Chloride fluxes activated by parathyroid hormone in human erythrocytes

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