Expression and localization of protein inhibitor of neuronal nitric oxide synthase in duchenne muscular dystrophy

In skeletal muscle fibers, nitric oxide is synthesized by neuronal nitric o xide synthase (nNOS), which normally associates with the dystrophin complex in close proximity to the sarcolemma. Many reports have documented that ve ry low levels of nNOS protein exist in muscle fibers of Duchenne muscular d ystrophy (DMD) patients. In this study we investigated the functional signi ficance of PIN (protein inhibitor of nNOS) in targeting of nNOS to the sarc olemma and the association between nNOS and the dystrophin complex in norma l and dystrophic muscle fibers. Northern blotting for PIN mRNA in normal mo use muscles and muscles of mdx mice (an animal model of DMD) revealed a sig nificant rise in PIN mRNA in dystrophic muscles compared with normal muscle s. Immunohistochemical analysis showed that, in normal mouse muscle fibers, PIN expression was localized at the sarcolemma, peripheral nuclei, and the sarcoplasm. By comparison, PIN protein in muscles from mdx mice was more c oncentrated around the sarcolemma and central nuclei. The presence of PIN p rotein expression in muscles from mdx mice was evident despite the signific ant reduction in nNOS and dystrophin protein expressions in these fibers. I n muscle sections of DMD patients, the absence of nNOS protein expression w as accompanied by maintained PIN expression. Prominent PIN expression was a lso detectable in macrophages infiltrating dystrophic muscle fibers both in mdx mice and DMD patients. These results suggest that PIN expression in mu scles from mdx mice and DMD patients is controlled by factors different fro m those involved in the regulation of nNOS and dystrophin. Moreover, our re sults indicate that PIN is not an integral component of the dystrophin comp lex inside skeletal muscle fibers. (C) 2001 John Wiley & Sons, Inc.