Vasoactive Intestinal Peptide Binding Sites Research Articles

Characterization of vasoactive intestinal peptide receptors in canine liver membranes

The binding characteristics of vasoactive intestinal peptide (VIP) in the liver membranes of the dog were examined using radioligand binding assay with 125I-VIP and unlabelled peptides and results were compared with those from the rat. The binding of VIP to canine liver membranes occurred in a reversible, saturable, specific and temperature-dependent manner. Guanine nucleotides dosedependently inhibited VIP binding. The order of potency in competition experiments with unlabelled peptide was: VIP > pituitary adenylate cyclase activating peptide (PACAP)-27 > PACAP-38 > peptide histidine isoleucine (PHI) = secretin in the dog, and PACAP-27 > PACAP-38 > VIP > PHI > secretin in the rat. PHI and secretin were about 5000 times less potent than VIP in the dog, but secretin was about 100 times less potent than VIP in the rat. The VIP binding sites in canine liver membranes have recognition sites for VIP which differ from those in rat liver membranes. As most of VIP in the portal vein was removed during its passage through the canine liver, the binding sites of canine liver may play a role in degradation of VIP.

VIP binding sites in adult rat hypothalamus: Nuclear distribution and daily variations

Vasoactive intestinal peptide (VIP) neurons are present in the suprachiasmatic nucleus (SCN) and project to various hypothalamic nuclei. Several results suggest that hypothalamic VIP is involved in the mediation of synchronizing information. [ 125I]Vasoactive intestinal peptide binding has been reported to be especially high in the hypothalamus. We show here by radioautography by dipping that [ 125I]VIP binding is high and homogenously distributed in the SCN. The correspondence between VIP binding and terminals densities supports the hypothesis that VIP acts through synapses in the SCN. Moderate and fairly homogenous VIP binding is also found in the paraventricular nucleus (PaVN), whereas VIP terminals are reported to be restricted to the subparaventricular area. This suggests that VIP could diffuse and thus function as a paracrine neuromodulator in the PaVN. [ 125I]Vasoactive intestinal peptide binding is stable along the nycthemer in the suprachiasmatic, peri-, and paraventricular nuclei as well as in all other areas investigated.

Characteristics of receptors for VIP in rat peritoneal macrophage membranes

Vasoactive intestinal peptide (VIP) receptors were investigated in rat peritoneal macrophage membranes (RPMM) using [ 125I]VIP as ligand. The receptor binding was rapid, reversible, saturable, specific, and dependent on time, temperature, and membrane concentration. The Scatchard analysis of binding data was consistent with the existence of two classes of VIP binding sites with K d values of 0.60 ± 0.08 and 275 ± 39 n M and binding capacities of 580 ± 71 and 72,500 ± 810 fmol VIP/mg protein, respectively. The interaction showed a high degree of specificity, as suggested by competitive displacement experiments with several peptides structurally or not structurally related to VIP. These pharmacological studies showed the following order of potency: VIP (IC 50 = 1 n M) > rGRF (IC 50 = 13 rmn M) > PHI (IC 50 = 421 n M) ⪢ secretin. Glucagon, somatostatin, insulin, octapeptide of cholecystokinin [CCK(26–33)], and pancreastatin were ineffective at concentrations up to 1 μ M. Binding of [ 125I]VIP to membranes is markedly reduced by increasing the ionic strength of incubation medium. Treatment of membranes with dithiothreitol, trypsin, and phospholipases A 2 and C resulted in a loss of the ability of these membranes to bind VIP. However, treatment with phospholipase D did not affect binding of VIP by membranes. The molecular characterization of VIP receptors in RPMM was performed after [ 125I]VIP cross-linking to membranes using the cross-linker dithiobis (succinimidyl propionate). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of membrane proteins revealed specific [ 125I]VIP-protein complexes of M r 55,000 ± 1700, 35,000 ± 900, and 22,000 ± 500.

Action of vasoactive intestinal peptide and distribution of its binding sites in vessels used for coronary artery bypass grafts

In an attempt to establish the possible role of vasoactive intestinal peptide (VIP) in the regulation of vasomotor tone of coronary artery bypass grafts, this study examined the action of this peptide and the distribution of [ 125I]VIP binding sites in isolated human gastroepiploic artery (GEA), internal mammary artery (IMA) and saphenous vein. VIP (10 −10 − 3 × 10 −7M) elicited concentration-dependent relaxations in ring segments that were preconstricted with the thromboxane analog U46619. The maximal response was mean ± SEM 79 ± 4%, 52 ± 8% and 23 ± 3% of glyceryl trinitrate (3 × 10 −5M)-induced maximal smooth muscle relaxation in the GEA, IMA and saphenous vein, respectively. Both receptor-binding and competition studies indicated that there was a higher density of [ 125I]VIP binding to smooth muscle cells of the GEA and IMA than to the saphenous vein. Total binding, at 50pM [ 125I]VIP, was 604 ± 89, 381 ± 64 and 87 ± 12 amol/mg wet weight in the GEA, IMA and saphenous vein, respectively. Dense binding of [ 125I]VIP was associated with the tunica media in all the vessels studied. There was also binding to perivascular regions with no obvious binding to endothelial cells. These data demonstrate that arterial grafts, particularly the GEA, are more sensitive to the relaxant effect of VIP and this may possibly be due to a higher receptor density.

Effect of Ovine Prolactin Administration on Hypothalamic Vasoactive Intestinal Peptide (VIP), Gonadotropin Releasing Hormone I and II Content, and Anterior Pituitary VIP Receptors in Laying Turkey Hens1

The effect of ovine prolactin (oPRL) administration to laying and incubating turkey hens was studied. In experiment one, five treatment groups (n = 6) of laying hens received injections of vehicle or oPRL (4 mg/bird/day) for 2, 4, 8, or 14 days. Hypothalamic vasoactive intestinal peptide (VIP) content decreased to its lowest level (p < 0.05) by Day 2 of oPRL injection. Serum turkey PRL decreased by Day 8 of injection, reaching nadir by Day 14. Similar to the decline in hypothalamic VIP content, the maximal decline in the number of anterior pituitary VIP binding sites was achieved by Day 2 of oPRL administration. Hypothalamic GnRH-I content decreased after 2 days of oPRL injection and remained low through Day 14 of the experiment. GnRH-II levels declined with time, reaching significantly lower levels after 8 days of injections, and remained low through Day 14. Plasma LH levels also declined (p < 0.05) after Day 14 of oPRL administration. In experiment 2, two groups (n = 6) of incubating birds were used: controls receiving injections of vehicle only and birds receiving injections of oPRL (4 mg/bird/day) for 10 days. Exogenous oPRL had no effect on hypothalamic VIP, GnRH-I or II, anterior pituitary VIP binding sites, or plasma turkey PRL or LH. The findings suggest that in laying hens, PRL inhibits its own secretion by acting on both the hypothalamus and the anterior pituitary; this phenomenon does not occur in incubating birds. Furthermore, we provide evidence that pRL acts centrally to reduce LH levels by reducing GnRH levels in the hypothalamus.

Changes in Vasoactive Intestinal Peptide Binding Site Densities in the Female Rat Central Nervous System and Pituitary During Lactation

Using quantitative autoradiography, vasoactive intestinal peptide (VIP) binding site densities were investigated in the female rat during lactation. Plasma prolactin levels were significantly (P<0.01) elevated on days 3 and 10 of lactation. In the brain, there was a significant (P<0.05) increase in VIP binding sites during lactation in the striatum, thalamus, anterior portion of the paraventricular nucleus of the hypothalamus, and several cortical areas compared to virgin female diestrous rats. VIP binding density in the anterior pituitary was significantly higher (P<0.01) when measured on day 3 of lactation, and decreased to diestrous levels by day 10. Pup removal resulted in a significant reversal of the lactation-induced increase in VIP binding density in the thalamus, parietal and insular cortices, and the anterior portion of the paraventricular nucleus. Taken together, modifications in VIP binding site density in the pituitary and in several central areas are associated with changes in the level of prolactin secretion. Moreover, the pattern of VIP receptor induction during lactation suggests a role for VIP in the brain that extends beyond the regulation of prolactin secretion.

Modulation of the expression of the VIP receptor by serum factors on the human melanoma cell line IGR39

IGR39 cells, isolated from a human superficial melanoma, display at their surface high and low affinity receptors for the vasoactive intestinal peptide (VIP). When grown in DME medium supplemented with 10% fetal calf serum, cells display 1.6 x 10(5) high affinity (Kd 0.74 nM) and 5.6 x 10(5) low affinity (Kd 55 nM) VIP binding sites per cell. When cultured in a chemically defined medium containing EGF, transferrin, and selenium, IGR39 cells display many neurite-like extensions. Following these morphological changes, the specific [125I]VIP binding is increased four- to fivefold after 6 days in culture. This phenomenon is reversible and is the result of an increased number of VIP binding sites available at the cell surface, without modification of their affinities. The molecular mass of the binding sites is also unchanged whatever cell culture conditions. Increase in [125I]VIP binding is inversely correlated to the serum concentration in the culture medium. When added to the chemically defined medium, sera from various origins as well as some serum substitutes reduce [125I]VIP binding to the same extent as that of the serum. The total cAMP production by VIP-stimulated IGR39 cells is enhanced by a factor of six to seven when cells are cultured in serum-free medium, in good correlation with the increase of VIP binding capacity. These data suggest that factor(s) present in fetal calf serum inhibit(s) the expression of VIP receptor, thus demonstrating the importance of a strict control of cell culture conditions for in vitro studies.

The effects of neuropeptides on mucous glycoprotein secretion from human nasal mucosa in vitro

The role of neuropeptides in the regulation of macromolecule secretion from human nasal mucosa is incompletely understood. Previous in vitro explant culture studies have demonstrated the effects of neuropeptides on lactoferrin release from serous cells and 3H-glucosamine labeled respiratory glycoconjugate secretion from mucus-containing cells. The generation of a new monoclonal antibody, 7F10, has led to the development of an ELISA for high molecular weight respiratory mucous glycoproteins (MGP). This ELISA was used to measure the ability of sensory, parasympathetic and sympathetic neuropeptides to stimulate MGP release from human nasal mucosal fragments in short term explant culture in vitro. Significant MGP release was stimulated by the sensory neuropeptides gastrin releasing peptide (10 μM GRP: 10.6% ± 2.4% increase, n = 8, P < 0.01 vs. control), substance P (1 μM SP: 12.5% ± 5.4%, n = 11, P < 0.05), neurokinin A (1 μM NKA: 17.8 ± 4.3%, n = 6, P < 0.01), while calcitonin gene related peptide (CGRP) was without effect. Vasoactive intestinal peptide (VIP), a neurotransmitter from parasympathetic nerves, induced significant dose dependent MGP secretion, but had no additive or inhibitory interaction with methacholine-induced secretion. Neuropeptide Y (NPY), present in sympathetic nerves, had no effect on MGP secretion. These observations correlate with the effects of neuropeptides on serous cell lactoferrin secretion, and the presence of specific GRP, SP, and VIP binding sites on human nasal submucosal glands that have been detected by autoradiography. GRP and tachykinins (SP and NKA) from sensory nerves, and VIP released during parasympathetic reflexes may signficantly stimulate mucous and serous cell secretion from human nasal mucosa in vivo.

Effect of vasoactive intestinal peptide and the distribution of receptors in human epicardial coronary arteries

Background The aim of this study was to elucidate a possible role for vasoactive intestinal peptide (VIP) in the regulation of coronary vascular tone. Its action and the distribution of [125I]-VIP binding sites were examined in isolated human coronary arteries. Methods A total of 97 segments were obtained from 23 patients undergoing cardiac transplantation. Results VIP (10−10 to 3 × 10−7 M) relaxed preconstricted coronary segments in a concentration-dependent manner, with a maximum response of 73.8% ± 8.5% of the maximum relaxation induced by 1 μg/mL of nitroglycerin. This action was inhibited by the specific receptor antagonist [4CI-D-Phe6, Leu17] VIP, with a reduction of the maximum effect from 73.8% ± 8.5% to 38.9% ± 5.4% (P < 0.001). Prior exposure to atropine and indomethacin, and removal of the endothelium had no effect on responses to VIP. Receptor autoradiography demonstrated that [125I]-VIP binding sites were displaced by unlabeled peptide and by [4CI-D-Phe6, Leu17] VIP in a concentration-dependent manner, with 50% inhibitory concentration values of 1.35 × 10−8 and 2.41 × 10−8 M, respectively. The binding was predominantly associated with smooth muscle and perivascular regions, with no binding to the endothelial cells. Conclusions Our results indicate that VIP elicits vasodilatation of the human coronary artery by acting mainly on specific receptors presumably present on the smooth muscle. This peptidergic control mechanism may play a role in the regulation of coronary artery tone.

Vasoactive intestinal peptide receptor antagonists in rat seminal vesicle membranes

Vasoactive intestinal peptide (VIP) receptor coupled to activation of adenylate cyclase have been previously identified in seminal vesicle membranes of rat. In the present study we demonstrate that the synthetic peptides [4-Cl-D-Phe 6, Leu 17]VIP and the growth hormone releasing factor (GRF) analog [Ac-Tyr 1,D-Phe 2]GRF 1–29-NH 2 inhibit in a competetive manner the specific 125I-VIP binding to the same membrane preparation. The order of potency of the two peptides compared to VIP was: VIP (IC 50 = 1.3± 0.5 nM) > [4-Cl-D-Phe 6, Leu 17]VIP (IC 50 = 160.0 ± 45.0 nM) > [Ac-Tyr 1, D-Phe 2]GRF 1–29-NH 2 (IC 50 = 290.0 ± 59.4 nM). Whereas VIP showed a stimulatory activity upon adenylate cyclase with a potency (ED 50 = 7.0 ± 0.7 nM) compatible with the affinity of the VIP binding sites previously described, the other two peptides tested showed no effect at that level. The behavior as antagonists of both [4-Cl-D-Phe 6, Leu 17]VIP and [Ac-Tyr 1,D-Phe 2]GRF 1–29-NH 2 was confirmed by: (a) the parallel shifts of the VIP dose-response curves for stimulation of adenylate cyclase activity in the presence of the antagonists; (b) the close agreement between the binding affinity and the inhibition of adenylate cyclase activity for the two peptides; and (c) the lack of effect of the two antagonists upon the adenylate cyclase activity stimulated by the β-adrenoceptor agonist isoproterenol which indicates the specificity of the interaction. The present results suggest that [4-Cl-D-Phe 6, Leu 17]VIP and [Ac-Tyr 1,D-Phe 2] GRF 1–29-NH 2 competitively antagonize VIP actions at the receptor level in rat seminal vesicle and provide a useful tool in the study of the physiological role of VIP in the genitourinary tract.

Glucocorticoids upregulate the high affinity receptors for vasoactive intestinal peptide (VIP) on human mononuclear leucocytes in vitro

Glucocorticoids were shown to induce a time- and dose-dependent increment of specific [ 125I]VIP-binding on human mononuclear leucocytes in culture. Cortisol (0.5 μM) increased specific [ 125I]VIP-binding to 132% of control after 48 h preincubation, to 162% after 96 h, and to 175% after 144 h. Dexamethasone (0.5 μM) increased specific [ 125I]VIP-binding to 140%, 194% and 210% after the same time periods. Analysis of the binding data revealed an increase in B max to 119% by cortisol (0.5 μM, 48 h) and to 194% by dexamethasone (0.5 μM, 48 h), and no change in K d for the high affinity receptor after preincubation. The number of low affinity binding sites for VIP was also increased by glucocorticoids. However, in contrast to the high affinity receptor, low affinity binding sites were initially downregulated in culture, and glucocorticoids induced a restitution to number and affinity close to those obtained for freshly isolated leucocytes. This increase in low affinity binding sites was blocked by actinomycin D, in contrast to the high affinity receptor upregulation which was independent of de novo protein synthesis. Furthermore, corresponding to the glucocorticoid induced high affinity receptor upregulation, an increase in VIP stimulated cyclic AMP production was observed. The results of this study suggest that leucocyte responsiveness to VIP can be influenced by glucocorticoids.

Molecular characterization and peptide specificity of two Vasoactive Intestinal Peptide (VIP) binding sites in the chicken pineal

Here we report that chicken pineal membranes express high affinity receptors for Vasoactive Intestinal Peptide (VIP), as revealed by competitive displacement analysis of [3-iodotyrosyl- 125I]-VIP by native VIP. These binding sites were further characterized by covalent cross-linking of radioiodinated VIP to chicken pineal cell membranes, using dithiobis(succinimidylpropionate) as a cross-linking reagent. Sodium dodecyl sulfate electrophoresis after solubilization of the cross-linked membranes, reveals the existence of two polypeptides, P 1 and P 2, with a similar labelling intensity and apparent molecular weights of Mr = 57 000 and Mr = 70 000 respectively. These two components behave like high affinity binding sites for VIP.

The effect of vasoactive intestinal peptide (VIP) on the contractile activity of human uterine smooth muscle.

1. In the present study we examined the in vitro effect of vasoactive intestinal peptide (VIP) on spontaneous contractions in both inner and outer layers of non-pregnant human myometrium. A dose-dependent relaxation was observed, but with a marked difference in sensitivity to VIP between the two layers, with an IC50 value of 1 x 10(-8) and 1 x 10(-5) mol L in the outer and inner layers, respectively. 2. We also established that VIP did not directly stimulate the adenylate cyclase activity. The only slight stimulations were observed in non-initial rate conditions. The maximal response of this indirect effect was obtained for VIP concentrations between 1 x 10(-9) and 1 x 10(-8) mol/L and this occurred to the same extent (an approximately 1.4-fold increase) in both layers. However this response is specific, since structurally related peptides such as glucagon, gastric inhibitory polypeptide (GIP), secretin, or human growth hormone-releasing factor (hGRF) had no effect in our preparations. 3. Autoradiographic studies revealed that specific VIP binding sites were located on the vascularization of the intermediate vascular layer and on arterioles and venules distributed in the inner and outer myometrial layers. They were also present in the endometrium, but not on smooth muscle cells of either layer. 4. Such observations could provide evidence for another signal transduction pathway to mediate the biological effect of VIP. An additional intermediate step on the vascularization distributed in all of the muscle cannot be excluded.

Vasoactive intestinal peptide binding sites and fibers in the brain of the pigeon Columba livia: An autoradiographic and immunohistochemical study

The distribution of vasoactive intestinal peptide (VIP) binding sites in the pigeon brain was examined by in vitro autoradiography on slide-mounted sections. A fully characterized monoiodinated form of VIP, which maintains the biological activity of the native peptide, was used throughout this study. The highest densities of binding sites were observed in the hyperstriatum dorsale, archistriatum, auditory field L of neostriatum, area corticoidea dorsolateralis and temporo-parieto-occipitalis, area parahippocampalis, tectum opticum, nucleus dorsomedialis anterior thalami, and in the periventricular area of the hypothalamus. Lower densities of specific binding occurred in the neostriatum, hyperstriatum ventrale and nucleus septi lateralis, dorsolateral area of the thalamus, and lateral and posteromedial hypothalamus. Very low to background levels of VIP binding were detected in the ectostriatum, paleostriatum primitivum, paleostriatum augmentatum, lobus parolfactorius, nucleus accumbens, most of the brainstem, and the cerebellum. The distribution of VIP-containing fibers and terminals was examined by indirect immunofluorescence using a polyclonal antibody against porcine VIP. Fibers and terminals were observed in the area corticoidea dorsolateralis, area parahippocampalis, hippocampus, hyperstriatum accessorium, hyperstriatum dorsale, archistriatum, tuberculum olfactorium, nuclei dorsolateralis and dorsomedialis of the thalamus, and throughout the hypothalamus and the median eminence. Long projecting fibers were visualized in the tractus septohippocampalis. In the brainstem VIP immunoreactive fibers and terminals were observed mainly in the substantia grisea centralis, fasciculus longitudinalis medialis, lemniscus lateralis, and in the area surrounding the nuclei of the 7th, 9th, and 10th cranial nerves. The correlation between the distribution of VIP binding sites and immunoreactive fibers and terminals was assessed in a restricted number of regions. A qualitatively good matching was found in the area corticoidea dorsolateralis, hyperstriatum dorsale, hyperstriatum accessorium, nucleus septi lateralis, nuclei dorsomedialis and dorsolateralis thalami, and in some hypothalamic areas. A striking mismatch occurred in the hyperstriatum ventrale, neostriatum, tectum opticum (high to moderate density of binding sites but only few immunoreactive profiles), and in the tuberculum olfactorium, median eminence, and spinal cord (lower density of binding sites but abundant immunoreactive profiles). The paleostriatum, lobus parolfactorius, and ectostriatum were virtually devoid of both binding sites and immunoreactive profiles. The results are discussed in relation to the known actions of VIP in the rodent and avian brain and are compared with previous observations on the distribution of VIP binding sites in the central nervous system of other vertebrates.

Autoradiographic characterization and localization of vasoactive intestinal peptide bindign sites in albino rat and rabbit eyes

Localization and pharmacological properties of vasoactive intestinal peptide (VIP) binding sites were investigated in eyes from albino rabbits and rats using an in vitro autoradiographic method. [125I]VIP was used as ligand, and various unlabelled peptides were studied to test the specificity of binding. Autoradiograms were generated by apposing 20-microns-thick cryostat eye sections to [3H]Hyperfilm or autoradiographic emulsion and quantified by means of image analysis procedures. Specific binding represented about 85% of total binding. Kinetic studies showed that equilibrium was reached after a 120-min incubation at room temperature. Biochemical investigations demonstrated that [125I-]VIP bound to a population of sites with high affinity (Kd = 2.27 +/- 0.25 nM). Inhibition of [125I]VIP binding with VIP and related peptides indicated the following rank order of potency: VIP greater than Peptide histidine isoleucine greater than secretin greater than human growth hormone-releasing factor, glucagon, VIP1-14, VIP14-28. In both species, specific binding was found in conjunctiva, iris, ciliary processes, choroid and retina. Moderate grain densities of VIP binding sites were also present in the rat cornea. Quantitative analysis of the autoradiograms revealed that the highest densities of [125I]VIP binding sites were located in the iris and ciliary epithelia in rabbits and in the inner retina in rats. Our findings suggest that VIP may play an important role in several ocular functions, especially in aqueous humor dynamics and retinal neuromodulation.

High-affinity binding sites for VIP in renal cortical membranes: Possible role of VIP in renal transport

We studied binding and degradation of vasoactive intestinal peptide (VIP) by highly purified brush border and basolateral membranes from rabbit kidney cortex. Brush border and basolateral membranes were capable of 73 and 49% degradation of VIP after 20 minutes, and the degradation was totally prevented by bacitracin. There was 66 and 87% specific binding of 125I-VIP to brush border and basolateral membranes, respectively. 125I-VIP binding to renal membrane was displaced in a dose dependent fashion by unlabeled VIP with half maximal displacement at 2 x 10(-7) M. Other related peptides failed to displace VIP. Scatchard analysis showed one single class of receptors for VIP in both membranes with similar Kd (0.5 x 10(-7) M), but higher number of binding sites (Bmax) in the basolateral membranes than in the brush border membranes (22.0 vs. 4.4 pmol/mg protein), respectively. Forty-eight percent of VIP binding to brush border membranes could be explained by cross contamination of these membranes with basolateral membranes. We examined the effect of VIP on Na-H antiporter, Na-dependent glucose uptake and Na-dependent phosphate uptake by isolated proximal tubule suspension. In acid loaded proximal tubules VIP (10(-6) M) inhibited total and amiloride-sensitive 22Na uptake by 35 and 75%, respectively, as compared to control. On the other hand VIP failed to inhibit Na-dependent methyl alpha-14C-glucopyranoside and Na-dependent 32phosphate uptake. VIP failed to stimulate cyclic AMP generation by proximal tubule suspension while PTH showed the expected stimulation. Our results demonstrate the presence of specific binding for VIP in highly purified cortical membranes and suggest an effect of VIP to inhibit the Na-H antiporter by a mechanism independent of cyclic AMP.

Characterization of vasoactive intestinal peptide receptors in retina

Vasoactive intestinal peptide (VIP) is a neuropeptide having a wide range of effects on a large number of tissues. To gain insight into the role VIP plays in retinal function, VIP receptors in bovine retinal membranes were analyzed in competition binding assays and by affinity labeling studies and compared to VIP receptors in rat liver membranes. In both membrane preparations, high affinity VIP binding sites (K D ∼ 1 n m) were detected. Secretin and glucagon, each having close structural homology to VIP, were found to have negligible effects on [ 125I]VIP binding in retina. In contrast, secretin (K D = 70 n m) was modestly effective in inhibiting [ 125I]VIP binding to rat liver membranes. Affinity labeling analysis revealed a VIP binding site of 59 kDa in both bovine retinal and rat liver membranes. Digestion of affinity-labeled receptor proteins with endoglycosidase F generated final cleavage products of approx. 45 kDa for both receptors. These results indicate that the retina expresses a high affinity, highly selective VIP receptor thereby supporting a specific function for VIP in this tissue.

PHI preferentially binds to VIP receptors in normal rat tissues

Vasoactive intestinal peptide (VIP) and peptide histidine isoleucine (PHI) are homologous neuropeptides with parallel biological actions. These similarities raise the question whether VIP and PHI have common or distinct mechanisms of action, including receptors. The present study attempted to distinguish specific binding sites for VIP and PHI in normal rat tissues using the homologous radioligands [Tyr( 125I) 10]VIP and [Tyr( 125I) 10]rat PHI. In rat brain, anterior pituitary, and liver membranes both radioligands identified a VIP-preferring receptor. Rat PHI had less than 10% the binding potency of VIP in these tissues irrespective of which radioligand was used. In rat uterine membranes [Tyr( 125I) 10]VIP bound to a receptor with approximately 100 times greater affinity for VIP over PHI. No specific binding of [Tyr( 125I) 10]rat PHI to rat uterus could be demonstrated. In conclusion, these results support the predominance of VIP-preferring receptors as opposed to PHI-preferring receptors in normal rat brain, anterior pituitary, liver and uterus.

Developmental and lactational changes in the rat anterior pituitary VIP receptor

The regulation of female rat anterior pituitary vasoactive intestinal peptide (VIP) receptors was examined during postnatal development and lactation. VIP receptor binding to anterior pituitary membranes from 1- to 60-week-old rats and lactating rats was examined using HPLC purified [Tyr( 125I) 10]VIP. Nonlinear regression of competitive binding studies indicated the presence of 2 VIP binding sites in 3-week and older animals, whereas only 1 site was identified in 1- and 2-week-old rats. The single site did not differ significantly in affinity or number when compared to the low affinity site of older animals. The guanine nucleotide, GTP-γ-S, decreased the specific binding of VIP by 60–80% in 3-week and older animals, but not in younger animals. Compared with adult nonlactating animals, the number of high affinity binding sites decreased significantly during lactation, with no change in receptor binding affinity.

Vasoactive intestinal peptide (VIP) and peptide histidine methionine (PHM) in human eccrine sweat glands: demonstration of innervation, specific binding sites and presence in secretions

Vasoactive intestinal peptide (VIP) and peptide histidine methionine (PHM) immunoreactivities have been detected in alcohol extracts of human axillary skin using sensitive and specific radioimmunoassays. VIP immunoreactivity (7.63 + 2.33 pmol/g, x + SE, n = 9) was more abundant than PHM immunoreactivity (3.86 + 0.56 pmol/g, x + SE, n = 9). Immunocytochemistry of sections of skin revealed a network of VIP/PHM immunoreactive nerve fibres around the perimeter of eccrine but not apocrine sweat glands. In vitro autoradiography of skin sections using 125I-labelled VIP and PHM, demonstrated binding sites on the membranes of eccrine gland secretory cells. The binding of each radiolabelled ligand was eliminated by the presence of a large molar excess of appropriate cold peptide but was unaffected when incubated with related peptide, indicating the presence of specific binding sites for both VIP and PHM. Radioimmunoassay of Sep-pak concentrated human sweat identified the presence of both VIP immunoreactivity (30.6 pmol/l) and PHM immunoreactivity (43.4 pmol/l). Reverse-phase HPLC analysis of axillary skin extracts and sweat, followed by radioimmunoassay of fractions, identified single resolved peaks of VIP and PHM immunoreactivity with identical retention times to synthetic standards. Eccrine sweat glands in human axillary skin have VIP and PHM peptidergic innervation and possess specific binding sites for each peptide which are both secreted to the surface of the skin.