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 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-sensitive 86Rb 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(+)-ATPase was assessed in the presence of amiloride. Expression of Na+/K(+)-ATPase alpha 1,alpha 2 and beta 1-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 uptake (cpm/10(4) 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 1mM amiloride reduced the activity of Na+/K(+)-ATPase but did not block the stimulatory effect of insulin. RT/PCR analysis 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.


CONCLUSIONS
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.
Witowski
J
J
Department of Pathophysiology, University Medical School, Poznan, Poland.
Breborowicz
A
A
Topley
N
N
Martis
L
L
Knapowski
J
J
Oreopoulos
D G
DG
D002478:Cells, Cultured; D004353:Drug Evaluation, Preclinical; D004848:Epithelium / Q000187:drug effects / Q000201:enzymology; D006801:Humans; D007004:Hypoglycemic Agents / Q000494:pharmacology*; D007328:Insulin / Q000494:pharmacology*; D010537:Peritoneum / Q000166:cytology / Q000187:drug effects* / Q000201:enzymology; D012333:RNA, Messenger / Q000096:biosynthesis; D000254:Sodium-Potassium-Exchanging ATPase / Q000235:genetics / Q000378:metabolism*; D013268:Stimulation, Chemical
D007004:Hypoglycemic Agents; D007328:Insulin; D012333:RNA, Messenger; D000254:Sodium-Potassium-Exchanging ATPase