Multiple G-protein involvement in parathyroid hormone regulation of acid production by osteoclasts

The involvement of multiple G-proteins in parathyroid hormone regulation of acid production was demonstrated in a highly enriched osteoclast population. Osteoclasts were isolated from the endosteum of 2.5 to 3-week-old chicken tibia using sequential enzymatic digestion. Single cell analysis of acid production was accomplished using microscope photometry and vital staining with acridine orange, a hydrogen ion concentration sensitive fluorescent dye. Lithium chloride, an uncoupler of G-proteins from their respective receptors, blocked parathyroid hormone stimulated production of acid. Cholera toxin, which permanently activates G_s-proteins, mimicked PTH stimulation. Pertussis toxin, which prevents receptor interaction with G_i- and G_o-proteins, blocked both 10 ⁸ M and 10 ¹¹ M PTH stimulated acid production, suggesting that the pertussis toxin-sensitive G-protein is utilized at both PTH concentrations. Immunoblots of osteoclast plasma membrane proteins, using a panel of antibodies generated against specific G-protein α subunits, revealed a 48 kDa G_sα, a 41 G_oα, a 34 kDa G_iα-3, and a unique 68 kDa Gα subunit, with the 41 kDa and 34 kDa bands being the most intense. Immunoblots of osteoblast plasma membrane proteins had a substantially different profile with the most intense bands being a G_sα (48 kDa) and a G_oα (36 and 38 kDa). The studies suggest the utilization of at least two different G-proteins in the parathyroid hormone regulation of acid formation by osteoclasts, a G_s and a pertussis toxin-sensitive G-protein (G_o and/or G_iα-3). J. Cell. Biochem. 64:161–170. © 1997 Wiley-Liss, Inc.