Effect of serum from chickens treated with clenbuterol on myosin accumulation, β-adrenergic receptor population, and cyclic AMP synthesis in embryonic chicken skeletal muscle cell cultures

Summary

Broiler chickens at 35 d of age were fed 1 ppm clenbuterol for 14 d. This level of dietary clenbuterol led to 5–7% increases in the weights of leg and breast muscle tissue. At the end of the 14-d 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 the breast muscle groups of 12-d chick embryos. Treatment groups included control chicken serum to which 10 nM, 50 nM, and 1 μM clenbuterol had been added, as well as cells grown in media containing 10% horse serum. Cultures were subjected to each treatment for 3 d, beginning on the seventh d in culture. Neither the percent fusion nor the number of nuclei in myotubes was significantly affected by any of the treatments. The quantity of myosin heavy chains (MHCs) was not increased by serum from clenbuterol-treated chickens in either breast or leg muscle cultures; however, the MHC quantity was 50–150% higher in cultures grown in control chicken serum to which 10 and 50 nM clenbuterol had also been added. The β-adrenergic receptor (βAR) population was 4000–7000 βARs 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 βAR population in leg and breast muscle cultures grown in the presence of 10% horse serum was 16,000–18,000 βARs per cell. Basal concentration of cyclic adenosine 3′∶5′ monophosphate (cAMP) was not significantly affected by the treatments. When cultures grown in chicken serum were stimulated for 10 min with 1 μM isoproterenol, limited increases of 12–20% in cAMP concentration above the basal levels were observed. However, when cultures grown in the presence of horse serum were stimulated with 1 μM isoproterenol, cAMP concentration was stimulated 5-to 9-fold above the basal levels. Thus, not only did cells grown in horse serum have a higher βAR population, but also each receptor had a higher capacity for cAMP synthesis following isoproterenol stimulation. Finally, the hypothesis that clenbuterol exerts its action on muscle protein content by changes in cAMP concentration was tested. No correlation was apparent between basal cAMP concentration and MHC content.