Serum regulates Na+/H+ exchange in Caco-2 cells by a mechanism which is dependent on F-actin.
Regulation of Na+/H+ exchange by fetal bovine serum was studied in Caco-2 cells, an established cell line derived from a human colon carcinoma. Cells were grown as polarized monolayers on collagen-coated filters and intracellular pH measured fluorometrically with 2',7'-bis(2-carboxymethyl)-5,6-carboxyfluorescein. Na+/H+ exchange was reduced 64% when cells were deprived of serum for 4 h. In contrast to other cell types, readdition of serum for 10 min did not activate Na+/H+ exchange; however, readdition of serum for 4 h restored Na+/H+ exchange to control values. This long-term effect of serum on Na+/H+ exchange activity could not be explained by changes in intracellular buffering capacity or intracellular [Na+]. 4-h serum deprivation reduced the K(t) of the exchanger for external Na+ from 21 to 6 mM, and reduced the V(max) by 57%, but did not alter the IC50 for amiloride in the presence of 140 mM Na+. Inhibition of protein synthesis with cycloheximide (5 microM) did not alter the effect of serum removal or readdition on Na+/H+ exchange. Low temperature (13 degrees C) completely prevented the inhibition of Na+/H+ exchange caused by the removal of serum. In addition, once Na+/H+ exchange was inhibited by serum removal at 37 degrees C, maintaining cells at 13 degrees C also blocked the recovery of Na+/H+ exchange caused by serum readdition. Conversely, cytochalasin D (0.1-20 microM) blocked the reduction of Na+/H+ exchange which occurred due to 4-h serum deprivation, but did not block the restoration of Na+/H+ exchange when the cells were re-exposed to serum for a further 4 h. Colchicine (20 microM) did not alter the effect of serum removal or readdition. These data suggest that serum regulates Na+/H+ exchange activity by a posttranslational mechanism which is dependent on F-actin.
Watson
A J
AJ
Department of Physiology, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205.
Levine
S
S
Donowitz
M
M
Montrose
M H
MH
D000199:Actins / Q000378:metabolism*; D000584:Amiloride / Q000494:pharmacology; D001769:Blood; D002352:Carrier Proteins / Q000378:metabolism*; D003078:Colchicine / Q000494:pharmacology; D003513:Cycloheximide / Q000494:pharmacology; D015638:Cytochalasin D / Q000494:pharmacology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D007930:Leucine / Q000378:metabolism; D011500:Protein Synthesis Inhibitors; D017923:Sodium-Hydrogen Exchangers; D014407:Tumor Cells, Cultured
D000199:Actins; D002352:Carrier Proteins; D011500:Protein Synthesis Inhibitors; D017923:Sodium-Hydrogen Exchangers; D015638:Cytochalasin D; D000584:Amiloride; D003513:Cycloheximide; D007930:Leucine; D003078:Colchicine