The proton gradient of secretory granules and glutamate transport in blood platelets during cholesterol depletion of the plasma membrane by methyl-β-cyclodextrin.

Abstract

Glutamate transport in blood platelets resembles that in brain nerve terminals because platelets contain neuronal Na(+)-dependent glutamate transporters, glutamate receptors in the plasma membrane, vesicular glutamate transporters in secretory granules, which use the proton gradient as a driving force, and can release glutamate during aggregation/activation. The acidification of secretory granules and glutamate transport were assessed during acute treatment of isolated platelets with cholesterol-depleting agent methyl-β-cyclodextrin (MβCD). Confocal imaging with the cholesterol-sensitive fluorescent dye filipin showed a quick reduction of cholesterol level in platelets. Using pH-sensitive fluorescent dye acridine orange, we demonstrated that the acidification of secretory granules of human and rabbit platelets was decreased by ∼15% and 51% after the addition of 5 and 15mM MβCD, respectively. The enrichment of platelet plasma membrane with cholesterol by the application of complex MβCD-cholesterol (1:0.2) led to the additional accumulation of acridine orange in secretory granules indicating an increase in the proton pumping activity of vesicular H(+)-ATPase. MβCD did not evoke release of glutamate from platelets that was measured with glutamate dehydrogenase assay. Flow cytometric analysis did not reveal alterations in platelet size and granularity in the presence of MβCD. These data showed that the dissipation of the proton gradient of secretory granules rather than their exocytosis caused MβCD-evoked decrease in platelet acidification. Thus, the depletion of plasma membrane cholesterol in the presence of MβCD changed the functional state of platelets affecting storage capacity of secretory granules but did not evoke glutamate release from platelets.