gradients by an intracellular divalent cation (Ca++ or also Mg++)-H+ exchange.     

The Effect of Antibodies to Gangliosides on Ca2+ Channel-Linked Release of γ-Aminobutyric Acid in Rat Brain Slices

Antibodies to GM1 ganglioside enhance the release of gamma-aminobutyric acid (GABA) from rat brain slices induced by depolarization with either 40 mM K+ or 200 microM veratrine. Three new observations are now reported. (a) GABA release induced by the Ca2+ ionophore A23187 was not affected by these antibodies. Because this Ca2+ ionophore causes transmitter release by bypassing depolarization-induced opening of Ca2+ channels, this result suggests that gangliosides participate either in the functioning of such Ca2+ channels or in the Na+ channels involved in depolarization. (b) The enhancement (by antibodies to GM1 ganglioside) of GABA release induced by high K+ levels occurred in the presence of tetrodotoxin (0.01 microM). (c) GABA release induced by veratrine in the absence of Ca2+ was not affected by the antibodies. These latter two observations indicate that Na+ channels are not involved in the action of the antibodies. We conclude that this evidence points to the participation of gangliosides in Ca2+ channel functions involved in GABA release in rat brain slices.     

Glucose 6-phosphate regulates Ca2+ steady state in endoplasmic reticulum of islets. A possible link in glucose-induced insulin secretion

Glucose stimulation of islets is coupled with the rapid intracellular release of myo-inositol 1,4,5-trisphosphate (IP3) and arachidonic acid which in turn mobilize Ca2+ stored in the endoplasmic reticulum (ER). The metabolism of glucose is required for insulin secretion although the link between glucose metabolism and the cellular events resulting in insulin release is unknown. In digitonin-permeabilized islets, glucose 6-phosphate (0.5-4 mM) increased significantly the ATP-dependent Ca2+ content of the ER at a free Ca2+ concentration of 1 microM. At 0.2 microM free Ca2+, glucose 6-phosphate (2-10 mM) had a smaller effect. Glucose, phosphate, mannose 6-phosphate, and fructose 1,6-diphosphate had no effect on the ATP-dependent Ca2+ content of the ER. Glucose 1-phosphate and fructose 6-phosphate also increased ATP-dependent Ca2+ content of the ER, presumably due to conversion to glucose 6-phosphate by islet phosphoglucomutase and phosphoglucoisomerase, respectively. The glucose 6-phosphate increase in the ATP-dependent Ca2+ content of the ER was shown to be mediated by glucose 6-phosphatase localized to the ER. Both arachidonic acid (10 microM) and the Ca2+ ionophore A23187 (2 microM) mobilized Ca2+ stored in the ER by glucose 6-phosphate. However, IP3-induced (10 microM) Ca2+ release from the ER was abolished in the presence of glucose 6-phosphate (0.5-10 mM). We propose that glucose 6-phosphate could provide a regulatory link between glucose metabolism and intracellular Ca2+ regulation by augmenting Ca2+ sequestered in the ER as well as attenuating IP3-induced Ca2+ release. Thus, glucose 6-phosphate would serve as an "off" signal leading to a decrease in intracellular Ca2+ when both the free Ca2+ and glucose 6-phosphate concentrations have increased following glucose stimulus.     

Matrix free Ca2+ in isolated chromaffin vesicles

Isolated secretory vesicles from bovine adrenal medulla contain 80 nmol of Ca2+ and 25 nmol of Mg2+ per milligram of protein. As determined with a Ca2+-selective electrode, a further accumulation of about 160 nmol of Ca2+/mg of protein can be attained upon addition of the Ca2+ ionophore A23187. During this process protons are released from the vesicles, in exchange for Ca2+ ions, as indicated by the decrease of the pH in the incubation medium or the release of 9-aminoacridine previously taken up by the vesicles. Intravesicular Mg2+ is not released from the vesicles by A23187, as determined by atomic emission spectroscopy. In the presence of NH4Cl, which causes the collapse of the secretory vesicle transmembrane proton gradient (delta pH), Ca2+ uptake decreases. Under these conditions A23187-mediated influx of Ca2+ and efflux of H+ cease at Ca2+ concentrations of about 4 microM. Below this concentration Ca2+ is even released from the vesicles. At the Ca2+ concentration at which no net flux of ions occurs the intravesicular matrix free Ca2+ equals the extravesicular free Ca2+. In the absence of NH4Cl we determined an intravesicular pH of 6.2. Under these conditions the Ca2+ influx ceases around 0.15 microM. From this value and the known pH across the vesicular membrane an intravesicular matrix free Ca2+ concentration of about 24 microM was calculated. This is within the same order of magnitude as the concentration of free Ca2+ in the vesicles determined in the presence of NH4Cl.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ionophore A23187 stimulates phosphorylation of the 40,000 dalton protein in human platelets without phospholipase C activation

The Ca2+ ionophore A23187 (0.2-5 microM) stimulates the phosphorylation of the substrates of protein kinase C (40,000 dalton protein) and myosin light chain kinase (20,000 dalton protein) in the presence or absence of cyclooxygenase inhibitors. In the presence of cyclooxygenase inhibitors or millimolar Ca2+ there is no stimulation of phospholipase C by A23187. Fingerprints of the 32P-labeled 40,000 dalton protein isolated from platelets that have been stimulated with A23187, thrombin, phorbol 12,13-dibutyrate and 1,2-didecanoylglycerol were identical. Higher concentrations of A23187 (1-5 microM) induced the loss of polyphosphoinositides through phosphomonoesterase activity.     

Potentiation by Ca2+ ionophores and inhibition by extracellular KCl of endothelin-induced phosphoinositide turnover in C6 glioma cells.

Interactions between endothelin-1 (ET)-induced phosphoinositide (PI) hydrolysis and agents that increase Ca2+ influx (i.e. A23187 and ionomycin) or induce depolarization (i.e. KCl) were investigated using C6 glioma. A23187 dose-dependently potentiated ET (30 nM)- and ATP (100 microM)-induced [3H]inositol phosphate (IP) accumulation. This potentiation was associated with an increase in the maximal stimulation elicited by both ET and ATP but their EC50 values were unchanged. This effect of A23187 occurred at concentrations that did not affect basal PI turnover; i.e. 10 nM-3 microM. Ionomycin within the range of 1 nM-1 microM also significantly enhanced ET-induced PI breakdown and this effect was associated with an increase of [Ca2+]i. KCl in a concentration-dependent manner (14.7-54.7 mM) markedly inhibited PI breakdown elicited by ET and ATP, but had much less inhibition on basal activity and no effect on A23187- and ionomycin-induced responses. In parallel, KCl added before or after ET, sharply attenuated the increase of ET-induced [Ca2+]i but did not affect basal level or ionomycin-induced [Ca2+]i response. Neither the potentiation by A23187 nor the inhibition by KCl of ET-induced PI turnover was observed in cultured cerebellar astrocytes. Our results suggest that the cell type-specific regulation by Ca2+ ionophores and KCl on ET-induced PI metabolism is closely related to perturbation of [Ca2+]i.     

Transport and control of Ca2+ by pigeon erythrocytes. I. Survey of some cell responses to a range of A23187 doses in the presence of Ca2+

Cells were subjected to a range of 45Ca2+ influx loads with A23187. We measured cell 45Ca2+ with time and A23187 dose, and the apparent 45Ca2+ influxes (identical to "J(in,app)") at Ca2+ steady state. We also measured endogeneous exchangeable and total cell Ca2+, which were 50 and 17-220 microM respectively. The effects of A23187 and Ca2+ on cell ATP, swelling, net Cl- permeability, and cell morphology were measured. These were modest and do not affect our conclusions. J(in,app) congruent to 3 X 10(-4) [A23187]2.9 X [Ca2+(o)]mumoles/l X min with 92-552 microM [Ca2+(o)] (identical to external Ca2+ concentration) and 0-7 microM A23187. J(in,app) was increased an order of magnitude by vanadate and is probably much less than the true influx. The least unlikely explanation found for the high [A23187] exponent, 2.9, was that most of the Ca2+ crossing the membrane is expelled by the pump before it can move deeper into the cell. Calcium pumping increased rapidly in response to increased influx, but the steady state cell 45Ca2+ was approximately proportional to J(in,app) rather than approximately constant between 10 and 120 mumoles/l X min with 184 microM [Ca2+(o)]. This is not the result expected from a simple feedback mechanism. At high A23187 doses the pump appears fully activated resulting in a linear relation between cell/medium 45Ca2+ and [A23187]-2. From the plot we calculated alpha identical to free/total exchangeable Ca2+ = 0.38 +/- 0.08 (n = 3) and a maximum pump rate, "Pmax" = 78 mumole/l X min. Pmax is underestimated insofar as J(in,app) is less than the true influx.     

The calcium signal and phosphatidylinositol breakdown in 2H3 cells

Phosphatidylinositol (PI) and its phosphorylated derivatives are rapidly broken down in 2H3 cells stimulated with antigen, with a time course which coincides with the generation of the Ca signal. Stimulated PI breakdown is absolutely dependent on Ca2+ in the medium with a concentration dependence similar to that of the Ca signal and histamine release described in the preceding paper. However, PI breakdown does not depend on the rise in free cytoplasmic Ca2+ concentration in stimulated cells over the range 100 nM to 1 microM. Thus, stimulation by the ionophore A23187 causes only a small increase in PI breakdown and the Ca signal stimulated by antigen can be selectively blocked with appropriate concentrations of Zn2+ (100 microM) or La3+ (10-100 microM) which have small or negligible effects on stimulated PI breakdown. Both PI breakdown and the Ca signal appear to depend on a common external Ca2+ site (or sites) with Km approximately equal to 0.4 mM, and the data are consistent with either independent activation of PI phosphodiesterase and the Ca signal after antigenic stimulation, or with PI breakdown as a component of the mechanism by which the Ca signal is generated.     

Involvement of Ca2+ and cGMP in bacterial taxis

Abstract The level of cGMP in a suspension of Escherichia coli cells increased transiently upon the addition of chemoattractants. Ca²⁺ (1 mM), but not Mg²⁺, produced constant tumbling of cells in the presence of the ionophore A23187. The effect was observed either in stationary-state cells, or in a logarithmic culture treated with EDTA to increase permeability by A23187. Under the same conditions, Ca²⁺ decreased the cytoplasmic level of cGMP. In Phormidium uncinatum , rapid ⁴⁵Ca²⁺ accumulation followed a light-dark stimulus, or the addition of tetramethylquinone (TMQ), a chemorepellent. La³⁺, which increases the reversal rate, also enhanced the level of cytoplasmic Ca²⁺, presumably by blocking the outward Ca²⁺ flux. In both E. coli and P. uncinatum Ca²⁺ inhibited methylaccepting chemotaxis protein (MCP) methylation. It is concluded that cGMP and Ca²⁺ are secondary messengers in taxis information-processing.     

The role of free calcium ion in calcium release in skinned muscle fibers

Major questions in excitation--contraction coupling of fast skeletal muscle concern the mechanism of signal transmission between sarcolemma and sarcoplasmic reticulum (SR), the mechanism of SR Ca release, and operation of the SR active transport system during excitation. Intracellular Ca movement can be studied in skinned muscle fibers with more direct control, analysis of 45Ca flux, and simultaneous isometric force measurements. Ca release can be stimulated by bath Ca2+ itself, ionic "depolarization," Mg2+ reduction, or caffeine. The effectiveness of bath Ca2+ has suggested a possible role for Ca2+ in physiological release, but this response is difficult to analyze and evaluate. Related evidence emerged from analysis of other responses: with all agents studied, stimulation of 45Ca efflux is highly Ca2+-dependent. The presence of a Ca chelator prevents detectable stimulation by ionic "depolarization" or Mg2+ reduction and inhibits the potent caffeine stimulus; inhibition is graded with chelator concentration and caffeine concentration, and is synergistic with inhibition by increased Mg2+. The results indicate that a Ca2+-dependent pathway mediates most or all of stimulated 45Ca efflux in skinned fibers, and has properties compatible with a function in physiological Ca release.     

小鼠卵受精和人工激活过程中胞质游离Ca^2＋变化及其在卵激活中的作用

休止于第二次成熟分裂中期（ＭＩ）的小鼠卵母细胞分别乙醇,钙离子载体Ａ２３１８７、电刺激或精子激活并用Ｃａ＾２＋特异荧光探针－Ｆｕｒａ２／ＡＭ测定细胞内游离Ｃａ＾２＋的变化。结果表明,受精诱导ＭⅡ卵内游离Ｃａ＾２＋浓度多次跃升（ｏｓｃｉｌｌａｔｉｏｎ）乙醇,钙离子载体及１次电刺激仅诱导胞内Ｃａ＾２＋１次升高,人工诱导激活的卵可象正常受精卵一样卵裂并发育至囊胚,用ＥＧＴＡ阻止受精和人工激活过程中卵内游     

The total and free concentrations of Ca2+ and Mg2+ inside platelet secretory granules. Measurements employing a novel double null point technique

The inorganic ion contents of platelet alpha-granules were determined by a combination of neutron activation analysis and flame photometry. Total concentrations were estimated using intragranular water spaces measured by isotope dilution. To measure the free concentrations of Ca2+ and Mg2+ we developed a novel double null point titration technique. The method requires independent determinations of transmembrane delta pH and the availability of two divalent cation-H+ exchange ionophores with different Ca2+/Mg2+ selectivity ratios. A23187 and the halogenated analog 4-bromo-A23187 were used for this purpose. Absolute delta pH was measured by methylamine distribution, while relative pH changes induced by the ionophores were monitored with 9-aminoacridine. The free concentrations of Ca2+ and Mg2+ were found to be 12 and 326 microM, respectively. These values are markedly lower than the calculated total concentrations of these cations, i.e. 32 mM for Ca2+ and 172 mM for Mg2+.     

Differential Calcium Dependence of γ-Aminobutyric Acid and Acetylcholine Release in Mouse Brain Synaptosomes

The dependence of gamma-aminobutyric acid (GABA) and acetylcholine (ACh) release on Ca2+ was comparatively studied in synaptosomes from mouse brain, by correlating the influx of 45Ca2+ with the release of the transmitters. It was observed that exposure of synaptosomes to a Na+-free medium notably increases Ca2+ entry, and this condition was used, in addition to K+ depolarization and the Ca2+ ionophore A23187, to stimulate the influx of Ca2+ and the release of labeled GABA and ACh. The effect of ruthenium red (RuR) on these parameters was also investigated. Of the three experimental conditions used, the absence of Na+ in the medium proved to be the most efficient in increasing Ca2+ entry. RuR inhibited by 60-70% the influx of Ca2+ stimulated by K+ depolarization but did not affect its basal influx or its influx stimulated by the absence of Na+ or by A23187. The release of ACh was stimulated by K+ depolarization, absence of Na+ in the medium, and A23187 in a strictly Ca2+-dependent manner, whereas the release of GABA was only partially dependent on the presence of Ca2+ in the medium. The extent of stimulation of ACh release was related to the extent of Ca2+ entry, whereas no such correlation was observed for GABA. In the presence of Na+, RuR did not affect the release of the transmitters induced by A23187. In the absence of Na+, paradoxically RuR notably enhanced the release of both ACh and GABA induced by A23187, in a Ca2+-dependent manner.(ABSTRACT TRUNCATED AT 250 WORDS)