INSULIN STIMULATES THE ACTIVITY OF NA+ K+-ATPASE IN HUMAN PERITONEAL MESOTHELIAL CELLS/

Objective: To assess the effect of insulin on the Na+/K+-ATPase expres sion and activity in human peritoneal mesothelial cells (HPMC). Method s: HPMC were isolated from the omental tissue of non-uremic patients, grown to confluence and rendered quiescent by serum deprivation for 24 hours. The activity of Na+/K+-ATPase was determined by measuring the ouabain-sensitive Rb-86 uptake. To assess whether the effect of insuli n was related to changes in [Na+](i) the sodium influx was measured wi th Na-22 and the activity of Na+/K+-ATPase was assessed in the presenc e of amiloride. Expression of Na+/K+-ATPase alpha(1), alpha(2), and be ta(1)-subunit mRNAs was determined by RT/PCR. Results: Exposure of HPM C to insulin resulted in a time- and dose-dependent increase in the Na +/K+-ATPase activity. After 60 minutes the ouabain-sensitive 86Rb UP, take (cpm/10(4) cells) was increased from 6650 +/- 796 in control cell s to 9763 +/- 1212 in HPMC exposed to 100 mU/ mL insulin (1.5-fold inc rease; n = 4, P < 0.05). In addition, incubation of HPMC with 100 mU/m L insulin resulted in a time-dependent increase in the Na-22 influx. P re-exposure of HPMC to 1 mM amiloride reduced the activity of Na+/K+-A TPase but did not block the stimulatory effect of insulin. RT/PCR anal ysis revealed that HPMC constitutively expressed alpha(1)- and beta(1) -subunit mRNAs while the alpha(2)-subunit mRNA was barely detectable. Exposure of HPMC to insulin for up to 24 hours was not associated with any changes in the expression of either alpha(1), alpha(2) or beta(1) -subunit. Conclusion: Insulin stimulates the Na+/K+-ATPase activity in HPMC in a time- and dose-dependent manner. This effect appears to med iated by an increase in [Na+](i) and is not related to alterations in Na+/K+-ATPase subunit mRNAs expression.