Evaluation of the effects of melatonin and melatonin receptors on aorta and HUVEC

Melatonin which its secretion from pineal gland decreases gradually in human, has not only interserebral and pheripheral impacts but also has vascular effects. In this study the vascular responces and intracellular mechanisms of melatonin were evaluated by comparing melatonin and melatonin receptor antagonists using rat aortae and HUVEC cell line. For this purpose we extracted the thoracal aortae of healhty male Wistar albino rats (350-400g) which has intact endothelia. We divided the aortic rings into four groups due their incubation material. 1st group was control aortae without any incubation, 2nd group was incubated in nonselective MT receptor agonist 10-5 M Melatonin (MLT), 3rd group was incubated in nonselective MT receptor antagonist 10-5 M Luzindol (LZL), 4th group was incubated in combination of 10-5 M MLT and 10-5 M LZL. After the incubation period, we evaluated the vasoconstrictive responces of cumulative potassium chlorure (KCl;10-50mM) or phenylephrine (Phe; 3x10-9-10-6M) and vasorelaxant responces of cumulative acetylcholine (Ach; 10-10-10-5 M) or sodyum nitroprussid (SNP; 3x10-9-10-6 M). Data were analysed by using two way ANOVA and Bonferroni Post Test (n:5) (Graph Pad). For endothelial experiments HUVEC cell line was cultured in F12 medium, and then, was divided into 5 groups depending on the incubation material: 1st group was control HUVECs without any incubation, 2nd group incubated in (10-10-7 M) MLT, 3rd group incubated in (10-10-7 M) MLT and (10-5 or 10-6 M) LZL, 4th group incubated in (10-10-7 M) MLT and (10-5or 10-6 M) 4P-PDOT, 5th group incubated in (10-10-7 M) MLT and (10-5 or 10-6 M) GR135531 for 24 hours. Then MTT, calcium assay and total nitrate/nitrit assays were carried out using these groups. Results were compared by using two way ANOVA and Bonferroni Post test (n:3) (Graph Pad). The vasoconstrictive responses of (3x10-7-10-5 M) Phe and (20mM) KCl were increased significantly by incubation of 10-5 M MLT and this increase was not observed by incubation of 10-5 M LZL or combination of 10-5 M MLT and 10-5 M LZL . Also, the EC50 values of KCl and Phe curves inreased significantly in the presence of 10-5 M MLT, in accordance with that, not in the presence of 10-5 M LZL or combination of 10-5 M MLT and 10-5 M LZL . But, there were no significant changes on vasorelaxant responces of Ach in any group. Incubation of 10-5 M MLT increased the vasorelaxant responses of (3x10-7 and 10-7 M) SNP in accordance to that 10-5 M LZL or both combination of 10- 5 M MLT and 10-5 M LZL reduced these increased responses to control levels. We performed MTT test on HUVEC cell line to find out if these is MT receptor-mediated cytotoxic effects of MLT. 10-0,001 mM concentrations of MLT reduced the percentage of cell viability significantly (60±4, 66±2, 55±1, 65±1, 64±4 %, respectively) but 10-4-10-7 mM concentrations of MLT slightly increased the percentage of cell viability compared to control cells. 10-5M LZL or 10-5M 4P-PDOT addition to 0,01 and 0,001 mM MLT increased the percentage of cell viability significantly to control levels. Similar results were obtained when 10-6 M LZL or 4P-PDOT were added to 0,1-0,001 mM MLT. However, incubation of (10-5 or 10-6 M) GR135531 with MLT (10-10-7 mM) did not make any difference when compared to only MLT. We have also observed that MLT (0,1-10-7 mM) incubation increased percentage of intracellular calcium (Ca2+) concentration and 10-5 M or 10-6 M LZL or 10-5 M or 10-6 M 4P-PDOT reduced intracellular Ca2+ levels significantly, but MT3 receptor agonist GR135531 did not exhibits any change when its compared to MLT. HUVEC were used to evaluate the effects of melatonin and melatonin receptors on NO production by total nitrite/nitrate amount assay. The reduction of total nitrite/nitrate amount by 10-4 mM MLT were increased by combined incubation of 10-5 M 4P-PDOT and 10-5 M GR135531. 10-5 mM MLT and 10-5 M luzindole incubation total nitrite/nitrate amount were reduced when it was compared with 10-5 mM MLT. Most of the studies that evaluate the vascular effects of MT receptors have done by using either rat caudal or cerebral arteries. It seems likely to oversimplify generalizing these effects to all blood vessels. In conclusion, 10-5 M MLT was increased the vasoconstrictive responces of KCl or Phe and the vasorelaxant responces of SNP through MT1 receptors in endothelium intact rat aortae.These data indicates that 10-10-3 mM MLT exhibits cytotoxic effects through MT1/MT2 receptors. Also MLT increased the intracellular Ca2+ levels through MT1/MT2 receptors. MLT was increased intercellular Ca2+ concentration by MT1/MT2 receptors but MT3 receptor did not change intercellular Ca2+ concentration. Due to the results that we got from the total nitrite nitrate assays, MLT slightly decreased NO by using MT2 receptors. Key Words: Melatonin, melatonin receptors, HUVEC, aortae, vasoreactivity, nitrik oksit, calcium.