Adaptation of murine inner medullary collecting duct (IMCD3) cell cultures to hypertonicity.
Recently, we have adapted IMCD3 cell cultures to survive under increasing hypertonic conditions (i.e., 600 and 900 mOsmol/kg H(2)O). In adapted cells, ATPase activity is increased by one order of magnitude, while the expression of the alpha and beta subunit is increased by a factor of 4 to 5 over controls (300 mOsmol/kg H(2)O). Corresponding increases in mRNAs were also detected. The gamma subunit has been described as being uniquely expressed in some areas of the kidney, but never in cell cultures (even those derived from kidney tissues). However, the gamma subunit was detected at the protein and mRNA levels in the adapted IMCD3 cells. In contrast to the alpha and beta subunits, the levels of gamma protein and mRNA expression continue to increase as a function of the media ion concentration. We have also demonstrated that signaling pathways that upregulate the alpha, beta, and gamma subunits are very different. Increasing concentrations of the PI3 kinase inhibitor, LY294002, resulted in a dose-dependent reduction in the expression of the gamma subunit, with total abolition at 10 micro M. However, LY294002 had no significant effect on the expression of the alpha subunit. Inhibition of the JNK2 (but not of the JNK1) pathways by dominant negative transfections abolished the upregulation of the gamma, but not the a subunit. Failure to upregulate the expression of the gamma subunit was associated with a marked decrease in cell viability upon stress.
Capasso
Juan M
JM
Department of Medicine, University of Colorado Health Sciences Center, Denver 80262, USA. juan.capasso@uchsc.edu
Rivard
Christopher J
CJ
Enomoto
Laura M
LM
Berl
Tomas
T
D000251:Adenosine Triphosphatases / Q000037:antagonists & inhibitors / Q000378:metabolism; D000818:Animals; D002460:Cell Line; D002867:Chromones / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006982:Hypertonic Solutions; D007679:Kidney Medulla / Q000166:cytology*; D007685:Kidney Tubules, Collecting / Q000166:cytology*; D007700:Kinetics; D051379:Mice; D009025:Morpholines / Q000494:pharmacology; D009994:Osmolar Concentration; D012333:RNA, Messenger / Q000235:genetics
D002867:Chromones; D004791:Enzyme Inhibitors; D006982:Hypertonic Solutions; D009025:Morpholines; D012333:RNA, Messenger; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; D000251:Adenosine Triphosphatases