CYCLIC-GMP INCREASES THE RATE OF THE CALCIUM EXTRUSION PUMP IN INTACT HUMAN PLATELETS BUT HAS NO DIRECT EFFECT ON THE DENSE TUBULAR CALCIUM ACCUMULATION SYSTEM

Sodium nitroprusside (SNP) and other agents that elevate cGMP levels are known to inhibit the aggregation of human platelets. Published data suggest that cGMP attenuation of agonist-induced Ca2+ transients is involved in this effect. The present study shows that elevation of cGMP increases the rate of the Ca2+ extrusion pump located in the plasma membrane (PM) but does not have a direct effect on the Ca2+ accumulating pump of the dense tubules (DT). The study verifies that SNP can specifically elevate the cGMP level in the platelet. The kinetics of the Ca2+ extrusion system were studied in situ in platelets overloaded with the cytoplasmic Ca2+ indicator quin2 according to a published protocol developed in this laboratory. Elevation of cGMP by means of (10-mu-M) SNP increased the V(m) of the Ca2+-ATPase pump by 63%, without affecting its K(m) (66-80 nM) or Hill coefficient (1.6-1.8). Dibutyryl-cGMP (Bt2-cGMP), preincubated for 45 min at 1 mM, increased the V(m) by a factor of 2.2 +/- 0.4. The experiments did not give any indication that SNP or Bt2-cGMP change the rate of the Na+/Ca2+ exchanger which makes a minor contribution to Ca2+ extrusion in the studied [Ca2+]cyt range. The rate constant for passive leakage of Ca2+ across the PM was increased by 32 +/- 4% by SNP and 90 +/- 34% by Bt2-cGMP. The net result is that the free Ca2+ in the cytoplasm ([Ca2+]cyt) at 'rest' is lowered from control values of 112 nM to 89 nM or 80 nM, respectively. The kinetics of Ca2+ uptake by the dense tubules were determined in situ using the fluorescence of chlorotetracycline (CTC) according to protocols developed in this laboratory. Analysis showed that SNP and Bt2-cGMP had no effect on the V(m) or K(m) of the dense tubular pump, and did not affect the rate constant for passive leakage. The agents did decrease resting [Ca2+]dt by 25% or 30%, respectively, but this result can be explained purely in terms of the reduced [Ca2+]cyt. The effects of cGMP (vs. cAMP) on the PM and DT pumps are closely correlated with reported effects of cGMP/cAMP induced phosphorylation of a protein of the molecular weight of the PM pump and a 22 kDa activator of the DT pump. Cyclic AMP increases the rate of both the PM and the DT pumps, whereas cGMP increases the rate of the PM pump only. In combination, treatment with maximally-effective doses of Bt2-cGMP and Bt2-cAMP had no greater effect on the PM pump than did either agent alone.