Study On Changes And Its Mechanisms Of NOS Gene Expression And NOS-NO System Activity In Injury Cardiomyocytes Induced By Cadmium

The present paper studied systematically, through primary cultured neonatal mouse cardiomyocyte model, eNOS mRNA expression, iNOS mRNA expression, total NOS, eNOS, iNOS activity as well as NO concentration in supernatant and cardiomyocytes Ca2+ changes in cardiomyocytes injury induced by cadmium and injuried cardiomyocytes pretreated with L-Arg, SNP and L-NMMA and its action mechanism by means of techniques from confocal laser microscope, cell culture in vitro and RT-PCR. Action of L-Arg, SNP and L-NMMA as well as cardiomyocyte [Ca2+]i homestasis in NOS-NO system activity changes of cardiomyocytes injuried by cadmium was discussed deeply. The results were summarized as follows:1. Establish of primary culture method in neonatal mouse cardiomyocytesCardiomyocytes from heart tissue in neonatal mouse were isolated and cultured by trypsinase digestion. The cardiomyocytes were confirmed by morphologic observation and trypan blue dye. Survival rate of myoctyes was above 90% and the survival myoctyes with intact and rod-shaped were motionlessly attached to the bottom of the culture flasks. The methods of isolation and culture of neonatal mouse cardiomyocytes were successful and perfect and make it important in a great deal of research fields.2. Studies on changes of NOS gene expression and NOS-NO system activity in cardiomyocytes induced by cadmiumPrimary cultured neonatal mouse cardiomyocytes were divided randomly into a control group (group C), a low cadmium loading group(group L-Cd), medium cadmium loading group (group M-Cd) and a high cadmium loading group (group H-Cd), and cardiomyocytes injury was induced by CdCla loading in the medium. The results showed that eNOS mRNA expression of groups L-Cd, M-Cd and H-Cd was remarkably lower than that of group C at 12h post cadmium loading, but at other time, there is no significant difference, however, during the 24h post cadmium loading, iNOS mRNA expression of groups L-Cd, M-Cd and H-Cd was remarkably increased than that of Group C. Total NOS and eNOS activity were lower or remarkably lower from 12h to 7d post cadmium loading, however, iNOS activity of group L-Cd, M-Cd and H-Cd were higher than group C at 6h and then decreased time-dependently, in particularly remarkably lower after the 3rd day. During 24h post cadmium loading, NO concentration in supernatant of various cadmium loading dose were lower than that of group C and lower or remarkably lower during 7 day periods. It was suggested that Cadmium loading caused cardiomyocyte injury, changed eNOS mRNA expression, and induced significant increase of iNOS mRNA expression, however, with a decrease of NOS-NO system activity.3. Effects of L-Arg , SNP and L-NMMA on NOS mRNA expression and NOS-NO system activity in injury cardiomyocytes induced by cadmiumPrimary cultured neonatal mouse cardiomyocytes were divided randomly into groups of control, L-Arg, L-Arg+CdCl2, L-Arg+CdCl2+SNP and L-Arg+CdCl2+ L-NMMA. The results showed that eNOS mRNA expression of groups L-Arg and L-Arg+CdCl2+SNP was remarkably higher than that of group C during 24h, however, groups L-Arg+ CdCl2 and L-Arg + CdCl2 + L-NMMA were lower or remarkably lower than groups C and L-Arg. iNOS mRNA expression of groups L-Arg , L-Arg+CdCl2+SNP and L-Arg+CdCl2+L-NMMA was remarkably increased than that of groups C and L-Arg. Total NOS and iNOS activity of groups L-Arg, L-Arg+CdCl2+SNP and L-Arg+CdCl2+L-NMMA were higher or remarkably higher than that of groups C and L-Arg from 12h to 7d, however, eNOS activity were lower than group L-Arg and higher than group C. During 7d period, NO concentration in supernatant of group L-Arg was higher or remarkably higher than that of group C, but NO levels of groups L-Arg+CdCl2 and L-Arg+CdCl2+L-NMMA were lower or remarkably lower than that of groups C and L-Arg, group L-Arg+CdCl2 +SNP was high to group L-Arg from 12h with the highest levels. It is concluded that L-Arg can increase the eNOS mRNA expression and eNOS activity of cardiomyocyte and synthesed a large amount of NO. Under pretreatment with L-Arg, inj...