[New biomarkers in serum and urine for detection of prostate cancer]. / Neue Biomarker im Serum und im Urin zur Detektion des Prostatakarzinoms.

Prostate-specific antigen (PSA) has revolutionized the management of prostate cancer (PCa) within the last 3 decades. This widely used tumour marker strongly correlates with the risk of harbouring a PCa but it lacks specificity. Therefore there is an urgent need for new biomarkers especially to detect clinically significant and aggressive PCa. Of all PSA-based markers, only the FDA-approved prostate health index phi shows improved specificity over percent free (%fPSA) and total PSA. Other serum kallikreins or sarcosine in serum or urine show more ambiguous data. In urine, the FDA-approved prostate cancer gene 3 (PCA3) has also proven its utility in the detection and management of early PCa with advantages as compared with PSA and %fPSA. However, some aspects of its correlation with aggressiveness and the low sensitivity at very high values have to be re-examined. The detection of alterations of the androgen regulated TMPRSS2 and ETS transcription factor genes in tissue of ~50% of all PCa patients was a milestone in PCa research. But only the combination of the urinary assays for TMPRSS2:ERG gene fusion and PCA3 (both use the same platform) show the expected improved accuracy for PCa detection. Comparisons of phi and PCA3, the best available PCa biomarkers so far, show an equal performance of both parameters.

Role of lipid droplet proteins in liver steatosis

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Five proteins of the perilipin (Plin) family such as Plin1 (perilipin) Plin2 (adipose differentiation-related protein), Plin3 (tail-interacting protein of 47 kDa), Plin4 (S3-12), and Plin5 (myocardial lipid droplet protein) are characterized as lipid droplet (LD) proteins in adipocytes. Recent reports have demonstrated that fat-specific protein 27 (FSP27) and hypoxia-inducible protein 2 (HIG2) are also thought to be novel LD proteins in addition to proteins of the Plin family. Growing evidence have shown that LD proteins play a role in the pathophysiology in the fatty liver disease which is characterized by hepatocytes containing LD with excessive neutral lipid. Studies showed LD proteins such as Plin1, Plin2, Plin3, Plin5, FSP27, and HIG2 are expressed in the liver steatosis. Among them, a high fat diet increases expression of Plin2 and/or FSP27 through activation of peroxisome proliferator-activated receptor ã to develop fatty liver. In this article, recent advances on the role of LD proteins in pathophysiology of fatty liver diseases are summarized (AU)

A peptidomics strategy to elucidate the proteolytic pathways that inactivate peptide hormones.

Proteolysis plays a key role in regulating the levels and activity of peptide hormones. Characterization of the proteolytic pathways that cleave peptide hormones is of basic interest and can, in some cases, spur the development of novel therapeutics. The lack, however, of an efficient approach to identify endogenous fragments of peptide hormones has hindered the elucidation of these proteolytic pathways. Here, we apply a mass spectrometry (MS) based peptidomics approach to characterize the intestinal fragments of peptide histidine isoleucine (PHI), a hormone that promotes glucose-stimulated insulin secretion (GSIS). Our approach reveals a proteolytic pathway in the intestine that truncates PHI at its C-terminus to produce a PHI fragment that is inactive in a GSIS assay, a result that provides a potential mechanism of PHI regulation in vivo. Differences between these in vivo peptidomics studies and in vitro lysate experiments, which showed N- and C-terminal processing of PHI, underscore the effectiveness of this approach to discover physiologically relevant proteolytic pathways. Moreover, integrating this peptidomics approach with bioassays (i.e., GSIS) provides a general strategy to reveal proteolytic pathways that may regulate the activity of peptide hormones.

Newly-identified receptors for peptide histidine-isoleucine and GHRH-like peptide in zebrafish help to elucidate the mammalian secretin superfamily.

A group of ten hormones in humans are structurally related and known as the secretin superfamily. These hormones bind to G-protein-coupled receptors that activate the cAMP pathway and are clustered as the secretin or B family. We used an evolutionary approach with zebrafish as a model to understand why some of these hormones, such as peptide histidine-methionine (PHM) and pituitary adenylate cyclase-activating polypeptide (PACAP)-related peptide (PRP) in humans lack a receptor. We used molecular techniques to clone two full-length receptor cDNAs in zebrafish, which were analyzed for amino acid sequence and ligand-binding motifs, phylogenetic position, synteny, tissue expression, functional response, and signaling pathway. Evidence is provided that the two cDNAs encoded the peptide histidine-isoleucine (PHI) receptor and PRP receptor, which is known as GHRH-like peptide (GHRH-LP) receptor in non-mammals. Further, we cloned a zebrafish cDNA encoding the peptides PHI and vasoactive intestinal peptide (VIP). The PHIR had been previously labeled as one type of a VIP-PACAP (VPAC2R) shared receptor based only on sequence data. The PHIR cDNA, transfected into COS7 cells, responded to zebrafish PHI in a sensitive and dose-dependent manner (EC(50)=1.8x10(-9) M) but not to PACAP and VIP. The GHRH-LP receptor responded to both zebrafish GHRH-LP1 and GHRH with a 3.5-fold greater response to the former. For comparison, two zebrafish receptors (PAC1R and VPAC1R) and two human receptors (VPAC2R and GHRHR) were tested with human and/or zebrafish peptides. Unexpectedly, zebrafish VIP activated its PAC1R suggesting that in evolution, PAC1R is not always a specific receptor for PACAP. We conclude that zebrafish, like goldfish, have a specific receptor for PHI and GHRH-LP. Our evidence that zebrafish PHI is more potent than human PHM in activating the human VPAC2R (EC(50)=7.4x10(-9) M) supports our suggestion that the VPAC2R and PHIR shared a common ancestral receptor.

Unconventional binding sites and receptors for VIP and related peptides PACAP and PHI/PHM: an update.

The 28-amino-acid neuropeptide VIP and related peptides PACAP and PHI/PHM modulate virtually all of the vital functions in the body. These peptides are also commonly recognized as major regulators of cell growth and differentiation. Through their trophic and cytoprotective functions, they appear to play major roles in embryonic development, neurogenesis and the progression of a number of cancer types. These peptides bind to three well-characterized subtypes of G-protein coupled receptors: VPAC1 and VPAC2 share a common high affinity in the nanomolar range for VIP and PACAP; a third receptor type, PAC1, has been characterized for its high affinity for PACAP but its low affinity for VIP. Complex effects and pharmacological behaviors of these peptides suggest that multiple subtypes of binding sites may cooperate to mediate their function in target cells and tissues. In this complex response, some of these binding sites correspond to the definition of the conventional receptors cited above, while others display unexpected pharmacological and functional properties. Here we present potential clues that may lead investigators to further characterize the molecular nature and functions of these atypical binding species.

Glucagon-related peptides in the mouse retina and the effects of deprivation of form vision.

BACKGROUND: In chickens, retinal glucagon amacrine cells play an important role in emmetropization, since they express the transcription factor ZENK (also known as NGFI-A, zif268, tis8, cef5, Krox24) in correlation with the sign of imposed image defocus. Pharmacological studies have shown that glucagon can act as a stop signal for axial eye growth, making it a promising target for pharmacological intervention of myopia. Unfortunately, in mammalian retina, glucagon itself has not yet been detected by immunohistochemical staining. To learn more about its possible role in emmetropization in mammals, we studied the expression of different members of the glucagon hormone family in mouse retina, and whether their abundance is regulated by visual experience. METHODS: Black wildtype C57BL/6 mice, raised under a 12/12 h light/dark cycle, were studied at postnatal ages between P29 and P40. Frosted hemispherical thin plastic shells (diffusers) were placed in front of the right eyes to impose visual conditions that are known to induce myopia. The left eyes remained uncovered and served as controls. Transversal retinal cryostat sections were single- or double-labeled by indirect immunofluorescence for early growth response protein 1 (Egr-1, the mammalian ortholog of ZENK), glucagon, glucagon-like peptide-2 (GLP-2), glucose-dependent insulinotropic polypeptide (GIP), peptide histidine isoleucine (PHI), growth hormone-releasing hormone (GHRH), pituitary adenylate cyclase-activating polypeptide (PACAP), secretin, and vasoactive intestinal polypeptide (VIP). In total, retinas of 45 mice were studied, 28 treated with diffusers, and 17 serving as controls. RESULTS: Glucagon itself was not detected in mouse retina. VIP, PHI, PACAP and GIP were localized. VIP was co-localized with PHI and Egr-1, which itself was strongly regulated by retinal illumination. Diffusers, applied for various durations (1, 2, 6, and 24 h) had no effect on the expression of VIP, PHI, PACAP, and GIP, at least at the protein level. Similarly, even if the analysis was confined to cells that also expressed Egr-1, no difference was found between VIP expression in eyes with diffusers and in eyes with normal vision. CONCLUSIONS: Several members of the glucagon super family are expressed in mouse retina (although not glucagon itself), but their expression pattern does not seem to be regulated by visual experience.

Expression and GTP sensitivity of peptide histidine isoleucine high-affinity-binding sites in rat.

High-affinity-binding sites for the vasoactive intestinal peptide (VIP) analogs peptide histidine/isoleucine-amide (PHI)/carboxyterminal methionine instead of isoleucine (PHM) are expressed in numerous tissues in the body but the nature of their receptors remains to be elucidated. The data presented indicate that PHI discriminated a high-affinity guanosine 5'-triphosphate (GTP)-insensitive-binding subtype that represented the totality of the PHI-binding sites in newborn rat tissues but was differentially expressed in adult animals. The GTP-insensitive PHI/PHM-binding sites were also observed in CHO cells over expressing the VPAC2 but not the VPAC1 VIP receptor.

PACAP- and PHI-mediated sustained relaxation in circular muscle of gastric fundus: findings obtained in PACAP knockout mice.

Mediators of neurogenic responses of the gastric fundus were studied in wild type and pituitary adenylate cyclase activating peptide (PACAP) knockout mice. Electrical field stimulation (EFS) to the circular muscle strips of the wild type mouse fundus induced a tri-phasic response, rapid transient contraction and relaxation, and sustained relaxation that was prolonged for an extended period after the end of EFS. The transient relaxation and contraction were completely inhibited by N(G)-nitro-L-arginine and atropine, respectively. The sustained relaxation was completely inhibited by a PACAP receptors antagonist, PACAP(6-38). The strips prepared from PACAP knockout mice exhibited a large contraction without rapid relaxation and unexpectedly, a sustained relaxation. However, the sustained relaxation was decreased to about a half of that observed in wild type mice. Anti-peptide histidine isoleucine (PHI) serum abolished the sustained relaxation in the knockout mice. The serum partially inhibited the sustained relaxation in wild type mice and PACAP(6-38) abolished the relaxation that remained after the antiserum-treatment. PHI relaxed the strips prepared from wild type mice. The relaxation was completely inhibited by PACAP(6-38). It was concluded that PACAP and PHI separately mediate the sustained relaxation in the mouse gastric fundus, and that nitric oxide and ACh mediate transient relaxation and contraction, respectively.

Roles of PACAP and PHI as inhibitory neurotransmitters in the circular muscle of mouse antrum.

Mediators of neurogenic responses of the gastric antrum were studied in wild-type and pituitary adenylate cyclase-activating polypeptide (PACAP) -knockout (KO) mice. Electrical field stimulation (EFS) to the circular muscle strips of the wild-type mouse antrum induced a triphasic response; rapid transient relaxation and contraction, and sustained relaxation that was prolonged for an extended period after the end of EFS. The transient relaxation and contraction were completely inhibited by L-nitroarginine and atropine, respectively. The sustained relaxation was significantly inhibited by a PACAP receptor antagonist, PACAP(6-38). The antral strips prepared from PACAP-KO mice unexpectedly exhibited a tri-phasic response. However, the sustained relaxation was decreased to about one-half of that observed in wild-type mice. PACAP(6-38) inhibited EFS-induced sustained relaxation (33.5% of control) in PACAP-KO mice. Anti-peptide histidine isoleucine (PHI) serum partially (the 30% inhibition) or significantly (the 60% inhibition) inhibited the sustained relaxations in the wild-type and PACAP-KO mice, respectively. The immunoreactivities to the anti-PACAP and anti-PHI serums were found in myenteric ganglia of the mouse antrum. These results suggest that nitric oxide and acetylcholine mediate the transient relaxation and contraction, respectively, and that PACAP and PHI separately mediate the sustained relaxation in the antrum of the mouse stomach.

VPAC2 receptors mediate vasoactive intestinal peptide-induced neuroprotection against neonatal excitotoxic brain lesions in mice.

Prepro-vasoactive intestinal peptide (VIP) mRNA codes for two neuropeptides: VIP and peptide histidine isoleucine (PHI). Two VIP receptors, shared with a similar affinity by pituitary adenylate cyclase-activating polypeptide (PACAP), have been cloned: VPAC(1) and VPAC(2). PHI binds to these receptors with a lower affinity. VPAC receptors are classically associated with a cAMP-dependent pathway, although other pathways, including calcium mobilization and protein kinase C activation have been described. We previously showed that intracerebral administration of the glutamate agonist ibotenate to postnatal day 5 mice induces white matter lesions mimicking human periventricular leukomalacia. In this model, coinjection of VIP protects against white matter lesions. This neuroprotection is independent from cAMP and is mediated by protein kinase C. Using this model, this study aimed to determine the receptor involved in VIP-induced neuroprotection. VIP effects were mimicked with a similar potency by VPAC(2) agonists and PHI but not by VPAC(1) agonists, PACAP 27, or PACAP 38. VIP neuroprotective effects were lost in mice lacking VPAC(2) receptor. In situ hybridization confirmed the presence of VPAC(2) mRNA in the postnatal day 5 white matter. When analyzed between embryonic life and adulthood, VIP-specific binding site density peaked at postnatal day 5. These data suggest that, in this model, VIP-induced neuroprotection is mediated by VPAC(2) receptors. The pharmacology of this VPAC(2) receptor seems unconventional because 1) PACAP does not mimic VIP effects, 2) PHI acts with a comparable potency, and 3) PACAP 27 modestly inhibited the VIP-specific binding, whereas for PHI or VIP, inhibition was complete.

[Neuroprotective role of PACAP, VIP, and PHI in the central nervous system]. / Neuroprotekcyjna rola peptydów PACAP, VIP oraz PHI w osrodkowym ukladzie nerwowym.

Pituitary adenylate cyclase-activating polypeptide (PACAP), vasoactive intestinal peptide (VIP), and peptide histidine-isoleucine (PHI) belong to a structurally related family of polypeptides present in many regions of the central and peripheral nervous system. The neuroprotective potential of PACAP, VIP, and PHI has become a matter of intensive investigations in many animal models. In vitro studies revealed that PACAP protects neurons against apoptosis occurring naturally during CNS development and apoptosis induced by a series of neurotoxins, such as ethanol, hydrogen peroxide (H2O2), prion protein, beta-amyloid, HIV envelope glycoprotein (gp120), potassium ion deficit, and high glutamate concentrations. Similarly, in vivo investigations conducted in models of ischemia and Parkinson's disease confirmed the neuroprotective properties of PACAP. It was revealed that the anti-apoptotic action of PACAP can be directly associated with the activation of signal transduction pathways preventing apoptosis in neurons or involve glial cells capable of releasing other neuroprotective factors affecting neurons. In contrast to PACAP, the neuroprotective action of VIP depends mainly on stimulation of astrocytes to produce and secrete factors of extremely high neuroprotective potential, including activity-dependent neurotrophic factor (ADNF) and activity-dependent neuroprotective protein (ADNP). It was shown that ADNF and ADNP, as well as their shortened derivatives ADNF-9 and NAP, prevent neurons from electrical blockade, excitotoxicity, apoE deficiency, glucose deficit, ischemia, toxic action of ethanol, beta-amyloid, and gp120. The neuroprotective potential of PHI has not been as thoroughly investigated yet, but recent data have confirmed that this peptide can also function as a neuroprotectant. It is thought that PACAP, VIP, and possibly PHI may serve as a goal of modern therapeutic strategies in various neurodegenerative disorders.

Demonstration of nerve fibers immunoreactive to peptide N-terminal histidine C-terminal isoleucine (PHI) and vasoactive intestinal peptide (VIP) in the pig pineal gland.

The pineal functions are modulated by some neuropeptides including PHI and VIP. The presence of PHI-immunoreactive and VIP-immunoreactive nerve fibers in the pineal gland has been shown in several mammalian species. Both peptides influence the pineal serotonin N-acetyltransferase activity and melatonin synthesis. The aim of the present study was to examine the localization of PHI- and VIP-immunoreactive nerve fibers in the pig pineal gland. Four three-month old female pigs housed in natural light conditions, with free access to food and water, were used in the study. The pineals were fixed by perfusion with 4% paraformaldehyde in 0.1 M phosphate buffer. An immunohistochemical ABC streptavidin-biotin-complex method was used for the demonstration of PHI and VIP. PHI- and VIP-immunopositive nerve fibers were found in the pineal gland as well as in the habenular and posterior commissural areas. In the pineal gland, the density of PHI-immunoreactive nerve fibers was considerably higher than that of the fibers containing VIP. PHI- and VIP-immunopositive nerve fibers were more abundant in the cortical than in the medullary part of the gland. The nerve fibers formed bundles in the pineal capsule, from where they penetrated to the connective tissue septa and formed a dense meshwork surrounding blood vessels. In the parenchyma, PHI- and VIP-immunoreactive nerve terminals created baskets around clusters of pinealocytes. No PHI- or VIP-immunopositive cells were found in the pig pineal gland.

Disrupted circadian rhythms in VIP- and PHI-deficient mice.

The related neuropeptides vasoactive intestinal peptide (VIP) and peptide histidine isoleucine (PHI) are expressed at high levels in the neurons of the suprachiasmatic nucleus (SCN), but their function in the regulation of circadian rhythms is unknown. To study the role of these peptides on the circadian system in vivo, a new mouse model was developed in which both VIP and PHI genes were disrupted by homologous recombination. In a light-dark cycle, these mice exhibited diurnal rhythms in activity which were largely indistinguishable from wild-type controls. In constant darkness, the VIP/PHI-deficient mice exhibited pronounced abnormalities in their circadian system. The activity patterns started approximately 8 h earlier than predicted by the previous light cycle. In addition, lack of VIP/PHI led to a shortened free-running period and a loss of the coherence and precision of the circadian locomotor activity rhythm. In about one-quarter of VIP/PHI mice examined, the wheel-running rhythm became arrhythmic after several weeks in constant darkness. Another striking example of these deficits is seen in the split-activity patterns expressed by the mutant mice when they were exposed to a skeleton photoperiod. In addition, the VIP/PHI-deficient mice exhibited deficits in the response of their circadian system to light. Electrophysiological analysis indicates that VIP enhances inhibitory synaptic transmission within the SCN of wild-type and VIP/PHI-deficient mice. Together, the observations suggest that VIP/PHI peptides are critically involved in both the generation of circadian oscillations as well as the normal synchronization of these rhythms to light.

A hVIPR transgene as a novel tool for the analysis of circadian function in the mouse suprachiasmatic nucleus.

A mouse bearing a novel transgene encoding the human VPAC2 receptor (hVIPR; Shen et al. (2000) PNAS, 97, 11575-11580) was used to investigate circadian function in the hypothalamic suprachiasmatic nuclei (SCN). Neurons expressing hVPAC2R, detected by a beta-galactosidase (beta-GAL) tag, have a distinct distribution within the SCN, closely matching that of neurophysin (NP) neurons and extending into the region of peptide histidine isoleucine (PHI) cells. In common with NP and PHI cells, neurons expressing hVPAC2R are circadian in nature, as revealed by synchronous rhythmic expression of mPERIOD (mPER) proteins. A population of SCN cells not expressing PHI, NP or hVPAC2R exhibited circadian PER expression antiphasic with the rest of the SCN. Nocturnal light exposure induced mPER1 in the ventral SCN and mPER2 widely across the nucleus. Induction of nuclear mPER2 in hVPAC2R cells confirmed their photic responsiveness. Having established their circadian properties, we tested the utility of SCN neurons expressing the hVIPR transgene as functionally and anatomically explicit markers for SCN tissue grafts. Prenatal SCN tissue from hVIPR transgenic pups survived transplantation into adult CD1 mice, and expressed beta-GAL, PER and PHI. Over a series of studies, hVIPR transgenic SCN grafts restored circadian activity rhythms to 17 of 72 arrhythmic SCN lesioned recipients (23.6%). By using heterozygous hVIPR transgenic grafts on a heterozygous Clock mutant background we confirmed that restored activity rhythms were conferred by the donor tissue. We conclude that the hVIPR transgene is a powerful and flexible tool for examination of circadian function in the mouse SCN.

A hVIPR transgene as a novel tool for the analysis of circadian function in the mouse suprachiasmatic nucleus.

A mouse bearing a novel transgene encoding the human VPAC2 receptor (hVIPR; Shen et al. (2000) PNAS, 97, 11575-11580) was used to investigate circadian function in the hypothalamic suprachiasmatic nuclei (SCN). Neurons expressing hVPAC2R, detected by a beta-galactosidase (beta-GAL) tag, have a distinct distribution within the SCN, closely matching that of neurophysin (NP) neurons and extending into the region of peptide histidine isoleucine (PHI) cells. In common with NP and PHI cells, neurons expressing hVPAC2R are circadian in nature, as revealed by synchronous rhythmic expression of mPERIOD (mPER) proteins. A population of SCN cells not expressing PHI, NP or hVPAC2R exhibited circadian PER expression antiphasic with the rest of the SCN. Nocturnal light exposure induced mPER1 in the ventral SCN and mPER2 widely across the nucleus. Induction of nuclear mPER2 in hVPAC2R cells confirmed their photic responsiveness. Having established their circadian properties, we tested the utility of SCN neurons expressing the hVIPR transgene as functionally and anatomically explicit markers for SCN tissue grafts. Prenatal SCN tissue from hVIPR transgenic pups survived transplantation into adult CD1 mice, and expressed beta-GAL, PER and PHI. Over a series of studies, hVIPR transgenic SCN grafts restored circadian activity rhythms to 17 of 72 arrhythmic SCN lesioned recipients (23.6%). By using heterozygous hVIPR transgenic grafts on a heterozygous Clock mutant background we confirmed that restored activity rhythms were conferred by the donor tissue. We conclude that the hVIPR transgene is a powerful and flexible tool for examination of circadian function in the mouse SCN.

Cyclic AMP formation in chicken brain: effect of vasoactive intestinal peptide, peptide histidine-isoleucine (PHI), and some PHI-related peptides.

Vasoactive intestinal peptide (chicken form; chVIP), peptide histidine-isoleucine (porcine and rat forms; pPHI and rPHI), D-Phe(4) derivative of porcine PHI (D-Phe(4)-pPHI), peptide histidine-methionine (PHM; human PHI), and helodermin, were tested for their ability to stimulate cAMP production in [(3)H]adenine-prelabeled slices of chick cerebral cortex (CCx) and hypothalamus (HTh). The chVIP (0.1-3 microM) concentration-dependently and potently stimulated cAMP production in HTh and CCx; the responses observed after 3 microM of chVIP were comparable to those produced by 0.1 microM PACAP38. Helodermin (5 microM) moderately but significantly stimulated cAMP formation in both HTh and CCx, whereas pPHI, rPHI, PHM at 5 microM concentration only weakly affected cAMP production in CCx, and were inactive in HTh; D-Phe(4)-pPHI was inactive in both tissues. These data demonstrate that chVIP, PACAP, and to a lesser extent helodermin were capable of potently stimulating cAMP generation in the avian central nervous system. PHI-related peptides showed only weak or no activity, depending on the tissue.

Involvement of peptide histidine isoleucine in non-adrenergic non-cholinergic relaxation of the rat gastric fundus induced by high-frequency neuronal firing.

The role of peptide histidine isoleucine (PHI) as a neurotransmitter of the inhibitory motor neurones, the physiological role of PHI and vasoactive intestinal polypeptide (VIP) in the non-adrenergic non-cholinergic (NANC) relaxation and the relative amounts of VIP- and PHI-like immunoreactivity (LI) co-released by neuronal activation were investigated in the rat gastric fundus. Longitudinal muscle strips from the rat gastric fundus precontracted by the thromboxane receptor agonist U46619 (0.1 micro M) were studied in organ baths under conditions of muscarinic receptor blockade by atropine (1 micro M) and adrenergic neurone blockade by guanethidine (5 micro M) ("NANC conditions"). Concentration-response curves were plotted for both amplitude and area under the curve (AUC) of the relaxant responses induced by VIP (0.3 nM-0.3 micro M), PHI (0.3 nM-1 micro M) and peptide histidine valine [PHV(1-42); 0.3 nM-1 micro M]. All three peptides were more potent in the curve based on amplitude than in that based on the AUC of relaxation. In addition, VIP was 5.3 and 7 times more potent than PHI and PHV(1-42), respectively, in producing relaxation expressed as amplitude, and 2.7 and 2.8 times, respectively, in producing relaxation expressed as AUC. PHI and PHV(1-42) behaved as partial agonists with respect to VIP in producing relaxation expressed as AUC. Electrical field stimulation (EFS; 120 mA, 1 ms, 4-32 Hz, pulse trains of 2 min) evoked frequency-dependent relaxant responses. Alpha-chymotrypsin (1 u/ml or 3 u/ml), an anti-VIP serum (1:100 or 1:50) and an anti-PHI serum (1:25), slightly reduced the amplitude, but greatly inhibited the AUC of the NANC relaxation induced by EFS (13 Hz) [approximately 72%, 47% and 28% less than that seen in time controls or with normal rabbit serum (1:100 or 1:25), respectively]. EFS (8-32 Hz) caused significant, frequency-dependent increases in the outflow of VIP- and PHI-LI from the strips. The EFS-induced release of VIP-LI was approximately 20% of the PHI-LI release. These findings indicate that PHI is involved in EFS-induced NANC relaxation of the rat gastric fundus, the major physiological role of VIP and PHI is the maintenance of smooth muscle relaxation and VIP is co-released in equimolar amounts mainly with PHI.

The VPAC(2) receptor is essential for circadian function in the mouse suprachiasmatic nuclei.

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.

Identification of a potential receptor for both peptide histidine isoleucine and peptide histidine valine.

Peptide histidine isoleucine (PHI), peptide histidine valine (PHV), and vasoactive intestinal polypeptide (VIP) are cosynthesized from the same precursor and share high levels of structural similarities with overlapping biological functions. In this study, the first PHI/PHV receptor was isolated and characterized in goldfish. To study this receptor using homologous peptides, we have also characterized the goldfish prepro-PHI/VIP, and, surprisingly, a shorter transcript lacking the VIP coding region was isolated. A PHI/VIP precursor without the VIP coding sequence has never before been reported. Initial functional expression of the PHI/PHV receptor in Chinese hamster ovary cells revealed that it could be activated by human PHV [50% effective concentration (EC(50)): 43 nM] and to a lesser extent human PHI (EC(50): 133 nM) and helodermin (EC(50): 166 nM) but not fish and mammalian pituitary adenylate cyclase-activating polypeptides and VIPs. Subsequent studies indicated that, similar to the pituitary adenylate cyclase-activating polypeptide receptors (PAC1-R, VPAC1-R, and VPAC2-R), the receptor isolated in this study is able to interact with goldfish PHI and its C-terminally extended form, PHV with EC(50) values 93 and 43 nM, respectively. Northern blot and RT-PCR/Southern blot analyses revealed that the PHI/VIP gene is expressed in the intestine, brain, and gall bladder and the PHI/PHV receptor gene is primarily expressed in the pituitary and to a lesser extend in the intestine and gall bladder, suggesting that PHI/PHV may play a role, notably in the regulation of pituitary function. In conclusion, our results demonstrate for the first time the existence of a PHI/PHV receptor, indicating that the functions of PHI and PHV could be mediated by their own receptor in addition to VIP receptors.

The polypeptide PHI discriminates a GTP-insensitive form of VIP receptor in liver membranes.

In early reports on 125I-VIP binding experiments in liver membranes, it has been proposed that, the VIP binding sites were partially sensitive to GTP. Here we confirm that the VIP binding sites of chicken liver membranes consisted mainly in bivalent VIP/PACAP receptors and that about 50% of the 125I-VIP binding capacity was not affected by the GTP analogue GppNHp. Part of these bivalent receptors also appeared to represent PHI binding sites. In GppNHp-treated membranes, the GTP-insensitive VIP binding sites displayed a 17-fold higher relative affinity than in control membranes for the VIP analogue PHI. Such data suggested that GTP-insensitive VIP receptors may correspond to a subclass of high-affinity PHI receptors. Cross-linking of 125 I-VIP or 125 I-PHI to their receptors, revealed 2 components of 48 and 60 kDa. The radiolabelling of the 60 kDa component was strongly affected by increasing concentrations of the GTP analogue but was modestly abolished by an excess of PHI. Conversely, the radiolabelling of the 48 kDa molecular form was not affected by the GTP analogue but was efficiently abolished by increasing concentrations of PHI. Taken together, the data suggest that the 48 kDa component expressed in chicken liver membranes display the properties of a GTP-insensitive VIP/PHI receptor that can be pharmacologically discriminated from the GTP-sensitive 60 kDa form, through its much higher affinity for PHI.