Tubular and glomerular L-arginine transport (uptake and transporters) and the nitric oxide synthases in ischemic acute renal failure (iARF) in streptozotocin-induced diabetic rats (STZ-DM)

BACKGROUND: L-arginine or its metabolites may be important pathogenetic factors in ischemic acute renal failure (iARF) in rats. It was found that the L-arginine-nitric oxide synthase-nitric oxide system plays an important role in the renal hemodynamic alterations in the early stages of diabetes. The iARF in diabetic rats is much more severe than the normal rats exposed to a same ischemia time. The purpose of the present study was to evaluated L-arginine uptake and its transporters and nitric oxide synthase isoform expression in tubuli and glomeruli of STZ-induced diabetic rats with iARF. METHODS: iARF was induced by right nephrectomy and left renal artery clamping for 60 min followed by a 60 min reflow period. iARF was induced in STZ diabetes rats two weeks after intraperitoneal streptozotocin (60 mg/kg body weight) and in normal control rats. L-arginine uptake, L-arginine transporters (CAT1 and CAT2) and nitric oxide synthases (iNOS, eNOS, and bNOS) were determined by RT-PCR) in both glomeruli and tubuli preparations. RESULTS: The STZ diabetic rats compared with the non diabetic normal rats have a higher glomerular L-arginine uptake, higher iNOS mRNA, lower eNOS mRNA, and lower tubular CAT1 mRNA, eNOS mRNA, and bNOS mRNA. The diabetic iARF after one hour of reperfusion had lower glomerular L-arginine uptake, lower CAT1 mRNA, lower eNOS mRNA, lower bNOS, and higher tubular iNOS mRNA compared with iARF in normal rats. CONCLUSIONS: Our findings suggest a prolonged and more severe post-glomerular vasoconstriction very early after the reflow in the iARF of STZ diabetic rats compared with the iARF in the normal control rats. That may be a plausible explanation to the very significant decline in GFR and tubular necrosis that characterize the iARF in diabetic rats.     

Tubular and Glomerular L-Arginine Transport (Uptake and Transporters) and the Nitric Oxide Synthases in Ischemic Acute Renal Failure (iARF) in Streptozotocin-Induced Diabetic Rats (STZ-DM)

Background. L-arginine or its metabolites may be important pathogenetic factors in ischemic acute renal failure (iARF) in rats. It was found that the L-arginine-nitric oxide synthase-nitric oxide system plays an important role in the renal hemodynamic alterations in the early stages of diabetes. The iARF in diabetic rats is much more severe than the normal rats exposed to a same ischemia time. The purpose of the present study was to evaluated L-arginine uptake and its transporters and nitric oxide synthase isoform expression in tubuli and glomeruli of STZ-induced diabetic rats with iARF. Methods. iARF was induced by right nephrectomy and left renal artery clamping for 60 min followed by a 60 min reflow period. iARF was induced in STZ diabetes rats two weeks after intraperitoneal streptozotocin (60 mg/kg body weight) and in normal control rats. L-arginine uptake, L-arginine transporters (CAT1 and CAT2) and nitric oxide synthases (iNOS, eNOS, and bNOS) were determined by RT-PCR) in both glomeruli and tubuli preparations. Results. The STZ diabetic rats compared with the non diabetic normal rats have a higher glomerular L-arginine uptake, higher iNOS mRNA, lower eNOS mRNA, and lower tubular CAT1 mRNA, eNOS mRNA, and bNOS mRNA. The diabetic iARF after one hour of reperfusion had lower glomerular L-arginine uptake, lower CAT1 mRNA, lower eNOS mRNA, lower bNOS, and higher tubular iNOS mRNA compared with iARF in normal rats.

Conclusions. Our findings suggest a prolonged and more severe post-glomerular vasoconstriction very early after the reflow in the iARF of STZ diabetic rats compared with the iARF in the normal control rats. That may be a plausible explanation to the very significant decline in GFR and tubular necrosis that characterize the iARF in diabetic rats.     

L-Arginine counteracts nitric oxide deficiency and improves the recovery phase of ischemic acute renal failure in rats

BACKGROUND: In ischemic acute renal failure (ARF), nitric oxide-dependent regulation of renal hemodynamics and glomerular function is disturbed. Previous studies indicate that the nitric oxide precursor l-arginine (l-Arg) has beneficial effects on renal function. Here we further analyzed the impact of l-Arg on functional and biochemical parameters of nitric oxide signaling during the course of ischemic ARF. METHODS: Ischemic ARF was induced in rats by bilateral clamping of renal arteries for 45 minutes. l-Arg was applied intraperitoneally during clamping, and orally during 14 days of follow-up. Glomerular filtration rate (GFR) and renal plasma flow (RPF) were measured, and biochemical parameters analyzed by protein immunoblots. RESULTS: Clamping resulted in 70% to 90% reduction of GFR and RPF, with a gradual recovery by day 14. Using an in situ assay with the oxidative fluorescent dye hydroethidine, increased tubular generation of O2- was detected in the early course of ischemic ARF, indicating enhanced oxidative stress. These findings were accompanied by up-regulation of the nitric oxide receptor, soluble guanylate cyclase, and by significant regulatory changes of inducible nitric oxide synthase (iNOS) and endothelial NOS expression. l-Arg had a beneficial effect on GFR and RPF, decreased O2- production, diminished up-regulation of soluble guanylate cyclase, and prevented up-regulation of iNOS. CONCLUSION: Ischemic ARF is accompanied by marked alterations in the expression of key enzymes of the nitric oxide pathway, indicative for deficiency of constitutive NOS activity. l-Arg supplementation reduces O2- generation and significantly improves the expression of nitric oxide signaling proteins as well as the recovery phase of ischemic ARF.     

Differential gene expression in the recovery from ischemic renal injury

Recovery from renal ischemia requires regeneration of damaged tubular epithelium. Previous studies have examined the expression of proto-oncogenes and growth factors after ischemia, but the response of genes coding for structural and functional genes has not been scrutinized. Rats were subjected to 40 minutes of renal artery occlusion and 60 minutes to 96 hours of reperfusion. Total RNA was isolated and mRNA for the structural protein actin, the enzymes superoxide dismutase and renin, the proto-oncogene c-fos, the nuclear protein histone H2b, and the putative marker for cell injury TRPM-2 was quantitated by Northern hybridization. Expression of the proto-oncogene c-fos was seen early but for only short duration. Histone gene expression was not markedly increased until 24 hours after ischemia, but remained increased for several days. Renin mRNA was undetectable one hour after ischemia, but was present in normal amounts at 24 and 48 hours. In contrast, superoxide dismutase mRNA was present in decreased amounts 24, 48, and 96 hours after ischemia. TRPM-2 gene expression was greatly increased 24 to 72 hours after ischemia and began decreasing at 96 hours. This selective sequence of gene expression or repression after renal ischemia might maximize the proliferative repair process. This information will be useful for designing therapies to further enhance recovery from acute renal injury.     

NF-kappaB inhibitors stabilize the mRNA of high-affinity type-2 cationic amino acid transporter in LPS-stimulated rat liver

BACKGROUND: Induction of inducible nitric oxide synthase (iNOS) results in nitric oxide (NO) overproduction during endotoxemia. Cellular uptake of L-arginine, modulated by the isozymes of type-2 cationic amino acid transporters (CAT), including CAT-2, CAT-2A and CAT-2B, has been reported to be a crucial factor in the regulation of iNOS activity. We sought to elucidate the expression of CAT-2 isozymes and the role of nuclear factor-kappaB (NF-kappaB) in this expression in lipopolysaccharide (LPS)-treated rat liver. METHODS: Adult male Sprague-Dawley rats were randomly given intravenous (i.v.) injections of normal saline (N/S), LPS, LPS preceded by an NF-kappaB inhibitor (PDTC, dexamethasone or salicylate) or an NF-kappaB inhibitor alone. After injection, rats were sacrificed at different times and enzyme expression and liver injury were examined. Hepatic and systemic NO production were also measured. RESULTS: CAT-2, CAT-2A and CAT-2B were constitutively expressed in un-stimulated rat liver. LPS stimulation not only significantly increased iNOS mRNA and NO concentrations but also decreased the mRNA concentrations of CAT-2 and CAT-2B, but not CAT-2A, in a time-dependent manner. LPS-induced hepatic and systemic NO overproduction was associated with hepatocellular injury. Pre-treatment with NF-kappaB inhibitors significantly attenuated LPS-induced iNOS induction as well as CAT-2/CAT-2B mRNA destabilization, which was associated with significant inhibition of NO biosynthesis and less liver injury. CONCLUSION: NF-kappaB inhibitors stabilize CAT-2 and CAT-2B mRNA in LPS-stimulated rat liver. The hepatic CAT-2/CAT-2B pathway may be a constitutive part of cytoprotective mechanisms against sepsis.     

Lipoteichoic acid from Staphylococcus aureus reduces renal ischemia/reperfusion injury

BACKGROUND: The aim of this study was to investigate whether in vivo administration of a low, sub-lethal dose of lipoteichoic acid (LTA), a bacterial wall-fragment derived from the Gram-positive bacterium Staphylococcus aureus, protects the kidney against the renal dysfunction and injury caused by ischemia/reperfusion (I/R). METHODS: Male Wistar rats were administered LTA from S. aureus (1 mg/kg, IP). After 24 hours, rats were subjected to bilateral renal ischemia (45 min) followed by reperfusion (6 h). Serum and urinary markers were measured for the assessment of renal function, tubular and reperfusion-injury. Renal sections were used for histological grading of renal injury and for immunohistochemical localization of P-selectin, inducible nitric oxide synthase (iNOS) and nitrotyrosine (indicative of peroxynitrite formation). Kidney myeloperoxidase (MPO) activity and malondialdehyde (MDA) levels were measured for assessment of polymorphonuclear (PMN) cell infiltration and lipid peroxidation, respectively. Nitric oxide (NO) production was determined by measurement of plasma nitrite/nitrate levels. RESULTS: LTA pretreatment significantly reduced renal dysfunction, tubular and reperfusion-injury caused by I/R of the kidney as well as histological evidence of renal injury. LTA also reduced the expression of P-selectin and kidney MPO activity associated with renal I/R. MDA levels were significantly reduced by LTA pretreatment suggesting a reduction in the lipid peroxidation and formation of reactive oxygen species (ROS). LTA pretreatment also markedly reduced both the expression of iNOS and the formation of nitrotyrosine associated with renal I/R. Although LTA significantly reduced plasma nitrite/nitrate levels associated with I/R, nitrite/nitrate levels remained at levels significantly higher than that measured from the plasma obtained from Sham-operated animals. CONCLUSIONS: These data suggest, to our knowledge for the first time, that LTA pretreatment for 24 hours significantly reduces renal I/R injury. We propose that the mechanism of the protective effect involves reduction of the production of NO, ROS and peroxynitrite subsequent to reduced P-selectin and iNOS expression and PMN recruitment. However, although LTA pretreatment resulted in a reduction of iNOS expression and NO production, we hypothesize that the remaining significant levels of NO contribute to the beneficial actions provided by LTA.     

GW274150, a potent and highly selective inhibitor of iNOS,reduces experimental renal ischemia/reperfusion injury

BACKGROUND: Generation of nitric oxide (NO) by inducible nitric oxide synthase (iNOS) may contribute to renal ischemia/reperfusion (I/R) injury. The aim of this study was to investigate the effects of GW274150, a novel, highly selective, potent and long-acting inhibitor of iNOS activity in rat and mouse models of renal I/R. METHODS: Rats were administered GW274150 (5 mg/kg intravenous bolus administered 30 minutes prior to I/R) and subjected to bilateral renal ischemia (45 minutes) followed by reperfusion (6 hours). Serum and urinary indicators of renal dysfunction, tubular and reperfusion injury were measured, specifically, serum urea, creatinine, aspartate aminotransferase (AST) and N-acetyl-beta-d-glucosaminidase (NAG) enzymuria. In addition, renal sections were used for histologic scoring of renal injury and for immunologic evidence of nitrotyrosine formation and poly [adenosine diphosphate (ADP)-ribose] (PAR). Nitrate levels were measured in rat plasma using the Griess assay. Mice (wild-type, administered 5 mg/kg GW274150, and iNOS-/-) were subjected to bilateral renal ischemia (30 minutes) followed by reperfusion (24 hours) after which renal dysfunction (serum urea, creatinine), renal myeloperoxidase (MPO) activity and malondialdehyde (MDA) levels were measured. RESULTS: GW274150, administered prior to I/R, significantly reduced serum urea, serum creatinine, AST, and NAG indicating reduction of renal dysfunction and injury caused by I/R. GW274150 reduced histologic evidence of tubular injury and markedly reduced immunohistochemical evidence of nitrotyrosine and PAR formation, indicating reduced peroxynitrite formation and poly (ADP-ribose) polymerase (PARP) activation, respectively. GW274150 abolished the rise in the plasma levels of nitrate (indicating reduced NO production). GW274150 also reduced the renal dysfunction in wild-type mice to levels similar to that observed in iNOS-/- mice subjected to I/R. Renal MPO activity and MDA levels were significantly reduced in wild-type mice administered GW274150 and iNOS-/- mice subjected to renal I/R, indicating reduced polymorphonuclear leukocyte (PMN) infiltration and lipid peroxidation. CONCLUSIONS: These results suggest that (1). an enhanced formation of NO by iNOS contributes to the pathophysiology of renal I/R injury and (2). GW274150 reduces I/R injury of the kidney. We propose that selective inhibitors of iNOS activity may be useful against renal dysfunction and injury associated with I/R of the kidney.     

Preconditioning of swine heart with monophosphoryl lipid A improves myocardial preservation

BACKGROUND: Ischemic preconditioning has been proven to be a powerful tool for myocardial protection in the setting of ischemia and reperfusion. A new drug to provide pharmacologic preconditioning, monophosphoryl lipid A (MLA), was administered 24 hours before an acute coronary occlusion in pigs to determine the effect on pharmacologic preconditioning. METHODS: Two studies were completed. In the first, swine were distributed into five groups: group I, control; group II,. aminoguanidine (AMG) (30 mg/kg), a selective inducible nitric oxide synthase (iNOS) blocker; group III, MLA (10 microg/kg); group IV, MLA (35 microg/kg); and group V, MLA and AMG (35 microg/kg and 30 mg/kg, respectively). Twenty-four hours after administration of the MLA, AMG, or both, regional left anterior descending coronary artery ischemia was induced for 15 minutes followed by one hour of global normothermic cardioplegic arrest and three hour reperfusion. Left ventricular function, tissue injury, and percentage of myocardial infarction were measured. Left ventricular myocardium in the left anterior descending coronary artery region was sampled for iNOS messenger RNA (mRNA) during ischemia and reperfusion. In the second study, pigs were sacrificed 0, 4, 6, 8, and 24 hrs after MLA/AMG administration for iNOS mRNA determination in nonischemic myocardium. RESULTS: Use of MLA significantly improved postischemic ventricular function, and reduced creatinine kinase release and percentage of infarction. Monophosphoryl lipid A induced expression of iNOS mRNA in nonischemic myocardium within four hours of administration which returned to base line by 24 hours. Normothermic regional ischemia then induced expression of iNOS mRNA, which returned to base line during reperfusion. Aminoguanidine completely abolished both MLA-induced and ischemia-induced iNOS mRNA and blocked the beneficial effects of MLA. CONCLUSIONS: Use of MLA can provide myocardial preservation through enhanced expression of iNOS mRNA.     

The tyrosine kinase inhibitor tyrphostin AG126 reduces renal ischemia/reperfusion injury in the rat

BACKGROUND: We investigate the effects of tyrphostin AG126, an inhibitor of tyrosine kinase activity, on the renal dysfunction and injury caused by ischemia/reperfusion (I/R) of the kidney. METHODS: Tyrphostin AG126 (5 mg/kg intraperitoneally) was administered to male Wistar rats 30 minutes prior to bilateral renal ischemia for 45 minutes followed by reperfusion for up to 48 hours. Biochemical markers of renal dysfunction and injury were measured and renal sections assessed for renal injury. Expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) and formation of nitrotyrosine and poly (ADP) ribose (PAR) were assessed using immunohistochemistry. Rat proximal tubular cells (PTCs) were incubated with interferon-gamma (100 IU/mL), bacterial lipopolysaccharide (10 microg/mL), and with increasing concentrations of tyrphostin AG126 (0.0001-1 mmol/L) for 24 hours. Nitric oxide production was measured in both plasma from rats subjected to I/R and in incubation medium from PTCs. RESULTS: After 6 hours of reperfusion, tyrphostin AG126 significantly reduced the increase in serum and urinary indicators of renal dysfunction and injury caused by I/R and reduced histologic evidence of renal injury. Tyrphostin AG126 also improved renal function (after 24 and 48 hours of reperfusion) and reduced the histologic signs of renal injury (after 48 hours of reperfusion). Tyrphostin AG126 reduced the expression of iNOS and nitric oxide levels in both rat plasma and in PTC cultures, as well as expression of COX-2. Tyrphostin AG126 also reduced nitrotyrosine and PAR formation, suggesting reduction of nitrosative stress and poly (ADP-ribose) polymerase (PARP) activation, respectively. CONCLUSION: Taken together, these results show that tyrphostin AG126 significantly reduces the renal dysfunction and injury caused by I/R of the kidney. We propose that inhibition of tyrosine kinase activity may be useful against renal I/R injury.     

Pharmacological induction of heme oxygenase-1 inhibits iNOS and oxidative stress in renal ischemia-reperfusion injury

Nitric oxide (NO), produced by nitric oxide synthase, is implicated in the pathophysiology of renal ischemia/reperfusion (I/R) injury. This study sought to elucidate the impact of pharmacological induction of heme oxygenase-1 (HO-1) on renal I/R injury. Rats were subjected to 45 minutes of renal ischemia followed by various times of reperfusion (30 minutes, 1 hour, or 3 hours). Plasma from sacrificed rats was obtained, and the kidneys processed for the expression of iNOS, cleaved caspase-3, p38MAPK and for immunohistochemical analysis. Furthermore, we determined renal and plasma levels of lipid hydroperoxides, total thiol groups, and plasmatic NO2-/NO3- formation. Our results showed a time-dependent increase in iNOS expression, which was also confirmed by increased plasma formation of NO2-/NO3-. Interestingly, this effect was reversed by pretreatment (12 hours) with SnCl2, a potent and specific inducer of renal HO-1 expression and activity, or by intraperitoneal injection of biliverdin (10 mg/kg). Furthermore, we observed a concomitant reduction in plasma and renal LOOH formation, a normalization of renal total thiol content, a reduction of caspase-3-mediated apoptosis, and a significant increase in p38MAPK phosphoration. Taken together, these results suggested that HO-1 and its byproduct biliverdin play major roles in the pathophysiological cascade leading to renal I/R injury.     

NF-kappaB involvement in the induction of high affinity CAT-2 in lipopolysaccharide-stimulated rat lungs

BACKGROUND: Endotoxemia stimulates nitric oxide (NO) biosynthesis through induction of inducible NO synthase (iNOS). Cellular uptake of L-arginine, the sole substrate for iNOS, is an important mechanism regulating NO biosynthesis by iNOS. The isozymes of type-2 cationic amino acid transporters, including CAT-2, CAT-2A, and CAT-2B, constitute the most important pathways responsible for trans-membrane L-arginine transportation. Therefore, regulation of CAT-2 isozymes expression may constitute one of the downstream regulatory pathways that control iNOS activity. We investigated the time course of enzyme induction and the role of nuclear factor-kappaB (NF-kappaB) in CAT-2 isozymes expression in lipopolysaccharide-(LPS) treated rat lungs. METHODS: Adult male Sprague-Dawley rats were randomly given intravenous injections of normal saline (N/S), LPS, LPS plus NF-kappaB inhibitor pre-treatment (PDTC, dexamethasone, or salicylate), or an NF-kappaB inhibitor alone. The rats were sacrificed at different times after injection and enzyme expression and lung injury were examined. Pulmonary and systemic NO production were also measured. RESULTS: LPS co-induced iNOS, CAT-2, and CAT-2B but not CAT-2A expression in the lungs. Furthermore, NF-kappaB actively participated in LPS-induction of iNOS, CAT-2, and CAT-2B. LPS induced pulmonary and systemic NO overproduction and resulted in lung injuries. Attenuation of LPS-induced iNOS, CAT-2, and CAT-2B induction significantly inhibited NO biosynthesis and lessened lung injury. CONCLUSION: NF-kappaB actively participates in the induction of CAT-2 and CAT-2B in intact animals. Our data further support the idea that CAT-2 and CAT-2B are crucial in regulating iNOS activity.     

缺血再灌流肾组织内皮素—1及其受体基因表达的实验研究

为了探究内皮素１（ＥＴ１）对肾功能的影响和作用方式，采用斑点杂交和原位杂交方法对大鼠缺血６０分钟再灌注肾组织ＥＴ１及其受体亚型（ＥＴＡ、ＥＴＢ）的基因表达进行了研究。结果发现：再灌流１小时，ＥＴ１、ＥＴＡ、ＥＴＢｍＲＮＡ均明显升高；再灌流２４小时仍维持较高水平。ＥＴ１和ＥＴＡｍＲＮＡ杂交信号再灌流３小时达高峰。ＥＴ１ｍＲＮＡ主要分布肾皮质小血管内皮细胞、髓质肾小管和集合管，ＥＴＡ受体ｍＲＮＡ则分布于上述小血管的平滑肌细胞。ＥＴＢ受体ｍＲＮＡ于再灌流６小时达高峰，主要分布髓质肾小管、集合管。说明缺血再灌流肾内皮素受体亚型上调在皮质以ＥＴＡ为主，在髓质以ＥＴＢ为主，分别与增强表达的ＥＴ１结合导致肾皮质缺血和水钠代谢异常。     

肾脏缺血预适应及细胞间黏附分子1的作用

目的建立肾脏缺血预适应大鼠模型,探讨缺血预适应对细胞间黏附分子(ICAM)-1mRNA表达的影响。方法摘除右肾后,对左肾采用2min缺血 5min再灌注,4个循环后再缺血45min,建立大鼠肾脏缺血预适应模型。RT-PCR检测肾脏ICAM-1mRNA表达。结果缺血预适应使肾缺血后Scr的升高幅度值减少,肾小管损伤减轻,髓质ICAM-1mRNA表达降低。结论肾脏缺血预适应可从组织学和功能上减轻肾脏的急性缺血性损伤,这可能与肾组织ICAM-1表达降低及局部炎症减轻有关。     

Role of nitric oxide in resveratrol-induced renal protective effects of ischemic preconditioning

BACKGROUND: Resveratrol, a natural antioxidant and polyphenol found in red wine and grapes, has been found to pharmacologically precondition the heart through upregulation of nitric oxide (NO). This study was designed to explore the involvement of NO in the renoprotective effect of resveratrol in renal ischemic preconditioning in rat kidney. METHODS: Ischemic preconditioning was induced by three cycles 2-minutes of ischemia followed by 5 minutes of reperfusion before 45 minutes of prolonged ischemia. Resveratrol was given 1 hour before the surgical procedures. RESULTS: Ischemic preconditioning and resveratrol treatment significantly improved the renal dysfunction, decrease in total NO levels, and oxidative stress induced by 45 minutes of ischemia followed by 24 hours of reperfusion. Histopatholgic examination of the kidneys of ischemic/reperfusion rats revealed severe renal damage, which was attenuated in both preconditioned and resveratrol-treated animals. Preconditioning and resveratrol administration led to a marked increase in NO levels in kidney. Renoprotective effects of resveratrol were abolished when animals were pretreated with NG-nitro-L-arginine methyl ester, a nonspecific NO synthase inhibitor. CONCLUSIONS: These findings demonstrate an important contributory role of NO in the protection afforded by resveratrol in renal ischemic preconditioning. CLINICAL RELEVANCE: It is now well established that brief periods of ischemia followed by reperfusion render a variety of tissues tolerant to subsequent ischemia/reperfusion-induced injury. This phenomenon, referred to as ischemic preconditioning, was first demonstrated in the dog myocardium. The potential for clinical application of such a powerful protective phenomenon has generated enormous interest in identifying the underlying intracellular signaling pathways, with the ultimate aim of pharmacologically exploiting these mechanisms to develop therapeutic strategies that can enhance tolerance to ischemia/reperfusion injury in patients. This study explored the possible involvement of nitric oxide in renal ischemic preconditioning.     

Differential regulation of L-arginine transporters (cationic amino acid transporter-1 and -2) by peroxynitrite in rat mesangial cells.

BACKGROUND: It has become evident that increased nitric oxide (NO) generation may be associated with production of reactive oxygen species, such as peroxynitrite (ONOO-). Peroxynitrite has been postulated to be responsible for several of the cytotoxic effects previously ascribed to NO. Since cellular arginine uptake has been shown to modulate nitric oxide synthase activity, we were intrigued to study the effect of ONOO- on arginine traffic in renal mesangial cells. METHODS: Arginine uptake, CAT-1 and CAT-2 mRNA expression by northern blotting analysis, and CAT-1 protein content using western blotting were determined in mesangial cells pre-treated with peroxynitrite (0.1 and 0.5 mM) for 2 h. RESULTS: Peroxynitrite induced a significant increase in arginine uptake and CAT-2 mRNA expression compared with untreated cells. In contrast, CAT-1 mRNA expression and protein abundance were diminished. CONCLUSIONS: In rat mesangial cells, peroxynitrite augments arginine uptake via augmentation of CAT-2 while decreasing CAT-1 expression.     

Catecholamines' enhancement of inducible nitric oxide synthase-induced nitric oxide biosynthesis involves CAT-1 and CAT-2A

Catecholamines enhance inducible nitric oxide synthase (iNOS) expression that results in nitric oxide (NO) overproduction in lipopolysaccharide (LPS)-stimulated macrophages. L-arginine transport mediated by cationic amino acid transporters (including CAT-1, CAT-2, CAT-2A, and CAT-2B) is crucial in regulating iNOS activity. We sought to assess the effects of catecholamines on L-arginine transport and CAT isozyme expression in stimulated macrophages. Confluent RAW264.7 cells were cultured with LPS with or without catecholamines (epinephrine or norepinephrine, 5 x 10(-6) M) for 18 h. NO production, L-arginine transport, and enzyme expression were determined. Our data revealed that LPS co-induced iNOS, CAT-2, and CAT-2B expression, whereas CAT-1 and CAT-2A expression remained unaffected. Significant increases in NO production and L-arginine transport (approximately eight-fold and three-fold increases, respectively) were found in activated macrophages. Catecholamines significantly enhanced NO production and L-arginine transport (approximately 30% and 20% increases, respectively) in activated macrophages. Catecholamines also enhanced the expression of iNOS, CAT-1, and CAT-2A but not CAT-2 or CAT-2B in LPS-stimulated macrophages. Furthermore, the enhancement effects of catecholamines were inhibited by either dexamethasone or propranolol. We provide the first evidence to indicate that L-arginine transport in activated macrophages could be enhanced by catecholamines. Furthermore, this catecholamine-enhanced L-arginine transport might involve CAT-1 and CAT-2A but not CAT-2 or CAT-2B.