Polyamines appear to be second messengers in mediating Ca2+ fluxes and neurotransmitter release in potassium-depolarized synaptosomes

High potassium (50 mM) depolarization induces a rapid (less than 15 sec) increase in the levels of the polyamines putrescine, spermidine and spermine and their rate-regulating synthetic enzyme ornithine decarboxylase in synaptosomes from rat cerebral cortex. The ornithine decarboxylase inhibitor alpha-difluoromethylornithine blocked the K+-stimulated increase in enzyme activity and polyamines and also suppressed the increase in 45Ca2+ influx and efflux and the Ca2+-dependent release of GABA and norepinephrine. Added putrescine attenuated or negated the effects of alpha-difluoromethylornithine. These results suggest that enhanced polyamine synthesis is required for potassium depolarized stimulation of synaptic function.     

The effect of psychotropic substances on synaptosomal uptake of gamma-aminobutyric H3-acid and the activity of Na,K-ATPase]

The centrally acting drugs belonging to different groups--fluphenazine, trifluperidol, phthoracyzine, imipramine, diazepam, apomorphine, fentanyl, diphneylhydantoin, nonachlazine displayed in vitro an inhibitory effect on the uptake of gamma-aminobutyric acid by rat brain synaptosomes. A decrease in the activity of synaptosomal Na,K-ATPase was found in most cases. Drugs that failed to alter GABA uptake were as a rule found to be ineffective in relation to the enzyme activity (carbidine, morphine). GABA uptake was not affected by certain drugs inhibiting the Na,K-ATPase activity (azabuperon, tetrabenazine). It is supposed that the drugs used had at least two possible sites of action - Na,K-ATPase itself and hypothetic GABA transmembrane carrier.     

Maitotoxin-Induced Release of γ-[3H]Aminobutyric Acid from Cultures of Striatal Neurons

The potent marine toxin, maitotoxin, induced the release of gamma-[3H]aminobutyric acid (GABA) from reaggregate cultures of striatal neurons in a dose-dependent manner. Maitotoxin-induced release occurred following a lag period of several minutes and was persistent. Release induced by 70 mM K+ on the other hand was immediate and transient in nature. Co2+ (3 mM) and Cd2+ (1 mM) inhibited maitotoxin-induced release of GABA as did removal of extracellular Ca2+. However, the organic calcium antagonists nisoldipine, nitrendipine, and D-600 at concentrations of 10(-6) M did not block maitotoxin-induced or 70 mM K+-induced release. High concentrations of D-600 (10(-4) M) partially blocked both maitotoxin- and 70 mM K+-induced release. The dihydropyridine calcium agonist BAY K8644 (10(-6) M) did not enhance maitotoxin-induced or 70 mM K+-induced release. Replacement of Na+ in the incubation medium with choline led to an increased basal output of GABA and an apparent inhibition of the effect of maitotoxin. These data are discussed with reference to the hypothesis that maitotoxin can directly activate voltage-sensitive calcium channels.     

Effects of Diltiazem on Bioenergetics, K+ Gradients, and Free Cytosolic Ca2+ Levels in Rat Brain Synaptosomes Submitted to Energy Metabolism Inhibition and Depolarization

Diltiazem was able to decrease the oxygen consumption rate and lactate production in synaptosomes isolated from rat forebrains, both under control and depolarized (40 microM veratridine) conditions, starting from a concentration of 250 microM. This effect was particularly evident when synaptosomes were depolarized by veratridine. This depolarization-counteracting action was evident also when transplasma membrane K+ diffusion potentials were measured after depolarization induced by veratridine and by rotenone with a glucose shortage. The concentrations of ATP, phosphocreatine, and creatine were less sensitive to diltiazem action. The concentration/response relationships were the same as those found for the oxygen consumption were the same as those found for the oxygen consumption rate, lactate production, and K+ diffusion potentials. The effects of 0.5 mM diltiazem in counteracting inhibition of energy metabolism induced by rotenone without glucose were no longer detectable when either Ca2+ or Na+ was absent from the incubation medium of synaptosomes. Diltiazem at the same concentrations (starting from 250 microM) was able to inhibit both the veratridine-induced and the rotenone-without-glucose-induced increase in intrasynaptosomal free Ca2+ levels evaluated with the fluorescent probe quin2. The results are discussed in view of a possible effect of diltiazem on voltage-dependent Na+ channels and the possibility of utilizing this approach for counteracting neuronal failure due to derangement of energy metabolism or hyperexcitation.     

Ca2+-Dependent Regulation of Presynaptic Stimulus-Secretion Coupling

In the present study, we have investigated the role of Ca2+ in the coupling of membrane depolarization to neurotransmitter secretion. We have measured (a) intracellular free Ca2+ concentration ([Ca2+]i) changes, (b) rapid 45Ca2+ uptake, and (c) Ca2+-dependent and -independent release of endogenous glutamate (Glu) and gamma-aminobutyric acid (GABA) as a function of stimulus intensity by elevating the extracellular [K+] to different levels in purified nerve terminals (synaptosomes) from rat hippocampus. During stimulation, Percoll-purified synaptosomes show an increased 45Ca2+ uptake, an elevated [Ca2+]i, and a Ca2+-dependent as well as a Ca2+-independent release of both Glu and GABA. With respect to both amino acids, synaptosomes respond on stimulation essentially in the same way, with maximally a fourfold increase in Ca2+-dependent (exocytotic) release. Ca2+-dependent transmitter release as well as [Ca2+]i elevations show maximal stimulation at moderate depolarizations (30 mM K+). A correlation exists between Ca2+-dependent release of both Glu and GABA and elevation of [Ca2+]i. Ca2+-dependent release is maximally stimulated with an elevation of [Ca2+]i of 60% above steady-state levels, corresponding with an intracellular concentration of approximately 400 nM, whereas elevations to 350 nM are ineffective in stimulating Ca2+-dependent release of both Glu and GABA. In contrast, Ca2+-independent release of both Glu and GABA shows roughly a linear rise with stimulus intensity up to 50 mM K+. 45Ca2+ uptake on stimulation also shows a continuous increase with stimulus intensity, although the relationship appears to be biphasic, with a plateau between 20 and 40 mM K+.(ABSTRACT TRUNCATED AT 250 WORDS)     

Neomycin inhibits K+- and veratridine-stimulated noradrenaline release in rat brain slices and rat brain synaptosomes

The possible involvement of phosphoinositides' turnover in the process of neurotransmitter release in the central nervous system (CNS) was studied using rat brain slices and synaptosomes. A depolarizing concentration of potassium chloride (25 mM) induces an 8.6 +/- 0.4% increase of [3H]noradrenaline [( 3H]NA) fractional release in cerebral cortical slices above spontaneous release, and 15 mM KCl induces a 3-fold increase of [3H]NA release in rat brain synaptosomes. Neomycin, an aminoglycoside which binds phosphoinositides, inhibits the potassium-induced release in cortical slices with an IC50 = 0.5 +/- 0.07 mM and with IC50 = 0.2 +/- 0.03 mM in synaptosomes. Veratridine, a veratrum alkaloid which increases membrane permeability to sodium ions and causes depolarization of neuronal cells, induces a net 13.4 +/- 0.3% increase of [3H]NA fractional release above spontaneous release in cortical slices. In analogy to K+ stimulation, neomycin inhibits the veratridine-stimulated release in cortical slices with an IC50 = 0.65 +/- 0.1 mM. It appears that the recycling of phosphoinositides, which is necessary for Ca2+ mobilization, participates in the Ca2+-dependent induced neurotransmitter release in the central nervous system.     

Effect of calcium ion on the release of gamma-aminobutyric acid from synaptosomal fraction

Brain synaptosomes released endogenous gamma-aminobutyric acid (GABA) in response to Ca2+. The release of GABA in response to 2.5 mM Ca2+ increased linearly with log[K+]0, showing that a membrane potential-dependent Ca2+ channel limits the GABA release. In the presence of Ca2+ ionophore, A23187, GABA release increased linearly with log[Ca2+]0 without altering the membrane potential of synaptosomes.     

Mercuric chloride inhibition of vasopressin release from the isolated neurointermediate lobe of the rat pituitary

The effects of HgCl2 and ouabain on vasopressin release and Ca2+ uptake and distribution was examined in the neurointermediate lobe of the rat pituitary. HgCl2 (0.5 mM) inhibited vasopressin release by approx. 90% in both basal and potassium depolarized states. With 0.1 mM HgCl2 vasopressin release was inhibited by 50% in the depolarized state, but release was not effected in basal state. On the other hand, ouabain (0.5 mM) caused a 3-fold stimulation of vasopressin release in the depolarized state. Both HgCl2 (0.5 mM) and ouabain (0.5 mM) increased net 45Ca+2 uptake by about 80% in groups of neurointermediate lobes. Following 45Ca+2 uptake, HgCl2 (0.5 mM), which is absorbed by the neurointermediate lobe, produced an increase in cytosolic 45Ca+2 content and a decrease in mitochondrial 45Ca+2 content compared to control. In comparison, ouabain (0.5 mM), which does not penetrate the neurointermediate lobe, gave no change in cytosolic 45Ca+2, but an increase in mitochondrial 45Ca+2. These results suggest that HgCl2 inhibits vasopressin release from the neurointermediate lobe of the rat pituitary at a point distal to Ca+2 uptake by the gland.     

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.     

ATP Release, Adenosine Formation, and Modulation of Dynorphin and Glutamic Acid Release by Adenosine Analogues in Rat Hippocampal Mossy Fiber Synaptosomes

Using a hippocampal subcellular fraction enriched in mossy fiber synaptosomes, evidence was obtained indicating that adenosine derived from a presynaptic pool of ATP may modulate the release of prodynorphin-derived peptides. and glutamic acid from mossy fiber terminals. Synaptosomal ATP was released in a Ca2+-dependent manner by K+-induced depolarization. The rapid hydrolysis of extracellular [14C]ATP in the presence of intact mossy fiber synaptosomes resulted in the production of [14C]adenosine. Micromolar concentrations of a stable adenosine analogue, 2-chloroadenosine, inhibited the K+-stimulated release of both dynorphin B and dynorphin A(1-8). 2-Chloroadenosine failed to suppress the evoked release of glutamic acid, measured in these same superfusates, unless the mossy fiber synaptosomes were pretreated with D-aspartic acid to deplete the cytosolic, Ca2+-independent, pool of this acidic amino acid. In synaptosomes pretreated in this manner, release of the remaining Ca2+-dependent pool of glutamic acid was significantly inhibited by NiCl2, 2-chloroadenosine, 5'-N-ethylcarboxamidoadenosine, cyclohexyladenosine, and R(-)-N6(2-phenylisopropyl)adenosine, but not by ATP. 2-Chloroadenosine-induced inhibition was reversed when the external CaCl2 concentration was raised from 1.8 mM to 6 mM. 8-Phenyltheophylline, an adenosine receptor antagonist, effectively blocked the inhibitory effects of 2-chloroadenosine on mossy fiber synaptosomes and significantly enhanced the K+-evoked release of both glutamic acid and dynorphin A(1-8) when added alone to the superfusion medium. These results support the proposition that depolarized hippocampal mossy fiber synaptosomes release endogenous ATP and are capable of forming adenosine from extracellular ATP, and that endogenous adenosine may act at a presynaptic site to inhibit the further release of glutamic acid and the prodynorphin-derived peptides.     

Effect of synaptosomal cytosolic [3H]GABA pool depletion on secretory ability of alpha-latrotoxin

alpha-Latrotoxin, a presynaptic neurotoxin from the venom of Latrodectus mactans tredecimguttatus, induces massive [3H]GABA release from rat brain synaptosomes as a result of interaction with either Ca(2+)-dependent (neurexin 1 alpha or Ca(2+)-independent (latrophilin) membrane receptor. The main aim of the study was to elucidate whether the binding of alpha-latrotoxin to different types of receptors led to [3H]GABA secretion from one pool or in each case the source of neurotransmitter differs: in the presence of Ca2+ exocytosis is induced, while in the absence of Ca(2+)--outflow by mobile membrane GABA transporter from cytoplasm. We examined the effect of the depletion of cytosolic [3H]GABA pool by competitive inhibitors of the GABA transporter (nipecotic acid and 2,4-diaminobutyric acid) on the alpha-latrotoxin-stimulated neurotransmitter release. We also compared the influence of these agents on neurosecretion, evoked by depolarization with that evoked by alpha-latrotoxin. Depolarization was stimulated by 4-aminopyridine in the Ca(2+)-containing saline and high KCl in Ca(2+)-free medium. In synaptosomes treated with nipecotic acid unstimulated [3H]GABA release was significantly augmented and high KCl-evoked Ca(2+)-independent [3H]GABA release was essentially inhibited. But under the same conditions neurosecretion stimulated by alpha-latrotoxin greatly raised with respect to the control response. The similar results were obtained with the synaptosomes treated with 2,4-diaminobutyric acid. Another way to determine which of GABA pool is the target of alpha-latrotoxin action lay in analysis of the toxin effects on the preliminary depolarized synaptosomes. alpha-Latrotoxin influence was diminished by the preceding depolarization by 4-aminopyridine in Ca2+ presence. But after the high KCl stimulation effect of alpha-latrotoxin didn't change. These data suggest that alpha-latrotoxin triggers neurotransmitter release from synaptic vesicles via exocytosis. We suppose that the type of membrane receptor does not determine the mechanism of GABA release evoked by the toxin.     

γ-Aminobutyric Acid and Glycine Modulate Each Other''s Release Through Heterocarriers Sited on the Releasing Axon Terminals of Rat CNS

The ability of gamma-aminobutyric acid (GABA) and glycine (Gly) to modulate each other's release was studied in synaptosomes from rat spinal cord, cerebellum, cerebral cortex, or hippocampus, prelabeled with [3H]GABA or [3H]Gly and exposed in superfusion to Gly or to GABA, respectively. GABA increased the spontaneous outflow of [3H]Gly (EC50, 20.8 microM) from spinal cord synaptosomes. Neither muscimol nor (-)-baclofen, up to 300 microM, mimicked the effect of GABA, which was not antagonized by either bicuculline or picrotoxin. However, the effect of GABA was counteracted by the GABA uptake inhibitors nipecotic acid and N-(4,4-diphenyl-3-butenyl)nipecotic acid. Moreover, the GABA-induced [3H]Gly release was Na+ dependent and disappeared when the medium contained 23 mM Na+. The effect of GABA was Ca2+ independent and tetrodotoxin insensitive. Conversely, Gly enhanced the outflow of [3H]GABA from rat spinal cord synaptosomes (EC50, 100.9 microM). This effect was insensitive to both strychnine and 7-chlorokynurenic acid, antagonists at Gly receptors, but it was strongly Na+ dependent. Also, the Gly-evoked [3H]GABA release was Ca2+ independent and tetrodotoxin insensitive. GABA increased the outflow of [3H]Gly (EC50, 11.1 microM) from cerebellar synaptosomes; the effect was not mimicked by either muscimol or (-)-baclofen nor was it prevented by bicuculline or picrotoxin. The GABA effect was, however, blocked by GABA uptake inhibitors and was Na+ dependent. Gly increased [3H]GABA release from cerebellar synaptosomes (EC50, 110.7 microM) in a strychnine- and 7-chlorokynurenic acid-insensitive manner. This effect was Na+ dependent. The effects of GABA on [3H]Gly release seen in spinal cord and cerebellum could be reproduced also with cerebrocortical synaptosomes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Uptake and release of [3H]gamma-aminobutyric acid by embryonic spinal cord neurons in dissociated cell culture

We have investigated the uptake and release of [3H]gamma-aminobutyric acid (GABA) by embryonic chick spinal cord cells maintained in culture. Cells dissociated from 4- or 7-d-old embryos were studied between 1 and 3 wk after plating. At 3 degrees C, [3H]GABA was accumulated by a high affinity (Km approximately equal to 4 microM) and a low affinity (Km approximately equal to 100 microM) mechanism. The high affinity transport was markedly inhibited in low Na+ media, by ouabain, at 0 degrees C, and by 2,4-diaminobutyric acid. Autoradiography, after incubation in 0.1 microM [3H]GABA, showed that approximately 50% (range = 30-70%) of the multipolar cells were labeled. These cells were neurons rather than glia; action potentials and/or synaptic potentials were recorded in cells subsequently found to be labeled. Non-neuronal, fibroblast-like cells and co-cultured myotubes were not labeled under the same conditions. The fact that not all of the neurons were labeled is consistent with the suggestion, based on studies of intact adult tissue, that high affinity transport of [3H]GABA may be unique to neurons that use GABA as a neurotransmitter. Our finding that none of fifteen physiologically identified cholinergic neurons, i.e., cells that innervated nearby myotubes, were heavily labeled after incubation in 0.1 microM [3H]GABA is significant in this regard. The newly taken up [3H]GABA was not metabolized in the short run. It was stored in a form that could be released when the neurons were depolarized in a high K+ (100 mM) medium. As expected for a neurotransmitter, the K+-evoked release was reversibly inhibited by reducing the extracellular Ca++/Mg++ ratio.     

Differential effects of zinc on native GABA(A) receptor function in rat hippocampus and cerebellum.

Hippocampal noradrenergic and cerebellar glutamatergic granule cell axon terminals possess GABA(A) receptors mediating enhancement of noradrenaline and glutamate release, respectively. The hippocampal receptor is benzodiazepine-sensitive, whereas the cerebellar one is not affected by benzodiazepine agonists, indicating the presence of an alpha6 subunit. We tested here the effects of Zn2+ on these two native GABA(A) receptor subtypes using superfused rat hippocampal and cerebellar synaptosomes. In the cerebellum, zinc ions strongly inhibited (IC50 approximately 1 microM) the potentiation of the K(+)-evoked [3H]D-aspartate release induced by GABA. In contrast, the GABA-evoked release of [3H]noradrenaline from hippocampal synaptosomes was much less sensitive to Zn2+ (IC50 > 30 microM). The effects of Zn2+ were then studied in two rat lines selected for high (ANT) and low (AT) alcohol sensitivity because granule cell GABA(A) receptors in ANT, but not AT, rats respond to benzodiazepine agonists due to a critical mutation in the alpha6 subunit. GABA increased the K(+)-evoked release of [3H]DCNS REGIONS-aspartate from cerebellar synaptosomes of AT and ANT rats, an effect prevented by the GABAA selective antagonist bicuculline. In AT rat cerebellum, the effect of GABA was strongly inhibited by Zn2+ (IC50 < or = 1 microM), whereas in ANT rats, the divalent cation was about 100-fold less potent. Thus, native benzodiazepine-sensitive GABAA receptors appear largely insensitive to functional inhibition by Zn2+ and vice versa. Changes in sensitivity to Zn2+ inhibition consequent to mutations in cerebellar granule cell GABA(A) receptor subunits may lead to changes in glutamate release from parallel fibers onto Purkinje cells and may play important roles in cerebellar dysfunctions.     

Functional characteristics of nerve endings isolated from brain by the Hajos method

A study was made of the functional potentialities of synaptosomes isolated from the brain cortex and lumbar enlargement of the spinal cord. The yield of synaptosomes from the brain cortex amounted to 10 mg (with reference to protein) from 1 g of wet tissue, and that of synaptosomes from the spinal cord was equal to 1/3 of the yield from the brain, with the preparation being strongly contaminated with myelin scraps. Brain synaptosomes were marked by high level of respiration whose magnitude was affected by the agents (ouabain, high concentrations of K+ and benzylpenicillin) that change ion membrane transport. Synaptosomes maintained higher GABA gradient across the plasmatic membrane. Ouabain and potassium depolarization produced a considerable release of GABA and 3H-GABA into the incubation medium. A conclusion is made that the method of Hajos should be rather used for rapid isolation of the synaptosomal fraction from the rat brain cortex.     

Rapid release of substance P and LH-RH from synaptosomes prepared from the medial basal hypothalamus and substantia nigra

We investigated Ca2+-dependent, depolarization-induced release of substance P (SP) and LH-RH from medial basal hypothalamic (MBH) and substantia nigra (SN) synaptosomes prepared from male rat brain. Depolarization of MBH synaptosomes evoked significant release of SP from 10.0 +/- 0.1 (5 mM K+) to 28.0 +/- 2.4 (75 mM K+) pg released/10 seconds. Fractional release was 1.0% and 2.7% respectively. In contrast, LH-RH was not released by depolarization of MBH synaptosomes: 11.6 +/- 0.9 (5 mM K+) to 11.0 +/- 0.7 (75 mM K+) pg released/10 seconds. Fractional release was 1.1 and 1.0% respectively. Depolarization-induced LH-RH release also did not occur in the presence of 10(-4) or 10(-6) M norepinephrine, 10(-7) M 12-O-tetradecanoylphorbol-13-acetate (TPA, PMA), 10(-5) M forskolin or in female rats. The inability of depolarizing concentrations of K+ to stimulate LH-RH release in physiological buffers remains an enigma. Significant depolarization-induced SP release was seen from MBH and SN synaptosomes at 20, 15, 10, 5 and only 1 second of release. Despite comparable basal release of SP from MBH and SN synaptosomes, the rate and magnitude of evoked release were much more pronounced in SN synaptosomes. The initial rate (0-1 second) of SP release was 4.5-fold greater from SN than from MBH synaptosomes [krel = 0.027(-1) (SN), krel = 0.006(-1) (MBH)]. The magnitude of SP release from SN synaptosomes was 2- to 3-fold greater at any given time interval compared with release from MBH synaptosomes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Binding of Lanthanum Ions and Ruthenium Red to Synaptosomes and Its Effects on Neurotransmitter Release

A technique for studying the binding of La3+ to synaptosomes in a double-beam spectrophotometer, using murexide as indicator, is described. The binding of La3+ was very rapid and Scatchard plots revealed two components, with KD values of 0.6 and 27 microM in a Na+-free medium (sucrose medium) and 2.3 and 63 microM in an ionic medium containing 135 mM Na+. The binding of the cationic dye ruthenium red (RuR) showed only one site, with a KD of 3.7 microM. La3+ binding was partially inhibited by RuR and vice versa, and La3+ was also capable of partially displacing RuR previously bound to the synaptosomes, particularly in the sucrose medium. The release of labeled gamma-aminobutyric acid (GABA) stimulated by K+ depolarization was inhibited by La3+ concentrations at or above 1 microM, in the ionic medium, whereas in the sucrose medium 2.5 microM or higher La3+ concentrations notably stimulated the spontaneous release of both GABA and glutamic acid. It is concluded that La3+ and RuR share at least one type of binding site, which is probably the high-affinity La3+ site. Since both La3+ and RuR at low concentrations have been shown to block the depolarization-induced Ca2+ entry in synaptosomes, this site might be related to the voltage-dependent Ca2+ entry involved in neurotransmitter release.     

CALCIUM-INDUCED RELEASE OF γ-AMINOBUTYRIC ACID FROM SYNAPTOSOMES: EFFECTS OF TRANQUILIZER DRUGS

Efflux of preloaded radioactive gamma-aminobutyric acid (GABA) from mouse brain fractions enriched in synaptosomes could be significantly and reproducibly increased by switching from Ca²⁺-free wash solution to one containing 2 mM-Ca²⁺. With a filtration apparatus equipped for simultaneous buffer changes of eight samples, effects of various drugs on Ca²⁺-dependent and Ca²⁺-independent efflux of GABA were studied. Several membrane-perturbing agents, including imipramine and neuroleptics like haloperidol and chlorpromazine, at 1 μM, potently inhibited Ca²⁺-dependent GABA release. Benzodiazepine drugs similarly inhibited Ca²⁺-dependent efflux, but also under appropriate conditions, enhanced Ca²⁺-independent efflux. Stimulation of Ca²⁺-independent efflux was observed with nonradioactive GABA and with GABA analogues which inhibit GABA transport, reflecting exchange of GABA via the high-affinity transport system. Diazepam was found to inhibit GABA transport (non-competitively; I₅₀, 50 μM); effects on the transport system might be related to observations of potentiated GABA action and anti-convulsant effects of this drug.     

Tricyclic antidepressants inhibit voltage-dependent calcium channels and Na(+)-Ca2+ exchange in rat brain cortex synaptosomes

We have used a resting (5 mM K+) or depolarizing (60 mM K+) choline-based medium, and a nondepolarizing sodium-based or choline-based medium, to characterize the inhibitory potential of tricyclic antidepressants against the voltage-dependent calcium channels or the Na(+)-Ca2+ exchange process, respectively, in synaptosomes from rat brain cortex. Imipramine, desipramine, amitriptyline, and clomipramine inhibited net K(+)-induced 45Ca uptake with similar IC50 values (26-31 microM), and this uptake was also inhibited by diltiazem with an IC50 of 36 microM; these results indicate an inhibition of voltage-dependent calcium channels by tricyclic antidepressants. The net uptake of 45Ca induced by Na(+)-Ca2+ exchange was also inhibited by the four tricyclic antidepressants tested, but not by diltiazem; imipramine (IC50 = 94 microM) was a more potent inhibitor of this process than desipramine (IC50 = 151 microM), and the IC50 values of amitriptyline (107 microM) and clomipramine (97 microM) were similar to that of imipramine. Some degree (approximately 25%) of brain calcium channel blockade could be present at the steady-state concentrations of tricyclic antidepressants expected to occur therapeutic use of these compounds to treat depression or panic disorder.     

Calcium-Independent Release of Acetylcholine from Electric Organ Synaptosomes and Its Changes by Depolarization and Cholinergic Drugs

Chemiluminescent detection was applied to measure the continuous spontaneous Ca2+-independent liberation of acetylcholine (ACh) from Torpedo electric organ synaptosomes. Differentiation between the release of ACh and choline was achieved by inhibiting cholinesterases with phospholine, and a way to quantify the continuous release was devised. The method permitted measurements during short time intervals from minute amounts of tissue and without an accumulation of ACh in the medium. Synaptosomes continuously liberated small amounts of ACh during incubations in the presence of 3 mM K+ and in the absence of Ca2+. The spontaneous liberation of ACh was similar both quantitatively and qualitatively at pH values of 8.6 and 7.8. It was unaltered by MgCl2 (10.4 mM), 2-(4-phenylpiperidino)cyclohexanol (10 microM), ouabain (104 microM), atropine (10 microM), and valinomycin (102 nM). Carbamoylcholine brought about a decrease, which could be partially reversed by atropine. The Ca2+-independent output of ACh was increased considerably when the concentration of K+ ions was raised (eightfold at 103 and 35-fold at 203 mM K+). Carbamoylcholine (104 microM) blocked the increase in ACh release produced by high K+; this effect of carbamoylcholine was not reversed by atropine (10 microM). When Ca2+ was added to synaptosomes depolarized by a high concentration of K+, the amount of ACh released during the first 1-3 min after the addition of Ca2+ was at least 20 times higher than in the absence of Ca2+, but the release returned rapidly to predepolarization values. Similarly high values of ACh release could be achieved by adding Ca2+ plus the ionophore A23187 and even higher values by adding Ca2+ plus gramicidin.(ABSTRACT TRUNCATED AT 250 WORDS)