Release of Acetylcholine from Rat Brain Synaptosomes Stimulated with Leptinotarsin, A New Neurotoxin

Abstract: Leptinotarsin is a neurotoxic protein found in the hemolymph of potato beetles of the genus Leptinotarsa. In order to study the action of leptinotarsin from two species, L. haldemani and L. decemlineata , synaptosomes were prelabeled with [³H]choline in order to synthesize [³H] acetylcholine (ACh). These synaptosomes were then immobilized on Millipore filters and used for assay. Toxins from both species induce the release of radioactivity in this system. Fractionation of the released radioactivity indicated that ACh was released in preference to choline. The toxin that caused release was heat-labile and was partially dependent on Ca²⁺ in the perfusing medium. Release followed apparent first order kinetics when stimulation was effected with leptinotarsin from L. haldemani (leptinotarsin-h), but was more complex when using leptinotarsin from L. decemlineata (leptinotarsin-d). Increasing the concentration of toxin increased the rate of release, but the shapes of the dose-release curves elicited by the leptinotarsins from the two species were different. While leptinotarsin-h exhibited a simple, saturating dose-release curve, leptinotarsin-d was characterized by a sigmoid function, which was well described, with a Hill coefficient of 1.8. Antibodies directed toward black widow spider venom glands had no effect upon the releasing activity of leptinotarsin-h but could partially neutralize that of leptinotarsin-d. Toxins from both species have been partially purified and do not appear to be identical. The purified toxins should be useful tools with which to study the release of acetylcholine.     

Acetylcholine Turnover and Compartmentation in Rat Brain Synaptosomes

Abstract: The turnover of acetylcholine (ACh) in rat brain synaptosomes and its compartmentation in the labile bound and stable bound pools were investigated. The P₂ fraction from rat brain was subjected to three sequential incubations, each terminated by centrifugation followed by determination of ACh concentrations by gas chromatography-mass spectrometry (GCMS): (1) Depletion phase: Incubation of synaptosomes at 37°C for 10 min in Na⁺-free buffer containing 35 mM-KCl reduced the content of both labile bound and stable bound ACh by 40%. (2) Synthesis phase: Incubation at 37°C with 2 μ M -[²H₄]choline resulted in accumulation of labeled and unlabeled ACh in both compartments. Addition of an anticholinesterase had little effect on stable bound ACh but greatly increased the content of labile bound ACh. This excess accumulated ACh was probably due to inhibition of intracellular acetylcholinesterase (AChE), because negligible uptake of ACh from the medium was observed. The effects on ACh synthesis of altered cation concentrations and metabolic inhibitors were examined. (3) Release phase: The tissue was incubated in the presence of 35 mM-KCl, 40 μM-paraoxon, and 20 μM-hemicholinium-3 (HC-3) (to inhibit further synthesis of ACh). Measurements of the compartmental localization of ACh at several time points indicated that ACh was being released from the labile bound fraction. In support of this conclusion, 20 mM-Mg²⁺ reduced ACh release and increased the labile bound ACh concentration.     

Inhibition by Quinacrine of Depolarization-Induced Acetylcholine Release and Calcium Influx in Rat Brain Cortical Synaptosomes

The effects of quinacrine on depolarization-induced [³H]acetylcholine (ACh) release and ⁴⁵Ca²⁺ influx were examined in rat brain cortical synaptosomes. Quinacrine significantly reduced the stimulated release of [³H]ACh by high K⁺ and veratridine without affecting the spontaneous efflux from the preloaded synaptosomes. Quinacrine had no effect on ionophore A23187-induced release of [³H]ACh from the synaptosomes. Quinacrine (100 μM) markedly diminished the stimulated Ca²⁺ influx by veratridine and high K⁺ but not that by “Na⁺-free.” Trifluoperazine, a potent calmodulin antagonist, inhibited both Ca²⁺ influx and ACh release induced by the depolarizing agents. Inhibitory potencies of the two drugs on ACh release and Ca²⁺ influx were compared with the antagonism of calmodulin by two drugs, suggesting that the inhibition of depolarization-induced Ca²⁺ influx and ACh release by these drugs could not be explained by the antagonism of calmodulin.     

Effects of In Vivo Hypoxia on Acetylcholine Synthesis by Rat Brain Synaptosomes

Abstract: Synaptosomes from normoxic and hypoxic rats were incubated aerobically in the presence and absence of veratridine. In the absence of veratridine, no significant difference was observed between the two types of preparation regarding either ATP/ADP ratio or ¹⁴CO₂ or [¹⁴C]acetylcholine synthesis from D-[U-¹⁴C]glucose. However, in the presence of veratridine, significant reductions in the output of ¹⁴CO₂ and [¹⁴C]acetylcholine by synaptosomes from hypoxic rats were apparent. It was concluded that irreversible metabolic lesions occur at the synapse as a result of hypoxia, which are apparent only when the metabolism of the preparation is accelerated to a level comparable with the maximal rate occurring in vivo. The presence of such lesions is further evidenced by the significant reductions in ATP/ADP ratio, ¹⁴CO₂ output, and [¹⁴C]acetylcholine synthesis that occur in synaptosomes from hypoxic rats made anoxic in vitro and permitted to recover. Such decreases are not seen when synaptosomes from normoxic rats are similarly treated.     

Effects of In Vitro Anoxia and Low pH on Acetylcholine Release by Rat Brain Synaptosomes

Acetylcholine and choline release from rat brain synaptosomes have been measured using a chemiluminescent technique under a variety of conditions set up to mimic anoxic insult, including conditions of low pH (6.2) and the presence of lactate plus pyruvate as substrate. Lactate plus pyruvate as substrate consistently gave higher respiration rates than glucose alone, but with either substrate (glucose or lactate plus pyruvate) the omission of Ca2+ caused an increase in respiration whereas a low pH caused a decreased respiration. Acetylcholine release under control conditions (glucose, pH 7.4) was Ca2+-dependent, stimulated by high K+ concentrations, and decreased significantly during anoxia but recovered fully after a period of postanoxic oxygenation. Low pH (6.2) suppressed K+ stimulation of acetylcholine release, and after a period of anoxia at low pH the recovery of acetylcholine release was only partial. With lactate plus pyruvate as substrate, the effects of anoxia and/or low pH on acetylcholine release and its subsequent recovery were exacerbated. Choline release from synaptosomes, however, was not affected by anoxic/ionic conditions in the same way as acetylcholine release. At low pH (6.2) there was a marked reduction in choline release both under aerobic and anoxic conditions. These results suggest that acetylcholine release per se from the nerve is very sensitive to anoxic insult and that the low pH occurring during anoxia may be an important contributory factor.     

Aging Decreases the Sensitivity of Rat Cortical Synaptosomes to Calcium Ionophore-Induced Acetylcholine Release

The capacity of calcium ions to trigger acetylcholine release was studied in cerebral cortical synaptosomes from adult (6-month-old) and senescent (24-month-old) rats, using a calcium ionophore, A23187, that bypasses voltage-sensitive calcium channels. The potency but not the efficacy of the A23187 was reduced with respect to releasing acetylcholine (ACh) in the aged animals. There was no age-related difference in the synthesis of ACh or potency of the ionophore with respect to increasing 45calcium uptake. These results suggest that aging reduces the sensitivity of cerebral cortical nerve terminals to calcium-triggered ACh-release.     

Oxygen Dependence of Glucose and Acetylcholine Metabolism in Slices and Synaptosomes from Rat Brain

Abstract: Previous studies have shown that a reduction in the O₂ tension of the blood from 120 torr to 57 torr (hypoxic hypoxia) decreases brain acetylcholine (ACh) synthesis. To determine if this decrease is due to a direct impairment of ACh metabolism or to an indirect effect mediated by other neurotransmitter systems, we studied ACh formation in rat brain slices and synaptosomes. At O₂ tensions ranging from 760 to less than 1 torr, ¹⁴CO₂ production and [¹⁴C]ACh synthesis from [U-¹⁴C]glucose, the levels of lactate and ATP, and the ATP/ADP ratio were determined. In slices, the first decreases were observed in the rate of ¹⁴CO₂ production and [¹⁴C]ACh synthesis at an O₂ tension of 152 torr. The ATP level started to decline at 53–38 torr, and a reduction in the ATP/ADP ratio was first found at and below 19 torr. Lactate formation was maximally stimulated at 38–19 torr. Synaptosomes responded differently than brain slices to reduced O₂ tensions. In synaptosomes, ¹⁴CO₂ production and [¹⁴C]ACh synthesis from [U-¹⁴C]glucose, the levels of lactate and ATP, and the ATP/ADP ratio were unaltered if a minimum O₂ tension of 19 torr was maintained. Despite the difference in sensitivities to decreases in O₂ levels, there is a curvilinear relationship between [U-¹⁴C]glucose decarboxylation and [¹⁴C]ACh synthesis at various O₂ tensions for both tissue preparations with a high coefficient of determination (R²= 0.970). The difference in the metabolic sensitivity of slices and synaptosomes to a reduced O₂ level may be explained by the greater distance O₂ must diffuse in slices. The results are discussed in comparison with hypoxia in vivo.     

Mode of Action of Palytoxin on the Release of Acetylcholine from Rat Cerebrocortical Synaptosomes

Palytoxin (PTX; 10(-14)-10(-6) M) caused a dose-dependent increase in the release of [3H]acetylcholine ([3H]ACh), cytosolic free Ca2+ concentration ([Ca2+]i), and uptake of 22Na+ and decrease in membrane potential in rat cerebrocortical synaptosomes. The dose-response curves for the PTX-induced increases in [3H]ACh release and in [Ca2+]i were depressed by removing extracellular Ca2+ or by decreasing extracellular Na+ concentrations. The release of [3H]ACh induced by concentrations of PTX less than 10(-10) M was more dependent on the simultaneous presence of both Ca2+ and Na+ than the release induced by higher concentrations of PTX. The PTX-induced increase both in [3H]ACh release and in [Ca2+]i was almost completely abolished by the combination of Ca2+ deprivation and Na+ concentration reduction. All responses to PTX were highly resistant to 10(-6) M tetrodotoxin. These results suggest that low concentrations of PTX cause depolarization as a result of an increase in Na+ permeability through tetrodotoxin-insensitive channels. This, in turn, increases Ca2+ influx and leads to an increase in the release of ACh. It appears that at high concentrations PTX increases the release of [3H]ACh by directly increasing the influx of Ca2+ into synaptosomes and by releasing Ca2+ from intracellular storage sites via an Na(+)-Ca2+ exchange mechanism.     

Effect of Liposomes Containing Various Divalent Cations on the Release of Acetylcholine from Synaptosomes

Abstract: The effect of increasing the cytoplasmic levels of various divalent cations on the release of [³H]acetylcholine ([³H]ACh) from synaptosomes was investigated. Synaptosomes prepared from rat brain and prelabeled with [³H]choline were incubated with liposomes containing Mg²⁺, Ca²⁺, Mn²⁺, Co²⁺, Sr²⁺, or Ba²⁺. This treatment allows the transfer of the aqueous contents of the liposomes to the cytoplasm of the synaptosomes. The efflux of radioactivity subsequent to this treatment was measured, and the relative proportions of [³H]ACh and [³H]choline were determined. The release of radioactivity from synaptosomes incubated with liposomes containing Mg²⁺, Mn²⁺, or Co²⁺ was not altered when compared with synaptosomes incubated either without liposomes or with liposomes containing isotonic K⁺/Na⁺. Synaptosomes incubated with liposomes containing Ca²⁺, Sr²⁺, or Ba²⁺, however, released significantly more radioactivity than did controls. Moreover, the released radioactivity consisted almost entirely of [³H]ACh. Liposomes containing either Ca²⁺ or Sr²⁺ were equally effective in promoting the release of [³H]ACh from synaptosomes, whereas liposomes containing Ba²⁺ were 2.5 times more effective in promoting the release of [³H]ACh than were liposomes containing either Ca²⁺ or Sr²⁺. Since liposomes introduce their aqueous contents into cytoplasm via a mechanism not involving plasma membrane channels, the increased release of [³H]ACh caused by liposomes containing Ca²⁺, Sr²⁺, or Ba²⁺ is attributable to an increase in the intrasynaptosomal concentration of these ions, and not to their passage through calcium channels.     

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.     

Distinct Muscarinic Receptor Subtypes Differentially Modulate Acetylcholine Release from Corticocerebral Synaptosomes

The effect of McN-A-343 and oxotremorine on acetylcholine (ACh) release and choline (Ch) transport was studied in corticocerebral synaptosomes of the guinea pig. The synaptosomes were preloaded with [3H]Ch after treatment with the irreversible cholinesterase inhibitor, diisopropyl fluorophosphate, and then tested for their ability to release isotope-labeled ACh and Ch in the presence and absence of these agents. The kinetics of release were determined at the resting state (basal release) and in the presence of 50 mM K+. Under either condition, McN-A-343 enhanced the release of isotope-labeled ACh, whereas oxotremorine inhibited the K(+)-evoked release but had no effect on the basal release. The enhancing effect of McN-A-343 on basal ACh release was fully blocked by the selective M1 muscarinic antagonist, pirenzepine (100 nM). In contrast to its enhancing effect on ACh release, McN-A-343 potently inhibited Ch efflux as well as Ch influx. These effects were not blocked by atropine, a nonselective muscarinic antagonist. Oxotremorine had no effect on Ch transport. Binding studies showed that McN-A-343 was 3.6-fold more potent in displacing radiolabeled quinuclidinyl benzilate from cerebral cortex muscarinic receptors (mostly M1 subtype) than from cerebellar receptors (mostly M2 subtype), whereas oxotremorine was 2.6-fold more potent in the cerebellum. The displacements of radio-labeled pirenzepine and cis-dioxolane confirmed the M1 subtype preference of McN-A-343 and the M2 subtype preference of oxotremorine.(ABSTRACT TRUNCATED AT 250 WORDS)     

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

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

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.     

The Rapid Uptake and Release of [3H]Adenosine by Rat Cerebral Cortical Synaptosomes

Abstract: Adenosine, a putative inhibitory transmitter or modulator in the brain, is rapidly transported by rat cerebral cortical synaptosomes. The uptake may represent a facilitated diffusion process, which is saturable and temperature-dependent. In this study, the uptake process was very rapid, reaching completion within 60 s of incubation at 37°C, and had an apparent K_m value of 0.9μM and a V_max value of 5.26 pmol/mg protein/ 30 s. Over 70% of the adenosine taken up remained unchanged, whereas 14% was metabolized to inosine. Twelve percent of the adenosine was converted to nucleotides. Rapid uptake of adenosine into rat cerebral cortical synaptosomes was partially inhibited by replacing Na⁺ with choline chloride in the medium. Ca²⁺ ion is important for the uptake process, as inhibition of adenosine uptake occurs in the presence of either Co²- or EGTA. Rapid uptake of adenosine is apparently mediated by a nucleoside carrier, a conclusion based on its inhibition by a variety of purine and pyrimidine nucleosides. Uptake was inhibited by dipyridamole, hexobendine, papaverine, flurazepam, and morphine. Over 60% of the adenosine taken up by the rapid uptake system (30 s) was released by depolarizing agents. In contrast, only 30% of the adenosine taken up during a 15-min incubation period was released under the same conditions. [³H]Adenosine was the predominant purine released in the presence or absence of depolarizing agents. The basal and KCl-evoked release mechanisms were found to be at least partially Ca²⁺-dependent, however, the release of adenosine by veratridine was increased in the presence of EGTA. This finding is in agreement with the reported Ca²⁺-independent release of ATP from brain synaptosomes. The present findings suggest that there are at least two functional pools of adenosine in synaptosomes. Adenosine taken up by different uptake systems may be destined for different uses (metabolism or release) in the neuron.     

Continuous Determination by a Chemiluminescent Method of Acetylcholine Release and Compartmentation in Torpedo Electric Organ Synaptosomes

Abstract: The detection of acetylcholine (ACh) with a chemiluminescent procedure enables one to follow continuously the release of transmitter from stimulated synaptosomes and to study the compartmentation of ACh in resting and active nerve terminals. A compartment of ACh liberated almost entirely by a single freezing and thawing could be directly measured and compared with a compartment of ACh resistant to several cycles of freezing and thawing but liberated by a detergent (60–70% of the total). It is the compartment liberated by freezing and thawing that is reduced when synaptosomes are stimulated. Up to half the total synaptosomal ACh content is readily releasable provided the calcium entry is maintained, or if a strong releasing agent such as the venom of Glycera convoluta is used. In addition, it is shown that synaptosomes contain only negligible amounts of choline, and that the proportion of the two ACh compartments is not influenced by changing extracellular calcium just before their determination.     

Effect of Acute Ethanol on Release of Endogenous Adenosine from Rat Cerebellar Synaptosomes

The effects of pharmacologically relevant concentrations of ethanol on the release of endogenous adenosine from rat cerebellar synaptosomes were investigated. Release was conducted for 5, 10, 30, or 60 s after which time the incubation medium (containing the released adenosine) was rapidly separated from the synaptosomal membranes by vacuum filtration. The adenosine content of the filtrate was measured by HPLC-fluorescence detection. Both basal and KCl-stimulated adenosine release consisted of an initial rapid phase, for the first 10 s, that was followed by a relatively slower phase. Basal endogenous adenosine release was estimated as 199 +/- 14 pmol/mg protein/5 s. Potassium (chloride) increased adenosine release from the basal level to 433 +/- 83 pmol/mg protein/5 s. Ethanol caused a dose-dependent increase of adenosine release. The interaction between dilazep and ethanol indicates that ethanol-stimulated release does not involve the dilazep-sensitive transport system. The results support previous findings that indicate that cerebellar adenosine is involved in the mediation of ethanol-induced motor disturbances in the rat.     

Stimulation of Immunoreactive Insulin Release by Glucose in Rat Brain Synaptosomes

The effect of glucose on the release of immunoreactive insulin (IRI) in synaptosomes isolated from rat brain was studied. In the absence of glucose synaptosomes release about 4% (0.77 IU/mg protein) of total content. Glucose increases significantly the IRI released by synaptosomes. Addition of the glycolytic inhibitor iodoacetic acid (IAA), decreased the glucose-induced release of IRI by about 50%, suggesting that glucose metabolism is involved. The observation that glucose provides a concentration related signal for IRI release indicates that this synaptosomal preparation may be useful as a model for research on the mechanism of insulin release in brain.     

Release of Immunoreactive Insulin from Rat Brain Synaptosomes Under Depolarizing Conditions

Synaptosome preparations were utilized to characterize the release and compartmentalization of immunoreactive insulin (IRI) in the adult rat brain. Depolarization of synaptosomes by elevation of the external potassium ion concentration elicited release of IRI from the synaptosomes into the incubation medium. This release was reduced or eliminated under three conditions known to prevent depolarization-induced Ca2+ flux: elevating the external MgCl2, adding CoCl2, and eliminating external Ca2+ with EGTA. Depolarization of synaptosomes by veratridine also elicited release of synaptosomal IRI. This release was inhibited by tetrodotoxin. The amount of IRI released under depolarizing conditions represented 3-7% of that contained in the synaptosomes. High levels of IRI release also were observed upon removal of external Na+ to allow depolarization-independent influx of external Ca2+ into the synaptosomal compartment. The Ca2+ dependency of synaptosomal IRI release suggests IRI is stored in the adult rat brain in synaptic vesicles within nerve endings from which it can be mobilized by exocytosis in association with neural activity.     

Acetylcholine Synthesis and CO2 Production from Variously Labeled Glucose in Rat Brain Slices and Synaptosomes

Abstract: The molecular basis of the close linkage between oxidative metabolism and acetylcholine (ACh) synthesis is still unclear. We studied this problem in slices and synaptosomes by measurement of ACh synthesis from [U-¹⁴C]glucose, and ¹⁴CO₂ production from [3,4-¹⁴C]- and [2-¹⁴C]glucose, an index of glucose decarboxylation by the pyruvate dehydrogenase complex (PDH) and the enzymes of the Krebs cycle, respectively. We examined both under conditions that either inhibited (low O₂ or antimycin) or stimulated (2,4- dinitrophenol [DNP] or 35 mm -K⁺) ¹⁴CO₂ production from [2-¹⁴C]- or [3,4-¹⁴C]glucose. Incorporation of [U-¹⁴C]glucose into ACh was reduced under low O₂ and by antimycin or DNP (by 51-93%) and stimulated by 35 mm -K⁺ (by 30-60%). Under all of these conditions, ACh synthesis and the decarboxylation of [3,4-¹⁴C]- and [2-¹⁴C]glucose were linearly related (r= 0.741 and 0.579, respectively). The difference in the rate of ¹⁴CO₂ production from [3,4-¹⁴C]- and [2-¹⁴C]glucose was used as a measure of the amount of glucose that was not oxidatively decarboxylated (efflux). We found that efflux was reduced (low 0₂ and antimycin), unchanged (DNP in slices), or increased (DNP in synaptosomes and K⁺ stimulation in slices) compared with control values under 100% O₂. ACh synthesis and efflux were more closely related (r= 0.860) than ACh synthesis and ¹⁴CO₂ production from variously labeled glucoses.     

Temporal Characteristics of Potassium-Stimulated Acetylcholine Release and Inactivation of Calcium Influx in Rat Brain Synaptosomes

Abstract: The time course of Ca²⁺-dependent [³H]acetylcholine ([³H]ACh) release and inactivation of ⁴⁵Ca²⁺ entry were examined in rat brain synaptosomes depolarized by 45 m M [K⁺]_o. Under conditions where the intrasynaptosomal stores of releasable [³H]ACh were neither exhausted nor replenished in the course of stimulation, the K⁺-evoked release consisted of a major (40% of the releasable [³H]ACh pool), rapidly terminating phase ( t _1/2 = 17.8 s), and a subsequent, slow efflux that could be detected only during a prolonged, maintained depolarization. The time course of inactivation of K⁺-stimulated Ca²⁺ entry suggests the presence of fast-inactivating, slow-inactivating, and noninactivating, or very slowly inactivating, components. The fast-inactivating component of the K⁺-stimulated Ca²⁺ entry into synaptosomes appears to be responsible for the rapidly terminating phase of transmitter release during the first 60 s of K⁺ stimulus. The noninactivating Ca²⁺ entry may account for the slow phase of transmitter release. These results indicate that under conditions of maintained depolarization of synaptosomes by high [K⁺]_o the time course and the amount of transmitter released may be a function of the kinetics of inactivation of the voltage-dependent Ca channels.