Unlabeled Vasoactive Intestinal Peptide Research Articles

Passage of vasoactive intestinal peptide across the blood–brain barrier

We investigated the ability of vasoactive intestinal peptide (VIP) to cross the blood–brain barrier (BBB), the interface between the peripheral circulation and central nervous system (CNS). VIP labeled with 131 I (I-VIP) and injected intravenously into mice was taken up by brain as determined by multiple-time regression analysis. Excess unlabeled VIP was unable to impede the entry of I-VIP, indicating that passage is by nonsaturable transmembrane diffusion. High pressure liquid chromatography (HPLC) showed the radioactivity entering the brain to be intact I-VIP. After intracerebroventricular (i.c.v.) injection, I-VIP was sequestered by brain, slowing its efflux from the CNS. In summary, VIP crosses the BBB unidirectionally from blood to brain by transmembrane diffusion.

Detection of VIP receptors in MNU-induced breast cancer in rats: implications for breast cancer targeting.

Vasoactive intestinal peptide (VIP) is a 28 amino acid neuropeptide with a wide range of biological activities. Receptors for VIP (VIP-R) are overexpressed in breast cancer, where they may have diagnostic and therapeutic implications. Although N-methyl nitrosourea (MNU)-induced breast cancer in rats is used extensively as a model to study mammary carcinogenesis, there is no information about the expression of VIP-R in this model. Therefore, the purpose of this study was to investigate the presence of VIP-R in MNU-induced breast cancer in rats so that this model can be used to perform studies involving VIP-R. Breast cancer was induced in 36-day-old virgin female Sprague-Dawley rats, by a single intravenous injection of MNU (50 mg/kg body weight). The breast tumors were detected 100-150 days after injection. The normal and cancerous rat breast tissue were excised and 20 micro sections were incubated with 40 nM fluorescein-labeled VIP (Fluo-VIP(TM)), in the presence and absence of 1000-fold excess unlabeled VIP, pituitary adenylate cyclase activating polypeptide (PACAP) or secretin. The sections were visualized under a fluorescence microscope and photographed. Fluo-VIP(TM) stained rat breast cancer tissue homogeneously and to a much greater extent than normal rat breast tissue (p < 0.05). This staining was specific as indicated by displacement of Fluo-VIP(TM) by excess unlabeled VIP and PACAP. Displacement of Fluo-VIP(TM) by secretin indicated the probable presence of VIP receptors of type VPAC1 (VIP receptor subtype 1) in the rat breast. These data suggest that, as in human breast cancer, VIP-R, predominantly of type VPAC1, are overexpressed in MNU-induced rat breast cancer tissue as compared to the normal rat breast tissue. Thus, MNU-induced rat breast cancer model can be used as a tool to study the functional role of VIP-R in human mammary carcinogenesis and VIP-R mediated active breast cancer targeting. This could have implications in the diagnosis, prognosis and therapy of human breast cancer.

Interaction between atrial natriuretic peptide and vasoactive intestinal peptide in guinea pig cecal smooth muscle

Backgrounds & Aims : The role of atrial natriuretic peptide (ANP) in gastrointestinal motility is still unclear. The aim of this study was to investigate the relationship between ANP and vasoactive intestinal peptide (VIP) in guinea pig cecal circular smooth muscle cells. Methods : The inhibition of 125I-ANP binding or 125I-VIP binding to cecal smooth muscle cells was assessed using unlabeled peptides (i.e., ANP, ANP fragments, VIP, secretin, and peptide histidine isoleucine); the effect of ANP, ANP fragments, and VIP on muscle contraction stimulated by 1 μmol/L carbachol was assessed; and the inhibitory effects of ANP 1–11 on VIP-induced relaxation, ANP 1–11 and VIP 10–28 (a VIP antagonist) on ANP-induced relaxation, and nitric oxide production inhibitors on ANP-induced relaxation were assessed. Results : The specific binding of 125I-ANP was inhibited completely by unlabeled ANP and VIP in a dose-dependent manner but only slightly inhibited by secretin and peptide histidine isoleucine. ANP 1–11 and C-atrial natriuretic factor inhibited the binding of 125I-ANP with a lower affinity than ANP. ANP only partly inhibited 125I-VIP binding. ANP and VIP inhibited 1 μmol/L carbachol-induced contraction in a dose-dependent manner. ANP 1–11 significantly inhibited VIP-induced relaxation. ANP 1–11, VIP 10–28, and NO production inhibitors completely inhibited ANP-induced relaxation. Conclusions : The results of the study showed that ANP 1–11 antagonized ANP-induced relaxation and that ANP stimulated NO production and subsequently induced relaxation via a receptor to which VIP binds.

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.

Effects of vasoactive intestinal peptide on adenosine 3',5'-monophosphate, ornithine decarboxylase, and cell growth in a human colon cell line.

Vasoactive intestinal peptide (VIP) is a widely distributed neuropeptide that has been considered a potential regulator of cell growth and differentiation in various tissues, including the gut. To examine this idea, we used a human colon carcinoma cell line (LoVo) as a model system and measured ornithine decarboxylase (ODC), because this is the rate-limiting enzyme for the formation of polyamines, which are thought to be key factors in regulating cell growth. LoVo cells, grown to about 80% confluence in F-12 medium containing 10% fetal bovine serum, were preincubated for 5 h in low serum medium (1% fetal bovine serum in F-12), and ODC activity was determined by measuring 14CO2 liberated from 14C-labeled ornithine. VIP caused a dose-related biphasic change in ODC, with activity increased at 10 pM, maximal (5-fold increase) at 10 nM, and decreased toward basal at 100 nM to 1 microM. Incubation of cells for 6 days with VIP in low serum medium showed similar changes in cell numbers, with growth being increased by doses in the 1 pM to 100 nM range and decreased at higher doses (greater than or equal to 100 nM). Exposure of cells to 5 mM alpha-difluoromethylornithine blocked both the VIP-induced increase in cell number and the VIP-induced increase in ODC activity. Increased ODC mRNA was detected after 2 h of exposure to VIP, a time at which ODC activity peaked after treatment, and the increase in ODC mRNA caused by VIP was dose-dependent. In related experiments LoVo cells were found to have high affinity VIP receptors (Kd = 0.4 nM), as assessed by examination of [125I]VIP binding in the presence of varying concentrations of unlabeled VIP. Studies of intracellular cAMP revealed a dose-related increase in cAMP in response to VIP (ED50 = 11 pM), and the adenylate cyclase activator forskolin increased both ODC activity and ODC mRNA. The findings support the idea that LoVo cells have VIP receptors linked to cAMP which can stimulate cell growth at least in part by increasing ODC synthesis and activity, thereby altering the production of polyamines. The decreased growth and ODC activity observed with high doses of VIP may involve a second messenger other than cAMP.

Vasoactive intestinal peptide (VIP) receptors positively coupled with adenylate cyclase activity in the human placenta: Vascular localization

The presence of Vasoactive Intestinal Peptide (VIP) has been reported in human placenta, a non-innervated organ. However, the exact origin of this neuropeptide, the precise localization of its binding sites, and the mechanism(s) of action remain tobe clarified. The 125 I-VIP binding to placental slices was saturable, and unlabeled VIP was able to compete in a dose-dependent manner with an IC 50 value of 5.2±1.3×10 −10 M. Light microscopic autoradiography on placental slices showed the association of high densities of labeling with blood vessel pathways, especially arteries of stem villi. When sections were treated by high resolution autoradiography, all cellular components contained silver grains, although a much higher density of labeling was associated with blood vessels, especially with endothelial cells. Consequently, adenylate cyclase activity was studied on placental slices and on isolated membranes of stem villi vessels. On placental slices, VIP produced a stimulatory effect of cAMP synthesis at a concentration as low as 10 −10 ×M. The dose response curve was monophasic with an ED 50 value of 2.9±1.6×10 −9 M. The specificity of the VIP effect was tested with peptides structurally related to VIP such as glucagon, secretin, gastric inhibitory polypeptide, and human growth hormone releasing factor. Only secretin in high concentrations (>10 −6 M) increased cAMP production. Leu-enkephalin or insulin were ineffective. Furthermore, the VIP stimulated, specifically and in a dose dependent manner, the adenylate cyclase activity of vascular membranes. These results confirm the presence of VIP receptors positively coupled with adenylate cyclase on the fetal vascularization and suggest a functional role for the peptide in placental hemodynamics.

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.

Inhibition of Mitogen-Stimulated Proliferation of Murine Splenocytes by a Novel Neuropeptide, Pituitary Adenylate Cyclase Activating Polypeptide: A Comparative Study with Vasoactive Intestinal Peptide*

Two novel polypeptides known as pituitary adenylate cyclase activating polypeptide with 38 residues (PACAP38) and a shorter form of the peptide corresponding to the N-terminal 27 residues (PACAP27) were isolated from ovine hypothalamus. The N-terminal 28 residues of PACAP show 68% homology with vasoactive intestinal peptide (VIP). VIP has been reported to have specific binding sites in lymphocytes and inhibit mitogen-stimulated lymphocyte proliferation through a receptor-mediated stimulation of cAMP-dependent protein kinase. Using concanavalin A-induced proliferation of murine splenocytes as a model system, we now report that both PACAP38 and PACAP 27 can inhibit the proliferation of these cells in the same dose-dependent manner as VIP. The minimal effective concentration of the PACAPs was 10(-10)-10(-9) M. However, neither PACAP affected lipopolysaccharide-induced proliferation of murine splenocytes. The binding of [125I]PACAP27 to these splenocytes was rapid, time dependent, reversible, and proportional to the numbers of murine splenocytes. Scatchard analysis of displacement of the bound tracer by unlabeled PACAP27 indicated the existence of two classes of binding sites. The dissociation constant (Kd) was 0.86 +/- 0.24 nM and the maximal binding capacity (Bmax) was 1.13 +/- 0.39 fmol/10(6) cells for the high affinity binding site. The low affinity binding site had a Kd of 0.13 +/- 0.03 microM with a Bmax of 73.5 +/- 9.5 fmol/10(6) cells. PACAP38 and VIP displaced the binding of [125I]PACAP27 in the same manner as PACAP27 and Scatchard analyses indicated the presence of two classes of binding sites with Kd and Bmax similar to those for PACAP27. Furthermore, when [125I]VIP was used as a radiolabeled ligand, PACAP27 and PACAP38 displaced the [125I]VIP binding to the same degree as unlabeled VIP. Scatchard analysis indicated that there was no significant difference of the Kd or Bmax between PACAP and VIP. Taken together, these data suggest that PACAPs bind to a site similar or identical to that used by VIP which inhibit the proliferation of murine splenocytes induced by concanavalin A.

Molecular identification of receptor for pituitary adenylate cyclase activating polypeptide

Pituitary adenylate cyclase activating polypeptide (PACAP) is a novel hypothalamic peptide with 38 (PACAP38) or 27 (PACAP27) amino acid residues, structurally related to vasoactive intestinal peptide (VIP). Bovine brain membrane has a PACAP specific receptor interacting with both PACAP27 and PACAP38. Affinity-labeling of the receptor with [ 125I]PACAP27 identified a dominant band of Mr = 60 k. The labeling density of the 60 k band decreased in the presence of unlabeled PACAP27 or PACAP38, whereas the 60 k band remained in the presence of unlabeled VIP. Binding of [ 125I]PACAP27 to the membrane decreased in the presence of GTP and the labeling density of the 60 k band decreased concomitantly. The results indicate that bovine brain has a specific PACAP receptor, whose apparent molecular weight is 57 k (substracting the molecular weight of [ 125I]PACAP27 from 60k).

Purification of vasoactive intestinal peptide receptor from porcine liver by a newly designed one-step affinity chromatography.

Vasoactive intestinal peptide (VIP) receptors were solubilized from porcine liver membrane using the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid. The solubilized VIP receptor has been purified approximately 50,000-fold to apparent homogeneity by a one-step affinity chromatography using a newly designed VIP-polyacrylamide resin. The purified receptor bound 125I-VIP with a Kd of 22.3 +/- 0.7 nM and retained its peptide specificity toward VIP-related peptides. The specific activity of the purified receptor (16,400 pmol/mg of protein) was very close to the theoretical value (18,900 pmol/mg of protein) calculated assuming one binding site/protein. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of purified receptor revealed a single band with an Mr of 53,000 after either silver staining or radioiodination. Affinity labeling of the purified receptor with 125I-VIP using dithiobis(succinimidyl propionate) gave a single radioactive band, the labeling of which was completely inhibited by an excess of unlabeled VIP. In conclusion, an Mr 53,000 protein containing the VIP-binding site was purified to homogeneity by a one-step affinity chromatography using immobilized VIP.

Solubilization and hydrodynamic characterization of guanine nucleotide-sensitive vasoactive intestinal peptide-receptor complexes from rat intestine

The purpose of this work was to solubilize vasoactive intestinal peptide (VIP) receptors from rat small intestinal plasma membranes and to analyze the nature and function of its molecular form(s) in a nondenaturing environment. Membranes were incubated with 3 nM 125I-VIP, washed, and treated with 1% Triton X-100. Chromatography on Sephadex G-50 showed that 60% of the extractable radioactivity was eluted with macromolecular components in the void volume. This radioactive material was dramatically reduced when 1 microM unlabeled VIP was present in the incubation medium or when membranes were pretreated with trypsin or dithiothreitol. Macromolecular components that had bound 125I-VIP were further chromatographed on Sephacryl S-300. Two peaks were observed: a major one (80%) and a minor one (20%) with Stokes radii of 5.2 and 3.1 nm, respectively. The labeling of both components was inhibited by unlabeled VIP or peptide with NH2-terminal histidine and COOH-terminal isoleucine amide (a VIP agonist). The presence of GTP (0.1 mM) in the incubation medium of membranes completely abolished the labeling of the 5.2-nm component but did not affect that of the 3.1-nm one. Moreover, GTP induced dissociation of 125I-VIP from the 5.2-nm component isolated by Sephacryl S-300 chromatography. This effect was time dependent and nucleotide specific. In contrast, GTP did not affect the stability of the 3.1-nm component. After cholera toxin catalyzed [32P]ADP-ribosylation of membranes, chromatography of solubilized material on Sephacryl S-300 showed that a peak of 32P radioactivity was coeluted with the 5.2-nm component.(ABSTRACT TRUNCATED AT 250 WORDS)

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 (IC 50) of 0.6 × 10 −9M 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 μM 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.

Autoradiographic localization of vasoactive intestinal peptide (VIP) binding sites in the human term placenta. Relationship with activation of adenylate cyclase

The presence of vasoactive intestinal peptide (VIP) binding sites and the adenylate cyclase activity in response to VIP were examined in the human term placenta. Slices were used in order to preserve the physicochemical environment and the structural integrity of this heterogeneous organ. 125I-VIP binding to placental slices was saturable. The steady state was reached after 90 min at 37°C and was maintained up to 3 h. Unlabeled VIP was able to compete in a dose-dependent manner with an IC 50 value of 5.2 ± 1.3 × 10 −10 M. Autoradiography and histological analysis showed that VIP binding sites were essentially located on fetal vascularization, especially arteries of stem villi. VIP produced a stimulatory effect on cAMP synthesis at a concentration as low as 10 −10 M. The dose-response curve was monophasic with an ED 50 value of 2.9 ± 1.6 × 10 −9 M. The specificity of the VIP effect was tested with peptides structurally related to VIP such as glucagon, secretin, gastric inhibitory polypeptide and human growth-hormone releasing factor. Only secretin at high concentrations (> 10 −6 M) increased cAMP production. Leu-enkephalin or insulin were ineffective. The presence of both VIP binding sites on fetal vascularization and VIP-induced adenylate cyclase activation would seem to suggest a regulatory role of the peptide on fetoplacental blood flow.

Autoradiographic mapping of [mono[ 125I]iodo-Tyr 10, MetO 17]vasoactive intestinal peptide binding sites in the rat brain

The distribution of vasoactive intestinal peptide binding sites in the rat brain was examined by in vitro autoradiography on slide-mounted sections. A fully characterized monoiodinated form of vasoactive intestinal peptide (M-[ 125I]VIP) previously shown 47 to maintain in the central nervous system the full biological activity of native vasoactive intestinal peptide was used for this study. In initial kinetic and pharmacological experiments the binding of M-[ 125I]vasoactive intestinal peptide to slide-mounted sections was shown to be time-dependent, saturable and reversible. Association of M-[ 125I]VIP specific binding was maximal within 90–120 minutes. Specific binding, corresponding to approximately 50% of total binding was saturable, of high affinity ( K d of 76.6 pM) and low capacity (fmol/mg prot range). Dissociation of M-[ 125I]VIP was maximal at 10 minutes. Unlabeled vasoactive intestinal peptide and the two structurally related peptides “peptide-histidine-isoleucine” (PHI) and secretin competed in a concentration-dependent manner for sites labeled by M-[ 125I]vasoactive intestinal peptide with the following rank order of potencies: vasoactive intestinal peptide > PHI > secretin. Vasoactive intestinal peptide receptors, as revealed by quantitative autoradiography, are present at various levels of the neuraxis. High densities were observed in olfactory bulb, cerebral cortex (highest in layers I, II, IV and VI), dentate gyrus, subiculum, various thalamic and hypothalamic nuclei, superior colliculus, locus coeruleus, area postrema, subependymal layer and pineal gland. Intermediate densities were found in the amygdala, nucleus accumbens, caudate-putamen, septum, bed nucleus of the stria terminalis, CA1 to CA4 fields of the hippocampus and central gray. No specific binding of M-[ 125I]vasoactive intestinal peptide was observed in white matter tracts such as corpus callosum, anterior commissure, medial forebrain bundle and fornix. The mapping of M-[ 125I]vasoactive intestinal peptide binding sites as revealed by autoradiography on slide-mounted sections indicates an association, although not exclusive, of vasoactive intestinal peptide receptors with brain regions involved in the processing of specific sensory inputs.

Hydrodynamic characterization of vasoactive intestinal peptide receptors extracted from rat lung membranes in Triton X-100 and n-octyl-beta-D-glucopyranoside.

Rat lung membrane vasoactive intestinal peptide (VIP) receptors were covalently labeled with 125I-VIP, extracted in Triton X-100 and n-octyl-beta-D-glucopyranoside, and analyzed by gel filtration and sucrose density gradient sedimentation. The fractions were characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography, and the identity of the 125I-VIP.receptor complex was demonstrated by its co-migration with the covalently labeled 55-kDa receptor unit identified previously. Furthermore, the radioactivity in the peak corresponding to the 125I-VIP.receptor complex was displaced in the presence of unlabeled VIP in a dose-dependent manner. The following hydrodynamic properties were determined for VIP receptors in each detergent solution: in Triton X-100, Stokes radius of 6.1 +/- 0.4 nm, sedimentation coefficient (S20,w) of 7.35 +/- 0.45 S, and partial specific volume (v) of 0.809 +/- 0.015 ml/g; in n-octyl-beta-D-glucopyranoside, Stokes radius of 5.6 +/- 0.00 nm, S20,w of 10.87 +/- 0.22 S, and partial specific volume of 0.783 +/- 0.020 ml/g. The apparent molecular weight of the 125I-VIP.receptor.detergent complex was calculated as 270,000 +/- 36,000 in Triton X-100 and 320,000 +/- 32,000 in n-octyl-beta-D-glucopyranoside. The amount of detergent bound to the receptor was estimated by using the two sets of hydrodynamic data and the significantly different partial specific volumes of the two detergents. Thus, the molecular weight of the receptor alone was calculated as 54,600 daltons, indicating that approximately 3.9 g of Triton X-100 and 4.9 g of n-octyl-beta-D-glucopyranoside were bound per g of receptor. This species contained the 55-kDa binding unit and appeared to be glycosylated as evidenced by its specific binding to wheat germ agglutinin-Sepharose. These results indicate that the rat lung VIP receptor is a glycoprotein with a single polypeptide chain of 55 kDa. The large amount of detergent bound suggests that the receptor is extensively embedded in the membrane.

Characterization and solubilization of vasoactive intestinal peptide receptors from rat lung membranes.

Receptors for vasoactive intestinal peptide (VIP) were characterized in rat lung membranes by binding and covalent cross-linking of [125I]VIP using ethylene glycolbis-(succinimidylsuccinate). Binding studies indicated the presence of two classes of binding sites for VIP in rat lung membranes: 0.28 +/- 0.11 pmol/mg protein high affinity receptors (Kd = 79.2 +/- 26.4 pM) and 3.3 +/- 0.9 pmol/mg protein lower affinity receptors (Kd = 4.8 +/- 2.1 nM). Furthermore, binding of [125I]VIP to rat lung receptors was inhibited by micromolar concentrations of GTP analogs, guanosine-5'-O-(3-thiotriphosphate) GTP gamma S), and guanylylimidodiphosphate, suggesting that VIP receptors in rat lung membranes were tightly coupled to the guanine nucleotide regulatory protein (Ns). Scatchard analysis of VIP binding in the presence of GTP gamma S revealed selective inhibition of binding to high affinity sites. A 58K band was specifically labeled when membranes covalently labeled with [125I]VIP were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions. The apparent size of this species was not altered when sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis was carried out in the absence of reducing agent. Unlabeled VIP inhibited the labeling, with an IC50 of about 1 nM. A related peptide, GH-releasing factor-(1-40)OH, exhibited a much lower binding affinity, and two unrelated peptides, insulin and atrial natriuretic factor, did not inhibit labeling of the 58K species, even at micromolar concentrations. Labeling of the 58K species was inhibited in a GTP gamma S-dependent manner, suggesting the involvement of this species in the coupling to Ns. These data collectively indicated that the 58K species was a VIP-binding unit of VIP receptors in rat lung membranes. Several nondenaturing detergents were tested for extraction of labeled receptors from the membrane; the best extraction was obtained using 1% n-octyl-beta-D-glucopyranoside.

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.

Solubilization of the liver vasoactive intestinal peptide receptor. Hydrodynamic characterization and evidence for an association with a functional GTP regulatory protein.

Vasoactive intestinal peptide (VIP) receptors were solubilized using the nondenaturing detergent Triton X-100 after occupancy of rat liver membrane-bound receptors with 125I-VIP. Gel filtration and ultracentrifugation on sucrose density gradients revealed the existence in the soluble macromolecular fraction of two labeled components: a major (80%) heavy component and a minor (20%) light one. The two components exhibit the following hydrodynamic parameters: Stokes radius, 5.8 nm: s20,w, 5.98 s; Mr, 150,000; frictional ratio, 1.52 for the major; and Stokes radius, 3.0 nm: s20,w, 3.98 s; Mr = 52,000; frictional ratio, 1.12 for the minor component. The labeling of these components was specific in that it dramatically decreased when unlabeled VIP was added together with 125I-VIP. The pharmacological specificity was also assessed by using 10 nM histidylisoleucineamide (a VIP agonist). Many lines of evidence indicate that the light component (Mr = 52,000) is the VIP-receptor complex while the heavy component (Mr = 150,000) is a ternary complex consisting of VIP, the receptor, and a guanine nucleotide regulatory protein, probably Ns. GTP is required to dissociate 125I-VIP from the heavy component whereas it is ineffective on the light component. This effect is nucleotide specific. After cholera toxin-induced [32P]ADP ribosylation of liver membranes, a high peak of 32P radioactivity containing the alpha subunit (Mr = 42,000) of the Ns protein is coeluted with the heavy component on Sephacryl S-300. By mild urea (2 M) treatment, the heavy component is converted into the light without significant dissociation of 125I-VIP. When a Triton extract of membranes prelabeled with 125I-VIP is treated with dithiobis(succinimidyl propionate) subsequent sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis reveals a major band corresponding to Mr = 150,000. Alternatively, when prelabeled membranes are directly treated with the cross-linker, a major complex of Mr = 51,000 is observed. This may be related to different accessibility of the cross-linker to the site at which the receptor and the Ns protein interact in the two conditions. In conclusion, these data represent initial reports on the successful solubilization of functional VIP-receptor complexes and provide evidence for an interaction between liver VIP-receptor complexes and a GTP-binding protein.

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)

Solubilization of active receptors for VIP from guinea pig lung

The zwitterionic detergent 3-[(3-cholamidopropyl)dimethyl-ammonio]-1-propane sulfonate (CHAPS) was used to solubilize functional receptors for vasoactive intestinal peptide (VIP) from guinea pig lung. The binding of [ 125I]-VIP to the soluble receptors was time-dependent, reversible and saturable. Trypsin destroyed the activity of the receptors, indicating their proteinic nature. Diothiothreitol reduced the binding in a dose-dependent manner, suggesting the importance of disulfide bond(s) in receptor-binding activity. Binding of [ 125I]-VIP to the receptors was reversible and was competitively inhibited by unlabeled VIP, with 50% inhibition obtained at 305 nM VIP.