Vasoactive Intestinal Peptide Binding Sites Research Articles

Heterogeneity of Vasoactive Intestinal Peptide Binding Sites in Hut‐78 T‐Lymphocytes

Vasoactive Intestinal Peptide (VIP) is a neuropeptide that possesses potent cell cycle arrest activity in primary healthy T‐Lymphocytes. Transformed T‐cells from human cancer patients and over 25 cell lines (human, mouse, and rat) have consistently shown reduced VIP receptor expression, suggesting that cancer cells obtain a growth advance with reduced VIP receptor signaling. Utilizing the human Hut‐78 T‐lymphoblastic T‐cell line that has reduced levels but functional VIP binding sites, we carried out growth curves +/‐ VIP utilizing alamar blue. Surprisingly, these results were inconclusive as they showed opposite effects of growth from exogenous addition of VIP ligand. To better understand the discrepancy in growth rates by VIP we measured the homogeneity of VIP binding sites in Hut‐78s by VIP‐FAM labeling and flow cytometry analysis. Curiously, three definable subpopulations possessing varying levels of VIP binding sites were observed. These three populations were sorted, cultured, and showed a stable homogenous population with intermediate levels of VIP binding sites. Collectively, we conclude that the heterogeneity of VIP binding sites in HUT‐78s may explain the disparate growth results collected above. Future studies will utilize the stable sorted homogenous VIP binding site sorted populations to investigate the cell cycle arresting abilities of VIP.

Production and Purification of Large Quantities of the Functional N-Terminal Ectodomain of Human VPAC1 Receptor

Vasoactive intestinal peptide (VIP) is implicated in many physiological and pathophysiological processes, and its receptors are promising targets for the development of new drugs. The human VPAC1 receptor for VIP and pituitary adenylate cyclase-activating polypeptide is a class II G protein coupled receptor. The N-terminal ectodomain (N-ted) of the VPAC1 receptor is a major VIP binding site. To determinate the high resolution structure of the VPAC1 receptor N-ted, large quantities of purified recombinant N-ted produced are required. The N-ted sequence (31-144), which is fused to thioredoxin protein and 6xHis tag, was expressed into Origami Escherichia coli strain. Purification of recombinant N-ted using Ni-NTA affinity column associated to Nu-polyacrylamide gel electrophoresis analysis reveals the presence of one single band of Mw 19,000 corresponding to the purified recombinant N-ted. The purified N-ted was able to recognize VIP and the selective antagonist PG96-269. About 5-10 mg of functional purified protein/liter of bacterial culture is currently produced. This is a crucial step to determine the structure of functional human VPAC1 receptor N-ted by nuclear magnetic resonance.

VIP regulation of embryonic growth.

Vasoactive intestinal peptide (VIP) plays a regulatory role in the growth of early postimplantation rodent embryos through its action on receptors localized to the central nervous system (CNS). However, the origin of the VIP influencing embryonic growth is unknown. VIP binding sites have been found prenatally; however, VIP mRNA was not detected in the rat CNS before birth and has been detected in peripheral organs only during the final third of gestation. Recent studies have revealed that VIP receptors were limited to the CNS in the embryonic day 11 (E11) rat embryo/trophoblast, which, in addition, had almost four times the VIP concentration of the E17 fetus. However, neither in situ hybridization or reverse transcriptase-polymerase chain reaction methods detected VIP mRNA in the E11 rat embryo or embryonic membranes. Rat maternal serum revealed a peak in VIP concentration at days E10-E12 of pregnancy, with VIP levels 6- to 10-fold higher than later during pregnancy. Radiolabeled VIP, administered intravenously to pregnant female mice, was found in the E10 embryo. These results suggest that VIP produced by extraembryonic tissues may regulate embryonic growth during the early postimplantation stage of development in the rodent.

Expression of vasoactive intestinal peptide and functional VIP receptors in human prostate cancer: Antagonistic action of a growth-hormone-releasing hormone analog

Vasoactive intestinal peptide (VIP) functions as a mitogenic agent in the human prostate gland acting by autocrine/paracrine mechanisms. Here we extend knowledge on the VIP system (expression of VIP and VIP receptors, functionality of VIP receptors) at this level by analyzing the differences between human normal prostate and prostate carcinoma specimens. RT-PCR showed the expression of mRNA for VIP in normal and malignant tissues, whereas VIP levels, as measured by enzyme immuno-analysis, were about two times higher in adenocarcinoma samples. Real-time RT-PCR indicated a minor expression of VPAC2 receptors in the prostate gland, as well as the overexpression of VPAC1 and PAC1 receptors in malignant tissue specimens. Radio-labeled binding experiments with [125I]VIP showed an increased number of VIP binding sites (2.5 times for the high- and 1.7 times for the low-affinity sites) during malignant transformation, whereas the affinity values were unaffected. The receptors were functional in control and cancer tissues as shown by the ability of increasing VIP doses to stimulate adenylate cyclase activity. Interestingly, JV-1-53 (a GHRH-related peptide analog) (at 0.1 microM) behaved as a potent VIP antagonist since it inhibited by 60% the maximal VIP effect upon the enzyme activity. The results further explain the mechanisms of the autocrine/paracrine actions of VIP in human prostate and prostatic carcinoma through the observation of differences between healthy tissue and malignant transformation. Moreover, present data support the potential usefulness of VIP and/or synthetic peptide analogs for diagnostic or radiotherapeutic purposes as well as for long-term peptide therapy in this malignancy.

Receptors for vasoactive intestinal peptide and pituitary adenylate cyclase-activating polypeptide in turkey cerebral cortex: characterization by 6125I9-VIP binding and effects on cyclic AMP synthesis

Receptors for vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP) in turkey cerebral cortex were characterized using two approaches: (1) in vitro radioreceptor binding of [125I]-VIP, and (2) effects of peptides from the PACAP/VIP/secretin family on cyclic AMP formation. The binding of [125I]-VIP to turkey cortical membranes was rapid, stable, and reversible. Saturation analysis resulted in a linear Scatchard plot, suggesting binding to a single class of high affinity receptor binding sites with a Kd of 0.70 nM and a Bmax of 52 fmol/mg protein. Various peptides displaced the specific binding of 0.12 nM [125I]-VIP to turkey cerebral cortical membranes in a concentration-dependent manner. The relative rank order of potency of the tested peptides to inhibit [125I]-VIP binding to turkey cerebrum was: PACAP38 ≈ PACAP27 ≈ chicken VIP ≈ mammalian VIP ⋙ PHI ≫ secretin, chicken VIP16-28 (inactive). About 65% of specific [125I]-VIP binding sites in turkey cerebral cortex was sensitive to Gpp(NH)p, a nonhydrolysable analogue of GTP. PACAP38, PACAP27, chicken VIP and, to a lesser extent, mammalian VIP potently stimulated cyclic AMP formation in turkey cerebral cortical slices in a concentration-dependent manner, displaying EC50 values of 8.7 nM (PACAP38), 21.3 nM (PACAP27), 67.4 nM (chicken VIP), and 202 nM (mammalian VIP). On the other hand, PHI and secretin very weakly affected the nucleotide production. The obtained results indicate that cerebral cortex of turkey contains VPAC type receptors that are positively linked to cyclic AMP-generating system and are labeled with [125I]-VIP.

Receptors for vasoactive intestinal peptide and pituitary adenylate cyclase-activating polypeptide in turkey cerebral cortex: characterization by [ 125I]-VIP binding and effects on cyclic AMP synthesis

Receptors for vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP) in turkey cerebral cortex were characterized using two approaches: (1) in vitro radioreceptor binding of [ 125I]-VIP, and (2) effects of peptides from the PACAP/VIP/secretin family on cyclic AMP formation. The binding of [ 125I]-VIP to turkey cortical membranes was rapid, stable, and reversible. Saturation analysis resulted in a linear Scatchard plot, suggesting binding to a single class of high affinity receptor binding sites with a K d of 0.70 nM and a B max of 52 fmol/mg protein. Various peptides displaced the specific binding of 0.12 nM [ 125I]-VIP to turkey cerebral cortical membranes in a concentration-dependent manner. The relative rank order of potency of the tested peptides to inhibit [ 125I]-VIP binding to turkey cerebrum was: PACAP38 ≈ PACAP27 ≈ chicken VIP ≈ mammalian VIP ⋙ PHI ≫ secretin, chicken VIP16-28 (inactive). About 65% of specific [ 125I]-VIP binding sites in turkey cerebral cortex was sensitive to Gpp(NH)p, a nonhydrolysable analogue of GTP. PACAP38, PACAP27, chicken VIP and, to a lesser extent, mammalian VIP potently stimulated cyclic AMP formation in turkey cerebral cortical slices in a concentration-dependent manner, displaying EC 50 values of 8.7 nM (PACAP38), 21.3 nM (PACAP27), 67.4 nM (chicken VIP), and 202 nM (mammalian VIP). On the other hand, PHI and secretin very weakly affected the nucleotide production. The obtained results indicate that cerebral cortex of turkey contains VPAC type receptors that are positively linked to cyclic AMP-generating system and are labeled with [ 125I]-VIP.

Functional and molecular characterization of VIP receptor–effector system in rat developing immunocompetent cells: G protein involvement

Changes in the functional characteristics for vasoactive intestinal peptide (VIP) receptor–effector system were evaluated in rat developing immunocompetent cells (from 1-week-old animals up to 12-week-old animals). These characteristics include [ 125 I ]VIP binding studies, cell cyclic AMP (cAMP) generation, analysis of [ 125 I ]VIP–receptor complexes by cross-linking experiments, as well as developed-associated G proteins assayed by cholera and pertussis toxin-catalyzed ADP-ribosylation and Western blot. The Scatchard analysis of binding data was consistent with the existence of two classes of VIP binding sites with K d values unaltered and B max increased during postnatal development. The efficiency of VIP stimulation of cAMP generation increased from 1-week-old rats to adult conditions. The VIP–receptor complex apparent molecular mass (52–55 kDa) remains unaltered, but it was significantly lower in 2-week-old than in 8-week-old rats. ADP-ribosylated material by cholera toxin (CTx) was higher from 8-week-old than from 2-week-old animals, while ADP-ribosylation by pertussis toxin (PTx) was quantitatively higher in 8-week-old rats. Results were confirmed when immunoblots for different G protein subunits were performed.

Regulation of Postimplantation Mouse Embryonic Growth by Maternal Vasoactive Intestinal Peptide

Vasoactive intestinal peptide (VIP) is an identified regulator of growth in the embryonic day (E) 9-11 mouse. Mouse embryonic and extra-embryonic tissues were studied to identify the source of VIP at this critical time. VIP and mRNA was detected in the decidua/trophoblast at E8 and declined until E10, after which it was not detectable. VIP mRNA was not apparent in the embryo until E11-E12. At E9, cells in decidua had VIP as well as lymphocyte marker (delta and CD3) immunoreactivity. VIP binding sites were dense in the decidua/trophoblast at E6, which gradually decreased until E10. VIP binding sites were detected in embryonic neuroepithelium by E9. The transient presence of VIP binding sites and mRNA in the decidua/trophoblast correlate with the identified period of VIP growth regulation, when VIP mRNA is absent in the embryo. Therefore, these findings suggest that maternal decidual lymphocytes are the source of VIP that regulate early postimplantation embryonic growth.

Distribution of vasoactive intestinal peptide and calcitonin gene-related peptide immunoreactive nerve fibers and binding sites in the hamster seminal vesicle during post-natal development.

The distribution of vasoactive intestinal peptide (VIP)- and calcitonin gene-related peptide (CGRP)-immunoreactive nerves and 125I-labeled VIP- and CGRP-binding sites was studied in the hamster seminal vesicle of 12-, 30- and 60-day-old animals. In addition, the general innervation of the seminal vesicle was examined using the general neuronal marker synaptophysin. Our results show that the densities of the overall (synaptophysin immunoreactive) and CGRP-immunoreactive innervation is constant during the post-natal development of the gland. However, a significant decrease in VIP-containing nerves is observed at the end of puberty. The autoradiographic study revealed that in 12-day-old animals, the epithelium presents VIP binding sites. However, in 30-day-old animals, VIP binding sites are observed in the epithelium of only a few clumps of acini. In 60-day-old animals, the gland is composed of acini with dilated lumina where VIP binding sites are not detected. In all groups studied the epithelium does not exhibit CGRP binding sites. The seminal vesicle muscle layer displays specific binding sites for both VIP and CGRP at all post-natal developmental times, but the density of VIP binding sites is higher in 12- than in 30- and 60-day-old animals. Our results, showing the presence of specific VIP and CGRP binding sites during the development of the hamster seminal vesicle, suggest that these neuropeptides may be involved in the growth and differentiation of the gland.

Actions of vasoactive intestinal peptide on the rat adrenal zona glomerulosa.

Previous studies, by this group and others, have shown that vasoactive intestinal peptide (VIP) stimulates aldosterone secretion, and that the actions of VIP on aldosterone secretion by the rat adrenal cortex are blocked by beta adrenergic antagonists, suggesting that VIP may act by the local release of catecholamines. The present studies were designed to test this hypothesis further, by measuring catecholamine release by adrenal capsular tissue in response to VIP stimulation. Using intact capsular tissue it was found that VIP caused a dose-dependent increase in aldosterone secretion, with a concomitant increase in both adrenaline and noradrenaline release. The effects of VIP on aldosterone secretion were inhibited by atenolol, a beta1 adrenergic antagonist, but not by ICI-118,551, a beta2 adrenergic antagonist. Binding studies were carried out to investigate VIP receptors. It was found that adrenal zona glomerulosa tissue from control rats contained specific VIP binding sites (Bmax 853+/-101 fmol/mg protein; Kd 2.26+/-0.45 nmol/l). VIP binding was not displaced by ACTH, angiotensin II or by either of the beta adrenergic antagonists. The response to VIP in adrenals obtained from rats fed a low sodium diet was also investigated. Previous studies have found that adrenals from animals on a low sodium diet exhibit increased responsiveness to VIP. Specific VIP binding sites were identified, although the concentration or affinity of binding sites in the low sodium group was not significantly different from the controls. In the low sodium group VIP was found to increase catecholamine release to the same extent as in the control group, however, in contrast to the control group, the adrenal response to VIP was not altered by adrenergic antagonists in the low sodium group. These data provide strong support for the hypothesis that VIP acts by the local release of catecholamines in adrenal zona glomerulosa tissue in normal animals. It does not appear that VIP acts through the same mechanism in animals maintained on a low sodium diet. The mechanism by which VIP stimulates aldosterone in this group remains to be determined.

Maternal regulation of embryonic growth: the role of vasoactive intestinal peptide.

Vasoactive intestinal peptide (VIP) is an important growth regulator of the embryonic day (E)9-E11 mouse. In comparably aged rat embryos, VIP messenger RNA (mRNA) is not detectable; however, peak concentrations of VIP in maternal rat serum indicate a nonembryonic source. In the current study, mouse maternal and embryonic tissues were examined from E6-E12. Although RT-PCR revealed VIP mRNA in E6-E7 conceptuses, by E8 (when extraembryonic tissues could be separated from the embryo), VIP mRNA was detected only in the decidua/trophoblast. Decidual/trophoblastic VIP mRNA decreased until E10, after which it was not detectable. VIP mRNA was not apparent in the embryo until E11-E12. At E9, VIP immunoreactivity was localized to abundant, diffuse cells in the decidua basalis, which were also immunoreactive for T cell markers. VIP binding sites were dense in the decidua/trophoblast at E6, but gradually decreased until E10, after which they were not apparent. VIP binding sites were detected in embryonic neuroepithelium by E9. The transient presence of VIP binding sites and mRNA in the decidua/trophoblast correlate with the critical period of VIP growth regulation, when VIP mRNA is absent in the embryo. These findings suggest that maternal lymphocytes are the source of VIP's regulating early postimplantation embryonic growth.

Growth factor properties of VIP during early brain development. Whole embryo culture and in vivo studies.

Vasoactive intestinal peptide (VIP), a 28 amino acid neuropeptide widely distributed in the mammalian nervous system, has potent growth-related actions that influence cell division, neuronal survival, and neurodifferentiation. To address the potential effects of VIP on embryonic growth, whole postimplantation embryo cultures were used. After a 4-hour incubation, VIP stimulated growth as assessed by the following increases from control: embryonic volume (63%), DNA (103%), and protein content (63%), as well as the number of cells in S-phase (490%). No apparent histological abnormalities are produced by VIP. To assess the in vivo function of VIP in early CNS growth, a VIP antagonist (VA) was injected i.p. between E9 and E11. VA induced a dose reduction of the DNA (84% of controls) and protein (80% of controls) contents of the E11 head and a decrease of E17 brain weight (87% of controls). In contrast, body growth was less affected by the antagonist. injections of VA for a longer period (E9 to E17) did not increase the severity of the microcephaly. By ex vivo autoradiography, GTP-sensitive VIP binding sites were detected in the germinative neuroepithelium between E9 and E11, but not between E13 and E15, during neuronal migration. These data demonstrate that VIP regulates mitogenic activity in the premigratory neuroepithelium. Although this effect is limited to a short ontogenic period, blockade of VIP by a specific antagonist induces a severe microcephaly.

Photoperiod Mediates the Ability of Serotonin to Release Prolactin in the Turkey

Photostimulation (PS) of turkeys increases the number of hypothalamic vasoactive intestinal peptide (VIP)-immunoreactive neurons, the number of anterior pituitary VIP binding sites, and prolactin (PRL) secretion. Serotonin (5-HT) was recently shown to stimulate PRL secretion through VIP. This study tested the hypothesis that 5-HT's ability to induce PRL secretion is mediated by reproductive status and/or photoperiod in normally cycling turkey hens. Initially, saline or 5-HT was infused into the third ventricle of nest-deprived, previously incubating (ND) hens for 60 min at rates of 0.1, 1.0, or 10 nmol/min. The results led to use of the 10 nmol/min infusion rate for the remaining 5-HT infusions. Next, 5-HT was infused into short-day (SD;6), laying (6), ND (5), and photorefractory (P/R;6) hens. Plasma PRL was elevated in all groups except for the SD hens (P<0.05). In Experiment 3, VIP was infused into the median eminence of SD (6), laying (5), and P/R (5) hens, increasing circulating PRL levels in all three groups (P<0.05). Finally, SD hens were photostimulated for 0, 3, or 10 days and then infused with 5-HT. Only the birds which were photostimulated for 10 days exhibited elevated plasma PRL (P<0.05). In conclusion, PS regulates PRL secretion at the hypothalamic level and more than 3 days of PS are required for 5-HT-ergic stimulation of PRL secretion.

Effect of castration on the VIPergic innervation and 125I-labelled vasoactive intestinal peptide (VIP) binding sites in the hamster seminal vesicle a quantitative immunohistochemical and receptor autoradiographic study

In the present work we have investigated the effects of medium-(15 days) and long-term (2 months) castration on vasoactive intestinal peptide (VIP)-immunoreactive nerve fibres and 125I-labelled VIP binding sites in the adult hamster seminal vesicle. The density of VIP- and synaptophysin (general neuronal marker)-containing nerve fibres was determined in immunofluorescently stained cryostat sections using a computerised image analysis system. The morphological analysis of 125I-VIP binding sites in seminal vesicle cryostat sections was performed by quantitative receptor autoradiography. Our results show that the densities of the overall (synaptophysin immunoreactive) and VIPergic innervation increase in both medium and long-term castrated animals. In absolute terms, the quantity of VIP- and synaptophysin- containing nerves is not altered in medium-term castrates, but decreases for synaptophysin in long-term castrates. Medium-term castration does not affect the density of 125I-VIP binding sites in the gland muscular coat, but a significant decrease is observed after long-term castration. In conclusion, our results indicate that whereas VIP nerves are apparently unaffected by castration. 125I-VIP binding sites in the muscular coat of hamster seminal vesicle are sensitive to androgen levels.

Vasoactive intestinal peptide in cirrhotic rats: hemodynamic effects and mesenteric arterial receptor characteristics.

Vasoactive intestinal peptide (VIP) blood levels in cirrhosis are elevated, but its hemodynamic and receptor characteristics remain unclarified. We aimed to quantify VIP receptor characteristics in mesenteric arteries, plasma VIP concentration by radioimmunoassay (RIA), and the hemodynamic effects of VIP infusion in bile duct-ligated (BDL) cirrhotic and sham-operated control rats. Mesenteric arterial membranes were prepared by ultracentrifugation, and receptor characteristics were studied using 125I-labeled VIP as a radioligand. For the hemodynamic study, there were four groups: cirrhotic and sham-operated rats were infused with either VIP (50 ng/kg/min for 15 minutes) or equivolumic isotonic saline. Regional blood flows were measured in conscious rats with radioactive microspheres. Receptor studies showed high- and low-affinity binding sites for VIP, which had similar equilibrium dissociation constants (binding affinities) and receptor densities for both the cirrhotic and control rats. Plasma VIP concentrations were significantly elevated in the cirrhotic rats. In both cirrhotic and sham-operated rats, VIP infusion produced plasma levels approximately twofold to threefold increased over the basal levels observed in cirrhotic rats. In cirrhotic rats, VIP infusion did not affect any hemodynamic parameter, whereas in the sham-operated rats VIP infusion significantly increased the mesenteric visceral blood flow. These results show that the hyporesponsiveness to VIP in cirrhotic rats is not attributable to receptor downregulation, implying postreceptor alterations. This suggests that VIP may not play a major role in the maintenance of splanchnic hyperemia in cirrhosis.

Vasoactive intestinal peptide in cirrhotic rats: Hemodynamic effects and mesenteric arterial receptor characteristics

Vasoactive intestinal peptide (VIP) blood levels in cirrhosis are elevated, but its hemodynamic and receptor characteristics remain unclarified. We aimed to quantify VIP receptor characteristics in mesenteric arteries, plasma VIP concentration by radioimmunoassay (RIA), and the hemodynamic effects of VIP infusion in bile duct-ligated (BDL) cirrhotic and sham-operated control rats. Mesenteric arterial membranes were prepared by ultracentrifugation, and receptor characteristics were studied using 125I-labeled VIP as a radioligand. For the hemodynamic study, there were four groups: cirrhotic and sham-operated rats were infused with either VIP (50 ng/kg/min for 15 minutes) or equivolumic isotonic saline. Regional blood flows were measured in conscious rats with radioactive microspheres. Receptor studies showed high-and low-affinity binding sites for VIP, which had similar equilibrium dissociation constants (binding affinities) and receptor densities for both the cirrhotic and control rats. Plasma VIP concentrations were significantly elevated in the cirrhotic rats. In both cirrhotic and sham-operated rats, VIP infusion produced plasma levels approximately twofold to threefold increased over the basal levels observed in cirrhotic rats. In cirrhotic rats, VIP infusion did not affect any hemodynamic parameter, whereas in the sham-operated rats VIP infusion significantly increased the mesenteric visceral blood flow. These results show that the hyporesponsiveness to VIP in cirrhotic rats is not attributable to receptor downregulation, implying postreceptor alterations. This suggests that VIP may not play a major role in the maintenance of splanchnic hyperemia in cirrhosis. (Hepatology 1996 May;23(5):1174-80)

Expression of vasoactive intestinal peptide binding sites in rat peritoneal macrophages is stimulated by inflammatory stimulus

Vasoactive intestinal peptide (VIP) binding to resident and stimulated-rat peritoneal macrophages was studied. No specific VIP binding was obtained with resident rat peritoneal macrophages. In contrast, VIP bound specifically to casein-elicited macrophages. The Scatchard analysis of binding data was consistent with the presence of two classes of VIP binding sites, but may represent a receptor site and internalized VIP. Both specific VIP binding and number of specific high affinity binding sites for VIP augmented progressively after sodium caseinate injection, reaching maximum at days 4–5. Macrophages obtained 1 day after injection showed a minimal specific VIP binding (0.3 ±0.1% of total), but cells obtained 4 days after injection showed a maximal binding to the peptide (3.1 ± 0.2% of total). The number of high affinity binding sites per cell raised also progressively after sodium caseinate injection: 2650 ±301 at day 2, 4939 ± 723 at day 3, 6684 ± 903 at day 4 and 9636± 1626 at day 5 ( P = 0.0035). The number of low affinity binding sites per cell exhibited the same changes. In contrast, the K d values of both high and low affinity VIP binding sites did not vary significantly ( P > 0.05). These results demonstrate that VIP binding sites are only displayed by stimulated macrophages, suggesting that VIP binding sites could be considered to be a pre-activation marker in macrophages and could be used to recognize inflammatory or stimulated macrophages.

Maternal vasoactive intestinal peptide and the regulation of embryonic growth in the rodent.

Vasoactive intestinal peptide (VIP) has been shown to regulate early postimplantation growth in rodents through central nervous system receptors. However, the source of VIP mediating these effects is unknown. Although VIP binding sites are present prenatally, VIP mRNA was not detected in the rat central nervous system before birth and was detected in the periphery only during the last third of pregnancy. In the present study, the embryonic day (E11) rat embryo/trophoblast was shown to have four times the VIP concentration of the E17 fetus and to have VIP receptors in the central nervous system. However, no VIP mRNA was detected in the E11 rat embryo or embryonic membranes by in situ hybridization or reverse transcriptase-PCR. RIA of rat maternal serum revealed a peak in VIP concentration at days E10-E12 of pregnancy, with VIP rising to levels 6-10-fold higher than during the final third of pregnancy. After intravenous administration of radiolabeled VIP to pregnant female mice, undegraded VIP was found in the E10 embryo. These results suggest that maternal tissues may provide neuroendocrine support for embryonic growth through a surge of VIP during early postimplantation development in the rodent.

Vasoactive intestinal peptide stimulates ACTH and corticosterone release after injection into the PVN

Vasoactive intestinal peptide (VIP), a neuropeptide originally isolated from the intestine, is widely distributed in the central and peripheral nervous systems and exhibits a broad range of biological actions. In the present study the effects of VIP on plasma ACTH and corticosterone (CORT) secretion were investigated in fasted, freely-moving male rats. Male rats, chronically implanted with a hypothalamic paraventricular nucleus (PVN) cannula, were injected with VIP doses (0.15–3.0 nmol/rat) of VIP or saline (control). Blood samples were collected (0.6 ml) from an intravenous catheter immediately preceding and at 15, 30, 60, 90 and 120 min following peptide administration. PVN administration of VIP increased the plasma ACTH and CORT levels in a dose-dependent manner and the maximal effect was obtained at 15 min after administration. At the highest dose tested, VIP increased ACTH and CORT to 167% and 342% of time-matched controls, respectively. These results demonstrate that the PVN is a sensitive site for VIP-induced elevation of plasma ACTH and CORT and imply that the VIP binding sites and immunoreactive terminals previously identified in this region may be involved in the central regulation of the pituitary-adrenal axis.

Distribution of VIP mRNA and two distinct VIP binding sites in the developing rat brain: relation to ontogenic events.

The peptide neurotransmitter vasoactive intestinal peptide (VIP) has neurotrophic properties and influences neurobehavioral development. To assess the role of VIP during neural ontogeny, the present work traces the development of VIP mRNA with in situ hybridization and VIP receptors with in vitro autoradiography in rat central nervous system (CNS) from embryonic day 14 (E14) to the adult. VIP mRNA was not evident in the CNS until birth. Postnatally, it was expressed in several distinct brain regions, but its distribution bore little relation to that of VIP receptors. VIP receptors were present and expressed changing patterns of distribution throughout CNS development. The changing patterns were the result of 1) the transient appearance of GTP-insensitive VIP receptors in several regions undergoing mitosis or glial fasciculation and 2) the transient appearance of GTP-sensitive VIP receptors homogeneously distributed throughout the CNS during the first 2 postnatal weeks, the period of the brain growth spurt. At E14-16 VIP binding was dense throughout the brainstem and spinal cord, but limited in the rest of the brain. From E19 to postnatal day 14 (P14), while VIP binding was higher in germinal zones, it tended to be uniformly dense throughout the remainder of the brain. By P21 the adult pattern began to emerge; VIP binding was unevenly distributed and was related to specific cytoarchitectural sites. Since the expression of VIP in the CNS is limited to postnatal development but VIP receptors are abundant prenatally, we suggest that extraembryonic VIP may act upon prenatal VIP receptors to regulate ontogenic events in the brain.