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Regulation of Cytosolic pH in Bovine Parathyroid Cells: Effect of Fluoride^*

TOSHITSUGU SUGIMOTO, ROBERT CIVITELLI, CYNTHIA RITTER, EDUARDO SLATOPOLSKY and JEREMIAH MORRISSEY

Renal Division, Department of Medicine, Washington University School of Medicine St. Louis, Missouri 63110

Address all correspondence and requests for reprints to: Jeremiah Morrissey, Washington University School of Medicine, Renal Division, Box 8126, 4949 Barnes Hospital Plaza, St. Louis, Missouri 63110.

Abstract

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 x 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 x 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. (Endocrinology 124: 149–156,1989)

Footnotes

^* This work was supported by USPHS NIDDK Grants DK-09976, DK-30178, and DK-07126.

Research Fellow of the St. Louis Kidney Foundation.

Received May 18, 1988.