α‐Thrombin‐induced inositol phosphate formation in go‐arrested and cycling hamster lung fibroblasts: Evidence for a protein kinase C‐mediated desensitization response

Abstract

In resting Chinese hamster fibroblasts (CCL39) α‐thrombin rapidly induces the breakdown, of phosphoinositides. Accumulation of inositol phosphates (IP), measured in the presence of Li⁺, is detectable within 5s (seconds) of thrombin stimulation. Formation of inositol tris‐ and bisphosphates slightly precedes that of inositol monophosphate, indicating that thrombin activates primarily the phospholipase C‐mediated generation of inositol trisphosphate from phosphatidylinositol 4,5‐bisphosphate. Initial rates of IP production increase with thrombin concentration, with no apparent saturability over the range 10⁻⁴‐10 U/ml. Thrombin‐induced phosphoinositide hydrolysis rapidly desensitizes (t_1/2 < 5 min), but a residual activity, corresponding to about 10% of the initial stimulation is sustained for at least 9 h, in contrast with the undetectable activity of G0‐arrested cells. This apparent desensitization may be due to a feedback regulation by protein kinase C, since pretreatment with the phorbol ester 12‐O‐tetradecanoyl phorbol 13‐acetate (TPA) markedly inhibits (by up to 70%) subsequent thrombin‐induced inositol phosphate formation. Conversely, growth factor deprivation of CCL39 cells results in a progressive increase of thrombin‐induced phosphoinositide hydrolysis, from the very low level of exponentially growing cells to the maximal level of G0‐arrested cells. This “up regulation” was found maximal in A51, a very well growth‐arrested CCL39 derivative, and reduced or virtually abolished in two tumoral and growth factor‐relaxed derivatives of CCL39. Although preliminary, this observation suggests that a persistent activation of phosphatidyl inositol breakdown might operate in variants selected for autonomous growth.