Role of Adenylate Cyclase in Presynaptic α2-Adrenoceptor- and μ-Opioid Receptor-Mediated Inhibition of [3H]Noradrenaline Release from Rat Brain Cortex Slices

Rat brain cortex slices, prelabelled with [3H]noradrenaline, were superfused and exposed to electrical biphasic block pulses (1 Hz; 12 mA, 4 ms) or to the Ca2+ ionophore A 23187 (10 microM) in the presence of 1.2 mM Ca2+. Forskolin (10 microM), 8-bromo-cyclic AMP (300 microM), and dibutyryl-cyclic AMP (300 microM) facilitated both the electrically evoked and A 23187-induced [3H]noradrenaline release, whereas the phosphodiesterase inhibitors 3-isobutyl-1-methylxanthine (IBMX, 300 microM) and 4-(3-cyclopentyloxy-4-methoxyphenyl)-2-pyrrolidone (ZK 62771, 30 microM) enhanced the electrically evoked release only. The inhibitory effects of clonidine (1 nM-1 microM) and the facilitatory effect of phentolamine (0.01-10 microM) on the electrically evoked [3H]noradrenaline release were strongly reduced in the presence of 8-bromo-cyclic AMP. Clonidine (1 microM) reduced and phentolamine (3 microM) enhanced A 23187-induced [3H]noradrenaline release, provided that the slices were simultaneously exposed to forskolin. The inhibitory effects of morphine (1 microM) and [D-Ala2-D-Leu5]enkephalin (DADLE, 0.3 microM), like that of the Ca2+ antagonist Cd2+ (15 microM), on the electrically evoked release of [3H]noradrenaline were not affected by 8-bromo-cyclic AMP. Moreover, morphine and DADLE did not inhibit A 23187-induced release in the absence or presence of forskolin. These data strongly suggest that in contrast to presynaptic mu-opioid receptors, alpha 2-adrenoceptors on noradrenergic nerve terminals are negatively coupled to adenylate cyclase and may thus reduce neurotransmitter release by inhibiting the feed-forward action of cyclic AMP on the secretion process.     

Regulation of calcium-dependent [3H]noradrenaline release from rat cerebrocortical synaptosomes by protein kinase C and modulation of the actin cytoskeleton.

The effects that active phorbol esters, staurosporine, and changes in actin dynamics, might have on Ca2+ -dependent exocytosis of [3H]-labelled noradrenaline, induced by either membrane-depolarizing agents or a Ca2+ ionophore, have been examined in isolated nerve terminals in vitro. Depolarization-induced openings of voltage-dependent Ca2+ channels with 30 mM KCl or 1 mM 4-aminopyridine induced limited exocytosis of [3H]noradrenaline, presumably from a readily releasable vesicle pool. Application of the Ca2+ ionophore calcimycin (10 microM) induced more extensive [3H]noradrenaline release, presumably from intracellular reserve vesicles. Stimulation of protein kinase C with phorbol 12-myristate,13-acetate increased release evoked by all secretagogues. Staurosporine (1 microM) had no effect on depolarization-induced release, but decreased ionophore-induced release and reversed all effects of the phorbol ester. When release was induced by depolarization, internalization of the actin-destabilizing agent DNAase I into the synaptosomes gave a slight increase in [3H]NA release and strongly increased the potentiating effect of the phorbol ester. In contrast, when release was induced by the Ca2+ ionophore, DNAase I had no effect, either in the absence or presence of phorbol ester. The results indicate that depolarization of noradrenergic rat synaptosomes induces Ca2+ -dependent release from a releasable pool of staurosporine-insensitive vesicles. Activation of protein kinase C increases this release by staurosporine-sensitive mechanisms, and destabilization of the actin cytoskeleton further increases this effect of protein kinase C. In contrast, ionophore-induced noradrenaline release originates from a pool of staurosporine-sensitive vesicles, and although activation of protein kinase C increases release from this pool, DNAase I has no effect and also does not change the effect of protein kinase C. The results support the existence of two functionally distinct pools of secretory vesicles in noradrenergic CNS nerve terminals, which are regulated in distinct ways by protein kinase C and the actin cytoskeleton.     

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.     

Enhancement of Stimulation-Evoked Catecholamine Release from Cultured Bovine Adrenal Chromaffin Cells by Forskolin

Acetylcholine (ACh) increased cyclic AMP levels in cultured bovine chromaffin cells with a peak effect at 1 min after the addition. Pretreatment with forskolin (0.3 microM) enhanced the ACh-evoked cyclic AMP increase. The catecholamine (CA) release induced by ACh was enhanced by forskolin, but forskolin alone did not enhance the CA release. The effect of forskolin increased dose-dependently up to 1 microM, but decreased at higher concentrations. Dibutyryl cyclic AMP (DBcAMP) also enhanced ACh-evoked CA release, but the effect was less potent than that of forskolin. Forskolin enhanced both [3H]norepinephrine ([3H]NE) and endogenous CA release evoked by 30 mM K+ from cells that were preloaded with [3H]NE. The effects of forskolin were substantial when CA release was evoked with low concentrations of ACh or excess K+, but decreased with higher concentrations of the stimulants. Forskolin also enhanced the CA release induced by ionomycin and veratrine, or by caffeine in Ca2+-free medium. The potentiation by forskolin of the ACh-evoked CA release was manifest in low Ca2+ concentrations in the medium, but decreased when Ca2+ concentration was increased. These results suggest that cyclic AMP may play a role in the modulation of CA release from chromaffin cells.     

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.     

ATP receptor. A putative receptor-operated channel in PC-12 cells.

External ATP induces [3H] dopamine [( 3H]DA) release in rat pheochromocytoma cells (PC-12 cells). The ATP-induced release is a saturable process with half-effective concentration of EC50 = 80 microM. ADP is a poor secretagogue of [3H]DA (one-sixth of ATP) and AMP is devoid of secretory capabilities. Adenosine and the non-hydrolyzable analogues of ATP, AppNHp and AppCp are ineffective as inducers of [3H]DA, release, or as inhibitors of the ATP-induced [3H]DA release. The most potent antagonist of ATP-induced release is Coomassie Blue (IC50 = 25 microM), compared to ADP beta S (IC50 = 500 microM). The overall rank order of potency is ATP greater than ADP much greater than AMP greater than adenosine, which is characteristic of the P2-purinergic receptor. ATP-induced secretion is absolutely Ca2+ dependent, indicating an exocytotic process and is independent of Mg2+ (up to 2 mM) suggesting that the active species is not ATP4-. (a) The ATP-induced 45Ca2+ influx into the cells is in good correlation to ATP induction of release (IC50 = 80 and 90 microM, respectively) and is carried over to ADP which has a diminished ability to induce both release and 45Ca2+ influx. (b) Divalent cations (Ba2+ greater than Sr2+ greater than Ln3+ greater than Mn2+) replace Ca2+ and support ATP-induced release similar to their effectiveness in supporting bradykinin- and K+ (50 mM)-induced release in PC-12 cells (Weiss, C., Sela, D., and Atlas, D. (1990) Neurosci. Lett. 119, 241-245). Combined together the absolute requirement of [Ca2+]ex for release, inhibition of release by Gd3+ (IC50 = 100 microM), Ni2+, and Co2+ (IC50 = 1 mM), and support of release by Ba2+, Sr2+, and Mn2+, we suggest that ATP induces Ca2+ entry via ligand-operated Ca2+ channels as previously suggested for ATP in smooth muscle cells (Benham, C.D., Bolton, T.B., Byren, N.G., and Large, W.A. (1987) J. Physiol. (Lond.) 387, 473-488). No significant inhibition by 1 microM verapamil, 10 microM nifedipine, or 2 mM Cd2+ argues against ATP activation of voltage-dependent Ca2+ channels as similarly shown for ATP-induced [3H]noradrenaline release (Inoue, K., Nakazawa, K., Fujimoro, K., and Takanaka, A. (1989) Neurosci. Lett. 106, 294-299). Thus, the widely distributed ATP receptor might play an essential role in Ca2+ homeostasis of the cell by introducing Ca2+ into the cell via specific ligand-gated Ca2+ channels.     

Effect of Organic and Inorganic Calcium Channel Blockers on γ-Amino-n-Butyiic Acid Release Induced by Monensin and Veratrine in the Absence of External Calcium

The effects of two organic Ca2+ antagonists (verapamil and nitrendipine) and of two inorganic Ca2+ channel blockers (Co2+ and ruthenium red) on the Na+-dependent release of gamma-amino-n-butyric acid (GABA) triggered by veratrine and monensin in the absence of external Ca2+ were studied in mouse brain synaptosomes. Ca2+-independent release of GABA stimulated by the Na+ channel activator veratrine was inhibited with micromolar concentrations of verapamil and nitrendipine. In contrast, GABA release induced by the Na+ ionophore monensin was insensitive to the organic Ca2+ antagonists. Verapamil also failed to modify A23187-stimulated release of GABA in the presence of Ca2+ but inhibited high K+-induced release of the transmitter. Co2+ partially diminished veratrine-induced release but did not change monensin-induced release. Releasing responses to monensin and veratrine were insensitive to ruthenium red, which inhibited the Ca2+-dependent component of GABA release evoked by high K+ depolarization. These data demonstrate that the mechanism of inducing GABA release is different for veratrine and monensin, as evidenced by their differing sensitivities to inhibition by Ca2+ channel antagonists and organic Ca2+ blockers. It is concluded that voltage-sensitive Ca2+ channels of the presynaptic membrane are not involved in the inhibitory action of Ca2+ antagonists on the Na+-dependent, Ca2+-independent mechanism of GABA release.     

Depolarization Increases Chloride-Dependent Glutamate Sequestration in Synaptic Membranes of Rat Cerebral Cortex

To assess the functions of Cl- -dependent glutamate "binding" (Cl- -dependent glutamate uptake) in synaptic membranes, possible effects of depolarization on the uptake were examined. When rat cerebral cortical slices were preincubated with depolarizing agents such as veratrine (7 micrograms/ml), 10 microM aconitine, 56 mM K+, and 50 microM monensin, [3H]glutamate uptake by the crude synaptic membranes, which were subsequently prepared from the pretreated slices, was increased by 60-85%. Stimulation of the glutamate uptake by predepolarization was dependent on Na+ but not on Ca2+. The bindings of gamma-[3H]aminobutyric acid and 5-[3H]hydroxytryptamine were not significantly affected by the predepolarization. Veratrine pretreatment increased the maximal density of the glutamate uptake sites without affecting the affinity for glutamate. Several characteristics of the uptake sites increased by the veratrine pretreatment coincided with those of Cl- -dependent glutamate uptake sites. Na+-dependent glutamate binding (Na+-dependent glutamate uptake) to the membranes was not affected by pretreatment with veratrine. The content of endogenous glutamate and the noninulin space in the membrane fractions were not changed by the predepolarization. The increase in the glutamate uptake induced by pretreatment with high K+ was reversible: it returned to the control level after a second incubation of the slices in control medium. These results suggest that the Cl- -dependent glutamate sequestration system in synaptic membranes is regulated by the membrane potential.     

Morphine and Enkephalins Potently Inhibit [3H]Noradrenaline Release from Rat Brain Cortex Synaptosomes: Further Evidence for a Presynaptic Localization of μ-Opioid Receptors

Synaptosomes prepared from rat cerebral cortex and labeled with [3H]noradrenaline (NA) were superfused with calcium-free Krebs-Ringer-bicarbonate medium and exposed to 10 mM K+ plus 0.1 mM Ca2+ so that [3H]NA release was induced. 6,7-Dihydroxy-N,N-dimethyl-2-aminotetralin (TL-99) strongly inhibited synaptosomal K+-induced [3H]NA release (EC50 = 5-10 nM) by activating alpha 2-adrenoceptors. Release was also inhibited (maximally by 40-50%) by morphine (EC50 = 5-10 nM), [Leu5]enkephalin (EC50 = approximately 300 nM), [D-Ala2,D-Leu5]enkephalin (DADLE), and Tyr-D-Ala-Gly-(NMe)Phe-Gly-ol (DAGO) (EC50 values = approximately 30 nM). In contrast to the mu-selective opioid receptor agonists morphine and DAGO, the highly delta-selective agonist [D-Pen2,D-Pen5]enkephalin (1 microM) did not affect [3H]-NA release. Furthermore, the inhibitory effect of DADLE, an agonist with affinity for both delta- and mu-opioid receptors, was antagonized by low concentrations of naloxone. The findings strongly support the view that, like alpha 2-adrenoceptors, mu-opioid receptors mediating inhibition of NA release in the rat cerebral cortex are localized on noradrenergic nerve terminals.     

Verapamil: influence upon basal and stimulated rat growth hormone and prolactin release in vitro

Verapamil is an organic calcium antagonist which is believed to prevent the passage of calcium (Ca2+) across the cell membrane into the cell. In a rat pituitary perifusion-immunoprecipitation system, verapamil (50 microM) prevents the inhibitory effect of increased extracellular Ca2+ (5.4 mM) on basal and stimulated release of stored, prelabeled [3H]GH and [3H]PRL. [3H]GH release from pituitary explants perifused in standard medium (GIBCO Minimum Essential Medium: 1.8 mM Ca2+) is transiently increased by 50 microM verapamil while [3H]PRL release is suppressed. With continued exposure to 50 microM verapamil, [3H]GH release rates fall below (89.8 +/- 2.1% of base) preverapamil levels while [3H]PRL release rates simply remain suppressed (48.2 +/- 7.3% of base). With 250 microM verapamil, poststimulatory inhibition of [3H]GH release occurs more quickly, and after its withdrawal rebound release of both GH and PRL occur. Inhibition of [3H]GH release by 25 nM somatostatin (SRIF) and post-SRIF rebound [3H]GH release is not prevented by 50 microM verapamil. The early, rapid [3H]GH release phase of 1 mM dibutyryl cyclic AMP (dbcAMP) stimulation is potentiated by verapamil pretreatment, but only if the verapamil is continued during dbcAMP stimulation. Potassium (21 mM K+)-stimulated release of both 3H-labeled hormones is inhibited after similar pretreatment 50 microM verapamil. Conclusions: (a) verapamil antagonizes the inhibitory effects of increased extracellular Ca2+ on basal or dbcAMP-stimulated [3H]GH and [3H]PRL release; (b) in standard medium (1.8 mM Ca2+), 50 microM verapamil increases basal [3H]GH release suggesting either a direct effect or an antagonism of 1.8 mM extracellular Ca2+; (c) although verapamil-sensitive Ca2+ movement is not necessary for dbcAMP stimulation of [3H]GH release, verapamil potentiates dbcAMP-stimulated release; (d) because verapamil also inhibits K+-stimulated [3H]GH and [3H]PRL release, these observations support previous suggestions that K+- and dbcAMP-stimulated rapid hormone release occurs from different intracellular sites; and (e) because verapamil does not prevent any phase of SRIF action and since these two agents differentially alter K+- and cAMP-stimulated release, their mechanisms of action must partially differ.     

Pharmacological characterization of the effects of methylmercury and mercuric chloride on spontaneous noradrenaline release from rat hippocampal slices

The environmental contaminants methylmercury (MeHg) and mercuric chloride (HgCl2) stimulated the spontaneous release of [3H]noradrenaline ([3H]NA) from hippocampal slices in a time- and concentration-dependent manner. Both MeHg and HgCl2 were similarly potent, with an EC50 of 88.4 microM and 75.9 microM, respectively. The releasing effects of MeHg and HgCl2 increased in the presence of desipramine, showing that the mechanism does not involve reversal of the transmitter transporter, and were completely blocked by reserpine preincubation, indicating a vesicular origin of [3H]NA release. The voltage-gated Na+ channel blocker tetrodotoxin (TTX) did not affect the response to mercury compounds. [3H]NA release elicited by MeHg was partially dependent on extracellular Ca2+, since it decreased significantly in a Ca2+-free EGTA-containing medium whereas HgCl2 induced a release of [3H]NA independent of extracellular Ca2+. Neither Ca2+-channels blockers, cobalt chloride (CoCl2) and (omega-conotoxin-GVIA, nor the Na+/Ca2+-exchanger inhibitor benzamil reduced MeHg-evoked [3H]NA release. Moreover, thapsigargin or caffeine, endoplasmic reticulum Ca2+-depletors, did not modify metal-evoked [3H]NA release, whereas ruthenium red, which inhibits the mitochondrial Ca2+ transport, decreased the effect of both MeHg and HgCl2. All these data indicate that, in hippocampal slices, mercury compounds release [3H]NA from the vesicular pool by a mechanism involving Ca2+ mobilization from mitochondrial stores.     

Intracellular calcium and hormone secretion in clonal AtT-20/D16-16 anterior pituitary cells

Intracellular ionized Ca2+ concentration was measured in clonal mouse anterior pituitary tumor cells with the fluorescent Ca2+ indicator Quin-2. In control physiological solution, free cytoplasmic Ca2+ concentration was found to be 139 +/- 11 nM. Replacement of 50 mM NaCl by 50 mM KCl in the extracellular fluid caused a 29 mV depolarization and a 4.2-fold increase in the concentration of free cytoplasmic Ca2+. Under comparable depolarizing conditions, a specific influx of 2.66 nmole of 45Ca2+ per mg protein was detected 1 min after addition of high K+, accompanied by a marked increase in the initial rate of beta-endorphin secretion. In the absence of external Ca2+, depolarization by K+ produced little or no increase in either intracellular free Ca2+ or hormone release. Incubation of AtT-20/D16-16 cells in the secretagogue norepinephrine led to a depolarization accompanied by an increase in spontaneous action potential frequency and a marked elevation in cytosolic Ca2+ concentration. Exposure of cells to somatostatin, an inhibitor of hormone release, led to only transient decreases in burst frequency and no significant reduction in intracellular Ca2+ levels. These results indicate that in addition to intracellular Ca2+, other factors also control secretory activity in AtT-20/D16-16 anterior pituitary cells.     

Adult astroglia is competent for Na+/Ca2+ exchanger-operated exocytotic glutamate release triggered by mild depolarization

Glutamate release induced by mild depolarization was studied in astroglial preparations from the adult rat cerebral cortex, that is acutely isolated glial sub-cellular particles (gliosomes), cultured adult or neonatal astrocytes, and neuron-conditioned astrocytes. K+ (15, 35 mmol/L), 4-aminopyridine (0.1, 1 mmol/L) or veratrine (1, 10 micromol/L) increased endogenous glutamate or [3H]D-aspartate release from gliosomes. Neurotransmitter release was partly dependent on external Ca2+, suggesting the involvement of exocytotic-like processes, and partly because of the reversal of glutamate transporters. K+ increased gliosomal membrane potential, cytosolic Ca2+ concentration [Ca2+]i, and vesicle fusion rate. Ca2+ entry into gliosomes and glutamate release were independent from voltage-sensitive Ca2+ channel opening; they were instead abolished by 2-[2-[4-(4-nitrobenzyloxy)phenyl]ethyl]isothiurea (KB-R7943), suggesting a role for the Na+/Ca2+ exchanger working in reverse mode. K+ (15, 35 mmol/L) elicited increase of [Ca2+]i and Ca2+-dependent endogenous glutamate release in adult, not in neonatal, astrocytes in culture. Glutamate release was even more marked in in vitro neuron-conditioned adult astrocytes. As seen for gliosomes, K+-induced Ca2+ influx and glutamate release were abolished by KB-R7943 also in cultured adult astrocytes. To conclude, depolarization triggers in vitro glutamate exocytosis from in situ matured adult astrocytes; an aptitude grounding on Ca2+ influx driven by the Na+/Ca2+ exchanger working in the reverse mode.     

The release of [3H]GABA formed from [3H]glutamate in rat hippocampal slices

to compare the storage and release of endogenous GABA, of [3H]GABA formed endogenously from glutamate, and of exogenous [14C]GABA, hippocampal slices were incubated with 5 microCi/ml [3,4-3H]1-glutamate and 0.5 microCi/ml [U-14C]GABA and then were superfused in the presence or absence of Ca+ with either 50 mM K+ or 50 microM veratridine. Endogenous GABA was determined by high performance liquid chromatography which separated labeled GABA from its precursors and metabolites. Exogenous [14C]GABA content of the slices declined spontaneously while endogenous GABA and endogenously formed [3H]GABA stayed constant over a 48 min period. In the presence of Ca+ 50 mM K+ and in the presence or absence of Ca2+ veratridine released exogenous [14C]GABA more rapidly than endogenous or endogenously formed [3H]GABA, the release of the latter two occurring always in parallel. The initial specific activity of released exogenous [14C]GABA was three times, while that of endogenously formed [3H]GABA was only 50% higher than that in the slices. There was an excess of endogenous GABA content following superfusion with 50 mM K+ and Ca2+, which did not occur in the absence of Ca2+ or after veratridine. The observation that endogenous GABA and [3H]GABA formed endogenously from glutamate are stored and released in parallel but differently from exogenous labelled GABA, suggests that exogenous [3H] glutamate can enter a glutamate pool that normally serves as precursor of GABA.     

Mechanism of vascular relaxation by thaligrisine: functional and binding assays

In the present study we examine the mechanism by which thaligrisine, a bisbenzyltetrahydroisoquinoline alkaloid, inhibits the contractile response of vascular smooth muscle. The work includes functional studies on rat isolated aorta and tail artery precontracted with noradrenaline or KCl. In other experiments rat aorta was precontracted by caffeine in the presence or absence of extracellular Ca2+. In order to assess whether thaligrisine interacts directly with calcium channel binding sites or with alpha-adrenoceptors we examined the effect of the alkaloid on [3H]-(+)-cis diltiazem, [3H]-nitrendipine and [3H]-prazosin binding to cerebral cortical membranes. The functional studies showed that the alkaloid inhibited in a concentration-dependent manner the contractile response induced by depolarization in rat aorta (IC50 = 8.9+/-2.9 microM, n=5) and in tail artery (IC50 = 3.04+/-0.3 microM, n=6) or noradrenaline induced contraction in rat aorta (IC50 = 23.0+/-0.39 microM, n=9) and in tail artery (IC50 = 3.8+/-0.9 microM, n=7). In rat aorta, thaligrisine concentration-dependently inhibited noradrenaline-induced contraction in Ca2+-free solution (IC50 = 13.3 microM, n=18). The alkaloid also relaxed the spontaneous contractile response elicited by extracellular calcium after depletion of noradrenaline-sensitive intracellular stores (IC50 = 7.7 microM, n=4). The radioligand receptor-binding study showed that thaligrisine has higher affinity for [3H]-prazosin than for [3H]-(+)-cis-diltiazem binding sites, with Ki values of 0.048+/-0.007 microM and 1.5+/-1.1 microM respectively. [3H]-nitrendipine binding was not affected by thaligrisine. The present work provides evidence that thaligrisine shows higher affinity for [3H]-prazosin binding site than [3H]-(+)-cis-diltiazem binding sites, in contrast with tetrandrine and isotetrandrine that present similar affinity for both receptors. In functional studies thaligrisine, acted as an alpha1-adrenoceptor antagonist and as a Ca2+ channel blocker, relaxing noradrenaline or KCl-induced contractions in vascular smooth muscle. This compound specifically inhibits the refilling of internal Ca2+-stores sensitive to noradrenaline, by blocking Ca2+-entry through voltage-dependent Ca2+-channels.     

Calcium permeability changes and neurotransmitter release in cultured brain neurons. II. Temporal analysis of neurotransmitter release

The coupling between depolarization-induced calcium entry and neurotransmitter release was studied in rat brain neurons in culture. The endogenous dopamine content of the cells was determined by high performance liquid chromatography utilizing electrochemical detection. The amount of dopamine in unstimulated cells was found to be about 16 ng/mg of protein. Depolarization of the neurons by elevated K+ caused a Ca2+-dependent release of dopamine from the cells. Following 1 min of depolarization, the cellular dopamine content and the amount of [3H]dopamine in cells preloaded with the radioactive transmitter were reduced by 35%. The release of [3H]dopamine by the neurons was measured at 1.5-6-s intervals by a novel rapid dipping technique. Depolarization in the presence of Ca2+ (1.8 mM) enhanced the rate of neurotransmitter release by 90-fold (0.072 +/- 0.003 s-1) over the basal release in the presence of Ca2+. The evoked release consisted of a major rapidly terminating phase (t1/2 = 9.6 s) which comprised about 40% of the neurotransmitter content of the cells and a subsequent slower efflux (t1/2 = 575 s) which was observed during following prolonged depolarization. Predepolarization of the cells in the absence of extracellular Ca2+ did not affect the kinetics of the evoked release. The fast evoked release could be re-elicited in the cells after 20 min "rest" in reference low K+ buffer. The effects of varying the extracellular Ca2+ concentrations on the kinetic parameters of the evoked release were measured. The amount of neurotransmitter released during the fast kinetic phase was very sensitive to the external Ca2+ (from 0% in the absence of Ca2+ to 40% of the neurotransmitter content at Ca2+ 0.3 mM). The rate constant of the fast release did not depend on the extracellular Ca2+, whereas the rate constant of the slow release increased from 0.0004 +/- 0.0001 s-1 at 0.4 mM Ca2+ to 0.0012 +/- 0.0002 s-1 at 0.8 mM Ca2+. The fast evoked release was inhibited by verapamil in a concentration-dependent manner. By contrast, verapamil enhanced the basal and the slow release independent of the presence of Ca2+. Both fast and slow phases of the evoked release were blocked by Co2+. Addition of Co2+ within the first 6 s after the onset of depolarization inhibited the fast release but failed to do so when added later on.(ABSTRACT TRUNCATED AT 400 WORDS)     

Multiple mechanisms of transmitter release evoked by "pathologically" elevated extracellular [K+]: involvement of transporter reversal and mitochondrial calcium

The release of [3H]GABA evoked by depolarization with various concentrations of KCl was studied using superfused rat cerebrocortex synaptosomes. Elevating [K+] produced release of [3H]GABA over basal which was increasingly less dependent on external Ca2+ but more sensitive to the GABA transporter blocker SKF 100330 A. Accordingly, the sensitivity to clostridial toxins of the depolarization-evoked amino acid release was inversely correlated to the concentration of KCl used. However, at 50 mM K+, one-third of the stimulated release remained which was external Ca2+-independent but insensitive to SKF 100330 A. This release was prevented by BAPTA, thapsigargin or dantrolene; it also was inhibited by blocking in mitochondria the ATP production with oligomycin, the H+-dependent Ca2+ uniporter with RU 360, the Na+/Ca2+ exchanger with CGP 37157 or by lowering extraterminal [Na+]. In fluorescence experiments with fura-2/AM, 50 mM K+ (in Ca2+ free medium) caused elevation of cytosolic [Ca2+] that was sensitive to thapsigargin or CGP 37157; these compounds produced partially additive effects. When exocytosis was monitored with the fluorescent dye acridine orange, the fluorescence elicited by 50 mM K+ was sensitive to thapsigargin or CGP 37157, which produced additive effects, and to low-Na+ media. To conclude, extracellular K+ concentrations occurring in the CNS in certain pathological conditions provoke GABA release by mechanisms different from classical exocytosis. These include carrier-mediated release and internal Ca2+-dependent exocytosis; in the latter, mitochondrial Ca2+ seems to play a primary role.     

Protein Kinase C Activation Enhances K+-Stimulated Endogenous Dopamine Release from Rat Striatal Synaptosomes in the Absence of an Increase in Cytosolic Ca2+

The possibility that protein kinase C modulates neurotransmitter release in brain was investigated by examining the effects of 12-O-tetradecanoylphorbol 13-acetate (TPA) on Ca2+ transport and endogenous dopamine release from rat striatal synaptosomes. TPA (0.16 and 1.6 microM) significantly increased dopamine release by 24 and 33%, respectively, after a 20-min preincubation with TPA followed by 60 s of depolarization with 30 mM KCl. Depolarization-induced 45Ca2+ uptake, measured simultaneously with dopamine release, was not significantly increased by TPA. Neither 45Ca2+ uptake nor dopamine release was altered under resting conditions. When the time course of K+-stimulated 45Ca2+ uptake and dopamine release was examined, TPA (1.6 microM) enhanced dopamine release after 15, 30, and 60 s, but not 1, 3, or 5 s, of depolarization. A slight increase in 45Ca2+ uptake after 60 s of depolarization was also seen. The addition of 30 mM KCl to synaptosomes which had been preloaded with the Ca2+-sensitive fluorophore fura-2 increased the cytosolic free Ca2+ concentration ([Ca2+]i) from 445 nM to 506 nM after 10 s of depolarization and remained elevated after 60 s. TPA had no effect on [Ca2+]i under depolarizing or resting conditions. Replacing extracellular Ca2+ with 100 microM EGTA reduced K+-stimulated (60 s) endogenous dopamine release by 53% and decreased [Ca2+]i to 120 nM. In Ca2+-free medium, 30 mM KCl did not produce an increase in the [Ca2+]i. TPA (1.6 microM) did not alter the [Ca2+]i under resting or depolarizing conditions, but did increase K+-stimulated dopamine release in Ca2+-free medium.(ABSTRACT TRUNCATED AT 250 WORDS)     

Glucose-, calcium- and concentration-dependence of acetylcholine stimulation of insulin release and ionic fluxes in mouse islets.

Mouse islets were used to define the glucose-dependence and extracellular Ca2+ requirement of muscarinic stimulation of pancreatic beta-cells. In the presence of a stimulatory concentration of glucose (10 mM) and of Ca2+, acetylcholine (0.1-100 microM) accelerated 3H efflux from islets preloaded with myo-[3H]inositol. It also stimulated 45Ca2+ influx and efflux, 86Rb+ efflux and insulin release. In the absence of Ca2+, only 10-100 microM-acetylcholine mobilized enough intracellular Ca2+ to trigger an early but brief peak of insulin release. At a non-stimulatory concentration of glucose (3 mM), 1 microM- and 100 microM-acetylcholine increased 45Ca2+ and 86Rb+ efflux in the presence and absence of extracellular Ca2+. However, only 100 microM-acetylcholine marginally increased 45Ca2+ influx and caused a small, delayed, stimulation of insulin release, which was abolished by omission of Ca2+. At a maximally effective concentration of glucose (30 mM), 1 microM- and 100 microM-acetylcholine increased 45Ca2+ influx and efflux only slightly, but markedly amplified insulin release. Again, only 100 microM-acetylcholine mobilized enough Ca2+ to trigger a peak of insulin release in the absence of Ca2+. The results thus show that only high concentrations of acetylcholine (greater than or equal to 10 microM) can induce release at low glucose or in a Ca2+-free medium. beta-Cells exhibit their highest sensitivity to acetylcholine in the presence of Ca2+ and stimulatory glucose. Under these physiological conditions, the large amplification of insulin release appears to be the result of combined effects of the neurotransmitter on Ca2+ influx, on intracellular Ca2+ stores and on the efficiency with which Ca2+ activates the releasing machinery.     

Cadmium-induced insulin release does not involve changes in intracellular handling of calcium

A possible interaction between Cd2+ and Ca2+ as a component in Cd2+-induced insulin release was investigated in beta cells isolated from obese hyperglycemic mice. The glucose stimulated Cd2+ uptake was dependent on the concentration of sugar. This uptake was sigmoidal with a Km for glucose of about 5 mM and was suppressed by both 50 microM of the voltage-activated Ca2+ channel blocker D-600 and 12 mM Mg2+. In the presence of 8 mM glucose 5 microM Cd2+ evoked a prompt and sustained stimulatory response, corresponding to about 3-fold of the insulin release obtained in the absence of the ion. Whereas 5 microM Cd2+ was without effect on the glucose-stimulated 45Ca efflux in the presence of extracellular Ca2+, 40 microM inhibited it. At a concentration of 5 microM, Cd2+ had no effect on the resting membrane potential or the depolarization evoked by either glucose or K+. In the absence of extracellular Ca2+ there was only a modest stimulation of 45Ca efflux by 5 microM Cd2+. Studies of the ambient free Ca2+ concentration maintained by permeabilized cells also indicate that 5 microM Cd2+ do not mobilize intracellularly bound Ca2+ to any great extent. On the contrary, at this concentration, Cd2+ even suppressed inositol 1,4,5-trisphosphate (IP3)-induced Ca2+ release. The present study suggests that Cd2+ stimulates insulin release by a direct mechanism which does not involve an increase in cytoplasmic free Ca2+ concentration.