The effects of monensin on the abundance of mRNA(alpha) and of sodium pumps in human cultured cells.
HeLa or MRC5-VI cells were grown for up to 1 day in media containing monensin at concentrations up to 10 microM. We measured the sodium pump density of the plasma membrane with [3H]ouabain and the mRNA for the alpha-subunit of the pump by hybridization to a cDNA probe. The sodium and potassium concentrations were measured under similar conditions, and in some experiments the rate of internalization of the sodium pumps estimated by using [3H]ouabain uptake into the cell. We found that the relationship between sodium pump density and [Na+]i was well described by Michaelis-Menten kinetics with a Km of 12 mM-[Na+]i and a Vmax twice the normal value. There was no obvious relationship between cell potassium and pump density. The relationship between sodium pump density and [Na+]i was the same as that found by growing the cells in low-potassium medium, so we conclude that the manner of raising [Na+]i is not important, merely the final value. In conditions of raised intracellular sodium there was an increase in the mRNA for the alpha-subunit of the pump, but there was no slowing of the rate of internalization of the pumps from the plasma membrane. We conclude that the increased density of pumps is due to an increased synthesis rather than a decreased internalization rate of pumps, suggesting that the cell can control the method of upregulation.
Cramb
G
G
Department of Biology and Preclinical Medicine, University of St Andrews, Fife.
Cutler
C P
CP
Lamb
J F
JF
McDevitt
T
T
Ogden
P H
PH
Owler
D
D
Voy
C
C
D002462:Cell Membrane / Q000032:analysis; D002478:Cells, Cultured; D006367:HeLa Cells; D006801:Humans; D066298:In Vitro Techniques; D008985:Monensin / Q000494:pharmacology*; D015221:Potassium Channels / Q000187:drug effects; D012333:RNA, Messenger / Q000378:metabolism*; D015222:Sodium Channels / Q000187:drug effects*; D013997:Time Factors
D015221:Potassium Channels; D012333:RNA, Messenger; D015222:Sodium Channels; D008985:Monensin