牛磺酸对果糖诱导的高血压胰岛素抵抗大鼠红细胞L-精氨酸/一氧化氮途径的影响

目的和方法:在4%果糖引起大鼠胰岛素抵抗高血压模型上,观察红细胞L-精氨酸(L-Arg)/一氧化氮(NO)途径的改变,并观察牛磺酸对其改变的影响。结果:饮用果糖大鼠血压、血糖、血浆胰岛素水平明显高于对照大鼠的同时,红细胞对L-Arg总的转运及Y⁺载体转运的最大转运速率(V_max)分别低于对照大鼠31%和37％(P＜0.01);米氏常数(Km)分别比对照大鼠高35%和30%(P＜0.01)。红细胞内一氧化氮合酶(NOS)活性、cGMP水平和血浆NO含量明显低于对照大鼠(P＜0.01)。应用牛磺酸治疗可明显逆转上述改变(P＜0.01)。结论:胰岛素抵抗高血压大鼠存在有红细胞L-Arg/NO系统的功能障碍,牛磺酸可显著提高红细胞对L-Arg的转运速率和NOS活性。     

自发性高血压大鼠肾脏L-精氨酸/一氧化氮系统与红细胞L-精氨酸转运

目的:研究自发性高血压大鼠(SHR)肾脏(L-精氨酸／一氧化氮)L-Arg／NO系统的改变及其与红细胞L-Arg转运的关系。方法:检测16周龄SHR、卡托普利治疗4周的16周龄SHR(CAP)和16周龄WKY大鼠肾脏L-Arg转运、NOS活性、NO₂^-和cGMP含量,红细胞L-Arg转运。结果: SHR肾脏高低亲和L-Arg转运体的Vmax均低于WKY组(P＜0.01,P＜0.05),Km值则无明显差异。NOS活性、NO₂^-、cGMP含量分别较WKY组低35.4%、36.2%和85.2%(P＜0.05或P＜0.01)。CAP组高亲和L-Arg转运体的Vmax、NOS活性均高于SHR组(+90%,P＜0.01;+58.6%,P＜0.05)。NOS活性与高亲和L-Arg转运体的Vmax呈正相关,r=0.585,P＜0.05。红细胞L-Arg转运的改 变与肾脏相似, SHR组的Vmax低于WKY组(－30%,P＜0.01),CAP组高于SHR组(+26.5%,P＜0.01),Km值组间比较无明显差异。红细胞L-Arg转运的Vmax与肾脏高或低亲和L-Arg转运体的Vmax均呈正相关,r=0.8434,P＜0.01(高亲和)和r=0.5255,P＜0.05(低亲和)。 结论:SHR肾脏L-Arg/NO系统活动抑制,卡托普利治疗明显解除此抑制状态。肾脏L-Arg转运的改变与红细胞L-Arg转运的改变基本一致。     

败血症休克大鼠血管外膜L-精氨酸/一氧化氮合酶/一氧化氮通路的变化

目的：观察败血症休克大鼠主动脉外膜L-精氨酸（L-Arg）转运，一氧化氮合酶（NOS）活性和一氧化氮(NO)生成的变化。方法:雄性Wistar大鼠盲肠结扎并穿孔复制败血症休克模型。测定大鼠主动脉外膜亚硝酸盐(NOx)含量、一氧化氮合酶(NOS)活性及L-精氨酸(L-Arg)转运；RT-PCR方法测定诱导型一氧化氮合酶(iNOS) mRNA水平。结果:严重感染休克大鼠呈现严重的血流动力学紊乱, 心功能抑制。败血症休克大鼠表现为严重的低血糖和高乳酸血症。血管外膜iNOS的mRNA水平均明显高于假手术组（均P<0.01），主动脉外膜NOx生成、NOS活性及L-Arg转运速率显著高于假手术组（P<0.01）。结论:败血症休克时血管外膜L-Arg/NOS/NO系统激活在败血症休克发病中可能起重要作用。     

L-精氨酸和一氧化氮抑制剂对大鼠血管钙化的影响

观察给予一氧化氮合酶(NOS)的抑制剂左旋硝基精氨酸(L-NNA)或底物L-精氨酸(L-Arg)对血管钙化的影响。利用维生素D3和尼古丁制备大鼠血管钙化模型，测定大鼠尾动脉压，血管钙含量、碱性磷酸酶活性及^45Ca沉积；并检测血管组织的L-Arg转运，NOS活性及NO2^-和cGMP含量。发现钙化大鼠血压略升高；动脉钙含量、ALP活性及^45Ca沉积明显增加，von Kossa染色可以看到明显的钙化颗粒；钙化血管组织NOS活性升高；NO和cGMP含量均减少。给予L-NNA或L-Arg后，与单纯钙化组相比，L-NNA干预组的L-Arg转运、NOS活性及NO和cGMP的含量均降低；而L-Arg干预组上述指标的变化正相反。同时，L-NNA干预组的钙化程度比钙化组加重，而L-Arg干预组的钙化程度比钙化组减轻；提示血管钙化时NO—NOS—cGMP途径发生紊乱，干预NO—NOS—cGMP途径可以影响钙化的进程。     

慢性低氧对大鼠肺血管L-精氨酸

目的:探讨慢性低氧对大鼠肺血管L-精氨酸/一氧化氮(L-Arg/NO)途径的影响。方法:采用慢性低氧性肺动脉高压(HPH)大鼠肺血管孵育,测定慢性HPH对大鼠肺动脉L-Arg转运,一氧化氮合酶(NOS)活性和NO生成释放的影响。结果:(1)低氧4周大鼠肺动脉平均压(mPAP)比对照组高33.7%(P＜0.01),右心室(RV)和左室加室间隔(LV+S)重量比值(RV/LV+S)高44.2%(P＜0.01)。(2)低氧对血浆L-Arg含量无明显影响。(3)低氧大鼠离体孵育的肺动脉摄取低浓度(0.2mmol/L)和高浓度(5.0mmol/L)[³H]-L-Arg分别低于对照组15.8%(P＜0.05)和27.2%(P＜0.01)。(4)低氧大鼠肺动脉tNOS、iNOS和cNOS活性较对照组高38.0%、32.8%和53.0%(P＜0.01)。(5)低氧大鼠血浆NO含量低于对照组,与mPAP和RV/LV+S呈负相关(P＜0.01)。结论:慢性HPH时NOS活性代偿性增强,但L-Arg转运受损使血浆NO生成仍减少,说明L-Arg转运是NO生成的重要限速步骤。     

败血症休克大鼠血管L-精氨酸/一氧化氮途径的变化

目的:观察败血症休克大鼠主动脉内膜、中膜和外膜一氧化氮合成途径的改变。方法:雄性Wistar大鼠盲肠结扎并穿孔复制败血症休克模型,分别测定假手术组、早期休克组和晚期休克组大鼠主动脉内膜、中膜和外膜的亚硝酸盐(NO^-₂)含量、一氧化氮合酶(NOS)活性及L-精氨酸(L-Arg)转运;免疫组化染色检测诱导型一氧化氮合酶(iNOS)在主动脉各层的分布。结果:早期及晚期败血症休克大鼠主动脉内膜产生的NO^-₂含量、NOS活性及L-Arg转运速率均低于假手术组,而中膜和外膜的NO^-₂、NOS活性及L-Arg转运速率则显著高于假手术组,外膜增加的程度尤为显著。免疫组织化学染色显示,败血症休克时血管中膜和外膜尤其是外膜iNOS阳性染色明显较强。结论:败血症休克时血管内膜NO合成受到抑制,而中膜和外膜NO合成显著增强,这一改变与休克状态下血管中L-Arg转运、iNOS表达及其活性的变化有关。     

L-精氨酸对大鼠蛛网膜下腔出血后一氧化氮含量及脑水肿的影响

目的:探讨一氧化氮(NO)与蛛网膜下腔出血(SAH)后脑水肿的关系和L-精氨酸(L-Arg)的影响。方法:检测假手术组(SO组)、SAH组和SAH+L-Arg组大鼠24 h内局部脑血流量(rCBF)动态变化,并测定不同时间的血清NO水平及脑组织水、Na⁺含量。结果:SAH组术后rCBF迅速而持续低于SO组,血清NO水平明显低于SO组、脑组织水、Na⁺含量显著高于SO组。L-Arg处理后上述异常改变均明显减轻。结论:SAH后血清NO水平下降与脑水肿的产生有关,L-Arg对SAH后脑水肿具有缓解作用。     

感染性休克下调大鼠血管内皮和血小板L-精氨酸/一氧化氮通路

目的：观察感染性休克对大鼠血小板及血管L-精氨酸(L-Arg)/一氧化氮合酶(NOS) /一氧化氮(NO)通路的影响及其相互联系,探讨感染性休克损伤的机制。方法:利用盲肠结扎并穿孔复制早期和晚期感染性休克大鼠模型,采用Greiss法测定血管各层及血小板孵育液亚硝酸盐(NO^-₂/NO^-₃)含量;以同位素示踪法检测其NOS活性及L -Arg转运。结果:早、晚期感染性休克大鼠血小板和主动脉内膜NO-2/NO-3水平、NOS 活性及低亲合L-Arg 转运量均显著低于假手术组(高亲合L- Arg 转运量在早期休克增加、晚期休克降低);而中膜和外膜的NO^-₂/NO^-₃水平、NOS 活性及L-Arg 转运量则显著高于假手术组,均以休克晚期改变为显著。血小板和主动脉内膜NO^-₂/NO^-₃生成、NOS 活性及高、低亲合L-Arg 转运的改变均呈正相关(均P< 0. 01)。结论:感染性休克下调血管内膜和血小板的L-Arg/NO通路,上调血管中膜和外膜L-Arg/ NO通路。提示检测血小板L-Arg/NO通路的变化可能反映休克时血管内皮功能的损伤。     

精-甘-天冬-丝氨酸肽对兔血管舒张功能及左旋精氨酸/一氧化氮途径的影响

本文观察了RGDS肽对兔离体股动脉舒张反应、主动脉一氧化氮(NO)生成、一氧化氮合酶(NOS)活性及左旋精氨酸(L-Arg)转运的影响。结果发现,RGDS肽呈剂量依赖性地引起内皮依赖和非依赖性的血管扩张;增加NOS活性及NO的产生,可被L-NNA所逆转。RGDS肽亦可增加血管组织高和低亲和性L-Arg的最大转运速率(与对照组相比分别增加126%和64%,P＜0.05和P＜0.01)。表明RGDS肽可显著增加L-Arg转运及NOS的活性,对内皮衍化舒张因子(EDRF)/NO系统具有重要调节作用。     

一氧化氮对成纤维细胞内游离Ca2+浓度的影响

目的: 研究一氧化氮(NO)对成纤维细胞内游离Ca²⁺浓度的影响。方法: 体外培养人胚肺成纤维细胞(HLF), 给予NO前体-L-精氨酸(L-Arg)及一氧化氮合酶(NOS)抑制剂-硝基-L-精氨酸(L-NNA), 采用比色法测定细胞培养液NO水平, 采用Fura-2/AM荧光法测定细胞内游离Ca²⁺浓度, 观察NO对成纤维细胞内游离Ca²⁺浓度的影响。结果: 随着细胞培养液NO水平逐渐升高, 成纤维细胞内游离Ca²⁺浓度逐渐升高[实验组NO水平、Ca²⁺ 浓度比正常对照组NO水平、Ca²⁺浓度分别为(3.82±0.53) mol/L比(2.62±0.55) mol/L和(894.48±93.01) nmol/L比(824.56±33.22) nmol/L, P<0.05]; 但进一步升高NO水平, Ca²⁺浓度却逐渐降低[实验组NO水平、Ca²⁺浓度比正常对照组NO水平、Ca²⁺浓度分别为(5.82±0.45) mol/L比(2.62±0.55) mol/L和(162.11±68.50) nmol/L比(824.56±33.22) nmol/L, P<0.01]。结论: NO对成纤维细胞内游离Ca²⁺浓度具有双向调节作用。即低水平NO升高细胞内游离Ca²⁺浓度, 高水平NO降低细胞内游离Ca²⁺浓度。     

Uridine transport in human erythrocytes: data from normal subjects and from patients with renal failure

Erythrocyte uridine transport has been studied in eight normal individuals and eight patients on haemodialysis for chronic renal failure. The initial rate of zero-trans uridine influx at 37 degrees C has been measured as a function of extracellular uridine concentration using [14C]-labelled uridine. The results are consistent with Michaelis-Menten kinetics. In normal humans the mean Vmax for uridine influx was 32.8 +/- 6.4 mumol (1 cells)-1 s-1 (mean +/- S.D.) and the mean Km was 190 +/- 12.3 microM. The measurements made in renal failure patients were not significantly different (mean Vmax 30.1 +/- 7.1 mumol (1 cells)-1 s-1, mean Km, 185 +/- 13.2 microM). These results are discussed with reference to the reported data on uridine transport in human erythrocytes at temperatures between 4 and 35 degrees C; it is suggested that zero-trans uridine influx shows a decrease in temperature dependence above 25 degrees C. The Vmax for zero-trans uridine influx at 37 degrees C in normal erythrocytes represents a turnover number for the nucleoside transporter of 180 uridine molecules per second.     

碱性成纤维细胞生长因子对兔血管舒张功能及左旋精氨酸/一氧化氮途径的影响

目的：本文观察了碱性成纤维细胞生长因子（ｂＦＧＦ）对离体主动脉舒张反应,一氧化氮（ＮＯ）生成及左旋精氨酸（Ｌ－Ａｒｇ）转运的影响。方法：离体大鼠主动脉环测定张力,主动脉薄片孵育测定ＮＯ生成和Ｌ－精氨酸（Ｌ－Ａｒｇ）转运。结果：ｂＦＧＦ呈剂量依赖性地诱导血管内皮依赖性舒张,增加血管低亲和Ｌ－Ａｒｇ的最大转运速度（与对照组比较增加４５％,Ｐ＜０．０１）,显著增加ＮＯ的产生（比对照组增加４３％）。结论：ｂＦＧＦ可增加Ｌ－Ａｒｇ转运,对内皮衍生舒张因子／一氧化氮（ＥＤＲＦ／ＮＯ）系统具有重要的调节作用     

Activation of L-arginine transport in peripheral blood mononuclear cells in chronic renal failure

Transport of LL-arginine, the precursor for nitric oxide (NO) synthesis, has been investigated in human peripheral blood mononuclear cells (PBMCs) obtained from healthy volunteers and chronic renal failure patients. Chronic renal failure patients were either on treatment by haemodialysis or continuous ambulatory peritoneal dialysis (CAPD). Saturable influx of L-arginine in PBMCs was mediated by the cationic amino acid transport systems y(+) and y(+)L. Initial rates of L-arginine transport (2 microM) via system y(+) were significantly increased in chronic renal failure patients, whereas transport via system y(+)L was unaffected. The increase in L-arginine transport via system y(+) was: 1.7-fold in uraemic patients on CAPD, 4.3-fold in uraemic patients pre-haemodialysis and 2.6-fold post-haemodialysis. When the intracellular PBMCs amino acid profile was analysed in chronic renal failure patients and control subjects, L-lysine and L-arginine concentrations were significantly increased in pre-haemodialysis uraemic patients and restored to normal values by haemodialysis and CAPD. The present study provides the first evidence that system y(+) mediates the increased transport of L-arginine in PBMCs from patients with chronic renal failure. The increased activity of system y(+) may provide the necessary supply of L-arginine to sustain NO synthesis in PBMCs exposed to increased levels of circulating cytokines in chronic renal failure.     

Increased nitric oxide synthesis in uraemic platelets is dependent on L-arginine transport via system y(+)L

Bleeding tendency in uraemic patients seems to be related to alterations in the activity of the L-arginine-nitric oxide (NO) signalling pathway in platelets. We have reported previously that L-arginine influx into human platelets is mediated by the high-affinity cationic amino acid transport system y(+)L. In the present study we examined the dependency of nitric oxide synthase (NOS) activity on L-arginine transport in platelets isolated from healthy controls and uraemic patients on haemodialysis. We investigated basal and ADP-stimulated NOS activity, as reflected by the conversion of L-[(3)H]arginine to L-[(3)H]citrulline, in platelets obtained from healthy controls and uraemic patients on haemodialysis. To determine whether NOS activity depended on L-arginine transport, we analysed the effects of competitive inhibitors of L-arginine transport via system y(+)L on NOS activity. Basal NOS activity was increased from 0.21+/-0.06 to 0.7+/-0.2 pmol/10(8) platelets ( n=9, P<0.05) in uraemic patients. Stimulation by ADP (10 micro M) significantly increased NOS activity (inhibitable by L-NAME) in control platelets (252%) but failed to increase further the elevated NOS activity in uraemic platelets. Homocysteine and L-leucine, competitive inhibitors of system y(+)L, markedly inhibited NOS activity in uraemic platelets. These observations indicate that platelets from uraemic patients on haemodialysis generate more NO than control platelets and that entry of L-arginine via system y(+)L is most likely rate-limiting for platelet NO production in chronic renal failure.     

Identification of system y+L as the high-affinity transporter for l-arginine in human platelets: up-regulation of l-arginine influx in uraemia

Kinetic studies of L-arginine transport in human platelets have identified a high-affinity, low-capacity transport system [Michaelis-Menten constant (K(m)) about 10 microM] for cationic amino acids that also transports neutral amino acids with high affinity in the presence of Na+ but not K+. These characteristics, together with our kinetic cis-inhibition studies, indicate that saturable L-arginine transport in human platelets is mediated via the system y+L and not the classic cationic transporter system y+. We present here the first evidence that L-arginine transport via system y+L is increased twofold in platelets from patients with chronic renal failure. System y+L has been described in human erythrocytes, peripheral blood mononuclear cells and placenta, and up-regulation of system y+L activity in human platelets could explain the paradox of increased nitric oxide (NO) production by uraemic platelets under conditions of decreased plasma L-arginine and elevated NG-monomethyl-L-arginine (L-NMMA) concentrations.     

一氧化氮对大鼠学习记忆和胆碱能系统的影响

目的:探讨一氧化氮(nitric oxide,NO)在大鼠空间学习和记忆过程中的作用及其对胆碱能受体作用机制。方法:大鼠侧脑室分别注射NO前体左旋精氨酸(L-arginine,L-Arg,L-Arg组)、α7烟碱型乙酰胆碱受体(α7nicotinic acetylcholine receptor,α7nAChR)拮抗剂甲基牛扁亭(methyllycaconitine,MLA,MLA组)、α7nAChR激动剂氯化胆碱(choline chloride,CC组)、一氧化氮合酶抑制剂Nω-硝基-L-精氨酸甲酯(nω-nitro-L-arginine methylester,L-NAME,L-NAME组)以及先注射MLA再注射L-Arg(ML组)、先注射L-NAME再注射氯化胆碱(NC组),并以等量生理盐水(NS组)作为对照。用Y型迷宫刺激器、硝酸还原酶法、免疫组织化学以及Western-Blot等技术分别检测大鼠空间学习和记忆行为能力、大脑皮质和海马NO含量和α7nAChR的表达。结果:与对照组比较,Y迷宫空间学习能力达标次数和24 h后30次测试记忆行为中错误反应次数在L-Arg组和CC组均减少,而在MLA组和L-NAME组均增多;大脑前额叶皮质和海马NO含量和α7nAChR阳性细胞数以及蛋白含量在L-Arg组和CC组均明显增多,而在MLA组和L-NAME组均明显减少。ML组和NC组分别与L-Arg和CC组相比较,大鼠学习和记忆行为能力均明显减弱,并且大脑前额叶皮质和海马NO含量以及α7nAChR的表达均减少。结论:侧脑室应用MLA或L-NAME可减弱L-Arg或氯化胆碱对大鼠空间学习和记忆行为能力的促进作用;NO通过α7nAChR促进大鼠空间学习和记忆能力。