体外培养的人腹主动脉瘤血管平滑肌细胞中一氧化氮的生成和诱生型一氧化氮合酶的表达

目的探讨体外培养的人腹主动脉瘤 (AAA)血管平滑肌细胞 (SMC)及其表达诱生型一氧化氮合酶 (iNOS)和培养液中生成一氧化氮 (NO)的情况。方法 0 0 2 %Ⅰ型胶原酶消化法进行AAA SMC原代培养 ,平滑肌α 肌动蛋白 (α SMA)鉴定SMC并绘制AAA SMC的增殖曲线 ;免疫细胞化学方法检测AAA SMC中iNOS蛋白的表达 ,并测定原代及 2代细胞培养液中亚硝酸盐和硝酸盐的浓度之和 (NO2 - NO3 -,NOX)。结果酶消化法成功培养AAA SMC ,α SMA阳性率为 4 5 %± 5 8% ,传代培养发现AAA SMC增殖力有限 ;AAA SMC中iNOS蛋白阳性率 86 7%± 4 6 % ,细胞培养液中存在高浓度的NOX。结论AAA SMC存在异常增殖但增殖力有限 ,且细胞可能存在表型变化 ;AAA SMC中iNOS蛋白的高表达及NO的过量生成 ,提示由SMC生成的过量NO可能在腹主动脉瘤发病机制中具有重要作用。     

Effects of estrogen on nitric oxide synthase expression in rat aorta allograft and smooth muscle cells.

BACKGROUND: We find that chronic estradiol treatment inhibits the development of transplant arteriosclerosis (TA). The mechanism of this inhibition remains unclear. The objective of this study is to investigate in a non-cyclosporin-requiring TA model whether estradiol-17beta treatment modulates the expression of both endothelial nitric oxide synthase (ecNOS) and inducible nitric oxide synthase (iNOS) in the early phase following transplantation. METHODS: Orthotopic abdominal aorta allograft transplantation was performed in male rats using Brown-Norway rats as donors and Lewis rats as recipients. The recipients (n = 50) were treated with estradiol 20 microg/kg/day or placebo by osmotic minipump from 2 days prior to surgery until sacrifice on post-operative days 1, 3, 7, 14, and 21. The allografts were harvested and cross-sections of the vascular tissues were used for immunohistochemical staining of ecNOS and iNOS. The effects of estradiol on cytokine-induced (tumor necrosis factor-alpha and interleukin-1 beta iNOS protein and messenger RNA (mRNA) expression were also evaluated on rat aorta smooth muscle cells by Western blotting and RT-PCR in vitro, respectively. RESULTS: The expression of ecNOS and iNOS was graded semiquantitatively from 0 to +3. Estrogen elevates ecNOS expression in the intima in the early phase following transplantation, 0.85 +/- 0.14 (day 7) and 1.08 +/- 0.11 (day 14) vs 1.53 +/- 0.25 (day 7) and 1.60 +/- 0.17 (day 14) for placebo and estradiol treated groups respectively, p < 0.01. Estrogen suppresses iNOS expression in neointima (0.67 +/- 0.17 vs 0.24 +/- 0.04, p < 0.01, day 14), media (1.03 +/- 0.15 vs 0.4 +/- 0.09, p < 0.01, day 7), and adventitia (1.55 +/- 0.12 vs 1.02 +/- 0.10, p < 0.05, day 14) in the same phase. Estradiol treatment inhibits cytokine-induced iNOS mRNA expression in cultured smooth muscle cells. CONCLUSIONS: Chronic estrogen treatment modulates both ecNOS and iNOS expression in the early phase following transplantation. This is associated with the estrogen-protective effects on TA.     

Activity and expression of nitric oxide synthase in the hypertrophied rat bladder and the effect of nitric oxide on bladder smooth muscle growth

PURPOSE: We investigated the expression and activity of nitric oxide synthase (NOS) and the localization of cyclic guanosine monophosphate (cGMP) in hypertrophied rat bladder. We also examined whether nitric oxide (NO) has a growth inhibitory effect in bladder smooth muscle cells. MATERIALS AND METHODS: The urethra was partly ligated and the bladder was removed 3 days, 3 or 6 weeks after obstruction. NOS activity was determined as the conversion of L-[14C]citrulline from L-[14C]arginine (Amersham Life Science, Solna, Sweden). Neuronal NOS (nNOS) expression was studied with Western blot analysis and immunohistochemistry. The expression of inducible NOS (iNOS) and cGMP was evaluated by immunohistochemistry. The effect of NO on isolated bladder smooth muscle cell growth was assessed as protein and DNA synthesis by [3H]-leucine and [3H]-thymidine (NEN Life Science Products, Zaventem, Belgium) incorporation, respectively. RESULTS: Ca independent iNOS activity increased after short-term obstruction. Immunohistochemical studies in obstructed bladders demonstrated iNOS expression primarily in urothelial and inflammatory cells. Ca dependent nNOS activity decreased after obstruction, as confirmed by Western blot analysis. The cGMP immunoreactive cells were mainly found within the serosal layer of obstructed bladders. The NO donor DETA-NONOate (Alexis Biochemicals, Lausen, Switzerland) (300 microM.) reduced [3H]-leucine and [ H]-thymidine incorporation by a mean of 29% +/- 2% and 95% +/- 2%, respectively, in cultured bladder smooth muscle cells. CONCLUSIONS: Bladder obstruction caused a small increase in iNOS activity and a decrease in nNOS activity. NO was found to have a growth inhibitory effect in bladder smooth muscle cells, suggesting that changes in NOS activity may influence the progress of bladder hypertrophy.     

Effects of intestinal transplantation on postprandial motility and regulation of intestinal transit

BACKGROUND: The effects of intestinal transplantation on gut motility have not been completely defined. In this study we examine the effects of ileal transplantation on ileal smooth muscle contractility, together with gastroduodenal emptying, intestinal flow, and transit rates in a canine model of short-gut syndrome. METHODS: Animals (n = 22) were instrumented with strain gauge transducers, collection cannulae, and infusion catheters to assess motility, intestinal flow and transit rates, and gastroduodenal emptying. Ten animals served to define normal parameters. Six animals underwent a 70% resection of the proximal small intestine to serve as short-gut controls. Six animals underwent removal of a 100-cm segment of the ileum, with cold storage, and autotransplantation the following day combined with a 70% resection of proximal bowel. RESULTS: Transplant animals exhibited delayed gastroduodenal emptying, reduced intestinal flow rates, and postprandial phasic contractions that were similar to short-gut controls. However, transplant animals experienced rapid intestinal transit compared with short-gut controls (4.8 +/- 0.4 cm/min vs 2.0 +/- 0.3 cm/min; mean +/- SEM; P <.05). CONCLUSIONS: The transplanted intestine, even with 18 hours of cold storage, exhibits a relatively normal postprandial motor response. However, adaptive responses of the transplanted intestine, such as regulation of intestine transit, may be impaired by neuromuscular injury associated with denervation or ischemia.     

Expression of inducible nitric oxide synthase in smooth muscle cells from rat penile corpora cavernosa

Nitric oxide (NO), the main mediator of penile erection, is assumed to be synthesized in the penis by the neuronal constitutive nitric oxide synthase (nNOS). However, nNOS has not been identified in the penile smooth muscle, the target of NO action. The other NOS isozymes, the inducible NOS (iNOS) and the endothelial NOS (eNOS) have not been reported in any penile tissue. The smooth muscle vascular and trabecular tissue from rat corpora cavernosa is represented in vitro by cell cultures designated RPSMC. To determine whether iNOS can be expressed in penile smooth muscle, RPSMC were treated with different lymphokines and/or bacterial lipopolysaccharide (LPS). The selected inducer, LPS/interferon, elicited at 48 hours up to a 50-fold increase in nitrites in the medium; the nitroarginine methyl ester (L-NAME), aminoguanidine, actinomycin D, cycloheximide, transforming growth factor-beta1 (TGF-beta1), and dexamethasone, but was resistant to nifedipine and platelet-derived growth factor AB (PDGF-AB). iNOS induction increased with cell passage. The [3H]L-arginine/citrulline measurement of NO synthesis with intact cells confirmed these results. Incubations of soluble and particulate fractions showed that the cytosol contained most of the activity (Km = 43 microM), which was partially inhibited by ethyleneglycal-bis-tetraacetic acid (EGTA). The 4.4-kb iNOS mRNA peaked at a late period (24-30 hours) and remained high for up to 72 hours. iNOS mRNA induction was strongly inhibited by actinomycin D and dexamethasone, partially inhibited by TGF-beta1, inhibited slightly by PDGF-AB, and unaffected by nifedipine. These results show that iNOS can be expressed in RPSMC in a cell passage-dependent fashion that has so far not been reported for other cell lines, and that the induction reaches much higher levels than in rat or human vascular smooth muscle cells. The expression pattern is also distinctive for the penile cells in time course of induction, Ca2+ dependence, response to certain agents, and mRNA stability.     

Halothane but not isoflurane attenuates interleukin 1beta-induced nitric oxide synthase in vascular smooth muscle

BACKGROUND: Inducible nitric oxide synthase (iNOS) is induced by endotoxin or cytokines, such as interleukin (IL)-1, through a protein synthesis pathway. Halothane reportedly inhibits protein synthesis in various tissues. The aim of the current study was to examine the effect of halothane on the IL-1beta-evoked induction of NOS in vascular smooth muscle. METHODS: After removal of the endothelium, arterial rings of rat aorta were mounted in an isometric force recording system. The effects of halothane (1.0-3.0%) or isoflurane (3.0%) on IL-1beta (20 ng/ml)-induced inhibition of the contractile responses to KCl (30 mM) and phenylephrine (10(-9)-10(-5) M) were studied. The cyclic guanosine monophosphate and cyclic adenosine monophosphate contents were determined by radioimmunoassay. Expression of iNOS and iNOS mRNA were measured by Western or Northern blot analysis, respectively. RESULTS: Halothane (1.0-3.0%) but not isoflurane (3%) significantly reduced the ML-1beta-induced inhibition of contraction in a concentration-dependent manner. The cyclic guanosine monophosphate content of the vascular smooth muscle increased significantly after a 5-h exposure to IL-1beta. Halothane at 3.0% significantly inhibited the increase in cyclic guanosine monophosphate content induced by IL-1beta. Halothane had no effect on cyclic adenosine monophosphate content. IL-1beta-induced expression of iNOS and iNOS mRNA in the rat aorta were inhibited significantly by halothane. CONCLUSION: The current study demonstrated that halothane but not isoflurane inhibits IL-1beta-stimulated hyporesponsiveness to vasoconstrictive agents in vascular smooth muscle and that this inhibitory effect of halothane involves the inhibition of iNOS mRNA expression. Thus, these findings suggest that halothane may have some sites to affect nitric oxide-signaling pathway.     

Relative roles of cyclooxygenase and nitric oxide synthase pathways in ischemia-induced increased contraction of cavernosal smooth muscle

PURPOSE: To study the mechanism of chronic ischemia-induced increased cavernosal smooth muscle contraction in an animal model of vasculogenic erectile dysfunction. MATERIALS AND METHODS: New Zealand White rabbits were divided into control (n = 6, fed with a regular diet), hypercholesterolemic (n = 9, fed with a diet containing 0.5% cholesterol) and chronic cavernosal ischemia (CCI, n = 10, underwent balloon de-endothelialization of iliac arteries and received a diet containing 0.5% cholesterol) groups. After 16 weeks, the relationship between iliac artery blood flow and cavernosal smooth muscle contraction was studied. The roles of cyclooxygenase and nitric oxide (NO) pathways in chronic ischemia-induced increased smooth muscle contraction were also examined. RESULTS: Iliac artery blood flow in the CCI group was significantly reduced compared with the control and hypercholesterolemic groups. Hypercholesterolemia alone did not affect cavernosal smooth muscle contraction. Atherosclerosis-induced chronic cavernosal arterial insufficiency did not affect contraction to norepinephrine while causing a significant increase in electrical field stimulation-induced neurogenic contraction. Inhibition of the cyclooxygenase pathway by indomethacin decreased electrical field stimulation-induced contraction in all animals but failed to normalize the differences between CCI and control groups. In the presence of indomethacin, L-arginine decreased electrical field stimulation-induced contraction in the control and hypercholesterolemic groups but not in the CCI group. In the presence of indomethacin, treatment with nitric oxide synthase (NOS) inhibitor, N(G)-monomethyl-L-arginine (L-NMMA), increased electrical field stimulation-induced contraction in all groups. This effect of L-NMMA on smooth muscle contraction was significantly greater in the control and hypercholesterolemic groups compared with the CCI group. After tissue treatment with L-NMMA, the magnitude of contraction in cavernosal tissue from control and hypercholesterolemic groups was similar to those observed in the CCI group. CONCLUSIONS: Mechanism of chronic ischemia-induced increased cavernosal smooth muscle contraction involves increased output of constrictor eicosanoids and impairment of the inhibitory influence of NO pathway in cavernosal tissue.     

Modulation of nitric oxide synthase isoenzymes inreperfused skeletal muscle

Objective:To investigate the modulation of nitric oxide synthase(NOS)isoenzymes in skeletal muscle during 3h ischemia/reperfusion (I/R,3h ischemia followed by 3h reperfusion).Methods:The extensor digitorum longuses(EDLs) from 20 adult rats were divided into 4 groups:the normal,the sham operation,the ischemia(3h),and the ischemia/reperfusion group.One normal EDL from each rat was used as the non-operated control,and the opposite ones are distributed into the 3 remaining groups.All the samples were studied with Western blotting technique and immunohistochemistry staining.Results:Three sizes or protein bands verified with the proteins of relative molecule to be of 155000,140000 and 135000,were detected in the EDL homogenate by Western blotting,which were comparable with the positive controls for nNOS,eNOS and iNOS,respectively.Immunostaining demonstrated that nNOS was present in the muscle fiber,with a similar location of the muscle stria,eNOS was found apparently in microvascular endothelia,but not found in muscle fibers,and iNOS was found in the leukocytes around the muscle fiber and some endothelia cells.Immunostaining paralleled the Western blotting results.Conclusions:It suggests that the constitutive nNOS and eNOS protein can be regulated by I/R,and I/R results in a down regulation of nNOW and up-regulation of eNOS and iNOS in reperfused skeletal muscle.The fact that nNOS is present around stria suggests that nNOS may have a close relationship with muscle function.The localization of eNOS in endothelial cell indicates its role in regulating blood supply of the muscle.Based on these findings,it is possible that No produced by distinct NOS may play a different role in I/R injury.     

人内皮型一氧化氮合成酶基因转染抑制人血管平滑肌细胞增殖及其机制

目的 探讨人内皮型一氧化氮合成酶基因(heNOS)转染抑制人血管平滑肌细胞(HVSMCs)增殖的机制.方法 以AdCMV-heNOS病毒感染复数分别为50、150、250、300、450 MOI,转染HVSMCs,放射免疫法检测转染HVSMCs中的环一磷酸鸟苷(cGMP)的表达变化;Western blot检测血管平滑肌细胞中p21、p27蛋白的变化,流式细胞术分析对细胞周期分布及凋亡的影响.结果　(1)转染120 h,A570值分别为1.410±0.081、1.357±0.150、1.303±0.311、0.995±0.248、0.731±0.101,其中感染复数300 MOI明显稳定抑制血管平滑肌细胞的增殖.(2)转染72 h,未转染组、Ad-LacZ转染组、Ad-heNOS转染组cGMP的含量分别为(7.91±0.39)、(8.36±0.34)、(12.89±2.06)μnol/L,差异有统计学意义(P＜0.01).(3)转染48 h,Westem blot检测转染组p27、p21表达明显上调,而未转染组虽也有p21、p27的表达,但两组差异有统计学意义(P＜0.05).(4)无血清转染48 h后血清刺激24 h,未转染组、Ad-LacZ转染组、Ad-heNOS转染组G0/G1期分别为(64.23±1.58)%、(64.96±1.36)%、(76.03±2.27)%,差异有统计学意义(P＜0.01).(5)转染3 d,第1天,未转染组、Ad-LacZ转染组、Ad-heNOS转染组细胞凋亡率分别为(4.70±0.56)%、(5.53±0.74)%、(8.53±1.06)%,差异无统计学意义(P＞0.05).第3天,细胞凋亡率分别为(5.40±0.62)%、(8.30±0.80)%、(9.30±0.90)%,差异无统计学意义(P＞0.05).结论　heNOS基因转染HVSMCs抑制细胞增殖,通过p21、p27上调导致细胞周期的阻滞,无诱导细胞凋亡.

Abstract：

Objectiye To invesigate the effect of human endothelial nitric oxide synthase (heNOS ) gene transfer on the proliferation of in vitro cultured human vascular smooth muscle cells (HVSMCs)and the mechanism. Methods The HVSMCs were transfeced with multiplicity of infection (MOI) of 50,150,250, 300,450. cGMP was measured by radioimmunoassay in HVSMCs. The expression levels of p21 and p27 were detected by Western blotting. Cell cycle and apoptosis were assayed by flow cytometry. Results (1) At 120th h, the A570values were respectively 1.410±0.081, 1.357 ±-0. 150, 1.303±0.311,0. 995 ±0. 248 and 0. 731 ±0. 101 at MOI of 50, 150,250,300,450. The proliferation of HVSMCs was significantly and stably inhibited with MOI 300 of AdCMV-heNOS; (2) Af72nd h after the gene transfer,cGMP levels were increased in heNOS-transduced ( 12. 89 ±2. 06) compared to LacZ- (8.36 ±0. 34) and non-tranduced (7. 91 ± 0. 39) cells ( P ＜ 0. 01 ); (3) At 48th h after the gene transfer, the expression of heNOS in HVSMCs up-regulated p21 and p27 (P＜0. 05); (4) After 48 h tronsfected with sersuln-depriveol and for 24 h serum stimulation, the cell cycle was significantly arrested in G0/G1 phase in heNOStransduced group, and G0/G1 was respectively ( 64. 23 ± 1.58 ) %, ( 64. 96 ± 1.36 ) %, ( 76. 03 ±2. 27 ) % in non-, LacZ- and heNOS-transduced cells; ( 5 ) At first and third day afer the gene transfer,there was no increase in apoptosis at the first day in all transduced cells ( P ＞ 0. 05 ). Conclusion Adenovirus-mediated heNOS gene transfer to HVSMCs inhibits cell proliferation via up-regulation of p21 and p27 resulting in a delay in cell progression not apoptosis.     

Involvement of endothelial nitric oxide synthase in the impaired endothelium-dependent relaxation of cavernous smooth muscle in hypercholesterolemic rabbit

The present study was designed to evaluate whether functional impairment and/or protein expression of constitutive nitric oxide synthase (cNOS; endothelial NOS [eNOS] and neuronal NOS[nNOS]) was involved in impairment of endothelium-dependent relaxation of cavernous smooth muscle in hypercholesterolemic rabbits. New Zealand White rabbits were randomly divided into control and experimental groups. The control group (n=20) received a regular diet, while the two experimental groups (n=20 for each) were fed a 2% cholesterol diet for 4 and 8 weeks, respectively. We conducted isometric tension studies with endothelium-dependent and endothelium-independent vasodilators with or without preincubation with L-arginine and nonadrenergic, noncholinergic (NANC)-selective electrical field stimulation on isolated strips of corpus cavernosum. Expression of cNOS (eNOS and nNOS) protein was assessed by Western blot analysis. cNOS activities in both cytosolic and particulate fractions were measured by determining the conversion of L-[U-14C] arginine to L-[U-14C] citrulline. Blood levels of cholesterol were significantly higher (P < 0.01) in the experimental groups than in the control group. The relaxation responses to endothelium-dependent agents (acetylcholine and adenosine 5'-diphosphate [ADP]) were significantly reduced (P < 0.05) in both experimental groups, regardless of their incubation with L-arginine, compared with the control group. However, no differences were found among the three groups in the relaxation response to endothelium-independent agents (papaverine and nitroprusside) and to NANC-selective electrical field stimulation. There was no difference in immunoreactive nNOS from cytosolic and particulate fractions between the cavernous tissues of the control and experimental groups. nNOS protein levels in the particulate fractions were markedly lower than in the cytosolic fractions. The particulate cNOS activity was significantly decreased (P < 0.05) in the experimental groups compared with the control group, while the cytosolic cNOS activity in the experimental groups was not different from that found in the control group. Therefore, it is concluded that functional impairment of eNOS, rather than of nNOS, may lead to impairment of cavernous smooth muscle relaxation in response to endothelium-mediated stimuli in hypercholesterolemic rabbits.     

The effect of nitric oxide and peroxynitrite on rabbit cavernosal smooth muscle relaxation

 Nitric oxide (NO) mediates penile erection by inducing cavernosal smooth muscle relaxation. Superoxide anion (O₂ ⁻) can influence the activity of NO by reacting with it to produce peroxynitrite (PN). This is a highly reactive species that is known to attack a variety of biological targets. It is far more reactive and damaging than its precursors. We therefore, investigated the effect of PN on rabbit cavernosal smooth muscle relaxation and compared it to NO. Cavernosal strips from nine adult New Zealand White rabbits were excised (n=12 strips for each arm of the study) and mounted in organ baths. After pre-contraction with phenylephrine (PE) (100 μM) the strips were exposed to either NO or PN (1–100 μM) and subsequent smooth muscle relaxations monitored. Some tissues were incubated with oxadiazoloquinoxalin-1-one (ODQ; 10 μM), an inhibitor of guanylyl cyclase, before the addition of NO or PN. NO and PN induced concentration-dependent relaxations in all strips. However, PN (IC₅₀: 26 ± 3.6 μM) was significantly less potent than NO (IC₅₀: 11 ± 0.7 μM) [P < 0.01]. Relaxation induced by NO was immediate and short-lived, with the tension returning to its original level. In contrast, PN-initiated relaxations were of a slower onset and more prolonged, with the tissues unable to recover tension. However, after several washouts the tissues were fully responsive to PE. Both NO- and PN-mediated relaxations were inhibited by ODQ, suggesting the involvement of cGMP in this process. Although PN mediates cavernosal smooth muscle relaxation, it is much less potent than NO. As PN is thought to play a role in a variety of pathologies where erectile dysfunction is prominent, it may also contribute to the pathogenesis of erectile dysfunction.     

门静脉高压症病人肠道通透性、NO和内毒素的研究

目的　观察门静脉高压症病人的门静脉压力、肠道通透性、NO和内毒素水平 ;并分析它们之间的相互关系。方法　门静脉高压症病人 2 0例 ,分别检测肠道通透性、门静脉压力和外周血中NO和内毒素浓度。另有 2 0位健康志愿者作对照。结果　门静脉高压症病人较健康志愿者的肠道通透性 (0 132± 0 110vs 0 0 32± 0 0 18,P <0 0 1) ,外周血的NO浓度 (38 77± 13 71vs 2 1 77±3 0 1μmol/L ,P <0 0 0 1)和内毒素浓度 (0 5 7± 0 18vs 0 11± 0 0 5EU/ml,P <0 0 0 1)均非常明显的升高。研究还发现门静脉压力和这三者均有显著相关性 (r >0 45 ,P <0 0 5 ) ,另外肠道通透性与内毒素浓度 (r=0 5 2 9,P <0 0 5 )也存在相关性 ,而与NO浓度则无相关性 ;内毒素和NO之间没有发现相关性。结论　研究表明门静脉高压症病人肠道通透性、NO和内毒素水平显著升高。门静脉压力和这三者的显著相关说明门静脉压力升高是导致肠道通透性升高的直接因素 ,而内毒素和NO的升高维持了门静脉的高动力循环。     

A possible role for nitric oxide in the regulation of human ureteral smooth muscle tone in vitro

There is ample evidence that nitric oxide (NO) is an important neurotransmitter in many tissues of the urogenital tract. The aim of the present study was to examine the possible role of NO in ureteral relaxation. Human ureteral rings were mounted in organ bath chambers and precontracted with KCl. Increasing doses of the NO donor linsidomine (SIN-1) were added with and without prior blockade of the NO/cGMP pathway by methylene blue and protein kinase (PK) inhibitors Rp-8-pCPT-cGMPS and Rp-8-CPT-cAMPS. Electrical field stimulation (EFS) was done before and after incubation with L-NOARD (N ^G-nitro-L-arginine) and TTX (tetratodoxin). For detection of neuronal NO synthase (NOS), ureters were stained immunohistochemically. Ureteral strips were dose dependently relaxed by SIN-1; preincubation with methylene blue and protein kinase G inhibitor significantly reduced the SIN-1-induced relaxations. No effects of L-NOARG and TTX on EFS-induced tone alterations were found. NOS-positive neuronal axons and nerve-ending-like structures were found in the muscular layers. Our in vitro findings suggest that ureteral relaxation may involve the NO pathway.     

转染内皮型一氧化氮合酶基因的内皮细胞抗血小板聚集和抗平滑肌细胞增殖的早期研究

目的　检测转染内皮型一氧化氮合酶 (eNOS)基因的内皮细胞功能。方法　将不同实验犬内皮细胞分为 4组 ,对照组为正常内皮细胞、β gal组为转染 β gal的内皮细胞、eNOS组为转染eNOS的内皮细胞、L 硝基 精氨酸甲酯组为转染eNOS的内皮细胞加L 硝基 精氨酸甲酯。分别采用四甲基偶氮唑盐比色法 (MTT)和氚 胸苷掺入法 ( 3 H TdR) ,检测各组的内皮细胞抗血小板聚集和抗平滑肌细胞增殖效用。结果　血小板聚集程度在不同时间eNOS组与正常组相比明显降低 ,12 0h时正常组为 4 2 2 ,eNOS组为 32 6 (P <0 0 5 ) ;平滑肌细胞增殖在不同时间eNOS组与正常组相比明显降低 ,12 0h正常组和eNOS组MTT吸光值分别为 0 2 8和 0 2 2 (P <0 0 5 ) ,3 H TdR掺入值分别为 4 6 91ccpm和3995ccpm(P <0 0 5 )。结论　体外培养已转染eNOS基因的内皮细胞 ,可明显抑制实验犬血小板聚集和平滑肌细胞增殖 ;抑制效果在培养至 4 8h时已非常明显 ,至少可持续至 12 0h ;抑制效果可被L 硝基 精氨酸甲酯阻遏     

VEGF regulation of endothelial nitric oxide synthase in glomerular endothelial cells

BACKGROUND: Vascular endothelial growth factor (VEGF) regulation of endothelial nitric oxide synthase (eNOS) and signaling pathways involved have not been well studied in glomerular endothelial cells (GENCs). METHODS: GENCs grown from tsA58 Immortomice were used. Immunoblotting and in-cell Western blot analysis were employed to assess changes in VEGF receptor signaling pathway and eNOS phosphorylation of ser1177. Immunokinase assay and immunoblotting with phosphospecific antibodies were performed to assess activity of kinases. RESULTS: VEGF rapidly induced tyrosine phosphorylation of type 1 and type 2 VEGF receptors. Physical association between VEGF-receptor 2 (VEGF-R2) and insulin receptor substrate (IRS-1) and phosphatidylinositol 3'-kinase (PI3K) was induced by VEGF, which augmented PI3K activity in VEGF-R2 immunoprecipitates. VEGF stimulated Akt phosphorylation in a PI3K-dependent manner. VEGF increased eNOS phosphorylation on Ser1177. Activation of eNOS was associated with nitric oxide generation as measured by medium nitrite content. Signaling mechanisms involved in VEGF stimulation of eNOS were explored. VEGF-induced eNOS phosphorylation was abolished by SU1498, a VEGF-R2 inhibitor, LY294002, a PI3K inhibitor, and infection of cells with an adenovirus carrying a dominant negative-mutant of Akt, demonstrating the requirement of the VEGF-R2/IRS-1/PI3K/Akt axis for activation of eNOS. VEGF also activated extracellular signal-regulated protein kinase (ERK) in a time-dependent manner; and VEGF-stimulated eNOS phosphorylation on Ser1177 was prevented by PD098059, an upstream inhibitor of ERK, demonstrating that ERK was involved in VEGF regulation of eNOS. ERK phosphorylation was abolished by LY294002, suggesting ERK was downstream of PI3K in VEGF-treated GENC. CONCLUSIONS: Our data demonstrate that in GENC, VEGF stimulates VEGF-R2/IRS-1/PI3K/Akt axis to regulate eNOS phosphorylation on Ser1177 in conjunction with the ERK signaling pathway.     

Nitric oxide synthase and nitric oxide-mediated effects in lower urinary tract smooth muscles

Summary In the lower urinary tract smooth muscles, both excitatory and inhibitory non-adrenergic, non-cholinergic (NANC) nerves and neurotransmission can be demonstrated. An inhibitory, relaxation-mediating system may serve not only the detrusor, the trigone, and the bladder neck/urethra, but may also be of importance for their integrated function. Available data suggest that nitric oxide synthase (NOS) is localized in nerve fibres of the lower urinary tract, preferably in the outflow region, and evidence has accumulated that l-arginine-derived nitric oxide (NO) is responsible for the main part of the inhibitory NANC response. Coinciding localization of NOS positive nerves with nerves expressing acetylcholine esterase, vasoactive intestinal peptide, and neuropeptide Y, suggests that NO may have a role both as a directly acting transmitter and as a modulator of efferent neurotransmission. In addition, NO may be involved in afferent neurotransmission. Theoretically, NO released from nerves in the detrusor, could be one factor keeping the bladder relaxed during filling. However, the detrusor has a low sensitivity to NO and agents acting via cyclic GMP, which makes it less likely that NO has a role as a relaxant neurotransmitter. This does not exclude that NO may modulate the effects of other transmitters, or that it has an afferent function. NO effectively relaxes isolated smooth muscle preparations from the outflow region, suggesting that it may be involved in the decrease in intraurethral pressure associated with normal micturition, and with the excessive urethral pressure variations (unstable urethra), which may be associated with certain voiding disturbances in women. The l-arginine/NO system may also control afferent activity in the outlet region, where lack of NO may lower the threshold for afferent firing leading to bladder instability. Another possible site where the l-arginine/NO pathway can have a functional role is in the urethral lamina propria. Here, NO-mediated relaxation may influence the inner urethral softness, and thereby the sealing function of the urethral mucosa. However, it should be stressed that the functional importance of the l-arginine/NO system in the central and peripheral pathways controlling micturition remains to be established.