Differential release of amino acids, neuropeptides, and catecholamines from isolated nerve terminals.

We have investigated transmitter release from small and large dense-core vesicles in nerve terminals isolated from guinea pig hippocampus. Small vesicles are found in clusters near the active zone, and large dense-core vesicles are located at ectopic sites. The abilities of Ca2+ channel activation and uniform elevation of Ca2+ concentration (with ionophores) to evoke secretion of representative amino acids, catecholamines, and neuropeptides were compared. For a given increase in Ca2+ concentration, ionophore was less effective than Ca2+ channel activation in releasing amino acids, but not in releasing cholecystokinin-8. Titration of the average Ca2+ concentration showed that the Ca2+ affinity for cholecystokinin-8 secretion was higher than that for amino acids. Catecholamine release showed intermediate behavior. It is concluded that neuropeptide release is triggered by small elevations in the Ca2+ concentration in the bulk cytoplasm, whereas secretion of amino acids requires higher elevations, as produced in the vicinity of Ca2+ channels.

Monitoring extracellular glutamate in hippocampal slices with a microsensor.

The direct local assessment of glutamate in brain slices may improve our understanding of glutamatergic neurotransmission significantly. However, an analytical technique that monitors glutamate directly in brain slices is currently not available. Most recording techniques either monitor derivatives of glutamate or detect glutamate that diffuses out of the slice. Microsensors provide a promising solution to fulfill this analytical requirement. In the present study we have implanted a 10 microm diameter hydrogel-coated microsensor in the CA1 area of hippocampal slices to monitor extracellular glutamate levels. The influence of several pharmacological agents, which facilitate glutamate release from neurons or astrocytes, was investigated to explore the applicability of the microsensor. It was observed that KCl, veratradine, alpha-latrotoxine (LTX), DL-threo-beta-benzyloxyaspartate (dl-TBOA) and L-cystine rapidly increased the extracellular glutamate levels. As far as we know this is the first study in which a microsensor is applied to monitor dynamic changes of glutamate in brain slices and in our opinion this type of research may contribute greatly to improve our understanding of the physiology of glutamatergic neurotransmission.

Rab3a is involved in transport of synaptic vesicles to the active zone in mouse brain nerve terminals.

The rab family of GTP-binding proteins regulates membrane transport between intracellular compartments. The major rab protein in brain, rab3A, associates with synaptic vesicles. However, rab3A was shown to regulate the fusion probability of synaptic vesicles, rather than their transport and docking. We tested whether rab3A has a transport function by analyzing synaptic vesicle distribution and exocytosis in rab3A null-mutant mice. Rab3A deletion did not affect the number of vesicles and their distribution in resting nerve terminals. The secretion response upon a single depolarization was also unaffected. In normal mice, a depolarization pulse in the presence of Ca(2+) induces an accumulation of vesicles close to and docked at the active zone (recruitment). Rab3A deletion completely abolished this activity-dependent recruitment, without affecting the total number of vesicles. Concomitantly, the secretion response in the rab3A-deficient terminals recovered slowly and incompletely after exhaustive stimulation, and the replenishment of docked vesicles after exhaustive stimulation was also impaired in the absence of rab3A. These data indicate that rab3A has a function upstream of vesicle fusion in the activity-dependent transport of synaptic vesicles to and their docking at the active zone.

Ca2+-stimulated, Mg2+-independent ATP hydrolysis and the high affinity Ca2+-pumping ATPase. Two different activities in rat kidney basolateral membranes.

The Mg2+-dependency of Ca2+-induced ATP hydrolysis is studied in basolateral plasma membrane vesicles from rat kidney cortex in the presence of CDTA and EGTA as Mg2+- and Ca2+-buffering ligands. ATP hydrolysis is strongly stimulated by Mg2+ with a Km of 13 microM in the absence or presence of 1 microM free Ca2+. At free Mg2+ concentrations of 1 microM and lower, ATP hydrolysis is Mg2+-independent, but is strongly stimulated by submicromolar Ca2+ concentrations (Km = 0.25 microM, Vmax = 24 mumol Pi/h per mg protein). The Ca2+-stimulated ATP hydrolysis strongly decreases at higher Mg2+ concentrations. The Ca2+-stimulated Mg2+-independent ATP hydrolysis is not affected by calmodulin or trifluoperazine and shows no specificity for ATP over ADP, ITP and GTP. In contrast, at high Mg2+ concentrations calmodulin and trifluoperazine affect the high affinity Ca2+-ATPase activity significantly and ATP is the preferred substrate. Control studies on ATP-dependent Ca2+-pumping in renal basolaterals and on Ca2+-ATPase in erythrocyte ghosts suggest that the Ca2+-pumping enzyme requires Mg2+. In contrast, a role of the Ca2+-stimulated Mg2+-independent ATP hydrolysis in active Ca2+ transport across basolateral membranes is rather unlikely.

1 alpha, 25-Dihydroxy-vitamin D-3 regulates ATP-dependent calcium transport in basolateral plasma membranes of rat enterocytes.

Basolateral plasma membrane vesicles of rat small intestinal epithelium accumulate calcium through an ATP-dependent pumping system. The activity of this system is highest in duodenum and decreases towards the ileum. This distribution along the intestinal tract is similar as the active calcium absorption capacity of intact intestinal epithelial segments. ATP-dependent calcium uptake in basolateral membrane vesicles from duodenum and ileum increased significantly after repletion of young vitamin D-3-deficient rats with 1 alpha,25-dihydroxy-vitamin D-3. Ca2+ -ATPase activity in duodenal basolateral membranes increased to the same extend as ATP-dependent calcium transport, but (Na+ + K+)-ATPase activity remained unaltered.

Kinetic properties of Na+/Ca2+ exchange in basolateral plasma membranes of rat small intestine.

The presence of an Na+/Ca2+ exchange system in basolateral plasma membranes from rat small intestinal epithelium has been demonstrated by studying Na+ gradient-dependent Ca2+ uptake and the inhibition of ATP-dependent Ca2+ accumulation by Na+. The presence of 75 mM Na+ in the uptake solution reduces ATP-dependent Ca2+ transport by 45%, despite the fact that Na+ does not affect Ca2+-ATPase activity. Preincubation of the membrane vesicles with ouabain or monensin reduces the Na+ inhibition of ATP-dependent Ca2+ uptake to 20%, apparently by preventing accumulation of Na+ in the vesicles realized by the Na+-pump.l It was concluded that high intravesicular Na+ competes with Ca2+ from intravesicular Ca2+ binding sites. In the presence of ouabain, the inhibition of ATP-dependent Ca2+ transport shows a sigmoidal dependence on the Na+ concentration, suggesting cooperative interaction between counter transport of at least two sodium ions for one calcium ion. The apparent affinity for Na+ is between 15 and 20 mM. Uptake of Ca2+ in the absence of ATP can be enhanced by an Na+ gradient (Na+ inside greater than Na+ outside). This Na+ gradient-dependent Ca2+ uptake is further stimulated by an inside positive membrane potential but abolished by monensin. The apparent affinity for Ca2+ of this system is below 1 microM. In contrast to the ATP-dependent Ca2+ transport, there is no significant difference in Na+ gradient-dependent Ca2+ uptake between basolateral vesicles from duodenum, midjejunum and terminal ileum. In duodenum the activity of ATP-driven Ca2+ uptake is 5-times greater than the greater than the Na+/Ca2+ exchange capacity but in the ileum both systems are of equal potency. Furthermore, the Na+/Ca2+ exchange mechanism is not subject to regulation by 1 alpha, 25-dihydroxy vitamin D-3, since repletion of vitamin D-deficient rats with this seco-steroid hormone does not influence the Na+/Ca2+ exchange system while it doubles the ATP-driven Ca2+ pump activity.

Dissociation between Ca2+-ATPase and alkaline phosphatase activities in plasma membranes of rat duodenum.

The presence of Ca2+-ATPase activities with high-affinity sites for Ca2+ in brush border as well as basolateral plasma membranes of rat duodenal epithelium has been reported previously (Ghijsen, W.E.J.M. and van Os, C.H. (1979) Nature 279, 802-803). Since both plasma membranes contain alkaline phosphatase (EC 3.1.3.1), which also can be stimulated by Ca2+, the substrate specificity of Ca2+-induced ATP-hydrolysis has been studied to determine whether or not alkaline phosphatase and Ca2+-ATPase are two distinct enzymes. In basolateral fragments, the rate of Ca2+-dependent ATP-hydrolysis was greater than that of ADP, AMP and p-nitrophenylphosphate at Ca2+ concentrations below 25 muM. At 0.2 mM Ca2+ the rates of ATP, ADO, AMO and p-nitrophenylphosphate hydrolysis were not significantly different. In brush border fragments the rates of ATP, ADP and AMP hydrolysis were identical at low Ca2+, but at 0.2 mM Ca2+, Ca2+-induced hydrolysis of ADO and AMO was greater than either ATP or p-nitrophenylphosphate. Alkaline phsophatase in brush border and basolateral membranes was inhibited by 75% after addition of 2.5 mM theophylline. Ca2+-stimulated ATP hydrolysis at 1 muM Ca2+ was not sensitive to theophylline in basolateral fragments while the same activity in brush border fragments was totally inhibited. At 0.2 mM Ca2+, Ca2+-induced ATP hydrolysis in both basolateral and brush border membranes was sensitive to theophylline. Oligomycin and azide had no effect on Ca2+-stimulated ATP hydrolysis, either at low or at high Ca2+ concentrations. Chlorpromazine fully inhibited Ca2+-stimulated ATP hydrolysis in basolateral fragments at 5 muM Ca2+, while it had no effect in brush border fragments. From these results we conclude that, (i) Ca2+-ATPase and alkaline phosphatase are two distinct enzymes, (ii) high-affinity Ca2+-ATPase is exclusively located in basolateral plasma membranes, (iii) alkaline phosphatase activity, present on both sides of duodenal epithelium is stimulated slightly by low Ca2+ concentrations, but this Ca2+-induced activity is inhibited by theophylline and shows no specificity with respect to ATP, ADP or AMP.

ATP-dependent calcium transport and its correlation with Ca2+ -ATPase activity in basolateral plasma membranes of rat duodenum.

Isolated basolateral plasma membrane vesicles from rat duodenum epithelial cells exhibit ATP-dependent calcium-accumulation and Ca2+ -dependent ATPase activity. Calcium accumulation stimulated by ATP is prevented by the calcium ionophore A23187, inhibited 80% by 0.1 mM orthovanadate but is not effected by oligomycin. Calcium accumulation is not observed with the substrate beta-gamma-(CH2)-ATP, ADP and p-nitrophenyl phosphate. Kinetic studies reveal an apparent Km of 0.2 microM Ca2+ and a Vmax of 5.3 nmol Ca2+/min per mg protein for the ATP-dependent calcium-uptake system. Calmodulin and phenothiazines have no effect on calcium accumulation in freshly prepared membranes, but small effects are inducible after a wash with a 5 mM EGTA. The kinetic parameters of Ca2+ -ATPase are: Km = 0.25 microM Ca2+ and Vmax = 19.2 nmol Pi/min per mg protein. Three techniques, osmotic shock, treatment with Triton X-100 or the channel-forming peptide alamethicin, reveal that about 40% of the vesicles are resealed. Assuming that half of the resealed vesicles have an inside-out orientation, the Vmax of ATP-dependent calcium uptake amounts to 25 nmol Ca2+/min per mg protein and of the Ca2+ -ATPase to 23 nmol Pi/min per mg protein. The close correlation between kinetic parameters of Ca2+ -ATPase and ATP-dependent calcium-transport strongly suggests that both systems are expressions of a Ca2+ -pump located in duodenal basolateral plasma membranes.

Separation of basolateral plasma membranes from smooth endoplasmic reticulum of the rat enterocyte by zonal electrophoresis on density gradients.

Basolateral plasma membranes of rat small intestinal epithelium were purified by density gradient centrifugation followed by zonal electrophoresis on density gradients. Crude basolateral membranes were obtained by centrifugation in which the marker enzyme, (Na+ + K+)-ATPase, was enriched 10-fold with respect to the initial homogenate. The major contaminant was a membrane fraction derived from smooth endoplasmic reticulum, rich in NADPH-cytochrome c reductase activity. The crude basolateral membrane preparation could be resolved into the two major components by subjecting it to zonal electrophoresis on density gradients. The result was that (Na+ + K+)-ATPase was purified 22-fold with respect to the initial homogenate. Purification with respect to mitochondria and brush border membranes was 35- and 42-fold, respectively. Resolution of (Na+ + K+)-ATPase from NADPH-cytochrome c reductase by electrophoresis was best with membrane material from adult rats between 180 and 250 g. No resolution between the two marker enzymes occurred with material from young rats of 125 to 140 g. These results demonstrate that zonal electrophoresis on density gradients, a simple and inexpensive technique, has a similar potential to free-flow electrophoresis.

Calcium uptake into rat small intestinal brush border membrane vesicles: characterization of transmembrane calcium transport at short initial incubation times.

Calcium transport into brush border vesicles from rat small intestine was investigated by determining uptake rates at very short incubation periods. At incubation times up to 1 second a linear relationship between calcium uptake and time was observed at free calcium concentrations ranging from 1 microM to 5 mM. At time points above 1 second calcium uptake deviates progressively from linearity. Several lines of evidences (EGTA-wash, dependency on membrane potential, temperature sensitivity and effect of the calcium ionophore A23187) suggest transmembrane transport rather than extravesicular binding of calcium as being responsible for calcium uptake. Saturation experiments performed under initial linear and curvilinear uptake conditions show a saturable transport component in the mu molar and only a tendency to saturate in the molar concentration range. It is concluded that uptake values far from equilibrium are characteristic for transmembrane flux of calcium. Transmembrane flux of calcium is mediated by multiple and potential-sensitive mechanisms.

Activity-dependent recruitment of endogenous glutamate for exocytosis.

The Ca(2+)-dependent release of the neurotransmitter glutamate from purified nerve terminals (synaptosomes) of the rat hippocampus was studied in a rapid perfusion apparatus. The response of the terminals was investigated with respect to the kinetics and duration of the release of endogenous glutamate upon brief and sustained stimulation and upon repetitive stimulation. The synaptosomes were stimulated by sustained chemical depolarization (0.5-3 min 30 mM K+). The cellular levels of glutamate, free Ca2+ and ATP in the nerve terminals were measured. The Ca(2+)-dependent release of glutamate showed an immediate elevation upon K(+)-depolarization. When the stimulation was maintained, a prolonged phase of glutamate release was observed. After 3 min, the Ca(2+)-dependent release stopped, although K(+)-depolarization was still effective. When synaptosomes were stimulated again after a relatively short stimulation period (30 s), the second response was similar to the previous one. After a longer stimulation period, maintained until termination of release, the second response did not show the immediate initial elevation of Ca(2+)-dependent glutamate release. Only 30 s after stimulation the release developed with a time profile comparable to the first response. This initial lack of response was not due to low cytosolic levels of glutamate or ATP or to changes in cellular Ca(2+)-buffering. It can be concluded that the capacity to release glutamate after brief depolarizations is fully restored during the repolarization period. However, if stimulation periods are of long duration (until termination of release), this capacity is no longer fully restored, especially with respect to a fast component of release. New glutamate is recruited only during the subsequent depolarization and with a delay.(ABSTRACT TRUNCATED AT 250 WORDS)

Sequential changes in synaptic vesicle pools and endosome-like organelles during depolarization near the active zone of central nerve terminals.

During periods of high-frequency stimulation the maintenance of synaptic transmission depends on a continued supply of synaptic vesicles. Local recycling in the terminals ensures synaptic vesicle replenishment, but the intermediate steps are still a matter of debate. We analyzed changes in synaptic vesicle pools and endosome-like organelles near the active zone in central nerve terminals during depolarization at the ultrastructural level by electron microscopy. A short, 100 ms, depolarization-induced recruitment of synaptic vesicles was observed from a reserve pool to a recruited pool, within 150 nm of the active zone, and the docked pool at the active zone was increased as well. Prolonged, 15 s or 3 min, depolarization decreased the total amount of synaptic vesicles, which was accompanied by a parallel increase in size and amount of endosome-like organelles. After a period of rest, the number of endosome-like organelles decreased and the amount of synaptic vesicles was restored to control level. The endocytotic nature of part of the endosome-like organelles after 15 s and 3 min depolarization was indicated by their labeling with extracellularly added horseradish peroxidase (HRP). In addition, a small number of synaptic vesicles entrapped HRP under these conditions. After repolarization, the number of HRP-loaded endosome-like structures decreased. Simultaneously, a strong increase in amount of HRP-loaded small vesicles did occur. These results indicate that during sub-second depolarization, synaptic vesicles were rapidly recruited from the reserve pool to replenish the releasable pool, whereas prolonged depolarization (s-min) induced local endocytosis in at least two ways, i.e. either directly as vesicles or via endosome-like organelles from which synaptic vesicles were reformed.

Regulation of cholecystokinin release from central nerve terminals.

The high abundance of the cholecystokinin octapeptide in various brain regions is expressed by involvement of this neuropeptide in diverse brain functions. This peptide is mostly, if not always, co-localized with classic transmitters in central nerve terminals. Since the functions of the coexisting transmitters are often different, differential regulation of their release is obvious. This differentiation is realized by differences in presynaptic localization, release dynamics, and calcium regulation. In addition, CCK release is locally modulated by receptors, kinases and phosphatases. The regulatory mechanisms of CCK release are placed into physiological perspective.

Characterization of the release of Met-enkephalin from isolated nerve terminals: release kinetics and cation-dependence.

The release of the neuropeptide Met-enkephalin (Met-ENK) from isolated nerve terminals (synaptosomes) of the rat forebrain was characterized with respect to the subcellular distribution, the release upon addition of various stimulatory agents, the release kinetics, the cation-dependence of release and the relationship between Met-ENK release and elevations of the intraterminal free Ca(2+)-concentration ([Ca]i). A highly specific radioimmunoassay was developed for determination of Met-ENK (H-Tyr-Gly-Gly-Phe-Met-OH). Truncated and elongated forms of Met-ENK, Leu-enkephalin, beta-endorphin and dynorphin displayed negligible cross-reactivity. Met-ENK-like immunoreactivity (Met-ENK-LI) is enriched in the purified synaptosomal fraction of rat forebrain homogenates and is released in a strictly Ca(2+)-dependent manner upon chemical depolarization or stimulation with the Ca2+ ionophore, ionomycin. A correlation exists between the release of Met-ENK-LI and the elevations of [Ca]i. Barium ions are able to replace Ca2+ in triggering Met-ENK-LI release. The release of Met-ENK-LI is initiated within 20 s after depolarization and is terminated after 3-5 min, although depolarization and [Ca]i elevation are maintained. At this time, > 90% of the initial Met-ENK-LI is still present inside the synaptosomes. Repolarization and renewed stimulation again evokes Ca(2+)-dependent release of this retained Met-ENK-LI. It is concluded that Met-ENK release from isolated nerve terminals is exocytotic, and that exocytosis is terminated by a regulatory mechanism in synaptosomes after 3-5 min of depolarization, a process which can be reversed by repolarization.(ABSTRACT TRUNCATED AT 250 WORDS)

Arachidonic acid inhibits uptake of amino acids and potentiates PKC effects on glutamate, but not GABA, exocytosis in isolated hippocampal nerve terminals.

Arachidonic acid (AA), the putative retrograde messenger in long-term potentiation, enhanced extracellular aspartate, glutamate, and GABA levels in rat hippocampus synaptosomes. Whether this effect was determined by stimulating the release and/or inhibiting the uptake of amino acids was further investigated using different experimental conditions. To approach physiological conditions, a static incubation assay was used where both release and uptake occur. Under these conditions, AA dose-dependently (10-25 microM) enhanced basal extracellular amino acid levels in a completely Ca2+-independent way. AA still exerted this effect in the presence of inhibitors of PKC or of AA metabolism. When using the superfusion release assay, in which amino acid uptake cannot occur, no potentiating effect of AA on superfusate amino acid levels was observed. Therefore, AA possibly enhances the extracellular levels of aspartate, glutamate and GABA by inhibiting the uptake of these amino acids and not their efflux. Indeed, AA reduced the Na+-dependent uptake of endogenously released amino acids, which were labelled with traces of tritiated D-aspartate and GABA. When stimulating hippocampus synaptosomes with 4-aminopyridine, AA (2 microM) potentiated the Ca2+-dependent release of glutamate, but not of GABA, synergistically with PKC activation by 4beta-phorbol-12,13-dibutyric acid. In rat hippocampus, AA exerts different presynaptic effects to regulate extracellular amino acid levels, by inhibiting carrier-mediated uptake and, for glutamate, by stimulating exocytosis.

Kindling increases the K(+)-evoked Ca2(+)-dependent release of endogenous GABA in area CA1 of rat hippocampus.

The release of endogenous amino acids from hippocampal CA1 subslices under basal conditions and the release evoked by high potassium (50 mM K+) depolarization was studied during kindling epileptogenesis. Emphasis was put on the release of the amino acid neurotransmitters gamma-aminobutyric acid (GABA) and glutamate. Kindling was induced by tetanic stimulation of the Schaffer-collaterals/commissural fibers of the dorsal hippocampus of the rat. The calcium-dependent GABA release in the presence of high K+ was significantly increased (40-46%) in fully kindled animals, 24 h after the last seizure, in comparison to controls. At long-term, 28 days after the last seizure, the calcium-dependent GABA release was still significantly increased (45-49%). An increased release of GABA in kindled animals was still found when GABA uptake was blocked by nipecotic acid. In contrast, no significant alterations were encountered in the basal or high potassium induced release of the excitatory amino acids aspartate and glutamate. These results suggest that kindling epileptogenesis is accompanied by a specific and long-lasting enhancement of GABA exocytosis which may lead to a desensitization of the GABA receptor, and thus determine the increase of seizure sensitivity.

Effects of uptake carrier blockers SK & F 89976-A and L-trans-PDC on in vivo release of amino acids in rat hippocampus.

This report describes the in vivo effects of the uptake carrier blockers 1-(4,4-diphenyl-3-butenyl)-3-piperidine carboxylic acid hydrochloride (SK & F 89976-A) and L-trans-pyrrolidine-2,4-dicarboxylate (L-trans-PDC) on basal and K(+)-evoked extracellular levels of gamma-aminobutyric acid (GABA), glutamate, aspartate and taurine in the hippocampus of anaesthetised rats, using the microdialysis technique. SK & F 89976-A increased extracellular GABA levels under K(+)-depolarised conditions and did not affect extracellular glutamate, aspartate and taurine levels, indicating its selective effect on GABA uptake L-trans-PDC dose dependently increased basal and K(+)-evoked extracellular glutamate levels, and did not affect extracellular GABA levels, but increased basal aspartate and taurine levels. The K(+)-evoked release of GABA and glutamate, measured in the presence of both SK & F 89976-A and L-trans-PDC, was Ca(2+)-dependent for about 50% and 65%, respectively. In contrast, the release of the putative amino acid transmitters aspartate and taurine was not Ca(2+)-dependent. These results indicate that (1) in rat hippocampus uptake carriers actively regulate extracellular GABA and glutamate levels, (2) the GABA and glutamate released by K+ was derived from both Ca(2+)-dependent (presumably vesicular) and Ca(2+)-independent (presumably cytosolic) pools, whereas aspartate and taurine release was exclusively from Ca(2+)-independent pools.

Ba2+ replaces Ca2+/calmodulin in the activation of protein phosphatases and in exocytosis of all major transmitters.

Exocytosis from nerve terminals is triggered by depolarization-evoked Ca2+ entry, which also activates calmodulin and stimulates protein phosphorylation. Ba2+ is believed to replace Ca2+ in triggering exocytosis without activation of calmodulin and can therefore be used to unravel aspects of presynaptic function. We have analysed the cellular actions of Ba2+ in relation to its effect on transmitter release from isolated nerve terminals. Barium evoked specific release of amino acid transmitters, catecholamines and neuropeptides (EC50 0.2-0.5 mM), similar to K-/Ca(2+)-evoked release both in extent and kinetics. Ba(2+)-and Ca(2+)-evoked release were not additive. In contrast to Ca2+, Ba2+ triggered release which was insensitive to trifluoperizine and hardly stimulated protein phosphorylation. These observations are in accordance with the ability of Ba2+ to replace Ca2+ in exocytosis without activating calmodulin. Nevertheless, calmodulin appears to be essential for regular (Ca(2+)-triggered) exocytosis, given its sensitivity to trifluoperizine. Both Ba(2+)-and Ca(2+)-evoked release were blocked by okadaic acid. Furthermore, anti-calcineurin antibodies decreased Ba(2+)-evoked release. In conclusion, Ba2+ replaces Ca2+/calmodulin in the release of the same transmitter pool. Calmodulin-dependent phosphorylation appears not to be essential for transmitter release. Instead, our data implicate both Ca(2+)-dependent and -independent dephosphorylation in the events prior to neurotransmitter exocytosis.

Anti-B-50 (GAP-43) antibodies decrease exocytosis of glutamate in permeated synaptosomes.

The involvement of the protein kinase C substrate, B-50 (GAP-43), in the release of glutamate from small clear-cored vesicles in streptolysin-O-permeated synaptosomes was studied by using anti-B-50 antibodies. Glutamate release was induced from endogenous as well as 3H-labelled pools in a [Ca(2+)]-dependent manner. This Ca(2+)-induced release was partially ATP dependent and blocked by the light-chain fragment of tetanus toxin, demonstrating its vesicular nature. Comparison of the effects of anti-B-50 antibodies on glutamate and noradrenaline release from permeated synaptosomes revealed two major differences. Firstly, Ca(2+)-induced glutamate release was decreased only partially by anti-B-50 antibodies, whereas Ca(2+)-induced noradrenaline release was inhibited almost completely. Secondly, anti-B-50 antibodies significantly reduced basal glutamate release, but did not affect basal noradrenaline release. In view of the differences in exocytotic mechanisms of small clear-cored vesicles and large dense-cored vesicles, these data indicate that B-50 is important in the regulation of exocytosis of both types of neurotransmitters, probably at stages of vesicle recycling and/or vesicle recruitment, rather than in the Ca(2+)-induced fusion step.

Cholecystokinin (CCK-8) modulates vesicular release of excitatory amino acids in rat hippocampal nerve endings.

The modulation of endogenous amino acid transmitter release by the sulphated octapeptide cholecystokinin (CCK-8S) was investigated in purified rat hippocampal synaptosomes. In the presence of extracellular Ca2+, CCK-8S increased the basal release of glutamate, but not of aspartate and GABA. In addition, CCK-8S dose-dependently increased the KCl-evoked Ca2+-dependent release of both glutamate and aspartate to about 1.4-fold at concentrations > or = 0.5 microM. CCK-8S did not change the KCl-evoked Ca2+-dependent GABA release, not even in the presence of the GABA uptake carrier blocker N-(4,4-diphenyl-3-butenyl)-3-piperidine carboxylic acid 89976-A (SK&F89976-A; 10 microM). The CCKB receptor antagonist L365,260 (1 microM) blocked the CCK-8S-induced release of glutamate by 70%, and of aspartate by 100%. In conclusion, CCK stimulates exocytosis of excitatory amino acids in rat hippocampus by activating a low-affinity presynaptic CCK receptor, presumably of the B-subtype. However, CCK does not modulate the release of GABA, which has been reported to be colocalized with this peptide.