RESUMEN
A gamma-aminobutyric acid (GABA)(B) receptor (named GABA(B)R1) has been recently cloned in the rat and human brain and two variants generated by alternative RNA splicing were identified. In the present study, we addressed the question as to whether these variants contribute to the diversity of GABA(B) receptor-mediated physiological responses and constitute real receptor subtypes with distinct functions. To this aim, we have mapped the GABA(B)R1 (R1a) and GABA(B)R1b (R1b) transcript distribution in the rat brain using in situ hybridization. We have compared the mRNA distribution with the distribution of [(3)H]CGP54626-labeled binding GABA(B)R1 receptor sites as assessed in adjacent cryosections by quantitative autoradiography. We found that GABA(B) receptor transcripts and binding sites are expressed in the brain in almost all neuronal cell populations. Expression in glial cells, if any, is marginal. We observed a good parallelism between GABA(B)R1 mRNA transcripts and binding sites in broad neuroanatomical entities with highest densities in hippocampus, thalamic nuclei, and cerebellum. By contrast, R1a and R1b transcripts exhibit marked differences in their regional and cellular distribution pattern. A typical example is the cerebellum with an almost exclusive expression of R1b in the Purkinje cells and of R1a in the granule, stellate, and basket cells. Data pointing at a pre- versus postsynaptic localization for R1a and R1b, respectively, at some neuronal sites are presented.
Asunto(s)
Química Encefálica/fisiología , Encéfalo/fisiología , ARN Mensajero/biosíntesis , Receptores de GABA-B/biosíntesis , Animales , Autorradiografía , Encéfalo/citología , Mapeo Encefálico , Agonistas del GABA/metabolismo , Hibridación in Situ , Masculino , Neuronas/metabolismo , Compuestos Organofosforados/metabolismo , Células de Purkinje/metabolismo , RatasRESUMEN
We have investigated whether the pathogenesis of spontaneous generalized non-convulsive seizures in rats with genetic absence epilepsy is due to an increase in the brain levels of gamma-hydroxybutyric acid (GHB) or in the rate of its synthesis. Concentrations of GHB or of its precursor gamma-butyrolactone (GBL) were measured with a new GC/MS technique which allows the simultaneous assessment of GHB and GBL. The rate of GHB synthesis was estimated from the increase in GHB levels after inhibition of its catabolism with valproate. The results of this study do not indicate significant differences in GHB or GBL levels, or in their rates of synthesis in rats showing spike-and-wave discharges (SWD) as compared to rats without SWD. Binding data indicate that GHB, but not GBL, has a selective, although weak affinity for GABAB receptors (IC50 = 150 microM). Similar IC50 values were observed in membranes prepared from rats showing SWD and from control rats. The average GHB brain levels of 2.12 +/- 0.23 nmol/g measured in the cortex and of 4.28 +/- 0.90 nmol/g in the thalamus are much lower than the concentrations necessary to occupy a major part of the GABAB receptors. It is unlikely that local accumulations of GHB reach concentrations 30-70-fold higher than the average brain levels. After injection of 3.5 mmol/kg GBL, a dose sufficient to induce SWD, brain concentrations reach 240 +/- 31 nmol/g (Snead, 1991) and GHB could thus stimulate the GABAB receptor. Like the selective and potent GABAB receptor agonist R(-)-baclofen, GHB causes a dose-related decrease in cerebellar cGMP. This decrease and the increase in SWD caused by R(-)-baclofen were completely blocked by the selective and potent GABAB receptor antagonist CGP 35348, whereas only the increase in the duration of SWD induced by GHB was totally antagonized by CGP 35348. The decrease in cerebellar cGMP levels elicited by GHB was only partially antagonized by CGP 35348. These findings suggest that all effects of R(-)-baclofen are mediated by the GABAB receptor, whereas only the induction of SWD by GHB is dependent on GABAB receptor mediation, the decrease in cGMP being only partially so. Taken together with the observations of Marescaux et al. (1992), these results indicate that GABAB receptors are of primary importance in experimental absence epilepsy and that GABAB receptor antagonists may represent a new class of anti-absence drugs.
Asunto(s)
4-Butirolactona/metabolismo , Encéfalo/metabolismo , Epilepsia Tipo Ausencia/fisiopatología , Receptores de GABA-A/fisiología , Oxibato de Sodio/metabolismo , 4-Butirolactona/aislamiento & purificación , Animales , Baclofeno/farmacología , Encéfalo/efectos de los fármacos , GMP Cíclico/metabolismo , Modelos Animales de Enfermedad , Epilepsia Tipo Ausencia/metabolismo , Antagonistas de Receptores de GABA-A , Masculino , Compuestos Organofosforados/farmacología , Ratas , Ratas Endogámicas/genética , Receptores de GABA-A/metabolismo , Valores de Referencia , Reproducibilidad de los Resultados , Oxibato de Sodio/aislamiento & purificación , Ácido Valproico/farmacologíaRESUMEN
CGP 20712 A (1-[2-((3-carbamoyl-4-hydroxy)phenoxy)ethylamino]-3- [4-(1-methyl-4-trifluoromethyl-2-imidazolyl) phenoxy]-2-propanol methanesulfonate), a specific beta 1-adrenoceptor antagonist, was tested for resolution of beta 1- and beta 2-adrenoceptors in an in vitro [3H]dihydroalprenolol ([3H]DHA) binding assay. Competition experiments, using rat neocortical and cerebellar membranes, yielded two dissimilar concentration-effect curves. A distinct biphasic curve was evident for neocortex, with a plateau at 100 nM CGP 20712 A (60% [3H]DHA displacement). This plateau indicated a differentiation between beta 1- and beta 2-adrenoceptors; the ratio of IC50-beta 2 to IC50-beta 1 was approximately 10,000. In contrast, only a monophasic curve was obtained for cerebellum. CGP 20712 A is a useful tool for estimating percentages of beta 1- and beta 2-adrenoceptors in a given tissue.