Distribution and functional characterization of pituitary adenylate cyclase-activating polypeptide receptors in the brain of non-human primates.

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.

Pharmacological, molecular and functional characterization of vasoactive intestinal polypeptide/pituitary adenylate cyclase-activating polypeptide receptors in the rat pineal gland.

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.