Regulation of the phosphoinositide cycle by Na+/H+ exchange and intracellular pH in human platelets.
We have found that thrombin-induced activation of protein kinase C (PKC) in platelets, measured by phosphorylation of the 47 kDa protein, is synergistically enhanced by the amiloride analogue ethylisopropylamiloride (EIA), a specific inhibitor of Na+/H+ exchange. This EIA effect was further synergistically enhanced by lowering intracellular pH (pHi) with either nigericin or sodium propionate, and reversed by raising pHi with monensin or ammonium chloride. The synergistic enhancement of thrombin-activated PKC by EIA plus nigericin was not observed when PKC was directly activated by phorbol esters. EIA and EIA plus nigericin caused a 3- to 6-fold increase in thrombin-induced diacylglycerol (DAG), but not phosphatidic acid (PA), production. EIA and nigericin also caused a marked increase in thrombin-induced breakdown and inhibition of resynthesis of phosphatidylinositol 4,5-bisphosphate (PIP2). In summary, we have presented evidence that inhibition of Na+/H+ exchange causes primarily a H(+)-mediated interruption of the phosphoinositide cycle in activated platelets, including the accumulation of DAG associated with the enhancement of PKC activation, the inhibition of conversion of DAG to PA, and increased PIP2 breakdown. These data suggest a model in which Na+/H+ and pHi play an important regulatory role in permitting the phosphoinositide cycle to proceed in thrombin-activated platelets.
Luzzatto
G
G
Department of Medicine, Brigham and Women's Hospital, Boston, MA.
Kroll
M H
MH
Zavoico
G B
GB
Schafer
A I
AI
D000584:Amiloride / Q000031:analogs & derivatives / Q000494:pharmacology; D001792:Blood Platelets / Q000187:drug effects / Q000378:metabolism*; D002352:Carrier Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D004075:Diglycerides / Q000032:analysis; D004789:Enzyme Activation / Q000187:drug effects; D006801:Humans; D006863:Hydrogen-Ion Concentration; D009550:Nigericin / Q000494:pharmacology; D010703:Phorbol Esters / Q000494:pharmacology; D010712:Phosphatidic Acids / Q000032:analysis; D019269:Phosphatidylinositol 4,5-Diphosphate; D010716:Phosphatidylinositols / Q000032:analysis / Q000378:metabolism*; D010766:Phosphorylation; D010974:Platelet Aggregation; D011493:Protein Kinase C / Q000378:metabolism; D017923:Sodium-Hydrogen Exchangers; D013917:Thrombin / Q000494:pharmacology
C011439:1,2-diacylglycerol; D002352:Carrier Proteins; D004075:Diglycerides; D010703:Phorbol Esters; D010712:Phosphatidic Acids; D019269:Phosphatidylinositol 4,5-Diphosphate; D010716:Phosphatidylinositols; D017923:Sodium-Hydrogen Exchangers; D000584:Amiloride; D011493:Protein Kinase C; D013917:Thrombin; D009550:Nigericin; C039614:ethylisopropylamiloride