Modulation of calcium uptake and D-aspartate release by GABAB receptors in cultured cerebellar granule cells

(-)Baclofen, a GABAB receptor agonist, and GABA attenuated by 60% the high K+-evoked 45Ca2+ uptake into cultured cerebellar granule cells with an EC50 of 110 +/- 18 nM and 2.4 +/- 0.2 microM, respectively. The attenuation by baclofen of 45Ca2+ uptake was stereospecific and the effect of GABA was unaffected by bicuculline. Moreover, muscimol, a GABAA receptor agonist did not affect the K+-evoked 45Ca2+ uptake. (-)Baclofen and GABA also decreased the K+-evoked and calcium-dependent release of preloaded [3H]D-aspartate from granule cells; however, their potency and efficacy appeared to be less than those for inhibiting the 45Ca2+ uptake. (+)Baclofen and muscimol failed to change this K+-evoked release. The release of [3H]D-aspartate induced by the calcium ionophore A23187 was unaffected by (-)-baclofen. The K+-evoked release of [3H]D-aspartate was effectively inhibited by nimodipine, a voltage sensitive calcium channel blocker. The results suggest that GABAB receptor in cultured cerebellar granule cells plays a crucial role in modulating the uptake of calcium and release of the excitatory transmitter. Moreover, these two effects mediated by GABAB receptor activation may be casually related.     

Release-regulating autoreceptors of the GABAB-type in human cerebral cortex.

1. The depolarization-evoked release of gamma-aminobutyric acid (GABA) and its modulation mediated by autoreceptors were investigated in superfused synaptosomes prepared from fresh human cerebral cortex. 2. The release of [3H]-GABA provoked by 15 mM K+ from human cortex nerve endings was almost totally (85%) calcium-dependent. 3. In the presence of the GABA uptake inhibitor SK&F 89976A (N-(4,4-diphenyl-3-butenyl)-nipecotic acid), added to prevent carrier-mediated homoexchange, GABA (1-10 microM) decreased in a concentration-dependent manner the K+-evoked release of [3H]-GABA. The effect of GABA was mimicked by the GABAB receptor agonist (-)-baclofen (1-100 microM) but not by the GABAA receptor agonist muscimol (1-100 microM). Moreover, the GABA-induced inhibition of [3H]-GABA release was not affected by two GABAA receptor antagonists, bicuculline or SR 95531 (2-(3'-carbethoxy-2'-propenyl)-3-amino-6-paramethoxy-phenyl-pyr idazinium bromide). 4. (-)-Baclofen also inhibited the depolarization-evoked release of endogenous GABA from human cortical synaptosomes. 5. It is concluded that GABA autoreceptors regulating the release of both newly taken up and endogenous GABA are present in human brain and appear to belong to the GABAB subtype.     

Human brain somatostatin release from isolated cortical nerve endings and its modulation through GABAB receptors.

1. The release of somatostatin-like immunoreactivity (SRIF-LI) in the human brain was studied in synaptosomal preparations from fresh neocortical specimens obtained from patients undergoing neurosurgery to remove deeply sited tumours. 2. The basal outflow of SRIF-LI from superfused synaptosomes was increased about 3 fold during exposure to a depolarizing medium containing 15 mM KCl. The K(+)-evoked overflow of SRIF-LI was almost totally dependent on the presence of Ca2+ in the superfusion medium. 3. The GABAB receptor agonist, (-)-baclofen (0.3 - 100 microM), inhibited the overflow of SRIF-LI in a concentration-dependent manner (EC50 = 1.84 +/- 0.20 microM; maximal effect: about 50%). The novel GABAB receptor ligand, 3-aminopropyl(difluoromethyl)phosphinic acid (CGP 47656) mimicked (-)-baclofen in inhibiting the SRIF-LI overflow (EC50 = 3.06 +/- 0.52 microM; maximal effect: about 50%), whereas the GABAA receptor agonist, muscimol, was ineffective up to 100 microM. 4. The inhibition by 10 microM (-)-baclofen of the K(+)-evoked SRIF-LI overflow was concentration-dependently prevented by two selective GABAB receptor antagonists, 3-amino-propyl (diethoxymethyl)-phosphinic acid (CGP 35348) (IC50 = 24.40 +/- 2.52 microM) and [3-[[(3,4-dichlorophenyl) methyl]amino]propyl] (diethoxymethyl) phosphinic acid (CGP 52432) (IC50 = 0.06 +/- 0.005 microM). 5. The inhibition of SRIF-LI overflow caused by 10 microM CGP 47656 was abolished by 1 microM CGP 52432. 6. When human synaptosomes were labelled with [3H]-GABA and depolarized in superfusion with 15 mM KCl, the inhibition by 10 microM (-)-baclofen of the depolarization-evoked [3H]-GABA overflow was largely prevented by 10 microM CGP 47656 which therefore behaved as an autoreceptor antagonist. 7. In conclusion: (a) the characteristics of SRIF-LI release from synaptosomal preparations of human neocortex are compatible with a neuronal origin; (b) the nerve terminals releasing the neuropeptide possess inhibitory receptors of the GABAB type; (c) these receptors differ pharmacologically from the GABAB autoreceptors present on human neocortex nerve terminals since the latter have been shown to be CGP 35348-insensitive but can be blocked by CGP 47656.     

Characteristics of GABAB receptor binding sites on rat whole brain synaptic membranes.

1 Saturable binding of (+/-)-[3H]-baclofen and [3H]-gamma- aminobutyric acid ([3H]-GABA) to rat brain crude synaptic membranes has been examined by means of a centrifugation assay. 2 The binding of [3H]-baclofen could be detected in fresh or previously frozen tissue and was dependent on the presence of physiological concentrations of Ca2+ or Mg2+ although a lower affinity Na+ -dependent component could also be observed. Both components probably reflect binding to receptor recognition sites. 3 The saturable portion of bound [3H]-baclofen formed 20.3 +/- 6.9% of total bound ligand. This could be displaced by GABA (IC50 = 0.04 microM), (-)-baclofen (0.04 microM) and to a much lesser extent by (+)-baclofen (33 microM). Isoguvacine, piperidine-4-sulphonic acid and bicuculline methobromide were inactive (up to 100 microM) and muscimol was only weakly active (IC50 = 12.3 microM). 4 Saturable binding of [3H]-GABA increased on adding CaCl2 or MgSO4 (up to 2.5 mM and 5.0 mM respectively) to the Tris-HCl incubation solution. This binding (GABAB site binding) was additional to the bicuculline-sensitive binding of GABA (GABAA site binding) and could be completely displaced by (-)-baclofen (IC50 = 0.13 microM). 5 Increasing the Ca2+ concentration (0 to 2.5 mM) increased the binding capacity of the membranes without changing their affinity for the ligand. 6 The binding of [3H]-GABA to GABAB sites could be demonstrated in fresh as well as previously frozen membranes with a doubling of the affinity being produced by freezing. Further incubation with the non-ionic detergent Triton-X-100 (0.05% v/v) reduced the binding capacity by 50%. 7 The pharmacological profile of displacers of [3H]-GABA from GABAB sites correlated well with that for [3H]-baclofen displacement. A correlation with data previously obtained in isolated preparations of rat atria and mouse vas deferens was also apparent. 8 It is concluded that [3H]-baclofen or [3H]-GABA are both ligands for the same bicuculline-insensitive, divalent cation-dependent binding sites in the rat brain.     

GABA terminal autoreceptors in the pars compacta and in the pars reticulata of the rat substantia nigra are GABAB

The depolarization-evoked release of gamma-aminobutyric acid (GABA) and its modulation mediated by autoreceptors were studied in superfused synaptosomes prepared from the pars compacta and from the pars reticulata of the rat substantia nigra. The release of [3H]GABA evoked by 9 mM KCl was almost totally calcium-dependent in both nigral subregions. In the presence of SK&F 89976A (N-(4,4-diphenyl-3-butenyl)nipecotic acid), a GABA uptake inhibitor added to minimize carrier-mediated homoexchange, GABA (0.3-10 microM) inhibited, in a concentration-dependent way, the K(+)-evoked overflow of [3H]GABA from both pars compacta and pars reticulata synaptosomes. Similarly to GABA, (-)-baclofen (0.3-10 microM) reduced the [3H]GABA overflow, being roughly equipotent to GABA in both nigral subregions. The (+) enantiomer of baclofen was ineffective. The overflow of [3H]GABA was not consistently affected by muscimol in either the pars compacta or the pars reticulata. The effects of GABA were bicuculline- and picrotoxin-insensitive. However, the inhibition by GABA of the [3H]GABA overflow was antagonized by phaclofen. It is concluded that (a) GABA autoreceptors are sited on GABAergic nerve endings in both the pars compacta and pars reticulata of the rat substantia nigra; (b) these autoreceptors belong to the GABAB type.     

Studies on [3H]GABA and endogenous GABA release in rat cerebral cortex suggest the presence of autoreceptors of the GABAB type

The presence of autoreceptors for gamma-aminobutyric acid (GABA) in the CNS was reinvestigated using rat cortex synaptosomes prelabeled with [3H]GABA and exposed to GABA by superfusion in the presence of a new GABA uptake inhibitor, N-(4,4-diphenyl-3-butenyl)-nipecotic acid (SK&F 89976A). This compound itself did not increase the basal or the depolarization-evoked release of [3H]GABA. GABA reduced in a concentration-dependent way the release of [3H]GABA evoked by 15 mM K+. The effect was not antagonized by bicuculline, picrotoxin or by the new GABAA antagonist SR 95531. The GABAA agonist muscimol did not affect [3H]GABA release. This was reduced by (-)baclofen (but not by the (+) isomer) and the concentration-inhibition curve of (-)baclofen was superimposable on to that of GABA. Also the K+-evoked release of endogenous GABA was stereoselectively and concentration dependently inhibited by the (-) enantiomer of baclofen. It is concluded that the release of GABA from rat cortical nerve endings may be inhibited through the activation of autoreceptors which appear to belong to the GABAB type.     

GABAB receptors are up-regulated by chronic treatment with lithium or carbamazepine. GABA hypothesis of affective disorders?

The effects of lithium and carbamazepine on GABAA and GABAB receptors were examined. The binding of [3H]muscimol and [3H](-)-baclofen to synaptic membranes from rat brain was used to label GABAA and GABAB receptors, respectively. Neither the [3H]muscimol nor the [3H](-)-baclofen binding site was displaced by lithium or carbamazepine even at a concentration of 100 microM. A single treatment with either of these drugs did not induce any change in [3H]muscimol and [3H](-)-baclofen binding sites in the frontal cortex and hippocampus. [3H](-)-Baclofen binding sites were up-regulated in the hippocampus but not in the frontal cortex following chronic treatment with lithium or carbamazepine. These results suggest that one common mechanism of action of lithium and carbamazepine is mediated by GABAB receptors and that GABA is involved in the pathophysiology of affective disorders.     

Paradoxical antagonism by bicuculline of the inhibition by baclofen of the electrically evoked release of [3H]GABA from rat cerebral cortex slices

The presynaptic regulation of the electrically evoked release of [3H]GABA was studied in the rat cerebral cortex. Among the GABA receptor agonists tested (GABA, SL 75102, muscimol, THIP, isoguvacine, (+/-)-baclofen), only (+/-)-baclofen inhibited the stimulation-evoked release of [3H]GABA. This effect of baclofen was stereoselective in favor of the (-) enantiomer. The inhibition by (+/-)-baclofen of the electrically evoked release of [3H]GABA was antagonized by bicuculline and picrotoxin. Our results suggest that the release of [3H]GABA in vitro can be modulated by a receptor-mediated mechanism which is sensitive to baclofen, bicuculline and picrotoxin but not to GABA, muscimol or THIP.     

Activation of alpha 6 GABAA receptors on depolarized cerebellar parallel fibers elicits glutamate release through anion channels.

Rat cerebellar synaptosomes labeled with [3H]D-aspartate ([3H]D-ASP) were exposed in superfusion to muscimol. The GABA(A) receptor agonist did not affect [3H]D-ASP basal release or the overflow provoked by 15mM K(+); muscimol potentiated the 35mM K(+)-evoked overflow of [3H]D-ASP or endogenous glutamate. Membrane potential measured by Rhodamine 6G fluorescence was -65mV under resting conditions and -32mV in the presence of 35mM K(+). The membrane potential was not significantly affected by muscimol. The muscimol effect on the K(+)(35mM)-evoked [3H]D-ASP overflow was not inhibited by omitting external Ca(2+) or by entrapping BAPTA to chelate cytosolic Ca(2+). Muscimol lost its ability to release glutamate following superfusion with D-aspartate to deplete cytosolic glutamate by heteroexchange suggesting that GABA(A) receptor activation elicits release of cytosolic glutamate. The non-transportable glutamate carrier blockers dihydrokainate or DL-TBOA did not reduce the muscimol potentiation. This was abolished by the anion channel blockers niflumic acid and NPPB. To conclude, when cerebellar parallel fiber terminals are sufficiently depolarized, activation of alpha6 GABA(A) receptors on these terminals mediates glutamate release in addition to that evoked by depolarization. This extra-release does not occur by exocytosis or transporter reversal but involves the opening of anion channels present on parallel fiber terminals.     

Effectiveness of GABAB antagonists in inhibiting baclofen-induced reductions in cytosolic free Ca concentration in isolated melanotrophs of rat.

1. The purpose of the present experiments was to assess the activities of GABAB receptor antagonists in mammalian isolated melanotrophs. 2. Cytosolic free Ca concentration ([Ca2+]i) in rat melanotrophs in primary culture was monitored with the fluorescent probe, fura-2. 3. (-)-Baclofen lowered [Ca2+]i in a concentration-dependent manner with an EC50 of 0.96 microM. The reduction in [Ca2+]i produced by (-)-baclofen at a maximally effective concentration (100 microM) was similar to that produced by the classic transmitter inhibitory to melanotroph secretion, dopamine, at a corresponding concentration (100 nM), or by perifusion with a nominally Ca-free solution. 4. The GABAB receptor antagonists, 3-aminopropyl(diethoxymethyl)phosphinic acid (CGP 35348), 2-hydroxy saclofen, phaclofen and 4-amino-3-(5-methoxybenzo[b]furan-2-yl) butanoic acid (9H), had inhibitory effects on the reduction in [Ca2+]i produced by (-)-baclofen (3 microM). Of the antagonists tested, CGP 35348 was the most potent with an IC50 of 60 microM, compared to 120 to 400 microM for the others. CGP 35348 acted competitively. 5. CGP 35348 alone had no effect on basal [Ca2+]i, or on the changes in [Ca2+]i produced by dopamine (10 nM) or the specific GABAA receptor agonist, muscimol (10 microM). 6. The evidence indicates that of the antagonists tested, CGP 35348 offers the greatest promise for pharmacological analysis of the functional significance of the GABAB receptors in melanotrophs.     

Autoreceptors modify the evoked release of [3H]GABA from cerebellar neurons in dissociated cell culture

We investigated the effect of GABA, muscimol and THIP on the K+ -stimulated and spontaneous release of [3H]GABA from neuron-enriched cell cultures of the rat cerebellum. Each agonist produced significant reductions in evoked [3H]GABA without causing marked changes in spontaneous release. The agonist-induced inhibition of K+ -stimulated [3H]GABA release was reversed by the GABA antagonists bicuculline and picrotoxin. It is suggested that GABAergic neurons in cerebellar cell cultures possess GABA receptors which are involved in the regulation of evoked transmitter release.     

Modulation of the GABA autoreceptor by benzodiazepine receptor ligands

The effects of various benzodiazepine receptor ligands on the GABA autoreceptor have been studied in slices of cerebral cortex of the rat. The GABAA receptor agonist muscimol inhibited the K+-stimulated release of [3H]GABA with a pIC25 of 7.65 +/- 0.11. This effect was antagonised by the GABAA receptor antagonist bicuculline, which had an IC50 of 0.36 +/- 0.03 microM. Small concentrations (less than 1 microM) of the benzodiazepine full agonist clonazepam did not significantly alter K+-evoked release of [3H]GABA but shifted the concentration-effect curve for muscimol to the left. This effect was blocked by the benzodiazepine antagonist flumazenil. By contrast, the benzodiazepine full inverse agonist methyl beta-carboline-3-carboxylate shifted the muscimol concentration-effect curve to the right and this too was blocked by flumazenil. The results suggest that the GABA autoreceptor in cortical slices from the rat is modulated by a benzodiazepine receptor.     

GABAB receptors modulate catecholamine secretion in chromaffin cells by a mechanism involving cyclic AMP formation.

1. The function of gamma-aminobutyric acidB (GABAB) receptors in modulation of catecholamine secretion by chromaffin cells and the possible mechanism involved in this action have been examined. 2. The GABAB agonists (-)-baclofen and 3-aminopropylphosphinic acid (3-APPA) were found to induce a dose-dependent increase of basal catecholamine secretion. The EC50s were 151 +/- 35 microM and 225 +/- 58 microM for baclofen and 3-APPA, respectively. This stimulatory effect was specific since it could be blocked by 0.5 mM of the specific GABAB antagonist CGP-35348. 3. In contrast, preincubation of chromaffin cells with the GABAB agonists was found to inhibit, in a dose-dependent manner, the catecholamine secretion evoked by 10 microM nicotine and 200 microM muscimol. 4. The effects of GABAB agonists on both basal and evoked catecholamine secretion were found to be accompanied by parallel changes in intracellular calcium concentration ([Ca2+]i). GABAB agonists produced a dose-dependent increase in [Ca2+]i which was partially blocked by CGP 35348, but they produced a strong inhibition of the [Ca2+]i increase induced by nicotine and muscimol. 5. The GABAB agonists also produced a dose-dependent increase in intracellular cyclic AMP levels, there being a direct correlation between both increase in catecholamine secretion and in intracellular cyclic AMP levels. 6. The pretreatment of chromaffin cells with pertussis toxin doubled the catecholamine secretion and increased by four times the intracellular cyclic AMP levels evoked by GABAB agonists. 7. The possible involvement of adenylate cyclase in the mechanism of GABAA receptor modulation of catecholamine secretion is discussed.     

Glycine stimulates [3H]noradrenaline release by activating a strychnine-sensitive receptor present in rat hippocampus

Rat hippocampus slices were prelabeled with [3H]noradrenaline ([3H]NA) and depolarized by superfusion with KCl. The release evoked by 12 mM K+ was totally calcium-dependent and more than 90% tetrodotoxin (TTX)-sensitive. Glycine (0.1-1 mM) increased the K(+)-evoked [3H]NA overflow in a concentration-dependent manner. The effect of 1 mM glycine reached 300%. Strychnine (0.3 microM) shifted to the right the concentration-response curve for glycine. The effect of glycine (0.1 or 1 mM) was totally abolished by 3 microM strychnine but was unaffected by the GABAA receptor antagonist, bicuculline (10 microM), or by 100 microM of 1-hydroxy-3-aminopyrrolidone-2 (HA-966), a proposed antagonist of glycine at the strychnine-insensitive site located on the N-methyl-D-aspartate (NMDA) receptor. The effect of glycine was mimicked by L-serine, although less potently; the release of [3H]NA was enhanced by 200% in presence of 3 mM L-serine. At this concentration D-serine was ineffective. Strychnine shifted to the right the concentration-response curve for L-serine. Glycine (1 mM) had only a minor effect (less than 20% potentiation) on the release of [3H]NA evoked by 12 mM KCl in hippocampal synaptosomes. While the effect of glycine in slices was increased by decreasing the depolarizing concentration of K+ (about 500% potentiation at 9 mM K+), the response of synaptosomes remained minimal, even in presence of 9 mM KCl. Hippocampal synaptosomes prelabeled with [3H]glycine released the radiolabeled amino acid when exposed to superfusion with 12 mM KCl. The release of [3H]glycine was more than 75% calcium-dependent. The results suggest that the release of NA in rat hippocampus may be enhanced by glycine through the activation of a strychnine-sensitive receptor. This receptor does not seem to be located on noradrenergic terminals.     

Diabetes-induced changes in cardiac beta-adrenoceptor responsiveness: effects of aldose reductase inhibition with ponalrestat.

1. Segments of the rat vena cava preincubated with [3H]-noradrenaline were superfused with [3H]-noradrenaline-free solution containing desipramine and corticosterone and the effects of gamma-aminobutyric acid (GABA) receptor ligands and of histamine on tritium overflow evoked by transmural electrical stimulation were studied. 2. GABA inhibited, and histamine failed to affect, the electrically (0.66 Hz) evoked tritium overflow both in the absence and presence of rauwolscine (which was present in the superfusion medium in the subsequent experiments). The effect of GABA was less pronounced at a stimulation frequency of 2 Hz. 3. The inhibitory effect of GABA (pIC35 5.83) on the electrically (0.66 Hz) evoked overflow was mimicked by the GABAB receptor agonist, R-(-)-baclofen (6.07) and less potently by S-(+)-baclofen (3.30) and the GABAA receptor agonist, muscimol (3.70). The concentration-response curve of GABA was shifted to the right by the GABAB receptor antagonist, CGP 35348 (P-(3-aminopropyl)-P- diethoxymethyl-phosphinic acid; apparent pA2 4.76), but not affected by the GABAA receptor antagonist, (-)-bicuculline methiodide 100 mumol l-1. Given alone, (-)-bicuculline methiodide slightly increased, and CGP 35348 did not affect, the evoked overflow. 4. The effect of GABA in veins from rats treated for 14 days with RS-baclofen (10 mg kg-1, i.p. once daily) did not differ from that in veins from rats which received the vehicle instead. The effect of GABA also did not differ in veins from rats treated once either with RS-baclofen or with its vehicle.(ABSTRACT TRUNCATED AT 250 WORDS)     

Baclofen and phaclofen modulate GABA release from slices of rat cerebral cortex and spinal cord but not from retina.

1. The effects of (-)-baclofen, muscimol and phaclofen on endogenous gamma-aminobutyric acid (GABA) release from rat cortical slices, spinal cord slices and entire retinas were studied. 2. The spontaneous resting release of GABA from the three tissues was 3 to 6 pmol mg-1 wet wt 10 min-1. Depolarization of cortical slices with KCl (50 mM) (high-K) produced an 8 fold increase in GABA release but high-K did not evoke an increased release of GABA from spinal slices or retinas. 3. When rats were injected with gamma-vinyl-GABA (250 mg kg-1 i.p.) (GVG) 18 h before death, the tissue GABA stores were increased 3 to 6 fold and high-K then evoked striking Ca-dependent releases of GABA from all three tissues. Thus, in subsequent experiments, unless otherwise stated, the nervous tissues were taken from GVG-treated rats. 4. (-)-Baclofen (10 microM) significantly reduced the K-evoked release of GABA from cortical and spinal slices but retinal release was not affected, even at a concentration of (+/-)-baclofen of 1 mM. For cortical slices, the IC50 for baclofen was approximately 5.2 microM. The inhibitory effect of baclofen on GABA release from cortical slices also occurred in slices prepared from saline-injected rats, indicating that GVG treatment did not qualitatively affect the results. 5. The inhibitory effect of (-)-baclofen on the K-evoked release of GABA from cortical and spinal slices was antagonised by phaclofen (500 microM), confirming that baclofen was producing its effects by acting at the GABAB-receptor.(ABSTRACT TRUNCATED AT 250 WORDS)     

Coexistence of carriers for dopamine and GABA uptake on a same nerve terminal in the rat brain.

The ability of gamma-aminobutyric acid (GABA) to affect the release of [3H]-dopamine in rat brain synaptosomes prepared from corpus striatum, frontal cortex and hypothalamus and prelabelled with the radioactive catecholamine in the presence of desipramine was examined. GABA (10-300 microM) increased in a concentration-dependent way the basal release of [3H]-dopamine from striatum and cortical synaptosomes; however, its effect was much less pronounced in hypothalamic nerve terminals. 2,4-Diaminobutyric acid (DABA) mimicked GABA although less potently. Neutral amino acids such as leucine, valine or alpha-aminoisobutyric acid (100-300 microM) did not affect or increased minimally the release of [3H]-dopamine. The GABA-induced [3H]-dopamine release was not prevented by the GABAA-receptor antagonists, bicuculline or picrotoxin. The GABAA-receptor agonist, muscimol (10-300 microM), displayed only a very weak, not significant, enhancing effect on [3H]-dopamine release. The GABAB-receptor agonist (-)-baclofen (100 or 300 microM) had no effect. Three novel and selective inhibitors of GABA uptake, N-(4,4-diphenyl-3-butenyl)-nipecotic acid (SK&F 89976A), N-(4,4-diphenyl-3-butenyl)-guvacine (SK&F 100330A) and N-(4,4-diphenyl-3-butenyl)-homo-beta-proline (SK&F 100561) potently counteracted the enhancing effect of GABA on [3H]-dopamine release. Nipecotic acid also reduced the effect of GABA. It is concluded that carriers for the uptake of dopamine and GABA may coexist on the same nerve terminal in the rat brain.     

Stimulation of high affinity gamma-aminobutyric acidB receptors potentiates the depolarization-induced increase of intraneuronal ionized calcium content in cerebellar granule neurons.

In the treatment of spasticity, the therapeutic cerebrospinal fluid levels of (+/-)-baclofen, a gamma-aminobutyric acid (GABA)B receptor agonist, are below 1 microM. However, the mechanism of the therapeutic action of (+/-)-baclofen remains unknown, because, for the most part, the action of (+/-)-baclofen on GABAB receptors requires micromolar concentrations. Using fura-2 fluorescence microscopy, intracellular ionized calcium was measured in cerebellar granule neurons. Stimulation of a high affinity GABAB receptor potentiated by 2-3-fold the rise in intracellular calcium observed after depolarization of the cell with a Krebs Ringer's buffered solution containing 40 mM K+. Both GABA (100 nM) and (+/-)-baclofen (10-100 nM) stimulated this high affinity receptor. The potentiation of the depolarization-induced rise in intracellular calcium by (+/-)-baclofen (100 nM) was completely blocked by the GABAB receptor antagonist CGP 35348 (200 microM). Also, the intracellular calcium response induced by the activation of high affinity GABAB receptors was prevented by dantrolene (10 microM). The cerebellar granule neurons contained calcium-induced calcium release (CICR) stores. Caffeine (3 mM) and ryanodine (100 microM) potentiated the depolarization-induced rise in intracellular calcium, and this response to both drugs was blocked by dantrolene (10 microM). Because dantrolene does not prevent the rise in intracellular calcium after cell depolarization (this calcium originated from the influx of extracellular calcium), (+/-)-baclofen acting via the high affinity GABAB receptor indirectly activates the CICR stores, allowing the influx of extracellular calcium to trigger the release of calcium from these dantrolene-sensitive CICR stores. Thus, this high affinity GABAB receptor might become activated during persistent depolarization caused by pathological states and could be a mechanism to be studied for the therapeutic action of (+/-)-baclofen in spasticity.     

Effect of depolarizing agents on the Ca(2+)-independent and Ca(2+)-dependent release of [3H]GABA from sheep brain synaptosomes.

The purpose of the present study was to compare the effects of several depolarizing agents on both the membrane potential and on the release of [3H] gamma-aminobutyric acid (GABA) from sheep brain cortex synaptosomes. We examined the effects of KCl, 4-aminopyridine (4-AP), veratridine, ouabain and tetraphenylphosphonium cation (TPP+) on Ca(2+)-independent (carrier-mediated) and Ca(2+)-dependent (exocytotic) release. We found that, in the absence of Ca2+, KCl at 40 mM releases 7.57 +/- 0.65%, veratridine at 50 microM releases 45.85 +/- 2.48%, ouabain at 1 mM releases 8.62 +/- 0.93% and TPP+ at 1 mM releases 4.09 +/- 0.37% of the total accumulated neurotransmitter, provided that the external medium contains Na+. These are about the maximal values of release obtained with each depolarizing agent in a Na+ medium and in the absence of Ca2+. Replacing external Na+ with choline blocks the release observed in the presence of the depolarizing agents in the absence of Ca2+, and this divalent ion can increase [3H]GABA release only for K+ or 4-AP. Synaptosomal depolarization requires Na+ except for K+ depolarization. Furthermore, although Ca2+ stimulates the release of [3H]GABA due to K+ depolarization (13.56 +/- 0.44%) or due to 4-AP (4.26 +/- 0.51%), it inhibits the release due to the other depolarizing agents. The amount of [3H]GABA released by 4-AP in Na+ medium (4.26 +/- 0.51%) is similar to that induced by KCl in the presence of Ca2+ in the absence of Na+ (3.39 +/- 0.29%) which represents only exocytotic release. This suggests that the Ca(2+)-dependent exocytotic release of [3H]GABA can be specifically induced by 4-AP in a Na+ medium, or by KCl in the absence of Na+, as reported by us earlier. The observation that Ca2+ inhibits the Ca(2+)-independent release is of interest because it suggests that Ca2+ may modulate the release of cytoplasmic GABA probably by inhibiting the carrier-mediated release of GABA. It is of interest as to whether Ca2+ regulation depends on intracellular Ca2+.     

The effects of GABAB receptor agonists and antagonists on potassium-stimulated [Ca2+]i in rat brain synaptosomes

The effects of GABAB agonists and putative antagonists on intrasynaptosomal calcium ion concentrations ([Ca2+]i) after stimulation with potassium ions were studied with the fluorescent probe Quin 2. gamma-Aminobutyric acid and (-)-baclofen, but not (+)-baclofen, produced a dose-dependent inhibition of the potassium-stimulated [Ca2+]i in cortical synaptosomes from the rat. This effect was mimicked by another GABAB agonist SL75102 and weakly by muscimol. It was not inhibited by the alpha-adrenoceptor antagonist phentolamine. This system thus appears to provide a useful test of GABAB receptor function. None of the putative GABAB antagonists, phaclofen, delta-aminovaleric acid or beta-phenyl GABA inhibited responses to (-)-baclofen. Indeed, all three compounds produced similar responses to that seen with (-)-baclofen, suggesting that they act as agonists in this system. These data suggest that those GABAB receptors modulating [Ca2+]i have a distinct pharmacology from post-synaptic GABAB receptors, defined in electrophysiological experiments.