Activation of Na/H Exchange in Erythrocytes of Frog Rana ridibunda

Abstract

Transport of Na+ in isolated erythrocytes of the frog Rana ridibunda was studied using radioactive isotope 2222Na. Treatment of erythrocytes with β-adrenergic agonist isoproterenol (ISP) or with a combination of ISP and phosphodiesterase blocker 3-isobutyl-methyl-xanthine (IBMX) did not affect the Na+ transport into the cells. These data indicated that cAMP-dependent protein kinase A did not participate in regulation of the Na+ transport into the frog erythrocytes. Incubation of erythrocytes with protein kinase C activator phorbol ester (PMA, 0.15 µM) led to a pronounced increase of 2222Na accumulation and intracellular Na+ concentration. These changes of the Na+ transport into the cells were completely blocked in the presence of 50 µM ethyl-isopropyl-amiloride (EIPA), a selective blocker of the NHE1-isoform of Na+/H+ exchanger. Hence, PMA produced activation of Na+/H+ exchange in frog erythrocytes. The unidirectional Na+ influx into erythrocytes amounted, on average, to 0.99 ± 0.12 and 147 ± 9 mmol/l cells/h for control and PMA-treated cells, respectively. The EIPA concentration producing a 50% inhibition of the PMA-induced Na+ influx (IC50) was 0.28 µM. A high sensitivity of the frog Na/H exchanger to EIPA indicates its similarity with the mammalian NHE1 isoform. The obtained data for the first time clearly indicate an important role of PKC in Na/H exchange regulation in the frog red blood cells.