The interaction of vasoactive intestinal polypeptide and secretin with liver-cell membranes.

Abstract

125I‐labelled derivatives of vasoactive intestinal polypeptide (VIP) and secretin have been used to examine the nature of the interaction of these hormones with isolated liver‐cell membranes of the rat.Incorporation of one atom of iodine per molecule of hormone does not appreciably affect the ability of VIP to stimulate the activity of adenylate cyclase in liver membranes, but causes a two to three‐fold decrease in the apparent affinity of secretin towards this enzyme.Specific binding of 125I‐labelled VIP to membranes is a saturatable process with respect to VIP. A maximum of 0.8 and 3.1 picomoles of hormone per milligram of protein, respectively, binds to microsomal and to plasma membranes. Native VIP and secretin compete with 125I‐labelled VIP for binding in a way expected from the respective affinities of these hormones towards adenylate cyclase activity; about 100 times greater concentrations of secretin are necessary to achieve effects similar to those observed with VIP. Synthetic secretin displaces the VIP‐membrane complex identically to natural secretin, indicating that the biological effects of the latter probably do not result from a contamination by VIP. Glucagon, 2–27‐gastrin, cholecystokinin‐pancreozymin, angiotensin and bradykinin do not compete for binding.The labelled material dissociated from the VIP‐membrane complex retains the ability of unused VIP to bind to membranes, but VIP in the incubation medium is rapidly inactivated upon exposure to membranes. Inactivation of the medium VIP is inhibited by certain polypeptides, such as bacitracin, and by the metal‐complexing agent 1,10‐phenantroline; these compounds increase the binding of 125I‐labelled VIP to membranes when subsaturating concentrations of hormone are used.The VIP‐membrane interaction has been characterized kinetically under conditions where inactivation of the medium VIP is inhibited. At 23°C, the rate of VIP‐membrane association is 1.3 × 107 M−1 s−1 and the rate of dissociation is 2.3 × 10−3 s−1. The dissociation constant based on these rate constantsis 0.17 nM; a value of 0.16 nM is obtained separately from equilibrium data. The rates of association and of dissociation are equally sensitive to temperature, resulting in a dissociation constant that is not temperature‐dependent.Binding of VIP to membranes is markedly reduced by increasing the ionic strength of the medium. Maximum binding occurs at pH 7.4. Nucleoside di‐ and triphosphates decrease the binding of VIP to membranes at equilibrium and increase the rate of dissociation of the VIP‐membrane complex; guanyl nucleotides are most effective. Digestion of the membranes with trypsin and phospholipase A results in a loss of the ability of these membranes to bind VIP. 125I‐labelled secretin binds to liver membranes in a way expected from the ability of native secretin to displace the 125I‐labelled VIP‐membrane complex; binding of iodinated secretin to membranes is inhibited by native secretin and VIP similarly to that of iodinated VIP.