REGULATION OF NA+ H+ EXCHANGE IN RAT ADIPOCYTES - EFFECTS OF INSULIN/

The activity of the Na+/H+ exchanger was examined by acidifying the in tracellular pH (pH(i)) with Na+ propionate (NaP) and monitoring the re covery in the absence of HCO3- in rat adipocytes. Acidification of pH( i), monitored with 2',7'-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein decreased the resting pH(i) from 7.25 +/- 0.01 to 6.70 +/- 0.01, ii sp ontaneous pH(i) recovery to 6.90 +/- 0.02 was inhibited by 300 mu M am iloride. This effect was Na+ specific, as recovery did not occur in ce lls exposed to K+ propionate (KP). The addition of NaCl (30 mM) to KP induced pH(i) alkalinization. Acidification of pH(i) increased Na-22() transport from 0.60 +/- 0.12 nmol/10(5) cells . min at resting pH(i) to 2.893 + 0.129 (P < 0.001) and 7.984 +/- 0.312 (P < 0.001) in the f irst and tenth minutes, respectively. Amiloride inhibited this 5- and 14-fold stimulation by 85% (P < 0.001). Insulin in the presence of 100 mu M ouabain stimulated Na+ influx by more than 15% (P < 0.01). Ethyl isopropylamiloride (10 mu M) 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+-depl eted cells (10.750 +/- 0.479 us. 1.045 +/- 0.100 mM; P < 0.001). Amilo ride decreased NaP-stimulated intracellular Na+ by 82% (P < 0.001). To our knowledge, this is the first report showing the presence of an in sulin-responsive and amiloride-sensitive Na+/H+ exchanger that regulat es pH(i) by a Na+-specific and pH(i)-dependent mechanism in rat adipoc ytes.