A neuropeptide was isolated from a frog brain extract by HPLC purification and characterized by mass spectrometry. This 26-aa neuropeptide, which belongs to the RFamide peptide family, was designated 26RFa, and its primary structure was established as VGTALGSLAEELNGYNRKKGGFSFRF-NH₂. Research in databases revealed the presence of sequences homologous to frog 26RFa in the human genome and in rat ESTs. On the basis of this sequence information, the cDNAs encoding the human and rat 26RFa precursors were cloned. The two preproteins show a similar organization, with the 26RFa sequence located in the C-terminal region of the precursor. Human preprotein (prepro)-26RFa encodes an additional putative RFamide peptide that is not found in the rat precursor. The primary structures of human, rat, and frog 26RFa exhibit ≈80% identity, and the C-terminal octapeptide has been fully conserved from amphibians to mammals.In situhybridization histochemistry revealed that, in the rat brain, the 26RFa gene is exclusively expressed in the ventromedial hypothalamic nucleus and in the lateral hypothalamic area. 26RFa induced a dose-dependent stimulation in cAMP production by rat pituitary cellsin vitroand markedly increased food intake in mice. The conservation of the primary structure of 26RFa during vertebrate evolution, the discrete localization of the mRNA encoding its precursor in hypothalamic nuclei involved in the control of feeding behavior, and the observation that 26RFa possesses orexigenic properties indicate that this neuropeptide may play important biological functions.

Zhang et al . (Research Articles, 11 November 2005, p. 996) reported that obestatin, a peptide derived from the ghrelin precursor, activated the orphan G protein–coupled receptor GPR39. However, we found that I ¹²⁵ -obestatin does not bind GPR39 and observed no effects of obestatin on GPR39-transfected cells in various functional assays (cyclic adenosine monophosphate production, calcium mobilization, and GPR39 internalization). Our results indicate that obestatin is not the cognate ligand for GPR39.

Alcohol exposure during development can cause brain malformations and neurobehavioral abnormalities. In view of the teratogenicity of ethanol, identification of molecules that could counteract the neurotoxic effects of alcohol deserves high priority. Here, we report that pituitary adenylate cyclase-activating polypeptide (PACAP) can prevent the deleterious effect of ethanol on neuronal precursors. Exposure of cultured cerebellar granule cells to ethanol inhibited neurite outgrowth and provoked apoptotic cell death. Incubation of granule cells with PACAP prevented ethanol-induced apoptosis, and this effect was not mimicked by vasoactive intestinal polypeptide, suggesting that PAC1 receptors are involved in the neurotrophic activity of PACAP. Ethanol exposure induced a strong increase of caspase-2, -3, -6, -8, and -9 activities, DNA fragmentation, and mitochondrial permeability. Cotreatment of granule cells with PACAP provoked a significant inhibition of all of the apoptotic markers investigated although the neurotrophic activity of PACAP could only be ascribed to inhibition of caspase-3 and -6 activities. These data demonstrate that PACAP is a potent protective agent against ethanol-induced neuronal cell death. The fact that PACAP prevented ethanol toxicity even when added 2 h after alcohol exposure, suggests that selective PACAP agonists could have potential therapeutic value for the treatment of fetal alcohol syndrome.

High concentrations of pituitary adenylate cyclase-activating polypeptide (PACAP) receptors are present in the external granule cell layer of the rat cerebellum during postnatal development. In vitro studies have shown that PACAP promotes cell survival and neurite outgrowth on immature cerebellar granule cells in primary culture. In the present study, we have investigated the effect of PACAP on the development of the cerebellar cortex of 8-day-old rats. Incubation of cultured granule cells for 12 or 18 h with PACAP provoked a significant increase in the rate of incorporation of [ ³ H]thymidine in cultured granule cells, suggesting that PACAP could stimulate the proliferation of granule cells. After 96 h of treatment, in vivo administration of PACAP provoked a transient increase in the number of granule cells in the molecular layer and in the internal granule cell layer. In contrast, PACAP did not affect the number of Purkinje cells. The augmentation of the number of granule cells evoked by PACAP was significantly inhibited by the PACAP receptor antagonist PACAP(6–38). Administration of PACAP also caused a significant increase in the volume of the cerebellar cortex. The present study provides evidence that PACAP can act in vivo as a trophic factor during rat brain development. Our data indicate that PACAP increases proliferation and/or inhibits programmed cell death of granule cells, as well as stimulating neuronal migration from the external granule cell layer toward the internal granule cell layer.

Caspase-3 knockout mice exhibit thickening of the internal granule cell layer of the cerebellum. Concurrently, it has been shown that intracerebral injection of pituitary adenylate cyclase-activating polypeptide (PACAP) induces a transient increase of the thickness of the cerebellar cortex. In the present study, we have investigated the possible effect of PACAP on caspase activity in cultured cerebellar granule cells from 8-day-old rat. Incubation of granule neurons with PACAP for 24 h promoted cell survival and prevented DNA fragmentation. Exposure of cerebellar granule cells to the specific caspase-3 inhibitor N-benzyloxycarbonyl-Asp-Glu-Val-Asp fluoromethylketone (Z-DEVD-FMK) for 24 h markedly enhanced cell survival and inhibited apoptotic cell death. Time-course studies revealed that PACAP causes a prolonged inhibition of caspase-3 activity without affecting caspase-1. Administration of graded concentrations of PACAP for 3 h induced a dose-dependent inhibition of caspase-3 activity. Incubation of granule cells with both dibutyryl-cAMP (dbcAMP) and phorbol 12-myristate 13-acetate (PMA) mimicked the inhibitory effect of PACAP on caspase-3. Cotreatment of cultured neurons with the protein kinase A inhibitor H89 and the protein kinase C inhibitor chelerythrine abrogated the effect of PACAP on caspase-3 activity. In contrast, the ERK kinase inhibitor U0126 did not affect the action of PACAP on caspase-3 activity. These data demonstrate that PACAP prevents cerebellar granule neurons from apoptotic cell death through a protein kinase A- and protein kinase C-dependent inhibition of caspase-3 activity.