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)     

Differential Calcium Dependence Between the Release of Endogenous Dopamine and Noradrenaline from Rat Brain Synaptosomes

The characteristics of the release of endogenous dopamine and noradrenaline from rat brain synaptosomes were studied using HPLC with an electrochemical detector. The spontaneous release of dopamine and noradrenaline was inhibited by approximately 50-60% in a Ca2(+)-free medium or a 100 microM La3(+)-containing medium. Also, the high-K+ (30 mM)-evoked release of dopamine and noradrenaline was inhibited by approximately 50-60% in a Ca2(+)-free medium or a 100 microM La3(+)-containing medium. From these results, the ratio of the Ca2(+)-dependent component to the total release of noradrenaline seemed to be similar to that of dopamine. On the other hand, 20 microM La3+ or 1 microM diltiazem inhibited both the spontaneous and 30 mM K(+)-evoked release of dopamine by approximately 50-60% but inhibited neither the spontaneous nor the 30 mM K(+)-evoked release of noradrenaline. The K(+)-evoked rise in intrasynaptosomal Ca2+ concentration was mostly blocked in Ca2(+)-free medium or 100 microM La3(+)-containing medium but was only partially blocked by 20 microM La3+ or 1 microM diltiazem. These data indicate alternative possibilities in that the Ca2(+)-dependent release of noradrenaline might be less sensitive to a change of intracellular Ca2+ concentration than that of dopamine and that the calcium channels directly involved in the noradrenaline release may be more resistant to diltiazem and La3+ than those involved in the dopamine release.     

Stimulation of Muscarinic Acetylcholine Receptors Increases Synaptosomal Free Calcium Concentration by Protein Kinase-Dependent Opening of L-Type Calcium Channels

In synaptosomes prepared from rat cerebral cortex, free cytosolic calcium concentration ([Ca2+]i) was measured using the fluorescent dye fura-2. Incubation of fura-2-loaded synaptosomes with carbachol increased [Ca2+]i in a dose-dependent manner (1-1,000 microM), with a maximum response of 22 +/- 2% at approximately 100 microM and an EC50 (calculated concentration producing 50% of the maximum response) of 30 microM. The effect of carbachol (100 microM) on [Ca2+]i was antagonised by atropine, but not by hexamethonium (10 microM). The calculated concentration of atropine needed for 50% inhibition (IC50) was 260 nM. The rise in [Ca2+]i produced by carbachol was reduced in the absence of extrasynaptosomal Ca2+ and effectively blocked by the L-type calcium channel blocker nifedipine (with an IC50 of 29 nM). The response to carbachol was reduced if the synaptosomes were preincubated with the protein kinase inhibitors H7 [1-(5-isoquinolinylsulfonyl)-2- methylpiperazine] (from 17% in the solvent control to 4%) and staurosporine (from 20% in the solvent control to 3%). These results show that stimulation of muscarinic acetylcholine receptors in synaptosomes increases [Ca2+]i by protein kinase-dependent activation of 1,4-dihydropyridine-sensitive calcium channels.     

Is Inositol Bisphosphate the Product of A23187 and Carbachol-Mediated Polyphosphoinositide Breakdown in Synaptosomes?

Synaptosomes have been isolated from rat cerebral cortex and labelled in vitro with [32P]orthophosphate and myo-[2-3H]inositol. Subsequent addition of the Ca2+ ionophore A23187 in the presence of 2 mM extrasynaptosomal Ca2+ raised intrasynaptosomal free [Ca2+] to greater than 2 microM from a resting level of 200 nM and led to rapid breakdown of polyphosphoinositides. This was accompanied by a small increase in the level of inositol monophosphate, greatly enhanced accumulation in inositol bisphosphate, but no detectable increase in inositol trisphosphate. Depolarising (25 mM) extrasynaptosomal K+ produced a smaller increase in intrasynaptosomal free [Ca2+] (to around 400 nM) and a proportional increase in inositol bisphosphate radioactivity. Carbachol (1 mM) alone elicited only limited polyphosphoinositide breakdown and inositol mono- and bisphosphate formation, but this was greatly increased in the presence of 25 mM K+. The effect of carbachol in the presence of depolarising K+ was time- and dose-dependent and was antagonised by atropine (10 microM). There was no detectable accumulation of inositol trisphosphate in the presence of carbachol, K+, or carbachol plus K+, even after short (30 s.) incubations. The lack of inositol trisphosphate accumulation does not appear to result from rapid formation of inositol tetrakisphosphate or from enhanced breakdown of the trisphosphate in synaptosomes.     

Nature of Extrasynaptosomal Accumulation of Endogenous Adenosine Evoked by K+ and Veratridine

When rat brain synaptosomes were incubated for 10 min at 37 degrees C, basal accumulation of adenosine in the medium was 66 pmol/mg of protein. An elevated K+ level (24 mM) evoked an additional accumulation of 200 pmol/mg of protein, and 50 microM veratridine evoked 583 pmol of adenosine accumulation/mg of protein. K+- and veratridine-evoked accumulation of adenosine did not arise from microsomal or mitochondrial contaminants of the synaptosomal preparation, because purified microsomes and mitochondria did not exhibit evoked accumulation of adenosine in the medium. K+-evoked accumulation of extrasynaptosomal adenosine was Ca2+-dependent, whereas veratridine-evoked accumulation of adenosine was increased in Ca2+-free medium. In the presence of alpha,beta-methylene ADP and GMP, which inhibit ecto-5'-nucleotidase, conversion of added ATP and AMP to adenosine was inhibited by 90% in synaptosomal suspensions. However, inhibition of ecto-5'-nucleotidase only reduced basal extrasynaptosomal accumulation of adenosine by 74%, veratridine-evoked accumulation of adenosine by 46%, and K+-evoked accumulation by 33%. Most of the basal accumulation of extrasynaptosomal adenosine appears to be derived from released nucleotide, probably ATP, but about half of the veratridine-evoked accumulation of adenosine and most of the K+-evoked accumulation may arise from adenosine released in its own right, rather than from a released nucleotide.     

Studies of blocking mechanisms of carbachol-induced polyphosphoinositide turnover in rat cortical synaptosomes by neuroactive drugs

1. The incorporation of 32Pi into 4 phospholipids of rat cortical synaptosomes was altered in the presence of carbachol (1 mM), viz. a decrease of phosphatidylinositol-4,5-bisphosphate and phosphatidyl-4-phosphate by 34 and 21%, and an increase of phosphatidylinositol and phosphatidic acid by 52 and 96% of basal controls respectively. 2. The IC30 values calculated from the dose-response curves for drugs affecting carbachol-induced 32Pi incorporation into these phospholipids, and [3H]QNB binding to the cortical synaptosomes were similar for the typical antimuscarinic agents (i.e. atropine, pirenzepine and trihexyphenidyl), and tricyclic antidepressants (i.e. amitriptyline, doxepin and imipramine) studied. 3. The IC30 values obtained for drugs affecting carbachol-induced 32Pi incorporation into these phospholipids, and high potassium-induced 45Ca2+-uptake by this preparation were similar for neuroselective calcium channel blockers (i.e. cinnarizine and flunarizine) studied. 4. Our results suggest that the neuroactive drugs studied can either act at, or beyond the receptor level, perhaps on the availability of calcium ion, to block carbachol-induced polyphosphoinositide turnover in rat cortical synaptosomes.     

Rapid Release of [3H]Dopamine from Median Eminence and Striatal Synaptosomes

Release of preaccumulated, tritium-labeled dopamine ([3H]DA) from preparations of isolated nerve terminals (synaptosomes) of rat median eminence (ME) and corpus striatum (CS) was examined over short time intervals (1-20 s). In both preparations, basal efflux of [3H]DA was linear with time. Depolarization with high K+ resulted in an initial rapid release of [3H]DA which stabilized by 20 s, whereas veratridine elicited an increased rate of release over basal levels that was linear over the first 20 s. The calculated rate constants of release for both the initial phase of K+- and the veratridine-stimulated release were approximately threefold greater in CS than in ME synaptosomes. The major component of the high K+-induced release of [3H]DA from both synaptosome preparations increased as a graded function of [Ca2+]o. However, a smaller component, independent of external Ca2+, existed in both ME and CS synaptosomes. Increasing the [Mg2+] in the external solution resulted in a right shift of both the [K+]o and the [Ca2+]o dose-response curves, consistent with actions of Mg2+ on screening surface membrane charges and blocking voltage-dependent Ca2+ channels. In all studies, steady-state uptake of the [3H]DA was about twofold greater into CS than into ME synaptosomes. Moreover, the fraction of incorporated [3H]DA released by stimulation from the CS was much greater than that released from ME synaptosomes. These data are consistent with differences between these two types of dopaminergic terminals with respect to packaging and/or distribution of the accumulated neurotransmitter in intraneuronal pools, as well as marked differences in the apparent kinetics of DA release.     

The Effect of Neurocatin on Protein Phosphorylation in Striatal Synaptosomes from Rat Brain

Abstract: Neurocatin, a neuroregulatory factor isolated from mammalian brain, is a powerful affector of protein phosphorylation in rat striatal synaptosomes. Two major synaptosomal phosphoproteins of ～80 and ～60 kDa, possibly synapsin I and tyrosine hydroxylase, were especially sensitive to neurocatin. Immunoprecipitation experiments confirmed that the 60-kDa protein is the enzyme tyrosine hydroxylase. At low concentrations of neurocatin (to ～7.5 ng/100 μl of suspension), incorporation of ³²P orthophosphate into these proteins increased with increasing neurocatin concentration. At 7.5 ng of neurocatin, incorporation of the label into the two proteins increased by 22 and 26%, respectively. Concentrations of neurocatin >7.5 ng/100 μl caused progressive decrease in incorporation of ³²P into many synaptosomal proteins; by a concentration of neurocatin of ～45 ng/100 μ/l, the level of ³²P incorporation into many proteins was ≤70% of control. The effects of neurocatin on synaptosomal protein phosphorylation were also dependent on the time of incubation. At a constant concentration of ～7.5 ng/100 μl of neurocatin, increased incorporation of 32P into many proteins was measurable within 0.5 min and was maximal by 1 min. Incubation times >2.0 min, showed progressive decrease in ³²P incorporation. Removing extrasynaptosomal Ca²⁺ with EGTA attenuated the increased ³²P incorporation induced by low neurocatin concentrations, suggesting that calcium plays a role in neurocatin-induced phosphorylation of rat striatal synaptosomal proteins. The reduced incorporation of label induced by high neurocatin concentrations, however, was not calcium dependent. The effects of neurocatin on the level of ³²P incorporation into proteins were observed only in intact synaptosomes, consistent with this compound acting through receptors on the plasma membrane.     

Phorbol Ester Enhancement of Neurotransmitter Release from Rat Brain Synaptosomes

Neurotransmitter release from rat brain synaptosomes was measured following pretreatment with various phorbol esters. Ca2+-dependent, evoked neurotransmitter release was increased by phorbol esters that were active in stimulating protein kinase C. Protein kinase C activation was demonstrated by increased incorporation of 32P into 87-kilodalton phosphoprotein, a specific substrate for that kinase. Inactive phorbol esters had no effect on neurotransmitter release or on the phosphorylation of 87-kilodalton phosphoprotein. The increased release was observed in either crude cortical synaptosomal fractions (P2) or purified cortical synaptosomal fractions. The enhancement was found for all neurotransmitters (norepinephrine, acetylcholine, gamma-aminobutyric acid, serotonin, dopamine, and aspartate), all brain regions (cerebral cortex, hippocampus, and corpus striatum), and all secretagogues (elevated extracellular K+ level, veratridine, or A23187) examined. It was also observed at all calcium concentrations present during stimulation of release. The phorbol ester enhancement of Ca2+-dependent release occurred whether or not calcium was present during pretreatment. These results indicate that stimulation of protein kinase C leads to an enhanced sensitivity of the stimulus-secretion coupling processes to calcium within the nerve terminal. The results support the possibility that presynaptic activation of protein kinase C modulates nerve terminal neurotransmitter release in the CNS.     

Free calcium concentrations in the nerve endings in the brain of rats with spontaneous hypertension

Fluorescent indicator Quin-2 was used for the determination of free calcium (Ca2+in) in synaptosomes incubated in the normal medium and media where sodium is replaced by potassium or choline. At external calcium concentration of 1 mM, Ca2+in in all three media was 20-30% higher in synaptosomes of spontaneously hypertensive rats (SHR) than in control animals. At external calcium concentration of 5 mM, the increase in Ca2+in values induced by K+-depolarization in sodium- or choline-containing media was 50-80% higher in synaptosomes of SHR. These differences are suggested to be the basis for the mechanism of increased peripheral chain activity in the sympathetic nervous system in primary hypertension.     

Dopamine Receptors on Adrenal Chromaffin Cells Modulate Calcium Uptake and Catecholamine Release

The presence of dopamine-containing cells in sympathetic ganglia, i.e., small, intensely fluorescent cells, has been known for some time. However, the role of dopamine as a peripheral neurotransmitter and its mechanism of action are not well understood. Previous studies have demonstrated the presence of D2 dopamine receptors on the surface of bovine adrenal chromaffin cells using radioligand binding methods and dopamine receptor inhibition of catecholamine release from perfused adrenal glands. In the present study, we provide evidence confirming a role of dopamine receptors as inhibitory modulators of adrenal catecholamine release from bovine chromaffin cell cultures and further show that the mechanism of modulation involves inhibition of stimulated calcium uptake. Apomorphine gave a dose-dependent inhibition (IC50 = 1 microM) of 45Ca2+ uptake stimulated by either nicotine (10 microM) or membrane depolarization with an elevated K+ level (60 mM). This inhibition was reversed by a series of specific (including stereospecific) dopamine receptor antagonists: haloperidol, spiperone, sulpiride, and (+)-butaclamol, but not (-)-butaclamol. In addition, the calcium channel agonist Bay K 8644 was used to stimulate uptake of 45Ca2+ into chromaffin cells, and this uptake was also inhibited by the dopamine receptor agonist apomorphine. The combined results suggest that dopamine receptors on adrenal chromaffin cells alter Ca2+ channel conductance, which, in turn, modulates catecholamine release.     

Correlation of Rates of Calcium Entry and Release of Endogenous Norepinephrine in Rat Brain Region Synaptosomes

Voltage-dependent 45Ca2+ uptake and endogenous norepinephrine (NE) release were measured simultaneously in synaptosomes isolated from rat hypothalamus, brainstem, and cerebellum at 1, 3, 5, 15, and 30 s. In synaptosomes depolarized by 125 mM KCl, 45Ca2+ uptake and NE release exhibited fast and slow components. Rates of NE release and 45Ca2+ uptake were fastest from 0 to 1 s. NE release and 45Ca2+ uptake rates from 1 to 5 s were less than 15% of 0-1 s rates. Both resting (5 mM KCl) and depolarization-induced (125 mM KCl) NE release paralleled 45Ca2+ uptake from 1 to 30 s. Voltage-dependent NE release was approximately 1% and 2% of total synaptosomal NE content at 1- and 30-s measurement intervals, respectively, and did not differ between the three brain regions studied. Calcium and potassium dependence studies showed that NE release was stimulated by increased potassium and that depolarization-induced NE release was dependent on the presence of external calcium. These results show that calcium-dependent NE release from synaptosomes is correlated with calcium entry. Both processes exhibit fast and slow temporal components.     

Dopamine receptor regulation of Ca2+ levels in individual isolated nerve terminals from rat striatum: comparison of presynaptic D1-like and D2-like receptors

We have directly observed the effects of activating presynaptic D1-like and D2-like dopamine receptors on Ca2+ levels in isolated nerve terminals (synaptosomes) from rat striatum. R-(+)-SKF81297, a selective D1-like receptor agonist, and (-)-quinpirole, a selective D2-like receptor agonist, induced increases in Ca2+ levels in different subsets of individual striatal synaptosomes. The SKF81297- and quinpirole-induced effects were blocked by R-(+)-SCH23390, a D1-like receptor antagonist, and (-)-sulpiride, a D2-like receptor antagonist, respectively. SKF81297- or quinpirole-induced Ca2+ increases were inhibited following blockade of voltage-gated calcium channels or sodium channels. In a larger subset of synaptosomes, quinpirole decreased baseline Ca2+. Quinpirole also inhibited veratridine-induced increases in intrasynaptosomal Ca2+ level. Immunostaining confirmed the presynaptic expression of D1, D5, D2 and D3 receptors, but not D4 receptors. The array of neurotransmitter phenotypes of the striatal nerve endings expressing D1, D5, D2 or D3 varied for each receptor subtype. These results suggest that presynaptic D1-like and D2-like receptors induce increases in Ca2+ levels in different subsets of nerve terminals via Na+ channel-mediated membrane depolarization, which, in turn, induces the opening of voltage-gated calcium channels. D2-like receptors also reduce nerve terminal Ca2+ in a different but larger subset of synaptosomes, consistent with the predominant presynaptic action of dopamine in the striatum being inhibitory.     

Calcium permeability of synaptosomes induced by alpha-latrotoxin

The paper deals with characteristics of ionic alpha-latrotoxin-induced permeability of rat brain synaptosomes. It has been shown that the addition of alpha-latrotoxin to synaptosomes in the Ca2+-containing media resulted in an extensive and rapid uptake of 45Ca2+ in synaptosomes. alpha -Latrotoxin was not able to enhance the 22Na+ and 86Rb+ uptake or efflux in the Ca2+-containing and Ca2+- and Mg2+-free media. The dye di-O-C3 was used to monitor the membrane potential changes as a consequence of alpha-latrotoxin treatment of synaptosomes. It has been found that alpha-latrotoxin increased synaptosomal fluorescence in the Ca2+-containing media, but failed to induce any increase of fluorescence in Ca2+- and Mg2+-free media. It has been also shown that the calcium uptake induced by alpha-latrotoxin depends on free calcium concentration in synaptosomes. Toxin-induced calcium flows are shown to be of the vector character.     

Glutamine and Aspartate Loading of Synaptosomes: A Reevaluation of Effects on Calcium-Dependent Excitatory Amino Acid Release

Guinea-pig cerebral cortical synaptosomes were preincubated for 60 min with 100 microM D-aspartate, L-aspartate, or L-glutamate. The total D- plus L-aspartate content of the synaptosomal fraction increased to 235%, 195%, or 164%, respectively, of the control. Despite this no increase was seen in the very low KCl evoked, Ca2+-dependent release of aspartate. Preincubation with the three amino acids changed the synaptosomal glutamate content to 78% (D-aspartate), 149% (L-aspartate), or 168% (L-glutamate) of control. However there was no statistically significant effect of these preincubations on the extent of Ca2+-dependent glutamate release. Thus the Ca2+-dependent release of aspartate and glutamate is not determined by the total synaptosomal content of these amino acids. The addition of 0.1-0.5 mM glutamine to the incubation caused a massive appearance of glutamate in the extrasynaptosomal medium. Analysis of specific activities showed that glutamine was hydrolysed directly by an extrasynaptosomal glutaminase, and that intrasynaptosomal glutamate was predominantly labelled by uptake of this glutaminase-derived glutamate. No increase was seen in the extent of Ca2+-dependent release of glutamate (by fluorimetry) either after preincubation with glutamine or in the continued presence of glutamine. Thus we are unable to confirm reports that glutamine expands the transmitter pool of glutamate. The extrasynaptosomal glutaminase activity in the synaptosomal preparation was inhibited by Ca2+ and activated by phosphate. Identical kinetics were obtained with "free" brain mitochondria, confirming the origin of the glutamine-derived glutamate.     

Non-genomic effect of L-triiodothyronine on calmodulin-dependent synaptosomal protein phosphorylation in adult rat cerebral cortex

The present study was undertaken to examine calmodulin-dependent effect of thyroid hormones (THs) on synaptosomal protein phosphorylation in mature rat brain. Effect of L-triiodothyronine (L-T3) on in vitro protein phosphorylation was measured in a hypotonic lysate of synaptosomes prepared from adult male rat cerebral cortex, incubated in presence and absence of calcium ion (Ca2+) and calmodulin. L-T3 significantly enhanced incorporation of 32P into synaptosomal proteins as compared to basal level of phosphorylation in the presence of Ca2+ and calmodulin. Under these conditions, increase in protein phosphorylation was 47, 74 and 52% for 10 nM, 100 nM and 1 microM L-T3, respectively. Chelation of Ca2+ using ethylene glycol-bis (2-aminoethylether)-N, N, N', N'-tetraacetic acid (EGTA) inhibited the effects of Ca2+/calmodulin on TH-stimulated protein phosphorylation levels. This study suggests that a high proportion of L-T3-stimulated protein phosphorylation involves Ca2+/calmodulin-dependent pathways in adult rat cerebrocortical synaptosomes.     

Diacylglycerol-Induced Stimulation of Neurotransmitter Release from Rat Brain Striatal Synaptosomes

These studies were undertaken to test the hypothesis that alterations in phosphatidylinositol metabolism can modulate neurotransmitter release in the central nervous system. The effects of 1,2-diacylglycerols (DAGs) on dopamine release in the rat central nervous system were determined by measuring dopamine release from rat striatal synaptosomes in response to two DAGs (sn-1,2-dioctanoylglycerol and 1-oleoyl-2-acetylglycerol) that can activate protein kinase C and one DAG (deoxydioctanoylglycerol) that does not activate this kinase. Dioctanoylglycerol and 1-oleoyl-2-acetylglycerol, at a concentration of 50 micrograms/ml, stimulated the release of labeled dopamine from striatal synaptosomes by 35-50 and 17%, respectively. Dioctanoylglycerol-induced release was also demonstrated for endogenous dopamine. In contrast, deoxydioctanoylglycerol (50 micrograms/ml) did not stimulate dopamine release. Dioctanoylglycerol-induced dopamine release was independent of external calcium concentration, indicating a utilization of internal calcium stores. Dioctanoylglycerol (50 micrograms/ml) also produced a 38% increase in labeled serotonin release from striatal synaptosomes. The addition of dioctanoylglycerol to the striatal supernatant fraction increased protein kinase C activity. These results are consistent with the concept that an increase in phosphatidylinositol metabolism can stimulate neurotransmitter release in the central nervous system via an increase in DAG concentration. The data suggest an involvement of protein kinase C in the DAG-induced release, but other sites for DAG action are also possible.     

Calcium dependent regulation of catecholamine and serotonin metabolism in human neuroblastoma cells

Three human neuroblastoma cell lines were shown to have markedly different contents of catecholamines and serotonin. Two of the cell lines (CHP-134 and IMR-5) have higher levels of dopamine and its metabolites, while CHP-404 cells have higher levels of serotonin and its metabolites. Each cell line responded to the addition of D,L-2-amino-5-phosphonovalerate, an agent which increases plasma membrane permeability to Ca2+ (Pastuszko and Wilson, 1988; with striking changes in the metabolism of the neurotransmitters. These changes were dependent on the extracellular calcium concentration and include activation of dopamine synthesis (tyrosine hydroxylase), increased levels of dihydroxyphenylacetic acid and increased formation of N-methylated dopamine derivatives. Catabolism of serotonin to 5-hydroxyindole acetic acid was inhibited while that to 5-hydroxytryptophol was stimulated. These data clearly identify several important sites for regulation of neurotransmitter metabolism by calcium. The mechanisms, direct or indirect, by which the enzyme activities are modulated by calcium remain to be established.     

Effects of lowering extracellular and cytosolic pH on calcium fluxes, cytosolic calcium levels, and transmitter release in presynaptic nerve terminals isolated from rat brain

We examined the effects of extracellular and intracellular pH changes on the influx of radioactive 45Ca, the concentration of ionized Ca (pCai) as monitored with the Ca-sensitive fluorescent indicator fura-2, and the efflux of dopamine in presynaptic nerve endings (synaptosomes) isolated from rat brain corpora striata and preloaded with [3H]dopamine. Cytosolic pH (pHi) was monitored by loading the synaptosomes with the H+-sensitive fluorescent indicator 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein (BCECF) (see Nachshen, D. A., and P. Drapeau, 1988, Journal of General Physiology, 91:289-303). An abrupt decrease of the pH of the external medium, from 7.4 to 5.5, produced a slow decrease of pHi (over a 5-min period) from an initial value of 7.2 to a steady state level of approximately 5.8. When 20 mM acetate was present in acidic media, pHi dropped as fast as could be measured (within 2 s) to a level similar to that reached (more slowly) in the absence of acetate. It was therefore possible to lower pHi over short time periods to different levels depending on whether or not acetate was present upon extracellular acidification. Extracellular acidification to pH 5.5 (in the absence of acetate) had no significant effect on pCai and dopamine release over a 30-s period (pHi = 6.4). Acidification in the presence of acetate lowered pHi to 5.8 without affecting pCai, but dopamine efflux increased approximately 20-fold. This increase in basal dopamine release was also observed in the absence of extracellular Ca. Thus, intraterminal, but not extracellular, acidification could stimulate the efflux of dopamine in a Ca-independent manner. The high Q10 (3.6) of acid-stimulated dopamine efflux in the presence of nomifensine (which blocks the dopamine carrier) was consistent with an activation of vesicular dopamine release by H+. When synaptosomes were both depolarized for 2 s in high-K (77.5 mM) solutions and acidified (in the absence of acetate), there was a parallel block of 45Ca entry and evoked dopamine release (50% block at pH 6.0 with 0.2 mM external Ca). When acetate was included in the acidic media to further reduce pHi, Ca entry remained blocked, but evoked dopamine release was increased. Therefore, extracellular, but not cytosolic, acidification inhibited the release of dopamine by blocking voltage-gated Ca channels. The stimulation by cytosolic acidification of both basal and evoked dopamine release suggests that vesicular release in resting and depolarized synaptosomes was directly activated by cytoplasmic H+.     

Histamine stimulated synaptosomal Ca2+ uptake through activation of calcium channels

Histamine stimulated Ca2+ uptake in synaptosomes was completely inhibited by the slow Ca2+ channel antagonists verapamil, cinnarizine and flunarizine, and slightly inhibited by nifedipine and diltiazem. Ca2+ uptake in synaptosomes depolarized or predepolarized with varying K+ concentrations was increased by histamine, in both conditions, until 30mM K+. At higher K+ concentrations histamine was not able to alter K+ effects in either conditions. 30mM K+ stimulated uptake of Ca2+ in the absence or presence of histamine was not inhibited by verapamil and diltiazem. However nifedipine slightly inhibited K+ and K+ +histamine effects. 3-Isobutyl-1-methyl-xanthine and dibutyryl cyclicAMP potentiated (10%) the uptake of Ca2+ in synaptosomes induced by histamine. Dibutyryl cyclicAMP alone however decreased the basal Ca2+ uptake in a concentration-dependent manner. Verapamil, but not diltiazem, antagonized the effects elicited by 3-isobutyl-1-methyl-xanthine and dibutyryl cyclicAMP in the presence of histamine. The data suggest that the increase in synaptosomal Ca2+ uptake induced by histamine is mediated by the activation of the voltage sensitive calcium channels, and possibly a cyclicAMP-dependent protein kinase phosphorylation can modulate the opening of Ca2+ channels.