Effect of P2 Nucleotide Receptor Activation on Phosphate Transport in Rabbit Proximal Tubular Cells

Background:Extracellular nucleotides act as agonists to regulate a broad range of physiological processes by interacting with P2 receptors in various tissues including the kidney tubules. This study was undertaken to evaluate the effect of P2 receptor activation on PTH-dependent regulation of phosphate transport in the renal proximal tubular cells. Methods:Proximal tubular cells were isolated from the rabbit kidney and grown as monolayers on 24 well culture plates. Phosphate uptake was determined by measuring the uptake of radiolabeled phosphate into cell monolayers. Cyclic AMP content was determined by radioimmunoassay using [cAMP assay kit. Results:Activation of P2 receptors with ATP exerted differential effects on phosphate uptake and cAMP generation. In the absence of PTH, it inhibited phosphate uptake and stimulated cAMP generation. In contrast, in the presence of PTH, it attenuated PTH-induced stimulation of cAMP generation and inhibition of phosphate uptake. The profile of the effects of different P2 agonists suggested that P2- and P2-like receptors are involved in the effects of ATP. The effect of ATP to interfere with the PTH-induced regulation was significantly blocked by calphostin C, pertussis toxin or PKC-depletion, whereas, the effects of ATP in the absence of PTH were abolished by indomethacin. Conclusion:Our results suggest that PKC-dependent modification of Gi proteins and, subsequently, reduced responsiveness of adenylate cyclases is responsible for the attenuating effect of ATP on the PTH-dependent regulation of phosphate transport in rabbit proximal tubule cells.