Molecular cloning and expression of nitric oxide synthase gene in chick embryonic muscle cells

The chick skeletal muscle nitric oxide synthase (NOS) gene was cloned in order to further define the involvement of NOS in the differentiation of skeletal muscle cells. The respective cDNA had an open reading frame of 1136 amino acid residues, predicting a protein of 129,709.85 Da, and recognition sites for FAD, FMN, NADPH, and a calmodulin-binding site like those of other mammalian NOS's. Alignment of the deduced amino acid sequence revealed high homology with mammalian inducible NOS (iNOS), but not other NOS isoforms, suggesting chick skeletal muscle NOS may be an iNOS isoform. Immunoblots showed that NOS expression was highly restricted in embryonic muscle, but not in adult skeletal muscle: NOS expression markedly increased from embryonic day 9, reached a maximum by embryonic day 13, and then gradually declined until it was no longer detectable on embryonic day 19. When muscle cells obtained on embryonic day 12 were cultured, NOS expression increased transiently prior to the onset of differentiation and decreased thereafter. Inhibition of NOS expression by PDTC completely prevented muscle cell differentiation, as indicated by the inhibition of expression of myosin heavy chain and creatine kinase. The inhibitory effect of PDTC was completely reversed by addition of sodium nitroprusside, a compound that produces NO. These results clearly indicate that NOS is significantly involved in the differentiation of chick skeletal muscle cells.