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Am J Physiol Cell Physiol 261: C482-C489, 1991;
0363-6143/91 $5.00
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AJP - Cell Physiology, Vol 261, Issue 3 C482-C489, Copyright © 1991 by American Physiological Society

ARTICLES

Effects of pH on Ca2+i, Na+i, and pHi of MDCK cells: Na(+)-Ca2+ and Na(+)-H+ antiporter interactions

A. B. Borle and C. Bender
Department of Physiology, University of Pittsburgh, School of Medicine, Pennsylvania.

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+ (Ca2+i) was measured with aequorin, while intracellular Na+ (Na+i) and H+ (Hi+ or pHi) 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 22Na or 45Ca, 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, pHi 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 (pHo) 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, pHi rose from 7.43 to 7.71 and Na+i increased from 15.6 to 19.7 mM. Finally, Ca2+i rose from 120 to 268 nM. These results suggest that the high pHo 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 Ca2+i.(ABSTRACT TRUNCATED AT 250 WORDS)


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