Endothelin receptor in osteoblastic cells is coupled to multiple messenger signals.

We analyzed the functional characteristics of endothelin (ET) peptides in the osteoblastic UMR-106 cells by studying receptor binding as well as dose-response curves for ET-1 and ET-3 on two biological responses: 1) induction of Ca2+ transients and 2) activation of the Na(+)-H+ exchanger. ET specifically binds to a single class of receptor with a rank order of affinity ET-1 >> ET-3. ET-1 and ET-3 dose dependently stimulated a rise in intracellular Ca2+ ([Ca2+]i), with ET-1 being two orders of magnitude more potent than ET-3 [50% effective concentration (EC50) = 8 x 10(-10) and 9 x 10(-8) M for ET-1 and ET-3, respectively; P < 0.01]. The effect of ET-1 on [Ca2+]i was 90% inhibitable by the ETA antagonist BQ-123. The activity of Na(+)-H+ exchange was studied by using the pH-sensitive dye 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein as well as by 22Na+ fluxes. ET-1 and ET-3 activated the exchange in a concentration-dependent manner and with similar potencies (EC50 approximately 10(-10) M). The action of ETs on Na(+)-H+ exchange was mimicked neither by phorbol esters nor by Ca2+ ionophores. It was, however, blocked by BQ-123 as well as by the protein tyrosine kinase inhibitor genistein. We conclude that in UMR-106 cells, a single ET receptor subtype is coupled to multiple effectors, a Ca2+ message system and a tyrosine-kinase system which, in turn, activates the Na(+)-H+ exchanger.

Acute phosphate depletion inhibits the Na+/H+ antiporter in a cultured renal cell line.

We studied the effect of acute Pi depletion on the regulation of intracellular pH (pHi) in the OK opossum kidney cell line by using the pH-sensitive dye 2'7'-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF). Cell recovery from an NH4Cl acid load in HCO3-free buffer disclosed an Na(+)-dependent component blocked by amiloride and a smaller Na(+)-independent component that increased on exposure of the cells to a high-K+ buffer. After 24-h incubation of the cells in phosphate-free medium, pHi recovery by the Na+/H+ exchanger was markedly inhibited, whereas the Na(+)-independent pHi recovery was not affected. The inhibition of Na+/H+ exchange was reversible on correction of cellular Pi deficit. A similar phenomenon was observed when cellular Pi depletion was induced by acute exposure (min) to fructose. Pi depletion shifted the pHi dependence of the exchanger and also reduced its maximal activity. Time-course studies revealed that the effect of Pi depletion could not be attributed to attenuation of Na(+)-K(+)-adenosinetriphosphatase activity and resultant diminution of the transmembrane gradient for the Na+ influx. We conclude that acute Pi depletion in cultured proximal tubular cells leads to reversible inhibition of the Na+/H+ exchanger. This in vitro finding may relate to the in vivo observation of impaired HCO3 reabsorption and bicarbonaturia in acute Pi depletion.