Robert S. Haworth

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 (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 mumol/L. In contrast, an inactive control peptide (FLLRN) was without effect at 25 mumol/L. In cells pretreated with 100 nmol/L GF109203X or 5 mumol/L chelerythrine (protein kinase C inhibitors), neither 5 U/mL thrombin nor 25 mumol/L SFLLRN produced a significant increase in JH. In the presence of 10 mumol/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 mumol/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 mumol/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.

Regulation of Cardiac Sarcolemmal Na+/H+ Exchanger Activity by Endogenous Ligands: Relevance to Ischemiaa

Abstract: The cardiac sarcolemmal Na+/H+ exchanger (NHE) extrudes one H+ in exchange for one Na+ entering the myocyte, utilizing for its driving force the inwardly directed Na+ gradient that is maintained by the Na+/K+ ATPase. The exchanger is quiescent at physiological values of intracellular pH but becomes activated in response to intracellular acidosis. Recent evidence suggests that a variety of extracellular signals (e.g., adrenergic agonists, thrombin, and endothelin) also modulate sarcolemmal NHE activity by altering its sensitivity to intracellular H+. Since sarcolemmal NHE activity is believed to be an important determinant of the extent of myocardial injury during ischemia and reperfusion, regulation of exchanger activity by endogenous ligands associated with ischemia is likely to be of pathophysiological importance.

Receptor-Mediated Regulation of the Cardiac Sarcolemmal Na+/H+ Exchanger

Intracellular pH (pHi) homeostasis in cardiac myocytes is achieved principally by the integrated action of 4 different sarcolemmal ion transporters (1). When the myocyte cytoplasm becomes acidic, the Na+/H+ exchanger (NHE) and the Na+/HCO3 - cotransporter (NBC) extrude acid from the cell, while under conditions of intracellular alkalosis, the Cl-/HCO3 - and Cl-/OH- exchangers effectively import acid. In order to investigate the function and regulation of NHE, experimental protocols are often performed in the absence of bicarbonate, which renders NBC inactive and thereby makes NHE the sole acid extrusion pathway. NHE activity is regulated primarily by pHi, and increases markedly in response to intracellular acidosis (1) through the interaction of H+ with an allosteric modifier site on the transport domain (2,3). The basal activity of the sarcolemmal NHE is low under physiological conditions, while increasing intracellular acidosis leads to a pHi-dependent increase in NHE activity, with a Hill coefficient of around 3 (4). This indicates that more than 1 proton binds to the NHE protein during the transport cycle, and has led to the suggestion that the NHE protein contains a non-transporting proton-binding site which allosterically modifies NHE activity. Thus, as pHi falls, the proton modifier site becomes increasingly occupied, leading to a greater increase in NHE activity than would be expected by simply in creasing the availability of transportable protons.

MECHANISM OF ARRHYTHMOGENESIS INDUCED BY FATTY ACIDS: A ROLE OF MODIFICATION OF MITOCHONDRIAL CREATINE KINASE AND CITRATE SYNTHASE?

There is evidence that mitochondrial depolarization and excess polyunsaturated fatty acids both promote arrhythmogenesis in ischemia/reperfusion (I/R) injury. We previously reported that the lipoxygenase (LOX) inhibitor baicalein inhibits both arachidonic acid (AA) induced mitochondrial