Neuropeptides Induce Release of Nitric Oxide from Human Dermal Microvascular Endothelial Cells

Nitric oxide is a potent mediator of endothelium-dependent vasodilatation, the synthesis of which is catalyzed by the constitutively expressed enzyme endothelial nitric oxide synthase. In this study we have investigated whether human dermal microvascular endothelial cells express endothelial nitric oxide synthase and whether the vasodilator neuropeptides, calcitonin gene-related peptide and substance P, stimulate the release of nitric oxide from these cells. Endothelial nitric oxide synthase was identified by immunohistochemistry in the blood vessels in both the papillary and deep dermis of normal skin, and also in monolayers of human dermal microvascular endothelial cells. On western blots of protein extracts prepared from both the dermis of normal human skin and human dermal microvascular endothelial cells, a 135-kDa band corresponding to endothellal nitric oxide synthase was identified. Nitric oxide was released from unstimulated human dermal microvascular endothelial cells as assessed by inhibition of platelet aggregation and nitrite formation. Endothelial cell-mediated inhibition of platelet aggregation was blocked by hemoglobin, which binds nitric oxide. Substance P (10 nM) potentiated micro-vascular endothelial cell inhibition of platelet aggregation, and this effect was also blocked by hemoglobin. Calcitonin gene-related peptide (100 pM to 100 nM) directly inhibited platelet aggregation, and this direct effect was not modulated by microvascular endotheial cells. Substance P (10 nM to 1 μM) and calcitonin gene-related peptide (100 pM to 10 nM) significantly (p < 0.05) increased nitrite formation, and this increase was blocked by the competitive nitric oxide synthase antagonist, N^Gmonomethyl-L-arginine. These results demonstrate that endothelial nitric oxide synthase is expressed in the microvascular endothelium of normal human skin and that human dermal microvascular endothelial cells release nitric oxide constitutively and in response to vasodilator neuropeptides.