Catecholamines synthesizing enzymes in vascular cells and their hypoxic regulation
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Catecholamines (dopamine, norepinephrine and epinephrine) are physiologically important as they are involved in a number of body functions and also act as neurotransmitters. Dopamine is a metabolic intermediate in the formation of norepinephrine and epinephrine and a well known neurotransmitter in the central nervous system. In most of the mammalian systemic arteries, dopamine acts as a potent vasodilator. The vascular effects of dopamine are mediated via specific G protein-coupled receptors (D1-D5). The present study aimed to look for the presence of enzymatic machinery (tyrosine hydroxylase = TH; DOPA-decarboxylase = DDC; dopamine-beta-hydroxylase = DbetaH; phenylethanolamine-N-methyltransferase = PNMT) required to synthesize catecholamines in rat arteries, and their regulation in response to hypoxia (1% O2) in endothelial (EC) and vascular smooth muscle cells (VSMC). Quantitative RT-PCR served to estimate mRNA content, and western blotting was used to test for TH protein expression. Furthermore, we also looked for the expression of the rate-limiting enzyme of catecholamine synthesis, TH, by quantitative RT-PCR, immunohistochemistry and western blotting in the intact vessels (rat thoracic aorta, abdominal aorta, superior mesenteric artery [SMA] and femoral artery). Expression of mRNA of all four enzymes of the catecholamine synthesis pathway was detected and quantified in rat thoracic and abdominal aorta, SMA and femoral artery. It was observed that these arteries express different levels of mRNA content of these enzymes with PNMT being least abundant. TH protein was also detected in these arteries by immunolabelling and western blot analysis. TH expression in these arteries suggests active synthesis of dopamine and/or other catecholamines in these vessels. Previously, TH mRNA expression has been reported in rat aorta and it has been suggested that this might be the result of paraganglia contamination during sampling process. No paraganglia were detectable in immunohistochemical preparation of rat aorta suggesting that this part of vascular tree is itself capable of synthesizing catecholamines. Rat pulmonary and aortic EC were isolated and cultured. These cells were characterized by DiI-Ac-LDL uptake and by immunolabeling with anti-vWF antibody. VSMC were isolated and cultured from thoracic and abdominal aorta and characterized by immunolabeling with anti alpha-smooth muscle actin antibody. Under normoxic conditions, EC and VSMC express mRNAs coding for all of the enzymes involved in catecholamine synthesis. These vascular cells were exposed to hypoxia (1% O2) for 6, 12 and 24 hours. Using real-time RT-PCR we detected the mRNAs for the complete or partial enzymatic machinery necessary for catecholamine synthesis in these cells. In rat pulmonary EC, hypoxia caused an up-regulation of TH mRNA by 25-fold after 24 h, of DbetaH mRNA by 8-fold after 6 and 24 h, whereas DDC and PNMT mRNA content were not significantly different after hypoxic exposure. In contrast, no significant effect of hypoxia on the regulation of the enzymatic machinery was observed in rat aortic EC. In VSMC, TH mRNA was qualitatively detectable while DDC mRNA content was too low to be detected. No statistically significant regulation of DbetaH and PNMT mRNA content was observed after hypoxic exposure in VSMC. This suggests that vascular cells are capable of catecholamine synthesis and this synthesis is differentially regulated under hypoxic conditions. This is the first study providing evidence of expression of catecholamine synthesis enzymes in isolated vascular cells. Previously, Kuncova et al., 2007, had shown that the presence of catecholamines in rat aorta and SMA. Chemical sympathectomy with the neurotoxin, 6-hydroxydopamine, significantly reduced norepinephrine and epinephrine levels, while dopamine content was unaffected. Functional studies using rat SMA showed an endothelium-dependent vasodilation in response to fall of PO2 approximately -25 mm Hg, which could be blocked by the specific dopamine D1 receptor antagonist SCH23990, suggesting possible role of locally produced dopamine in the vasodilation. This study suggests that there are two sources of catecholamines, in particular dopamine - neuronal and non-neuronal - and the non-neuronal synthesis is carried out by both principal cell types of vascular wall, i.e. EC and VSMC. Catecholamines produced by vascular cells are released in the event of hypoxia. This locally synthesized and released dopamine mediates vasodilation and might help in an increased O2 and nutrient supply to the affected organ.Verknüpfung zu Publikationen oder weiteren Datensätzen
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Giessen : VVB Laufersweiler