To assess the effect of insulin on the Na+/ K+-ATPase expression and activity in human peritoneal mesothelial cells (HPMC).
Methods
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-sensitive86Rb uptake. To assess whether the effect of insulin was related to changes in [Na+]i the sodium influx was measured with 22Na and the activity of Na+/K+ -A TPase was assessed in the presence of amiloride. Expression of Na+/K+ -A TPaseα1’ α2 and β1-subunit mRNAs was determined by RT/PCR.
Results
Exposure of HPMC 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/104 cells) was increased from 6650±796 in control cells to 9763±1212 in HPMC exposed to 100 mU/ mL insulin (1.5-fold increase; n=4, P<0.05). In addition, incubation of HPMC with 100 mU/mL insulin resulted in a time-dependent increase in the 22Na influx. Pre-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 analysis revealed that HPMC constitutively expressed α1 and β1-subunit mRNAs while the α2-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 α1’ α2 or B1-subunit.
Conclusion
Insulin stimulates the Na+/K+-ATPase activity in HPMC in a time and dose-dependent manner. This effect appears to mediated by an increase in [Na+]i and is not related to alterations in Na+/K+-ATPase subunit mRNAs expression.
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