Thrombin Activates the Sarcolemmal Na + -H + Exchanger

Abstract

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 NH 4 Cl) 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 μmol/L. In contrast, an inactive control peptide (FLLRN) was without effect at 25 μmol/L. In cells pretreated with 100 nmol/L GF109203X or 5 μmol/L chelerythrine (protein kinase C inhibitors), neither 5 U/mL thrombin nor 25 μmol/L SFLLRN produced a significant increase in J H . In the presence of 10 μmol/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 μmol/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 μmol/L SFLLRN affected resting pH i and Ca 2+ 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.