David Alexandre

PACAP and NGF regulate common and distinct traits of the sympathoadrenal lineage: effects on electrical properties, gene markers and transcription factors in differentiating PC12 cells

To determine the possible role of pituitary adenylate cyclase‐activating polypeptide (PACAP) in the development of the sympathoadrenal cell lineage, we have examined the effects of this neurotrophic peptide, in comparison to nerve growth factor (NGF), on the morphology, electrophysiological properties, expression of neuronal and neuroendocrine marker genes, and activity of transcription factors during differentiation of sympathoadrenal‐derived cells, using the rat pheochromocytoma PC12 cell model. Both PACAP and NGF elicited rapid neurite outgrowth, which was accompanied by induction of cell excitability and the development of both sodium and calcium currents. Concurrently, PACAP and NGF increased the expression of a marker of synaptic vesicles. By contrast, PACAP, but not NGF, regulated the expression of different constituents of neuroendocrine large dense core vesicles in PC12 cells. Furthermore, PACAP and NGF differentially regulated the expression of mammalian achaete‐scute homologue and paired homeobox 2b genes, transcription factors instrumental for sympathoadrenal development. To compare downstream effectors activated by PACAP and NGF, we studied the effects of these factors on the binding activity of consensus 12‐O‐tetradecanoylphorbol‐13‐acetate‐ and cAMP‐responsive elements to nuclear extracts of differentiating PC12 cells. We found that both PACAP and NGF markedly increase the binding activity of these cis‐regulatory sequences and that PACAP preferentially recruits activator protein‐1‐like transcription factors to these elements. Taken together, these results show that PACAP and NGF exert common as well as different effects on neuronal and neuroendocrine traits in differentiating PC12 cells, strongly suggesting that these two trophic factors could play complementary roles in the development of the sympathoadrenal cell lineage.

Structure and distribution of the mRNAs encoding pituitary adenylate cyclase-activating polypeptide and growth hormone-releasing hormone-like peptide in the frog, Rana ridibunda

The structure of the neuropeptide pituitary adenylate cyclase‐activating polypeptide (PACAP) has been characterized in several species including protochordates, fish, amphibians, birds, and mammals. Although PACAP has been shown to stimulate frog pituitary and adrenal cell activity, the structure of the PACAP precursor and the expression of its gene have not yet been reported in any amphibian species. In this study, we have characterized two cDNA variants encoding PACAP of the frog Rana ridibunda, one of which encodes a second peptide exhibiting strong homologies to growth hormone‐releasing hormone (GHRH) of fish and mammals. Northern blot and reverse transcriptase‐polymerase chain reaction (RT‐PCR) analyses revealed that PACAP/GHRH‐like peptide mRNAs are predominantly expressed in the brain and spinal cord and, to a lesser extent, in the neurointermediate lobe of the pituitary. Other tissues including the testis and the distal lobe of the pituitary do not express the PACAP precursor gene. The distribution of PACAP/GHRH‐like peptide mRNAs in the frog brain has been determined by in situ hybridization histochemistry. High levels of expression were found in the accessory olfactory bulb, the distal pallium, the ventral part of the magnocellular preoptic nucleus, the ventral hypothalamic nucleus, the posterior tuberculum, and the ventral habenular nucleus. These data contribute to the understanding of the evolution of the PACAP and GHRH genes in vertebrates and provide the anatomical bases to elucidate the roles of PACAP and the GHRH‐like peptide in amphibians. J. Comp. Neurol. 421:234–246, 2000.

Distribution of 26RFa binding sites and GPR103 mRNA in the central nervous system of the rat

The novel RFamide peptide 26RFa, the endogenous ligand of the orphan receptor GPR103, affects food intake, locomotion, and activity of the gonadotropic axis. However, little is known regarding the localization of 26RFa receptors. The present report provides the first detailed mapping of 26RFa binding sites and GPR103 mRNA in the rat central nervous system (CNS). 26RFa binding sites were widely distributed in the brain and spinal cord, whereas the expression of GPR103 mRNA was more discrete, notably in the midbrain, the pons, and the medulla oblongata, suggesting that 26RFa can bind to a receptor(s) other than GPR103. Competition experiments confirmed that 26RFa interacts with an RFamide peptide receptor distinct from GPR103 that may be NPFF2. High densities of 26RFa binding sites were observed in olfactory, hypothalamic, and brainstem nuclei involved in the control of feeding behavior, including the piriform cortex, the ventromedial and dorsomedial hypothalamic nuclei, the paraventricular nucleus, the arcuate nucleus, the lateral hypothalamic area, and the nucleus of the solitary tract. The preoptic and anterior hypothalamic areas were also enriched with 26RFa recognition sites, supporting a physiological role of the neuropeptide in the regulation of the gonadotropic axis. A high density of 26RFa binding sites was detected in regions of the CNS involved in the processing of pain, such as the dorsal horn of the spinal cord and the parafascicular thalamic nucleus. The wide distribution of 26RFa binding sites suggests that 26RFa has multiple functions in the CNS that are mediated by at least two distinct receptors. J. Comp. Neurol. 503:573–591, 2007.

Molecular cloning of frog secretogranin II reveals the occurrence of several highly conserved potential regulatory peptides

Secretogranin II (SgII) is an acidic secretory protein present in large dense core vesicles of neuronal and endocrine cells. Based on the sequence of a peptide derived from the processing of SgII in the brain of the frog Rana ridibunda, degenerate oligonucleotides were used to clone the cDNA encoding frog SgII from a pituitary cDNA library. This cDNA encodes a 574 amino acid protein which exhibits 46–48% sequence identity with mammalian SgII and contains 11 pairs of basic amino acids. Four potential processing products delimited by pairs of basic residues exhibited a much higher degree of identity (68–82%) with the corresponding mammalian SgII sequences. The frog SgII mRNA is ∼4 kb in length and is differentially expressed in the brain and endocrine tissues. The present data reveal that several SgII‐derived peptides have been highly conserved during evolution, suggesting that these peptides may play important neuroendocrine regulatory functions.

The Orexigenic Activity of the Hypothalamic Neuropeptide 26RFa Is Mediated by the Neuropeptide Y and Proopiomelanocortin Neurons of the Arcuate Nucleus

26RFa is a hypothalamic RFamide neuropeptide that was identified as the endogenous ligand of the orphan G protein-coupled receptor, GPR103, and that stimulates appetite in mice. Up until now, the mechanism of action of 26RFa in the hypothalamic control of food intake remains unknown. The high density of GPR103 in the arcuate nucleus (Arc) prompted us to investigate, in the present study, the effects of 26RFa on the rat neuropeptide Y (NPY)/proopiomelanocortin (POMC) system. Intracerebroventricular injection of 26RFa stimulated NPY expression and release in the basal hypothalamus, whereas it decreased POMC expression and alpha-MSH release, and these effects were associated with an increase in food intake. A double in situ hybridization procedure indicated that the 26RFa receptor is present in NPY neurons of the Arc, but not in POMC neurons. Central administration of NPY Y1 and Y5 receptor antagonists abolished the inhibitory effects of 26RFa on POMC expression and alpha-MSH release, and reversed 26RFa-induced food consumption. Finally, 26RFa antagonized the effects of leptin on NPY expression and release, POMC expression and alpha-MSH release, and food intake. Altogether, the present data demonstrate for the first time that 26RFa exerts its orexigenic activity by stimulating the release of NPY in the Arc, which in turn inhibits POMC neurons by activating the Y1 and Y5 receptors. It is also suggested that the balance 26RFa/leptin is an important parameter in the maintenance of energy homeostasis.

Vasotocin and Mesotocin Stimulate the Biosynthesis of Neurosteroids in the Frog Brain

The neurohypophysial nonapeptides vasopressin (VP) and oxytocin (OT) modulate a broad range of cognitive and social activities. Notably, in amphibians, vasotocin (VT), the ortholog of mammalian VP, plays a crucial role in the control of sexual behaviors. Because several neurosteroids also regulate reproduction-related behaviors, we investigated the possible effect of VT and the OT ortholog mesotocin (MT) in the control of neurosteroid production. Double immunohistochemical labeling of frog brain sections revealed the presence of VT/MT-positive fibers in close proximity of neurons expressing the steroidogenic enzymes 3β-hydroxysteroid dehydrogenase/Δ5-Δ4 isomerase (3β-HSD) and cytochrome P450 17α-hydroxylase/c17, 20-lyase (P450C17). High concentrations of VT and MT receptor mRNAs were observed in diencephalic nuclei containing the 3β-HSD and P450C17 neuronal populations. Exposure of frog hypothalamic explants to graded concentrations of VT or MT produced a dose-dependent increase in the formation of progesterone, 17-hydroxypregnenolone, 17-hydroxyprogesterone, and dehydroepiandrosterone. The stimulatory effect of VT and MT on neurosteroid biosynthesis was mimicked by VP and OT, as well as by a selective V1b receptor agonist, whereas V2 and OT receptor agonists had no effect. VT-induced neurosteroid production was completely suppressed by selective V1a receptor antagonists and was not affected by V2 and OT receptor antagonists. Concurrently, the effect of MT on neurosteroidogenesis was markedly attenuated by selective OT and V1a receptor antagonists but not by a V2 antagonist. The present study provides the first evidence for a regulatory effect of VT and MT on neurosteroid biosynthesis. These data suggest that neurosteroids may mediate some of the behavioral actions of VT and MT.

Novel Splice Variants of Type I Pituitary Adenylate Cyclase-Activating Polypeptide Receptor in Frog Exhibit Altered Adenylate Cyclase Stimulation and Differential Relative Abundance

Pituitary adenylate cyclase-activating polypeptide (PACAP) exerts its various effects through activation of two types of G protein-coupled receptors, a receptor with high affinity for PACAP named PAC1-R and two receptors exhibiting similar affinity for both PACAP and vasoactive intestinal polypeptide named VPAC1-R and VPAC2-R. Here, we report the characterization of PAC1-R and novel splice variants in the frog Rana ridibunda. The frog PAC1-R has 78% homology with human PAC1-R and is highly expressed in the central nervous system. Two splice variants of the frog receptor that display additional amino acid cassettes in the third intracellular loop were characterized. PAC1-R25 carries a 25-amino acid insertion that matches the hop cassette of the mammalian receptor, whereas PAC1-R41 carries a cassette with no homology to any mammalian PAC1-R variant. A third splice variant of PAC1-R, exhibiting a completely different intracellular C-terminal domain, named PAC1-Rmc has also been identified. Determination of c...

The RFamide neuropeptide 26RFa and its role in the control of neuroendocrine functions.

Identification of novel neuropeptides and their cognate G protein-coupled receptors is essential for a better understanding of neuroendocrine regulations. The RFamide peptides represent a family of regulatory peptides that all possess the Arg-Phe-NH2 motif at their C-terminus. In mammals, seven RFamide peptides encoded by five distinct genes have been characterized. The present review focuses on 26RFa (or QRFP) which is the latest member identified in this family. 26RFa is present in all vertebrate phyla and its C-terminal domain (KGGFXFRF-NH2), which is responsible for its biological activity, has been fully conserved during evolution. 26RFa is the cognate ligand of the orphan G protein-coupled receptor GPR103 that is also present from fish to human. In all vertebrate species studied so far, 26RFa-expressing neurons show a discrete localization in the hypothalamus, suggesting important neuroendocrine activities for this RFamide peptide. Indeed, 26RFa plays a crucial role in the control of feeding behavior in mammals, birds and fish. In addition, 26RFa up-regulates the gonadotropic axis in mammals and fish. Finally, evidence that the 26RFa/GPR103 system regulates steroidogenesis, bone formation, nociceptive transmission and arterial blood pressure has also been reported. Thus, 26RFa appears to act as a key neuropeptide in vertebrates controlling vital neuroendocrine functions. The pathophysiological implication of the 26RFa/GPR103 system in human is totally unknown and some fields of investigation are proposed.

Orexigenic Neuropeptide 26RFa: New Evidence for an Adaptive Profile of Appetite Regulation in Anorexia Nervosa

CONTEXT Restrictive anorexia nervosa (AN) presents an adaptive appetite regulating profile including mainly high levels of ghrelin. Because this adaptive mechanism is not effective on food intake, other appetite-regulating peptides need to be explored. 26RFa is a hypothalamic neuropeptide that stimulates appetite, gonadotropin release, and bone metabolism. OBJECTIVE The objective of the study was to evaluate the circadian levels of 26RFa in AN patients compared with healthy subjects, other eating disorders, and constitutional thinness (CT). DESIGN AND SETTINGS This was a cross-sectional study performed in an endocrine unit and an academic laboratory. INVESTIGATED SUBJECTS Five groups of age-matched young women were included in the study: 19 restrictive AN, 10 AN with bingeing/purging episodes, 14 with CT, 10 bulimic, and 10 normal-weight controls. MAIN OUTCOME MEASURES Twelve-point circadian profiles of plasma 26RFa levels were measured in each subject. RESULTS Significant circadian variations of 26 RFA were noticed in controls with higher values in the morning and abrupt decrease at noon. Twenty-four-hour mean 26RFa levels were significantly increased in restrictive AN and AN with bingeing/purging episodes (P < 0.001), predominantly in the afternoon and evening when compared with controls. Preprandial rises of 26 RFA were noticed in AN patients. Mean 26RFa levels trend to be higher in CT than in controls (P = 0.06) and significantly lower than in AN. The bulimic patients presented a circadian profile of 26RFa similar to that of controls. CONCLUSION High levels of circulating 26RFa observed in AN patients might reflect an adaptive mechanism of the organism to promote energy intake and to increase fat stores in response to undernutrition.

The orexin type 1 receptor is overexpressed in advanced prostate cancer with a neuroendocrine differentiation, and mediates apoptosis

AIM In the present study, we have examined the presence of orexins and their receptors in prostate cancer (CaP) and investigated their effects on the apoptosis of prostate cancer cells. METHODS We have localised the orexin type 1 and 2 receptors (OX1R and OX2R) and orexin A (OxA) in CaP sections of various grades and we have quantified tumour cells containing OX1R. Expression of OX1R was evaluated in the androgeno-dependent (AD) LNCaP and the androgeno-independent (AI) DU145 prostate cancer cells submitted or not to a neuroendocrine differentiation. The effects of orexins on the apoptosis and viability of DU145 cells were also investigated. RESULTS OX1R is strongly expressed in carcinomatous foci exhibiting a neuroendocrine differentiation, and the number of OX1R-stained cancer cells increases with the grade of the CaP. In contrast, OX2R is only detected in scattered malignant cells in high grade CaP. OX1R is expressed in the AI DU145 cells but is undetectable in the LNCaP cells. Acquisition of a neuroendocrine phenotype by the DU145 cells is associated with an overexpression of OX1R. Orexins induce the apoptosis of DU145 cells submitted to a neuroendocrine differentiation. CONCLUSION The present data indicate that OX1R-driven apoptosis is overexpressed in AI CaP exhibiting a neuroendocrine differentiation opening a gate for novel therapies for these aggressive cancers which are incurable until now.