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Abstract

Human platelets were loaded with the fluorescent Na⁺-sensitive dye sodium-binding benzofuran isophtalate (SBFI), and changes in the fluorescence excited at 345 and 385 nm were analyzed after manipulations that evoked predictable changes in the cytosolic Na⁺ concentration ([Na⁺]_i). Raising [Na⁺]_i by either gramicidin D or monensin specifically increased the fluorescence excited at 345 nm and decreased that excited at 385 nm. Hence, calculation of changes in the 345/385 nm excitation ratio yields an estimate of actual changes in [Na⁺]_i. A transient activation of Na⁺/H⁺ exchange evoked by addition of acidified platelets to buffer, pH 7.4, evoked a transient rise in [Na⁺]_i. The re-establishment of basal [Na⁺]_i could be prevented by ouabain, indicating an involvement of the Na⁺,K⁺-ATPase. Upon stimulation by 0.5 unit/ml of thrombin, [Na⁺]_i immediately increased by 16 ± 4 mM and this rise continued for at least 60 min after addition of agonist, albeit at a lower rate. This latter sustained rise could not be curtailed by scavenging thrombin by means of hirudin. Addition of ouabain or the phorbol ester 12-O-tetradecanoylphorbol-13-acetate induced a comparable slow rise in the 345/385 excitation ratio. This may indicate a protein kinase C-mediated inhibition by thrombin of the Na⁺,K⁺-ATPase. In the absence of extracellular Ca²⁺ (Ca²⁺_o), the [Na⁺]_i gain was augmented to 38 ± 9 mM. This additional uptake of Na⁺ was prevented by (i) Mn²⁺ ions, (ii) La³⁺ ions, (iii) the blocker of receptor-mediated Ca²⁺ entry (1-[beta[3-(4-methoxyphenyl)propoxyl]-4-methoxyphenethyl]-1H-im ida zole hydrochloride), and (iv) by hirudin which reversed receptor occupancy by thrombin. These findings suggest that the additional thrombin-induced [Na⁺]_i gain in the absence of Ca²⁺ is due to Na⁺ influx through a Ca²⁺ entry pathway. The increase in [Na⁺]_i in the presence of Ca²⁺ results from Na⁺ influx via Na⁺/H⁺ exchange.