Effect of glucose on pHin and [Ca2+]in in NIH-3T3 cells transfected with the yeast P-type H(+)-ATPase.
NIH-3T3 cells transfected with yeast H(+)-ATPases (RN1a cells) are tumorigenic (Perona and Serrano, 1988, Nature, 334:438). We have previously shown that RN1a cells maintain a chronically high intracellular pH (pHin) under physiological conditions. We have also shown that RN1a cells are serum-independent for growth, maintain a higher intracellular Ca2+ ([Ca2+]in), and glycolyze more rapidly than their non-transformed counterparts (Gillies et al., Proc. Natl. Acad. Sci., 1990, 87:7414; Gillies et al., Cell. Physiol. Biochem., 1992, 2:159). The present study was aimed to understand the interrelationships between glycolysis, pHin, and [Ca2+]in in RN1a cells and their non-transformed counterparts, NIH-3T3 cells. Our data show that the higher rate of glycolysis observed in RN1a cells is due to the presence of low affinity glucose transporters. Consequently, the higher rate of glycolysis is exacerbated at high glucose concentration in RN1a cells. Moreover, the maximal velocity (Vmax) for glucose utilization is up to sixfold higher in RN1a cells than in the NIH-3T3 cells, suggesting that the number of glucose transporters is higher in RN1a than NIH-3T3 cells. Glucose addition to NIH-3T3 cells results in modest decreases in both pHin and [Ca2+]in. In contrast, RN1a cells respond to glucose with a large decrease in pHin, followed by a large decrease in [Ca2+]in. The decrease in [Ca2+]in observed upon glucose addition is likely due to activation of Ca(2+)-ATPase by glycolysis, since the Ca2+ decrease is abolished by the Ca2+ ATPase inhibitors thapsigargin and cyclopiazonic acid. Glucose addition to ATP-depleted cells results in a decrease in [Ca2+]in, suggesting that ATP furnished by glycolysis is utilized by this pump.
Martínez
G M
GM
Department of Biochemistry, University of Arizona, College of Medicine, Tucson 85724.
Martínez-Zaguilán
R
R
Gillies
R J
RJ
D016475:3T3 Cells; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors / Q000378:metabolism; D002461:Cell Line, Transformed; D005947:Glucose / Q000494:pharmacology*; D006019:Glycolysis; D006863:Hydrogen-Ion Concentration; D007425:Intracellular Membranes / Q000378:metabolism; D051379:Mice; D009994:Osmolar Concentration; D006180:Proton-Translocating ATPases / Q000037:antagonists & inhibitors / Q000145:classification / Q000235:genetics*; D017923:Sodium-Hydrogen Exchangers / Q000037:antagonists & inhibitors; D014162:Transfection; D015003:Yeasts / Q000201:enzymology / Q000235:genetics*
D017923:Sodium-Hydrogen Exchangers; D006180:Proton-Translocating ATPases; D000252:Calcium-Transporting ATPases; D005947:Glucose; D002118:Calcium