ABSTRACT
Objective:To investigate the regulatory effects of endogenous nitric oxide (NO) on the activity of superoxide dismutase-1 (SOD1) and apoptosis of human umbilical vein endothelial cells (HUVECs).Methods:HUVECs were taken as the research object.The endothelial NO synthase (eNOS) short hairpin RNA(shRNA) lentivirus was employed to transfect HUVECs to knock down eNOS.HUVECs were divided in 4 groups: the scramble group, the eNOS shRNA group, the eNOS shRNA + sodium nitroprusside(SNP) group and the eNOS shRNA+ SNP+ tris (2-carboxyethyl) phosphine hydrochloride (TCEP) group.The protein expressions of eNOS and SOD1 dimer/monomer in cells were detected by western blot.The activity of SOD was detected by the enzyme-linked immunosorbent assay.The NO content in cells was detected with NO fluorescence probe.The level of superoxide anion in HUVECs was detected with dihydropyridine (DHE). The terminal deoxynucleotidyl transferase (TdT) dUTP nick-end labeling (TUNEL) assay was adopted to detect the apoptosis of HUVECs in situ.Results:Compared with the scramble group, the endogenous NO content (2.690±0.420 vs.15.029±2.193, P<0.01), eNOS protein expression (1.000±0.778 vs.3.141±0.199, P<0.01), SOD1 dimer/monomer ratio (4.6±1.0 vs.7.6±2.0, P<0.05) and SOD activity [(0.432±0.254) Carmen′s unit/10 4 cell vs.(1.000±0.116) Carmen′s unit/10 4 cell, P<0.01] were significantly decreased, while the level of intracellular superoxide anion (11.180±1.560 vs.6.146±1.007, P<0.01) and HUVECs apoptosis [75.0 (55.0, 100.0)% vs.0 (0, 0)%, P<0.01] were significantly increased in the eNOS shRNA group.Compared with the eNOS shRNA group, the content of endogenous NO (16.705±0.116 vs.2.690±0.420, P<0.01), the ratio of SOD1 dimer/monomer (7.3±2.0 vs.4.6±1.0, P<0.05) and the activity of SOD [(0.737±0.060) Carmen′s unit/10 4 cell vs.(0.432±0.254) Carmen′s unit/10 4 cell, P<0.05] were significantly increased, while the level of superoxide anion (6.897±1.648 vs.11.180±1.560, P<0.01) and the HUVECs apoptosis [0 (0, 0)% vs.75.0 (55.0, 100.0)%, P<0.01] were significantly decreased in the eNOS shRNA+ SNP group.Compared with the eNOS shRNA + SNP group, the ratio of SOD1 dimer/monomer (4.4±0.9 vs.7.3±2.0, P<0.05) and the activity of SOD [(0.214±0.084) Carmen′s unit/10 4 cell vs.(0.737±0.060) Carmen′s unit/10 4 cell, P<0.01] were significantly decreased, while the level of superoxide anion (10.917±1.552 vs.6.897±1.640, P<0.01) and the apoptosis level of HUVECs[63.6 (55.0, 90.0)% vs.0 (0, 0)%, P<0.01] were significantly increased in the eNOS shRNA+ SNP+ TCEP group.However, there was no significant difference in the NO content (16.112±0.926 vs.16.705±0.116, P>0.05). Conclusions:Endogenous NO could effectively antagonize the apoptosis of endothelial cells by increasing the cysteine-dependent SOD1 dimer/monomer ratio, enhancing SOD activity and inhibiting the accumulation of reactive oxygen species.
ABSTRACT
Summary: The changes in the expression of cardiac bradykinin B2 receptors (BKB2Rs) and endogenous nitrix oxide synthase (eNOs) mRNA were studied in rats with remnant kidneys. Thirty-two rats were divided into sham-operated and experimental groups randomly (n=16 in each group). The remnant kidney model was established by 2-stage 5/6 nephrectomy. Blood pressure and serum Cr were measured before operation and 15, 30, 60, 120 days after 5/6 nephrectomy. Eight animals in each group were killed at the first month and 4th month after the operation. The expression of BKB2Rs and eNOs mRNAs was detected by using RT-real time PCR from isolated left ventricle, and their correlation was also analyzed. The results showed that blood pressure and serum Cr were increased significantly 15 days after 5/6 nephrectomy (both P<0.01), and the hypertension and azomia existed constantly till 120 days but had no significant fluctuation. Cardiac BKB2Rs and eNOs mRNA was declined time-dependently (both P<0.05). And there was a close positive correlation between cardiac BKB2Rs and eNOs mRNA (r=0.82, P<0.01). It was suggested that a significant chronic renal failure can be produced at least 15 days after 5/6 nephrotomy and can sustain more than 4 months. The expression of BKB2Rs and eNOs was down-regulated time-dependently in this model, and there was a significant correlation between them.
ABSTRACT
The present study was designed to investigate whether endogenous nitric oxide (EDNO) is involved in submandibular vasodilation and salivation induced by parasympathetic nerve stimulation. Effects of Nw-nitro-L-arginine-methyl ester (L-NAME) which blocks the synthesis of EDNO from L-arginine on the submandibular vasodilation and salivation induced by chorda stimulation or administration of various vasodilators were examined in anesthetized cats. Effect of L-NAME on K+ efflux induced by carbachol was also examined using the excised submandibular slice in vitro. In the submandibular slices, acetylcholine (10(-5) mol/L) or vasoactive intestinal polypeptide (VIP, 10(-5) mol/L) increased NO2 contents, which was prevented by pretreatment with L-NAME. Salivary secretion in response to the chorda stimulation (3 V, 1 msec, 10 ~ 20 Hz) was completely blocked by treatment with atropine (1 mg/kg). Increased blood flow response to the low frequency (1, 2, 5 Hz) stimulation was significantly reduced, whereas the blood flow induced by the higher frequency (10, 20 Hz) stimulation was not affected. Lingual-arterial infusion of L-NAME (100 mg/kg) significantly diminished the vasodilatory and salivary responses to the chorda stimulation at all stimuli frequencies used. Intra-arterial infusion of L-NAME (100 mg/kg) markedly diminished the vasodilatory responses to acetylcholine (5 mug/kg), VIP (5 mug/kg) or bradykinin (5 mug/kg). In the excised submandibular slice, K+ efflux in response to carbachol (10(-5) mol/L) was significantly decrease by pretreatment with L-NAME (10(-5) mol/L). In the isolated submandibular artery precontracted with phenylephrine (10(-5) mol/L), the vasorelaxation induced by ACh (10-7 mol/L) was reversed into a contraction by methylene blue (10(-4) mol/L). These results suggest that EDNO may play an important role in vasodilation and secretion of the submandibular gland.