AN NMR-STUDY OF CELLULAR PHOSPHATES AND MEMBRANE-TRANSPORT IN RENAL PROXIMAL TUBULES

Technical limitations in the measurement of cellular phosphates have hindered studies of interrelationships between cellular P-i, its transport, and its metabolism in renal proximal tubule (PT) cells. We have developed a noninvasive P-31-nuclear magnetic resonance (NMR) probe-perifusion system to measure cellular P-i and have utilized this system to investigate relationships in canine PT cells between the membrane transport and the cellular content of P-i. With 1.2 mM P-i in the extracellular medium, the cellular P-i content of PT averaged 4.94 +/- 0.55 nmol/mg protein. Inhibition of P-i uptake by removal of extracellular P-i rapidly decreased all cellular phosphate compounds to values that were between 55 and 85% of control. Partial replacement of extracellular P-i (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 P-i or organic phosphates. Reducing the basolateral membrane potential with the addition of barium chloride increased cellular P-i content by nearly 30%. Maximal contents of cellular P-i and ATP were achieved at 0.4 mM P-i in the presence of an inwardly directed Na+ gradient and at 0.8 mM P-i in its absence. These data indicate that cellular P-i 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 P-i content over a physiological range.