INTERACTION OF VASOACTIVE INTESTINAL PEPTIDE WITH ISOLATED INTESTINAL EPITHELIAL-CELLS FROM RAT .1. CHARACTERIZATION, QUANTITATIVE ASPECTS AND STRUCTURAL REQUIREMENTS OF BINDING-SITES

Specific binding sites for porcine vasoactive intestinal peptide have been characterized in isolated intestinal epithelial cells from rat using 125I‐labeled vasoactive intestinal peptide and a variety of physicochemical conditions. At 15°C binding of the peptide reached a plateau between 90 and 240 min incubation and was a linear function of the cell concentration up to about 8.8 × 105 cells/ml. Inactivation of the peptide and of binding sites after 120 min did not exceed 30% and 15% of the controls, respectively. Optimal pH for binding was around 7.5. At 30°C an apparent steady‐state of vasoactive intestinal peptide binding could not be obtained, and the inactivation of both peptide and binding sites were higher than those at 15°C. Native vasoactive intestinal peptide competitively inhibited the binding of the 125I‐labeled peptide in the range 0.1 nM–0.1 μM; half‐maximal inhibition was observed at about 3.1 nM vasoactive intestinal peptide, and a concentration as low as 0.15 nM showed a significant effect. Kinetics studies and Scatchard analysis indicated the existence of two functionally independent classes of binding sites. There are approximately 1.4 × 105 high affinity sites/cell with a dissociation constant (Kd) of 1.6 nM and 1.1 × 106 low affinity sites/cell with a Kd of 74 nM. Chicken vasoactive intestinal peptide exhibited about seven times more affinity than the porcine peptide in competing with the binding of the 125I‐labeled peptide to intestinal epithelial cells, suggesting the possible role of the aminothreonine at residue 28 in enhancing the affinity. Secretin inhibited the binding of the 125I‐labeled peptide in a continuous and parallel fashion as to the unlabeled peptide but with a seventy times lower affinity. Competition experiments using several synthetic analogs of secretin indicated that binding affinity is partially related to the presence of hydrophobic amino acids at the NH2‐terminal hexapeptide sequence of the vasoactive intestinal peptide molecule. Glucagon and gastric inhibitory peptide were ineffective in displacing 125I‐labeled vasoactive intestinal peptide at 10 and 0.1 μM, respectively. These properties of the vasoactive intestinal peptide receptors in intestinal epithelial cells, together with earlier reports on biological actions of the peptide, strongly support the concept that this peptide may act under physiological conditions as a regulator of small intestinal function. Copyright © 1979, Wiley Blackwell. All rights reserved