Characterization of Na(+)-H+ exchange in segments of rat mesenteric artery

To develop a technique for measuring Na(+)-H+ exchange activity and intracellular pH (pH(i)) "on line" in resistance vessels, we utilized strips of rat mesenteric arteries loaded with the pH-sensitive dye 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein. Strips were held at a fixed length within a 3-ml cuvette, and fluorescence emission was monitored at 530 nm. The spectrofluorimeter was monitored in the ratio mode, and the excitation wavelength was alternated between 440 and 505 nm. Tissues were maintained by perfusing with N-2-hydroxyethylpiperazine-N'-2-ethane-sulfonic acid containing buffers. The introduction of ammonium chloride produced a rapid alkalinization. Washout of ammonium caused rapid acidification. Restoration of pH(i) was Na+ dependent and inhibited by dimethyl amiloride (concentration that produces half-maximal inhibition, K0.5 = 30 microM), features characteristic of Na(+)-H+ exchange. Further studies assessed the transport rate of the exchanger, which averaged 0.19 +/- 0.02 pH U/min (means +/- SE, n = 8). An estimate of the dependence of Na(+)-H+ exchange on external Na+ gave an apparent Michaelis constant for external Na+ of 10 mM and an apparent maximal velocity of 0.1 mM H+/s. Intracellular H+ was found to have a cooperative effect (Hill coefficient = 4) on Na(+)-H+ exchange.