Modulation of the Ca2+,Mg2+-ATPase Activity of Synaptosomal Plasma Membrane by the Local Anesthetics Dibucaine and Lidocaine

It has been previously shown that local anesthetics inhibit the total Ca2+, Mg2(+)-ATPase activity of synaptosomal plasma membranes. We have carried out kinetic studies to quantify the effects of these drugs on the different Ca2(+)-dependent and Mg2(+)-dependent ATPase activities of these membranes. As a result we have found that this inhibition is not altered by washing the membranes with EDTA or EGTA. We have also found that the Ca2(+)-dependent ATPase activity is not significantly inhibited in the concentration range of these local anesthetics and under the experimental conditions used in this study. The inhibition of the Mg2(+)-dependent ATPase activities of these membranes was found to be of a noncompetitive type with respect to the substrate ATP-Mg2+, did not significantly shift the Ca2+ dependence of the Ca2+, Mg2(+)-ATPase activity, and occurred in a concentration range of local anesthetics that does not significantly alter the order parameter (fluidity) of these membranes. Modulation of this activity by the changes of the membrane potential that are associated with the adsorption of local anesthetics on the synaptosomal plasma membrane is unlikely, on the basis of the weak effect of membrane potential changes on the Ca2+,Mg2(+)-ATPase activity. It is suggested that the local anesthetics lidocaine and dibucaine inhibit the Ca2+, Mg2(+)-ATPase of the synaptosomal plasma membrane by disruption of the lipid annulus.     

Effects of Ca2+ Channel Blockers on Ca2+ Translocation Across Synaptosomal Membranes

The binding of [3H]nimodipine to purified synaptic plasma membranes (SPM) isolated from sheep brain cortex was characterized, and the effects of nimodipine, nifedipine, and (+)-verapamil on the [3H]nimodipine binding were compared to the effects on 45Ca2+ translocation under conditions that separate 45Ca2+ fluxes through Ca2+ channels from 45Ca2+ uptake via Na+/Ca2+ exchange. [3H]Nimodipine labels a single class of sites in SPM, with a KD of 0.64 +/- 0.1 nM, a Bmax of 161 +/- 27 fmol X mg-1 protein, and a Hill slope of 1.07, at 25 degrees C. Competition of [3H]nimodipine binding to purified SPM with unlabelled Ca2+ channel blockers shows that: nifedipine and nimodipine are potent competitors, with IC50 values of 4.7 nM and 5.9 nM, respectively; verapamil and (-)-D 600 are partial competitors, with biphasic competition behavior. Thus, (+)-verapamil shows an IC50 of 708 nM for the higher affinity component and the maximal inhibition is 50% of the specific binding, whereas for (-)-verapamil the IC50 is 120 nM, and the maximal inhibition is 30%; (-)-D 600 is even less potent than verapamil in inhibiting [3H]nimodipine binding (IC50 = 430 nM). However, (+)-verapamil, nifedipine, and nimodipine are less potent in inhibiting depolarization-induced 45Ca2+ influx into synaptosomes in the absence of Na+/Ca2+ exchange than in competing for [3H]nimodipine binding. Thus, (+)-verapamil inhibits Ca2+ influx by 50% at about 500 microM, whereas it inhibits 50% of the binding at concentrations 200-fold lower, and the discrepancy is even larger for the dihydropyridines. The Na+/Ca2+ exchange and the ATP-dependent Ca2+ uptake by SPM vesicles are also inhibited by the Ca2+ channel blockers verapamil, nifedipine, and d-cis-diltiazem, with similar IC50 values and in the same concentration range (10(-5)-10(-3) M) at which they inhibit Ca2+ influx through Ca2+ channels. We conclude that high-affinity binding of the Ca2+ blockers by SPM is not correlated with inhibition of the Ca2+ fluxes through channels in synaptosomes under conditions of minimal Na+/Ca2+ exchange. Furthermore, the relatively high concentrations of blockers required to block the channels also inhibit Ca2+ translocation through the Ca2+-ATPase and the Na+/Ca2+ exchanger. In this study, clear differentiation is made of the effects of the Ca2+ channel blockers on these three mechanisms of moving Ca2+ across the synaptosomal membrane, and particular care is taken to separate the contribution of the Na+/Ca2+ exchange from that of the Ca2+ channels under conditions of K+ depolarization.     

Dose-Dependent, K+-Stimulated Efflux of Endogenous Taurine from Primary Astrocyte Cultures Is Ca2+-Dependent

The K+-stimulated efflux of endogenous taurine from primary rat cerebellar astrocyte cultures prepared from 7-9-day-old rats was studied at 16-18 days in vitro using HPLC analysis. Taurine efflux was dose-dependent at K+ concentrations between 10 mM and 80 mM, with an EC50 of approximately 50 mM. Maximum stimulation of efflux above basal levels ranged from 56% at 10 mM K+ (204 pmol/min/mg protein) to 470% at 80 mM K+ (960 pmol/min/mg protein). Removal of Ca2+ from the buffer and the addition of either 1 mM EGTA or 10 mM Mg2+ abolished K+-stimulated efflux. Taurine efflux peaked and fell in parallel with the K+ concentration, but with an approximate lag of 3-5 min. The time course and amount of preloaded [3H]taurine released did not differ significantly from that seen for endogenous efflux. Basal taurine efflux varied inversely with the extracellular concentration of Ca2+ over the concentration range 0-5.0 mM. The observed Ca2+ dependence is consistent with a role for Ca2+ in the regulation of taurine release. Furthermore, taurine release from astrocytes in response to elevated K+ may reflect a neuromodulatory role for this amino acid in the CNS.     

Ryanodine Inhibits Caffeine-Evoked Ca2+ Mobilization and Catecholamine Secretion from Cultured Bovine Adrenal Chromaffin Cells

The effects of ryanodine, a selective inhibitor of the Ca(2+)-induced Ca2+ release mechanism, on caffeine-evoked changes in cytosolic Ca2+ concentration ([Ca2+]i) and catecholamine secretion were investigated using cultured bovine adrenal chromaffin cells. Caffeine (5-40 mM) caused a concentration-dependent transient rise in [Ca2+]i and catecholamine secretion in Ca2+/Mg(2+)-free medium containing 0.2 mM EGTA. Ryanodine (5 x 10(-5) M) alone had no effect on either [Ca2+]i or catecholamine secretion. Although the application of ryanodine plus caffeine caused the same increase in both [Ca2+]i and catecholamine secretion as those induced by caffeine alone, ryanodine (4 x 10(-7) - 5 x 10(-5) M) irreversibly prevented the increase in both [Ca2+]i and catecholamine secretion resulting from subsequent caffeine application over a range of concentrations. The secretory response to caffeine was markedly enhanced by replacement of Na+ with sucrose in Ca2+/Mg(2+)-free medium, and this enhanced response was also blocked by ryanodine. Caffeine was found to decrease the susceptibility of the secretory apparatus to Ca2+ in digitonin-permeabilized cells. These results indicate that caffeine mobilizes Ca2+ from intracellular stores, the function of which is irreversibly blocked by ryanodine, resulting in the increase in catecholamine secretion in the bovine adrenal chromaffin cell.     

Effect of Monovalent Cations on Na+/Ca2+ Exchange and ATP-Dependent Ca2+ Transport in Synaptic Plasma Membranes

Two Ca2+ transport systems were investigated in plasma membrane vesicles isolated from sheep brain cortex synaptosomes by hypotonic lysis and partial purification. Synaptic plasma membrane vesicles loaded with Na+ (Na+i) accumulate Ca2+ in exchange for Na+, provided that a Na+ gradient (in leads to out) is present. Agents that dissipate the Na+ gradient (monensin) prevent the Na+/Ca2+ exchange completely. Ca2+ accumulated by Na+/Ca2+ exchange can be released by A 23187, indicating that Ca2+ is accumulated intravesicularly. In the absence of any Na+ gradient (K+i-loaded vesicles), the membrane vesicles also accumulate Ca2+ owing to ATP hydrolysis. Monovalent cations stimulate Na+/Ca2+ exchange as well as the ATP-dependent Ca2+ uptake activity. Taking the value for Na+/Ca2+ exchange in the presence of choline chloride (external cation) as reference, other monovalent cations in the external media have the following effects: K+ or NH4+ stimulates Na+/Ca2+ exchange; Li+ or Cs+ inhibits Na+/Ca2+ exchange. The ATP-dependent Ca2+ transport system is stimulated by increasing K+ concentrations in the external medium (Km for K+ is 15 mM). Replacing K+ by Na+ in the external medium inhibits the ATP-dependent Ca2+ uptake, and this effect is due more to the reduction of K+ than to the elevation of Na+. The results suggest that synaptic membrane vesicles isolated from sheep brain cortex synaptosomes possess mechanisms for Na+/Ca2+ exchange and ATP-dependent Ca2+ uptake, whose activity may be regulated by monovalent cations, specifically K+, at physiological concentrations.     

Stimulation of D-2 Dopamine Receptors Decreases the Evoked In Vitro Release of [3H] Acetylcholine from Rat Neostriatum: Role of K+ and Ca2+

Reportedly, stimulation of D-2 dopamine receptors inhibits the depolarization-induced release of acetylcholine from the neostriatum in a cyclic AMP-independent manner. In the present study, we investigated the role of K+ and Ca2+ in the D-2 receptor-mediated inhibition of evoked [3H]acetylcholine release from rat striatal tissue slices. It is shown that the D-2 receptor-mediated decrease of K+-evoked [3H]acetylcholine release is not influenced by the extracellular Ca2+ concentration. However, increasing extracellular K+, in the presence and absence of Ca2+, markedly attenuates the effect of D-2 stimulation on the K+-evoked [3H]acetylcholine release. Furthermore, it is shown that activation of D-2 receptors in the absence of Ca2+ also inhibits the veratrine-evoked release of [3H]acetylcholine from rat striatum. These results suggest that the D-2 dopamine receptor mediates the decrease of depolarization-induced [3H]acetylcholine release from rat striatum primarily by stimulation of K+ efflux (opening of K+ channels) and inhibition of intracellular Ca2+ mobilization.     

Effects of Opiates on High-Affinity Ca2+,Mg2+-ATPase in Brain Membrane Subfractions

The effect of a single administration of morphine sulfate (15 mg/kg, s.c. or 30 mg/kg, i.p., 30 min) on Ca2+-stimulated Mg2+-dependent ATPase activity was investigated in synaptosomal plasma membranes (SPM) prepared from rat cortex. Morphine produced a significant decrease in Ca2+,Mg2+-ATPase activity in synaptosomal fractions (SPM 1 + 2) known to contain a high density of opiate receptors and calmodulin-dependent Ca2+,Mg2+-ATPase. However, in another subpopulation (SPM 3) that contains fewer opiate receptors and less enzyme activity, no such decrease in the enzyme activity was observed after the opiate administration. The decrease in Ca2+,Mg2+-ATPase activity seen in SPM 1 + 2 was specifically antagonized by the opiate antagonist naloxone hydrochloride (2 mg/kg, s.c.) when given 15 min before morphine administration. Mg2+-ATPase was not altered either by morphine or by a naloxone-morphine combination. These findings give further evidence for the role of intracellular Ca2+ in mediating many of the acute effects of opiates.     

Calmodulin Stimulation of Ca2+-Dependent ATP Hydrolysis and ATP-Dependent Ca2+ Transport in Synaptic Membranes

We report here characterization of calmodulin-stimulated Ca2+ transport activities in synaptic plasma membranes (SPM). The calcium transport activity consists of a Ca2+-stimulated, Mg2+-dependent ATP hydrolysis coupled with ATP-dependent Ca2+ uptake into membraneous sacs on the cytosolic face of the synaptosomal membrane. These transport activities have been found in synaptosomal subfractions to be located primarily in SPM-1 and SPM-2. Both Ca2+-ATPase and ATP-dependent Ca2+ uptake require calmodulin for maximal activity (KCm for ATPase = 60 nM; KCm for uptake = 50 nM). In the reconstituted membrane system, KCa was found to be 0.8 microM for Ca2+-ATPase and 0.4 microM for Ca2+ uptake. These results demonstrate for the first time the calmodulin requirements for the Ca2+ pump in SPM when Ca2+ ATPase and Ca2+ uptake are assayed under functionally coupled conditions. They suggest that calmodulin association with the membrane calcium pump is regulated by the level of free Ca2+ in the cytoplasm. The activation by calmodulin, in turn, regulates the cytosolic Ca2+ levels in a feedback process. These studies expand the calmodulin hypothesis of synaptic transmission to include activation of a high-affinity Ca2+ + Mg2+ ATPase as a regulator for cytosolic Ca2+.     

Effects of Substance P on Carbachol-Stimulated 45Ca2+ Uptake into Cultured Adrenal Chromaffin Cells

Substance P is known to modulate acetylcholine-induced catecholamine release from adrenal chromaffin cells. To investigate the mechanisms involved in this modulation, the present study examined the effects of substance P on net 45Ca2+ fluxes in cultures of bovine adrenal chromaffin cells. Two effects of substance P were observed: (1) Substance P inhibited carbachol-induced 45Ca2+ uptake and 45Ca2+ efflux and (2) substance P protected against desensitization of carbachol-induced 45Ca2+ uptake and 45Ca2+ efflux. Thus substance P modulates two other cholinergic responses, 45Ca2+ uptake and 45Ca2+ efflux, in a manner similar to its modulation of catecholamine release. The results also indicate that substance P's inhibition of net carbachol-induced 45Ca2+ uptake is due to inhibition of 45Ca2+ uptake rather than enhancement of 45Ca2+ efflux. Substance P almost completely inhibited carbachol-induced 45Ca2+ uptake in both Na+-containing and Na+-free media, suggesting that substance P can inhibit the uptake of 45Ca2+ induced by carbachol regardless of whether 45Ca2+ is taken up through voltage-sensitive or acetylcholine receptor-linked channels. However, substance P produced only a small inhibition of K+-induced 45Ca2+ uptake, indicating that substance P does not interact directly with voltage-sensitive Ca2+ channels. In addition, substance P's inhibition of carbachol-induced 45Ca2+ uptake was noncompetitive with respect to Ca2+, were unable to overcome substance P's inhibition of [3H]-norepinephrine ( [3H]NE) release. It is concluded that substance P does not interact directly with Ca2+ channels in bovine adrenal chromaffin cells.     

Transmitter-Like Basal and K+-Evoked Release of 3,4-Dihydroxyphenylalanine from the Striatum in Conscious Rats Studied by Microdialysis

Using microdialysis and HPLC, characteristics of the release of endogenous 3,4-dihydroxyphenylalanine (DOPA) from striatum in conscious rats were studied in comparison with those of 3,4-dihydroxyphenylethylamine (dopamine; DA). Purified L-aromatic amino acid decarboxylase (AADC) converted a putative peak of DOPA to DA. The retention time of DOPA differed from that of DA and major metabolites of DA and norepinephrine. The DOPA peak of dialysates comigrated with that of authentic DOPA when the pH of the HPLC buffer was modified. The ratio of the basal release of DOPA:DA was 1:2. 3-Hydroxybenzylhydrazine (NSD-1015; 100 mg/kg, i.p.), an AADC inhibitor, markedly increased the basal release of DOPA but produced no effect on DA. The basal release of DOPA was markedly decreased by alpha-methyl-p-tyrosine (200 mg/kg, i.p.), substantially tetrodotoxin (1 microM) sensitive, and Ca2+ (removal plus 12.5 mM Mg2+ addition) dependent. Fifty millimolar K+ released DOPA and this release was also Ca2+ dependent. These characteristics of the basal and evoked release of DOPA were similar to those of DA. The ratio of the evoked release of DOPA:DA was 1:3. These results indicate that DOPA is released under physiological conditions and by K(+)-induced depolarization in a manner similar to that for transmitter DA from striatum in freely moving rats.     

Kinetic Characterization of Ca2+ Transport in Synaptic Membranes

Lysed synaptosomal membranes were prepared from brain cortices of HA/ICR Swiss mice, and the ATP-stimulated Ca2+ uptake, Ca2+-stimulated Mg2+-dependent ATPase activity, and the Ca2+-stimulated acyl phosphorylation of these membranes were studied. The Km values for free calcium concentrations ([Ca2+]f) for these processes were 0.50 microM, 0.40 microM, and 0.31 microM, respectively. Two kinetically distinct binding sites for ATP were observed for the ATP-stimulated Ca2+ uptake and the Ca2+-stimulated Mg2+-ATPase activity. The high-affinity Km values for ATP for these two processes were 16.3 microM and 28 microM, respectively. These results indicate that the processes studied operate in similar physiological concentration ranges for the substrates [Ca2+]f and ATP under identical assay conditions and, further, that these processes may be functionally coupled in the membrane.     

Characterization of a High-Affinity Mg2+-Independent Ca2+-ATPase from Rat Brain Synaptosomal Membranes

A high-affinity Mg2+-independent Ca2+-ATPase (Ca2+-ATPase) has been differentiated from the Mg2+-dependent, Ca2+-stimulated ATPase (Ca2+,Mg2+-ATPase) in rat brain synaptosomal membranes. Using ATP as a substrate, the K0.5 of Ca2+ for Ca2+-ATPase was found to be 1.33 microM with a Km for ATP of 19 microM and a Vmax of 33 nmol/mg/min. Using Ca-ATP as a substrate, the Km for Ca-ATP was found to be 0.22 microM. Unlike Ca2+,Mg2+-ATPase, Ca2+-ATPase was not inhibited by N-ethylmaleimide, trifluoperazine, lanthanum, zinc, or vanadate. La3+ and Zn2+, in contrast, stimulated the enzyme activity. Unlike Ca2+, Mg2+-ATPase activity, ATP-dependent Ca2+ uptake was negligible in the absence of added Mg2+, indicating that the Ca2+ transport into synaptosomal endoplasmic reticulum may not be a function of the Ca2+-ATPase described. Ca2+-ATPase activity was not stimulated by the monovalent cations Na+ or K+. Ca2+, Mg2+-ATPase demonstrated a substrate preference for ATP and ADP, but not GTP, whereas Ca2+-ATPase hydrolyzed ATP and GTP, and to a lesser extent ADP. The results presented here suggest the high-affinity Mg2+-independent Ca2+-ATPase may be a separate form from Ca2+,Mg2+-ATPase. The capacity of Mg2+-independent Ca2+-ATPase to hydrolyze GTP suggests this protein may be involved in GTP-dependent activities within the cell.     

Modulation of GABA Receptor Binding by Ca+2

In frozen-thawed repeatedly washed rat cortical synaptic membranes, Ca2+ (1-5 mM) decreased the binding of [3H]muscimol whereas it increased the binding of [3H]gamma-aminobutyric acid (GABA). However, the binding of [3H]GABA was decreased by the same extent as the binding of [3H]muscimol when the membranes were incubated with baclofen (a selective ligand for the GABAB binding site) and Ca2+. Scatchard analysis of [3H]muscimol binding revealed that Ca2+ reduced the density of GABA binding sites without affecting the dissociation constant. Ca2+ was more potent than Ba2+, Mg2+ was ineffective, and the Ca2+ antagonist La3+ stimulated [3H]muscimol binding. The inhibition of [3H]muscimol binding by Ca2+ was not influenced by calmodulin (50 micrograms/ml), trifluoperazine (10(-5) M), verapamil (10(-6) M), quinacrine (10(-4) M), cordycepin (0.1 mM), leupeptin (20 microM), or soybean trypsin inhibitor (0.1 mg/ml). Moreover, the effect of Ca2+ was additive to that of GABA-modulin. These results indicate that Ca2+ decreases the number of GABAA binding sites while unveiling GABAB binding sites.     

Store-Operated Ca2+ Influx and Voltage-Gated Ca2+ Channels Coupled to Exocytosis in Pheochromocytoma (PC12) Cells

Microamperometry was used to monitor quantal catecholamine release from individual PC12 cells in response to raised extracellular K+ and caffeine. K+-evoked exocytosis was entirely dependent on Ca2+ influx through voltage-gated Ca2+ channels, and of the subtypes of such channels present in these cells, influx through N-type was primarily responsible for triggering exocytosis. L-type channels played a minor role in mediating K+-evoked secretion, whereas P/Q-type channels did not appear to be involved in secretion at all. Caffeine also evoked catecholamine release from PC12 cells, but only in the presence of extracellular Ca2+. Application of caffeine in Ca2+-free solutions evoked large, transient rises of [Ca2+]i, but did not trigger exocytosis. When Ca2+ was restored to the extracellular solution (in the absence of caffeine), store-operated Ca2+ influx was observed, which evoked exocytosis. The amount of secretion evoked by this influx pathway was far greater than release triggered by influx through L-type Ca2+ channels, but less than that caused by Ca2+ influx through N-type channels. Our results indicate that exocytosis may be regulated even in excitable cells by Ca2+ influx through pathways other than voltage-gated Ca2+ channels.     

ATP-Evoked Ca2+ Mobilisation and Prostanoid Release from Astrocytes: P2-Purinergic Receptors Linked to Phosphoinositide Hydrolysis

Astrocyte cultures prelabelled with either [3H]inositol or 45Ca2+ were exposed to ATP and its hydrolysis products. ATP and ADP, but not AMP and adenosine, produced increases in the accumulation of intracellular 3H-labelled inositol phosphates (IP), efflux of 45Ca2+, and release of thromboxane A2 (TXA2). Whereas ATP-stimulated 3H-IP accumulation was unaffected, its ability to promote TXA2 release was markedly reduced by mepacrine, an inhibitor of phospholipase A2 (PLA2). ATP-evoked 3H-IP production was also spared following treatment with the cyclooxygenase inhibitor, indomethacin. We conclude that ATP-induced phosphoinositide (PPI) breakdown and 45 Ca2+ mobilisation occurred in parallel with, if not preceded, the release of TXA2. Following depletion of intracellular Ca2+ with a brief preexposure to ATP in the absence of extracellular Ca2+, the release of TXA2 in response to a subsequent ATP challenge was greatly reduced when compared with control. These results suggest that mobilisation of cytosolic Ca2+ may be the stimulus for PLA2 activation and, thus, TXA2 release. Stimulation of alpha 1-adrenoceptors also caused PPI breakdown and 45 Ca2+ efflux but not TXA2 release. The effects of ATP and noradrenaline (NA) on 3H-IP accumulation were additive, but their combined ability to increase 45Ca2+ efflux was not. Interestingly, in the presence of NA, ATP-stimulated TXA2 release was reduced. Our data provide evidence that functional P2-purinergic receptors are present on astrocytes and that ATP is the first physiologically relevant stimulus found to initiate prostanoid release from these cells.     

Effects of Hypoxia on the Catecholamine Release, Ca2+ Uptake, and Cytosolic Free Ca2+ Concentration in Cultured Bovine Adrenal Chromaffin Cells

The purpose of the present study is to clarify the effects of hypoxia on catecholamine release and its mechanism of action. For this purpose, using cultured bovine adrenal chromaffin cells, we examined the effects of hypoxia on high (55 mM) K(+)-induced increases in catecholamine release, in cytosolic free Ca2+ concentration ([Ca2+]i), and in 45Ca2+ uptake. Experiments were carried out in media preequilibrated with a gas mixture of either 21% O2/79% N2 (control) or 100% N2 (hypoxia). High K(+)-induced catecholamine release was inhibited by hypoxia to approximately 40% of the control value, but on reoxygenation the release returned to control levels. Hypoxia had little effect on ATP concentrations in the cells. In the hypoxic medium, [Ca2+]i (measured using fura-2) gradually increased and reached a plateau of approximately 1.0 microM at 30 min, whereas the level was constant in the control medium (approximately 200 nM). High K(+)-induced increases in [Ca2+]i were inhibited by hypoxia to approximately 30% of the control value. In the cells permeabilized by digitonin, catecholamine release induced by Ca2+ was unaffected by hypoxia. Hypoxia had little effect on basal 45Ca2+ uptake into the cells, but high K(+)-induced 45Ca2+ uptake was inhibited by hypoxia. These results suggest that hypoxia inhibits high K(+)-induced catecholamine release and that this inhibition is mainly the result of the inhibition of high K(+)-induced increases in [Ca2+]i subsequent to the inhibition of Ca2+ influx through voltage-dependent Ca2+ channels.     

Prostaglandin E2 Activates Ca2+ Channels in Bovine Adrenal Chromaffin Cells

We have demonstrated that prostaglandin E2 (PGE2) treatment of bovine adrenal chromaffin cells results in a sustained elevation of intracellular Ca2+ concentration ([Ca2+]i) in these cells. Because the continued elevation of [Ca2+]i was dependent on extracellular Ca2+ concentration, it can be assumed that the PGE2-induced [Ca2+]i increase is due, at least in part, to an opening of membrane Ca2+ channels. In this study, we used electrophysiological methods to examine the mechanism of the PGE2-induced [Ca2+]i increase directly. Puff application of PGE2 to the external medium resulted in a prolonged depolarization in about half of the chromaffin cells examined. In whole-cell voltage-clamp recordings, an increase in inward current was observed over a 6-7 min period following bath application of PGE2 (greater than or equal to 10 microM), even in the absence of external Na+. This inward current was abolished when the recordings were made with the cells in a Ca2(+)-free medium, but it was not inhibited by Mn2+, a blocker of voltage-dependent Ca2+ channels. In cell-attached patch-clamp configuration, PGE2 produced an increase in the opening frequency of inward currents. The reversal potential of the PGE2-induced currents was about +40 mV, which is close to the reversal potential of the Ca2+ channel. The opening frequency was not affected by membrane potential changes. In inside-out patch-clamp configuration, inositol 1,4,5-trisphosphate (2 microM) added to the cytoplasmic side activated the Ca2(+)-channel currents, but PGE2 was ineffective when applied to the cytoplasmic side. These results suggest that PGE2 activates voltage-independent Ca2+ channels in chromaffin cells through a diffusible second messenger, possibly inositol 1,4,5-trisphosphate.     

Ca2+/Calmodulin Distinguishes Between Guanyl-5''-yl-Imidodiphosphate-and Opiate-Mediated Inhibition of Rat Striatal Adenylate Cyclase

The inhibition of adenylate cyclase from rat striatal plasma membranes by guanyl-5'-yl-imidodiphosphate [Gpp(NH)p] and morphine was compared to determine whether Gpp(NH)p-mediated inhibition accurately reflected hormone-mediated inhibition in this system. Inhibition of adenylate cyclase activity by Gpp(NH)p and morphine was examined with respect to temperature, divalent cation concentration, and the presence of Ca2+/calmodulin (Ca2+/CaM). Gpp(NH)p-mediated inhibition was dependent on the presence of Ca2+/CaM at 24 degrees C; the inhibition was independent of Ca2+/CaM at 18 degrees C; and inhibition could not be detected in the presence, or absence, of Ca2+/CaM at 30 degrees C. In contrast, naloxone-reversible, morphine-induced inhibition of adenylate cyclase was independent of both temperature and the presence of Ca2+/CaM. Mg2+ dose-response curves also reinforced the differences in the Ca2+/CaM requirement for Gpp(NH)p- and morphine-induced inhibition. Because Gpp(NH)p-mediated inhibition was independent of Ca2+/CaM at low basal activities (i.e., 18 degrees C, or below 1 mM Mg2+) and dependent on the presence of Ca2+/CaM at higher basal activities (24 degrees C, or above 1 mM Mg2+), the inhibitory effects of Gpp(NH)p were examined at 1 mM Mg2+ in the presence of 100 nM forskolin. Under these conditions, both Gpp(NH)p- and morphine-induced inhibition of adenylate cyclase were independent of Ca2+/CaM. The results demonstrate that the requirement for Ca2+/CaM to observe Gpp(NH)p-mediated inhibition depends on the basal activity of adenylate cyclase, whereas hormone-mediated inhibition is Ca2+/CaM independent under all conditions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ca2+-Surrogate Action of Pb2+ on Acetylcholine Release from Rat Brain Synaptosomes

The effect of lead ions on the release of acetylcholine (ACh) was investigated in intact and digitonin-permeabilized rat cerebrocortical synaptosomes that had been prelabeled with [3H]choline. Release of ACh was inferred from the release of total 3H label or by determination of [3H]ACh. Application of 1 microM Pb2+ to intact synaptosomes in Ca2(+)-deficient medium induced 3H release, which was enhanced by K+ depolarization. This suggests that entry of Pb2+ into synaptosomes and Pb2(+)-induced ACh release can be augmented by activation of the voltage-gated Ca2+ channels in nerve terminals. The lead-induced release of [3H]ACh was blocked by treatment of synaptosomes with vesamicol, which prevents uptake of ACh into synaptic vesicles without affecting its synthesis in the synaptoplasm. This indicates that Pb2+ selectively activates the release of a vesicular fraction of the transmitter with little or no effect on the leakage of cytoplasmic ACh. Application of 1-50 nM (EC50 congruent to 4 nM) free Pb2+ to digitonin-permeabilized synaptosomes elicited release of 3H label that was comparable with the release induced by 0.2-5 microM (EC50 congruent to 0.5 microM) free Ca2+. This suggests that Pb2+ triggers transmitter exocytosis directly and that it is a some 100 times more effective activator of exocytosis than is the natural agonist Ca2+.     

Characterization of Prostaglandin E2-Induced Ca2+ Mobilization in Single Bovine Adrenal Chromaffin Cells by Digital Image Microscopy

We recently reported that prostaglandin E2 (PGE2) stimulates phosphoinositide metabolism accompanied by an increase in intracellular free Ca2+ concentration ([Ca2+]i) in cultured bovine adrenal chromaffin cells. In the present study, temporal and spatial changes in [Ca2+]i induced by PGE2 in fura-2-loaded individual cells were investigated by digital image microscopy and were compared with those induced by nicotine and histamine. Image analysis of single cells revealed that responses to PGE2 showed asynchrony with the onset of [Ca2+]i changes. After a lag time of 10-30 s, PGE2-induced [Ca2+]i changes took a similar prolonged time course in almost all cells: a rapid rise followed by a slower decline to the basal level over 5 min. Few cells exhibited oscillations in [Ca2+]i. In contrast, nicotine and histamine induced rapid and transient [Ca2+]i changes, and these [Ca2+]i changes were characteristic of each stimulant. Whereas pretreatment of the cells with pertussis toxin (100 ng/ml, 6 h) did not block the response to any of these stimulants, treatment with 12-O-tetradecanoylphorbol 13-acetate (100 nM, 10 min) completely abolished [Ca2+]i changes elicited by PGE2 and histamine. In a Ca2(+)-free medium containing 3 mM EGTA, or in medium to which La3+ was added, the [Ca2+]i response to nicotine disappeared, but that to histamine was not affected significantly. Under the same conditions, the percentage of the cells that responded to PGE2 was reduced to 37% and the prolonged [Ca2+]i changes induced by PGE2 became transient in responding cells, suggesting that the maintained [Ca2+]i increase seen in normal medium is the result of a PGE2-stimulated entry of extracellular Ca2+. Whereas the organic Ca2(+)-channel blocker nicardipine inhibited [Ca2+]i changes by all stimulants at 10 microM, these [Ca2+]i changes were not affected by any of the organic Ca2(+)-channel blockers, i.e., verapamil, diltiazem, nifedipine, and nicardipine, at 1 microM, a concentration high enough to inhibit voltage-sensitive Ca2+ channels. These results demonstrate that PGE2 may promote Ca2+ entry with concomitant release of Ca2+ from intracellular stores and that the mechanism(s) triggered by PGE2 is apparently different from that by histamine or nicotine.