Chloride fluxes activated by parathyroid hormone in human erythrocytes

We used the chloride fluorescent probe, 6-methoxy-N-(3-sulfopropyl)quinolinium (SPQ), to study chloride fluxes in human erythrocytes. The SPQ load was made by hypotonic buffer (150 mOsm, 10 min). Intracellular fluorescence was monitored continuously at 360 nm excitation and 410 nm emission wavelengths. The leakage of SPQ out of cells was <5% h(-1) and the Stern-Volmer constant for quenching of intracellular SPQ by Cl was 0.023 mM(-1). The time course of intracellular [Cl] was measured and the influence of PTH, forskolin, and phorbol 12-myristate 13-acetate (PMA) on erythrocyte Cl transport was examined. The results establish a direct method to measure intracellular [CI] continuously in erythrocytes and show that PTH induces a Cl efflux inhibited by 4,4'-diisothiocyanatostilbene-2,2'-disulfonate, This effect was similar to those induced by forskolin, which stimulates cAMP generation, and by PMA, which stimulates protein kinase C. (C) 2000 Academic Press.