Laurence Desrues

Isolation, characterization, and distribution of a novel neuropeptide, Rana RFamide (R-RFa), in the Brain of the european green frog Rana esculenta

A novel neuropeptide of the RFamide peptide family was isolated in pure form from a frog (Rana esculenta) brain extract by using reversed‐phase high performance liquid chromatography in combination with a radioimmunoassay for mammalian neuropeptide FF (NPFF). The primary structure of the peptide was established as Ser‐Leu‐Lys‐ Pro‐Ala‐Ala‐Asn‐Leu‐Pro‐Leu‐ Arg‐Phe‐NH2. The sequence of this neuropeptide, designated Rana RFamide (R‐RFa), exhibits substantial similarities with those of avian LPLRFamide, gonadotropin‐inhibitory hormone, and human RFRP‐1. The distribution of R‐RFa was investigated in the frog central nervous system by using an antiserum directed against bovine NPFF. In the brain, immunoreactive cell bodies were primarily located in the hypothalamus, i.e., the anterior preoptic area, the suprachiasmatic nucleus, and the dorsal and ventral hypothalamic nuclei. The most abundant population of R‐RFa–containing neurons was found in the periependymal region of the suprachiasmatic nucleus. R‐RFa– containing fibers were widely distributed throughout the brain from the olfactory bulb to the brainstem, and were particularly abundant in the external layer of the median eminence. In the spinal cord, scattered immunoreactive neurons were found in the gray matter. R‐RFa–positive processes were found in all regions of the spinal cord, but they were more abundant in the dorsal horn. This study provides the first characterization of a member of the RFamide peptide family in amphibians. The occurrence of this novel neuropeptide in the hypothalamus and median eminence and in the dorsal region of the spinal cord suggests that, in frog, R‐RFa may exert neuroendocrine activities and/or may be involved in the transmission of nociceptive stimuli. J. Comp. Neurol. 448:111–127, 2002.

Pituitary Adenylate Cyclase‐Activating Polypeptide (PACAP) Stimulates Adenylyl Cyclase and Phospholipase C Activity in Rat Cerebellar Neuroblasts

Abstract: The presence of receptors for the novel neuropeptide pituitary adenylate cyclase‐activating polypeptide (PACAP) has been recently demonstrated in the external granule cell layer of the cerebellum, a germinative matrix that generates the majority of cerebellar interneurons. In the present study, we have taken advantage of the possibility of obtaining a culture preparation that is greatly enriched in immature cerebellar granule cells to investigate the effect of PACAP on the adenylyl cyclase and phospholipase C transduction pathways. The two molecular forms of PACAP, i.e., 27‐(PACAP27) and 38‐(PACAP38) amino‐acid forms of PACAP, induced a dose‐dependent stimulation of cyclic AMP production in granule cells. The potencies of PACAP27 and PACAP38 were similar (ED50 = 0.12 ± 0.01 and 0.23 ± 0.07 nM, respectively), whereas vasoactive intestinal polypeptide (VIP) was ∼100 times less potent. PACAP27 and PACAP38 also induced a dose‐dependent stimulation of polyphosphoinositide breakdown (ED50 = 19.1 ± 6.3 and 13.4 ± 6.0 nM, respectively), whereas VIP had no effect on polyphosphoinositide metabolism. The effect of PACAP38 on inositol phosphate formation was significantly reduced by U‐73122 and by pertussis toxin, indicating that activation of PACAP receptors causes stimulation of a phospholipase C through a pertussis toxin‐sensitive G protein. In contrast, forskolin and dibutyryl cyclic AMP did not affect PACAP‐induced stimulation of inositol phosphates. Taken together, the present results demonstrate that PACAP stimulates independently the adenylyl cyclase and the phospholipase C transduction pathways in immature cerebellar granule cells. These data favor the concept that PACAP may play important roles in the control of proliferation and/or differentiation of cerebellar neuroblasts.

Central-type benzodiazepines and the octadecaneuropeptide modulate the effects of GABA on the release of α-melanocyte-stimulating hormone from frog neurointermediate lobe in vitro

The involvement of the GABA-benzodiazepine receptor complex in the regulation of melanotropin secretion has been investigated using perfused frog neurointermediate lobes. The GABAA agonist 3-amino-1 propane sulfonic acid mimicked the biphasic effect of GABA on alpha-melanocyte-stimulating hormone secretion: a brief stimulation followed by an inhibition of melanotropin secretion. The GABAA antagonist SR 95531 (10(-4) M) inhibited both stimulation and inhibition of alpha-melanocyte-stimulating hormone release induced by GABA (10(-4) M). Since the inhibitory effect of baclofen (10(-4) M) was partially antagonized by SR 95531 (10(-4) M), it appears that the GABAergic control of alpha-melanocyte-stimulating hormone release is mainly achieved through activation of GABAA receptors. GABA-induced stimulation of alpha-melanocyte-stimulating hormone release was inhibited by tetrodotoxin (10(-5) M), an Na+ -channel blocker, or nifedipine (10(-5) M), a voltage-dependent Ca2+ -channel blocker, suggesting that Na+ and Ca2+ ions are involved in the stimulatory phase of GABA action. Only central-type benzodiazepine binding site agonists such as clonazepam (10(-4) M) modified alpha-melanocyte-stimulating hormone release. In fact, clonazepam (10(-7) to 10(-5) M) led to a dose-dependent potentiation of both GABA-induced stimulation and inhibition of alpha-melanocyte-stimulating hormone release. This potentiating effect was antagonized by the GABAA antagonist SR 95531 (10(-4) M) or by the central-type benzodiazepine binding site antagonist flumazenil (10(-4) M), whereas picrotoxin (10(-4) M) abolished only the stimulatory phase.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemical and functional characterization of high-affinity urotensin II receptors in rat cortical astrocytes

The urotensin II (UII) gene is primarily expressed in the central nervous system, but the functions of UII in the brain remain elusive. Here, we show that cultured rat astrocytes constitutively express the UII receptor (UT). Saturation and competition experiments performed with iodinated rat UII ([125I]rUII) revealed the presence of high‐ and low‐affinity binding sites on astrocytes. Human UII (hUII) and the two highly active agonists hUII4‐11 and [3‐iodo‐Tyr9]hUII4‐11 were also very potent in displacing [125I]rUII from its binding sites, whereas the non‐cyclic analogue [Ser5,10]hUII4‐11 and somatostatin‐14 could only displace [125I]rUII binding at micromolar concentrations. Reciprocally, rUII failed to compete with [125I‐Tyr0,D‐Trp8]somatostatin‐14 binding on astrocytes. Exposure of cultured astrocytes to rUII stimulated [3H]inositol incorporation and increased intracellular Ca2+ concentration in a dose‐dependent manner. The stimulatory effect of rUII on polyphosphoinositide turnover was abolished by the phospholipase C inhibitor U73122, but only reduced by 56% by pertussis toxin. The GTP analogue Gpp(NH)p caused its own biphasic displacement of [125I]rUII binding and provoked an affinity shift of the competition curve of rUII. Pertussis toxin shifted the competition curve towards a single lower affinity state. Taken together, these data demonstrate that rat astrocytes express high‐ and low‐affinity UII binding sites coupled to G proteins, the high‐affinity receptor exhibiting the same pharmacological and functional characteristics as UT.

The endozepine ODN stimulates polyphosphoinositide metabolism in rat astrocytes

Astrocytes synthesize a series of peptides called endozepines which act as endogenous ligands of benzodiazepine receptors. The present study demonstrates that the endozepine ODN causes a dose‐dependent increase in inositol trisphosphate and a parallel decrease in phosphatidylinositol bisphosphate in cultured rat astrocytes. Pre‐incubation of astrocytes with the phospholipase C inhibitor U 73122 or with pertussis toxin totally blocked polyphosphoinositide metabolism. These data show that, in rat astrocytes, ODN stimulates a phospholipase C coupled to a pertussis toxin‐sensitive G protein.

The vasoactive peptides urotensin II and urotensin II-related peptide regulate astrocyte activity through common and distinct mechanisms: Involvement in cell proliferation

UII (urotensin II) and its paralogue URP (UII-related peptide) are two vasoactive neuropeptides whose respective central actions are currently unknown. In the present study, we have compared the mechanism of action of URP and UII on cultured astrocytes. Competition experiments performed with [125I]UII showed the presence of very-high- and high-affinity binding sites for UII, and a single high-affinity site for URP. Both UII and URP provoked a membrane depolarization accompanied by a decrease in input resistance, stimulated the release of endozepines, neuropeptides specifically produced by astroglial cells, and generated an increase in [Ca2+]c (cytosolic Ca2+ concentration). The UII/URP-induced [Ca2+]c elevation was PTX (pertussis toxin)-insensitive, and was blocked by the PLC (phospholipase C) inhibitor U73122 or the InsP3 channel blocker 2-APB (2-aminoethoxydiphenylborane). The addition of the Ca2+ chelator EGTA reduced the peak and abolished the plateau phase, whereas the T-type Ca2+ channel blocker mibefradil totally inhibited the Ca2+ response evoked by both peptides. However, URP and UII induced a mono- and bi-phasic dose-dependent increase in [Ca2+]c and provoked short- and long-lasting Ca2+ mobilization respectively. Similar mono- and bi-phasic dose-dependent increases in [3H]inositol incorporation into polyphosphoinositides in astrocytes was obtained, but the effect of UII was significantly reduced by PTX, although BRET (bioluminescence resonance energy transfer) experiments revealed that both UII and URP recruited Galphao-protein. Finally, UII, but not URP, exerted a dose-dependent mitogenic activity on astrocytes. Therefore we described that URP and UII exert not only similar, but also divergent actions on astrocyte activity, with UII exhibiting a broader range of activities at physiological peptide concentrations.

The Pituitary-Skin Connection in Amphibians: Reciprocal Regulation of Melanotrope Cells and Dermal Melanocytes

ABSTRACT: In amphibians, α‐MSH secreted by the pars intermedia of the pituitary plays a pivotal role in the process of skin color adaptation. Reciprocally, the skin of amphibians contains a number of regulatory peptides, some of which have been found to regulate the activity of pituitary melanotrope cells. In particular, the skin of certain species of amphibians harbours considerable amounts of thyrotropin‐releasing hormone, a highly potent stimulator of α‐MSH release. Recently, we have isolated and sequenced from the skin of the frog Phyllomedusa bicolor‐a novel peptide named skin peptide tyrosine tyrosine (SPYY), which exhibits 94% similarity with PYY from the frog Rana ridibunda. For concentrations ranging from 5 × 10−10 to 10−7 M, SPYY induces a dose‐related inhibition of α‐MSH secretion. At a dose of 10−7 M, SPYY totally abolished α‐MSH release. These data strongly suggest the existence of a regulatory loop between the pars intermedia of the pituitary and the skin in amphibians.

Multihormonal Regulation of Pituitary Melanotrophs

The pars intermedia of the pituitary is composed of one predominant category of endocrine cells, the melanotrophs, which synthesize the precursor protein proopiomelanocortin (POMC). 1,2 In lower vertebrates a-melanocyte stimulating hormone (a-MSH), which plays a pivotal role in skin color adaptation, is considered as the major peptide hormone secreted by melanotrope cells. Early experiments demonstrated that the release of MSH from the intermediate lobe is primarily under an inhibitory control from the hypothalamus. In particular, hypoth~amic lesions, pituitary stalk section, and autotransplantation of the pituitary all induce darkening of the animal^.^.^ However, Wilson and Morgan have observed that the concentration of MSH in the plasma of black-adapted Xenopus laevis was higher than in pituitary stalk-sectioned animals,’ suggesting the involvement of stimulatory hypothalamic factor(s) in the control of MSH release. The purpose of the present review is to summarize our current knowledge on the regulatory mechanisms that are involved in the control of the activity of amphibian pituitary meianotrophs.

The octadecaneuropeptide ODN prevents 6-hydroxydopamine-induced apoptosis of cerebellar granule neurons through a PKC-MAPK-dependent pathway.

Oxidative stress, induced by various neurodegenerative diseases, initiates a cascade of events leading to apoptosis, and thus plays a critical role in neuronal injury. In this study, we have investigated the potential neuroprotective effect of the octadecaneuropeptide (ODN) on 6‐hydroxydopamine (6‐OHDA)‐induced oxidative stress and apoptosis in cerebellar granule neurons (CGN). ODN, which is produced by astrocytes, is an endogenous ligand for both central‐type benzodiazepine receptors (CBR) and a metabotropic receptor. Incubation of neurons with subnanomolar concentrations of ODN (10−18 to 10−12 M) inhibited 6‐OHDA‐evoked cell death in a concentration‐dependent manner. The effect of ODN on neuronal survival was abrogated by the metabotropic receptor antagonist, cyclo1–8[DLeu5]OP, but not by a CBR antagonist. ODN stimulated polyphosphoinositide turnover and ERK phosphorylation in CGN. The protective effect of ODN against 6‐OHDA toxicity involved the phospholipase C/ERK MAPK transduction cascade. 6‐OHDA treatment induced an accumulation of reactive oxygen species, an increase of the expression of the pro‐apoptotic gene Bax, a drop of the mitochondrial membrane potential and a stimulation of caspase‐3 activity. Exposure of 6‐OHDA‐treated cells to ODN blocked all the deleterious effects of the toxin. Taken together, these data demonstrate for the first time that ODN is a neuroprotective agent that prevents 6‐OHDA‐induced oxidative stress and apoptotic cell death.

Beta-amyloid peptide stimulates endozepine release in cultured rat astrocytes through activation of N-formyl peptide receptors

Astroglial cells synthesize and release endozepines, a family of neuropeptides derived from diazepam‐binding inhibitor (DBI). The authors have recently shown that β‐amyloid peptide (Aβ) stimulates DBI gene expression and endozepine release. The purpose of this study was to determine the mechanism of action of Aβ in cultured rat astrocytes. Aβ25–35 and the N‐formyl peptide receptor (FPR) agonist N‐formyl‐Met‐Leu‐Phe (fMLF) increased the secretion of endozepines in a dose‐dependent manner with EC50 value of ≈2 μM. The stimulatory effects of Aβ25–35 and the FPR agonists fMLF and N‐formyl‐Met‐Met‐Met (fMMM) on endozepine release were abrogated by the FPR antagonist N‐t‐Boc‐Phe‐Leu‐Phe‐Leu‐Phe. In contrast, Aβ25–35 increased DBI mRNA expression through a FPR‐independent mechanism. Aβ25–35 induced a transient stimulation of cAMP formation and a sustained activation of polyphosphoinositide turnover. The stimulatory effect of Aβ25–35 on endozepine release was blocked by the adenylyl cyclase inhibitor somatostatin, the protein kinase A (PKA) inhibitor H89, the phospholipase C inhibitor U73122, the protein kinase C (PKC) inhibitor chelerythrine and the ATP binding cassette transporter blocker glyburide. Taken together, these data demonstrate for the first time that Aβ25–35 stimulates endozepine release from rat astrocytes through a FPR receptor positively coupled to PKA and PKC.