RESUMEN
The distribution and density of pituitary adenylate cyclase-activating polypeptide (PACAP) binding sites have been investigated in the brain of the primates Jacchus callithrix (marmoset) and Macaca fascicularis (macaque) using [(125)I]-PACAP27 as a radioligand. PACAP binding sites were widely expressed in the brain of these two species with particularly high densities in the septum, hypothalamus and habenula. A moderate density of recognition sites was seen in all subdivisions of the cerebral cortex with a heterogenous distribution, the highest concentrations occurring in layers I and VI while the underlying white matter was almost devoid of binding sites. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis revealed intense expression of the mRNAs encoding the short and hop-1 variants of pituitary adenylate cyclase-activating polypeptide-specific receptor (PAC1-R) in the cortex of both marmoset and macaque, whereas vasoactive intestinal polypeptide/pituitary adenylate cyclase-activating polypeptide mutual receptor, subtype 1 (VPAC1-R) and vasoactive intestinal polypeptide/pituitary adenylate cyclase-activating polypeptide mutual receptor, subtype 2 (VPAC2-R) mRNAs were expressed at a much lower level. In situ hybridization histochemistry showed intense expression of PAC1-R and weak expression of VPAC1-R mRNAs in layer IV of the cerebral cortex. Incubation of cortical tissue slices with PACAP induced a dose-dependent stimulation of cyclic AMP formation, indicating that PACAP binding sites correspond to functional receptors. Moreover, treatment of primate cortical slices with 100 nM PACAP significantly reduced the activity of caspase-3, a key enzyme of the apoptotic cascade. The present results indicate that PACAP should exert the same neuroprotective effect in the brain of primates as in rodents and suggest that PAC1-R agonists may have a therapeutic value to prevent neuronal cell death after stroke or in specific neurodegenerative diseases.
Asunto(s)
Mapeo Encefálico , Encéfalo/metabolismo , Polipéptido Hipofisario Activador de la Adenilato-Ciclasa/metabolismo , Receptores del Polipéptido Activador de la Adenilato-Ciclasa Hipofisaria/metabolismo , Animales , Callithrix , Femenino , Habénula/metabolismo , Hipotálamo/metabolismo , Macaca fascicularis , Masculino , Polipéptido Hipofisario Activador de la Adenilato-Ciclasa/genética , ARN Mensajero/análisis , Receptores del Polipéptido Activador de la Adenilato-Ciclasa Hipofisaria/clasificación , Receptores del Polipéptido Activador de la Adenilato-Ciclasa Hipofisaria/genética , Tabique del Cerebro/metabolismo , Especificidad de la Especie , Distribución TisularRESUMEN
Most of the actions of neurosteroids on the central nervous system are mediated through allosteric modulation of the gamma-aminobutyric acid type A (GABA(A)) receptor, but a direct effect of GABA on the regulation of neurosteroid biosynthesis has never been investigated. In the present report, we have attempted to determine whether 3beta-hydroxysteroid dehydrogenase (3beta-HSD)-containing neurons, which secrete neurosteroids in the frog hypothalamus, also express the GABA(A) receptor, and we have investigated the effect of GABA on neurosteroid biosynthesis by frog hypothalamic explants. Double immunohistochemical labeling revealed that most 3beta-HSD-positive neurons also contain GABA(A) receptor alpha(3) and beta(2)/beta(3) subunit-like immunoreactivities. Pulse-chase experiments showed that GABA inhibited in a dose-dependent manner the conversion of tritiated pregnenolone into radioactive steroids, including 17-hydroxy-pregnenolone, progesterone, 17-hydroxy-progesterone, dehydroepiandrosterone, and dihydrotestosterone. The effect of GABA on neurosteroid biosynthesis was mimicked by the GABA(A) receptor agonist muscimol but was not affected by the GABA(B) receptor agonist baclofen. The selective GABA(A) receptor antagonists bicuculline and SR95531 reversed the inhibitory effect of GABA on neurosteroid formation. The present results indicate that steroid-producing neurons of the frog hypothalamus express the GABA(A) receptor alpha(3) and beta(2)/beta(3) subunits. Our data also demonstrate that GABA, acting on GABA(A) receptors at the hypothalamic level, inhibits the activity of several key steroidogenic enzymes, including 3beta-HSD and cytochrome P450(C17) (17alpha-hydroxylase).