Effect of Serum from Chickens Treated with Clenbuterol on Myosin Accumulation, Beta-Adrenergic Receptor Population, and Cyclic AM Synthesis in Embryonic Chicken Skeletal Muscle Cell Cultures
Broiler chickens at 35 days of age were fed 1 ppm clenbuterol for 14 days. This level of dietary clenbuterol led to 5-7% increases in weights of leg and breast muscle tissue. At the end of the 14-day period, serum was prepared from both control and clenbuterol-treated chickens and was then employed as a component of cell culture media at a final concentration of 20% (v/v). Muscle cell cultures were prepared from both the leg and breast muscle groups of twelve-day chick embryos. Treatment groups included control chicken serum to which 10 nM, 50 nM, and 1 micron clenbuterol had been added, as well as cells grown in media containing 10% horse serum. Cultures were subjected to each treatment for 3 days beginning on the seventh day in culture. Neither the percent fusion nor the number of nuclei in myotubes were significantly affected by any of the treatments. The quantity of MHC was not increased by serum from clenbuterol-treated chickens in either breast and leg muscle cultures; however, MHC quantity was 50- 100% higher in cultures grown in control chicken serum to which 10 nM and 50 nM clenbuterol had also been added. The Beta-AR population was 4,000-7,000 Beta-AR per cell in cultures grown in chicken serum, with leg muscle cultures having approximately 25-30% more receptors than breast muscle cultures. Receptor population was not significantly affected by the presence of clenbuterol or by the presence of serum from clenbuterol-treated chickens. In contrast, the Beta-AR population in leg and breast muscle cultures grown in the presence of 10% horse serum was 18,000-20,000 Beta-AR per cell. Basal concentration of cAMP was not significantly affected by any of the treatments. When cultures grown in chicken serum were stimulated for 10 min with 1 micron isoproterenol, limited increases of 12-20% in cAMP concentration above basal levels were observed. However, when cultures grown in the presence of horse serum were stimulated with 1 micron isoproterenol, increases of 600-800 % in cAMP concentration above basal levels were observed. Thus, not only did cells grown in horse serum have a higher Beta-AR population, each receptor had a higher capacity for cAMP synthesis following isoproterenol stimulation. Finally, the hypothesis was tested that clenbuterol exerts its action on muscle protein content by changes in cAMP concentration. No correlation was apparent between basal cAMP concentration and MHC content.