Interaction of Vasoactive Intestinal Peptide with Isolated Intestinal Epithelial Cells from Rat

Abstract

Porcine vasoactive intestinal peptide stimulated adenosine 3′:5′‐monophosphate (cyclic AMP) production in rat intestinal epithelial cells. The stimulation was dependent on time and temperature and was potentiated by the phosphodiesterase inhibitor 3‐isobutyl‐1‐methylxanthine. Under optimal conditions (at 15°C, with 0.2 mM 3‐isobutyl‐1‐methylxanthine, at a cell concentration up to 18 μg DNA/ml), the cyclic AMP production produced by vasoactive intestinal peptide was constant for 10 min and stopped after 15 min incubation, at either low (1 nM) or high (30 nM) concentration of the peptide. This plateau effect was demonstrated not to be due to an inactivation of vasoactive intestinal peptide in the medium nor to an alteration of receptors for the peptide. Cyclic AMP production was sensitive to a concentration as low as 0.1 nM vasoactive intestinal peptide. Maximal stimulation of cyclic AMP levels by vasoactive intestinal peptide was observed with 30 nM vasoactive intestinal peptide and represented an 11‐fold increase above basal. The dose‐response curve was monophasic with a K_m of 2.3 × 10⁻⁹ M. No cooperative effects were detected by Hill analysis. The positive non‐linear relationship observed between stimulation of cyclic AMP production and occupancy of binding sites was not time‐dependent as indicated by experiments performed after 15, 45 and 120 min incubation. Maximal and half‐maximal responses were obtained at about 70% and 7% occupation of binding sites, respectively. Chicken vasoactive intestinal peptide and porcine secretin were agonists of porcine vasoactive intestinal peptide with a 6‐times higher and a 120‐times lower potency, respectively. Among secretin analogs that were found to have low affinity for vasoactive intestinal peptide binding sites, [4‐alanine, 5‐valine]secretin, that resembles vasoactive intestinal peptide at the first seven amino acids at the N‐terminal end, was a partial agonist of vasoactive intestinal peptide and others failed to stimulate cyclic AMP production. Glucagon (10 μM), gastric inhibitory peptide (0.1 μM), substance P, neurotensin, octapeptide of cholecystokinin, bovine pancreatic polypeptide, human gastrin I with leucine at residue 15, Leu‐enkephalin and somatostatin (1 μM) did not alter cyclic AMP levels. Non‐peptide mediators such as dopamine, serotonin, acetylcholine and histamine, tested at 10 μM, were also ineffective. Prostaglandins E₂, E₁ and isoproterenol, tested at 10 μM, induced an increase of cyclic AMP levels above basal but were 9.5, 13.7 and 17.5 times less efficient than vasoactive intestinal peptide, respectively. Thus vasoactive intestinal peptide is a unique stimulus of cyclic AMP production in rat intestinal epithelial cells.