Distribution of Constitutive Nitric Oxide Synthase Immunoreactivity and NADPH-Diaphorase Activity in Murine Telogen and Anagen Skin

The freely diffusible radical nitric oxide is generated by nitric oxide synthase, and is a pleiotropic, bioregulatory molecule that regulates, e.g., the vascular tone, functions as a major neurotransmitter, and is involved in macrophage-mediated cytotoxicity and platelet aggregation. Constitutive nitric oxide synthase exhibits NADPH-diaphorase activity that can be demonstrated histochemically. To study whether this enzyme is present in mammalian skin during distinct phases of the murine hair cycle, we have examined cryosections of C 57 BL-6 mouse skin in telogen and depilation-induced anagen VI. Histochemical analysis of NADPH-diaphorase activity was complemented by immunohistology, using two specific rabbit antisera against constitutive neuronal nitric oxide synthase. Epidermis and the outer root sheath showed both immunoreactivity for the enzyme and NADPH-diaphorase activity, whereas dermal papilla and sebaceous glands displayed only strong NADPH-diaphorase activity, suggesting that this enzyme histochemical test measures additional enzymes besides nitric oxide synthase. Intrinsic nitric oxide synthase immunoreactivity was also detected by immunoblot in mouse skin homogenates, staining proteins of an apparent 160-kDa molecular weight. Compared to telogen skin, these immunoreactive proteins were quantitatively increased in anagen VI skin. Thus, our study suggests that defined epithelial compartments of normal murine skin are capable of synthesizing nitric oxide and that the molecule may be involved in skin physiology, growth, and remodeling.