1 alpha,25-(OH)2-vitamin D3 stimulates the adenylyl cyclase pathway in muscle cells by a GTP-dependent mechanism which presumably involves phosphorylation of G alpha i.
To further understand the mechanism underlying 1,25(OH)2D3 activation of the cAMP pathway, the effect of the hormone on adenylyl cyclase (AC), GTPase and protein kinase A (PKA) activities as well as on the phosphorylation of G alpha i was studied in membranes from chick skeletal muscle cells. The sterol stimulated AC activity in a dose (0.1-10 nM) and time (1-5 min.) dependent fashion, provided GTP (10 microM) was present in the assay. High affinity GTPase activity was unaffected by the hormone. In the absence of GTP or in the presence of Mn2+ (20 mM), 1,25(OH)2D3 effects on AC were abolished. PKA activity was increased (+120%) in cells pretreated (1 nM, 5 min.) with the sterol. Moreover, immunoprecipitation of G alpha i from [32P]-labeled myoblast membranes showed that 5 min. exposure to 1 nM 1,25(OH)2D3 increased (1.5-2 fold) the phosphorylation of its alpha subunit. The present data suggest that in muscle cells, 1,25(OH)2D3 activates AC by a non direct, GTP-dependent action which could imply amelioration of Gi function by sterol-induced alpha i phosphorylation.
Vazquez
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G
Dpto. Biología, Bioquímica y Farmacia, Universidad Nacional del Sur, San Juan, Bahía Blanca, Argentina.
de Boland
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Boland
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D000262:Adenylyl Cyclases / Q000378:metabolism*; D000818:Animals; D002117:Calcitriol / Q000494:pharmacology*; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D002478:Cells, Cultured; D002642:Chick Embryo; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D020558:GTP Phosphohydrolases / Q000378:metabolism; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000378:metabolism*; D018482:Muscle, Skeletal / Q000166:cytology / Q000378:metabolism*; D010766:Phosphorylation; D015398:Signal Transduction / Q000502:physiology
D006160:Guanosine Triphosphate; D017868:Cyclic AMP-Dependent Protein Kinases; D020558:GTP Phosphohydrolases; D019206:GTP-Binding Protein alpha Subunits, Gi-Go; D000262:Adenylyl Cyclases; D002117:Calcitriol