Martínez G M, Martínez-Zaguilán R, Gillies R J
Department of Biochemistry, University of Arizona, College of Medicine, Tucson 85724.
J Cell Physiol. 1994 Oct;161(1):129-41. doi: 10.1002/jcp.1041610116.
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.
用酵母H(+)-ATP酶转染的NIH-3T3细胞（RN1a细胞）具有致瘤性（佩罗纳和塞拉诺，1988年，《自然》，334:438）。我们之前已经表明，RN1a细胞在生理条件下维持细胞内pH值（pHin）长期处于较高水平。我们还表明，RN1a细胞生长不依赖血清，细胞内Ca2+浓度（[Ca2+]in）较高，且糖酵解速度比未转化的对应细胞更快（吉利斯等人，《美国国家科学院院刊》，1990年，87:7414；吉利斯等人，《细胞生理学与生物化学》，1992年，2:159）。本研究旨在了解RN1a细胞及其未转化的对应细胞NIH-3T3细胞中糖酵解、pHin和[Ca2+]in之间的相互关系。我们的数据表明，RN1a细胞中观察到的较高糖酵解速率是由于存在低亲和力葡萄糖转运体。因此，在高葡萄糖浓度下，RN1a细胞中的糖酵解速率更高。此外，RN1a细胞中葡萄糖利用的最大速度（Vmax）比NIH-3T3细胞高多达六倍，这表明RN1a细胞中葡萄糖转运体的数量比NIH-3T3细胞多。向NIH-3T3细胞中添加葡萄糖会导致pHin和[Ca2+]in适度降低。相比之下，RN1a细胞对葡萄糖的反应是pHin大幅下降，随后[Ca2+]in大幅下降。添加葡萄糖后观察到的[Ca2+]in下降可能是由于糖酵解激活了Ca(2+)-ATP酶，因为Ca2+的下降被Ca2+ ATP酶抑制剂毒胡萝卜素和环匹阿尼酸消除。向ATP耗尽的细胞中添加葡萄糖会导致[Ca2+]in下降，这表明糖酵解提供的ATP被该泵利用。
