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 upta ke, and mitochondrial respiration. Recently, a novel 89-amino acid protein, designated protein inhibitor of nNOS (PIN), has been shown to interact wit h and specifically inhibit nNOS activity. In this study, we investigated th e distribution, localization, and regulation of PIN expression in ventilato ry and limb muscles of various species. Amplified PIN cDNA from the rat dia phragm 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 i n 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 expre ssed at much higher levels in the embryonic rat diaphragm than in adult mus cle. Immunohistochemistry revealed that PIN protein was localized in close proximity to the sarcolemma and nuclei. PIN protein was also abundant in mu scle spindles and axons of nerves supplying skeletal muscle fibers. We conc lude that PIN is expressed in various skeletal muscle fibers and that its e xpression is regulated during muscle development. The localization of PIN i n muscle regions containing abundant nNOS protein suggests that it plays a role in the regulation of NO synthesis in skeletal muscle fibers.