Regulation of Na+/H+ exchange in rat adipocytes; effects of insulin.

The activity of the Na+/H+ exchanger was examined by acidifying the intracellular pH (pHi) with Na+ propionate (NaP) and monitoring the recovery in the absence of HCO3- in rat adipocytes. Acidification of pHi, monitored with 2',7'-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein, decreased the resting pHi from 7.25 +/- 0.01 to 6.70 +/- 0.01. A spontaneous pHi recovery to 6.90 +/- 0.02 was inhibited by 300 microM amiloride. This effect was Na+ specific, as recovery did not occur in cells exposed to K+ propionate (KP). The addition of NaCl (30 mM) to KP induced pHi alkalinization. Acidification of pHi increased 22Na+ transport from 0.60 +/- 0.12 nmol/10(5) cells.min at resting pHi to 2.893 +/- 0.129 (P < 0.001) and 7.984 +/- 0.312 (P < 0.001) in the first and tenth minutes, respectively. Amiloride inhibited this 5- and 14-fold stimulation by 85% (P < 0.001). Insulin in the presence of 100 microM ouabain stimulated Na+ influx by more than 15% (P < 0.01). Ethylisopropylamiloride (10 microM) inhibited the effect of insulin by 85% (P < 0.001). Intracellular Na+, measured with a Na(+)-specific electrode, increased by 10-fold in acid-loaded cells compared to that in Na(+)-depleted cells (10.750 +/- 0.479 vs. 1.045 +/- 0.100 mM; P < 0.001). Amiloride decreased NaP-stimulated intracellular Na+ by 82% (P < 0.001). To our knowledge, this is the first report showing the presence of an insulin-responsive and amiloride-sensitive Na+/H+ exchanger that regulates pHi by a Na(+)-specific and pHi-dependent mechanism in rat adipocytes.