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 Gs-proteins, mimicked PTH stimulation. Pertussis toxin, which prevents receptor interaction with Gi- and Go-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 alpha subunits, revealed a 48 kDa Gs alpha, a 41 kDa Go alpha, a 34 kDa Gi alpha-3, and a unique 68 kDa G alpha 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 Gs alpha (48 kDa) and a Go alpha (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 Gs and a pertussis toxin-sensitive G-protein (Go and/or Gi alpha-3).