Acute inhibition of brain-specific Na(+)/H(+) exchanger isoform 5 by protein kinases A and C and cell shrinkage

Little is known of the functional properties of the mammalian,brain-specific Na⁺/H⁺ exchanger isoform 5 (NHE5). Rat NHE5 was stably expressed in NHE-deficient PS120 cells, andits activity was characterized using the fluorescent pH-sensitive dye2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein. NHE5was insensitive to ethylisopropyl amiloride. The transport kinetics displayed a simple Michaelis-Menten relationship for extracellular Na⁺ (apparent K_Na = 27 ± 5 mM) and a Hill coefficient near 3 for the intracellularproton concentration with a half-maximal activity at an intracellularpH of 6.93 ± 0.03. NHE5 activity was inhibited by acute exposureto 8-bromo-cAMP or forskolin (which increases intracellular cAMP byactivating adenylate cyclase). The kinase inhibitor H-89 reversed thisinhibition, suggesting that regulation by cAMP involves a proteinkinase A (PKA)-dependent process. In contrast, 8-bromo-cGMP did nothave a significant effect on activity. The protein kinase C (PKC)activator phorbol 12-myristrate 13-acetate inhibited NHE5, and the PKCantagonist chelerythrine chloride blunted this effect. Activity wasalso inhibited by hyperosmotic-induced cell shrinkage but wasunaffected by a hyposmotic challenge. These results demonstrate thatrat brain NHE5 is downregulated by activation of PKA and PKC and bycell shrinkage, important regulators of neuronal cell function.