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).
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Department of Biochemistry and Molecular Biology, Pennsylvania State University, University Park 16802, USA.
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D000143:Acids / Q000378:metabolism*; D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D002645:Chickens; D002772:Cholera Toxin / Q000494:pharmacology; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000187:drug effects / Q000378:metabolism; D019205:GTP-Binding Protein alpha Subunits, Gs / Q000187:drug effects / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000187:drug effects / Q000276:immunology / Q000378:metabolism*; D015151:Immunoblotting; D018021:Lithium Chloride / Q000494:pharmacology; D008297:Male; D009206:Myocardium / Q000378:metabolism; D010006:Osteoblasts / Q000378:metabolism; D010010:Osteoclasts / Q000187:drug effects / Q000378:metabolism*; D010281:Parathyroid Hormone / Q000378:metabolism / Q000494:pharmacology*; D037342:Pertussis Toxin; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
D000143:Acids; D010281:Parathyroid Hormone; D010566:Virulence Factors, Bordetella; D002772:Cholera Toxin; D037342:Pertussis Toxin; D019204:GTP-Binding Proteins; D019206:GTP-Binding Protein alpha Subunits, Gi-Go; D019205:GTP-Binding Protein alpha Subunits, Gs; D018021:Lithium Chloride