Glutathione deficiency intensifies ischaemia-reperfusion induced cardiac dysfunction and oxidative stress.

The efficacy of glutathione (GSH) in protecting ischaemia-reperfusion (I-R) induced cardiac dysfunction and myocardial oxidative stress was studied in open-chest, stunned rat heart model. Female Sprague-Dawley rats were randomly divided into three experimental groups: (1) GSH-depletion, by injection of buthionine sulphoxamine (BSO, 4 mmol kg(-1), i.p.) 24 h prior to I-R, (2) BSO injection (4 mmol kg(-1), i.p.) in conjunction with acivicin (AT125, 0.05 mmol kg(-1), i.v.) infusion 1 h prior to I-R, and (3) control (C), receiving saline treatment. Each group was further divided into I-R, with surgical occlusion of the main left coronary artery (LCA) for 30 min followed by 20 min reperfusion, and sham. Myocardial GSH content and GSH : glutathione disulphide (GSSG) ratio were decreased by approximately 50% (P < 0.01) in both BSO and BSO + AT125 vs. C. Ischaemia-reperfusion suppressed GSH in both left and right ventricles of C (P < 0.01) and left ventricles of BSO and BSO + AT125 (P < 0.05). Contractility (+dP/dt and -dP/dt) in C heart decreased 55% (P < 0.01) after I and recovered 90% after I-R, whereas +/-dP/dt in BSO decreased 57% (P < 0.01) with ischaemia and recovered 76 and 84% (P < 0.05), respectively, after I-R. For BSO + AT125, +/-dP/dt were 64 and 76% (P < 0.01) lower after ischaemia, and recovered only 67 and 61% (P < 0.01) after I-R. Left ventricular systolic pressure in C, BSO and BSO + AT125 reached 95 (P > 0.05) 87 and 82% (P < 0.05) of their respective sham values after I-R. Rate-pressure double product was 11% (P > 0.05) and 25% (P < 0.05) lower in BSO and BSO + AT125, compared with Saline, respectively. BSO and BSO + AT125 rats demonstrated significantly lower liver GSH and heart Mn superoxide dismutase activity than C rats after I-R. These data indicate that GSH depletion by inhibition of its synthesis and transport can exacerbate cardiac dysfunction inflicted by in vivo I-R. Part of the aetiology may involve impaired myocardial antioxidant defenses and whole-body GSH homeostasis.     

Glutathione-dependent modulation of exhausting exercise-induced changes in neutrophil function of rats

Reduced glutathione (GSH) plays a central role in maintaining an effective synergism between various physiological and exogenous antioxidants. We tested the effects of GSH andN-acetylcysteine (NAC, a pro-GSH clinical drug), intraperitoneal (i.p.) supplementation and GSH deficiency on exercise-induced leucocyte margination and neutrophil oxidative burst activity. GSH, NAC (1g · kg^–1) or placebo saline was i.p. injected (one or eight times) to male rats (n seven per group). The GSH-deficient rats were prepared by i.p. injections ofl-buthionine-[SR]-sulphoximine (BSO, 6 mmol · 1^–1 · kg^–1) twice daily for 4 days. Exercised animals were subjected to treadmill run to exhaustion. Exhausting treadmill exercise significantly decreased peripheral blood leucocyte count in the controls (P < 0.001). Such exercise-associated leucocyte margination was prevented by GSH supplementation. Peripheral blood neutrophil counts were significantly higher (P < 0.02) in the GSH-supplemented groups compared to the placebo control groups. Exercise-induced increase in peripheral blood neutrophil oxidative burst activity as measured by luminol-enhanced chemiluminescence per volume of blood tended to be higher in the GSH-supplemented group (P < 0.10), and lower in the GSH-deficient rats (P < 0.02). In these experiments, for the first time we have shown that GSH supplementation can induce neutrophil mobilization and decrease exercise-induced leucocyte margination, and that exogenous and endogenous GSH can regulate exercise-induced stimulation of the neutrophil oxidative burst.     

Glutathione deficiency intensifies ischaemia‐reperfusion induced cardiac dysfunction and oxidative stress

The efficacy of glutathione (GSH) in protecting ischaemia‐reperfusion (I‐R) induced cardiac dysfunction and myocardial oxidative stress was studied in open‐chest, stunned rat heart model. Female Sprague–Dawley rats were randomly divided into three experimental groups: (1) GSH‐depletion, by injection of buthionine sulphoxamine (BSO, 4 mmol kg^–1, i.p.) 24 h prior to I‐R, (2) BSO injection (4 mmol kg^–1, i.p.) in conjunction with acivicin (AT125, 0.05 mmol kg^–1, i.v.) infusion 1 h prior to I‐R, and (3) control (C), receiving saline treatment. Each group was further divided into I‐R, with surgical occlusion of the main left coronary artery (LCA) for 30 min followed by 20 min reperfusion, and sham. Myocardial GSH content and GSH : glutathione disulphide (GSSG) ratio were decreased by ?50% (P < 0.01) in both BSO and BSO + AT125 vs. C. Ischaemia‐reperfusion suppressed GSH in both left and right ventricles of C (P < 0.01) and left ventricles of BSO and BSO + AT125 (P < 0.05). Contractility (+dP/dt and –dP/dt) in C heart decreased 55% (P < 0.01) after I and recovered 90% after I‐R, whereas ±dP/dt in BSO decreased 57% (P < 0.01) with ischaemia and recovered 76 and 84% (P < 0.05), respectively, after I‐R. For BSO + AT125, ±dP/dt were 64 and 76% (P < 0.01) lower after ischaemia, and recovered only 67 and 61% (P < 0.01) after I‐R. Left ventricular systolic pressure in C, BSO and BSO + AT125 reached 95 (P > 0.05) 87 and 82% (P < 0.05) of their respective sham values after I‐R. Rate‐pressure double product was 11% (P > 0.05) and 25% (P < 0.05) lower in BSO and BSO + AT125, compared with Saline, respectively. BSO and BSO + AT125 rats demonstrated significantly lower liver GSH and heart Mn superoxide dismutase activity than C rats after I‐R. These data indicate that GSH depletion by inhibition of its synthesis and transport can exacerbate cardiac dysfunction inflicted by in vivo I‐R. Part of the aetiology may involve impaired myocardial antioxidant defenses and whole‐body GSH homeostasis.     

Effects of age and glutathione levels on oxidative stress in rats after chronic exposure to stretch-shortening contractions

We investigated effects of age and glutathione synthesis inhibition on the oxidative stress status of tibialis anterior muscles from young and old Fisher 344 × Brown Norway male rats after chronic administration of stretch-shortening contractions. Oral supplementation of l-buthionine-(S,R)-sulfoximine (BSO) inhibited glutathione synthesis. Dorsiflexor muscles in the hindlimb were exposed to 80 maximal stretch-shortening contractions (SSCs) three times per week for 4.5 weeks. We measured malondialdehyde, hydrogen peroxide (H₂O₂), and free isoprostanes to determine oxidative stress. Glutathione peroxidase activity was measured as an indicator of H₂O₂ scavenging. Glutathione measurements confirmed the effectiveness of BSO treatment. In young rats, the SSC exposure protocol prevented oxidative stress and enhanced H₂O₂ scavenging. In old rats, malondialdehyde was increased in the exposed muscle and a BSO-induced increase in H₂O₂ was not alleviated with SSC exposure as seen in young rats. In addition, glutathione peroxidase activity and total glutathione were increased in old rats relative to their young counterparts. All comparisons were significant at the 0.05 level. Overall, BSO administration was effective in decreasing total glutathione levels and increasing H₂O₂ levels in old and young rats exposed to SSCs. In addition, effects of chronic exposure to high-force resistive loading SSCs in active muscle from old animals are: (1) antioxidant capacity is enhanced similar to what is seen with endurance training and (2) oxidative stress is increased, probably as a consequence of the enhanced vulnerability due to aging.     

The Na+-dependent binding of [3H]l-aspartate in thaw-mounted sections of rat forebrain

Binding of [³H]l-aspartate to thaw-mounted coronal sections of frozen rat forebrain was strong in grey regions of telencephalon (neocortex, hippocampus and neostriatum), but it was weaker and unevenly distributed in diencephalon. At low nanomolar concentrations of ligand used in the present studies, [³H]l-aspartate binding was strongly inhibited by l-threo-3-hydroxyaspartate and l-trans-pyrrolidine-2,4-dicarboxylate, compounds known to be substrate/inhibitors of the high affinity uptake of l-glutamate and l-aspartate. None of the typical ligands for the glutamate and aspartate receptors, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), N-methyl-d-aspartate and kainate, produced a strong enough inhibition (only CNQX at 100 M weakly inhibited) of the Na⁺-dependent [³H]l-aspartate binding to suggest that [³H]l-aspartate was bound to the receptor binding sites. Furthermore, the binding was absolutely dependent on the presence of Na⁺ in the incubation medium. It is concluded that [³H]l-aspartate is a ligand suitable for autoradiographic studies of the distribution of Na⁺-dependent, high affinity uptake of acidic amino acids in the central nervous system (CNS). However, feasibility of using [³H]l-aspartate as a specific marker of glutamatergic and/or aspartergic synapses in the CNS requires further investigation.     

The effect of acute hypoxia on heat shock protein 72 expression and oxidative stress in vivo

The inducible human stress protein HSP72 performs vital roles within the body at rest and during periods of stress. Recently, a previously disclosed quadratic trend in basal HSP72 expression was shown to be reliable and repeatable. The notion of a physiological stressor such as hypoxia disrupting this basal quadratic trend is an interesting one. Monocyte-expressed HSP72 and TBARS were determined every 3 h, over a 12-h period in 12 healthy male subjects on two separate days, with trial day one ascertaining control values. A hypoxic intervention consisting of 75 min at a simulated altitude of 2,980 m, commencing and ceasing at 0930 and 1045, respectively, was incorporated on trail day 2. The hypoxic condition induced significantly (elevated) HSP72 values at 1100 (P = 0.002), 1400 (P < 0.001), 1700 (P = 0.034) and 2000 (P = 0.041) compared to control. Significant increases in plasma TBARS were seen in the hypoxic condition compared to control at 1100 (P = 0.006) and 1400 (P = 0.032). The results demonstrate that a 75-min bout of normobaric hypoxia is sufficient to induce significant increases in HSP72 expression, which disrupts the basal quadratic trend shown by others and here in the control condition. This increase may be linked to the observed changes in oxidative stress. These results may provide a tool for manipulating basal monocyte HSP72 expression within human heat acclimation exercise protocols.     

Mutation and haplotype analyses of the <Emphasis Type="Italic">MUT</Emphasis> gene in Japanese patients with methylmalonic acidemia

Methylmalonic acidemia (MMA) is caused by a deficiency in the activity of l-methylmalonyl-CoA mutase (MCM), a vitamin B12 (or cobalamin, Cbl)-dependent enzyme. Apoenzyme-deficient MMA (mut MMA) results from mutations in the nuclear gene MUT. Most of the MUT mutations are thought to be private or restricted to only a few pedigrees. Our group elucidated the spectrum of mutations of Japanese mut MMA patients by performing mutation and haplotype analyses in 29 patients with mut MMA. A sequence analysis identified mutations in 95% (55/58) of the disease alleles. Five mutations were relatively frequent (p.E117X, c.385 + 5G > A, p.R369H, p.L494X, and p.R727X) and four were novel (p.M1V, c.753_753 + 5delGGTATA, c.1560G > C, and c.2098_2099delAT). Haplotype analysis suggested that all of the frequent mutations, with the exception of p.R369H, were spread by the founder effect. Among 46 Japanese patients investigated in the present and previous studies, 76% (70/92) of the mutations were located in exons 2, 6, 8, and 13. This finding – that a limited number of mutations account for most of the mutations in Japanese mut MMA patients – is in contrast with results of a previous study in Caucasian patients.     

Nucleotide Sequence and Phylogenetic Analysis of Cucurbit Yellow Stunting Disorder Virus RNA 2

The complete nucleotide sequence of Cucurbit yellow stunting disorder virus (CYSDV) RNA 2, a whitefly (Bemisia tabaci)-transmitted closterovirus with a bi-partite genome, is reported. CYSDV RNA 2 is 7,281 nucleotides long and contains the closterovirus hallmark gene array with a similar arrangement to the prototype member of the genus Crinivirus, Lettuce infectious yellows virus (LIYV). CYSDV RNA 2 contains open reading frames (ORFs) potentially encoding in a 5 to 3 direction for proteins of 5 kDa (ORF 1; hydrophobic protein), 62 kDa (ORF 2; heat shock protein 70 homolog, HSP70h), 59 kDa (ORF 3; protein of unknown function), 9 kDa (ORF 4; protein of unknown function), 28.5 kDa (ORF 5; coat protein, CP), 53 kDa (ORF 6; coat protein minor, CPm), and 26.5 kDa (ORF 7; protein of unknown function). Pairwise comparisons of CYSDV RNA 2-encoded proteins (HSP70h, p59 and CPm) among the closteroviruses showed that CYSDV is closely related to LIYV. Phylogenetic analysis based on the amino acid sequence of the HSP70h, indicated that CYSDV clusters with other members of the genus Crinivirus, and it is related to Little cherry virus-1 (LChV-1), but is distinct from the aphid- or mealybug-transmitted closteroviruses.     

Increased FXYD1 and PGC‐1α mRNA after blood flow‐restricted running is related to fibre type‐specific AMPK signalling and oxidative stress in human muscle

Aim

This study explored the effects of blood flow restriction (BFR) on mRNA responses of PGC‐1α (total, 1α1, and 1α4) and Na⁺,K⁺‐ATPase isoforms (NKA; α_1‐3, β_1‐3, and FXYD1) to an interval running session and determined whether these effects were related to increased oxidative stress, hypoxia, and fibre type‐specific AMPK and CaMKII signalling, in human skeletal muscle.

Methods

In a randomized, crossover fashion, 8 healthy men (26 ± 5 year and 57.4 ± 6.3 mL kg^?1 min^?1) completed 3 exercise sessions: without (CON) or with blood flow restriction (BFR), or in systemic hypoxia (HYP, ~3250 m). A muscle sample was collected before (Pre) and after exercise (+0 hour, +3 hours) to quantify mRNA, indicators of oxidative stress (HSP27 protein in type I and II fibres, and catalase and HSP70 mRNA), metabolites, and α‐AMPK Thr¹⁷²/α‐AMPK, ACC Ser²²¹/ACC, CaMKII Thr²⁸⁷/CaMKII, and PLBSer¹⁶/PLB ratios in type I and II fibres.

Results

Muscle hypoxia (assessed by near‐infrared spectroscopy) was matched between BFR and HYP, which was higher than CON (~90% vs ~70%; P < .05). The mRNA levels of FXYD1 and PGC‐1α isoforms (1α1 and 1α4) increased in BFR only (P < .05) and were associated with increases in indicators of oxidative stress and type I fibre ACC Ser²²¹/ACC ratio, but dissociated from muscle hypoxia, lactate, and CaMKII signalling.

Conclusion

Blood flow restriction augmented exercise‐induced increases in muscle FXYD1 and PGC‐1α mRNA in men. This effect was related to increased oxidative stress and fibre type‐dependent AMPK signalling, but unrelated to the severity of muscle hypoxia, lactate accumulation, and modulation of fibre type‐specific CaMKII signalling.

     

Insulin enhances l-dopa renal proximal tubule uptake: a regulatory mechanism impaired in insulin resistance

A stimulatory role for insulin in the uptake of neutral amino acids has been reported for a variety of tissues. Here we examine the effect of insulin on l-dopa uptake by proximal tubule cells (PT cells) isolated from control and fructose-fed rats (FR-rats, 10% w/v fructose solution in tap water), a model of insulin resistance. Insulin (200 U/ml) increased l-dopa uptake into PT cells by about 50% (705±186 vs.1117±140 pmol l-dopa/mg protein per minute) (p<0.05). The higher uptake correlated with a 40% increase in the number of high-affinity l-dopa transport sites (l-dopa 0.2 M) (0.59±0.05 vs. 0.82±0.09 pmol l-dopa/mg protein per minute), without changing their affinity. The effect of insulin was not modified by ouabain (1 mM), nocodazole (1–10 M) or colchicine (50–100 M), whereas it was abolished by cytochalasin D or latrunculin B (both 1 M). This suggests that the process is independent of Na⁺,K⁺-ATPase activity or the microtubule network but that it requires the integrity of the actin cytoskeleton. l-dopa transport by the low-affinity transport sites (l-dopa 5 M) was not affected by insulin, neither was the effect of insulin observed in PT cells isolated from FR-rats. In line with this, FR-rats showed lower renal l-dopa reabsorption as compared to control animals (81±4 vs. 51±9%). Taken together, our results support the involvement of insulin in the multifactorial regulation of renal l-dopa reabsorption.     

肾透明细胞癌中氧化应激蛋白的表达

目的:探讨人肾透明细胞癌细胞株RLC-310与人正常肾近曲小管上皮细胞株HK-2,肾癌及其癌旁组织中氧化应激蛋白表达的差异。方法:体外培养RLC-310和HK-2,采用PF-2D蛋白分级分离系统分离细胞总蛋白,选取差异蛋白组分进行毛细管LC-ESI-MS/MS分析,结合蛋白质数据库检索对差异蛋白进行鉴定,并对代表性氧化应激差异蛋白采用免疫组织化学方法进行验证。结果:两个细胞株经串联质谱分析共鉴定出12个氧化应激差异表达蛋白,分别是peroxiredox-in-1(PRX-1)、peroxiredoxin-6(PRX-6)、superoxide dismutase[Cu-Zn]SOD1、glutathione peroxidase 1、catalase、glutathionesynthetase、glutathione S-transferase Pi(GSTPi)、thioredoxin、热休克蛋白10(heat shock protein,HSP10)、HSP 60、HSP 70和HSP 90。其中PRX-6、HSP 60、GSTPi三种代表性差异蛋白在人肾透明细胞癌细胞株RLC-310和人正常肾近曲小管上皮细胞株HK-2中均有表达,前者的表达水平较后者显著升高(P<0.05),这三种蛋白在肾癌组织中表达也较癌旁组织显著升高(P<0.05)。结论:人肾透明细胞癌中存在抗氧化应激蛋白的差异表达,这些氧化应激蛋白的异常表达在阻止肾癌细胞氧化损伤中起一定作用。     

Blocked production of thermal shock proteins prevents the cardioprotective effect of adaptation to exercise

The resistance of isolated rat heart to ischemia and reperfusion increases after adaptation to exercise (swimming, 30 sessions 1 h/day), which correlated with accumulation of HSP70 cytoprotector proteins in the myocardium. Quercetin blockage of HSP70 production during adaptation prevents the development of adaptation defense of the heart. It was hypothesized that the accumulation of HSP70 in the myocardium is an important mechanism of local adaptation defense of the heart. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 126, No. 9, pp. 299–301, September, 1998     

Evaluation of the role of the endocytic receptor L-SIGN for cytoadhesion of Plasmodium falciparum-infected erythrocytes

Hepatic cell populations play an important role during the malaria life cycle. L-SIGN, a homologue of DC-SIGN, mediating leukocyte and pathogen binding, is selectively expressed on liver endothelial cells. Here, we present evidence that L-SIGN acts as an endocytic cell surface receptor. However, P. falciparum-infected erythrocytes did not cytoadhere to L-SIGN. Thus, L-SIGN contributes to elimination of mannosylated ligands but does not participate in hepatic clearance of P. falciparum-infected erythrocytes.     

Metformin protects the brain against the oxidative imbalance promoted by type 2 diabetes

We aimed to investigate whether metformin protects the brain against the oxidative imbalance promoted by type 2 diabetes. This study analyzed the effect of metformin on oxidative stress markers (thiobarbituric acid reactive substances (TBARS), malondialdehyde (MDA) and carbonyl groups), hydrogen peroxide (H(2)O(2)) levels, non-enzymatic antioxidant defenses [reduced (GSH) and oxidized (GSSG) glutathione and vitamin E] and enzymatic antioxidant defenses [glutathione peroxidase (GPx), glutathione reductase (GRed) and manganese superoxide dismutase (MnSOD)] in brain homogenates of diabetic GK rats, a model of type 2 diabetes. For this purpose we compared brain homogenates obtained from untreated GK rats versus GK rats treated with metformin during a period of 4 weeks. Brain homogenates obtained from Wistar rats were used as control. The MDA levels, GPx and GRed activities are significantly higher in untreated GK rats, while TBARS levels, carbonyl groups, glutathione content and vitamin E levels remain statistically unchanged when compared with control rats. In contrast, MnSOD activity and the levels of H(2)O(2) are significantly decreased in untreated GK rats when compared with control animals. However, metformin treatment normalized the majority of the parameters altered by diabetes. We observed that metformin, besides its antihyperglycemic action, induces a significant decrease in TBARS and MDA levels, GPx and GRed activities and a significant increase in GSH levels and MnSOD activity. These results indicate that metformin protects against diabetes-associated oxidative stress suggesting that metformin could be an effective neuroprotective agent.     

Arguments for revision of classification of hypoxic states

Hypoxia always leads to dysfunction of organs and culminates in the fatal outcome. The principles of classification of the hypoxic states were formulated in 1930-s. The first successful cardiac transplantation posed the problem of dividing of circulatory cardiovascular hypoxia into two subdivisions: hypoxia associated with impaired cardiac contractility (cardiac insufficiency) and hypoxia resulting from vascular smooth muscle cell. Here we attempted to improve classification of hypoxic states on the basis of new medical achievements. The proposed classification considers the following hypoxic states: 1) exogenous hypoxia; 2) respiratory hypoxia; 3) hypoxia resulting from cardiac insufficiency; 4) hypoxia provoked by vascular smooth muscle dysfunction; 5) hemic hypoxia; 6) tissue hypoxia; and 7) combined hypoxia. There are specific and pathogenically substantiated methods for correction of all elements of the “hypoxic chain” that regulate tissue metabolism at the cellular and subcellular level both in the whole organism and in individual organs. These methods open new vistas in biology and medicine, in particular, in transplantology. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 127, No. 2, pp. 146–151, February, 1999     

Overloaded training increases exercise-induced oxidative stress and damage.

We hypothesized that overloaded training (OT) in triathlon would induce oxidative stress and damage on muscle and DNA. Nine male triathletes and 6 male sedentary subjects participated in this study. Before and after a 4-week OT, triathletes exercised for a duathlon. Blood ratio of reduced vs. oxidized glutathione (GSH/GSSG), plasma thiobarbituric acid reactive substances (TBARS), leukocyte DNA damage, creatine kinase (CK), and CK-MB mass in plasma, erythrocyte superoxide dismutase (SOD) activity, erythrocyte and plasma glutathione peroxidase (GSH-Px) activities, and plasma total antioxidant status (TAS) were measured before and after OT in pre- and postexercise situations. Triathletes were overloaded in response to OT. In rest conditions, OT induced plasma GSH-Px activity increase and plasma TAS decrease (both p < 0.05). In exercise conditions, OT resulted in higher exercise-induced variations of blood GSH/GSSG ratio, TBARS level (both p < 0.05), and CK-MB mass (p < 0.01) in plasma; and decreased TAS response (p < 0.05). OT could compromise the antioxidant defense mechanism with respect to exercise-induced response. The resulting increased exercise-induced oxidative stress and further cellular susceptibility to damage needs more study.     

Behavior of microflow and localP O 2 of the brain cortex during and after direct electrical stimulation

Summary Microflow was continuously recorded at four sites of the brain cortex (cat) during and after direct electrical stimulation of the brain. In some experiments local oxygen partial pressure (P o ₂) was additionally measured with a new combined element in the same capillary area where microflow was determined. This simultaneous measurement of both microflow and localP o ₂ in the tissue enabled us to analyze the kinetics of microflow and its dependence on localP o ₂ during activation. Microflow increased at all sites measured, in most cases within 1–2 s after the beginning of stimulation, reached the maximum of hyperemia after the end of stimulation and then gradually returned to the initial level within 30 s up to several minutes according to the intensity of the stimulation. The reaction pattern of microflow was uniform. As localP o ₂ normally did not decrease and did not even show an initial decrease after the onset of stimulation, the hyperemia could not be caused by local hypoxia. On the contrary, localP o ₂ always increased with the increase of microflow. ThisP o ₂ increase is necessary, because the tissue which consumes more oxygen needs higherP o ₂ gradients to transport the oxygen to the mitochondria. The results were presented in part at “45. Tagung der Deutschen Physiologischen Gesellschaft”, September 23–26, 1975, in Vienna     

Localization and capacity of the last step of mercapturic acid biosynthesis and the reabsorption and acetylation of cysteine S-conjugates in the rat kidney

We investigated the capacity and the localization of N-acetylation of the mercapturic acid precursor S-benzyl-l-cysteine (BC), as well as the tubular reabsorption of this compound in the rat kidney in vivo et situ by renal clearance and continuous microinfusion and microperfusion experiments. In renal clearance experiments, 450 mol BC was infused intravenously for 180 min. During the time of BC infusion and the following 180 min, the two kidneys excreted 400 mol or 90% of the infused BC dose as the mercapturate N-acetyl-S-benzyl-l-cysteine (AcBC). Comparison of the amounts of BC and AcBC entering the left kidney via the renal artery with those leaving it via the renal vein and the ureter showed that 0.13±0.04 mol BC/min (mean ± SEM) was extracted and 0.24±0.08 mol AcBC/min was formed by one kidney. The intrarenal acetylation can account for the formation of 38% of the mercapturate excreted in the final urine. In additional experiments, 50 pmol/min [¹⁴C]BC was microinfused into single superficial tubules at three different sites. During microinfusion into early proximal tubules, the final urine contained 16.3±1.8% of the microinfused radioactivity as AcBC, but no BC. When [¹⁴C]BC was microinfused into late proximal tubules, 13.0±2.3% of the infused label was recovered as BC, 28.1±2.3% as AcBC. During microinfusion into early distal tubules, the final urine contained no AcBC, but 90.3±2.1% of the infused [¹⁴C]BC was recovered. As the infused BC dose was reabsorbed completely by the high-capacity carrier for neutral amino acids in the proximal convolution, our results show acetylation of the cysteine S-conjugate both by the convoluted and the straight part of the proximal nephron, the capacity for acetylation and/or secretion being almost twice as high in the straight part.     

N G-nitro-l-arginine antagonizes endothelium-dependent dilator responses by inhibiting endothelium-derived relaxing factor release in the isolated rabbit heart

The effects of a recently described inhibitor of endothelial NO synthesis, N ^G-nitro-l-arginine (l-NNA), on the vasomotor responses to endothelium-dependent and independent vasodilators, and on the release of endothelium-derived relaxing factor (EDRF), were studied in the isolated saline-perfused rabbit heart. Infusion of l-NNA (30 M) resulted in a 52±12% increase in basal coronary perfusion pressure. The vasomotor responses to 1 M acetylcholine (ACh) and serotonin after l-NNA became biphasic, showing a small transient dilation followed by a pronounced vasoconstriction. In contrast, the dilation observed with sodium nitroprusside was not affected by l-NNA. None of the above-mentioned effects was elicited by the Stereo-isomer d-NNA. Similarly, an increase in the basal coronary perfusion pressure by endothelin-1 (0.3 nM) to the same level as observed with l-NNA did not alter the vasomotor responses to ACh and sodium nitroprusside. The increase in cyclic GMP (cGMP) in platelets passing through the coronary vascular bed was used as an index of EDRF release. Platelet cGMP amounted to 0.50±0.10 pmol/mg protein after passage through the coronary bed of the unstimulated heart. When platelets were injected during an ACh infusion (1 M), a 2.7 fold increase in cGMP was observed (P<0.01). After a 30-min infusion with l-NNA, the cGMP content of platelets passing through the unstimulated heart was reduced by 62%. Likewise, the ACh-induced increase in platelet cGMP was totally blocked. These results show that l-NNA inhibits EDRF release, and is thus a potent and selective inhibitor of EDRF-mediated dilation in the isolated rabbit heart.     

Endogenous nitric oxide and pulmonary circulation response to hypoxia in high-altitude adapted Tibetan sheep

Nitric oxide (NO) is important for the pulmonary circulation response to acute and chronic hypoxia. We examined effects of endogenous nitric oxide synthase (NOS) inhibition on pulmonary vascular tone in response to hypoxia in Tibetan sheep dwelling at 3,000 m above sea level using a pressure chamber. Unanaesthetized male sheep living at 2,300 m above sea level (n=7) were prepared for vascular monitoring. Pulmonary artery (P_pa), pulmonary artery wedge (P_cwp) and systemic artery pressures together with cardiac output (CO) were measured, and pulmonary vascular resistance (PVR) was calculated as (P_pa–P_cwp)/CO. A non-selective NOS inhibitor, N-nitro-l-arginine (NLA; 20 mg kg^–1), and a selective NOS inhibitor, ONO-1714 (0.1 mg kg^–1), were used and measurements were made at 0 m, 2,300 m, and 4,500 m, with and without the NOS inhibitors. After NLA, P_pa increased slightly and CO decreased in animals at baseline (2,300 m). The increased PVR after NLA at 4,500 m was greater than that at 2,300 m (P<0.05). Selective NOS inhibition increased PVR at baseline, but not at 4,500 m. The enhanced pulmonary vasoconstriction after NO inhibition at basal and hypoxic conditions suggests a modulating role of NOS bioactivity in the pulmonary circulation and that augmented endothelial NOS plays a counterregulatory role in the pulmonary vasoconstrictor response to acute hypoxia in high-altitude adapted Tibetan sheep.