EFFECT OF DIABETES ON NA+-H+ EXCHANGE BY SINGLE ISOLATED HEPATOCYTES

We used the fluorescent dye 2',7'-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF) to examine intracellular pH (pH(i)) regulation in single hepatocytes isolated from control rats and rats with either spontaneous or drug-induced diabetes mellitus (DM). In the absence of CO2-HCO3-, both control and DM cells recovered from cellular acid loads applied by the NH4+ prepulse technique. Because the pH(i) recovery was blocked by either Na+ withdrawal or ethylisopropylamiloride in both control and DM cells, it was presumbly mediated by Na+-H+ exchange. In the control cells, the pH(i) threshold above which the rate of change of pH(i) (dpH(i)/dt) was zero was 7.06, and the slope of the dpH(i)/dt-pH(i) relationship was -0.030 s-1. In the DM cells, the pH(i) threshold was 7.22 and the slope was -0.017 s-1. Thus, at pH(i) values below approximately 6.9, the pH(i) recovery was slower in the DM cells. Inasmuch as we observed no difference in the cellular buffering power between control and DM cells, diabetes inhibits Na+-H+ exchange within this low pH(i) range. At pH(i) values above approximately 6.9, however, Na+-H+ exchange was apparently stimulated by diabetes. Thus diabetes induces two distinct alterations of Na+-H+ exchange, an alkaline shift in pH(i) threshold and decrease in slope. Treatment of diabetic rats with insulin for 48 h restored both Na+-H+ exchange parameters to normal. On the other hand, insulin added in vitro to DM cells for 2-5 h shifted the threshold toward the control value without affecting the slope, thus leading to a further inhibition of Na+-H+ exchange over the entire pH(i) range. In contrast, fetal calf serum added in vitro to DM cells had no effect on the threshold but increased the slope to the control value, resulting in a stimulation of Na+-H+ exchange compared with both control and DM cells over the entire pH(i) range.