Sarcoplasmic-reticulum biogenesis in contraction-inhibited skeletal-muscle cultures.
We have previously shown that inhibition of the spontaneous contractile activity of cultured embryonic-chick skeletal-muscle fibres with tetrodotoxin (TTX) leads to decreased sarcoplasmic-reticulum Ca(2+)-transport rates and steady-state concentrations of the high-energy Ca(2+)-ATPase phosphoenzyme intermediate [Charuk & Holland (1983) Exp. Cell Res. 144, 143-157]. In the present study we used a monoclonal antibody to the Ca(2+)-ATPase to show that there is a decreased amount of enzyme accumulated by contraction-inhibited myotubes. Indirect immunofluorescence microscopy using the monoclonal antibody to the Ca(2+)-ATPase also revealed a disordered subcellular organization of the sarcotubular system in contraction-inhibited myotubes. The biogenesis of sarcoplasmic-reticulum proteins in TTX-paralysed myofibres was studied by labelling cells with [35S]methionine before isolation of the active Ca(2+)-pump membrane fraction. Protein turnover was selectively increased in that fraction from TTX-treated muscle cultures. Electrophoretic analysis and quantitative fluorography confirmed that decreased accumulation of the Ca(2+)-ATPase enzyme in contraction-inhibited myotubes was associated with increased turnover of this protein. The present results demonstrate that biogenesis of the sarcoplasmic-reticulum Ca(2+)-ATPase is regulated by the contractile activity of skeletal-muscle fibres.
Charuk
J H
JH
Department of Neurology and Neurosurgery, Montreal Neurological Institute, Quebec, Canada.
Guerin
C
C
Holland
P C
PC
D000818:Animals; D001692:Biological Transport; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism; D000252:Calcium-Transporting ATPases / Q000378:metabolism; D002413:Cations, Divalent; D002478:Cells, Cultured; D002642:Chick Embryo; D004591:Electrophoresis, Polyacrylamide Gel; D005455:Fluorescent Antibody Technique; D008715:Methionine / Q000378:metabolism; D008856:Microscopy, Fluorescence; D009119:Muscle Contraction; D009132:Muscles / Q000187:drug effects / Q000502:physiology*; D009218:Myosins / Q000378:metabolism; D012519:Sarcoplasmic Reticulum / Q000502:physiology*; D013779:Tetrodotoxin / Q000494:pharmacology
D002413:Cations, Divalent; D013779:Tetrodotoxin; D008715:Methionine; D009218:Myosins; D000252:Calcium-Transporting ATPases; D002118:Calcium