Specification of hypothalamic neurons by dual regulation of the homeodomain protein Orthopedia.

In the developing hypothalamus, a variety of neurons are generated adjacent to each other in a highly coordinated, but poorly understood process. A critical question that remains unanswered is how coordinated development of multiple neuronal types is achieved in this relatively narrow anatomical region. We focus on dopaminergic (DA) and oxytocinergic (OT) neurons as a paradigm for development of two prominent hypothalamic cell types. We report that the development of DA and OT-like neurons in the zebrafish is orchestrated by two novel pathways that regulate the expression of the homeodomain-containing protein Orthopedia (Otp), a key determinant of hypothalamic neural differentiation. Genetic analysis showed that the G-protein-coupled receptor PAC1 and the zinc finger-containing transcription factor Fezl act upstream to Otp. In vivo and in vitro experiments demonstrated that Fezl and PAC1 regulate Otp at the transcriptional and the post-transcriptional levels, respectively. Our data reveal a new genetic network controlling the specification of hypothalamic neurons in vertebrates, and places Otp as a critical determinant underlying Fezl- and PAC1-mediated differentiation.

Expression of vasoactive intestinal peptide receptor messenger RNA in the hypothalamus and pituitary throughout the turkey reproductive cycle.

Vasoactive intestinal peptide (VIP) has been implicated in the regulation of avian reproductive activity and appears to act at the level of the hypothalamus and pituitary. This in situ hybridization histochemistry study describes the distribution of VIP receptor mRNA expression in the hypothalamus and the pituitary of reproductively active (laying) and quiescent (nonphotostimulated, incubating, and photorefractory) female turkeys and characterizes the differences observed in VIP receptor gene expression. VIP receptor mRNA, while expressed throughout the hypothalamus, was specifically expressed in areas known to contain GnRH-I neurons in the chicken, i.e., the lateral septum, medial preoptic area, anterior hypothalamus, and paraventricular nucleus. Significant differences in VIP receptor mRNA expression between different reproductive states was observed only within the infundibular nuclear complex. VIP receptor mRNA was markedly less in nonphotostimulated and photorefractory hens as compared with laying and incubating hens. The most dense VIP receptor mRNA was found in the anterior pituitary, where it was 2.4- and 3.0-fold greater in laying and incubating hens, respectively, as compared with that in nonphotostimulated ones. Hens that stopped incubating and became photorefractory displayed pituitary VIP receptor mRNA levels similar to those of nonphotostimulated birds. The changes in pituitary VIP receptor mRNA expression were positively correlated with known changes in pituitary prolactin (PRL) mRNA expression and PRL content and release. These findings indicate that the variations in PRL secretion observed across the turkey reproductive cycle are, in part, regulated by changes in VIP receptors at the pituitary level.

Vasoactive intestinal peptide (VIP)/pituitary adenylate cyclase-activating peptide receptor subtypes in mouse calvarial osteoblasts: presence of VIP-2 receptors and differentiation-induced expression of VIP-1 receptors.

Three distinct complementary DNAs for vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating peptide (PACAP) receptors have been cloned and designated VIP-1 receptor (VIP-1R), VIP-2 receptor (VIP-2R), and PACAP receptor (PACAP-R). In the present study, we have characterized the binding sites on primary mouse calvarial osteoblasts for VIP and related peptides. By analyzing the cAMP response, the rank order of response observed was PACAP 38 > PACAP 27 > helodermin > VIP > helospectin > glucagon > PHI >>> secretin. The VIP-2R/PACAP-R antagonist, PACAP 6-38, inhibited both VIP- and PACAP-stimulated cAMP formation. Binding studies using an atomic force microscopy (AFM) technique showed high affinity binding for VIP and PACAP 38, but not for secretin. Radioligand binding studies using (125)I-VIP and (125)I-PACAP 38 demonstrated a more specific and higher affinity binding for PACAP 38 than for VIP. Secretin failed to inhibit both (125)I-VIP and (125)I-PACAP 38 binding. RT-PCR demonstrated that undifferentiated mouse calvarial osteoblasts express messenger RNA for VIP-2R, but not for VIP-1R or PACAP-R. When the osteoblasts were cultured for 20 days to induce bone noduli formation, VIP-1R, in addition to VIP-2R, were expressed when the nodules started to mineralize at 12 days. Taken together, these data demonstrate that mouse calvarial osteoblasts express functional VIP-2R with higher affinity binding for PACAP than for VIP and that the VIP-1R expression is induced during osteoblastic differentiation.

Role of second extracellular loop in the function of human vasoactive intestinal polypeptide/pituitary adenylate cyclase activating polypeptide receptor 1 (hVPAC1R).

To elucidate the functional role of the second extracellular loop of human vasoactive intestinal polypeptide/pituitary adenylate cyclase-activating polypeptide (VIP/PACAP) receptor (hVPAC1R), surface expression, ligand binding, and receptor activation were analyzed. Amino acids in the entire second extracellular loop were individually substituted by alanine by site-directed mutagenesis. The mutant and wild-type receptors were transiently expressed in HEK293 cells and purified cell membranes were tested for the ability to bind VIP, while the receptor activity was measured as potency of cAMP production analysed on intact cells. Surface expression of the substituted conserved residues, W286A, I289A, W294A, and W295A, was evidently decreased to 20-30% compared to the wild-type expression. W286A also showed an significantly reduced potency of cAMP production. Substituted residues as F280A, E281A, and G284A showed a significant reduction in the potency of stimulated cAMP production amounting to 8-46-fold, compared to the wild-type with unaffected surface expression and VIP binding. These results indicate that some residues in the second extracellular loop of the human VPAC1R participate in the active mechanism of a ligand-mediated response without being directly involved in the binding of VIP.

The augmentation of pituitary adenylate cyclase-activating polypeptide (PACAP) in streptozotocin-induced diabetic rats.

Pituitary adenylate cyclase activating polypeptide (PACAP), which was isolated from ovine hypothalamic extract, has been shown to have a physiological role in the regulation of insulin or islet functions. In streptozotocin (STZ)-induced diabetic rats, we examined the content of PACAP immunoreactivity and gene expression of three specific receptors. Four weeks after administration of STZ (50 mg/kg), plasma glucose levels increased 3.3-fold, and plasma insulin levels decreased to one-tenth as compared with the control. The content of PACAP immunoreactivity in the pancreas potently increased by 30%, but the content of vasoactive intestinal polypeptide (VIP) immunoreactivity was not changed. In the other tissues, the content of PACAP immunoreactivity did not significantly change except in the hypothalamus, which showed a 10% increment. In the expression level of PACAP/VIP receptors, semi-quantitative RT-PCR analysis revealed that VIP1/PACAP receptor mRNA significantly increased as compared with the other two types of receptors in the pancreas of STZ-induced diabetic rats. These findings suggest that PACAP and VIP1/PACAP receptor might be involved in the pathophysiology of diabetes mellitus.

Pharmacological, molecular and functional characterization of vasoactive intestinal polypeptide/pituitary adenylate cyclase-activating polypeptide receptors in the rat pineal gland.

Melatonin secretion from the mammalian pineal gland is strongly stimulated by noradrenaline and also by vasoactive intestinal polypeptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP). Three types of receptors for VIP and PACAP have been characterized so far: VIP1/PACAP receptors and VIP2/PACAP receptors, which possess similar high affinities for VIP and PACAP, and PACAP1 receptors which exhibit a 100-1000-fold higher affinity for PACAP. The aim of the present study was to characterize the receptor subtype(s) mediating the stimulatory effects of VIP and PACAP on melatonin synthesis in the rat pineal gland. Autoradiographic studies showed that PACAP and VIP were equally potent in displacing binding of radioiodinated PACAP27 from pineal sections. Amplification of pineal complementary DNAs by polymerase chain reaction using specific primers for the different receptor subtypes revealed that all three receptor messenger RNAs are expressed and that VIP1/PACAP receptor messenger RNA was predominant over VIP2/PACAP receptor messenger RNA. In vitro, VIP and PACAP stimulated melatonin synthesis with similar high potency and the effect of the two peptides were not additive. The selective VIP1/PACAP receptor agonists [R16]chicken secretin (1-25) and [K15, R16, L27]VIP(1-7)/growth hormone releasing factor(8-27) were significantly more potent than the selective VIP2/PACAP receptor agonist RO 25-1553 in stimulating melatonin secretion. The stimulatory effects of VIP and PACAP were similarly inhibited by the VIP1/PACAP antagonist [acetyl-His1, D-Phe2, K15, R16, L27]VIP(3-7)/growth hormone releasing factor(8-27). These data strongly suggest that VIP and PACAP exert a stimulatory effect on melatonin synthesis mainly through activation of a pineal VIP1/PACAP receptor subtype.

Vasoactive intestinal peptide (VIP)1 receptor. Three nonadjacent amino acids are responsible for species selectivity with respect to recognition of peptide histidine isoleucineamide.

Vasoactive intestinal peptide (VIP)1 receptors in rats and humans recognize peptide histidine isoleucineamide (PHI) with high and low affinity, respectively. We took advantage of this phenotypic difference to identify the domain responsible for the selective recognition of PHI by rat and human receptors which display >80% sequence identity. After transfection of human and rat receptors in COS cells, the ratio of IC50 for PHI/IC50 for VIP (referred to as P/V) in inhibiting 125I-VIP binding was shown to be >1,000 and <40, respectively. Construction of eight rat/human receptor chimerae by overlap polymerase chain reaction and determination of their P/V ratios demonstrated that the critical domain for PHI recognition is present within a sequence comprising part of the first extracellular loop and third transmembrane domain. This domain contains three different amino acids numbered according to human and rat sequences, respectively, e.g. Gln207 (human) versus His208 (rat), Gly211 versus Ala212 and Met219 versus Val220. Site-directed mutagenesis introducing individual, double, or triple mutations in a chimeric construct revealed that all three amino acids were involved in the recognition of PHI. Triple mutations were then introduced in the wild-type receptors i.e. Q207H, G211A, M219V human VIP1 receptor and H208Q, A212G, V220M rat VIP1 receptor, resulting in a complete change in their phenotype from human to rat and from rat to human, respectively. The results demonstrate that three nonadjacent amino acids are responsible for the selective recognition of PHI by human and rat VIP1 receptors.