EFFECTS OF PH ON CAI2+, NAI+, AND PHI OF MDCK CELLS - NA+-CA2+ AND NA+-H+ ANTIPORTER INTERACTIONS

The effects of high and low extracellular pH (8.0 and 6.8) on the intracellular concentration of Ca2+, Na+, and H+ were measured in perfused Madin-Darby canine kidney (MDCK) cells cast in agarose gel threads. Cytosolic free Ca2+ (Ca(i)2+) was measured with aequorin, while intracellular Na+ (Na(i)+) and H+ (H(i)+ or pH(i)) were determined with the fluorescent indicators SBFI and BCECF, respectively. In addition, H+ secretion was assayed by the pH-stat method, and Na+ or Ca2+ fluxes were measured with Na-22 or Ca-45, respectively. H+ secretion was significantly depressed by several experimental conditions that are known to inhibit the Na+-H+ antiporter: H+ secretion decreased 44% in the presence of 10(-5) M ethylisopropylamiloride, 49% in Na(o)+-free media, 44% in the presence of 10(-4) M ouabain, and 32% in the presence of 10(-4) M 8-bromoadenosine 3',5'-cyclic monophosphate. In addition, pH(i) decreased by 0.2 pH units in Na(o)+-free media. Finally, recovery from an intracellular acidosis evoked by 20 mM NH4Cl pulse required the presence of extracellular Na+. When the extracellular pH (pH(o)) was increased from 7.4 to 8.0, H+ secretion increased 58% from 17.5 to 27.7 nmol.min-1.mg protein-1 and Na+ influx increased 48%. As a result, pH(i) rose from 7.43 to 7.71 and Na(i)+ increased from 15.6 to 19.7 mM. Finally, Ca(i)2+ rose from 120 to 268 nM. These results suggest that the high pH(o) stimulated the Na+-H+ antiporter, and the subsequent rise in Na(i)+ decreased the Na+ electrochemical potential, thereby activating the reverse mode of Na+-Ca2+ exchange (Ca2+ influx vs. Na+ efflux) which led to the rise in Ca(i)2+. When the pH(o) was decreased from 7.4 to 6.8, H+ secretion decreased 63% and Na+ influx fell 41%. As a result, pH(i) fell from 7.43 to 7.11 and Na(i)+ decreased from 15.6 to 9.5 mM. At the same time, Ca(i)2+ fell from 120 to 14 nM. These results can be explained by an inhibition of Na+-H+ exchange following the decrease in pH(o). The resulting fall in Na(i)+, by increasing the Na+ potential, activated the forward mode of Na+-Ca2+ exchange, which depressed Ca(i)2+. These experiments suggest that Ca(i)2+, Na(i)+, and H(i)+ are interrelated, presumably through the interaction of the Na+-H+ and Na+-Ca2+ antiporters.