Regulation of pH in rat papillary tubule cells in primary culture.

Abstract

To investigate the mechanisms responsible for urinary acidification in the terminal nephron, primary cultures of cells isolated from the renal papilla were grown as monolayers in a defined medium. Morphologically, cultured cells were epithelial in type, and similar to collecting duct principal cells. Cell pH measured fluorometrically in monolayers grown on glass slides showed recovery from acid loads in Na+-free media. Recovery was inhibited by cyanide, oligomycin A, and N-ethylmaleimide. Cyanide and oligomycin inhibited recovery less in the presence than in the absence of glucose. When cells were first acid loaded in a Na+-free medium and then exposed to external Na+, pH recovery also took place. This recovery exhibited first-order dependence on Na+ concentration and was inhibited by 5- (N-ethyl-N-isopropyl)amiloride. These studies demonstrate that in culture, collecting duct principal cells possess at least two mechanisms for acid extrusion: a proton ATPase and an Na+-H+ exchanger. The former may be responsible for some component of the urinary acidification observed in the papillary collecting duct in vivo; the role of the latter in acidbase transport remains uncertain.

Dual regulatory mechanisms of proton transport in rat papillary collecting duct cells in culture.

“…Especially, the value of Km in luminal-membrane vesicles from rabbit proximal convoluted and straight tubules was 29 mM (JACOBSEN et al, 1986). In contrast, KLEINMAN et al (1987) have revealed that the value of Km (46mM) was more than two fold higher than Km values of the current studies. The individual values of Km ranged from 9 to 115 mM; therefore, their data do not seem reliable enough.…”

“…LABELLE (1984) has revealed a I(5 value of 60 µM for amiloride in vesicles from rabbit medullary tissue. Although KLEINMAN et al (1987) have demonstrated a K; value of 0.8 µM for 5-(N-ethyl-N-isopropyl) amiloride, this compound has been shown to be a 50 to 140 times more potent inhibitor for Na+-H+ exchange systems in a variety of tissues (VIGNE et al, 1983). In addition, our results suggest that the effect of amiloride on Na+-H+ exchanger in PCD cells is quite different compared with other mammalian cells.…”

“…4B) reveal that two major processes are involved in cytoplasmic pH regulation in the absence of HCO3 -in PCD cells: a Na+-H + exchange and a plasma membrane proton ATPase. A recent study has also demonstrated that in culture, collecting duct principal cells possess at least two mechanisms for acid extrusion (KLEINMAN et al, 1987). On the other hand, outer medullary collecting duct cell suspensions freshly isolated from rabbit kidneys failed to demonstrate Na + dependent of pH regulation in studies performed utilizing techniques similar to those used in the current experiments (ZEIDEL et al, 1986).…”

“…Although the exchangers appear to be less active under normal conditions than acid-loaded cells, the Na + -H + exchanger might contribute in a part to the regulation of resting pH; in HCO3 --free solution in PCD cells. In contrast, KLEINMAN et al (1987) were unable to demonstrate significant Na +-H + exchange activity at resting state under similar experimental conditions. An ATP-driven proton pump has been described in toad urinary bladder (AL-AWQATI et al, 1977), bovine renal medulla (CLUCKS and AL-AWQATI, 1984), rat renal cortex and medulla, rabbit medullary collecting duct cells (LAsKI and KURTZMAN, 1983;LOMBARD et al, 1983;STONE et al, 1983;SABOLIC and BURCKHARDT, 1986;ZEIDEL et al, 1986), and human renal medulla (DIAz-DIAz et al, 1986).…”

“…Based on these observations, it is highly probable that the rat PCD cells in culture consist of principal cells in the current experiments. Although we have not studied these problems, a recent study has demonstrated that the cultured cells were principal cells rather than intercalated cells (KLEINMAN et al, 1987).…”