Possible role of intracellular vacuolar-ATPases in hepatocyte pH regulation.

摘要

This thesis has studied cytosolic pH regulation in suspensions of isolated hepatocyte with emphasis on the contribution of the intracellular, organellar vacuolar ATPases (V-ATPases) and their interaction with the plasma membrane Na/H exchanger in the maintenance of the steady-state cytosolic pH.; pH was measured with the fluorescent dye, BCECF and the radiolabeled tracer, DMO. To distinguish the activity of the V-ATPase and Na/H exchanger the inhibitors nitrate, amiloride, and ouabain were used. Nitrate was substituted for an equimolar amount of chloride over a concentration range of 12.5-75mM. At concentrations of 16-37.5mM, nitrate produced a cytosolic acidification attributed to inhibition of proton removal by the V-ATPase, followed by a recovery attributed to the amiloride-sensitive Na/H exchanger. Ouabain, an inhibitor of the plasma membrane Na/K-ATPase, causes an increase in cytosolic pH and a concurrent increase in chloride uptake. It is concluded that the increase in cytosolic chloride levels stimulated the V-ATPase to produce an increase in cytosolic pH removal. This conclusion is supported by the effect of nitrate, which prevented the pH increase.; Hepatocytes were given an acid load by a CO{dollar}sb2{dollar} pulse pH recovery was determined in control and inhibitor-treated cells. Control cclls recovered to an average of 68% of the initial prepulse values, whereas nitrate or ouabain reduced the recovery to 13% of the initial prepulse values, indicating that the plasma membrane Na/H exchanger and V-ATPases interact in the regulation of cytosolic pH. The pH sensitivity of the V-ATPase was also determined; it was found to be active when the cytosolic pH was 6.8 or above, but showed minimal activity below 6.8. By contrast, the hepatocyte plasma membrane Na/H exchanger was reported to show its greatest activity at cytosolic pH of 6.4 with activity decreasing until the cytosolic pH reaches 7.2. It appears that the intracellular V-ATPases do contribute to cytosolic pH regulation in the hepatocyte and that their activity overlaps that of the Na/H exchanger. (Abstract shortened by UMI.)