Alpha-thrombin-induced inositol phosphate formation in G0-arrested and cycling hamster lung fibroblasts: evidence for a protein kinase C-mediated desensitization response.
In resting Chinese hamster fibroblasts (CCL39) alpha-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 (t1/2 less than 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.
D000818:Animals; D006224:Cricetinae; D004305:Dose-Response Relationship, Drug; D005347:Fibroblasts / Q000166:cytology; D006868:Hydrolysis; D007295:Inositol Phosphates / Q000096:biosynthesis*; D007399:Interphase; D008168:Lung / Q000166:cytology*; D010716:Phosphatidylinositols / Q000378:metabolism; D011493:Protein Kinase C / Q000378:metabolism*; D013403:Sugar Phosphates / Q000096:biosynthesis*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D013917:Thrombin / Q000494:pharmacology*
D007295:Inositol Phosphates; D010716:Phosphatidylinositols; D013403:Sugar Phosphates; D011493:Protein Kinase C; D013917:Thrombin; D013755:Tetradecanoylphorbol Acetate