Peptides stimulate phosphoinositide hydrolysis in human retinal pigment epithelium

Abstract

Purpose: This study examined the effects of peptides on inositol lipid turnover and intracellular calcium (Ca2+) levels in cultured human retinal pigment epithelium (RPE).

Methods: Cultured human RPE were stimulated with bradykinin, arginine vasopressin (AVP), or bombesin. Accumulation of 3H-inositol phosphates in the presence of 10 mmol/l lithium reflected phosphoinositide (PPI) hydrolysis. Peptide-coupled changes in intracellular Ca2+ levels were determined by fura-2 fluorescence.

Results: Bradykinin increased PPI hydrolysis to greater than 300% of basal with an EC50 of 300 nM. Bradykinin-coupled PPI turnover was linear up to 60 min and was blocked by > 90% by the B2 antagonist [Thi5,8,D-Phe7]-bradykinin. AVP stimulated PPI turnover in a linear manner by 260% with an EC50 of 800 nM. The V1 receptor antagonist [beta-mercapto-beta,beta-cyclopentamethylenepropionyl1,-O-Me-Tyr2, A rg8]- vasopressin completely inhibited AVP-induced PPI hydrolysis. Bombesin enhanced PPI hydrolysis by 185% with an EC50 of 1 nM. Stimulation was linear up to 60 min and was blocked by > 90% by the bombesin antagonist [Leu13-psi(CH2NH)-Leu14] bombesin. In fura-2-loaded human RPE cells, the resting intracellular Ca2+ concentration was 105 +/- 32 nM (n = 29). Bradykinin increased peak intracellular Ca2+ to 764 +/- 71 nM, AVP to 310 +/- 42 nM, and bombesin to 234 +/- 20.4 nM.

Conclusions: Peptide-stimulated inositol lipid turnover is coupled to cytosolic Ca2+ flux in cultured human RPE.