vasoactive-intestinal-peptide and 3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate

Anion-exchange chromatography of solubilized pig liver cell membranes on DEAE-Sepharose gave a fraction with high affinity binding proteins for VIP and glucagon distinct from each other. Scatchard analysis indicated the presence of one binding site for VIP (Kd 1.5 +/- 0.6 nM and Bmax 1.3 +/- 0.4 pmol/mg). The order of potency for VIP-related peptides to inhibit [125I]VIP binding was: VIP > peptide histidine isoleucine amide (PHI) > rat growth hormone releasing factor (rGRF) > secretin. GTP-gamma-S inhibited [125I]VIP binding and reduced the affinity of VIP binding sites to 6.5 nM. In the same isolated fraction, [125I]glucagon binding was displaced by glucagon preferentially to oxyntomodulin, and GTP did not affect this [125I]glucagon binding. Scatchard analysis indicated the presence of one binding site for glucagon (Kd 0.08 +/- 0.03 nM and Bmax 0.31 +/- 0.01 pmol/mg). A low-affinity VIP binding protein (IC50 0.7 microM) was detected in a fraction eluting later and exhibited a peptide specificity: rGRF > VIP > VIP(10-28) > secretin > PHI. This rGRF-preferring protein (18 kDa) was purified and had a partial amino-acid sequence identical to that of calmodulin. Its [125I]VIP binding was competitively inhibited by VIP and calmidazolium in a manner similar to that for pig brain calmodulin. Thus we have co-solubilized VIP and glucagon high affinity receptors from pig liver cell membranes and separated them from VIP-binding calmodulin.

Topics: Amino Acid Sequence; Animals; Calmodulin; Cholic Acids; Chromatography, DEAE-Cellulose; Detergents; Electrophoresis, Polyacrylamide Gel; Glucagon; Guanosine 5'-O-(3-Thiotriphosphate); Molecular Sequence Data; Receptors, Gastrointestinal Hormone; Receptors, Glucagon; Receptors, Vasoactive Intestinal Peptide; Solubility; Swine; Vasoactive Intestinal Peptide

Relaxation of penile corpus cavernosum smooth muscle is controlled by nerve and endothelium derived substances. In this study, endothelium-dependent relaxation of corporal smooth muscle was characterized and the role of arachidonic acid products of cyclooxygenase in endothelium-dependent relaxation was examined. Endothelium removal from rabbit corpora was performed by infusion with 3-[(3-cholamidopropyl)-dimethylammonio]-1-propane sulfonate and was confirmed by transmission electron microscopy. Strips of human and rabbit corporal tissues were studied in the organ chambers for isometric tension measurement. The accumulation of cyclic guanosine monophosphate (cGMP) and the release of eicosanoids from corporal tissue was measured by radioimmunoassay and correlated to smooth muscle relaxation. Our study showed that relaxation of corpus cavernosum tissue to acetylcholine, bradykinin and substance P was endothelium-dependent; potentiated by indomethacin; and inhibited by NG-monomethyl-L-arginine, methylene blue or LY83583. Relaxation to papaverine and sodium nitroprusside was endothelium-independent, and unaffected by NG-monomethyl-L-arginine. Relaxation to vasoactive intestinal polypeptide was partially endothelium-dependent; potentiated by indomethacin; attenuated by NG-monomethyl-L-arginine or methylene blue. The tissue level of cGMP was enhanced by acetylcholine and nitric oxide. Methylene blue inhibited both basal and drug-stimulated levels of cGMP. The release of eicosanoids was enhanced by acetylcholine and blocked by indomethacin. In conclusion, nitric oxide or a closely related substance accounts for the activity of endothelium-derived relaxing factor in the corporal tissue. Inhibition of the release of eicosanoids potentiates the relaxing effect of nitric oxide. Nitric oxide increases tissue cGMP which appears to modulate corporal smooth muscle relaxation.

Topics: Acetylcholine; Animals; Bradykinin; Cholic Acids; Cyclic GMP; Eicosanoids; Endothelium, Vascular; Guanylate Cyclase; Humans; In Vitro Techniques; Indomethacin; Male; Muscle Relaxation; Muscle, Smooth, Vascular; Neurotransmitter Agents; Nitric Oxide; Nitroprusside; Papaverine; Penis; Prostaglandin-Endoperoxide Synthases; Rabbits; Substance P; Vasoactive Intestinal Peptide

Vasoactive intestinal peptide (VIP) receptors were solubilized from porcine liver membranes using CHAPS. The binding of 125I-VIP to solubilized receptors was reversible, saturable and specific. Scatchard analysis indicated the presence of one binding site with a Kd of 6.5 +/- 0.3 nM and a Bmax of 1.20 +/- 0.15 pmol/mg protein. Solubilized and membrane-bound receptors displayed the same pharmacological profile since VIP and VIP-related peptides inhibited 125I-VIP binding to both receptor preparations with the same rank order of potency e.g. VIP greater than helodermin greater than rat GRF greater than rat PHI greater than secretin greater than human GRF. GTP inhibited 125I-VIP binding to membrane-bound receptors but not to solubilized receptors supporting functional uncoupling of VIP receptor and G protein during solubilization. Affinity labeling of solubilized and membrane-bound VIP receptors with 125I-VIP revealed the presence of a single molecular component with Mr 55,000 in both cases. It is concluded that VIP receptors from porcine liver can be solubilized with a good yield, in a GTP-insentive, G protein-free form. This represents a major advance towards the purification of VIP receptors.

Topics: Animals; Cell Membrane; Cholic Acids; GTP-Binding Proteins; Guanosine Triphosphate; Iodine Radioisotopes; Kinetics; Liver; Receptors, Gastrointestinal Hormone; Receptors, Vasoactive Intestinal Peptide; Substrate Specificity; Succinimides; Swine; Time Factors; Vasoactive Intestinal Peptide

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.

Topics: Animals; Cholic Acids; Chromatography, Affinity; Electrophoresis, Polyacrylamide Gel; Indicators and Reagents; Kinetics; Liver; Molecular Weight; Receptors, Gastrointestinal Hormone; Receptors, Vasoactive Intestinal Peptide; Swine; Vasoactive Intestinal Peptide

Topics: Adenylyl Cyclases; Animals; Cell Membrane; Cholic Acids; Cyclic AMP; Enzyme Activation; Growth Hormone-Releasing Hormone; GTP-Binding Proteins; Guanosine Triphosphate; Guinea Pigs; Lung; Phospholipases A; Receptors, Gastrointestinal Hormone; Receptors, Vasoactive Intestinal Peptide; Solubility; Type C Phospholipases; Vasoactive Intestinal Peptide

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.

Topics: Animals; Binding, Competitive; Chemical Phenomena; Chemistry, Physical; Cholic Acids; Chromatography, Gel; Guanosine Triphosphate; Guanylyl Imidodiphosphate; Guinea Pigs; Lung; Receptors, Gastrointestinal Hormone; Receptors, Vasoactive Intestinal Peptide; Solubility; Trypsin; Vasoactive Intestinal Peptide

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. Dithiothreitol 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.

Topics: Animals; Cholic Acids; Guinea Pigs; Lung; Receptors, Cell Surface; Receptors, Vasoactive Intestinal Peptide; Solubility; Vasoactive Intestinal Peptide