Effect of alpha-lipoic acid supplementation on markers of protein oxidation in post-mitotic tissues of ageing rat

In the present study, we investigated whether DL-alpha-lipoic acid (LA) supplementation could have prooxidant or antioxidant effects on oxidative protein damage parameters such as protein carbonyl (PCO), nitrotyrosine (NT), advanced oxidation protein products (AOPP), and protein thiol (P-SH), as well as oxidative stress parameters such as total thiol (T-SH), non-protein thiol (Np-SH), and lipid hydroperoxide (LHP) in the brain and the skeletal muscle tissue of aged rats. PCO, and NT levels were increased, AOPP and P-SH levels were not changed in the brain tissue of aged rats given LA supplementation. On the other hand, TSH, Np-SH, and LHP levels were decreased in the brain tissue of aged rats given LA supplementation. The levels of the same parameters were not significantly different in the skeletal muscle tissue of aged rats given LA supplementation. The increased levels of protein oxidation markers such as PCO, and NT in the brain tissue of LA-supplemented aged rats compared with non-supplemented aged rats may suggest that oxidative protein damage is increased in LA-supplemented aged rats. We assume that an explanation for our findings regarding LA supplementation on protein oxidation markers in the brain tissue of aged rats may be due to the prooxidant effects of LA. Depending on post-mitotic tissue type and dosage of LA, the prooxidant effects of LA supplementation, should be considered in future studies.     

糖基化终产物对人肾小球系膜细胞氧化应激及MCP-1表达的影响及机制

目的：探讨体外培养条件下糖基化终产物（AGEs）对人肾小球系膜细胞（HRMCs）中糖基化终产物受体（RAGE）、氧化应激及单核细胞趋化因子-1（MCP-1）表达的影响。方法：将HRMCs与不同浓度的糖化牛血清白蛋白（AGE-BSA）和牛血清白蛋白（BSA）共同培养,或与同一质量浓度的AGE-BSA和BSA共同培养不同时间,以中和抗RAGE抗体封闭细胞膜上RAGE;采用细胞免疫化学法检测AGEs对HRMCs中RAGE表达的影响,流式细胞术检测细胞内活性氧（ROS）,半定量逆转录-聚合酶链反应（RT-PCR）法检测MCP-1 mRNA的表达。结果：在HRMCs中AGE-BSA能够促进RAGE的表达,并以时间和剂量依赖方式促进HRMCs中ROS及MCP-1的表达;ROS及MCP-1的表达水平在加入不同浓度（50、100、200、400 mg/L）的AGE-BSA作用48 h后以及加入质量浓度为200 mg/L的AGE-BSA作用不同时间（12、24、48、72 h）后,较相应质量浓度或时间的BSA组和对照组均明显升高（P〈0.05）;抗RAGE抗体干预后能够部分抑制AGE-BSA诱导ROS及MCP-1的表达,而人IgG没有这种作用。结论：AGEs通过RAGE激活氧化应激效应诱导MCP-1的表达上调,是糖尿病肾病发生发展的可能机制。     

Effects of erythropoietin on ICAM-1 and PECAM-1 expressions on human umbilical vein endothelial cells subjected to oxidative stress

The protective effect of erythropoietin (Epo) is based on its ability to reduce oxidation and to stabilize the cells. The aim of the study was to evaluate the influence of Epo on malonyl dialdehyde (MDA), intercellular adhesion molecule‐1 (ICAM‐1) (CD54) and platelet–endothelial cell adhesion molecule‐1 (PECAM‐1) (CD31) levels on human umbilical vein endothelial cells (HUVECs) stimulated by tumour necrosis factor‐α (TNF‐α). HUVECs were incubated with Epo (10–40 IU ml⁻¹) or TNF‐α (10–40 ng ml⁻¹) alone or preincubated with Epo (20 IU ml⁻¹) and subsequently stimulated with TNF‐α (10–40 ng ml⁻¹). MDA concentrations were measured using the high‐performance liquid chromatography, whereas ICAM‐1 and PECAM‐1 expressions were evaluated by flow cytometry. Incubation with Epo resulted in a decrease in MDA and the increased expressions of ICAM‐1 and PECAM‐1. Exposure to TNF‐α reflected an increase in MDA, ICAM‐1 and PECAM‐1 levels. These changes were inhibited by preincubation with Epo. The cytoprotective activity proven in this study points to new applications and therapeutic possibilities for Epo. Copyright © 2011 John Wiley & Sons, Ltd.     

IGF-1 increases laminin, cyclin D1, and p21Cip1 expression in glomerular mesangial cells: an investigation of the intracellular signaling pathway and cell-cycle progression

Insulin-like growth factor (IGF)-1 is accumulated in the diabetic kidney and is considered to be involved in the development of glomerular sclerosis. Here, we investigate IGF-1 regulation of laminin, an extracellular matrix (ECM) component, and cyclin D1 and p21Cip1, cell-cycle progression factor, expressions in glomerular mesangial cells. We show that IGF-1 increases the level of laminin gamma1 and beta1 subunits approximately 1.5- and 2.5-fold, respectively, in a time-dependent manner. IGF-1 also stimulates protein kinase Akt/PKB phosphorylation at Thr 308, which correlates with its activity, up to 24 h. The Akt activation is coupled with Ser 9 phosphorylation of its downstream target, glycogen synthase kinase-3beta (GSK-3beta), which inhibits its kinase activity. Laminin beta1 is reduced significantly (P < 0.03) by inhibitors of Akt and p38MAPK whereas laminin gamma1 is not affected. Surprisingly, IGF-1 activates the expression of both cyclin D1 and cell-cycle arrest factor, p21Cip1 parallely. Pharmacological inhibition of calcineurin by cyclosporin A blocks IGF-1-induced cyclin D1 and p21Cip1expression significantly (P < 0.05). IGF-1 enhances cellular metabolic activity and viability of rat mesangial cells; however, they are arrested at the G1 phase of cell cycle as revealed by the FACS analysis. These results indicate that IGF-1 mediates mesangial cell-cycle progression, hypertrophy, and ECM protein synthesis. The Akt/GSK-3beta, p38MAPK, and calcineurin pathways may play an important role in IGF-1 signaling, cell-cycle regulation, and matrix gene expression in mesangial cells leading to the development of diabetic glomerulopathy.     

Downregulation of sirtuin 3 by palmitic acid increases the oxidative stress,impairment of mitochondrial function,and apoptosis in liver cells

Elevated levels of saturated fatty acids show a strong cytotoxic effect in liver cells. Sirtuin 3 (SIRT3), a mitochondrially localized member of NAD⁺‐dependent deacetylase has been shown to protect hepatocytes against the oxidative stress. The role of SIRT3 on the cytotoxicity caused by fatty acids in liver cells is not fully understood. The aim of this study was to evaluate the expression level of SIRT3, oxidative stress, and mitochondrial impairments in human hepatoma HepG2 cells exposed to palmitic acid (PA). Our results showed that PA treatment caused the deposition of lipid droplets and resulted in an increased expression of tumor necrosis factor‐α in a dose‐dependent manner. Excessive accumulation of PA induces the reactive oxygen species formation and apoptosis while dissipating the mitochondrial transmembrane potential. The level of SIRT3 expression in both nuclear and mitochondrial fractions in HepG2 cells was decreased with the increase in PA concentrations. However, in the cytosolic fraction, the SIRT3 was undetectable. In conclusion, our results showed that PA caused an increase in inflammation and oxidative stress in HepG2 cells. The exposure of PA also resulted in the decline in transmembrane potential and an increase in apoptosis. The underexpression of nuclear and mitochondrial SIRT3 by PA suggests that the PA target the process that regulates the stress‐related gene expression and mitochondrial functions.     

Mutations in amyloid precursor protein and presenilin-1 genes increase the basal oxidative stress in murine neuronal cells and lead to increased sensitivity to oxidative stress mediated by amyloid beta-peptide (1-42), HO and kainic acid: implications for Alzheimer's disease

Oxidative stress is observed in Alzheimer's disease (AD) brain, including protein oxidation and lipid peroxidation. One of the major pathological hallmarks of AD is the brain deposition of amyloid beta-peptide (Abeta). This 42-mer peptide is derived from the beta-amyloid precursor protein (APP) and is associated with oxidative stress in vitro and in vivo. Mutations in the PS-1 and APP genes, which increase production of the highly amyloidogenic amyloid beta-peptide (Abeta42), are the major causes of early onset familial AD. Several lines of evidence suggest that enhanced oxidative stress, inflammation, and apoptosis play important roles in the pathogenesis of AD. In the present study, primary neuronal cultures from knock-in mice expressing mutant human PS-1 and APP were compared with those from wild-type mice, in the presence or absence of various oxidizing agents, viz, Abeta(1-42), H2O2 and kainic acid (KA). APP/PS-1 double mutant neurons displayed a significant basal increase in oxidative stress as measured by protein oxidation, lipid peroxidation, and 3-nitrotyrosine when compared with the wild-type neurons (p < 0.0005). Elevated levels of human APP, PS-1 and Abeta(1-42) were found in APP/PS-1 cultures compared with wild-type neurons. APP/PS-1 double mutant neuron cultures exhibited increased vulnerability to oxidative stress, mitochondrial dysfunction and apoptosis induced by Abeta(1-42), H2O2 and KA compared with wild-type neuronal cultures. The results are consonant with the hypothesis that Abeta(1-42)-associated oxidative stress and increased vulnerability to oxidative stress may contribute significantly to neuronal apoptosis and death in familial early onset AD.     

Induction of necrotic cell death by oxidative stress in retinal pigment epithelial cells

Age-related macular degeneration (AMD) is a degenerative disease of the retina and the leading cause of blindness in the elderly. Retinal pigment epithelial (RPE) cell death and the resultant photoreceptor apoptosis are characteristic of late-stage dry AMD, especially geographic atrophy (GA). Although oxidative stress and inflammation have been associated with GA, the nature and underlying mechanism for RPE cell death remains controversial, which hinders the development of targeted therapy for dry AMD. The purpose of this study is to systematically dissect the mechanism of RPE cell death induced by oxidative stress. Our results show that characteristic features of apoptosis, including DNA fragmentation, caspase 3 activation, chromatin condensation and apoptotic body formation, were not observed during RPE cell death induced by either hydrogen peroxide or tert-Butyl hydroperoxide. Instead, this kind of cell death can be prevented by RIP kinase inhibitors necrostatins but not caspase inhibitor z-VAD, suggesting necrotic feature of RPE cell death. Moreover, ATP depletion, receptor interacting protein kinase 3 (RIPK3) aggregation, nuclear and plasma membrane leakage and breakdown, which are the cardinal features of necrosis, were observed in RPE cells upon oxidative stress. Silencing of RIPK3, a key protein in necrosis, largely prevented oxidative stress-induced RPE death. The necrotic nature of RPE death is consistent with the release of nuclear protein high mobility group protein B1 into the cytoplasm and cell medium, which induces the expression of inflammatory gene TNFα in healthy RPE and THP-1 cells. Interestingly, features of pyroptosis or autophagy were not observed in oxidative stress-treated RPE cells. Our results unequivocally show that necrosis, but not apoptosis, is a major type of cell death in RPE cells in response to oxidative stress. This suggests that preventing oxidative stress-induced necrotic RPE death may be a viable approach for late-stage dry AMD.     

Protective effects of R-alpha-lipoic acid and acetyl-L-carnitine in MIN6 and isolated rat islet cells chronically exposed to oleic acid

Mitochondrial dysfunction due to oxidative stress and concomitant impaired beta-cell function may play a key role in type 2 diabetes. Preventing and/or ameliorating oxidative mitochondrial dysfunction with mitochondria-specific nutrients may have preventive or therapeutic potential. In the present study, the oxidative mechanism of mitochondrial dysfunction in pancreatic beta-cells exposed to sublethal levels of oleic acid (OA) and the protective effects of mitochondrial nutrients [R-alpha-lipoic acid (LA) and acetyl-L-carnitine (ALC)] were investigated. Chronic exposure (72 h) of insulinoma MIN6 cells to OA (0.2-0.8 mM) increased intracellular oxidant formation, decreased mitochondrial membrane potential (MMP), enhanced uncoupling protein-2 (UCP-2) mRNA and protein expression, and consequently, decreased glucose-induced ATP production and suppressed glucose-stimulated insulin secretion. Pretreatment with LA and/or ALC reduced oxidant formation, increased MMP, regulated UCP-2 mRNA and protein expression, increased glucose-induced ATP production, and restored glucose-stimulated insulin secretion. The key findings on ATP production and insulin secretion were verified with isolated rat islets. These results suggest that mitochondrial dysfunction is involved in OA-induced pancreatic beta-cell dysfunction and that pretreatment with mitochondrial protective nutrients could be an effective strategy to prevent beta-cell dysfunction.     

Alpha lipoic acid prevents doxorubicin‐induced nephrotoxicity by mitigation of oxidative stress,inflammation, and apoptosis in rats

This study aimed to evaluate the protective effects of alpha lipoic acid (ALA) against doxorubicin (DOX)‐induced nephrotoxicity in rats. A single dose of DOX (7.5 mg/kg i.v.) induced nephrotoxicity evidenced by significant elevations in kidney weight, kidney/body weight ratio, serum urea, creatinine, tumor necrosis factor alpha, and renal contents of malondialdehyde, nitric oxide, cyclooxygenase‐2, and caspase‐3. Also, it causes significant reduction in final body weight, serum albumin, renal contents of reduced glutathione and superoxide dismutase activity. Histopathological changes in the kidney tissue confirmed the nephrotoxic effect. In contrast, pretreatment with ALA (50 mg/kg, orally) for 14 days before DOX and for 7 days after DOX administration mitigated renal toxicity evidenced by greater improvement in the examined oxidative stress, inflammation, and apoptosis parameters. In conclusion, ALA had promising protective effects against DOX‐induced nephrotoxicity that might be attributed to its antioxidant, anti‐inflammatory, and antiapoptoic activities.     

Ferulic acid exerts a protective effect against cyclosporine-induced liver injury in rats via activation of the Nrf2/HO-1 signaling,suppression of oxidative stress,inflammatory response,and halting the apoptotic cell death

Drug-induced liver injury is one of the main challenges that leads to the withdrawal of several drugs in the clinical setting. Cyclosporine is one of the drugs that its long-term administration exerts devastating effects on the hepatocytes. In the present study, we aimed to evaluate the effect of ferulic acid, a natural compound found in plants, on cyclosporine-mediated hepatotoxicity. Forty-eight male Wistar rats were treated with cyclosporine and/or ferulic acid to evaluate the function as well as the morphology of liver cells. We found that ferulic acid dose-dependently recovered the functional as well as histopathological parameters of liver cells, as revealed by the improvement of hepatocellular vacuolation, portal fibroplasia, and necrosis. Moreover, this phenolic compound was able to restore the balance of the redox system in cyclosporine-treated rats by activating the nuclear factor (NF) erythroid 2-related factor 2 (Nrf2)/hemeoxygenase-1 (HO-1) signaling axis. Of note, the protective effects of ferulic acid against cyclosporine-mediated liver toxicity were not restricted only to induction of the potential antioxidant property, as in the presence of this agent, the expression of pro-inflammatory cytokines such as NF-κB, tumor necrosis factor (TNF)-α, and interleukin-1β was also diminished. Ferulic acid also shifted the equilibrium between the expression levels of proapoptotic to antiapoptotic proteins and thereby prevented the development of cyclosporine-induced liver injury. Overall, these findings highlighted that ferulic acid can reduce cyclosporine-induced liver injury due to its antioxidant properties.     

HGF-mediated inhibition of oxidative stress by 8-nitro-cGMP in high glucose-treated rat mesangial cells

Abstract

Hepatocyte growth factor (HGF) is a potential therapeutic agent for diabetic nephropathy. The mechanisms for the renoprotective effect of HGF have been studied extensively, but antioxidant signalling of HGF in diabetic nephropathy is minimally understood. Our observations indicated that a nitrated guanine nucleotide, 8-nitroguanosine 3′5′-cyclic monophosphate (8-nitro-cGMP) diminished in high glucose (HG)-treated rat mesangial cells (RMC). However, HGF obviously lifted intracellular 8-nitro-cGMP level, which was accompanied by remarkably suppressed oxidative stress as evidenced by decreased reactive oxygen species and malondialdehyde levels and elevated glutathione level. Inhibitor of soluble guanylyl cyclase (sGC) NS-2028 and inhibitor of nitric oxide synthase (NOS) l-NMMA could block increased 8-nitro-cGMP level and repress oxidative stress by HGF. Accordingly, these two inhibitors abrogated HGF-induced nuclear accumulation of NF-E2 related factor 2 (Nrf2) and up-regulation of Nrf2 downstream glutamate-cysteine ligase catalytic subunit (GCLC) expression. In conclusion, HGF ameliorated HG-mediated oxidative stress in RMC at least in part by enhancing nitric oxide and subsequent 8-nitro-cGMP production.     

Beta-carotene storage, conversion to retinoic acid, and induction of the lipocyte phenotype in hepatic stellate cells

Hepatic stellate cells (HSCs) are the major site of retinol (ROH) metabolism and storage. GRX is a permanent murine myofibroblastic cell line, derived from HSCs, which can be induced to display the fat-storing phenotype by treatment with retinoids. Little is known about hepatic or serum homeostasis of beta-carotene and retinoic acid (RA), although the direct biogenesis of RA from beta-carotene has been described in enterocytes. The aim of this study was to identify the uptake, metabolism, storage, and release of beta-carotene in HSCs. GRX cells were plated in 25 cm(2) tissue culture flasks, treated during 10 days with 3 micromol/L beta-carotene and subsequently transferred into the standard culture medium. beta-Carotene induced a full cell conversion into the fat-storing phenotype after 10 days. The total cell extracts, cell fractions, and culture medium were analyzed by reverse phase high-performance liquid chromatography for beta-carotene and retinoids. Cells accumulated 27.48 +/- 6.5 pmol/L beta-carotene/10(6) cells, but could not convert it to ROH nor produced retinyl esters (RE). beta-Carotene was directly converted to RA, which was found in total cell extracts and in the nuclear fraction (10.15 +/- 1.23 pmol/L/10(6) cells), promoting the phenotype conversion. After 24-h chase, cells contained 20.15 +/- 1.12 pmol/L beta-carotene/10(6) cells and steadily released beta-carotene into the medium (6.69 +/- 1.75 pmol/ml). We conclude that HSC are the site of the liver beta-carotene storage and release, which can be used for RA production as well as for maintenance of the homeostasis of circulating carotenoids in periods of low dietary uptake.     

缬沙坦对人肾小球系膜细胞糖基化终产物受体表达的影响

目的：本实验探讨缬沙坦对糖基化终产物诱导的人肾小球系膜细胞氧化应激水平及糖基化终产物受体（RAGE）表达的影响。方法：体外常规培养人肾小球系膜细胞,运用糖基化修饰的牛血清白蛋白（AGE-BSA）和缬沙坦进行干预,流式细胞术检测细胞内活性氧（ROS）,RT-PCR法检测NADPH氧化酶的亚基p47^phox的mRNA表达,RT-PCR和细胞免疫化学法检测RAGE的表达量。结果：缬沙坦干预组人肾小球系膜细胞的ROS产生量、NADPH氧化酶的亚基p47^phox mRNA表达量、RAGE表达量均低于AGE-BSA组（P〈0．05）,且缬沙坦的抑制作用呈浓度和时间依赖性。结论：缬沙坦可能通过降低氧化应激水平来抑制RAGE的表达。     

Antioxidant effect of homogenetisic acid on hydrogen peroxide induced oxidative stress in human lung fibroblast cells

Homogentisic acid was found to scavenge intracellular reactive oxygen species (ROS), and 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals and thus prevented lipid peroxidation in human fibroblast (WI 38) cells. The radical scavenging activity of homogentisic acid was found to protect WI 38 cells against hydrogen peroxide (H₂O₂) induced oxidative stress, via the activation of extracellular signal regulated kinase (ERK) protein. Homogentisic acid increased the activity of catalase. Hence, from the present study, it is suggested that homogentisic acid protects WI 38 cells against H₂O₂ damage by enhancing the intracellular antioxidative activity.     

Alpha-lipoic acid affects the oxidative stress in various brain structures in mice with methionine and choline deficiency

Deficiency in methionine or choline can induce oxidative stress in various organs such as liver, kidney, heart, and brain. This study was to examine the effects of alpha-lipoic acid (LA) on oxidative stress induced by methionine and choline deficiency (MCD) in several brain structures. Male mice C57BL/6 (n = 28) were divided into four groups: (1) control – continuously fed with standard chow; (2) LA – fed with standard chow and receiving LA; (3) MCD2 – fed with MCD diet for two weeks, and (4) MCD2+LA – fed with MCD diet for two weeks and receiving LA (100 mg/kg/day intraperitonealy [i.p.]). Brain tissue (cortex, hypothalamus, striatum and hippocampus) was taken for determination of oxidative stress parameters. MCD diet induced a significant increase in malondialdehyde and NOx concentration in all brain regions, while LA restored their content to normal values. Similar to this, in MCD2 group, activity of total SOD, MnSOD, and Cu/ZnSOD was reduced by MCD diet, while LA treatment improved their activities in all brain structures. Besides, in MCD2 group a decrease in catalase activity in cortex and GSH content in hypothalamus was evident, while LA treatment induced an increase in catalase activity in cortex and striatum and GSH content in hypothalamus. LA treatment can significantly reduce lipid peroxidation and nitrosative stress, caused by MCD diet, in all brain regions by restoring antioxidant enzymes activities, predominantly total SOD, MnSOD, and Cu/ZnSOD, and to a lesser extent by modulating catalase activity and GSH content. LA supplementation may be used in order to prevent brain oxidative injury induced by methionine and choline deficiency.     

The effects of alpha-lipoic acid on MMP-2 and MMP-9 activities in a rat renal ischemia and re-perfusion model

Matrix metalloproteinases (MMPs) are enzymes that are responsible for degradation of extracellular matrix (ECM); they are involved in the pathogenesis of ischemia-re-perfusion (I-R) injury. We investigated the possible preventive effect of alpha-lipoic acid (LA) in a renal I-R injury model in rats by assessing its reducing effect on the expression and activation of MMP-2 and MMP-9 induced by I-R. Rats were assigned to four groups: control, sham-operated, I-R (saline, i.p.) and I-R+ LA (100 mg/kg, i.p.). After a right nephrectomy, I-R was induced by clamping the left renal pedicle for 1 h, followed by 6 h re-perfusion. In the sham group, a right nephrectomy was performed and left renal pedicles were dissected without clamping and the entire left kidney was excised after 6 h. LA pretreatment was started 30 min prior to induction of ischemia. Injury to tubules was evaluated using light and electron microscopy. The expressions of MMP-2 and MMP-9 were determined by immunohistochemistry and their activities were analyzed by gelatin zymography. Serum creatinine was measured using a quantitative kit based on the Jaffe colorimetric technique. Malondialdehyde (MDA) and glutathione (GSH) were analyzed using high performance liquid chromatography. Tissue inhibitor of metalloproteinase (TIMP)-2 and TIMP-1 were assessed using enzyme-linked immunosorbent assay (ELISA). I-R caused tubular dilatation and brush border loss. LA decreased both renal dysfunction and abnormal levels of MDA and GSH during I-R. Moreover, LA decreased significantly both MMP-2 and MMP-9 expressions and activations during I-R. TIMP-1 and TIMP-2 levels were increased significantly by LA administration. LA modulated increased MMP-2 and MMP-9 activities and decreased TIMP-1 and TIMP-2 levels during renal I-R.     

Oxidative stress, nitric oxide, and the mechanisms of cell death in Lurcher Purkinje cells

Oxidative stress is postulated to play a role in cell death in many neurodegenerative diseases. As a model of neonatal neuronal cell death, we have examined the role of oxidative stress in Purkinje cell death in the heterozygous Lurcher mutant (+/Lc). Lurcher is a gain of function mutation in the delta2 glutamate receptor (GluRdelta2) that turns the receptor into a leaky membrane channel, resulting in chronic depolarization of +/Lc Purkinje cells starting around the first week of postnatal development. Virtually, all +/Lc Purkinje cells die by the end of the first postnatal month. To investigate the role of oxidative stress in +/Lc Purkinje cell death, we have examined nitric oxide synthase (NOS) activity and the expression of two markers for oxidative stress, nitrotyrosine and manganese super oxide dismutase (MnSOD), in wild type and +/Lc Purkinje cells at P10, P15, and P25. The results show that NOS activity and immunolabeling for nitrotyrosine and MnSOD are increased in +/Lc Purkinje cells. To determine whether peroxynitrite formation is a prerequisite for +/Lc Purkinje cell death, +/Lc mutants were crossed with an alpha-nNOS knockout mutant (nNOSalpha(-/-)) to reduce the production of NO. Analysis of the double mutants showed that blocking alpha-nNOS expression does not rescue +/Lc Purkinje cells. However, we present evidence for sustained NOS activity and nitrotyrosine formation in the GluRdelta2(+/Lc):nNOS(-/-) double mutant Purkinje cells, which suggests that the failure to rescue GluRdelta2(+/Lc):nNOS(-/-) Purkinje cells may be explained by the induction of alternative nNOS isoforms.