VIP in inflammatory bowel disease: state of the art.

The pathogenesis of inflammatory bowel syndrome (IBD), which includes Crohn's disease (CD) and ulcerative colitis (UC) is poorly understood. However, an inflammatory component is a common hallmark. It has been suggested that CD principally involves Th1 and/or Th17 cells, while UC is considered to be more Th2 driven. Because vasoactive intestinal peptide (VIP) has emerged in the last decade as a putative candidate for the treatment of inflammatory diseases with a Th1 component, it may as well serve as a therapeutic target in CD. In addition, experiments using mice deficient in VIP or its receptors have revealed that the endogenously-produced VIP may participate in the regulation of immunity. The aim of the present review is to summarize the quite considerable array of data which suggests that the VIP-receptor system plays a key role in modulating multiple molecular and cellular players involved in IBD.

Vasoactive intestinal peptide/vasoactive intestinal peptide receptor relative expression in salivary glands as one endogenous modulator of acinar cell apoptosis in a murine model of Sjögren's syndrome.

Sjögren's syndrome (SS) is a chronic autoimmune disease characterized by a progressive oral and ocular dryness that correlates poorly with the autoimmune damage of the glands. It has been proposed that a loss of homeostatic equilibrium in the glands is partly responsible for salivary dysfunction with acinar cells involved actively in the pathogenesis of SS. The non-obese diabetic (NOD) mouse model of Sjögren's syndrome develops secretory dysfunction and early loss of glandular homeostatic mechanisms, with mild infiltration of the glands. Based on the vasodilator, prosecretory and trophic effects of the vasoactive intestinal peptide (VIP) on acini as well as its anti-inflammatory properties we hypothesized that the local expression of VIP/vasoactive intestinal peptide receptor (VPAC) system in salivary glands could have a role in acinar cell apoptosis and macrophage function thus influencing gland homeostasis. Here we show a progressive decline of VIP expression in submandibular glands of NOD mice with no changes in VPAC receptor expression compared with normal mice. The deep loss of endogenous VIP was associated with a loss of acinar cells through apoptotic mechanisms that could be induced further by tumour necrosis factor (TNF)-α and reversed by VIP through a cyclic adenosine-5'-monophosphate (cAMP)/protein kinase A (PKA)-mediated pathway. The clearance of apoptotic acinar cells by macrophages was impaired for NOD macrophages but a shift from inflammatory to regulatory phenotype was induced in macrophages during phagocytosis of apoptotic acinar cells. These results support that the decline in endogenous VIP/VPAC local levels might influence the survival/apoptosis intracellular set point in NOD acinar cells and their clearance, thus contributing to gland homeostasis loss.

Investigation of cancer cell lines for peptide receptor-targeted drug development.

Many tumors highly express specific populations of G-protein-coupled receptors (GPCRs) that could be utilized for receptor-targeted therapy. We confirmed significant quantities of mRNAs specific for certain somatostatin (SST), vasoactive intestinal peptide (VIP), and bombesin (BN) receptors in various commercially available tumor cell lines. Very few of the tumor cell lines examined displayed the high receptor-binding affinity despite exhibiting the expression of appropriate mRNAs and proteins of the cognate receptors. However, binding assays establish that some tumor cell lines, such as pancreatic cancer CFPAC-1, prostate cancer DU-145, and pancreatic carcinoid BON, demonstrate high BN receptor binding. BON cells also demonstrate high somatostatin receptor (SSTR) affinity binding. We also found that tumor cell lines, such as BON and host cells expressing SST receptor subtypes 1 or 2 (CHO-R1 or CHO-R2), underwent a decrease in cell surface receptor density in multiple passages. BON and CHO-R2 cells also rapidly internalize a significant proportion of cell surface ligand-receptor complexes. The tumor cells CFPAC-1, DU-145, and BON with high receptor binding could be useful for peptide drug studies. BON cells were further applied to test SST/BN analogs and cytotoxic conjugates. Furthermore, the in vivo antitumor assay showed that the cytotoxic conjugate CPT-SST targeting all SSTR subtypes displayed a potent tumor-suppressive ability to BON tumors expressing multiple SSTR subtypes.

Regulation of human polymorphonuclear leukocytes functions by the neuropeptide pituitary adenylate cyclase-activating polypeptide after activation of MAPKs.

Anti-inflammatory activities of pituitary adenylate cyclase-activating protein (PACAP) are mediated in part through specific effects on lymphocytes and macrophages. This study shows that in human polymorphonuclear neutrophils (PMNs), PACAP acts as a proinflammatory molecule. In PMNs, vaso-intestinal peptide/PACAP receptor 1 (VPAC-1) was the only receptor found to be expressed by RT-PCR. Using VPAC-1 Ab, we found that VPAC-1 mRNA was translated into proteins. In PMNs, PACAP increases cAMP, inositol triphosphate metabolites, and calcium. It activates two of the three members of the MAPK superfamily, the ERK and the stress-activated MAPK p38. U73122, an inhibitor of phospholipase C (PLC), inhibits PACAP-induced ERK activation, whereas p38 MAPK phosphorylation was unaffected. Using specific pharmalogical inhibitors of ERK (PD098059) and p38 MAPK (SB203580), we found that PACAP-mediated calcium increase was ERK and PLC dependent and p38 independent. PACAP primes fMLP-associated calcium increase; it also primes fMLP activation of the respiratory burst as well as elastase release, these last two processes being ERK and PLC dependent and p38 MAPK independent. PACAP also increases membrane expression of CD11b and release of lactoferrin and metallo proteinase-9 (MMP-9). These effects were PLC dependent (CD 11b, lactoferrin, MMP-9), ERK dependent (CD 11b, lactoferrin, MMP-9), and p38 dependent (CD11b, lactoferrin). We conclude that PACAP is a direct PMN activator as well as an effective PMN priming agent that requires PLC, ERK, and p38 MAPK activities.

Expression of VIP and/or PACAP receptor mRNA in peptide synthesizing cells within the suprachiasmatic nucleus of the rat and in its efferent target sites.

The suprachiasmatic nucleus (SCN) contains the predominant circadian pacemaker in mammals. Considerable evidence indicates that VPAC(2) and PAC(1), receptors for vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating peptide (PACAP), play critical roles in maintaining and entraining circadian rhythms. Retinal projections to the rat SCN contain PACAP and terminate mostly in the ventral SCN, the site of VIP neurons. The incidence of VPAC(2) and PAC(1) mRNAs within distinct neuronal populations of the rat SCN has been determined using double-label in situ hybridization. VPAC(2) mRNA was detected in almost all arginine-vasopressin (AVP) neurons of the dorsomedial SCN and in 41% of the VIP neurons; somatostatin (SST) neurons, predominantly in dorsomedial and intermediate regions, showed a decreased incidence (23%). PAC(1) mRNA was present in nearly half of the VIP and SST neurons (45% and 40%, respectively) and in one-third of the AVP neurons (32%). Cells expressing VPAC(2) mRNA also were detected in diencephalic areas that receive VIP-immunoreactive SCN efferents, such as the peri-suprachiasmatic region, lateral subparaventricular zone, parvocellular hypothalamic paraventricular subdivisions, dorsomedial hypothalamic nucleus, and anterior thalamic paraventricular and paratenial nuclei. The extensive distribution of PAC(1) mRNA within the SCN suggests that actions of PACAP are not restricted to the predominantly retinorecipient region. The presence of VPAC(2) mRNA in nearly half the VIP neurons, in almost all the AVP neurons, and at sites receiving VIP-immunoreactive SCN efferents suggests that the SCN VIP neurons are coupled and/or autoregulated and also influence the AVP-containing dorsomedial SCN and distal sites via VPAC(2).

[The temporal and spatial distribution of vasoactive intestinal peptide and its receptor in the development of airway hyperresponsiveness].

OBJECTIVE: To determine the possible involvement of vasoactive intestinal peptide (VIP) in the development of airway hyper responsiveness (AHR). METHODS: Twenty-five rabbits were randomly divided into five groups (5 animals each). Four groups were exposed to 2.0 ppm ozone 1 h/day for 1 (group B(0)), 2 (group B(1)), 4 (group B(2)), and 8 (group B(3)) days, respectively. The control group (group A) breathed only filtered room air. The changes of the VIP level and the mRNA expression of VIP receptor 1 (VIPR1) in the lung were detected at various ozone-stressing time points. In situ hybridization was performed to examine the distribution of VIPR1 in the lung. RESULTS: (1) The concentration of VIP in the lung increased slowly and were maximal at day 4, then returned to the normal level. (2) The changing pattern of the VIPR1 mRNA in the lung was similar to those observed for VIP. Increases in VIPR1 mRNA were detectable by 1 day and maximal by 2 - 4 days, and then decreased slowly. (3) In group A, VIPR1 was expressed on airway epithelium, in pulmonary interstitial and focal areas of airways and vascular smooth muscles. By days 2 to 4, hybridization staining increased and the majority of VIPR1-positive cells was located in the perivascular and peribronchiolar area. On day 8, very few positive cells were seen in the lung. CONCLUSION: VIP may play an important role in the development of AHR by binding with VIPR1.

Transgenic approach reveals expression of the VPAC2 receptor in phenotypically defined neurons in the mouse suprachiasmatic nucleus and in its efferent target sites.

Circadian rhythms in mammals depend on the properties of cells in the suprachiasmatic nucleus (SCN). The retino-recipient core of the mouse SCN is characterized by vasoactive intestinal peptide (VIP) neurons. Expression within the SCN of VPAC2, a VIP receptor, is required for circadian rhythmicity. Using transgenic mice with beta-galactosidase as a marker for VPAC2, we have phenotyped VPAC2-expressing cells within the SCN and investigated expression of the VPAC2 marker at sites previously shown to receive VIP-containing SCN efferents. In situ hybridization and immunohistochemistry demonstrated identical distributions for VPAC2 mRNA and beta-galactosidase and coexpression of the two signals in the SCN. Double-label confocal immunofluorescence identified beta-galactosidase in 32% of the VIP and 31% of the calretinin neurons in the SCN core. Of the arginine-vasopressin neurons that characterize the SCN shell, 45% expressed beta-galactosidase. In contrast, this marker was not apparent in astrocytes within the SCN core or shell. Cell bodies containing beta-galactosidase were detected at sites reportedly receiving VIP-containing SCN efferents, including the subparaventricular zone and lateral septum and the anteroventral periventricular, preoptic suprachiasmatic, medial preoptic and paraventricular hypothalamic nuclei. The detection of a marker for VPAC2 expression in the SCN in almost one-third of the VIP and calretinin core neurons and nearly half of the arginine-vasopressin shell neurons and also in cell bodies at sites receiving VIP-immunoreactive projections from the SCN indicates that VPAC2 may contribute to autoregulation and/or coupling within the SCN core and to the control of the SCN shell and sites distal to this nucleus.

Roles of vasoactive intestinal peptide (VIP) in the expression of different immune phenotypes by wild-type mice and T cell-targeted type II VIP receptor transgenic mice.

Vasoactive intestinal peptide (VIP) and its two G protein-coupled receptors, VPAC1 and VPAC2, are quantitatively prominent and functionally critical in the immune system. Transgenic (T) mice constitutively expressing VPAC2 selectively in CD4 T cells, at levels higher than those found after maximal induction in CD4 T cells of wild-type (N) mice, have elevated blood concentrations of IgE, IgG1, and eosinophils; enhanced immediate-type hypersensitivity; and reduced delayed-type hypersensitivity. In contrast, VPAC2-null (K) mice manifest decreased immediate-type hypersensitivity and enhanced delayed-type hypersensitivity. The phenotypes are attributable to opposite skewing of the Th2/Th1 cytokine ratio, but no studies were conducted on the roles of T cell-derived VIP and altered expansion of the Th subsets. Dependence of the Th phenotype of T mice, but not of N or K mice, on T cell-derived VIP now is proven by showing that eliminating VIP from TCR-stimulated T cell cultures with VIPase IgG normalizes the elevated number of IL-4-secreting CD4 T cells, decreases the secretion of IL-4 and IL-10, and increases the secretion of IFN-gamma. Flexible responsiveness of CD4 T cells from N and K mice, but not T mice, to exogenous VIP in vitro and in vivo is shown by increased numbers of IL-4-secreting CD4 T cells, greater secretion of IL-4 and IL-10, and lesser secretion of IFN-gamma after TCR stimulation with VIP. The level of VIP recognized by CD4 T cells thus is a major determinant of the relative contributions of Th subsets to the immune effector phenotype.

Vasoactive intestinal peptide (VIP) inhibits the proliferation of bone marrow progenitors through the VPAC1 receptor.

OBJECTIVE: The cellular and molecular mechanisms of hematopoietic stimulation have been studied. However, an understanding of negative effects in the hematopoietic system remains elusive. To this end, we studied the effects of vasoactive intestinal peptide (VIP) on bone marrow (BM) progenitors. MATERIALS AND METHODS: Different BM cell subsets were used to perform clonogenic assay for granulocytic (CFU-GM) or erythroid (BFU-E and CFU-E) progenitors with 10(-7)-10(-13) M VIP. The relevant receptor was verified with specific antagonists, or agonists, semi-quantitative RT-PCR, and chemical cross-linking studies with stromal membranes. RESULTS: Assays performed with unfractionated mononuclear cells and enriched CD34(+) cells showed dose-dependent inhibition on BM progenitors with significant inhibition up to 10(-10) M. Nylon wool separated cells, which depleted stroma, reversed the inhibitory effects of VIP between 10 and 20%. Combined experimental evaluation indicated that the effects of VIP on BM functions are mediated through the type 1 receptor (VPAC1). VIP induced the production of TGF-beta and TNF-alpha in BM mononuclear cells and stroma. These cytokines are partly involved in reversing the suppressive effects of VIP on CFU-GM. CONCLUSIONS: The effect of VIP on BM progenitors could be mediated through direct and indirect mechanism. Direct effects were evident by the suppressive effects of VIP on clonogenic assays with highly purified CD34(+) cells. Indirect effects were mediated through putative functions of the stromal cells and the production of TGF-beta and TNF-alpha.

Expression and regulatory role of receptors for vasoactive intestinal peptide in bone cells.

An intense network of nerve fibers can be demonstrated in skeletal tissues, not only in the periosteum but also within cortical bone, growth plate, and bone marrow. This neuro-osteogenic network expresses a restricted number of signalling molecules, including neuropeptides, neurotransmitters, and neurotrophins. Several lines of evidence indicate that receptors for these molecules are present on bone cells and that activation of these receptors leads to changes in bone cell activities. In addition, deletion of signalling molecules has been shown to alter bone metabolism. In the present review, these studies are summarized with a focus on distribution and effects of vasoactive intestinal peptide.

The neuropeptide PACAP attenuates beta-amyloid (1-42)-induced toxicity in PC12 cells.

Pituitary adenylate cyclase activating polypeptide (PACAP) modulates neurotransmission in the central and peripheral nervous systems. In vitro and in vivo studies have shown the protective effects of PACAP against neuronal damage induced by ischemia and agonists of NMDA-type glutamate receptors. Here, we demonstrated that PACAP also protected against neuronal toxicity induced by beta-amyloid (Abeta) peptide, aggregation of which is a causative factor for Alzheimer's disease. PACAP (10(-9)M) rescued 80% of decreased cell viability and 50% of elevated caspase-3 activity that resulted from exposure of PC12 cells to Abeta. PACAP was at least 10(4)-fold more effective than other neuropeptides including vasoactive intestinal peptide (VIP) and humanin, which correlated with the level of cAMP accumulation. Thus, our results suggested that PACAP attenuates Abeta-induced cell death in PC12 cells through an increase in cAMP and that caspase-3 deactivation by PACAP is involved in the signaling pathway for this neuroprotection.

Pituitary adenylate cyclase-activating polypeptide and vasoactive intestinal peptide attenuate glutamate-induced nNOS activation and cytotoxicity.

Both vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP) act as neurotransmitters in the central and peripheral nervous systems. Attention has been focused on these neuropeptides because among their numerous biological activities, they have been confirmed to show neuroprotective effects against ischemia and glutamate-induced cytotoxicity. It is well established that glutamate has excitatory effects on neuronal cells, and that excessive glutamate shows potent neurotoxicity, especially in neuronal nitric oxide synthase-containing neurons. Glutamate stimulates the production of nitric oxide (NO) in neurons, and the NO generated is tightly associated with the delayed death of neurons. We examined the effects of these neuropeptides on the glutamate-induced neural actions using PC12 cells, and we confirmed the important activities of PACAP/VIP on the production of NO as well as the delayed cell death stimulated by glutamate.

Pituitary adenylate cyclase activating polypeptide regulates the basal production of nitric oxide in PC12 cells.

Vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP), two members of the VIP/secretin/glucagon family, modulate neurotransmission via stimulation of protein kinases including cAMP-dependent protein kinase (PKA) and protein kinase C (PKC) in the central and peripheral nervous systems. They are reported to co-exist with nitric oxide synthases (NOSs) and other neuropeptides within the nervous system and peripheral tissues. In the present study, we investigated the neuronal role of these peptides in NO production in PC12 cells. We showed that PACAP decreased NO production in a dose-dependent manner, and the activators of protein kinase A and C also inhibited the NO production in PC12 cells. RT-PCR experiments demonstrated that PC12 cells constitutively express the mRNAs for neuronal NOS and the PACAP-specific (PAC1) receptor, and we concluded that PACAP plays an important role in the regulation of nNOS activity through PAC1 receptor in PC12 cells.

Gene expression profiling of testosterone and estradiol-17 beta-induced prostatic dysplasia in Noble rats and response to the antiestrogen ICI 182,780.

We previously demonstrated that 1) treatment of Noble rats for 16 wk with testosterone (T) and estradiol-17 beta (E2) led to 100% incidence of dorsolateral prostate (DLP) dysplasia and hyperprolactinemia and 2) blockade of PRL release with bromocriptine cotreatment significantly lowered the incidence of DLP dysplasia. In the current study, we sought to determine whether E2 exerts direct effects, independent of PRL, in this model system. The pure antiestrogen ICI 182,780 (ICI), reported to have no effect on PRL release in female rats, was administered biweekly to T + E2-treated rats at 3 mg/kg body weight. ICI cotreatment completely prevented DLP dysplasia development but it also blocked hyperprolactinemia in the dual hormone-treated rats. Gene profiling with an 1185 gene rat cDNA array identified approximately 100 genes displaying > or = 3-fold changes in rat lateral prostates (LPs) following T + E2 treatment. Significantly more genes were up-regulated (77) than down-regulated (14), reflecting cellular/molecular changes associated with enhanced cell proliferation, DNA damage, heightened protein and RNA synthesis, increased energy metabolism, and activation of several proto-oncogenes and intracellular signaling pathways. Post hoc analyses, using quantitative real-time RT-PCR, corroborated differential expression of eight genes, exhibiting three different patterns of altered expression. Genes encoding the early growth response protein 1 and metalloendopeptidase meprin beta-subunit were similarly altered in T + E2- and T + E2 + ICI-treated animals when compared with untreated controls. In contrast, transcripts of fos-related antigen-2, growth arrest and DNA damage-inducible protein-45, and signal transducer and activator of transcription-3 were significantly increased in the LPs of T + E2-treated animals, but the increases were reversed by cotreatment with ICI. Differential expression of fos-related antigen-2 and growth arrest and DNA damage-inducible protein-45 were further confirmed at the protein level by immunohistochemistry. Lastly, levels of A-RAF, VIP-1 receptor, and calpastatin mRNA were distinctly lessen in rat LPs under T + E2 influence, but rebound with ICI cotreatment. In conclusion, our findings further implicated pituitary PRL in the induction of dysplasia in rat LP. Gene profiling provided clues that molecular events related to enhancement of cell proliferation, DNA damage, and activation of proto-oncogenes and transforming factors may be causally linked to the genesis of LP dysplasia in this rat model.

[Effects of vasoactive intestinal peptide on chemotaxis of bronchial epithelial cells].

To investigate the influence of vasoactive intestinal peptide (VIP) on chemotaxis of bronchial epithelial cells (BECs). Rabbit chemotactic migration of primary BEC was assessed in a blind-well Boyden chamber. Radioimmunoassay and radio-ligand affinity analysis were used for determining VIP secretion and vasoactive intestinal peptide receptor (VIPR) expression. The results showed: (1) the method for determining chemotaxis of BECs by using insulin as chemotactic factor was stable and reproducible (r=0.9703, P<0.01). (2) VIP (0.001-1 micromol/L) elicited chemotaxis of BECs which was substantial and concentration-dependent. The effects of VIP were inhibited by W-7 and H-7 (P<0.01). (3) Heat stress enhanced the secretion of VIP (P<0.01) and upregulated the expression of VIPR on BECs (P<0.05). These results indicate that VIP in the lungs may play an important role in the repair of damaged epithelium, accelerating restoration of the airway to its normal state. Calmodulin and protein kinase C may be involved in the signal transduction of VIP effects.

Distribution of mRNA for VIP and PACAP receptors in human cerebral arteries and cranial ganglia.

The distribution of mRNA for pituitary adenylate cyclase-activating polypeptide (PACAP) type 1 (PAC1) and vasoactive intestinal polypeptide (VIP) types 1 and 2 (VPAC1 and VPAC2, respectively) receptors was examined by reverse transcriptase polymerase chain reaction (RT-PCR) in human cerebral arteries and in trigeminal, otic, sphenopalatine and superior cervical ganglia. RT-PCR products of the expected sizes were detected in the arteries, in both the presence and absence of endothelium. In the majority (80%) of the trigeminal, otic and superior cervical ganglia, mRNA for all three receptors were found (n = 5). However, in the sphenopalatine ganglion neither VPAC2 nor PAC1 was detected (n = 2). This finding indicates the occurrence of both prejunctional (ganglia) and postjunctional (vessels) location of VIP and PACAP receptors.

Pituitary adenylate cyclase-activating polypeptide receptors mediating insulin secretion in rodent pancreatic islets are coupled to adenylate cyclase but not to PLC.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a potentiator of glucose-induced insulin secretion. PACAP binds to a PACAP-specific receptor (PAC1) and to VPAC receptors (VPAC1 and VPAC2), which share high affinity for vasoactive intestinal polypeptide (VIP). In the present study, the molecular expression of PACAP receptor isoforms and the signaling pathways involved in the insulin secretory effect of PACAP were investigated in isolated rat and mouse pancreatic islets. mRNA encoding PAC1-short, -hop, and -very short variants, as well as VPAC1 and VPAC2, were expressed in pancreatic islets. PACAP and VIP were equipotent in potentiating glucose-induced insulin release. Both peptides were also equipotent in increasing cAMP production, but PACAP was more efficient than VIP. Unlike carbachol, PACAP and VIP had no effect on inositol phosphate production. In the PAC1-deficient mouse, the insulinotropic effect of PACAP was reduced, and its differential effect on cAMP production was abolished, whereas the effects of VIP remained unchanged. These results clearly show that the insulinotropic effect of PACAP involved both VPAC and PAC1. The PAC1 variants expressed in rat and mouse pancreatic islets seem to be coupled to adenylate cyclase but not to PLC.

Elucidation of vasoactive intestinal peptide pharmacophore for VPAC(1) receptors in human, rat, and guinea pig.

Vasoactive intestinal peptide (VIP) is a neurotransmitter involved in a number of pathological and physiological processes. VIP is rapidly degraded and simplified stable analogs are needed. VIP's action was extensively studied in rat and guinea pig. However, it is largely unknown whether its pharmacophore in these species resembles human. To address this issue we investigated the VIP pharmacophore for VPAC(1) (the predominant receptor subtype in cancers and widely distributed in normal tissues) by using alanine and D-amino acid scanning. Interaction with rat, guinea pig, and human VPAC(1) was assessed using transfected Chinese hamster ovary (CHO) and PANC1 cells and cells possessing native VPAC(1). Important species differences existed in the VIP pharmacophore. The human VPAC(1) expressed in CHO cells, which were used almost exclusively in previous studies, differed markedly from the native VPAC(1) in T47D cells. The most important amino acids for determining affinity are His(1), Asp(3), Phe(6), Arg(12), Arg(14), and Leu(23). Ser(2), Asp(8), Asn(9), Thr(11), Val(19), Asn(24), Ser(25), Leu(27), and Asn(28) are not essential for high-affinity interaction/activation. [Ala(2,8,9,11,19,24,25,27,28)]VIP, which contained 11 alanines, was synthesized and it was equipotent to VIP at VPAC(1) receptors in all species and was metabolically stable. Our results show in any design of simplified VIP analogs for VPAC(1) it will be important to consider species differences and it is essential to use transfected systems that reflect the native receptor's pharmacophore. Last, with our results a simplified, metabolically stable VIP analog was identified that should be useful as a prototype for design of selective agonists/antagonists that could be useful therapeutically.

Molecular cloning and expression analysis of the turkey vasoactive intestinal peptide receptor.

Vasoactive intestinal peptide (VIP) is a prolactin (PRL)-releasing factor whose activity in avian species is believed to be mediated by a specific VIP receptor (VIP-R). Circulating PRL levels are closely related to hypothalamic VIP immunoreactivity, hypothalamic VIP mRNA content, and hypophysial-portal blood VIP concentrations in turkeys. In the present study, a turkey VIP-R (tVIP-R) cDNA was cloned and its mRNA abundance was quantified in various tissues during different reproductive stages. The 2347-bp tVIP-R cDNA encoded a 457 amino acid protein, with a predicted Mr of 52 kDa. The full-length cDNA shares approximately 55% similarity with the mammalian VIP receptor-1. Northern blot analysis revealed that a major 2.7-kb transcript was expressed in laying hen pituitaries. Furthermore, two minor tVIP-R transcripts of 3.7 and 3.4 kb were observed. Semiquantitative reverse transcription polymerase chain reaction (RT-PCR) was performed using RNA from various turkey brain and peripheral tissues throughout the reproductive cycle. The steady-state levels of pituitary tVIP-R mRNA changed during the reproductive cycle, whereas mRNA expression in other tissues was not affected. The steady-state levels of tVIP-R mRNA were only affected in the pituitary, whereas mRNA expression in any of the other tissues examined following the immunization of turkeys against VIP were not affected.

Vasoactive intestinal peptide fibers innervate neuroendocrine dopaminergic neurons.

Hypothalamic neuroendocrine dopaminergic neurons exhibit a diurnal rhythm. Higher level input to these neurons has not been described. In the present study, we identified fibers known to originate in the suprachiasmatic nucleus (SCN), which were associated with neuroendocrine dopaminergic neurons. Hypothalamic sections were obtained from either ovariectomized (OVX) female rats or OVX female rats implanted with estrogen and progesterone (E+P). Confocal microscopic images were acquired from the periventricular nucleus, as well as the rostral, dorsomedial, ventrolateral, and caudal regions of the arcuate nucleus. Using antibodies directed against vasoactive intestinal peptide (VIP) and tyrosine hydroxylase (TH) the rate-limiting enzyme in dopamine synthesis, fine VIP fibers in close apposition to TH-immunoreactive (IR) soma and proximal dendrites were revealed. Of the antibodies for the two VIP receptor subtypes (VIP1R and VIP2R), only VIP2R was found on TH-IR neurons. E+P significantly increased the incidence and density of neuroendocrine dopaminergic neurons expressing VIP2R, when compared to OVX animals. E+P did not affect the percent of neuroendocrine dopaminergic neurons associated with VIP fibers. No VIP fibers or VIP2R were found on dopaminergic neurons in the zona incerta. Brain sections triple labeled for Synapsin (a protein localized in synaptic vesicles) VIP, and TH demonstrated that Synapsin was colocalized with VIP fibers that were associated with TH-IR neurons in the arcuate nucleus. Double-label immuno-electron microscopy of hypothalamic sections labeled with antibodies for VIP and TH revealed VIP boutons associated with TH-IR soma and proximal dendrites. These results suggest VIPergic neurons may directly regulate neuroendocrine dopaminergic neuron activity, and ovarian steroids may play a modulatory role.