Chobanian M C, Anderson M E, Brazy P C
Department of Pediatrics, University of Wisconsin-Madison 53792.
Am J Physiol. 1995 Mar;268(3 Pt 2):F375-84. doi: 10.1152/ajprenal.1995.268.3.F375.
Technical limitations in the measurement of cellular phosphates have hindered studies of interrelationships between cellular Pi, its transport, and its metabolism in renal proximal tubule (PT) cells. We have developed a noninvasive 31P-nuclear magnetic resonance (NMR) probe-perifusion system to measure cellular Pi and have utilized this system to investigate relationships in canine PT cells between the membrane transport and the cellular content of Pi. With 1.2 mM Pi in the extracellular medium, the cellular Pi content of PT averaged 4.94 +/- 0.55 nmol/mg protein. Inhibition of Pi uptake by removal of extracellular Pi rapidly decreased all cellular phosphate compounds to values that were between 55 and 85% of control. Partial replacement of extracellular Pi (0.4 mM) increased cellular phosphates up to 84-100% of control values. Inhibition of Na(+)-K(+)-adenosinetriphosphatase uptake by the addition of ouabain failed to change either cellular Pi or organic phosphates. Reducing the basolateral membrane potential with the addition of barium chloride increased cellular Pi content by nearly 30%. Maximal contents of cellular Pi and ATP were achieved at 0.4 mM Pi in the presence of an inwardly directed Na+ gradient and at 0.8 mM Pi in its absence. These data indicate that cellular Pi content in canine PT is regulated by Na(+)-dependent and -independent transport mechanisms and by the membrane potential across the basolateral membrane. Lastly, cellular ATP content was found to be directly proportional to the cellular Pi content over a physiological range.
细胞磷酸盐测量中的技术限制阻碍了对肾近端小管（PT）细胞中细胞内无机磷酸盐（Pi）、其转运及其代谢之间相互关系的研究。我们开发了一种非侵入性的31P-核磁共振（NMR）探针灌流系统来测量细胞内Pi，并利用该系统研究犬类PT细胞中Pi的膜转运与细胞内含量之间的关系。细胞外培养基中Pi浓度为1.2 mM时，PT细胞内Pi含量平均为4.94±0.55 nmol/mg蛋白。去除细胞外Pi抑制Pi摄取后，所有细胞磷酸盐化合物迅速降至对照值的55%至85%之间。部分替代细胞外Pi（0.4 mM）可使细胞磷酸盐增加至对照值的84 - 100%。添加哇巴因抑制Na(+)-K(+)-三磷酸腺苷酶摄取未能改变细胞内Pi或有机磷酸盐。添加氯化钡降低基底外侧膜电位可使细胞内Pi含量增加近30%。在存在内向Na+梯度时，细胞内Pi和ATP的最大含量在0.4 mM Pi时达到；在不存在内向Na+梯度时，在0.8 mM Pi时达到。这些数据表明，犬类PT细胞内Pi含量受Na+依赖性和非依赖性转运机制以及基底外侧膜跨膜电位的调节。最后，发现在生理范围内细胞内ATP含量与细胞内Pi含量成正比。
