Sequential sodium-proton exchange in thrombin-induced human platelets.
Thrombin stimulation of human platelets initiates a membrane depolarization attributable to a Na+ influx into, and an alkalinization of, the cytoplasm, both of which follow a similar rapid time scale and thrombin-dose dependence. These responses precede secretion of the contents of the dense granules (serotonin) and, after 1 minute, of lysosomes (beta-glucuronidase). We have evaluated these parameters in the presence of 2H2O in order to determine if the Na+ influx and H+ efflux are sequential or simultaneous. NMR evidence indicates that 2H2O equilibration in rapid, and virtually complete within the 3 min prestimulation platelet equilibration period. In response to an 0.05 U/ml addition of thrombin, the rate of depolarization is 70-80% slower in 2H2O than in H2O. The time to reach maximal depolarization is 5 to 10 seconds longer in 2H2O, the extent of depolarization 60% inhibited, and the pH change 85% inhibited. The serotonin secretion is unaltered, while the beta-glucuronidase secretion is 130-180% enhanced. Dimethylamiloride inhibits the Na+ influx and the pH change completely. These results suggest that the Na+ and H+ fluxes across the plasma membrane are interdependent but neither simultaneous nor electroneutral. Furthermore, granule secretion, previously shown by us to be independent of the existent Na+ gradient, depends on the cytoplasmic K+ and H+ concentrations.
Davies
T A
TA
Department of Biochemistry, Boston University School of Medicine, MA 02118.
Katona
E
E
Vasilescu
V
V
Cragoe
E J
EJ
Simons
E R
ER
D000584:Amiloride / Q000494:pharmacology; D001792:Blood Platelets / Q000187:drug effects / Q000502:physiology*; D002462:Cell Membrane / Q000502:physiology; D003593:Cytoplasm / Q000378:metabolism; D003903:Deuterium; D005966:Glucuronidase / Q000097:blood; D006801:Humans; D006863:Hydrogen-Ion Concentration; D009682:Magnetic Resonance Spectroscopy; D008564:Membrane Potentials / Q000187:drug effects; D010042:Ouabain / Q000494:pharmacology; D011188:Potassium / Q000097:blood; D011522:Protons; D012701:Serotonin / Q000097:blood; D012964:Sodium / Q000097:blood*; D013917:Thrombin / Q000494:pharmacology*; D014634:Valinomycin / Q000494:pharmacology
D011522:Protons; D014634:Valinomycin; D012701:Serotonin; D010042:Ouabain; D000584:Amiloride; D012964:Sodium; D003903:Deuterium; D005966:Glucuronidase; D013917:Thrombin; D011188:Potassium