© 1996 American Heart Association, Inc.
Articles |
Thrombin Activates the Sarcolemmal Na+-H+ Exchanger
Evidence for a Receptor-Mediated Mechanism Involving Protein Kinase C
Cardiovascular Research, The Rayne Institute, St. Thomas' Hospital, London, UK.
Correspondence to Dr Metin Avkiran, Cardiovascular Research, The Rayne Institute, St. Thomas' Hospital, London SE1 7EH, UK. E-mail m.avkiran@umds.ac.uk.
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 (JHs) 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 pHi of 6.9, JH 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, JH 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 JH 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 JH. In the presence of 10 µmol/L HOE-694 (a Na+-H+ exchanger inhibitor), pHi 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 pHi 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.
Key Words: thrombin receptor • protein kinase C • Na+-H+ exchanger • heart • myocyte
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