Thrombin activates the sarcolemmal Na+-H+ exchanger - Evidence for a receptor-mediated mechanism involving protein kinase C

Thrombin can activate the plasma membrane Na+- H+ exchanger in a variety of noncardiac cells. We have studied (1) the effect of thrombin on the activity of the sarcolemmal Na+-H+ exchanger in freshly isolated quiescent ventricular myocytes from the adult rat heart and (2) the signaling mechanism(s) underlying any effect. Reverse-transcription polymerase chain reaction analysis revealed thrombin receptor mRNA expression in a myocyte-enriched cell preparation. As an index of Na+-H+ exchanger activity, acid efflux rates (J(H)s) were determined in single myocytes (n=4 to 11 per group) loaded with the pH-sensitive fluoroprobe carboxy-seminaphthorhodafluor-1 after two consecutive intracellular acid pulses (induced by transient exposure to 20 mmol/L NH4Cl) in bicarbonate-free medium. At a pH(i) of 6.9, J(H) did not change significantly during the second pulse relative to the first in control cells. However, when the second pulse occurred in the presence of 0.2, 1, or 5 U/mL thrombin, J(H) increased by 30%, 62% (P<.05), and 87% (P<.05), respectively. A hexameric thrombin receptor-activating peptide (SFLLRN) mimicked the effect of thrombin and increased J(H) by 73% (P<.05) at 25 mu mol/L. In contrast, an inactive control peptide (FLLRN) was without effect at 25 mu mol/L. In cells pretreated with 100 nmol/L GF109203X or 5 mu mol/L chelerythrine (protein kinase C inhibitors), neither 5 U/mL thrombin nor 25 mu mol/L SFLLRN produced a significant increase in J(H). In the presence of 10 mu mol/L HOE-694 (a Na+-H+ exchanger inhibitor), pH(i) did not recover after an acid load, even during exposure to 5 U/mL thrombin or 25 mu mol/L SFLLRN, confirming that the Na+-H+ exchanger was the primary acid efflux mechanism under the conditions used. Neither 5 U/mL thrombin nor 25 mu mol/L SFLLRN affected resting pH(i), and Ca2+ or background acid loading. We conclude that (1) adult rat ventricular myocytes express a functional thrombin receptor, whose stimulation results in increased activity of the sarcolemmal Na+-H+ exchanger, and (2) this effect appears to occur through a protein kinase C-mediated mechanism.