Intracellular calcium mobilization and activation of the Na+/H+ exchanger in platelets.
The aim of the present study was to evaluate the regulatory relationship between the cytosolic free calcium concentration ([Ca2+]i and cytosolic pH (pHi). [Ca2+]i and pHi were measured using the fluorescent dyes fura-2 and BCECF [2',7'-bis-(carboxyethyl)-5,6-carboxyfluorescein] respectively. In a medium with 1 mmol/l extracellular calcium, thrombin (2.5 units/ml) induced an increment in [Ca2+]i of 638 +/- 31 nmol/l (n = 5) and an intracellular alkalinization of 0.14 +/- 0.01 pH units (n = 8). Both responses were dependent on the concentration of thrombin, displaying a sigmoidal dose-response pattern. The intracellular alkalinization was dependent upon extracellular Na+ and was amiloride-sensitive, indicating that it was mediated by activation of the Na+/H+ exchanger. When extracellular calcium was chelated with EGTA prior to the addition of thrombin, the intracellular alkalinization was not affected (0.15 +/- 0.02 at 2.5 units/ml thrombin, n = 8). Under these circumstances, the [Ca2+]i increment represents mobilization from internal stores, reaching 157 +/- 42 nmol/l at 2.5 units/ml thrombin. When platelets were preloaded with the intracellular calcium chelator MAPTAM (1,2-bis-5-methylaminophenoxylethane-NNN'-tetraacetoxymethyl acetate) to block the increase in [Ca2+]i induced by thrombin, no increment in pHi was observed. Moreover, MAPTAM-loaded calcium-depleted platelets had a basal pHi that was more acidic than in the presence of 1 mmol/l extracellular calcium (6.93 +/- 0.09 versus 7.14 +/- 0.01, n = 26, P < 0.001). Ionomycin induced an elevation of [Ca2+]i that was accompanied by a concomitant increase in pHi, which was Na(+)-dependent and amiloride-sensitive. [Ca2+]i and pHi increases induced by ionomycin were both dependent on the concentration of ionomycin. In conclusion, an increase in [Ca2+]i is necessary for the agonist-induced activation of the Na+/H+ exchanger in platelets. Non-agonist-induced increases in [Ca2+]i seems to prompt activation of the exchanger. In addition, Ca(2+)-depleted platelets have a more acidic basal pHi, indicating that the basal level of [Ca2+]i is also important for maintaining the basal pHi.
Poch
E
E
Nephrology Service, Hospital Clinic I Provincial, University of Barcelona, Catalonia, Spain.
Botey
A
A
Gaya
J
J
Cases
A
A
Rivera
F
F
Revert
L
L
D001792:Blood Platelets / Q000187:drug effects / Q000378:metabolism*; D002118:Calcium / Q000737:chemistry / Q000378:metabolism*; D002352:Carrier Proteins / Q000378:metabolism*; D002478:Cells, Cultured; D002614:Chelating Agents; D004533:Egtazic Acid / Q000031:analogs & derivatives / Q000737:chemistry; D006801:Humans; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D017923:Sodium-Hydrogen Exchangers; D013917:Thrombin / Q000494:pharmacology
D002352:Carrier Proteins; D002614:Chelating Agents; D017923:Sodium-Hydrogen Exchangers; D004533:Egtazic Acid; C058180:5,5'-dimethyl-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetate; D013917:Thrombin; D002118:Calcium