Cells of the microvascular vessel system play an important role in physiological and pathophysiological processes. They are involved in the initiation and maintenance of inflammatory reactions. Nitric oxide (NO) acts as an autocrine and paracrine mediator in the inter- and intracellular communication between various cell systems. NO is also involved in cutaneous active vasodilatation in the skin. Although an abundance of knowledge exists about the regulation of NO release in human macrovascular endothelial cells, only little is known about the cellular control of NOS in microvascular endothelial cells. In the present study we set out to demonstrate possible regulatory mechanisms of L-arginine/NO-pathway in human dermal microvascular endothelial cells. In the centre of investigations was on the one hand, the availability of the cationic amino acid L-arginine, the natural substrat of NO synthase and also the cofactors, tetrahydrobiopterin and calcium/calmodulin which are essential for enzym catalyse. On the other hand, the aim of the study was to analyse the expression of endothelial and inducible nitric oxide synthase (eNOS and iNOS) in HDMEC. The results show that application of L-arginine at non-toxic concentrations (up to 25 mM) does not lead to a stimulation of NO release. The dependence of the enzyme activity on the intracellular availability of calcium/calmodulin-complex is a typical feature of the eNOS. We supposed, that extracellulary added Ca2+ enters the cells and therefore causes an increase of activity. Only a slight increase of NO production following an treatment with 2 mM CaCl2 (72 h) could be shown. The application of the essential cofactor tetrahydrobiopterin (BH4) led to a concentration dependent increase of NO production after 24 h incubation. Furthermore, 60 - 80% of the transport of L-arginine occurs via the y+ system, which also transports the cationic amino acids, L-ornithine and L-lysine. An incubation with these amino acids (each 2 mM) decreased the NO release of HDMEC. Following the simultaneous application of L-ornithine plus L-arginine or L-lysine plus L-arginine, the inhibitory effect of L-ornithine or L-lysine with regards to NO production was reduced by L-arginine. In addition, using RT-PCR we investigated the expression of endothelial and inducible nitric oxide synthase in HDMEC on mRNA level. It could be shown that microvascular endothelial cells express as well eNOS as iNOS. The application of various L-arginine concentrations caused an increase of transcription of iNOS genes. This effect appeared only after 24 h. Finally it can be concluded, that a modulation of L-arginine/NO pathway in human dermal microvascular endothelial cells was possible in vitro, and the present results give further indications about the regulation of nitric oxide production and its function in the vasodilatory processes in human cutaneous vessels. |