title: Studies of endogenous polyphosphoinositide hydrolysis in human platelet membranes. Evidence that polyphosphoinositides remain inaccessible to phosphodiesterase in the native membrane.
abstract: Human platelet plasma membranes incubated in the presence of [gamma-32P]ATP and 15 mM MgCl2 incorporated radioactivity mostly into phosphatidylinositol 4,5-bisphosphate (PIP2) and phosphatidylinositol 4-phosphate (PIP), which represented together over 90% of the total lipid radioactivity. After washing, reincubation of prelabelled membranes revealed some hydrolysis of the two compounds by phosphomonoesterase(s), as detected by the release of radioactive inorganic phosphate (Pi) from the two phospholipids. This degradation attained 40%/30 min for PIP in the presence of 2 mM calcium and cytosol. The effect of calcium was observed at concentrations equal to or greater than 10(-4) M. In no case did calcium alone facilitate the formation of inositol 1,4,5-trisphosphate (IP3) and inositol 1,4-bisphosphate (IP2). In contrast, simultaneous addition of 2 mM calcium and 2 mg/ml sodium deoxycholate promoted the formation of IP3 and IP2, indicating phosphodiesteratic cleavage of PIP2 and PIP. Phospholipase C activity was detected at calcium concentrations as low as 10(-7) M, in which case PIP2 hydrolysis was slightly more pronounced compared to PIP. Addition of cytosol increased to some extent the phospholipase C activity, suggesting that the low amount of enzyme remaining in the membrane is sufficient to promote submaximal degradation of PIP2 and PIP. We conclude that platelet polyphosphoinositides are present in the plasma membrane in a state where they remain inaccessible to phospholipase C, which is still fully active even at basal calcium concentrations, i.e., 10(-7) M. These results support the view that phosphodiesteratic cleavage of PIP2 promotes and thus precedes calcium mobilization brought about by IP3. The in vitro model presented here may prove very useful in future studies dealing with the mechanism rendering polyphosphoinositides accessible to phospholipase C attack upon agonist-receptor binding.
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D001792:Blood Platelets / Q000166:cytology*; D002118:Calcium / Q000494:pharmacology; D002462:Cell Membrane / Q000378:metabolism; D003840:Deoxycholic Acid / Q000494:pharmacology; D004533:Egtazic Acid / Q000494:pharmacology; D006801:Humans; D006868:Hydrolysis; D008274:Magnesium / Q000494:pharmacology; D019269:Phosphatidylinositol 4,5-Diphosphate; D018129:Phosphatidylinositol Phosphates; D010716:Phosphatidylinositols / Q000378:metabolism*; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism*; D013997:Time Factors; D010738:Type C Phospholipases / Q000378:metabolism
chemical_list: D019269:Phosphatidylinositol 4,5-Diphosphate; D018129:Phosphatidylinositol Phosphates; D010716:Phosphatidylinositols; C037178:phosphatidylinositol 4-phosphate; D003840:Deoxycholic Acid; D004533:Egtazic Acid; D000255:Adenosine Triphosphate; D010727:Phosphoric Diester Hydrolases; D010738:Type C Phospholipases; D008274:Magnesium; D002118:Calcium