Regulation of cytosolic ph in bovine parathyroid cells: Effect of fluoride

In the present investigation, sodium fluoride (NaF) was employed to explore the role of guanine nucleotidebinding proteins (G-proteins), protein kinase-C, or cytosolic calcium ([Ca]i) in the regulation of cytosolic pH ([pH]i) in dispersed bovine parathyroid cells, using the pH-sensitive fluorescent dye BCECF. When cells acidified by nigericin in Nafree medium were resuspended in Na-containing buffer, [pH]i returned to basal levels. This recovery was blocked by continued removal of Na⁺ or the addition of amiloride. NaF (10 mM) increased [³²P]phosphate incorporation into phosphatidylinositol bisphosphate, suggesting an increase in phosphatidylinositol bisphosphate turnover. NaF caused an initial acidification, followed by an alkaline recovery in a dose-dependent manner (1†10 mM). Amiloride blocked the NaF-induced alkaline recovery. The protein kinase-C activator phorbol 12-myristate 13-acetate (10^-7 M) caused cytosolic alkalinization, while the protein kinase- C inhibitor H7 (6 Ã-10^-5 M) significantly inhibited the NaF-induced alkaline recovery. Pertussis toxin (1 μg/nA) did not affect the NaF-induced changes in [pH]i. Removal of extracellular Ca²⁺ with EGTA blocked the NaF-induced increase in [Ca]i and alkaline recovery. Ionomycin (5 Ã-10^-7 M) caused cytosolic alkalinization, but pretreatment with EGTA inhibited the ionomycin-induced cytosolic alkalinization. The present studies clearly demonstrated the presence of an amiloride-sensitive Na⁺/H⁺ exchanger in parathyroid cells. Our findings suggest that the NaF-induced cytosolic alkaline recovery was via two complementing pathways: 1) activation of protein kinase-C, followed by stimulation of a Na⁺/H⁺ exchanger, and 2) existence of extracellular calcium and/or an increase in [Ca]i.