Expression and regulation of protein inhibitor of neuronal nitric oxide synthase in ventilatory muscles

In skeletal muscle fibers, nitric oxide (NO) is synthesized by neuronal NO synthase (nNOS) and regulates excitation-contraction coupling, glucose uptake, and mitochondrial respiration. Recently, a novel 89-amino acid protein, designated protein inhibitor of nNOS (PIN), has been shown to interact with and specifically inhibit nNOS activity. In this study, we investigated the distribution, localization, and regulation of PIN expression in ventilatory and limb muscles of various species. Amplified PIN cDNA from the rat diaphragm revealed an open reading frame identical to that of human PIN. Among muscles of adult rats, PIN mRNA was strongly expressed in muscles rich in type I fibers, whereas much weaker expression was evident in muscles rich in type II fibers. By comparison, PIN protein expression was not related to fiber-type distribution. Similarly, PIN protein was equally expressed among rat, mouse, and human diaphragms. Both PIN mRNA and PIN protein were expressed at much higher levels in the embryonic rat diaphragm than in adult muscle. Immunohistochemistry revealed that PIN protein was localized in close proximity to the sarcolemma and nuclei. PIN protein was also abundant in muscle spindles and axons of nerves supplying skeletal muscle fibers. We conclude that PIN is expressed in various skeletal muscle fibers and that its expression is regulated during muscle development. The localization of PIN in muscle regions containing abundant nNOS protein suggests that it plays a role in the regulation of NO synthesis in skeletal muscle fibers.