Effect of resistance training on Na,K pump and Na+/H+ exchange protein densities in muscle from control and patients with type 2 diabetes

Ten patients with type 2 diabetes and seven controls were strength-trained with one leg for 30 min three times per week for 6 weeks. The training-induced changes in the protein densities of the Na,K-pump subunits and the Na⁺/H⁺ exchanger protein NHE1 were quantified with Western blotting of needle biopsy material obtained from trained and untrained legs of both groups. Training increased the bench press and knee-extensor force by 77±15 and 28±1%, respectively, in the control subjects, and by 75±7 and 42±8%, respectively, in the diabetics. In the control subjects the Na,K-pump isoform ₁ was increased by 37% (P<0.05) in trained compared to untrained leg, and in the diabetics the ₁ content was 45% higher (P=0.052) in trained compared to untrained leg. For the ₂ isoform the corresponding values were 21% and 41% (P<0.05), respectively. The content of the ₁ subunit in the control subjects was 33% higher (P<0.05) in trained compared to untrained leg, and 47% higher (P=0.06) in trained compared to untrained leg in the diabetics. Thus, a limited amount of strength-training is able to increase the Na,K-pump subunit and isoform content both in controls and in patients with type 2 diabetes.     

Regulation of sodium pump endocytosis by cardiotonic steroids: Molecular mechanisms and physiological implications

We have previously shown that ouabain and other cardiotonic steroids interact with the plasmalemmal Na/K-ATPase and cause a time and dose dependent endocytosis of the Na/K-ATPase. This endocytosis is demonstrable using fluorescence imaging as well as conventional biochemical and biophysical cell separation methods. In proximal tubule cells, this process appears to regulate the density of basolateral Na/K-ATPase expression directly as well as indirectly modulate transepithelial sodium transport.

Work with genetic manipulations, as well as pharmacological agents with cell culture models, have demonstrated that the cardiotonic steroid stimulated endocytosis of the plasmalemmal Na/K-ATPase requires caveolin and clathrin as well as the activation of c-Src, transactivation of the EGFR and activation of PI3K. Interestingly c-Src, EGFR and ERK1/2 all appear to be endocytosed along with the plasmalemmal Na/K-ATPase. These observations suggest a close analogy between a subset of plasmalemmal Na/K-ATPase and signaling companions with conventional receptor tyrosine kinases. While further studies are necessary to delineate the role of this endocytosis in the generation as well as the limit of signal transduction through the Na/K-ATPase signal cascade, we propose that it has an important role in the regulation of renal sodium handling as well as other important processes.     

Cyclooxygenase-2 and inducible nitric oxide synthase expression in thyroid neoplasms and their clinicopathological correlation

To evaluate the expressions of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) in thyroid neoplasms in a Korean population, we studied a total of 154 cases: papillary carcinoma of classical type (PTC), 86; follicular adenoma (FA), 21; follicular carcinoma (FC), 35; medullary carcinoma (MC), 3; undifferentiated carcinoma (UC), 5; and Hurthle cell neoplasm (HN), 4. Using immunohistochemical staining, COX-2 expression was detected in 62 (72.1%) PTC specimens, 5 (23.8%) FA specimens, 10 (28.6%) FC specimens, 0 (0.0%) MC specimens, 1 (20.0%) UC specimen, and 3 (75%) HN specimens. iNOS expression was detected in 66 (76.7%) PTC specimens, 4 (19.0%) FA specimens, 13 (37.1%) FC specimens, 0 (0.0%) MC specimens, 3 (60.0%) UC specimens, and 4 (100%) HN specimens. The results showed that COX-2 and iNOS were frequently expressed in the PTC and HN specimens, and iNOS was more frequently overexpressed in the FC specimens than in the FA specimens. In PTC, COX-2 and iNOS were significantly overexpressed in patients over 45 yr of age (p=0.029, p=0.041), and iNOS expression was increased in patients with a large primary tumor (p=0.028). These results suggest that the upregulation of COX-2 and iNOS may contribute to the tumor progression of thyroid gland, particularly in PTC and HN, and iNOS may play an adjuvant role during the tumor progression of FC.     

一氧化氮在肾缺血再灌注肾小球损伤中的作用

目的:探讨一氧化氮(NO)对肾缺血再灌注（ischemia-reperfusion injury，I-RI）时大鼠肾小球超微结构及负电荷位点的影响。 方法:SD大鼠15只，建立肾缺血再灌注模型，动物随机分为5组：（1）假手术（sham）组（n=6）；（2）I-RI组（n=6），缺血前20 min舌静脉注入生理盐水0.3 mL；（3）SNP+I-RI组（n=6），缺血前20 min舌静脉注入2.5 μg/kg硝普钠（SNP）；（4）AG+I-RI组（n=6），缺血前20 min舌静脉注入10 mg/kg氨基胍（AG）；（5）L-NNA+I-RI组（n=6），缺血前20 min舌静脉注入10 mg/kg L-硝基精氨酸（L-NNA）。以聚乙烯亚胺（PEI）为阳离子探针标记肾小球滤过膜负电荷位点，透射电镜观察肾I-RI对大鼠肾小球超微结构及负电荷位点的影响。 结果:(1) sham组电镜下见肾小球结构正常，肾小球基底膜（GBM）外透明层负电荷位点（AS）清晰，呈连续的规则点线状排列［（19.3±1.7）个/1 000 nm］。I-RI组肾小球足细胞足突有明显的融合现象；GBM外透明层AS排列稀疏［（16.6±1.0）个/1 000 nm，P<0.05］，PEI颗粒小。（2）与I-RI组相比，给予SNP使肾I-RI大鼠肾小球滤过膜上皮细胞足突融合现象加重，肾小球GBM的AS［（11.7±3.2）个/1 000 nm］显著少于假手术组（P<0.05），且PEI颗粒的电子致密度也明显低于假手术组；而AG的应用使I-RI大鼠肾小球滤过膜损伤减轻，可见清晰的足突间隙；L-NNA+I-RI组大鼠肾小球上皮细胞足突融合也明显加重，但和I-RI组相比，L-NNA+I-RI组大鼠GBM的AS数量［（14.7±0.9）个/1 000 nm］无显著差异（P>0.05）。 结论:肾I-RI时出现肾小球上皮细胞足突融合、肾小球滤过膜的负电荷位点减少等病理性损伤，NO可加重这些损伤；肾I-RI时肾小球滤过膜超微结构的损伤与NO的生成及其作用有关。     

一氧化氮对慢性缺氧高二氧化碳大鼠肺血管尾加压素Ⅱ的影响

目的： 探讨一氧化氮/L-精氨酸（NO/L-Arg）系统和尾加压素Ⅱ（UⅡ）在大鼠慢性缺氧（O2）高二氧化碳（CO2）肺动脉高压病理过程的作用及关系。 方法： 40只大鼠随机分成4组（每组各10只）：正常对照组（A组）、慢性缺O2高CO2 加生理盐水4周组（B组）、慢性缺O2高CO2 加L-Arg脂质体4周组（C组）、慢性缺O2高CO2 加N-硝基-L-精氨酸甲酯（L-NAME）4周组（D组）。免疫组化法和组织原位杂交法检测肺小动脉UⅡ和UⅡ mRNA、UⅡ受体（UT）mRNA的表达，并观察肺小动脉显微结构的变化。 结果： （1）肺动脉平均压(mPAP)、右心室(RV)和左心室+室间隔(LV+S)重量比值［RV/(LV+S)］:Ｂ组高于A组（均P<0.05）；C组低于B组（均P<0.01）；D组两指标不仅高于A组（P<0.01和<0.05），且mPAP也高于B组（P<0.01）。（2）肺小动脉管壁面积/管总面积（WA/TA）和中膜厚度（PAMT）：B组显著大于A组（P<0.05）；C组与B组的差异也有显著性（P<0.01）；而D组WA/TA也显著高于A组。（3）肺小动脉UⅡ、UⅡ mRNA、UT mRNA表达：同A组比较，Ｂ组、D组各指标都显著增高（均P<0.01）；C组UⅡ、UⅡ mRNA的表达较Ｂ组明显下调（P<0.01），同A组比较，其UⅡ表达下调但UT mRNA表达增加（均P<0.01）；D组UⅡ表达较Ｂ组低，而UT mRNA表达较Ｂ组高（均P<0.01）。 结论： 慢性缺O2高CO2肺动脉高压的发生发展可能与UⅡ的异常表达增加有关，而外源性NO可能有抑制UⅡ的作用。     

慢性低氧性肺动脉高压大鼠内源性一氧化氮代谢及其外源性吸入的研究

目的:研究一氧化氮(NO)在大鼠慢性低氧性肺动脉高压发生发展中的代谢变化,观察外源性吸入NO对肺血流动力学的疗效。方法:67只SD雄性成年大鼠,随机分为7组:常氧对照组(n=9),慢性间断性低氧(6h/d,7d/周)1周组(n=7),低氧2周组(n=11),低氧3周组(n=11),L-NAME(NO合成酶抑制剂,30mg/kg,灌饲)处理组(n=10),L-Arg(NO合成前质,10mg/kg,灌饲)处理组(n=9),NO吸入(0.0004%,20min)组(n=10)。插管测肺动脉平均压(MPAP),分离右心室(R)、室间膈+左心室(S+L),计算R/(S+L)(g/g)和R/Wt(Wt:体重,g/kg)。结果:①低氧1周组MPAP显著高于对照组,低氧2周时更高,R/(S+L)和R/Wt也显著高于对照组;②血浆NO₂^-/NO₃^-含量在低氧2周组显著高于对照组,而在低氧3周组显著低于低氧2周组和低氧1周组;低氧1周,血浆ET-1含量显著高于对照组,血浆ET-1含量与MPAP和R/(S+L)均呈显著正相关,r分别为0.43和0.46,P值均＜0.01;③L-NAME组大鼠血浆NO₂^-/NO₃^-含量降低33.2%,R/(S+L)显著增加15.2%,P＜0.05;④L-Arg组大鼠血浆NO₂^-/NO₃^-含量和PAPM无显著改变,但R/(S+L)降低8.7%,P＜0.05;⑤吸入NO,MPAP降低17.8%,P＜0.01。结论:内源性NO在慢性低氧早期(1-2周)继发性增加,但进一步低氧时则减少,血浆ET-1含量的增加在低氧性心肺血管重建中可能具有重要意义,吸入低浓度NO对以肺血管增生和右心室肥大为主要基础的肺动脉高压仍具有明显降低作用。     

Effect of focal cerebral ischemia on nitric oxide synthase expression in rats

Time- and cell-type-dependent immunohistochemical activity of nitric oxide synthase (NOS) was investigated in rat cerebral cortex following focal ischemia and the local concentration of nitric oxide (NO) was measured. NO concentration increased 2 min after the ischemia. Brain NOS-immunoreactive neurons increased in number 5 min after the ischemia. Endothelial cell NOS immunoreactivity was first detected in vascular endothelial cells and astrocytes 5 min after the ischemia, and it increased again during 60 min to 4 days after the ischemia in reactive astrocytes. Inducible NOS immunoreactivity was detected in astrocytes, vascular endothelium, and microglia/macrophages at the periphery of the ischemic core during 2–4 days after the ischemia.This study was presented at the 28th Annual Meeting of the Clinical Electron Microscopy Society of Japan, Osaka, October 17–19, 1996.     

The expression and activity of renal nitric oxide synthase and circulating nitric oxide in polycystic kidney disease rats

Nitric oxide (NO) influences tubular fluid and electrolyte transport, and hence possibly also fluid accumulation in renal cysts. The expression and activity of intrarenal constitutive NO synthase (cNOS) [neuronal NOS, nNOS and endothelial NOS, eNOS] and inducible NOS (iNOS) and plasma nitrite/nitrate (PNOx) concentration were assessed in homozygous Han:SPRD polycystic kidney disease (PKD) rats (cy/cy), heterozygous Han:SPRD PKD rats (cy/+), homozygous normal Han:SPRD littermates (+/+) and Sprague Dawley rats (sd). The results showed: 1) nNOS expression was decreased in proximal tubules and thick ascending limbs of the loop of Henle in cy/cy and cy/+ rats compared to +/+ and sd rats (p<0.05). nNOS was weakly expressed in the epithelium of small cysts and unexpressed in epithelium of large cysts. 2) iNOS expression was increased in proximal tubular epithelial cells in cy/+ rats compared to +/+ rats and sd rats (p<0.01). iNOS expression in cyst epithelium was decreased in cy/+ rats (p<0.05) and absent in cy/cy rats. 3) eNOS expression was similar in the endothelium of intrarenal arteries in all groups. 4) The activity of renal cNOS was decreased in cy/cy and cy/+ rats; the activity of iNOS was decreased only in cy/cy rats, with no significant difference among the other three groups. 5) PNOx concentration was higher in cy/cy rats than in the other three groups, and correlated positively with plasma creatinine and urea. In conclusion, NOS expression and activity decreased as cysts developed, suggesting that NO downregulation is involved in the pathogenesis of PKD.     

PKC抑制剂灯盏花素乙对甲醛炎性痛大鼠脊髓NOS表达及NO含量的影响

目的：观察蛋白激酶C（PKC）选择性抑制剂灯盏花素乙（CH）对甲醛炎性痛时大鼠自发痛反应、脊髓一氧化氮合酶（NOS）表达和一氧化氮（NO）含量的影响，探讨炎性痛时脊髓内NO产生是否受PKC调控。 方法： 采用右后掌足底注射甲醛复制炎性痛模型；计数缩足反射次数反映自发痛程度；应用NADPH-d组织化学法测定脊髓NOS表达；硝酸/亚硝酸还原法测定脊髓腰膨大部位NO2-/NO3-含量。 结果： 甲醛炎性痛大鼠L5脊髓后角浅层和中央管周围灰质NADPH-d阳性细胞的数目、阳性细胞胞体及纤维的染色深度均明显大于正常对照组，脊髓腰膨大部位 NO2-/NO3-含量明显增高。预先鞘内给予CH，可明显抑制甲醛炎性痛诱导的大鼠第二相自发痛反应以及脊髓NOS表达和NO2-/NO3-的含量。 结论： 炎性痛时，脊髓伤害性感受神经元内PKC激活可以促进NOS表达和NO的产生。     

Effect of skin sensitizers on inducible nitric oxide synthase expression and nitric oxide production in skin dendritic cells: role of different immunosuppressive drugs

Nitric oxide (NO) is involved in the pathogenesis of acute and chronic inflammatory conditions, namely in allergic contact dermatitis (ACD). However, the mechanism by which NO acts in ACD remains elusive. The present study focuses on the effects of different contact sensitizers (2,4-dinitrofluorbenzene, 1,4-phenylenediamine, nickel sulfate), the inactive analogue of DNFB, 2,4-dichloronitrobenzene, and two irritants (sodium dodecyl sulphate and benzalkonium chloride) on the expression of the inducible isoform of nitric oxide synthase (iNOS) and NO production in skin dendritic cells. It was also studied the role of different immunosuppressive drugs on iNOS expression and NO production. Only nickel sulfate increased the expression of iNOS and NO production being these effects inhibited by dexamathasone. In contrast, cyclosporin A and sirolimus, two other immunosuppressive drugs tested, did not affect iNOS expression triggered by nickel.     

苯妥英钠对应激大鼠海马NO变化的影响

目的:探讨应激对大鼠海马CA3区锥体细胞神经型一氧化氮合酶(nNOS)和诱导型一氧化氮合酶(iNOS)表达及海马匀浆液中亚硝酸盐(NO₂^-)/硝酸盐(NO₃^-)含量的影响及苯妥英钠对它们的效应。方法:采用强迫游泳作为应激模型。利用免疫组织化学方法及计算机图像分析技术,定量测定各组大鼠海马CA3区神经元nNOS和iNOS的表达水平;采用硝酸还原酶法测定海马匀浆液中NO₂^-/NO₃^-的含量。结果:应激组大鼠海马CA3区锥体细胞nNOS表达的平均灰度值(155.42±3.77)显著低于对照组(164.54±4.62)和应激给药组(164.27±2.55)(P<0.01);应激组iNOS表达的相对切面面积比(5.87%±2.90%)显著高于对照组(0.90%±0.89%)和应激给药组(0.90%±0.88%)(P<0.01);应激组海马匀浆液中NO₂^-/NO₃^-含量(42.75μmol/L±14.49μmol/L)显著高于对照组(21.23μmol/L±6.99μmol/L)和应激给药组(18.40μmol/L±8.11μmol/L)(P<0.01),后两组间差异无显著性。结论:应激可诱导海马CA3区锥体细胞nNOS和iNOS表达水平增加及海马中NO含量升高,苯妥英钠能阻止慢性应激所致的海马CA3区锥体细胞nNOS和iNOS异常表达,从而抑制NO的过度生成。     

Vesicular glutamate transporters and neuronal nitric oxide synthase colocalize in aortic depressor afferent neurons

The aortic depressor nerve (ADN) primarily transmits baroreceptor signals from the aortic arch to the nucleus tractus solitarii. Cell bodies of neurons that send peripheral fibers to form the ADN are located in the nodose ganglion (NG). Studies have implicated glutamate and nitric oxide in transmission of baroreflex signals; therefore, we tested the hypothesis that ADN neurons contain either vesicular glutamate transporters (VGLUTs) or neuronal nitric oxide synthase (nNOS) or both. We applied a fluorescent tracer, tetramethyl rhodamine dextran (TRD), to rat ADN to identify ADN neurons and then performed immunofluorescent labeling for nNOS and VGLUTs 1, 2, and 3 in NG sections. We found that VGLUT2-immunoreactivity (IR) and VGLUT3-IR was present in a significantly higher proportion of TRD positive neurons than in TRD negative neurons. In contrast, the percentage of TRD positive neurons containing VGLUT1-IR or nNOS-IR did not differ from that of TRD negative neurons. We also observed that the percentage of TRD positive neurons containing both VGLUT2-IR and nNOS-IR and the percentage of TRD positive neurons containing both VGLUT3-IR and nNOS-IR were significantly higher than that of TRD negative neurons. On the other hand, colocalization of VGLUT1-IR and nNOS-IR in TRD positive neurons did not differ from that of TRD negative neurons. These results support our hypothesis and suggest prominent roles of VGLUT2-IR containing neurons and VGLUT3-IR containing neurons in transmitting cardiovascular signals via the ADN to the brain stem.     

吸入一氧化氮对婴幼儿体外循环中肺功能的影响

目的探讨吸入一氧化氮（Nitric oxide,NO）对婴幼儿体外循环手术中肺功能的影响。方法将30例患室间隔缺损的婴幼儿随机分为对照组和NO组,NO组在体外循环期间吸入40 ppm NO直至关胸。体外循环前和术后气管插管未拔前0-1h、1-2h、2-3h测定气道压、吸入氧浓度和呼气末二氧化碳浓度（ETCO2）,并分别在同时点采动脉血进行血气分析,计算肺泡死腔率（VD/VT）肺、泡动脉血氧分压差（P（A-a）O2）、动脉血氧含量（CaO2）和肺泡氧合指数（OI）,记录术后呼吸机支持时间。体外循环前、主动脉开放后1、5、10min分别取右上肺静脉血和右心房血用于测定丙二醛（MDA）、超氧化物歧化酶（SOD）。结果与NO组相比,对照组再灌注后VD/VT、P（A-a）O2,OI明显升高（P〈0.05）,CaO2下降（P〈0.01）;MDA明显升高（P〈0.05）;SOD明显降低（P〈0.01）。结论婴幼儿体外循环术中存在明显肺损害,表现为一些亚临床性肺功能损伤。吸入40 ppm NO对体外循环期间肺功能有保护作用。     

Angiotensin II AT1 receptor blockade changes expression of renal sodium transporters in rats with chronic renal failure

We aimed to examine the effects of angiotensin II AT(1) receptor blocker on the expression of major renal sodium transporters and aquaporin-2 (AQP2) in rats with chronic renal failure (CRF). During 2 wks after 5/6 nephrectomy or sham operation, both CRF rats (n=10) and sham-operated control rats (n=7) received a fixed amount of low sodium diet and had free access to water. CRF rats (n=10) were divided into two groups which were either candesartan-treated (CRF-C, n=4) or vehicle-treated (CRF-V, n=6). Both CRF-C and CRF-V demonstrated azotemia, decreased GFR, polyuria, and decreased urine osmolality compared with sham-operated rats. When compared with CRF-V, CRF-C was associated with significantly higher BUN levels and lower remnant kidney weight. Semiquantitative immunoblotting demonstrated decreased AQP2 expression in both CRF-C (54% of control levels) and CRF-V (57%), whereas BSC-1 expression was increased in both CRF groups. Particularly, CRF-C was associated with higher BSC-1 expression (611%) compared with CRF-V (289%). In contrast, the expression of NHE3 (25%) and TSC (27%) was decreased in CRF-C, whereas no changes were observed in CRF-V. In conclusion, 1) candesartan treatment in an early phase of CRF is associated with decreased renal hypertrophy and increased BUN level; 2) decreased AQP2 level in CRF is likely to play a role in the decreased urine concentration, and the downregulation is not altered in response to candesartan treatment; 3) candesartan treatment decreases NHE3 and TSC expression; and 4) an increase of BSC-1 is prominent in candesartan-treated CRF rats, which could be associated with the increased delivery of sodium and water to the thick ascending limb.     

Zonal differences in nitric oxide synthesis by bovine chrondrocytes exposed to interleukin-1

To determine the role of nitric oxide (NO) in the inhibition of aggrecan synthesis, we measured levels of NO produced by bovine chondrocytes from different layers of articular cartilage in the presence of interleukin-1 (IL-1). Chondrocytes from the superficial layer showed a large increase in NO synthesis in response to IL-1. Although chondrocytes from the deep layer also produced NO in response to IL-1, the amount was less than that from the superficial layer. Enhanced NO production evoked by IL-1 was accompanied by a significant inhibition of aggrecan synthesis. These data suggest that chondrocytes in both superficial and deep layer of articular cartilage inhibit aggrecan synthesis with IL-1 via NO production. In addition, superficial layer cells respond to lower amounts of IL-1 with respect to NO-production and inhibition of proteoglycan synthesis.Abbreviations interleukin-1-IL-1 nitric - oxide-NO L-NG-monomethylarginine-NMA     

硫氢化钠对烧伤大鼠血清中一氧化氮含量的影响

目的观察硫氢化钠（NaHS）干预烧伤大鼠血清中一氧化氮（NO）含量的变化,探讨硫化氢（H2S）对烧伤后NO的影响。方法将64只雄性SD大鼠,随机分为烧伤组和干预组,每组32只;两大组又分为伤前与伤后24h、48h、96h小组,每小组8只;其中伤后各小组大鼠制成30%Ⅲ度烫伤模型。测定烧伤组与干预组大鼠血清中NO含量,应用统计学方法分析相关数据。结果烧伤组与干预组的伤前NO含量比较差异没有有统计学意义（t=1.822,P〉0.05）;烧伤组和干预组伤后NO含量均较伤前增高,比较差异有统计学意义（t=14.587~28.786,P〈0.05）。烧伤组和干预组伤后组NO含量均在伤后24h达最高值。干预组伤后各时相点NO含量均明显低于烧伤组,比较差异有统计学意义（t=3.144~24.762,P〈0.05）。结论给予硫氢化钠后烧伤大鼠血清NO含量上升幅度明显降低,提示硫化氢可以影响烧伤大鼠NO含量。     

Altered renal sodium transporter expression in an animal model of type 2 diabetes mellitus

Hemodynamic factors play an important role in the development and/or progression of diabetic nephropathy. We hypothesized that renal sodium transporter dysregulation might contribute to the hemodynamic alterations in diabetic nephropathy. Otsuka Long Evans Tokushima Fatty (OLETF) rats were used as an animal model for type 2 diabetes. Long Evans Tokushima (LETO) rats were used as controls. Renal sodium transporter regulation was investigated by semiquantitative immunoblotting and immunohistochemistry of the kidneys of 40-week-old animals. The mean serum glucose level in OLETF rats was increased to 235+/-25 mg/dL at 25 weeks, and the hyperglycemia continued up to the end of 40 weeks. Urine protein/ creatinine ratios were 10 times higher in OLETF rats than in LETO rats. At 40th week, the abundance of the epithelial sodium channel (ENaC) beta-subunit was increased in OLETF rats, but the abundance of the ENaC gamma-subunit was decreased. No significant differences were observed in the ENaC alpha-subunit or other major sodium transporters. Immunohistochemistry for the ENaC beta-subunit showed increased immunoreactivity in OLETF rats, whereas the ENaC gamma-subunit showed reduced immunoreactivity in these rats. In OLETF rats, ENaC beta-subunit upregulation and ENaC gamma-subunit downregulation after the development of diabetic nephropathy may reflect an abnormal sodium balance.     

24-Hour rhythm in gene expression of nitric oxide synthase and heme-peroxidase in anterior pituitary of ethanol-fed rats

Chronic exposure of rats to ethanol results in significant changes in pituitary hormone secretion. However, identification of the site(s) and mechanism of action of ethanol to induce these effects remains elusive. Free radical damage at the adenohypophyseal level may play a role in the decline in serum gonadotropin levels in ethanol-fed rats. Since 24-h changes in redox state occurred, we analyzed the 24-h changes in pituitary gene expression of the prooxidant enzymes nitric oxide synthase (NOS) 1 and 2, and of heme oxygenase-1 (HO-1) enzyme, and in plasma NO(2)(-) and NO(3)(-) (NO(x)) levels, in ethanol and control rats. Male rats, 35-day-old, received a liquid diet for 4 weeks. The ethanol-fed group received a similar diet to controls except for that maltose was isocalorically replaced by ethanol. Animals were killed at six time intervals during a 24-h cycle. Anterior pituitary mRNA levels encoding NOS1, NOS2 and HO-1 were measured by real-time PCR analysis. Plasma NO(x) concentration was determined by the Griess reaction. Ethanol feeding of prepubertal rats changed significantly the 24-h pattern of expression of NOS1, NOS2 and HO-1 in the adenohypophysis and augmented NOS2 and HO-1 mRNA levels. Peak values for the three enzymes in ethanol-fed rats occurred at the beginning of the scotophase (i.e., at 21:00 h). Ethanol feeding augmented mean values plasma NO(x) levels with a maximum at 13:00 h while in controls a biphasic pattern was observed, with peaks at 09:00 h and 17:00-21:00 h. One of the mechanisms by which ethanol augments oxidative damage in the adenohypophysis may include overproduction of nitric oxide and carbon monoxide.