Effects of ciliary neurotrophic factor (CNTF) on protein turnover in cultured muscle cells.
The goal of the study was to evaluate the mechanism by which ciliary neurotrophic factor (CNTF) regulated protein metabolism in skeletal muscle. L8 myotubes were cultured and effects of various times and doses of CNTF on protein synthesis and degradation were evaluated. Effects of CNTF on turnover of specific pools of proteins (myofibrillar and non-myofibrillar) were also evaluated. Protein synthesis was assayed by incorporation of radioactive tyrosine into muscle proteins. Degradation was assessed by release of labelled tyrosine from pre-labelled myotubes. Effects of CNTF on protein turnover were found to be time- and dose-dependent. CNTF (1 and 10 ng/ml) increased myofibrillar protein synthesis after 12 h of exposure but had no effect on non-myofibrillar protein synthesis. Longer exposures of CNTF (24 h) reduced non-myofibrillar protein synthesis and had no effect on myofibrillar protein synthesis. High concentrations of CNTF (10 and 20 ng/ml) reduced myofibrillar protein degradation but had no effect on degradation of non-myofibrillar proteins. To evaluate the mechanism by which CNTF exerts control of protein turnover, we completed a Northern blot for CNTF receptor alpha-subunit (CNTFRalpha). This was non-detectable via conventional northern analysis. Use of RT-PCR, however, confirmed expression of CNTFRalpha, albeit at a low level compared to rat skeletal muscle. This low expression of the receptor in L8 myotubes may explain the limited effect of CNTF in vitro compared to the larger effects typically detected in vivo. CNTF regulated protein turnover through control of protein synthesis and degradation. Effects were dose and timedependent. These observations may explain ability of CNTF to exert both anabolic and catabolic actions in vivo.
Wang
M C
MC
Department of Animal Sciences, Oregon State University, Corvallis, OR, 97331-6702, USA.
Forsberg
N E
NE
D000818:Animals; D015152:Blotting, Northern; D002478:Cells, Cultured; D020934:Ciliary Neurotrophic Factor / Q000235:genetics / Q000378:metabolism*; D007470:Monoiodotyrosine; D009124:Muscle Proteins / Q000378:metabolism*; D018482:Muscle, Skeletal / Q000166:cytology / Q000378:metabolism*; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats; D020133:Reverse Transcriptase Polymerase Chain Reaction
D020934:Ciliary Neurotrophic Factor; D009124:Muscle Proteins; D012333:RNA, Messenger; D007470:Monoiodotyrosine