Abstract
We have analyzed the mechanism of Na(+)-dependent pHi recovery from an acid load in A6 cells (an amphibian distal nephron cell line) by using the intracellular pH indicator 2'7'-bis(2-carboxyethyl)5,6 carboxyfluorescein (BCECF) and single cell microspectrofluorometry. A6 cells were found to express Na+/H(+)-exchange activity only on the basolateral membrane: Na+/H(+)-exchange activity follows simple saturation kinetics with an apparent Km for Na+ of approximately 11 mM; it is inhibited in a competitive manner by ethylisopropylamiloride (EIPA). This Na+/H(+)-exchange activity is inhibited by pharmacological activation of protein kinase A (PKA) as well as of protein kinase C (PKC). Addition of arginine vasopressin (AVP) either at low (subnanomolar) or at high (micromolar) concentrations inhibits Na+/H(+)-exchange activity; AVP stimulates IP3 production at low concentrations, whereas much higher concentrations are required to stimulate cAMP formation. These findings suggest that in A6 cells (i) Na+/H(+)-exchange is located in the basolateral membrane and (ii) PKC activation (heralded by IP3 turnover) is likely to be the mediator of AVP action at low AVP concentrations.
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