Application of microcalorimetry for recording basal metabolic and Na+, K+-ATPase activity in LLC-PK1 cells, a model for the renal tubular epithelial cell.
In the present study we have employed a microcalorimetric procedure to measure the heat generated by a porcine renal tubule cell line (LLC-PK1) and its Na+, K+-ATPase. Microplates with an area of 2.2 cm2 were found to be optimal in terms of producing sufficient heat and a steady-state power curve. We compared the rate of heat production by cells in suspension and on monolayers and found a much lower value in suspension, that is, 1.42+/-0.2 versus 2.54+/-0.19 microW/microg DNA. Ouabain, the specific Na+, K+-ATPase inhibitor, caused a reduction in this heat output. The maximal inhibition in cell suspensions was 40% and remained unchanged with as much as 100 microM ouabain, the highest concentration tested. With cells cultured on microplates, ouabain in the concentration interval 0.1-3 microM caused a 25% inhibition of heat output. With 25-100 microM ouabain, a 50% inhibition was observed and at higher concentrations, no further inhibition occurred. Furthermore, upon removal of ouabain, full recovery of the Na+, K+-ATPase was observed, a process that could easily be monitored by using cell monolayers cultured on microplates.
Xie
Y
Y
Department of Biochemistry, Wallenberg Laboratory, Stockholm University, Sweden.
Karlsson
H
H
DePierre
J W
JW
Nässberger
L
L
D000818:Animals; D001481:Basal Metabolism; D002151:Calorimetry / Q000379:methods*; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D004847:Epithelial Cells / Q000378:metabolism; D007684:Kidney Tubules / Q000378:metabolism*; D018374:LLC-PK1 Cells; D008832:Microchemistry; D010042:Ouabain / Q000494:pharmacology*; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism*; D013535:Suspensions; D013552:Swine
D004791:Enzyme Inhibitors; D013535:Suspensions; D010042:Ouabain; D000254:Sodium-Potassium-Exchanging ATPase