High-affinity receptors for vasoactive intestinal peptide on human myeloma cells

Cultured human myeloma cells of the U266 line and leukemic T cells of the Jurkat line bound synthetic [125I]Tyr10-vasoactive intestinal peptide1-28 ([125I]VIP1-28) specifically and with an affinity similar to that of neuroendocrine cells. Specific binding reached equilibrium after 2 h at 22 degrees C for both myeloma cells and T cells, attained a maximum of 57 to 71% of total binding, and was reversed in 1.5 to 3 h by an excess of non-radioactive VIP1-28. Analyses of the ligand concentration-dependence of binding of the ligand concentration-dependence of binding of [125I]VIP1-28 revealed a mean Kd of 7.6 nM for a mean of 41,207 receptors per myeloma cell and 5.2 nM for 12,266 receptors per T cell. The relative affinity of binding of mast cell-derived VIP10-28 free acid and synthetic analogues suggested differences in specificity between lymphocyte and neuroendocrine receptors. Distinct sets of receptors thus appear to mediate the effects of VIP on functions of both antibody-producing cells and T cells.     

Molecular characteristics and evidence for internalization of vasoactive-intestinal-peptide (VIP) receptors in the tumoral rat-pancreatic acinar cell line AR 4-2 J

1. Vasoactive intestinal peptide (VIP) receptors were investigated in the tumoral acinar cell line AR 4-2 J derived from rat pancreas [125I]Iodo-VIP binding to cell membranes showed the following IC50 values for unlabeled peptides: VIP, 0.3 nM; peptide His-IleNH2, 2 nM; helodermin, 30 nM; secretin, 100 nM. After incubation with 20 nM dexamethasone, the binding capacity increased twofold but affinities were unchanged. External [125I]iodo-VIP binding to intact cells reached steady state after 5 min at 37 degrees C, while the sequestration-internalization of the [125I]iodo-VIP-receptor complex (tested by cold acid washing) increased progressively, reaching 75% of total binding after 1 h. This phenomenon was blocked at 4 degrees C. Further data with dexamethasone, tunicamycin, cycloheximide, low temperature, and/or phenylarsine oxide, suggested a half-life of 2 days for VIP receptors and the necessity of N-glycosylation for proper translocation. 2. For chemical [125I]iodo-VIP cross-linking bis[2-(succinimidooxycarbonyloxy)ethyl]sulfone gave the best yield when compared with five other bifunctional reagents. In membranes, the main specifically cross-linked peptide had Mr 66,000 under nonreducing conditions, and migrated with lower velocity (-5%) under reducing conditions. Cross-linking was suppressed by VIP, peptide His-IleNH2 and helodermin (competitively) and also by GTP. In intact cells, the Mr of [125I]iodo-VIP-cross-linked peptides depended on the mode of cell solubilization. After direct solubilization, the major cross-linked radioactivity migrated as a smear of Mr 130,000-180,000 but an Mr-66,000 peptide was also detectable. In contrast, the solubilization of cross-linked cells detached by mild trypsinisation gave mainly the Mr-66,000 labeled peptide. This suggests that most VIP receptors in intact, attached cells were in a high-Mr complex and that mild cell treatment was sufficient to disrupt this complex.     

Molecular Characteristics and Peptide Specificity of Vasoactive Intestinal Peptide Receptors from Rat Cerebral Cortex

Vasoactive intestinal peptide (VIP) receptors have been identified in CNS by their chemical specificity and molecular size. Using synaptosomes isolated from rat cerebral cortex, it was shown that central VIP receptors discriminated among natural and synthetic VIP-related peptides, because half-maximal inhibition of [125I]VIP binding to synaptosomes was obtained for 0.6 nM VIP, 9 nM peptide histidine isoleucineamide (PHI), 50 nM VIP 2-28, 70 nM secretin, 100 nM rat growth hormone-releasing factor (GRF), and 350 nM human GRF. Other peptides of the VIP family, such as glucagon and gastric inhibitory polypeptide, did not interact with cortical VIP receptors. The molecular components of VIP receptors in rat cerebral cortex were identified after [125I]VIP cross-linking to synaptosomes using the cross-linker dithiobis(succinimidyl propionate). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of synaptosomal proteins revealed two major [125I]VIP-protein complexes of Mr 49,000 and 18,000. The labeling of the Mr 49,000 component was specific, because it was abolished by native VIP, whereas the labeling of the Mr 18,000 component was not. Natural VIP agonists reduced the labeling of the Mr 49,000 component with the following order of potency: VIP greater than PHI greater than secretin approximately equal to rat GRF. In contrast, glucagon and octapeptide of cholecystokinin were without effect, a result indicating its peptide specificity. Densitometric scanning of autoradiographs showed that the labeling of the Mr 49,000 component was inhibited by low VIP concentrations between 10(-10) and 10(-6) M (IC50 = 0.8 nM), a result indicating the component's high affinity for VIP.(ABSTRACT TRUNCATED AT 250 WORDS)     

D-Phe4]peptide histidine-isoleucinamide ([D-Phe4]PHI), a highly selective vasoactive-intestinal-peptide (VIP) agonist, discriminates VIP-preferring from secretin-preferring receptors in rat pancreatic membranes

The capacity of vasoactive intestinal peptide (VIP), peptide histidine-isoleucinamide (PHI), secretin, and a series of analogs to discriminate between VIP-preferring and secretin-preferring receptors that coexist in rat pancreatic plasma membranes was evaluated by their ability to inhibit [125I]iodo-VIP and [125I]iodo-secretin binding and to activate adenylate cyclase. VIP, the VIP analogs [D-His1]VIP, [D-Ser2]VIP, [D-Asp3]VIP and [D-Ala4]VIP, PHI, [D-Phe4]PHI, and secretin inhibited the binding of both ligands in a concentration range of 10(-11) M to 10(-5) M and with a selectivity factor varying from 18,000 to 0.1. The only exception was [D-Phe4]PHI that inhibited 125I-VIP binding only, with an IC50 of 7 nM, and with no inhibition of 125I-secretin binding at 10 microM. The peptides tested stimulated adenylate cyclase in the same membranes and the slope of the dose-effect curves indicated that all peptides, except [D-Phe4]PHI, interacted with at least two classes of receptors: VIP-preferring and secretin-preferring receptors. By contrast, the dose-effect curve of [D-Phe4]PHI activation of adenylate cyclase was monophasic and competitively modified by [D-Phe2]VIP (a VIP antagonist) but not by secretin(7-27) (a secretin antagonist), indicating an interaction with VIP-preferring receptors only. Thus, [D-Phe4]PHI appears to be a highly selective tool to characterize these receptors.     

Demonstration of [125I]VIP binding sites and effects of VIP on cAMP-formation in rat insulinoma (RINm5F and RIN14B) cells.

Vasoactive intestinal polypeptide (VIP)-immunoreactive nerves have been demonstrated in close association with the islets of Langerhans, and VIP has been shown to stimulate insulin and somatostatin secretion. Using [125I]VIP and membranes prepared from rat insulinoma (RIN) cells, i.e., the subclones m5F (m5F; mainly insulin-secreting) and 14B (14B; mainly somatostatin-secreting), it was found that VIP (10(-10)-10(-7) M) competitively inhibited the binding of [125I]VIP. A single class of high affinity binding sites with Kd values of 0.40 +/- 0.06 nM and 0.36 +/- 0.08 nM for m5F and 14B, respectively, with a corresponding number of binding sites (Bmax) of 163 +/- 20 and 254 +/- 51 fmol/mg protein was observed. The rank order of potency in inhibiting [125I]VIP binding was in both cell lines: VIP greater than helodermin greater than pituitary adenylate cyclase activating polypeptide 1-27 (PACAP27) greater than peptide histidine isoleucine (PHI) greater than secretin. VIP caused a dose-dependent increase in cAMP-formation in both m5F and 14B cell membranes with EC50 values of 3.0 and 3.5 nM, respectively, but VIP (1.10(-9)-3.10(-6) M) had no effect on insulin secretion (over 2 h) from the m5F cells. Thus, the data suggest that the VIP-receptors in these neoplastic rat cell lines, despite an apparent coupling to adenylate cyclase activity, seem to be functionally uncoupled to an effect on insulin secretion following an acute exposure to VIP.     

Direct demonstration of guanine nucleotide sensitive receptors for vasoactive intestinal peptide in the anterior lobe of the rat pituitary gland

The vasoactive intestinal peptide (VIP) receptors were identified on the membranes from the rat anterior pituitary gland with [125I]VIP. The dissociation constant (Kd) and the maximal binding capacity (Bmax) values were estimated from the competitive inhibition data. The Kd and Bmax values were 1.05 +/- 0.75 nM and 103 +/- 11 fmol/mg protein, respectively. The order of molar potency of related peptides to inhibit [125I]VIP binding was VIP greater than peptide histidine isoleucine (PHI) greater than secretin greater than glucagon. Glucagon was not effective to inhibit the binding. [125I]VIP binding was effectively inhibited by the addition of guanine nucleotides. The order of molar potency to inhibit the binding was Gpp(NH)p greater than GTP greater than GDP greater than GMP greater than ATP. These results directly suggest the coupling of VIP receptors with guanine nucleotide binding proteins in the anterior pituitary gland.     

Presence of highly selective receptors for PACAP (pituitary adenylate cyclase activating peptide) in membranes from the rat pancreatic acinar cell line AR 4-2J

We characterized highly selective receptors for PACAP, the pituitary adenylate cyclase activating peptide, in the tumoral acinar cell line AR 4-2J derived from the rat pancreas. PACAP, a novel hypothalamic peptide related to vasoactive intestinal peptide (VIP), was tested as the full natural 38-residue peptide (PACAP-38) and as an N-terminal amidated 27-residue derivative (PACAP-27). The binding sites showed considerable affinity for [125I]PACAP-27 (Kd = 0.4 nM) and PACAP-38, while their affinity for VIP and the parent peptide helodermin was 1000-fold lower. These receptors were coupled to adenylate cyclase, the potency of PACAP-38 and PACAP-27 (Kact = 0.2 nM) being much higher than that of VIP (Kact = 100 nM) and helodermin (Kact = 30 nM). Chemical cross-linking of [125I]PACAP-27 followed by SDS-PAGE and autoradiography revealed a specifically cross-linked peptide with an Mr of 68,000 (including 3000 for one PACAP-27 molecule).     

Autoantibody to vasoactive intestinal peptide in human circulation

In a radioassay for Vasoactive Intestinal Peptide (VIP)-binding, eight out of 33 plasma samples from healthy human subjects exhibited specific binding ranging from 2.6% to 46.7% of total [125 I]VIP. This binding was competitively displaced by unlabeled VIP. The structurally homologous peptides, Peptide Histidine Isoleucine (PHI) and secretin, were, respectively, 72-fold and 413-fold less potent than VIP in displacing bound [125 I]VIP, whereas the unrelated peptides, neurotensin, eledoisin, bombesin and metenkephalin, were without effect on the binding. The antibody nature of the VIP-binding factor was suggested by its precipitation with ammonium sulfate, attenuation after absorption with Staphylococcus aureus preparations, precipitation with antisera against human IgG and IgM, and coelution with standard IgG and IgM on anion-exchange and high-performance gel-filtration columns. Pepsin treatment of purified IgG fraction yielded a VIP-binding species with apparent molecular weight of 108 +/- 13 kDa that was precipitated by antiserum against the F(ab)2 fragment of the IgG molecule. These results demonstrate the existence in some human plasmas of an autoantibody that binds VIP.     

A human melanoma-derived cell line (IGR39) with a very high number of vasoactive-intestinal-peptide (VIP) receptors. 1. Molecular characterization of the binding site

Using mono[125I]iodinated vasoactive intestinal peptide (125I-VIP), a very high number of specific binding sites for VIP were identified at the surface of the human melanoma cell line IGR39. The Scatchard analysis of competitive displacement experiments between native VIP and 125I-VIP was consistent with the existence of two classes of VIP-binding sites. IGR39 cells possess 0.54 x 10(6) high-affinity sites with a dissociation constant (Kd) of 0.66 nM and 1.3 x 10(6) sites of moderate affinity with a Kd of 4.7 nM. Pharmacological studies indicated that the order of potency in inhibiting 125I-VIP binding of the VIP/secretin family peptides was VIP much greater than peptide histidine methioninamide greater than human growth-hormone-releasing factor(1-44) greater than secretin. Glucagon has no effect on the binding of the labelled peptide. By means of photoaffinity labelling a polypeptide of Mr 63,000 was characterized. The labelling of this species was completely abolished by native VIP. The order of potency of VIP-related peptides in inhibiting 125I-VIP cross-linking to its receptor was the same as in the competition experiments. The glycoprotein nature of the VIP-binding sites of IGR39 cells has been investigated by affinity chromatography on wheat-germ-agglutinin-Sepharose.     

Interaction of vasoactive intestinal peptide (VIP) with rat lymphoid cells

Receptors for vasoactive intestinal peptide (VIP) have been characterized in rat lymphoid cells. The interaction of [125I] VIP with blood mononuclear cells was rapid, reversible, specific and saturable. At apparent equilibrium, the binding of [125I] VIP was competitively inhibited by native VIP in the 0.01-100 nM range concentration. The binding data were compatible with the existence of two classes of receptors: a high-affinity class with a Kd = 0.050 +/- 0.009 nM and a low binding capacity (2.60 +/- 0.28 fmol/10(6) cells), and a low-affinity class with a Kd = 142 +/- 80 nM and a high binding capacity (1966 +/- 330 fmol/10(6) cells). Secretin, glucagon, insulin and somatostatin did not show any effect at a concentration as high as 100 nM. With spleen lymphoid cells, stoichiometric studies were performed. The binding data were compatible with the existence of two classes of receptors: a high-affinity class with a Kd = 0.100 +/- 0.033 nM and a low binding capacity (4.60 +/- 1.07 fmol/10(6) cells), and low-affinity class with a Kd = 255 +/- 110 nM and high binding capacity (2915 +/- 1160 fmol/10(6) cells). With thymocytes, no binding was obtained under different conditions.     

Characterization of Functional Receptors for Vasoactive Intestinal Peptide in Bovine Cerebral Arteries

This study reports the characterization of receptors for vasoactive intestinal peptide (VIP) on membranes prepared from bovine cerebral arteries. By use of HPLC we prepared two purified monoiodinated VIP radioligands with nearly equivalent cerebral vasorelaxant potency as native VIP, [Tyr(125I)10 )VIP and [Tyr(125I)22]VIP. The former resulted in a higher proportion of specific binding to arterial membranes than the latter and was therefore thought to be the superior radioligand for receptor characterization. The binding of [Tyr(125I)10]VIP to cerebral arterial membranes was saturable, specific, reversible, and dependent on time and temperature. Scatchard analysis suggested the presence of a high- and a low-affinity binding site with KD values of 0.2 and 11 nM and receptor concentrations of 79 and 737 fmol/mg of protein, respectively. The dose-response curves for binding to the VIP receptor by the VIP-homologous peptides PHI, PHM, and rat growth hormone-releasing factor (GRF) were very similar to their dose-response curves for relaxation of cerebral arteries. The order of potency was VIP greater than PHM greater than PHI greater than rat GRF. It is suggested that the characteristics of the vascular VIP binding sites and the close correlation between the binding and vasorelaxant properties of VIP and its related peptides argue for the vascular binding sites being functional receptors for VIP.     

Evidence for vasoactive intestinal peptide receptors in apical membranes from tracheal epithelium

[125I]VIP (vasoactive intestinal peptide) bound to apical membranes isolated from the bovine tracheal epithelium with a half maximal inhibition by unlabeled VIP (IC50) of 0.6 x 10(-9)M and binding was reversible. Glucagon did not affect [125I]VIP binding to the membranes. [125I]VIP was covalently cross-linked to tracheal membrane proteins using disuccinimidyl suberate. SDS-polyacrylamide gel electrophoresis of labeled tracheal membranes revealed one major [125I]-receptor complex of Mr = 71,000 to which binding of [125I]VIP was inhibited by 10 microM unlabeled VIP. These results are consistent with the presence of a specific, high-affinity receptor for VIP, with a Mr = 71,000, in apical membrane vesicles isolated from the bovine tracheal epithelium.     

Influence of synthetic peptide corresponding to the ACTH-like sequence of human immunoglobulin G1 on proliferation of lymphoblastoid cells

Influence of the ACTH-like peptide H-Val-Lys-Lys-Pro-Gly-Ser-Ser-Val-Lys-Val-OH corresponding to the sequence 11-20 of the variable part of human immunoglobulin G1 (IgG1) heavy chain on growth of MT-4 human T-lymphoblastoid cell line was investigated. It was found that the ACTH-like peptide at concentration range 10(-11) -10(-7) M inhibits the proliferation of MT-4 cells. Labeled ACTH 'address segment' [(125)I]ACTH (13-24) was used to establish that MT-4 cells express specific receptors for ACTH (K(d) = 97 pM). The ACTH-like peptide and human ACTH (but not IgG1 heavy chain) were shown to compete with [(125)I]ACTH (13-24) for binding to these receptors (K(i1) = 0.38 nM and K(i2) = 0.34 nM).     

The novel VIP-like hypothalamic polypeptide PACAP interacts with high affinity receptors in the human neuroblastoma cell line NB-OK

We investigated the ability of two forms of Pituitary Adenylate Cyclase Activating Polypeptide [PACAP-38, the 38 amino acid peptide isolated from ovine hypothalamus, and PACAP-27, a shorter N-terminal (1-27) amidated version] to interact with specific receptors in membranes from the human neuroblastoma cell line NB-OK. [125I]PACAP-27 bound rapidly and specifically to one class of high affinity sites (Kd 0.5 nM). VIP inhibited [125I]PACAP-27 binding 300- to 1000-fold less potently than PACAP-27 and PACAP-38. One microM PHI prevented tracer binding only partially and secretin, glucagon and GRF(1-29)NH2 were ineffective in this respect. PACAP-27 and PACAP-38 stimulated adenylate cyclase activity dose dependently and with similar efficacy (Kact 0.2-0.3 nM), this activation being compatible with the occupancy of specific high affinity PACAP receptor. VIP was markedly less potent and less efficient on this enzyme than PACAP. Chemical cross-linking of [125I]PACAP-27 followed by SDS-PAGE and autoradiography revealed specific cross-linking with a 68 kDa protein.     

HT 29, a model cell line: stimulation by the vasoactive intestinal peptide (VIP); VIP receptor structure and metabolism

HT 29, a cell line derived from a human colonic adenocarcinoma, is highly responsive to the vasoactive intestinal peptide (VIP) as shown by a more than 100-fold intracellular cAMP increase (Ka = 0.3 nM), the stimulations of protein kinase A (Ka = 0.1 nM) and the low-Km cAMP phosphodiesterase (Ka = 40 nM). Remarkably, adenylate cyclase, cAMP-dependent kinase and cAMP-specific phosphodiesterase are activated in a sequential manner. Binding studies with [125I]-labeled VIP indicate a high affinity site with a Kd value (0.5 nM) close to the activation constant value (Ka) of the three enzymes. The molecular structure of the VIP receptor was studied by immunological and chemical approaches. A monoclonal antibody (mAb 109-10-16) which partially decreased the binding of VIP to its receptor allowed the characterization of Mr = 53,000 and Mr = 48-49,000 polypeptides. More precise identification of protein components of the VIP receptor resulted from covalent cross-linking on intact HT 29 cells by four bifunctional reagents: dithiobis-(succinimidyl propionate) and its non-cleavable analog disuccinimidyl suberate, the photoactivable azido phenyl glyoxal and dimethylpimelimidate. Analysis by SDS-polyacrylamide gel electrophoresis demonstrated a major band of Mr = 67,000 regardless of which cross-linker was used. The same band and an Mr = 49,000 species were found in experiments using a crude membrane fraction of HT 29 cells. Assuming one molecule of VIP (Mr = 3326) linked per polypeptide, these observations suggest that an Mr = 64,000 species belongs to the VIP specific plasma membrane receptor. This protein contains an Mr = 20,000 N-linked sialic acid rich oligosaccharidic moiety.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identity of a membrane-bound vasoactive intestinal peptide-binding protein with calmodulin.

A vasoactive intestinal peptide (VIP)-binding protein purified from guinea pig lung membranes (p18) was digested with trypsin, and the amino acid sequence of the peptide fragments was determined. The sequence of six tryptic fragments of p18 was identical with subsequences present in mammalian calmodulin. Authentic porcine brain calmodulin and p18 co-migrated on an sodium dodecyl sulfate-electrophoresis gel and displayed identical chromatographic behavior on a reverse phase high performance liquid chromatography column. The VIP-binding properties of p18 and calmodulin were indistinguishable. Both proteins displayed saturable and apparent high affinity binding of VIP, evidenced by potent inhibition of complexation with [Tyr10-125I]VIP by unlabeled VIP (IC50 = 6.0-8.1 nM). Rat growth hormone releasing factor and a C terminally extended form of VIP ([Leu17]VIP-GKR) also displayed potent inhibition of the binding (IC50 = 6.4 and 4 nM, respectively). These neuropeptides are potential modulators of calmodulin function.     

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.     

A new type of functional VIP receptor has an affinity for helodermin in human SUP-T1 lymphoblasts

A new type of VIP receptor was characterized in human SUP-T1 lymphoblasts. The order of potency of unlabeled peptides, in the presence of [125I]helodermin, was: helodermin(1-35)-NH2 = helodermin(1-27)-NH2 greater than helospectin greater than VIP = PHI greater than [D-Ser2]VIP greater than [D-Asp3]VIP greater than [D-His1]VIP greater than or equal to [D-Ala4]VIP greater than or equal to secretin = GRF. This specificity was distinct from that of all VIP receptors described so far in that: (i) the affinity for helodermin (Kd = 3 nM) was higher than that of VIP (Kd = 15 nM) and PHI (Kd = 20 nM); and (ii) position 4 played an important role in ligand binding. The labeled sites were likely to be functional receptors as adenylate cyclase in crude lymphoblastic membranes (200-10,000 x g pellets) was stimulated by peptides, in the presence of GTP, with the following order of potency: helodermin(1-35)-NH2 greater than helodermin(1-27)-NH2 greater than helospectin = VIP = PHI.     

A human melanoma-derived cell line (IGR39) with a very high number of vasoactive-intestinal-peptide (VIP) receptors. 2. Effect of VIP on cAMP production and on cell-surface VIP-binding sites

Vasoactive intestinal peptide (VIP) stimulated in a dose-dependent manner the accumulation of cAMP in human melanoma-derived cell line IGR39. The maximal effect (about 100 times the basal level) was observed with 10 nM VIP. Half-maximum cAMP production was obtained at 0.78 nM VIP. VIP-related peptides were also potent in stimulating the cAMP production in IGR39 cells. The order of potency was VIP much greater than peptide histidine-methioninamide greater than human growth-hormone-releasing factor(1-44) greater than secretin greater than glucagon. Using the same conditions, IGR37 cells, a metastasic counterpart of IGR39 cells, displayed a weak stimulation of cAMP production. After exposure of IGR39 cells to 10 nM VIP, the cAMP response to a new stimulation by VIP was strongly reduced. This desensitization of IGR39 cells to VIP was rapid (t1/2 less than 2 min) and homologous. Preincubation of IGR39 cells in the presence of native VIP induced disappearance of the VIP-binding sites at the cell surface. This phenomenon was dependent on time and VIP concentration. Maximum effect (loss of 80% of binding capacity) was obtained after exposure of the cells at 37 degrees C with a VIP concentration of 1 microM. The t1/2 of maximum disappearance was less than 2 min and the concentration of VIP giving half-maximum decrease in binding of mono[125I]iodinated VIP (125I-VIP) was 8 nM. This phenomenon was also reversible since 85% of the VIP-binding capacity could be restored in less than 1 h by incubating IGR39 cells in a VIP-free medium. The IGR39 cell line should be a useful model for further study of the structure and function of the human VIP receptor.     

mono[125I]iodo-Tyr10,MetO17]-vasoactive intestinal polypeptide. Preparation, characterization, and use for radioimmunoassay and receptor binding

Vasoactive intestinal polypeptide (VIP) was labeled with sodium [125I]iodide using the chloramine-T method and subsequently purified by reverse-phase high performance liquid chromatography.Three main 125I-labeled peaks designated A, B, and C resulted from the radioiodination and purification procedures. They were characterized by electrophoresis of tryptic fragments; Edman degradation (for Peaks A and C); enzymatic digestion to amino acids by leucine aminopeptidase, carboxypeptidase Y and Pronase; and treatment with cyanogen bromide. Peak A corresponds to VIP monoiodinated on Tyr10 and with the Met17 residue oxidized to methionine sulfoxide. This [mono[125I]iodo-Tyr10,MetO17]VIP displays the following characteristics. 1) It constitutes quantitatively the major product of the iodination procedure (62.5%); 2) it is well resolved from other labeled and unlabeled products; 3) it is stable (2 months at -20 degrees C); 4) it possesses a high specific activity (2050 Ci/mmol); 5) it maintains the biological activity of native VIP; and 6) it binds to antibody and membrane recognition sites in a specific, saturable, and reversible manner. Reduction of [mono[125I]iodo-Tyr10, Met-O17]VIP to [mono[125I]iodo-Tyr10]VIP does not improve the performance of the tracer in a radioimmunoassay. The method described in this article is simple and rapid and yields a molecular form of 125I-labeled VIP that has been fully characterized and is suitable for use in biological studies.