title: Further characterization of the mechanisms mediating the rise in cytosolic free Na+ in thrombin-stimulated platelets. Evidence for inhibition of the Na+,K(+)-ATPase and for Na+ entry via a Ca2+ influx pathway.
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+o is due to Na+ influx through a Ca2+ entry pathway. The increase in [Na+]i in the presence of Ca2+o results from Na+ influx via Na+/H+ exchange.
authors.affiliation: Max-Planck-Institut für Biophysik, Frankfurt/Main, Federal Republic of Germany.
authors.forename: M
authors.initials: M
authors.lastname: Borin
authors.forename: W
authors.initials: W
authors.lastname: Siffert
mesh_terms: D001792:Blood Platelets / Q000187:drug effects / Q000378:metabolism*; D002118:Calcium / Q000097:blood*; D002352:Carrier Proteins / Q000097:blood; D003600:Cytosol / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007093:Imidazoles / Q000494:pharmacology; D066298:In Vitro Techniques; D007700:Kinetics; D010042:Ouabain / Q000494:pharmacology; D010975:Platelet Aggregation Inhibitors / Q000494:pharmacology; D012964:Sodium / Q000097:blood*; D017923:Sodium-Hydrogen Exchangers; D000254:Sodium-Potassium-Exchanging ATPase / Q000037:antagonists & inhibitors / Q000097:blood*; D013050:Spectrometry, Fluorescence; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D013779:Tetrodotoxin / Q000494:pharmacology; D013917:Thrombin / Q000494:pharmacology*
chemical_list: D002352:Carrier Proteins; D007093:Imidazoles; D010975:Platelet Aggregation Inhibitors; D017923:Sodium-Hydrogen Exchangers; D013779:Tetrodotoxin; D010042:Ouabain; D012964:Sodium; D013917:Thrombin; D000254:Sodium-Potassium-Exchanging ATPase; C063159:1-(2-(3-(4-methoxyphenyl)propoxy)-4-methoxyphenylethyl)-1H-imidazole; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium