HIGH CONCENTRATIONS OF EXOGENOUS ARACHIDONATE INHIBIT CALCIUM MOBILIZATION IN PLATELETS

Exogenous arachidonic acid (AA) induces platelet aggregation and secretion which are inhibited at higher AA concentrations.To define the mechanisms for platelet inhibition at high AA,we studied the effect of itS2$odium salt on cytoplasmic ionized calcium concentration [Ca ]i, a key modulator of several platelet responses. In platelets suspended in Hepes buffer containing no albumin, peak aggregation and secretion occurred at 2-5 pM AA with partial inhibition above 10^-15 pM AA and complete inhj^ition around 25-50 pM AA. In platelets ^aded with quin2, [Ca⁺² ]i rose, in presence of 1 mM external Ca , from basal levels of 70-80 nM to peak of 300-500 nM at 2-5 μM AA; this was followed by inhibition to basal levels at 25-50 μM AA. At these AA concentrations there was no cell lysis. Thromboxane B. production, measured using a radioimmunoassay, was not inhibited even at 25 pM AA. Elevated celjlar levels of cAMP inhibit platelet responses including Ca²⁺ signals and were therefore measured using a radioimmunoassay. Platelet cAMP levels rose, in the presence of theophylline (7mM), from basal levels of 3.4 pmol/10₂₊ plat to 5.5 at 5 μM AA and to 6.8 pmol/10⁸ plat at 50 pM AA; Ca signals, aggregation, and secretion were inhibited with doubling of cAMP levels. On incubation of platelets with adenylate cyclase inhibitor, 2'5' dideoxgdenosine (DDA, 200 μM, 2 min) there was enhancement of peak [Ca ⁺²]and aggregation noted with 15 pM AA; at 25 μM AA peak [Ca²⁺ ]i rose from 126 nM to 205 nM and aggregation was restored. Incubation with SQ 22,536 (500 μM, 5min), another adenylate cyclase inhibitor, also attenuated the inhibition by high AA levels. Treatment of platelets with aspirin or_BW 755C, a combined lipooxygenase/cyclooxygenase inhibitor, did not pryent the inhibition by high AA levels of aggregation and Ca responses induced by thromboxane analog, U46619 In conclusion, high AA concentrations inhibit cytoplasmic Ca₊₂ mobilization in platelets which is related to elevation of platelet cAMP through stimulation of adenylate cyclase. We propose that high AA levels inhibit aggravation and secretion by modulating cytoplasmic levels of Ca²⁺ and cAMP, two major messenger molecules in platelets.