Importance of disulfide bonds in receptors for vasoactive intestinal peptide and secretin in rat pancreatic plasma membranes.
Vasoactive intestinal peptide (VIP), secretin, and C-terminal octapeptide of cholecystokinin (CCK-8) receptors were identified in rat pancreatic plasma membranes by the ability of these peptides to stimulate adenylate cyclase activity. The membrane preparation procedure was conducted through a series of steps including discontinuous sucrose density gradient fractionation. 5 mM beta-mercaptoethanol was added stepwise. Membrane preparations obtained stepwise were preincubated for 10 min at 25 degrees C in the presence of various concentrations of beta-mercaptoethanol or dithiothreitol before assaying adenylate cyclase. The use of the reducing agents exerted no effect on p[NH]ppG-, NaF-, and CCK-8- stimulated activities. By contrast, stimulation of adenylate cyclase by low VIP concentrations was specifically altered when beta-mercaptoethanol was used during tissue homogeneization at 5 degrees C. In addition, both VIP and secretin responses were highly sensitive towards a preincubation of 10 min at 25 degrees C in the presence of dithiothreitol. These results were likely to reflect alterations at the receptor level. 125I-VIP binding was, indeed, reduced after dithiothreitol preincubation, low concentrations of the thiol reagent decreasing the apparent number of high-affinity VIP receptors and higher dithiothreitol concentrations reducing the affinity of VIP receptors.
D000262:Adenylyl Cyclases / Q000378:metabolism; D000818:Animals; D002462:Cell Membrane / Q000378:metabolism*; D004220:Disulfides / Q000032:analysis; D004789:Enzyme Activation; D007700:Kinetics; D008623:Mercaptoethanol / Q000494:pharmacology; D010179:Pancreas / Q000378:metabolism*; D051381:Rats; D011956:Receptors, Cell Surface / Q000378:metabolism*; D011949:Receptors, Cholecystokinin; D043562:Receptors, G-Protein-Coupled; D011964:Receptors, Gastrointestinal Hormone; D018005:Receptors, Vasoactive Intestinal Peptide; D012633:Secretin / Q000378:metabolism* / Q000494:pharmacology; D012844:Sincalide / Q000378:metabolism*; D014660:Vasoactive Intestinal Peptide / Q000378:metabolism* / Q000494:pharmacology
D004220:Disulfides; D011956:Receptors, Cell Surface; D011949:Receptors, Cholecystokinin; D043562:Receptors, G-Protein-Coupled; D011964:Receptors, Gastrointestinal Hormone; D018005:Receptors, Vasoactive Intestinal Peptide; C029937:secretin receptor; D012633:Secretin; D014660:Vasoactive Intestinal Peptide; D008623:Mercaptoethanol; D000262:Adenylyl Cyclases; D012844:Sincalide