Ontogenesis of nitric oxide synthases in the ventilatory muscles.

Nitric oxide (NO) acts as an endogenous mediator in mature skeletal muscle. In this study, we investigated the regulation of the endothelial (eNOS) and neuronal (nNOS) isoforms of nitric oxide synthase (NOS) in skeletal-muscle development (rat diaphragm). Muscle NOS activity, nNOS and eNOS protein, and mRNA expressions were markedly increased during the late gestational and early postnatal periods. Expression of both isoforms, however, declined progressively thereafter. Similarly, argininosuccinate lyase and argininosuccinate synthetase, both involved in the recycling of L-citrulline to L-arginine, were expressed at high levels in rat embryonic and neonatal diaphragms, with gradual reduction in their expression during late postnatal development. Immunostaining revealed extensive nNOS expression at the sarcolemma in neonatal and mature diaphragms, whereas eNOS expression was limited to the endothelium. Both neonatal and adult diaphragms expressed an alternatively spliced nNOS isoform with an insert of 34 amino acids between exons 16 and 17. In vitro-generated muscle force rose significantly after NOS inhibition in both neonatal and adult diaphragms, but the magnitude of force augmentation was larger in adult than in neonatal diaphragm. These results indicate that constitutive NOS isoforms are developmentally regulated in skeletal muscles, suggesting multiple roles for NO in developing and mature skeletal-muscle fibers.