The increase of wall shear stress in capillaries by oral administration of the α1-adrenergic receptor antagonist prazosin induces angiogenesis in skeletal muscles. Because endothelial nitric oxide synthase (eNOS) is upregulated in response to elevated wall shear stress, we investigated the relevance of eNOS for prazosin-induced angiogenesis in skeletal muscles. Prazosin and/or the NOS inhibitor Nω-nitro-l-arginine methyl ester (l-NAME) were given to C57BL/6 wild-type mice and eNOS-knockout mice for 14 days. The capillary-to-fiber (C/F) ratio and capillary density (CD; no. of capillaries/mm2) were determined in frozen sections from extensor digitorum longus (EDL) muscles of these mice. Immunoblotting was performed to quantify eNOS expression in endothelial cells isolated from skeletal muscles, whereas VEGF (after precipitation with heparin-agarose) and neuronal NOS (nNOS) concentrations were determined in EDL solubilizates. In EDL muscles of C57BL/6 mice treated for 14 days, the C/F ratio was 28% higher after prazosin administration and 11% higher after prazosin and l-NAME feeding, whereas the CD increased by 21 and 13%, respectively. The C/F ratio was highest after day 4 of prazosin treatment and decreased gradually to almost constant values after day 8. Prazosin administration led to elevation of eNOS expression. VEGF levels were lowest at day 4, whereas nNOS values decreased after day 8. In EDL muscles of eNOS-knockout mice, no significant changes in C/F ratio, CD, or VEGF and nNOS expression were observed in response to prazosin administration. Our data suggest that the presence of eNOS is essential for prazosin-induced angiogenesis in skeletal muscle, albeit other signaling molecules might partially compensate for or contribute to this angiogenic activity. Furthermore, subsequent remodeling of the capillary system accompanied by sequential downregulation of VEGF and nNOS in skeletal muscle fibers characterizes shear stress-dependent angiogenesis.

Nitric oxide synthase-1 (NOS-1) is found in high concentrations in skeletal muscles, where its synthesis product nitric oxide (NO) is reported to be involved in a number of processes, including the modulation of the oxidative metabolism of myofibers. Performing immunoblot analysis and quantification of formazan produced by its specific NADPH diaphorase activity, we found NOS-1 to be enriched in rat skeletal muscles with a high proportion of fast-twitch myofibers. Since these myofibers represent a metabolically heterogeneous subpopulation, we extended our investigation to the level of individual myofibers. Using serial sections we combined myosin heavy chain-based fiber-typing with quantitative succinate dehydrogenase histochemistry to determine three groups of fiber-types, comprising fast-oxidative, fast-glycolytic and slow-oxidative myofibers. Image analysis showed that NOS-1 diaphorase activity is significantly enriched in fast-oxidative myofibers compared with fast-glycolytic and slow-oxidative ones. In order to characterize potential biological effects of the fiber-type-specific enrichment of NOS-1, we performed cytochrome oxidase histochemistry in the presence of the NO donors NOC-9 and SNAP. Both NO donors reduced cytochrome oxidase activity in all myofibers investigated with almost identical semi-maximal inhibition rates, although fast-oxidative and slow-oxidative myofibers contained twice as much basal catalytic activity than fast-glycolytic ones. In summary, we suggest that the NOS-1/NO system of skeletal muscles exerts its biological role especially in fast-oxidative myofibers, since these myofibers express more NOS-1 than fast-glycolytic or slow-oxidative ones and also contain the highest concentrations of cytochrome oxidases as potential target molecules of NO.

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An impact of nitric oxide (NO) on lactation and milk secretion in mammary glands has previously been documented, but the underlying molecular mechanisms for this modulatory effect remain unclear. Therefore, we investigated the expression patterns of NO synthase (NOS)-1, NOS-3 and the NO receptor soluble guanylyl cyclase (sGC) in mammary glands of lactating and non-lactating female C57/Bl6 mice. RT-PCR demonstrated the existence of NOS-1-mRNA and NOS-3-mRNA in both lactating and resting mammary tissue. Immunoblots loaded with equal amounts of homogenate proteins from lactating and resting mammary tissues revealed comparable intensities of NOS-1 and sGC bands. Performing catalytic NADPH diaphorase histochemistry and immunohistochemistry, NOS-1 was only detected in myoepithelial cells (MEC), while sGC was localized in alveolar epithelial cells (lactocytes) and MEC in both lactating and non-lactating mammary glands. The non-modulated co-expression of both enzymes suggests that NOS-1 and sGC contribute to the constitutive regulation of tone in MEC.

The subcellular appearance of NADPH diaphorase activity in different rat skeletal muscles has been analyzed. Both a sarcolemma-associated as well as a non-sarcolemma-associated NADPH diaphorase-dependent generation of formazan was observed. The sarcolemma-associated NADPH diaphorase staining appeared regularly in two manifestations: one observed in longitudinal sections as dotted costameres at the cell surface which accordingly appeared in transversal sections as rings surrounding the myofibre surface. At this site, nitric oxide synthase (NOS)-1 was located. The second sarcolemma-associated site of NADPH diaphorase staining was found as bundles of longitudinal-orientated stripes of hitherto unidentified origin. The non-sarcolemma-associated production of formazan was likewise manifested at two sites: the first was found regularly in longitudinal sections as intense sarcomere-like striations occurring parallel to the I-bands and indicating mitochondria. The second non-sarcolemma-associated NADPH diaphorase staining was realized as fine longitudinal filaments of variable occurrence connecting the mitochondria and presumably belonging to the sarcoplasmic reticulum. Attempts to identify single NADPH diaphorase(s) existing in skeletal muscles by incubation with specific inhibitors failed but showed the presence of two different subpopulations of NADPH diaphorases in myofibres: a urea-resistant fraction in the sarcolemma region containing NOS-1 and a non-sarcolemma-associated, urea-sensitive fraction depleted of NOS-1.