The neuropeptides pituitary adenylate cyclase-activating polypeptide (PACAP) and vasoactive intestinal peptide (VIP) are implicated in the photic entrainment of circadian rhythms in the suprachiasmatic nuclei (SCN). We now report that mice carrying a null mutation of the VPAC(2) receptor for VIP and PACAP (Vipr2(-/-)) are incapable of sustaining normal circadian rhythms of rest/activity behavior. These mice also fail to exhibit circadian expression of the core clock genes mPer1, mPer2, and mCry1 and the clock-controlled gene arginine vasopressin (AVP) in the SCN. Moreover, the mutants fail to show acute induction of mPer1 and mPer2 by nocturnal illumination. This study highlights the role of intercellular neuropeptidergic signaling in maintenance of circadian function within the SCN.

We have cloned and sequenced a cDNA (RPR4) encoding a new member of the secretin/calcitonin/parathyroid hormone (PTH) receptor family. RPR4 was identified by PCR of rat pituitary cDNA, and a full-length clone was isolated from a rat olfactory bulb cDNA library. When RPR4 was functionally expressed in COS 7 cells, cyclic adenosine monophosphate (cAMP) production was stimulated by vasoactive intestinal peptide (VIP), pituitary adenylate cyclase activating peptides (PACAP-38 and PACAP-27) and helodermin, with equal potency. Peptide histidine isoleucine (PHI) and rat growth hormone releasing hormone (rGHRH) also stimulated cAMP production at lower potency. This suggests that RPR4 encodes a novel VIP receptor which we have designated the VIP 2 receptor. In situ hybridisation showed that mRNA for this receptor was present mainly in the thalamus, hippocampus and in the suprachiasmatic nucleus.Vasoactive intestinal peptide (VIP); G protein-linked receptor; Rat pituitary; Rat olfactory bulb; cDNA cloning

Abstract-Heightened expression of the S100 calcium-binding protein, S100A4/Mts1, is observed in pulmonary vascular disease. Loss of serotonin (5-hydroxytryptamine [5-HT]) receptors or of the serotonin transporter (SERT) attenuates pulmonary hypertension in animals, and polymorphisms causing gain of SERT function are linked to clinical pulmonary vascular disease. Because 5-HT induces release of S100␤, we investigated the codependence of 5-HT receptors and SERT in regulating S100A4/Mts1 in human pulmonary artery smooth muscle cells (hPA-SMC). 5-HT elevated S100A4/Mts1 mRNA levels and increased S100A4/Mts1 protein in hPA-SMC lysates and culture media. S100A4/Mts1 in the culture media stimulated proliferation and migration of hPA-SMC in a manner dependent on the receptor for advanced glycation end products. Treatment with SB224289 (selective antagonist of 5-HT 1B ), fluoxetine (SERT inhibitor), SERT RNA-interference, and iproniazid (monoamine oxidase-A inhibitor), blocked 5-HT-induced S100A4/ Mts1. 5-HT signaling mediated phosphorylation (p) of extracellular signal-regulated kinase 1/2 (pERK1/2), but pERK1/2 nuclear translocation depended on SERT, monoamine oxidase activity, and reactive oxygen species. Nuclear translocation of pERK1/2 was required for pGATA-4 -mediated transcription of S100A4/Mts1. These data provide evidence for a mechanistic link between the 5-HT pathway and S100A4/Mts1 in pulmonary hypertension and explain how the 5-HT 1B receptor and SERT are codependent in regulating S100A4/Mts1. (Circ Res. 2005;97:227-235.)Key Words: smooth muscle cells Ⅲ pulmonary hypertension Ⅲ S100A4/Mts1 Ⅲ serotonin Ⅲ ERK1/2 A berrations in serotonin (5-hydroxytryptamine [5-HT])-mediated signaling events have been linked to pulmonary vascular disease (PVD). 1,2 Studies in transgenic mice indicate that both the serotonin transporter (SERT) 2,3 and 5-HT receptors 4 -6 are necessary in the development of pulmonary hypertension (PAH). An insertion/deletion polymorphism in the SERT promoter, resulting in 2-to 3-fold higher levels of SERT gene transcription, has been observed in 65% of idiopathic PAH (IPAH) versus 27% of control patients. 7 Additional studies have shown that patients using the anorectic drug dexfenfluramine, a 5-HT uptake inhibitor and SERT substrate that results in increased circulating 5-HT levels and exaggerated receptor signaling, 8 have a 23-fold increased risk of developing IPAH. 9 Similarly, patients with abnormal 5-HT platelet storage and elevated circulating 5-HT levels 10 similar to the Fawn-hooded rat, 11 are at increased risk of developing PAH.A recent study has linked SERT-mediated phosphorylation of ERK1/2 to phosphorylation of the transcription factor GATA-4 and to heightened expression of cyclin D2, a gene expressed in proliferating cells. 12 Transport of serotonin via SERT results in monoamine oxidase-A (MAO-A) activity, necessary for Rho kinase (ROCK)-mediated nuclear translocation of ERK1/2. 13 How SERT interacts with 5-HT receptor signaling in regulating genes specifically associated with P...

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Expression of coherent and rhythmic circadian (approximately 24 h) variation of behaviour, metabolism and other physiological processes in mammals is governed by a dominant biological clock located in the hypothalamic suprachiasmatic nuclei (SCN). Photic entrainment of the SCN circadian clock is mediated, in part, by vasoactive intestinal polypeptide (VIP) acting through the VPAC2 receptor. Here we used mice lacking the VPAC2 receptor (Vipr2-/-) to examine the contribution of this receptor to the electrophysiological actions of VIP on SCN neurons, and to the generation of SCN electrical firing rate rhythms SCN in vitro. Compared with wild-type controls, fewer SCN cells from Vipr2-/- mice responded to VIP and the VPAC2 receptor-selective agonist Ro 25-1553. By contrast, similar proportions of Vipr2-/- and wild-type SCN cells responded to gastrin-releasing peptide, arginine vasopressin or N-methyl-D-aspartate. Moreover, VIP-evoked responses from control SCN neurons were attenuated by the selective VPAC2 receptor antagonist PG 99-465. In firing rate rhythm experiments, the midday peak in activity observed in control SCN cells was lost in Vipr2-/- mice. The loss of electrical activity rhythm in Vipr2-/- mice was mimicked in control SCN slices by chronic treatment with PG 99-465. These results demonstrate that the VPAC2 receptor is necessary for the major part of the electrophysiological actions of VIP on SCN cells in vitro, and is of fundamental importance for the rhythmic and coherent expression of circadian rhythms governed by the SCN clock. These findings suggest a novel role of VPAC2 receptor signalling, and of cell-to-cell communication in general, in the maintenance of core clock function in mammals, impacting on the cellular physiology of SCN neurons.

The neuropeptides vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP) belong to a superfamily of structurally related peptide hormones that includes glucagon, glucagon-like peptides, secretin, and growth hormonereleasing hormone. Microinjection of VIP or PACAP into the rodent suprachiasmatic nucleus (SCN) phase shifts the circadian pacemaker and VIP antagonists, and antisense oligodeoxynucleotides have been shown to disrupt circadian function. VIP and PACAP have equal potency as agonists of the VPAC 2 receptor (VPAC2R), which is expressed abundantly in the SCN, in a circadian manner. To determine whether manipulating the level of expression of the VPAC 2R can influence the control of the circadian clock, we have created transgenic mice overexpressing the human VPAC 2R gene from a yeast artificial chromosome (YAC) construct. The YAC was modified by a strategy using homologous recombination to introduce (i) the HA epitope tag sequence (from influenza virus hemagglutinin) at the carboxyl terminus of the VPAC 2R protein, (ii) the lacZ reporter gene, and (iii) a conditional centromere, enabling YAC DNA to be amplified in culture in the presence of galactose. High levels of lacZ expression were detected in the SCN, habenula, pancreas, and testis of the transgenic mice, with lower levels in the olfactory bulb and various hypothalamic areas. Transgenic mice resynchronized more quickly than wild-type controls to an advance of 8 h in the light-dark (LD) cycle and exhibited a significantly shorter circadian period in constant darkness (DD). These data suggest that the VPAC 2R can influence the rhythmicity and photic entrainment of the circadian clock. E vidence is accumulating to suggest that the neuropeptides vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP) may play important roles in the control of the circadian clock in the suprachiasmatic nucleus (SCN). VIP is synthesized in cells of the retinorecipient region of the rodent SCN (1, 2), at least some of which may have a direct retinal input (3). These cells form synapses with vasopressin-containing cells in the dorsomedial SCN as well as contacts with other VIP cells (4). There is diurnal variation of VIP immunoreactivity (5) and prepro VIP mRNA (6, 7) in the SCN. Exogenous VIP can mimic the phase-advancing and -delaying effects of light pulses (8, 9). VIP antagonists (10) and VIP antisense oligodeoxynucleotides (11) have been shown to disrupt circadian function. Elevated SCN levels of VIP mRNA have been found in two mutant rodent strains [the tau mutant hamster (12) and the spontaneously hypertensive (SHR) rat (13)] that exhibit a significantly shorter free-running period in constant darkness (DD), consistent with an involvement of VIP in establishing the free-running period of the circadian clock.In the SCN, PACAP originates almost exclusively from a subpopulation of glutamate-containing retinal ganglion cells (14, 15) that terminate in the retinorecipient region of the SC...

The endopeptidase EC 3.4.24.15 (EP24.15) is a zinc metalloendopeptidase that is widely distributed in a variety of tissues, including the testes, pituitary and the central nervous system. Among its numerous roles in metabolizing and processing biologically-active peptides, the enzyme degrades gonadotropin-releasing hormone (GnRH) by cleaving the central Tyr5-Gly6 bond. The aim of the present studies was to determine whether EP24.15 can modulate the concentrations of GnRH within the hypothalamo-hypophysial portal blood and thereby play a physiological role in reproduction. Our data suggest the presence of immunoreactive EP24.15 in the perivascular space of the median eminence and that this enzyme is secreted into portal blood. We have also shown a physiological role for this enzyme in that an inhibition of its activity with a specific inhibitor augmented the steroid-induced LH increase in ovariectomized rats. The present results suggest that secretory and post-secretory mechanisms are important in shaping the GnRH signal from the central nervous system; GnRH metabolism by EP24.15 may be one such mechanism.

The master clock driving mammalian circadian rhythms is located in the suprachiasmatic nuclei (SCN) of the hypothalamus and entrained by daily light/dark cycles. SCN lesions abolish circadian rhythms of behavior and result in a loss of synchronized circadian rhythms of clock gene expression in peripheral organs (e.g., the liver) and of hormone secretion (e.g., corticosterone). We examined rhythms of behavior, hepatic clock gene expression, and corticosterone secretion in VPAC 2 receptor-null (Vipr2 Ϫ/Ϫ ) mice, which lack a functional SCN clock. Unexpectedly, although Vipr2 Ϫ/Ϫ mice lacked robust circadian rhythms of wheel-running activity and corticosterone secretion, hepatic clock gene expression was strongly rhythmic, but advanced in phase compared with that in wild-type mice. The timing of food availability is thought to be an important entrainment signal for circadian clocks outside the SCN. Vipr2 Ϫ/Ϫ mice consumed food significantly earlier in the 24 h cycle than wild-type mice, consistent with the observed timing of peripheral rhythms of circadian gene expression. When restricted to feeding only during the daytime (RF), mice develop rhythms of activity and of corticosterone secretion in anticipation of feeding time, thought to be driven by a food-entrainable circadian oscillator, located outside the SCN. Under RF, mice of both genotypes developed food-anticipatory rhythms of activity and corticosterone secretion, and hepatic gene expression rhythms also became synchronized to the RF stimulus. Thus, food intake is an effective zeitgeber capable of coordinating circadian rhythms of behavior, peripheral clock gene expression, and hormone secretion, even in the absence of a functional SCN clock.