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
Melatonin secretion from the mammalian pineal gland is strongly stimulated by noradrenaline and also by vasoactive intestinal polypeptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP). Three types of receptors for VIP and PACAP have been characterized so far: VIP1/PACAP receptors and VIP2/PACAP receptors, which possess similar high affinities for VIP and PACAP, and PACAP1 receptors which exhibit a 100-1000-fold higher affinity for PACAP. The aim of the present study was to characterize the receptor subtype(s) mediating the stimulatory effects of VIP and PACAP on melatonin synthesis in the rat pineal gland. Autoradiographic studies showed that PACAP and VIP were equally potent in displacing binding of radioiodinated PACAP27 from pineal sections. Amplification of pineal complementary DNAs by polymerase chain reaction using specific primers for the different receptor subtypes revealed that all three receptor messenger RNAs are expressed and that VIP1/PACAP receptor messenger RNA was predominant over VIP2/PACAP receptor messenger RNA. In vitro, VIP and PACAP stimulated melatonin synthesis with similar high potency and the effect of the two peptides were not additive. The selective VIP1/PACAP receptor agonists [R16]chicken secretin (1-25) and [K15, R16, L27]VIP(1-7)/growth hormone releasing factor(8-27) were significantly more potent than the selective VIP2/PACAP receptor agonist RO 25-1553 in stimulating melatonin secretion. The stimulatory effects of VIP and PACAP were similarly inhibited by the VIP1/PACAP antagonist [acetyl-His1, D-Phe2, K15, R16, L27]VIP(3-7)/growth hormone releasing factor(8-27). These data strongly suggest that VIP and PACAP exert a stimulatory effect on melatonin synthesis mainly through activation of a pineal VIP1/PACAP receptor subtype.
Asunto(s)
Adenilil Ciclasas/metabolismo , Glándula Pineal/química , Glándula Pineal/enzimología , Receptores de la Hormona Hipofisaria/análisis , Receptores de Péptido Intestinal Vasoactivo/análisis , Agonistas Adrenérgicos beta/farmacología , Animales , Autorradiografía , Broncodilatadores/farmacología , Activación Enzimática/efectos de los fármacos , Expresión Génica/fisiología , Radioisótopos de Yodo , Isoproterenol/farmacología , Masculino , Melatonina/metabolismo , Neuropéptidos/farmacología , Neurotransmisores/farmacología , Fragmentos de Péptidos/farmacología , Péptidos Cíclicos/farmacología , Polipéptido Hipofisario Activador de la Adenilato-Ciclasa , ARN Mensajero/análisis , Ratas , Ratas Wistar , Receptores del Polipéptido Activador de la Adenilato-Ciclasa Hipofisaria , Receptores de la Hormona Hipofisaria/genética , Receptores de la Hormona Hipofisaria/metabolismo , Receptores de Péptido Intestinal Vasoactivo/genética , Receptores de Péptido Intestinal Vasoactivo/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Péptido Intestinal Vasoactivo/análogos & derivados , Péptido Intestinal Vasoactivo/farmacologíaRESUMEN
The radiolabelled somatostatin analogs [125I-Tyr0,DTrp8]S14 and [Leu8,DTrp22,125I-Tyr25]S28 were used as radioligands to study the distribution of somatostatin receptors in the rat hypothalamus. Previous studies have detected very few somatostatin-binding sites in the hypothalamus using in vitro autoradiography. Since the lack of autoradiographic labelling has been ascribed to the occupancy of the receptors by endogenous ligands, we have developed a method using guanosine triphosphate (GTP) pretreatment to unmask somatostatin receptors. Preincubation of brain slices with 10(-6) M GTP, by desaturating the occupied receptors, made it possible to reveal the wide distribution of somatostatin-binding sites in the rat hypothalamus. Somatostatin-14 binding site populations were observed in numerous hypothalamic areas including the preoptic area where the receptors likely account for self-inhibition of somatostatin release, the supraoptic nucleus, the bed nucleus of the stria terminalis, the anterior hypothalamic nucleus, the perifornical area, the zona incerta and a mediolateral area located laterally to the ventromedian and dorsomedian nuclei and limited laterally by the mammillo-thalamic tract, the fornix and the optic tract. All structures showing S14-binding sites were labelled by the S28 radioligand. In addition, the paraventricular parvocellular nucleus contained exclusively S28-binding sites, which could be involved in the inhibitory effect of S28 on CRF-mediated endocrine and sympathetic responses. A moderate density of S28-preferring sites was also detected in the periventricular nucleus. In summary, GTP preincubation of brain slices appeared to be a useful technique to reveal multiple somatostatin receptors populations in the brain. The widespread distribution of somatostatin receptors in the hypothalamus is in total agreement with the variety of physiological effects of the somatostatin peptide family.