Effects of aqueous leaf extract of Tridax procumbens on contractile activity of corpus cavernosum in N-nitro-L-arginine methyl ester-induced hypertensive male rats

Objective： This study investigated the effects of aqueous leaf extract of Tridax procumbens （ALETP） on con- tractile activity of corpus cavernosum in N-nitro-L-arginine methyl ester （L-NAME）-induced hypertensive male rats. Methods： Twenty normal, adult male rats （130-150 g） were divided into four groups of five rats each. Group I （control） was given normal saline （0.6 mL/kg） and group II was given L-NAME （40 mg/kg） for 6 weeks. Groups Ill and IV also received L-NAME （40 mg/kg） for 6 weeks but were further co-treated with 100 and 200 mg/kg of ALETP, respectively, from week 4 to week 6. All treatments were given orally. Strips of corpus cavernosum from each of the four groups were exposed to increasing concentrations of acetyl~ choline （ACh） and sodium nitroprusside （SNP） （10^-9-10^-5 tool/L） after contraction with phenylephrine （10^-7 mol/L） to test for a dose-response effect, Response to potassium and calcium was also measured after cumulatively adding potassium and calcium （10-50 mmol/L） to potassium- and calcium-free organ chamber. Isometric contractions were recorded through an Ugo Basile data capsule acquisition system. Results： Mean arterial blood pressure was significantly reduced in the ALETP co-treated group compared to the control and L-NAME-only groups （P 〈 0.05）. Cavernosa strips from ALETP co-treated rats exhibited significant inhibition of contraction in response to phenylephrine, potassium chloride, and calcium chlo- ride （P 〈 0.05）. Relaxation in response to Ach and SNP was also significantly impaired in cavernosa strips from the L-NAME-only treated group （P 〈 0.05）, while ALETP co-treated groups showed enhanced per- centage relaxation. Conclusion： ALETP treatment of L-NAME-induced hypertensive rats promotes a relaxant effect on isolated cavernosa strips. ALETP shows potential in correcting erectile dysfunction in hypertension.

Chloroquine Relieves Acute Lung Injury in Rats with Acute Hemorrhagic Necrotizing Pancreatitis

Summary： This study preliminarily investigated the mechanism by which chloroquine （CQ） relieves acute lung injury （ALI） complicated in acute hemorrhagic necrotizing pancreatitis （AHNP）. Sixty male Wistar rats were randomized into sham-operated group （group A, n=10）, AHNP group （group B, n=10）, L-arginine-treated group （group C, n=10）, L-N-nitro-L-arginine methyl ester （NAME）-treated group （group D, n=10）, CQ-treated group （group E, n=10） and CQ＋L-NAME-treated group （group F, n=10）. TLR4 expression was measured by using real time-PCR and Western blotting respectively. The results showed that, in the group B, the expression of TLR4 and the levels of TNF-α and IL-6 in the lungs were significantly increased, and the nitric oxide （NO） concentration was reduced, as compared with those in the group A （P〈0.05 or P〈0.01）. Lung injury was aggravated with the increased expression of TLR4. When the inhibitor and stimulator of TLR4, namely L-Arg and L-NAME, were added respectively, lung injury was correspondingly relieved or aggravated （P〈0.05 or P〈0.01）. In the group E, TLR4 expres- sion was substantially lower and NO concentration higher than those in the group B （P〈0.05 or P〈0.01）. However, in the group F, NO concentration was markedly decreased, and the inhibitory effect of CQ on TLR4 expression and the relief of lung injury were weakened when compared with those in the group E （P〈0.05 or P〈0.01）. It was concluded that TLR4 may play an important role in the pathogenesis and development of ALl complicated in AHNP. CQ could relieve ALl by decreasing the TLR4 expression and increasing the NO release.

Implications of alpha-synuclein nitration at tyrosine 39 in methamphetamine-induced neurotoxicity in vitro and in vivo

Methamphetamine is an amphetamine-type psychostimulant that can damage dopaminergic neurons and cause characteristic pathological changes similar to neurodegenerative diseases such as Parkinson's disease. However, its specific mechanism of action is still unclear. In the present study, we established a Parkinson's disease pathology model by exposing SH-SY5 Y cells and C57 BL/6 J mice to methamphetamine. In vitro experiments were performed with 0, 0.5, 1.0, 1.5, 2.0 or 2.5 mM methamphetamine for 24 hours or 2.0 mM methamphetamine for 0-, 2-, 4-, 8-, 16-, and 24-hour culture of SH-SY5 Y cells. Additional experimental groups of SH-SY5 Y cells were administered a nitric oxide inhibitor, 0.1 mM N-nitro-L-arginine, 1 hour before exposure to 2.0 mM methamphetamine for 24 hours. In vivo experiments: C57 BL/6 J mice were intraperitoneally injected with N-nitro-L-arginine(8 mg/kg), eight times, at intervals of 12 hours. Methamphetamine 15 mg/kg was intraperitoneally injected eight times, at intervals of 12 hours, but 0.5-hour after each N-nitro-L-arginine injection in the combined group. Western blot assay was used to determine the expression of nitric oxide synthase, α-synuclein(α-Syn), 5 G4, nitrated α-synuclein at the residue Tyr39(nT39 α-Syn), cleaved caspase-3, and cleaved poly ADP-ribose polymerase(PARP) in cells and mouse brain tissue. Immunofluorescence staining was conducted to measure the positive reaction of NeuN, nT39 α-Syn and 5 G4. Enzyme linked immunosorbent assay was performed to determine the dopamine levels in the mouse brain. After methamphetamine exposure, α-Syn expression increased; the aggregation of α-Syn 5 G4 increased; nT39 α-Syn, nitric oxide synthase, cleaved caspase-3, and cleaved PARP expression increased in the cultures of SH-SY5 Y cells and in the brains of C57 BL/6 J mice; and dopamine levels were reduced in the mouse brain. These changes were markedly reduced when N-nitro-L-arginine was administered with methamphetamine in both SH-SY5 Y cells and C57 BL/6 J mice. These results suggest that nT39 α-Syn aggregation is involved in methamphetamine neurotoxicity.

Quantitative Assessment of the Effect of Nitric Oxide Synthase Inhibition on Tumor Vascular Activity Using Dynamic Contrast-Enhanced Computed Tomography

Purpose: The purpose of this study was to develop a method to quantitatively assess the effect of nitric oxide synthase (NOS) inhibition on tumor vascular activity using dynamic contrast-enhanced computed tomography (DCE-CT) and to investigate its usefulness using animal experiments. Mate-rials and Methods: The DCE-CT studies were performed in anesthetized Fisher rats bearing tumors using a 4-row multi-slice CT. The scanning started 4 s before a bolus injection of iodinated contrast agent (CA) (150 mgI/kg) from the tail vein using an automatic injector and lasted 60 s at 1-s in-tervals. The contrast enhancement (CE) images were generated by subtracting the CT images before and after the administration of CA. First, the DCE-CT studies were performed before and 15, 30, and 45 min after administration of N-nitro-L-arginine (L-NNA) (1, 3, and 10 mg/kg) or vehicle, and the relative CE values were calculated by normalizing the CE image at each time point by that obtained from the first DCE-CT study. Second, we investigated the case when L-arginine (L-ARG) (200 mg/kg) and L-NNA (1, 3, and 10 mg/kg) were administered after the first and second DCE-CT studies, respectively. Third, we investigated the case when L-NNA (1, 3, and 10 mg/kg) and L-ARG (200 mg/kg) were administered after the first and second DCE-CT studies, respectively. Finally, we investigated the case when L-NNA (1, 3, and 10 mg/kg) and L-ARG (200 mg/kg) were administered simultaneously after the first DCE-CT study. Results: The relative CE value significantly decreased after L-NNA administration in a dose-dependent manner (p-values = 0.0074 and <0.0001 for 0 vs. 3 mg/kg and 0 vs. 10 mg/kg, respectively, at 15 min, 0.0003 and <0.0001 for 0 vs. 3 mg/kg and 0 vs. 10 mg/kg, respectively, at 30 min, and 0.0367 and 0.0004 for 0 vs. 3 mg/kg and 0 vs. 10 mg/kg, respectively, at 45 min). When L-ARG was administered prior to the administration of 1 mg/kg L-NNA, the relative CE value at 45 min was significantly higher than that at 15 min. When L-ARG was administered after L-NNA administration, there was no significant difference between the relative CE values at 15 min and 45 min. These results suggest that when using L-NNA in combination with L-ARG, their effect on tumor vascular activity differs depending on the order of their administration. When L-NNA and L-ARG were administered simultaneously, there was a tendency for the relative CE value to be higher than that when only L-NNA was administered, at all injected doses of L-NNA. Conclusion: Our method using DCE-CT is useful for monitoring the effect of NOS inhibition on tumor vascular activity and for determining the optimal injected dose and timing of NOS inhibitors for anticancer therapy.

Effects of L-arginine and N~ω-nitro-L-arginine methylester on learning and memory and α7 nAChR expression in the prefrontal cortex and hippocampus of rats

Nitric oxide （NO） is a novel type of neurotransmitter that is closely associated with synaptic plasticity, learning and memory. In the present study, we assessed the effects of Larginine and NnitroL arginine methylester （LNAME, a nitric oxide synthase inhibitor） on learning and memory. Rats were assigned to three groups receiving intracerebroventricular injections of LArg （the NO precursor）, LNAME, or 0.9% NaCI （control）, once daily for seven con secutive days. Twelve hours after the last injection, they underwent an electric shockpaired Y maze test. Twentyfour hours later, the rats＇ memory of the safe illuminated arm was tested. After that, the levels of NO and a7 nicotinic acetylcholine receptor （a7 nAChR） in the prefrontal cortex and hippocampus were assessed using an NO assay kit, and immunohistochemistry and Western blots, respectively. We found that, compared to controls, LArgtreated rats received fewer foot shocks and made fewer errors to reach the learning criterion, and made fewer errors during the memorytesting session. In contrast, LNAMEtreated rats received more foot shocks and made more errors than controls to reach the learning criterion, and made more errors during the memorytesting session. In parallel, NO content in the prefrontal cortex and hippocampus was higher in LArgtreated rats and lower inLNAME rats, compared to controls. Similarly, （]7 nAChR immunoreactivity and protein expression in the prefrontal cortex and hippocampus were higher in LArgtreated rats and lower in LNAME rats, compared to controls. These results suggest that the modulation of NO content in the brain correlates with a7 nAChR distribution and expression in the prefrontal cortex and hippocampus, as well as with learning and memory performance in the Ymaze.

Role of nitric oxide in biological effects of vascular endothelial growth factor

To evaluate the role of nitric oxide in the biological effects of vascular endothelial growth factor(VEGF)and the possible mechanism of VEGF,the in vitro cultured vascular endothelial cells of rabbit aorta were divided into control group,VEGF-treated group and VEGF+N-nitro-L-arginine methyl ester(L-NAME)-treated group.The absorbance(A)value of vascular endothelial cells and the levels of prostaglandin(PGI2),endothelin-1(ET-1)and von Willebrand factor(vWF)in the supernatant were observed by water-soluble tetrazo-lium salt assay,radioimmunoassay and enzyme-linked immunosorbent assay.The A values and PGI2 in VEGF-treated group and VEGF+L-NAME-treated group were higher than those in control group(P<0.05 and P<0.01).The ET-1 and vWF were significantly decreased in VEGF-treated group and VEGF+L-NAME-treated group com-pared with the control(P<0.05 and P<0.01).These results indicate that VEGF could promote the proliferation and secretion of PGI2 and inhibit the secretion of ET-1 and vWF in vascular endothelial cells and that L-NAME could inhibit the effect of VEGF partially.Nitric oxide is an important mediator in the process of stimulating prolifera-tion and regulating secretion of vascular endothelial cells by VEGF.