125I-VIP Binding Research Articles

Characterization of receptors for VIP on pancreatic acinar cell plasma membranes using covalent cross-linking

Vasoactive intestinal peptide (VIP) receptors on guinea pig pancreatic acini differ from those on all other tissues in containing a high-affinity VIP receptor and a low-affinity VIP receptor that has a high affinity for secretin. To characterize the molecular components of these receptors, 125I-VIP was covalently cross-linked to these receptors by four different cross-linking agents: disuccinimidyl suberate, ethylene glycol bis (succinimidyl succinate), dithiobis (succinimidylpropionate), and m-maleimidobenzoyl N-hydroxysuccinimide ester. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated a single major polypeptide band of Mr 45,000 and a minor polypeptide band of Mr 30,000 were cross-linked to 125I-VIP. Covalent cross-linking only occurred when a cross-linking agent was added, was inhibited by GTP, was inhibited by VIP receptor agonists or antagonists that interact with VIP receptors, and not by other pancreatic secretagogues that interact with different receptors. For inhibiting both cross-linking and binding of 125I-VIP to the major polypeptide Mr 45,000 and the minor polypeptide Mr 30,000 components, the relative potencies were VIP greater than helodermin greater than rat growth hormone releasing factor greater than peptide histidine isoleucine greater than secretin. The apparent molecular weight of the cross-linked polypeptides were unchanged by dithiothreitol. Thus the high-affinity VIP receptor on pancreatic acinar cell membranes consists of a single major polypeptide of Mr 45,000, and this polypeptide is not a subunit of a larger disulfide-linked structure. Furthermore, either the low-affinity VIP/secretin-preferring receptor was not covalently cross-linked under the experimental conditions or it consists of a major polypeptide with the same molecular weight as the high-affinity VIP receptor.

Analysis of the immunomodulatory properties of the secretin-glucagon family of peptides on mouse lymphoid cell functions and the demonstration of specific receptors on T cells.

The ability of members of the secretin-glucagon family of peptides to modulate the responses of mouse lymphoid cells stimulated with Concanavalin A (Con A), Lipopolysaccharide (LPS) and alloantigens was determined. It was observed that vasoactive intestinal peptide (VIP) and peptide having NH2-terminal histidine and COOH-terminal Isoleucine (PHI) inhibited the incorporation of 3H-methyl-thymidine by cells stimulated with Con A (55% inhibition) or alloantigen-bearing cells (40% inhibition). Secretin was approximately 10,000 less effective as an immunomodulator. Other members of the neuropeptide family, including glucagon and gastric inhibitory peptide, were ineffective in affecting mitogenesis elicited by Con A (20% inhibition). Lipopolysaccharide stimulated spleen cells were refractory to modulation by all members of the secretin-glucagon family of peptides (less than 5% modulation). The inhibition measured was concentration dependent over the range of 10(-6) to 10(-16) M. A peptide fragment of VIP encompassing amino acid residues 10-28, although capable of modulating in vitro responses, was 30-50% less effective than intact VIP. In addition, a VIP specific binding assay for mouse lymphoid cells was described. The binding of 125I-VIP to lymph node cells was rapid, saturable and reversible. Apparent equilibrium was reached within 15 minutes and nonspecific binding, measured as 125I-VIP binding in the presence of an excess (2 x 10(-7) M) of native VIP, did not exceed 25% of the total binding. In competitive experiments using VIP related peptides, PHI but not gastric inhibitory peptide, glucagon or secretin was able to significantly inhibit 125I-VIP binding. PHI had only one-eighth of the competitive capacity of native VIP. Scatchard analyses indicated the existence of a single class of high affinity receptors on lymph node cells (KD = 3.46 nM; 26,000 sites/cell). 125I-VIP specific binding to purified T cells (14%) was markedly higher than to B cells (3% binding). Thymocytes bound less than 2% of the label and had relatively few VIP binding sites (8,000) as compared with purified T cells (45,000 sites/cell). There was variability in the ability of various T cells tumors and functional T cell clones to bind 125I-VIP. The role of VIP as a physiological modulator of T cell activation is discussed.

Characterization of receptors for vasoactive intestinal peptide solubilized from the lung.

The zwitterionic detergent CHAPS was used to solubilize functional receptors for vasoactive intestinal peptide (VIP) from guinea pig lung. The solubilized receptors were resolved by high performance gel filtration in 3 mM CHAPS into two active fractions with apparent Stokes radii of 5.9 +/- 0.1 and 2.3 +/- 0.1 nm. The binding of 125I-VIP to the two receptor fractions was time-dependent, reversible, and saturable. Trypsin destroyed the binding activity of the receptor fractions, indicating their proteinic nature. Unlabeled VIP competitively displaced the binding of 125I-VIP to the 5.9-nm fraction (IC50 = 240 pM) and the 2.3-nm fraction (IC50 = 1.2 microM). Scatchard analysis indicated a single class of binding sites in each receptor fraction, with Kd values 300 pM and 0.97 microM for the 5.9- and 2.3-nm Stokes radii fractions, respectively. When the high affinity, 5.9-nm Stokes radius fraction was rechromatographed in 9 nM CHAPS, 46% of the binding activity eluted in the low affinity, 2.3-nm Stokes radius fraction, indicating that the latter is a product of dissociation of the high affinity receptor complex. GTP inhibited the binding of 125I-VIP to the high affinity complex but not the low affinity species. Scatchard plots of VIP binding by the high affinity receptors treated with GTP suggested the presence of two distinct binding sites (Kd 4.4 and 153 nM), compared to a single binding site (Kd = 0.3 nM) obtained in untreated receptors. The nonhydrolyzable GTP analog, guanyl-5'-yl-imidodiphosphate, inhibited VIP binding by the high affinity receptor fraction with potency nearly equivalent to that of GTP. These observations suggest that GTP-binding regulatory proteins are functionally coupled to the VIP-binding subunit in the high affinity receptor complex. The peptide specificity characteristics of the two receptor fractions were different. Peptide histidine isoleucine and growth hormone releasing factor, peptides homologous to VIP, were 87.5- and 22.9-fold less potent than VIP in displacing 125I-VIP binding by the high affinity receptor complex, respectively. On the other hand, growth hormone-releasing factor was more potent (22.7-fold) and peptide histidine isoleucine was less potent (31.3-fold) than VIP in displacing the binding by the low affinity species.

Characterization and autoradiographic distribution of vasoactive intestinal peptide binding sites in the rat central nervous system

Biochemical characteristics and topographical distribution of mono-[125I )vasoactive intestinal peptide (VIP) binding sites in rat brain were studied on tissue sections and by quantitative autoradiography. Biochemical investigations show two classes of binding sites with a dissociation constant of 1.03 +/- 0.11 nM and 68 +/- 14 nM and a maximal binding capacity of 43.3 +/- 5.1 fmol/mg protein and 713 +/- 117 fmol/mg protein respectively. The order of potency of various peptides to inhibit 125I-VIP binding to brain sections is: VIP greater than PHI greater than secretin greater than VIP greater than hGRF. Autoradiography reveals the highest densities of binding sites in the pineal gland, the dentate gyrus of the hippocampus, the central amygdaloid nucleus and in various thalamic nuclei such as the mediodorsal, lateral posterior, submedius, dorsolateral and medial geniculate nuclei. Similar high densities are observed in the olfactory bulbs as well as in the suprachiasmatic and dorsomedial nuclei of the hypothalamus and in the superior colliculus. These data together with the distribution of the endogenous peptide suggest a physiological role for VIP both in the regulation of CNS activities and pituitary functions.

Characterization of Vasoactive Intestinal Peptide (VIP) receptors in mammalian lung

125I-VIP bound specifically to sites on human, rat, guinea pig, and rabbit lung membranes with a dissociation constant (K D) of 60–200 pM and binding site maxima of 200–800 fmol/mg of protein. The presence of a second lower affinity site was detected but not investigated further. High affinity 125I-VIP binding was reversible and displaced by structurally related peptides with an order of potency: VIP > rGRF > PHI > hGRF > secretin=Ac Tyr 1 D Phe 2 GRF. 125I-VIP has been covalently incorporated into lung membranes using disuccinimidyl suberate. Sodium dodecyl sulfate-polyacrilamide gel electrophoresis of labeled human, rat, and rabbit lung membranes revealed major 125I-VIP-receptor complexes of: Mr=65,000 , 56,000, and 64,000 daltons, respectively. Guinea pig lung membranes exhibited two 125I-VIP-receptor complexes of Mr=66,000 and 60,000 daltons. This labeling pattern probably reflects the presence of differentially glycosylated forms of the same receptor since treatment with neuroaminidase resulted in a single homogeneous band ( Mr=57,000 daltons ). Soluble covalently labeled VIP receptors from guinea pig and human lung bound to and were specifically eluted from agarose-linked wheat germ agglutinin columns. Our studies indicate that mammalian lung VIP receptors are glycoproteins containing terminal sialic acid residues.

Mechanism of action of calcitonin gene-related peptide in stimulating pancreatic enzyme secretion

In guinea pig pancreatic acini rat calcitonin gene-related peptide (CGRP) caused an eightfold increase in amylase release with various phosphodiesterase inhibitors present. Rat CGRP and rat [Tyro]CGRP caused half-maximal effect at 2 nM, and were threefold more potent than human CGRP. CGRP-stimulated amylase release was not inhibited by VIP-(10-28) or secretin-(5-27). CGRP stimulated cAMP and was augmented by phosphodiesterase inhibitors with the order of sensitivity being Ro-20-1724 greater than isobutylmethylxanthine greater than theophylline. CGRP did not increase 45Ca outflux or effect of 125I-VIP binding. CGRP specifically inhibited 125I-CGRP binding. The dose-response curves were broad and each peptide accelerated dissociation of bound 125I-CGRP. Computer analysis demonstrated two classes of CGRP-binding sites. Occupation of a high-affinity class (Kd 20 nM) correlated with stimulation of enzyme secretion and cAMP, and occupation of the low-affinity class (Kd 1 microM) correlated with accelerated dissociation. These studies demonstrate that CGRP interacts with specific pancreatic receptors, CGRP-stimulated cAMP is in a different compartment from that stimulated by other secretagogues, and CGRP differs from other agents that increase cAMP and amylase release in the relationship among receptor occupation, cAMP generation, and enzyme secretion.

SINDING, INTERNAL IZATION AND DEGRADATION OF VIP BY PIG JENNUM EPITHELIAL CELLS

As VIP is involved in the intestinal water and electrolyte secretion and a VIP-ergic innervation of the epithelial layer has been demonstrated, we investigated binding, internalization and degredation of VIP by pig jejunum epithelial cells. Enterocytes were isolated by a non-enzymatic dissociation procedure. 125I-VIP binding was time- and temperature dependent and was inhibited dose-depend-ently by VIP(KD=1.5±0.2 nM, mean±SD;n=5) and secretin(half maximal binding 10nM) but not by other peptides. Internalized radioactivity was separated from cell surface-bound radioactivity by washing the cells with isotonic saline, PH2.5.At 37°C the amount of internalized radioactivity increased till 30 min and represented 30% of cell bound radioactivity. At 10°C, all radioactivity bound to the cells was acid dissociable. To investigate degredation of VIP, cells were incubated with VIP(5-20 nmol/1) and the reaction mixture was analysed by HPLC and metabolites were identified by amino acid analysis. After only 30 sec incubation, des(His)VIP(fragment 2-28) was identified as a major metabolite, representing 30% of the substrate In conclusion normal enterocytes internalized VIP but, in view of the rapid cell-surface degradation, it is suggested that VIP is not internalized as intact peptide. As VIP(2-2B) has only 1% of the biological activity of VIP, its action in the gut may be terminated by aminopeptidases.

Secretin stimulates cyclic AMP formation in the rat brain.

The effects of secretin on cyclic AMP levels in the rat brain were determined. Incubation of rat brain frontal cortex slices with secretin or the structurally related peptides peptide histidine leucine (PHI) or vasoactive intestinal polypeptide (VIP) in the presence of 10 mM theophylline resulted in a dose-dependent increase in the cyclic AMP levels. The half-maximal increase in cyclic AMP occurred using a 1 microM dose of secretin or a 2 microM dose of PHI or VIP. Preincubation of slices with secretin-(5-27) produced a dose-dependent inhibition of the secretin but not VIP- or PHI-stimulated increase in the cyclic AMP content. Also, in receptor binding studies, secretin-(5-27) produced a dose-dependent inhibition (Ki = 400 nM) of 125I-secretin but not of 125I-VIP binding to rat brain membranes. Guanyl-5'-yl imidodiphosphate decreased the affinity of radiolabelled secretin binding as a result of an increased rate of dissociation of bound 125I-secretin. These data suggest that secretin receptors in the rat brain may be coupled to adenylate cyclase in a stimulatory manner and that secretin-(5-27) may function as a central secretin receptor antagonist.

Cycle of the vasoactive intestinal peptide and its binding site in a human adenocarcinoma cell line (HT 29).

The disappearance of vasoactive-intestinal-peptide (VIP) binding sites at the cell surface of a cultured target cell, originating from a human colonic adenocarcinoma (HT 29 cell line), was studied, after preexposition of the cell to the peptide, as a function of time, VIP concentration and temperature. Maximum effect (60-80% loss of binding capacity) was obtained after a 5-10 min exposure of the cells at 37 degrees C with a VIP concentration of 100 nM. The t1/2 of maximum disappearance was less than 2 min and the concentration of native VIP giving half-maximum decrease in 125I-VIP binding was 6 nM. The affinity of remaining binding sites for VIP was not affected compared to that of control cells (Kd = 0.3 nM). Disappearance of VIP binding sites was specific since, with the same conditions of preincubation, the specific binding of 125I-labeled epidermal growth factor to HT 29 cells was not modified. The phenomenon was reversible and 90% of binding capacity could be restored in less than 60 min by incubating cells in VIP-free medium. Correlatively we showed, by two independent experimental procedures, that 125I-VIP, initially bound to HT 29 cells, was maximally internalized after 10 min of incubation at 37 degrees C. All the data strongly suggest that: internalization of VIP is receptor-mediated; upon exposure to native VIP, VIP receptors are down-regulated or at least sequestered within HT 29 cells.

Autoradiographic localisation of VIP receptors in human lung

Localisation and pharmacological properties of the VIP receptor in human lung sections are described. The receptor density determined by saturation analysis using 125I-VIP is approx. 100 fmol mg protein, with a k d of approx. 600 pM. Inhibition of 125I-VIP binding with VIP and related peptides gives a rank order of potency: VIP /s> peptide histidine methionine /s> secretin. Light microscope autoradiography reveals specific VIP binding sites, with a high density over the pulmonary artery smooth muscle and the alveolar walls and with a lower density over the bronchial epithelium.

Autoradiographic visualization of CNS receptors for vasoactive intestinal peptide

Receptors for VIP were characterized in the rat CNS. 125I-VIP bound with high affinity to rat brain slices. Binding was time dependent and specific. Pharmacology studies indicated that specific 125I-VIP binding was inhibited with high affinity by VIP and low affinity by secretin and PHI. Using in vitro autoradiographic techniques high grain densities were present in the dentate gyrus, pineal gland, supraoptic and suprachiasmatic nuclei, superficial gray layer of the superior colliculus and the area postrema. Moderate grain densities were present in the olfactory bulb and tubercle, cerebral cortex, nucleus accumbens, caudate putamen, interstitial nucleus of the stria terminalis, paraventricular thalamic nucleus, medial amygdaloid nucleus, subiculum and the medial geniculate nucleus. Grains were absent in the corpus callosum and controls treated with 1 μM unlabeled VIP. The discrete regional distribution of VIP receptors suggest that in may function as an important modulator of neural activity in the CNS.

Vasoactive intestinal peptide receptor on liver plasma membranes: characterization as a glycoprotein.

The receptor for vasoactive intestinal peptide (VIP) was identified in rat liver plasma membranes after covalent cross-linking to 125I-VIP by three different agents [disuccinimido dithiobis(propionate), disuccinimido suberate, and succinimido 4-azidobenzoate] and examined by sodium dodecyl sulfate-acrylamide electrophoresis. Regardless of the presence of reducing conditions, two molecular species of the putative VIP binding unit were identified as broad autoradiographic bands of 80,000 and 56,000 daltons (Da). Both the large and small species showed the same high affinity for 125I-VIP binding and subsequent cross-linking (half-maximal inhibition at 3 nM unlabeled VIP). The 80-kDa species was partially converted to the 56-kDa form by denaturing conditions and was extensively degraded when incubated at 20 degrees C for 30 min with 1 microgram/mL chymotrypsin, trypsin, or elastase to fragments that that migrated similarly to the 56-kDa unit. In contrast, the 56-kDa moiety was resistant to attack by serine proteases. Both the 80- and 56-kDa species were microheterogeneous due at least in part to the presence of carbohydrate chains, each species binding fractionally to wheat germ agglutinin (WGA)-agarose (approximately 50%). The WGA-bound fraction (eluted with N-acetylglucosamine) was relatively retarded on acrylamide gels as compared to the WGA-unbound fraction. Exposure of the 80- and 56-kDa species to endo-beta-acetylglucosaminidase F reduced the apparent molecular mass of each by 19 kDa, indicating the presence of complex N-linked carbohydrate chains. The receptor species do not appear to have high-mannose N-linked chains since they did not interact with concanavalin A and were not cleaved by endo-beta-acetylglucosaminidase H.(ABSTRACT TRUNCATED AT 250 WORDS)

VIP binding sites on synaptosomes from rat cerebral cortex: Structure-binding relationship

The structural requirements for VIP interaction with receptors on synaptosomes from rat cerebral cortex was investigated by the ability of VIP and VIP fragments, secretin analogues and fragments, peptides of the VIP/secretin family and several other regulatory peptides to inhibit specific 125I-VIP binding. Only large VIP fragments interacted with the VIP receptors with potencies relative to VIP ranging from 0.9–0.006%. The rank order of inhibition was: VIP 7–27 > VIP 11–28 > VIP 1–22-NH 2 > VIP 16–28. Shorter fragments: VIP 18–28; VIP 18–28-NH 2; VIP 19–28; VIP 21–28; VIP 22–28; VIP 1–18; VIP 1–18-NH 2; VIP 1–10-NH 2; VIP 1–6; VIP 16–20 and VIP 16–19 had no effect. Secretin fragments and analogues inhibited 125I-VIP binding with potencies of 2.2–0.01% relative to VIP in the order: secretin > (Ala 4, Val 5) secretin > (D-Ala 4) secretin > (D-Phe 6) secretin > secretin 5–27 > secretin 14–27. Other peptides of the VIP/secretin family inhibited 125I-VIP binding with potencies of 200-1%; avian VIP > porcine VIP > PHI = secretin > human GRF, whereas glucagon and GIP showed no inhibition. Among twenty-five other regulatory peptides only avian PP and somatostatin were inhibitors with relative potencies of 0.02% and 0.03%, respectively. In conclusion it may be emphasized that the intact VIP molecule is essential for VIP interaction with its receptors in the rat brain cortex. The central amino acid sequence in the molecule (residues 11–22), which forms the α-helix, seems to be required for VIP receptor recognition, whereas the N- and C-terminal sequences (residues 1–10 and 23–28) seems to be important for the affinity of VIP receptor binding.

Interaction of synthetic N- and C-terminal fragments of helodermin with rat liver VIP receptors

Synthetic helodermin was more potent than natural helodermin (purified from the venom of Gila monster) in activating adenylate cyclase in rat liver membranes. Two possible reasons for this discrepancy were discussed. Comparing adenylate cyclase activation to the binding of 125I-natural helodermin and 125I-VIP in the presence of six synthetic helodermin fragments (1–27, 7–35, 13–35, 17–35, 18–35 and 22–35), we conclude that the effects of both synthetic and natural helodermin were mediated through an interaction with VIP receptors. The C-terminal (28–35) extension of these peptides favored VIP receptor recognition. Their N-terminal extremity was not necessary for binding and for ensuing adenylate cyclase activation, illustrating again the atypical coupling of VIP receptors with the effector enzymatic system, in rat liver membranes.

Characterization of the vasoactive intestinal peptide (VIP) binding sites: A biochemical and an immunological approach

The initial event of VIP action is its interaction with a specific receptor at the surface of a target cell. The understanding of the fine mechanism of action of VIP requires the characterization and of course the purification of the receptor. The understanding of the mechanism which regulates the number of receptor sites at the cell surface, if it occurs, is also a way to characterize the properties of VIP receptor. In this paper we report a number of data which represent several attempts to characterize VIP receptor in a human colonic adenocarcinoma cell (HT 29 cells). (1) We have characterized a monoclonal antibody which partially inhibits 125I-VIP binding to HT 29 cells. (2) We have specifically cross-linked, on intact HT 29 cells, a major polypeptide of M r-64,000 with 125I-VIP using DTSP or DSS as cross-linking reagents. This polypeptide behaves like a high affinity binding site for VIP. (3) We have demonstrated that VIP is rapidly internalized in HT 29 cells (in less than 10 minutes) and that simultaneously VIP receptors were no more detectable on the cell surface by cross-linking experiments. This suggests that VIP is internalized together with its receptor.

Molecular identification and structural requirement of vasoactive intestinal peptide (VIP) receptors in the human colon adenocarcinoma cell line, HT-29.

The human colon adenocarcinoma cell line HT-29 in culture exhibits a cyclic AMP production system highly sensitive to vasoactive intestinal peptide (VIP), making HT-29 cells a unique cultured cell system for studying the mechanism of VIP action [Laburthe, Rousset, Boissard, Chevalier, Zweibaum & Rosselin (1978) Proc. Natl. Acad. Sci. U.S.A. 75, 2772-2775]. The quantitative characteristics of VIP receptors in HT-29 cells and their structural requirement and molecular size were studied. 125I-labeled VIP bound in a time-dependent manner to HT-29 cell homogenates. At equilibrium (60 min incubation at 30 degrees C), unlabelled VIP in the 0.01-10 nM concentration range competed with 125I-VIP for binding to cell homogenates. Scatchard analysis of binding data gave a straight line, indicating that VIP bound to a single population of sites with a KD of 0.12 +/- 0.02 nM and a capacity of 120 +/- 9 fmol/mg of protein. The structural requirement of these receptors was studied with peptides structurally related to VIP, either natural or synthetic. Several peptides inhibited 125I-VIP binding to HT-29 cell homogenates with the following order of potency, which is typical of the human VIP receptor: VIP (IC50 = 0.1 nM) greater than VIP-(2-28)-peptide (IC50 = 13 nM) greater than human growth hormone releasing factor (IC50 = 56 nM) greater than peptide histidine isoleucine amide (IC50 = 80 nM) greater than secretin (IC50 greater than 10 000 nM). To characterize the molecular component(s) of the VIP receptor in HT-29 cells, 125I-VIP was covalently bound to cell homogenates by using the cross-linker dithiobis(succinimidyl propionate). Sodium dodecyl sulphate/polyacrylamide-gel autoradiographic studies of affinity-labelled cell homogenates revealed two major bands, corresponding to 125I-VIP-protein complexes of Mr 66 000 and 16 000. The labelling of the Mr-66 000 component was specific, since it was abolished by native VIP, whereas that of the Mr-16 000 component was not. Densitometric scanning of autoradiographs indicated that the labelling of the Mr-66 000 complex was inhibited by low VIP concentrations in the 0.1-10 nM range (IC50 = 0.6 nM), but was unaffected by 1 microM-glucagon or octapeptide of cholecystokinin. It was also decreased by VIP-(2-28)-peptide with a potency 1% that of VIP. Assuming that one molecule of 125I-VIP bound per molecule of protein, one protein of Mr 63 000 was identified as a component of the VIP receptor in HT-29 cells.

Covalent cross-linking of vasoactive intestinal peptide (VIP) to its receptor in intact colonic adenocarcinoma cells in culture (HT 29).

[125I]Monoiodinated vasoactive intestinal peptide (125I-VIP) was cross-linked with human colonic adenocarcinoma cells (HT29 cells) grown as a monolayer using dithiobis(succinimidylpropionate) as cross-linking reagent. The cross-linked polypeptides were separated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. A major polypeptide of Mr = 67 000 was characterized and it behaved like a high-affinity binding site for VIP according to the following data. The concentration of native VIP (0.5 nM) giving half-maximum inhibition of 125I-VIP covalent cross-linking with this polypeptide was very similar to that giving half-maximum displacement of 125I-VIP on HT 29 cells (0.6 nM). Glucagon or insulin was unable to inhibit the labelling of the Mr-67 000 component. In our experimental conditions neither specific 125I-VIP binding nor covalent labelling was observed with monolayers of Madin Darby canine kidney epithelial cells (MDCK cells) or African green monkey kidney fibroblasts (Vero cells) while the Mr-67 000 polypeptide was also characterized with human rectal adenocarcinoma cells (HRT 18 cells), known to possess the VIP receptor. Preincubation of HT 29 cells with native VIP at 37 degrees C, before 125I-VIP binding and subsequent cross-linking reaction, decreased the labelling of the Mr-67 000 polypeptide up to 80%. Assuming one molecule of 125I-VIP cross-linked per polypeptide, we have characterized, for the first time, a major polypeptide of Mr = 64 000, which belongs to the high-affinity VIP binding site of an intestinal human cell line.

Preferential binding of vasoactive intestinal peptide to hepatic nonparenchymal cells.

The binding of 125I-vasoactive intestinal peptide (125I-VIP) and 125I-insulin has been examined in highly enriched populations of rat hepatocytes and hepatic nonparenchymal cells. 125I-VIP bound to high-affinity sites (Ka = 1.7 X 10(9) M-1) in nonparenchymal cells. Specific binding in these cells was nearly fivefold greater than in hepatocytes (15.0 +/- 0.6% vs. 3.6 +/- 0.7% radioactivity bound per 4 X 10(5) cells). In contrast, 125I-insulin binding was similar in both cell populations (18.2 +/- 2.4% per 4 X 10(5) cells in hepatocytes vs. 17.1 +/- 1.0% in nonparenchymal cells). Glucagon and insulin had no effect on 125I-VIP binding in nonparenchymal cells. Secretin inhibited 125I-VIP binding but was only about 1% as potent as unlabeled VIP. VIP had no apparent effect on cAMP levels in either cell population, whereas glucagon increased cAMP levels in both cell types. Our findings suggest that VIP binds preferentially to hepatic nonparenchymal cells and that these cells are primarily responsible for the clearance of VIP from the portal circulation.

Cyclic AMP-dependent protein kinase in Molt 4b lymphoblasts: identification by photoaffinity labeling and activation in intact cells by vasoactive intestinal polypeptide (VIP) and peptide histidine isoleucine (PHI).

Molt 4b lymphoblasts have previously been shown to possess a single class of pharmacologically specific, high affinity receptors for vasoactive intestinal polypeptide (VIP). This study further explores the molecular basis for modulation of human lymphocyte function by VIP. Dose-dependent stimulation of adenylate cyclase was observed in Molt lymphoblasts over the range of 0.1 nM to 1 microM VIP. VIP-mediated by guanine nucleotide. Accumulation of intracellular cAMP was observed in the presence of either VIP or the diterpene, forskolin. The effects of these two agonists were synergistic. Two neuropeptides that share sequence homology with VIP were also studied; both peptide histidine isoleucine (PHI) and human pancreatic growth hormone releasing factor (1-44 GHRF) competed for 125I-VIP binding to Molt cells. PHI stimulated intracellular cAMP accumulation and demonstrated synergism with forskolin, whereas GHRF had no effect on cAMP. Photoaffinity labeling of 100,000 X G soluble proteins with 8-N3-[32P]cAMP followed by SDS gel electrophoresis demonstrated the presence of cAMP-dependent protein kinases I and II. Cyclic AMP-dependent protein kinase II predominated in the soluble fraction and was the only isozyme observed in particulate fractions. Protein phosphorylation was studied in Molt 4b cells preincubated with [32P]PO43- followed by addition of media alone, 1 microM peptide, or 10 microM forskolin. Cells were lysed and subjected to two-dimensional electrophoresis. Increased phosphorylation of a specific 41,000 Mr protein was observed after addition of forskolin, VIP, or PHI. A much lower concentration of VIP (1 nM) also caused a significant net increase in phosphorylation, which was of a lower magnitude. In contrast, no net effect on protein phosphorylation was seen with GHRF. These data demonstrate the presence of a functional VIP receptor that is linked to the G protein-adenylate cyclase complex. The demonstration of cAMP-dependent protein kinase and of VIP- and PHI-mediated protein phosphorylation in Molt 4b lymphoblasts provides evidence on a molecular level for neuropeptide modulation of human lymphocyte function.

Role of phospholipids in the binding activity of vasoactive intestinal peptide receptors.

Phospholipase digestion of rat intestinal epithelial cell membranes was performed in order to study the influence of membrane phospholipids on the binding activity of VIP receptors. Phospholipases A2 and C strongly (ED50 congruent to 4 X 10(-2) and 4 X 10(-1) micrograms/ml, respectively) and rapidly reduced 125I-VIP binding to membranes whereas phospholipase D was ineffective. This suggests an important role of both hydrophobic and hydrophilic groups of phospholipids on VIP receptor binding activity.