Peptide heterogeneity of GABAA/benzodiazepine receptors in bovine cerebral cortex and cerebellum.

The GABA_A/benzodiazepine receptor complex has been purified from both bovine cerebral cortex and cerebellum by immunoaffinity chromatography on immobilized monoclonal antibody 62-3G1. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified receptor from either cerebral cortex or cerebellum revealed 3 main bands corresponding to 51 000, 55 000 and 57 000M_r silver-stained peptides In addition, a minor band corresponding to a 53 000M_r peptide was also found. The difference between the two receptor preparations were: (1) that the main silver-stained 55 000M_r subunit was present in a relative smaller quantity in cerebellum than in cerebral cortex, and (2 when the membrane-bound receptor was photoaffinity-labeled with [³H]flunitrazepam and subsequently immunoaffinity-purified, two photolabeled peptide bands of 51 000 and 57 000M_r were found in cerebral cortex while only the 51 000M_r photolabeled peptide was detected cerebellum following one-dimension sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Peptide maps of the 57 000M_r [³H]flunitrazepam photoaffinity-labeled peptide indicated that it was composed of two closely migrating photolabeled peptides of 55 000M_r and 57 000M_r 0899 Peptide mapping and deglycosylation experiments using the [³H]flunitrazepam photolabeled receptor suggested that the photolabeled peptides commonly present in cerebellum and cerebral cortex are qualitatively similar if not identical. The results suggest that there are subunits of some type(s) of GABA_AR/BZDR complex(es) which are more abundant in cerebral cortex than in cerebellum. Photoaffinity labeling with [³H]muscimol showed similar photolabeled peptides in both cerebral cortex and cerebellum: two main peptides of 54 000 and 57 000M_r wer photolabeled with [³H]muscimol to a similar extent in both receptor preparations. Following deglycosylation, the mobility shifts of the peptides that were photolabeled with [³H]flunitrazepam or [³H]muscimol were different, suggesting that the co-migrating 54 000 – 57 000M_r peptides that have high affinity binding sites for [³H]flunitrazepam or [³H]muscimol are different receptor subunits.     

The GABAA/benzodiazepine receptor complex in rat brain neuronal cultures. Characterization by immunoprecipitation

The expression of the GABA_A/benzodiazepine receptor (GABAR/BZDR) complex in primary neuronal cultures from rat brain embryos has been investigated. The GABAR/BZDR complex was photoaffinity labeled with [³H]flunitrazepam [³H]FNZ and immunoprecipitated with subunit specific antibodies. These were the mAb 62-3G1 which is specific for the 57-kDa GABA binding subunit, and the rabbit antiserum A which recognizes the 51-kDa [³H]FNZ binding subunit. The results indicate that the cultured neurons express 5 different peptides of 51, 53, 54, 57 and 59 kDa that can be photoaffinity labeled with [³H]FNZ and that all of them are physically coupled to the GABA_A receptor. Most of the [³H]FNZ photolabeled peptides have similar mobilities to those found in the brain of the newborn rat. Nevertheless, some of the quantitative changes in the photolabeled peptides observed during the normal development of the rat brain were not observed or occurred at much slower pace in the cultured neurons.     

Clathrin-coated vesicles from bovine brain contain uncoupled GABAA receptors

Clathrin-coated vesicles are thought to be a vehicle for the sequestration of GABA_A receptors. For coated vesicles from bovine cerebrum, we examined the binding properties of [³H]muscimol, a GABA_A-specific agonist, [³H]flunitrazepam, a benzodiazepine agonist, and [³⁵S]t-butylbiocyclophosphorthionate (TBPS), a ligand for GABA_A receptor channels. Under standard conditions, the binding level of [³H]muscimol, [³H]flunitrazepam, and [³⁵S]TBPS to coated vesicles represented 12.3±1.8%, 7.9±1%, and 10.2±1.8%, respectively, of that in crude synaptic membranes. Coated vesicles showed a single [³H]flunitrazepam binding site with a K_D value (12 nM) which was 9-fold that for synaptic membranes. The allosteric coupling between binding sites was measured by the addition of GABA to [³H]flunitrazepam and [³⁵S]TBPS binding assays. For [³H]flunitrazepam binding to synaptic membranes, GABA gave an EC₅₀=2.0 μM and at saturation (100 μM) an enhancement of 122%. This stimulation was completely blocked by the GABA antagonist SR95531. In contrast, neither GABA nor SR95531 had a significant effect on [³H]flunitrazepam binding to CCVs, indicating that the allosteric interaction between GABA and benzodiazepine binding sites is abolished. Likewise, GABA displaced nearly all of the [³⁵S]TBPS binding to synaptic membranes but had no effect on binding to coated vesicles, indicating that coupling between the GABA binding sites and chloride channel is also impaired. Thus GABA_A receptors appear to be uncoupled during normal intracellular trafficking via coated vesicles. The presence of major GABA_A receptor subunits on these particles was verified by quantitative immunoblotting. Relative to the levels in synaptic membranes, CCVs contained 110±14% and 29.5±3.8%, respectively, of the immunoreactivity for GABA_A receptor β2 and α1 subunits. Thus, in comparison to GABA_A receptors on synaptic membranes, those on CCVs have a reduced α1/β2-subunit ratio. It may be suggested that a selective decline in the content of α1 subunits in coated vesicles could in part account for GABA_A receptor uncoupling.     

An investigation on the role of nitric oxide in the modulation of the binding characteristics of various radioligands of GABAA receptors by 5α-pregnan-3α-ol-20-one in the rat brain regions

Chronic administration of N^ω-nitro- -arginine methyl ester ( -NAME), an inhibitor of nitric oxide synthase, diminished the ability of 5α-pregnan-3α-ol-20-one, a neurosteroid, to potentiate the [³H]muscimol (5 nM) binding in the rat hippocampus but not in the cerebellum or cerebral cortex. This effect of NAME was stereospecific and susceptible to reversal by the pre-treatment of rats with -arginine. However, chronic administration of -NAME did not affect the modulation of the [³H]flunitrazepam (2 nM) or [³⁵S]TBPS (4 nM) binding by the neurosteroid in any of the brain regions investigated in this study. These results suggest that nitric oxide may be involved in some of the effects of neurosteroids in hippocampus.     

Age-related modifications on the GABAA receptor binding properties from Wistar rat prefrontal cortex

In the present communication we have investigated the pharmacological properties of the GABA_A receptor from adult (3 months old) and aged (24 months old) Wistar rat prefrontal cortex. The prefrontal cortex is implicated in cognitive functions and stress and both processes seem to be altered during aging. These changes could be mediated by modifications in the GABA_A receptor properties. Our results indicated the absence of generalized age-related modifications on the pharmacological properties of the GABA_A receptor from prefrontal cortical membranes. Saturation experiments using the non-selective benzodiazepine [³H]flunitrazepam revealed that neither the K_d values or the B_max were modified during aging. Moreover, Cl 218 872 displacement of [³H]flunitrazepam showed no age-related modifications on either the K_is or the relative proportion between the Type I and Type II benzodiazepine binding sites. Therefore, the benzodiazepine binding sites are well preserved in aged prefrontal cortex. On the other hand, saturation experiments using the GABA agonist [³H]muscimol demonstrated a decrease in the B_max of the low affinity [³H]muscimol binding sites in aged rats (4.3 ± 0.8 pmol/mg protein vs. 2.3 ± 0.2 pmol/mg protein in adult and aged rats, respectively). However, no age-dependent modifications were observed in the allosteric interaction between GABA and benzodiazepine binding sites. These results demonstrate that the benzodiazepine binding sites and the GABA binding sites of the GABA_A receptor complex from rat prefrontal cortical membranes are differentially affected by the aging process.     

Activation of protein kinase C by phorbol dibutyrate modulates GABAA receptor binding in rat brain slices

Effects of protein kinase C (PKC) activation on the function of the GABA/benzodiazepine receptor-chloride complex were analyzed by quantitative autoradiography using [³H]muscimol, [³H]flunitrazepam and [³⁵S]TBPS in rat brain slices. The density of [³H]muscimol binding was highest in cerebellar granular layers and high in both the frontal cortex and thalamus, but binding levels in the hippocampus were low. After activation of PKC by 100 nM phorbol-12,13-dibutyrate (PDBu), [³H]muscimol binding was decreased in the frontal cortex, striatum and thalamus, but binding levels were not changed in the hippocampus or cerebellum. The density of [³H]flunitrazepam binding was high in the cortex, hippocampus and molecular layers of cerebellum but was low in thalamus. PDBu increased the [³H]flunitrazepam binding only in the striatum and in part of the cortex and thalamus after activation of PKC. After activation of PKC by PDBu, [³⁵S]TBPS binding was increased in most areas, but binding levels were not changed in the brainstem or cerebellum. The receptor binding was markedly decreased in almost all areas by the addition of 2.5 mM Mg²⁺. Elevated [³⁵S]TBPS binding produced by PDBu was significantly inhibited by the addition of Mg²⁺. These results suggest that the activation of PKC potentiates benzodiazepine and TBPS binding, but decreases muscimol binding in a region-specific manner in the rat brain.     

Changes of GABAA receptor binding and subunit mRNA level in rat brain by infusion of subtoxic dose of MK-801

In the present study, we have investigated the effects of prolonged inhibition of NMDA receptor by infusion of subtoxic dose of MK-801 to examine the modulation of GABA_A receptor binding and GABA_A receptor subunit mRNA level in rat brain. It has been reported that NMDA-selective glutamate receptor stimulation alters GABA_A receptor pharmacology in cerebellar granule neurons in vitro by altering the levels of selective subunit. However, we have investigated the effect of NMDA antagonist, MK-801, on GABA_A receptor binding characteristics in discrete brain regions by using autoradiographic and in situ hybridization techniques. The GABA_A receptor bindings were analyzed by quantitative autoradiography using [³H]muscimol, [³H]flunitrazepam, and [³⁵S]TBPS in rat brain slices. Rats were infused with MK-801 (1 pmol/10 μl per h, i.c.v.) for 7 days, through pre-implanted cannula by osmotic minipumps (Alzet, model 2ML). The levels of [³H]muscimol binding were highly elevated in almost all of brain regions including cortex, caudate putamen, thalamus, hippocampus, and cerebellum. However, the [³H]flunitrazepam binding and [³⁵S]TBPS binding were increased only in specific regions; the former level was increased in parts of the cortex, thalamus, and hippocampus, while the latter binding sites were only slightly elevated in parts of thalamus. The levels of β2-subunit were elevated in the frontal cortex, thalamus, hippocampus, brainstem, and cerebellar granule layers while the levels of β3-subunit were significantly decreased in the cortex, hippocampus, and cerebellar granule layers in MK-801-infused rats. The levels of α6- and δ-subunits, which are highly localized in the cerebellum, were increased in the cerebellar granule layer after MK-801 treatment. These results show that the prolonged suppression of NMDA receptor function by MK-801-infusion strongly elevates [³H]muscimol binding throughout the brain, increases regional [³H]flunitrazepam and [³⁵S]TBPS binding, and alters GABA_A receptor subunit mRNA levels in different directions. The chronic MK-801 treatment has differential effect on various GABA_A receptor subunits, which suggests involvement of differential regulatory mechanisms in interaction of NMDA receptor with the GABA receptors.     

Chronic ethanol administration alters the modulatory effect of 5α-pregnan-3α-ol-20-one on the binding characteristics of various radioligands of GABAA receptors

In this study, we investigated the modulatory effect of 5α-pregnan-3α-ol-20-one, a neurosteroid, on the binding characteristics of [ ]flunitrazepam (2 nM), [ ]muscimol (5 nM), and 4 nM [ ]t-butylbicyclophosphorothionate (TBPS) in cerebral cortex, cerebellum, and hippocampus of control, ethanol-dependent, and ethanol-withdrawn rats. 5α-Pregnan-3α-ol-20-one potentiated the binding of [ ]flunitrazepam and [ ]muscimol in all the rat brain regions investigated in this study. There was a significant increase in the maximal potentiation of [ ]flunitrazepam as well as [ ]muscimol binding (E_max) in the ethanol-dependent rat cerebellum as compared to control group (p<0.025). Furthermore, 5α-pregnan-3α-ol-20-one elicited a biphasic response, i.e., it potentiated the binding of [ ]TBPS at lower concentrations (100 nM) and inhibited the binding at higher concentrations (>100 nM). There was a significant higher inhibition of [ ]TBPS binding (−E_max) by 5α-pregnan-3α-ol-20-one in the hippocampus of ethanol-dependent as well as ethanol-withdrawn rats (p<0.025). These observations suggest that the neurosteroid binding site associated with the γ-aminobutyric acid_A (GABA_A) receptors in cerebellum and hippocampus plays an important role during ethanol-dependence and ethanol-withdrawal, and some of the changes following ethanol dependence and its withdrawal may be mediated through the neurosteroid binding site.     

Autoradiographic localization of the GABAA receptor agonist [H]muscimol in rat cerebral vessels

By the use of combined in vitro radioreceptor binding and autoradiographic techniques with [³H]muscimol as a ligand, we analyzed the distribution of GABA_A receptor sites in the arteries of the circle of Willis as well as in the arteries and arterioles of the pial-arachnoid membrane in the rat. [³H]Muscimol was bound by sections of rat cerebral vessels in a manner consistent with the existence of GABA_A receptors, with K_d and B_max values of 46 nM and 0.60 pmol/mg tissue respectively. [³H]Muscimol was bound by the medial layer of cerebral arteries, while no specific binding was observed in the intima, the adventitia and the adventitial-medial border. These findings suggest that the vasodilatory action of GABA on in vitro preparations of cerebral vessels is mediated by muscular receptor sites. The posterior cerebral arteries are richer in [³H]muscimol binding sites than the anterior ones. Pial-arachnoid arterioles, which are of critical importance in controlling local cerebral blood flow, did not exhibit any significant binding of [³H]muscimol. These results may explain the difficulty in manipulating pharmacologically the cerebral tissue perfusion in intact animals using GABAergic agonists.     

Glucocorticoids are modulators of GABAA receptors in brain

Modulation of binding of [³H]muscimol, a GABA_A receptor agonist, by natural and synthetic glucocorticoids was investigated in crude synaptosomal membranes and in brain sections of rat. In adrenalectomized (Adx) rats, muscimol binding was reduced by 30–50% in cerebral cortex, cerebellum, thalamus and hippocampus, as compared to sham-operated controls. This decrease was due to reduced binding affinities of GABA receptors for muscimol. In contrast muscimol binding was increased by 38% in the hypothalamus and did not change in the pons-medulla after Adx. Nanomolar concentrations of corticosterone and pregnenolone-sulfate, but not dexamethasone, enhanced muscimol binding in brain regions that were characterized by reduced binding following Adx. This steroid-induced increase in muscimol binding was due to enhanced affinities of GABA receptors.     

Decreased forebrain [S]TBPS binding and increased [H]muscimol binding in rats that do not develop stress-induced behavioral depression

Recent evidence suggests that anxiety and its biological concomitants may be involved in the pathophysiology of depression. In the present study, the in vitro radioligand binding of [³H]flunitrazepam, [³H]muscimol and[³⁵S]t-butylbicyclophosphorothionate (TBPS) sites on the benzodiazepine /GABA chloride ionophore receptor complex (BGRC) was examined using the learned helplessness paradigm. Only rats which did not develop the syndrome showed a significant increase in [³H]muscimol binding in cerebral cortex and a decrease in [³⁵S]TBPS binding in cerebral cortex and hippocampus in comparison to naive controls. For both ligands, this represented a change inB_max rather than a change in affinity. Adrenalectomy had no impact on these alterations indicating that critical endogenous factors are not manufactured by the adrenal glands. These findings suggest that the BGRC in the forebrain may be a site mediating the ‘coping’ ability of rats that do not develop the learned helplessness syndrome. The possible involvement of neurosteroids in the effect is discussed.     

Receptor binding sites and uptake activities mediating GABA neurotransmission in chronic alcoholics with Wernicke encephalopathy

Superior frontal cortex (SFC) and primary motor cortex tissue was obtained at autopsy from thirteen severe chronic alcoholics with neuropathologically confirmed Wernicke Encephalopathy (WE) and 22 controls. Cases with both WE and cirrhosis showed markedly fewer neurones in SFC than did WE cases without cirrhosis. The extent of the apparent neuronal loss corresponded to an increase in post-synaptic GABA_A receptor sites, as assessed by the binding of [³H]muscimol to synaptic membranes. Increased [³H]muscimol binding was not accompanied by an increase in ‘central-type’ benzodiazepine binding sites: as assessed by [³H]flunitrazepam binding, these sites were apparently unaltered, while as assessed by [³H]diazepam binding, they were decreased. The affinities of the two benzodiazepine ligands varied differently with disease. These discrepancies between [³H]flunitrazepam and [³H]diazepam binding could not be accounted for, either by the presence of a second, diazepam-preferring, ‘central-type’ benzodiazepine binding site, or by loss of ‘peripheral-type’ sites. The changes in the post-synaptic GABA_A benzodiazepine receptor sites did not reflect any regional, disease-related deficit of afferent GABAergic terminals, as assessed by synaptosomal high-affinity [³H]GABA uptake. On a number of indices, it appears most likely that the data reflect both a loss of receptor sites, and a change in the population of receptor sub-types.     

Prevalence of the GABAA receptor assemblies containing alpha1-subunit in the rat cerebellum and cerebral cortex as determined by immunoprecipitation: lack of modulation by chronic ethanol administration

The anti-alpha1 antibody elicited higher immunoprecipitation (%) values of the [3H]flunitrazepam and [3H]muscimol binding activity in the rat cerebellum vs. cerebral cortex, whereas immunoprecipitation values for [3H]Ro 15-4513 and [3H]zolpidem were comparable in these brain regions. Chronic ethanol administration neither changed the radioligand binding to the immunoprecipitated pellet nor the percentage immunoprecip-itation values, thereby indicating that chronic ethanol did not result in down-regulation of the GABAA receptor assemblies containing alpha1-subunit.     

Biochemical and functional alterations of central GABA receptors during chronic estradiol treatment

The characteristics of GABA and benzodiazepine receptors were examined in the hippocampus, striatum and cerebral cortex of female rats at various times (up to 9 months) after the subcutaneous implantation of an estradiol pellet (10 mg). A significant decrease in the B_max of the high-affinity binding of [³H]muscimol to membranes from these 3 regions was detected as soon as one week after the implantation. Although the characteristics of the high-affinity binding of [³H]flunitrazepam remained unaffected during the whole treatment, the stimulatory effect of GABA (and muscimol) on this binding was significantly reduced by estrogenization. The changes in GABA receptor binding appeared functionally relevant since the elevation of striatal acetylcholine levels normally induced by the peripheral administration of muscimol (5 mg/kg) was significantly lower in estradiol-treated than in control female rats.In contrast to that observed in intact female rats, the implantation of estradiol in hypophysectomized animals did not affect the characteristics of [³H]muscimol binding to hippocampal, striatal and cortical membranes. [³H]muscimol binding was also unchanged in female rats implanted with estradiol and treated chronically with bromocriptine for 3 weeks. Since both hypophysectomy and the chronic administration of bromocriptine suppressed the hyperprolactinemia normally induced by estrogenization, the down-regulation of central GABA receptors very likely involved prolactin in intact animals implanted with 17-β-estradiol.     

Autoradiographic comparison of D1-specific binding of [H]SCH39166 and SCH23390 in the primate cerebral cortex

Quantitative autoradiography was used to compare the binding of the novel dopamine D₁ receptor antagonist, [³H]SCH39166, with that of the widely used radioligand, [³H]SCH23390 (in the presence of ritanserin), in the primate cerebral cortex. Specific binding of both radioligands, determined using SCH23390 or cis-flupentixol as displacing agents, had very similar densities and distributions throughout the cortex. However, the specific binding of [³H]SCH39166 obtained with SCH39166 as a blank was significantly higher than that obtained using SCH23390 or cis-flupentixol as displacing agents in some layers of motor, somatosensory and occipital cortices. In addition, the non-specific binding of [³H]SCH39156 obtained in the presence of an excess of SCH23390 of cis-flupentixol displayed a complex laminar pattern very different from that of the specific binding. These observations suggest that [³H]SCH39166 may have a high affinity to more than the D₁ receptor subtype bound by SCH23390 or cis-flupentixol. Also, these additional sites are likely to be different from 5-HT₂ or 5-HT_1C receptors since the latter sites were not displaced by 1 μM SCH23390.     

Neurosteroid modulation of the GABAA receptor in the developing guinea pig cerebral cortex

Developmental changes in 5alpha-pregnan-3alpha-ol-20-one (allopregnanolone; 5alpha-3alpha-P) potentiation of muscimol and benzodiazepine binding to the GABAA receptor were studied in the guinea pig cerebral cortex at three prenatal ages (gestational day (GD) 40, GD 50, GD 62), and three postnatal ages (postnatal day (PD) 11, PD 21, PD 61) (term, about GD 68). The number and affinity of [3H]flunitrazepam binding sites, and 5alpha-3alpha-P potentiation of [3H]muscimol and [3H]flunitrazepam binding to the GABAA receptor were determined at each age. There was no age effect on the affinity (Kd) for [3H]flunitrazepam. However, the number (Bmax) of [3H]flunitrazepam binding sites doubled between GD 40 and GD 62, and then declined slightly to reach adult levels by PD 11. 5alpha-3alpha-P produced a concentration-dependent potentiation of [3H]muscimol and [3H]flunitrazepam binding at each developmental age examined. The potency (high-affinity) for 5alpha-3alpha-P potentiation of both [3H]muscimol and [3H]flunitrazepam binding was lowest at GD 40, and increased to adult levels by GD 62. In contrast, the efficacy for 5alpha-3alpha-P potentiation of both [3H]muscimol and [3H]flunitrazepam binding was greatest at GD 40, and decreased to adult levels between GD 50 and GD 62. The percentage of high-affinity zolpidem binding sites increased in an age-dependent manner from 34.2+/-2.2% at GD 40, to reach adult levels by GD 62 (59. 4+/-2.5%). These data suggest that 5alpha-3alpha-P can modulate GABAA receptors in the immature cerebral cortex, and that changes in 5alpha-3alpha-P action are temporally related to changes in GABAA receptor benzodiazepine pharmacology late in gestation in the guinea pig.     

The GABAA/benzodiazepine receptor complex in rat brain neuronal cultures. Characterization by immunoprecipitation

The expression of the GABAA/benzodiazepine receptor (GABAR/BZDR) complex in primary neuronal cultures from rat brain embryos has been investigated. The GABAR/BZDR complex was photoaffinity labeled with [3H]flunitrazepam [3H]FNZ and immunoprecipitated with subunit specific antibodies. These were the mAb 62-3G1 which is specific for the 57-kDa GABA binding subunit, and the rabbit antiserum A which recognizes the 51-kDa [3H]FNZ binding subunit. The results indicate that the cultured neurons express 5 different peptides of 51, 53, 54, 57 and 59 kDa that can be photoaffinity labeled with [3H]FNZ and that all of them are physically coupled to the GABAA receptor. Most of the [3H]FNZ photolabeled peptides have similar mobilities to those found in the brain of the newborn rat. Nevertheless, some of the quantitative changes in the photolabeled peptides observed during the normal development of the rat brain were not observed or occurred at much slower pace in the cultured neurons.     

Effect of NG-nitro-L-arginine methyl ester a nitric oxide synthase inhibitor on neurotransmitter receptor systems in aged rats

In order to examine the effect of age and nitric oxide synthase inhibitor N^G-nitro-L-arginine methl ester (l -NAME) we studied the changes on major neurotransmitter receptor systems in 6 (adult and 24-month-old (aged) Fischer male rats using receptor autoradiography. l -name was administrated intraperitoneally in aged rats once a day for 4 weeks. [³H]QNB (quinuclidinyl benzilate) ³HC (hemicholinium-3) [³H] muscimol ³H] SCH 23390 ([N-methyl-³H] N-methyl-³H]R[+]-8-chloro-2 3 4 5-tetrahydro-3-methyl-5-phenyl-7-il-benzazepine) ³H] mazindol were used as markers of muscarinic acetylcholine receptors high-high-affinity choline uptake sites GABA_A (γ-aminobutyric acid (SP2)_A) receptors dopamine D₁ receptors dopamine D₂ receptors and dopamine uptake sites respectively. The age-related change in ³H muscimol binding in the brain was more pronounced than that in [³H] QNB ³H]HC ³H]SCH 23390 ³H] nemonapride and ³H] nemonapride and ³H] mazindol binding.Chronic treatment (4 weeks) with l -NAME caused no significant changes in [sp1)³H] muscimol ³H SCH 23390 and [³H] nemonapride binding in most areas of aged rat brain as compared with vehicle-treated aged animals. However chronic treatment with l -NAME caused a significant reduction in ³H] HC and ³H] mazindol binding in any brain regions of aged rats in comparison with the vehicle-treated aged animals. These results demonstrate that the GABAergic system is more susceptible to aging processes than cholinergic and dopaminergic systems in teh brain. Furthermore our findings suggest that nitric oxide may play some role in the regulation of choline uptake and dopamine uptake systems during aging processes.     

Distribution of GABA binding site subtypes in rat pituitary gland

GABA_A and GABA_B binding sites in rat pituitary gland were investigated using equilibrium binding assays in vitro. Specific binding of both [³H]GABA and [³H]muscimol could be detected in both anterior and neurointermediate lobes, with a relative concentration in the anterior lobe. [³H]GABA binding was discriminated into GABA_A and GABA_B receptor type binding using baclofen. GABA_B sites were detectable in the anterior but not in the neurointermediate lobe. Saturation analysis of [³H]muscimol binding to whole pituitary gland membranes demonstrated that the pituitary contains two classes of GABA_A sites differing in affinity, as found in the CNS, although the number of sites is considerably lower than in the CNS.     

Ca-binding proteins in rat synaptic fractions surveyed by the Ca overlay method

Ca²⁺-binding proteins in the synaptic and subsynaptic fractions (P₂, synaptosome, synaptic plasma membrane, and postsynaptic density [PSD]-enriched fractions) and soluble fraction of rat brain were surveyed by a ⁴⁵Ca²⁺ overlay method. The PSD-enriched fraction from cerebral cortex contained two major Ca²⁺-binding proteins (55,000 M_r and 19,000 M_r) and a distinct group (in 140,000 M_r region)m and two minor ones (66,000 M_rand16,000 M_r); and the fraction from cerebellum contained two (55,000 M_rand19,000 M_r). The proteins with 55,000 M_rand19,000 M_r were identified as tubulin and calmodulin, respectively, and present in all the fractions investigated. The Ca²⁺-binding proteins of 140,000 M_r region were found only in the PSD-enriched fraction isolated from cerebral cortex: neither the PSD-enriched fraction isolated from cerebellum nor other subcellular fractions prepared from cerebral cortex and cerebellum contained the proteins. The 140,000 M_r Ca²⁺-binding proteins were the substrates for the Ca²⁺/calmodulin-dependent protein kinase II associated with PSD, and no change in the Ca²⁺-binding was detected by the ⁴⁵Ca²⁺ overlay method after phosphorylation of the proteins by the protein kinase. The 16,000 M_r Ca²⁺-binding protein might be the β-subunit of calcineurin. Calretinin and calbindin-D_28k were also detected as Ca²⁺-binding proteins in the soluble fractions of both cerebral cortex and cerebellum.