DDAH1 plays dual roles in PM2.5 induced cell death in A549 cells

BackgroundDimethylarginine dimethylaminohydrolase 1 (DDAH1) is an enzyme that can degrade asymmetric dimethylarginine (ADMA), an endogenous nitric oxide synthase (NOS) inhibitor. Emerging evidence suggests that alterations in the ADMA–DDAH1 pathway are involved in environmental pollution induced airway inflammation. However, the role of DDAH1 in protection against cytotoxicity of ambient airborne particulate matter is unclear.MethodsWe examined the influence of DDAH1 expression on oxidative stress and cell apoptosis in human type II alveolar epithelial A549 cells exposed to PM_2.5 (particulate matter with an aerodynamic diameter less than 2.5 μM).ResultsWe found that PM_2.5 exposure for 48 h significantly decreased DDAH1 expression. However, knockdown of DDAH1 prior to PM_2.5 exposure actually attenuated the cytotoxicity of PM_2.5. Cytoprotection in DDAH1 deficient cells was due to increased reactive oxygen species, activation of PI3K–AKT and mitogen-activated protein kinase (MAPK) pathways, subsequent activation of nuclear factor erythroid-2-related factor 2 (Nrf2) and this caused a subsequent reduction in PM_2.5 induced oxidative stress relative to control. DDAH1 depletion also repressed the induction of inducible NOS (iNOS) in PM_2.5-exposed cells and knockdown of iNOS protected cells against PM_2.5 induced cell death. Interestingly, overexpression of DDAH1 also exerted a protective effect against the cytotoxicity of PM_2.5 and this was associated with a reduction in oxidative stress and upregulation of the anti-apoptotic protein Bcl-2.ConclusionsOur data indicate that DDAH1 plays dual roles in protection against cytotoxicity of PM_2.5 exposure, apparently by limiting PM_2.5 induced oxidative stress.General significanceOur findings reveal new insights into the role(s) of the DDAH1/ADMA in pulmonary protection against airborne pollutants. This article is part of a Special Issue entitled Air Pollution, edited by Wenjun Ding, Andrew J. Ghio and Weidong Wu.     

dl-trans-3,4-Dihydroxy-1-selenolane (DHSred) heals indomethacin-mediated gastric ulcer in mice by modulating arginine metabolism

BackgroundThe importance of the arginine metabolism in gastric ulcer-healing is given relatively less attention. Hence the role of controlling this pathway by dl-trans-3,4-dihydroxy-1-selenolane (DHS_red) and omeprazole against indomethacin-induced stomach ulceration in mouse was investigated.MethodsSwiss albino mice were ulcerated with indomethacin followed by treatment with the test samples, and the activities of myeloperoxidase (MPO), total nitric oxide synthase (NOS) and arginase, the expressions of inducible nitric oxide synthase (iNOS) and endothelial nitric oxide synthase (eNOS), and the pro-/anti-inflammatory cytokine levels were assayed. NOS-inhibitors were also used to establish the biochemical mechanism.ResultsIndomethacin induced maximum ulceration in mice on the 3rd day, associated with reduced arginase activity, eNOS expression, along with increased MPO and total NOS activities, nitric oxide (NO) generation, iNOS expression, and pro-/anti-inflammatory (Th₁/Th₂₎ cytokine ratio. Treatment with DHS_red (2.5 mg kg^− 1 × 3 days) restored the cytokine balance to shift the iNOS/NO axis to the arginase/polyamine axis as revealed from the increased arginase activity and eNOS expression, and reduced iNOS expression, total NOS activity and NO level.ConclusionsThe ulcer-healing property of DHS_red, but not of omeprazole was due to a favorable pro-/anti-inflammatory cytokine ratio that shifted the arginine metabolism to the polyamine pathway and increased the eNOS/iNOS ratio. The healing action of omeprazole was not significantly associated with these parameters.General significanceThe shift in the ariginine-metabolism from the iNOS/NO axis to the arginase/polyamine axis is guided by Th_1/Th₂ cytokines ratio and plays an important role in gastric ulcer-healing. The favourable effects of the non-toxic and water-soluble compound, DHS_red on these pathways and other COX-dependent and antioxidative parameters suggested it to be a promising anti-ulcer formulation for further studies.     

Macrophages expressing arginase 1 and nitric oxide synthase 2 accumulate in the small intestine during Giardia lamblia infection

Nitric oxide (NO) has been shown to inhibit Giardia lamblia in vitro and in vivo. This study sought to determine if Giardia infection induces arginase 1 (ARG1) expression in host macrophages to reduce NO production. Stimulations of RAW 264.7 macrophage-like cells with Giardia extract induced arginase activity. Real-time PCR and immunohistochemistry showed increased ARG1 and nitric oxide synthase 2 (NOS2) expression in mouse intestine following infection. Flow cytometry demonstrated increased numbers of macrophages positive for both ARG1 and NOS2 in lamina propria following infection, but there was no evidence of increased expression of ARG1 in these cells.     

Chromosomal localization, gene structure, and expression pattern of DDAH1: comparison with DDAH2 and implications for evolutionary origins

Endogenously produced asymmetrically methylated arginine residues are competitive inhibitors of all three isoforms of nitric oxide synthase (NOS). The enzyme dimethylarginine dimethylaminohydrolase (DDAH) specifically hydrolyzes these asymmetrically methylated arginine residues to citrulline and methylamines. Previously we have proposed that regulation of asymmetric methylarginine concentration by DDAH may provide a novel mechanism for the regulation of NOS activity in vivo. Recently we reported the cloning of human DDAH and identified a novel human DDAH isoform (DDAH I and DDAH II, respectively). Here we report that the DDAH1 gene maps to chromosome 1p22 and confirm that DDAH2 maps to the MHC III region of chromosome 6p21.3. Extensive analysis of the distribution of DDAH1 and DDAH2 mRNA in 50 human tissues indicates differential expression of DDAH isoforms in brain regions, in immune cells, and during development. DDAH2 expression predominates in highly vascularized tissues that express the endothelial NOS isoform and in immune tissues that can express iNOS. Whereas DDAH2 is expressed at relatively high levels in all fetal tissues examined, DDAH1 expression varies little between fetal and adult tissues. The chromosomal localization of the DDAHs is consistent with gene duplication, and consistent with this, comparison of the gene structures indicates that the intron/exon organization is highly conserved. Phylogenetic analysis of DDAH sequences from diverse species suggests that DDAH gene duplication occurred prior to the emergence of bony fish some 400 million years ago. Overall the data suggest that DDAH2 may be the more ancient of the two genes.     

The role of asymmetric dimethylarginine in the regulation of nitric oxide level in rats with acute renal injury

Nitric oxide (NO) is synthesized from arginine (ARG) by NO synthase (NOS). Asymmetric dimethylarginine (ADMA), a competitive inhibitor of NOS, participates in the endogenous regulation of NO synthesis. The main amount of ADMA is enzymatically degraded by dimethylarginine dimethylaminohydrolase (DDAH) widely expressed in renal tissue. The aim of our study was to compare the changes in DDAH activity and ARG synthesis in kidneys, ADMA and ARG concentration in plasma and their urinary excretion under physiological conditions and in acute renal injury (ARI) induced by glycerol in rats. Urinary nitrite/nitrate excretion (NOx) was estimated as an indicator of whole-body NO synthesis. DDAH activity was decreased, ADMA excretion was increased and plasma ADMA did not change in ARI. Plasma ARG concentration, renal ARG synthesis and urinary NOx excretion were decreased. In conclusion, the diminished enzymatic hydrolysis of the NOS inhibitor ADMA and the reduced synthesis of the NOS substrate ARG might affect NO production in ARI.     

Roles of accumulated endogenous nitric oxide synthase inhibitors, enhanced arginase activity, and attenuated nitric oxide synthase activity in endothelial cells for pulmonary hypertension in rats

Nitric oxide (NO) has been suggested to play a key role in the pathogenesis of pulmonary hypertension (PH). To determine which mechanism exists to affect NO production, we examined the concentration of endogenous nitric oxide synthase (NOS) inhibitors and their catabolizing enzyme dimethylarginine dimethylaminohydrolase (DDAH) activity and protein expression (DDAH1 and DDAH2) in pulmonary artery endothelial cells (PAECs) of rats given monocrotaline (MCT). We also measured NOS and arginase activities and NOS protein expression. Twenty-four days after MCT administration, PH and right ventricle (RV) hypertrophy were established. Endothelium-dependent, but not endothelium-independent, relaxation and cGMP production were significantly impaired in pulmonary artery specimens of MCT group. The constitutive NOS activity and protein expression in PAECs were significantly reduced in MCT group, whereas the arginase, which shares l-arginine as a common substrate with NOS, activity was significantly enhanced in PAECs of MCT group. The contents of monomethylarginine (MMA) and asymmetric dimethylarginine (ADMA), but not symmetric dimethylarginine (SDMA), were increased in PAECs of MCT group. The DDAH activity and DDAH1, but not DDAH2, protein expression were significantly reduced in PAECs of MCT group. These results suggest that the impairment of cGMP production as a marker of NO production is possibly due to the blunted endothelial NOS activity resulting from the downregulation of endothelial NOS protein, accumulation of endogenous NOS inhibitors, and accelerated arginase activity in PAECs of PH rats. The decreased overall DDAH activity accompanied by the downregulation of DDAH1 would bring about the accumulation of endogenous NOS inhibitors.     

Inducible nitric oxide synthase-mediated decrease of intestinal P-glycoprotein expression under streptozotocin-induced diabetic conditions

AimsP-glycoprotein (P-gp), one of the important drug-efflux pumps, is known to be affected by pathological conditions such as inflammation or infection. Recently, it is reported that high glucose or hyperglycemia can alternate P-gp expression levels at the blood-brain barrier or in the kidney, although the details are still unknown. Here, we analyzed the alteration of intestinal P-gp expression and function in the development of diabetes and elucidated the mechanisms.Main methodsType 1 diabetes was induced in male ddY mice by an i.p. injection of streptozotocin (STZ) (230 mg/kg). We analyzed ileal P-gp expression and function using Western blot analysis and an in situ closed loop method, respectively.Key findingsA significant reduction of P-gp expression level in ileum was found 9 days after STZ administration. In contrast, a remarkable decrease in P-gp function was observed on the 3rd and 9th days. Interestingly, nitric oxide synthase (NOS) activity in ilea was significantly increased on the 9th day. The decrease of P-gp expression levels observed on the 9th day was completely suppressed by L-N^G-nitroarginine methyl ester (L-NAME), a broad range NOS inhibitor, or aminoguanidine, a specific inducible NOS (iNOS) inhibitor.SignificanceThese results indicate the possibility that nitric oxide (NO), produced by iNOS in the ileum, is involved in the reduction of ileal P-gp expression under STZ-induced diabetic conditions.     

Dimethylarginine dimethylaminohydrolase-2 overexpression improves impaired nitric oxide synthesis of endothelial cells induced by glycated protein.

Nitric oxide (NO) synthesis is modulated by dimethylarginine dimethylaminohydrolase (DDAH) via metabolizing asymmetric dimethylarginine (ADMA), an endogenous NO synthase (NOS) inhibitor. This study investigated whether glycosylated bovine serum albumin (GBSA) could impair NO synthesis by inhibition of DDAH expression and activity, and whether DDAH2 overexpression could reverse the impaired NO synthesis induced by GBSA in endothelial cells. Overexpression of DDAH2 gene was established by liposome-mediated gene transfection in ECV304 endothelial cell line. Cells were incubated with 1.70 mmol/L GBSA for 48h. And the expressions of DDAH1 and DDAH2, gene activities of DDAH and NOS in cells, as well as concentrations of ADMA and NO in media were assayed. The activity of DDAH and expression of DDAH2 gene but not DDAH1 gene were inhibited in endothelial cells after exposure to GBSA, whereas the concentrations of ADMA were increased concomitantly with the decrease of NOS activity in cells and NO production in media. Overexpression of DDAH2 gene could prevent the inhibition of DDAH activity induced by GBSA (0.55+/-0.02 vs 0.42+/-0.02U/g pro; n=3; P<0.05), decrease ADMA concentration (0.59+/-0.04 vs 1.13+/-0.11 micromol/L; n=3; P<0.01), and increase NOS activity and NO production (53.77+/-3.40 vs 34.59+/-2.57 micromol/L; P<0.05) compared with untransfected cells treated with GBSA. These results suggest that decreased DDAH activity and subsequent elevated endogenous ADMA are implicated in the inhibition of NO synthesis induced by GBSA, and overexpression of DDAH2 gene can prevent these changes in DDAH/ADMA/NOS/NO pathway of endothelial cells exposed to GBSA.     

Cardioprotective effects of lipoic acid,quercetin and resveratrol on oxidative stress related to thyroid hormone alterations in long-term obesity

This study investigated possible mechanisms for cardioprotective effects of lipoic acid (LA), quercetin (Q) and resveratrol (R) on oxidative stress related to thyroid hormone alterations in long-term obesity. Female C57BL/6 mice were fed on high-fat diet (HFD), HFD + LA, HFD + R, HFD + Q and normal diet for 26 weeks. Body weight, blood pressure, thyroid hormones, oxidative stress markers, angiotensin converting enzyme (ACE), nitric oxide synthase (NOS) and ion pump activities were measured, and expression of cardiac genes was analyzed by real-time polymerase chain reaction. HFD induced marked increase (P < .05) in body weight, blood pressure and oxidative stress, while plasma triidothyronine levels reduced. ACE activity increased (P < .05) in HFD mice (0.69 ± 0.225 U/mg protein) compared with controls (0.28 ± 0.114 U/mg protein), HFD + LA (0.231 ± 0.02 U/mg protein) and HFD + Q (0.182 ± 0.096 U/mg protein) at 26 weeks. Moreover, Na⁺/K⁺-ATPase and Ca^2 +-ATPase activities increased in HFD mice whereas NOS reduced. A 1.5-fold increase in TRα1 and reduction in expression of the deiodinase iodothyronine DIO1, threonine protein kinase and NOS3 as well as up-regulation of AT1α, ACE, ATP1B1, GSK3β and Cja1 genes also occurred in HFD mice. Conversely, LA, Q and R inhibited weight gain; reduced TRα1 expression as well as increased DIO1; reduced ACE activity and AT1α, ATP1B1 and Cja1 gene expression as well as inhibited GSK3β; increased total antioxidant capacity, GSH and catalase activity; and reduced blood pressure. In conclusion, LA, resveratrol and quercetin supplementation reduces obesity thereby restoring plasma thyroid hormone levels and attenuating oxidative stress in the heart and thus may have therapeutic potential in heart diseases.     

The effect of hyperbaric oxygen on neuroregeneration following acute thoracic spinal cord injury

AimsAlthough hyperbaric oxygen (HBO) treatment following spinal cord injury (SCI) have been studied in terms of neurological function and tissue histology, there is a limited number studies on spinal cord tissue enzyme levels.Main methodsThe effect of HBO treatment in SCI was investigated by measuring superoxide dismutase (SOD), catalase, glutathione peroxidase (GPx), nitric oxide synthase (NOS) and nitric oxide (NO) activity in the injured tissue. SCI was induced by applying an aneurysm clip extradurally at the level of T9-T11 vertebrae. Preoperative HBO (preopHBO) treatment was applied for 5 days and postoperative HBO (postopHBO) for 7 days.Key findingsIn the preopHBO group, a significant decrease was observed in NOS and NO compared to the SCI group. There was a decrease in SOD, NOS and NO in the postopHBO group when compared to the SCI group. In the pre–postHBO group SOD, GPx, NOS and NO decreased significantly. There was a decrease in SOD in postopHBO compared to preopHBO. In the prepostopHBO, SOD decreased significantly compared to that in the preopHBO group. The prepostopHBO presented a significant decrease in GPx compared to postopHBO (p < 0.05 for all parameters). No significant difference was observed for catalase for all groups. Significant improvement was found in BBB scores for both postopHBO and prepostHBO groups when compared to the SCI group (p < 0.05).SignificanceHBO treatment was found to be beneficial following SCI in terms of biochemical parameters and functional recovery in the postoperative period.     

Rb1 postconditioning attenuates liver warm ischemia-reperfusion injury through ROS-NO-HIF pathway

AimsGinsenoside Rb1 could prevent ischemic neuronal death and focal cerebral ischemia, but its roles to liver warm I/R injury remain to be defined. We determined if Rb1 would attenuate warm I/R injury in mice.Main methodsMice were divided into sham, I/R, Rb1 + I/R (Rb1 postconditioning, 20 mg/kg, i.p. after ischemia), sham + L-NAME, I/R + L-NAME, and Rb1 + I/R + L-NAME groups using 60 min of the liver median and left lateral lobes ischemia. Serum levels of alanine aminotransferase (ALT) were measured and morphology changes of livers were evaluated. Contents of nitric oxide (NO) and nitric oxide synthase (NOS), malondialdehye (MDA) and activity of superoxide dismutase (SOD) were measured. Expressions of Akt, p-Akt, iNOS, HIF-1alpha, tumor necrosis factor-a (TNF-α) and intercellular adhesion molecule-1 (ICAM-1) were also determined by western blot or immunohistochemistry.Key findingsRb1 postconditioning attenuated the dramatically functional and morphological injuries. The levels of ALT were significantly reduced in Rb1 group (p < 0.05). Rb1 upregulated the concentrations of NO, iNOS in serum, iNOS, and activity of SOD in hepatic tissues (p < 0.05), while it dramatically reduced the concentration of MDA (p < 0.05). Protein expressions of p-Akt, iNOS and HIF-1alpha were markedly enhanced in Rb1 group. Protein and mRNA expressions of TNF-α and ICAM-1 were markedly suppressed by Rb1 (p < 0.05).SignificanceWe found that Rb1 postconditioning could protect liver from I/R injury by upregulating the content of NO and NOS, and also HIF-1alpha protein expression. These protective effects could be abolished by L-NAME. These findings suggested Rb1 may have the therapeutic potential through ROS-NO-HIF pathway for management of liver warm I/R injury.     

l-NAME prevents GM1 ganglioside-induced vasodilation in the rat brain

Monosialoganglioside (GM1) is a glycosphingolipid present in most cell membranes that displays antioxidant and neuroprotective properties. It has been recently described that GM1 induces vasodilation. However, the mechanisms underlying GM1-induced vasodilation were not evaluated to date. Therefore, in this study we investigated whether the nonspecific NOS inhibitor l-NAME prevents GM1-induced vasodilation in rats. The systemic injection of GM1 (50 mg/kg, i.p.) increased the outer diameter of pial vessels by 50% in anesthetized animals at 30 min, and this effect was fully prevented by the administration of the nitric oxide synthase inhibitor N^G-nitro-l-arginine methyl ester (l-NAME, 60 mg/kg, i.p. 15 min before GM1 injection).A 30 min exposure of cerebral cortex slices to GM1 (100 μM) increased the content of nitrite plus nitrate (NOx) by 50%. Addition of l-NAME (100 μM) to the incubation medium fully prevented GM1-induced NOx increase. Conversely, a 60 min exposure of slices to GM1 (100 μM) decreased NOx content, revealing a biphasic effect of GM1. Our results suggest that NO plays an important role in the vasodilation induced by GM1.     

Overexpression of Dimethylarginine Dimethylaminohydrolase 1 Attenuates Airway Inflammation in a Mouse Model of Asthma

Levels of asymmetric dimethylarginine (ADMA), an endogenous inhibitor of nitric oxide synthase, are increased in lung, sputum, exhaled breath condensate and plasma samples from asthma patients. ADMA is metabolized primarily by dimethylarginine dimethylaminohydrolase 1 (DDAH1) and DDAH2. We determined the effect of DDAH1 overexpression on development of allergic inflammation in a mouse model of asthma. The expression of DDAH1 and DDAH2 in mouse lungs was determined by RT-quantitative PCR (qPCR). ADMA levels in bronchoalveolar lavage fluid (BALF) and serum samples were determined by mass spectrometry. Wild type and DDAH1-transgenic mice were intratracheally challenged with PBS or house dust mite (HDM). Airway inflammation was assessed by bronchoalveolar lavage (BAL) total and differential cell counts. The levels of IgE and IgG1 in BALF and serum samples were determined by ELISA. Gene expression in lungs was determined by RNA-Seq and RT-qPCR. Our data showed that the expression of DDAH1 and DDAH2 was decreased in the lungs of mice following HDM exposure, which correlated with increased ADMA levels in BALF and serum. Transgenic overexpression of DDAH1 resulted in decreased BAL total cell and eosinophil numbers following HDM exposure. Total IgE levels in BALF and serum were decreased in HDM-exposed DDAH1-transgenic mice compared to HDM-exposed wild type mice. RNA-Seq results showed downregulation of genes in the inducible nitric oxide synthase (iNOS) signaling pathway in PBS-treated DDAH1-transgenic mice versus PBS-treated wild type mice and downregulation of genes in IL-13/FOXA2 signaling pathway in HDM-treated DDAH1-transgenic mice versus HDM-treated wild type mice. Our findings suggest that decreased expression of DDAH1 and DDAH2 in the lungs may contribute to allergic asthma and overexpression of DDAH1 attenuates allergen-induced airway inflammation through modulation of Th2 responses.     

Synthesis of new heterocyclic lupeol derivatives as nitric oxide and pro-inflammatory cytokine inhibitors

A series of heterocyclic derivatives including indoles, pyrazines along with oximes and esters were synthesized from lupeol and evaluated for anti-inflammatory activity through inhibition of lipopolysaccharide (LPS) induced nitric oxide (NO) production in RAW 264.7 and J774A.1 cells. All the synthesized molecules of lupeol were found to be more active in inhibiting NO production with an IC₅₀ of 18.4–48.7 μM in both the cell lines when compared to the specific nitric oxide synthase (NOS) inhibitor, L-NAME (IC₅₀ = 69.21 and 73.18 μM on RAW 264.7 and J774A.1 cells, respectively). The halogen substitution at phenyl ring of indole moiety leads to potent inhibition of NO production with half maximal concentration ranging from 18.4 to 41.7 μM. Furthermore, alkyl (11, 12) and p-bromo/iodo (15, 16) substituted compounds at a concentration of 20 μg/mL exhibited mild inhibition (29–42%) of LPS-induced tumor necrosis factor alpha (TNF-α) and weak inhibition (10–22%) towards interleukin 1-beta (IL-1β) production in both the cell lines. All the derivatives were found to be non-cytotoxic when tested at their IC₅₀ (μM). These findings suggest that the derivatives of lupeol could be a lead to potent inhibitors of NO.     

Cytoprotective induction of nitric oxide synthase in a cellular model of 5-aminolevulinic acid-based photodynamic therapy

Photodynamic therapy (PDT) employs a photosensitizing agent, molecular oxygen, and visible light to generate reactive species that kill tumor and tumor vasculature cells. Nitric oxide produced by these cells could be procarcinogenic by inhibiting apoptosis or promoting angiogenesis and tumor growth. The purpose of this study was to determine whether tumor cells upregulate NO as a cytoprotective measure during PDT. Breast tumor COH-BR1 cells sensitized in their mitochondria with 5-aminolevulinic acid (ALA)-derived protoporphyrin IX died apoptotically after irradiation, ALA- and light-only controls showing no effect. Western analysis revealed that inducible nitric oxide synthase (iNOS) was upregulated > 3-fold within 4 h after ALA/light treatment, whereas other NOS isoforms were unaffected. Exposing cells to a NOS inhibitor (L-NAME or 1400W) during photochallenge enhanced caspase-3/7 activation and apoptotic killing up to 2- to 3-fold while substantially reducing chemiluminescence-assessed NO production, suggesting that this NO was cytoprotective. Consistently, the NO scavenger cPTIO enhanced ALA/light-induced caspase-3/7 activation and apoptotic kill by > 2.5-fold. Of added significance, cells could be rescued from 1400W-exacerbated apoptosis by an exogenous NO donor, spermine-NONOate. This is the first reported evidence for increased tumor cell resistance due to iNOS upregulation in a PDT model. Our findings indicate that stress-elicited NO in PDT-treated tumors could compromise therapeutic efficacy and suggest NOS-based pharmacologic interventions for preventing this.     

Nonspecific inhibition of nitric oxide synthesis evokes endothelin-dependent increases in myocardial contractility

The role of endogenous nitric oxide (NO) in modulating myocardial contractility is still unclear, in part because of unknown, secondary effects of blocking NO release. We hypothesized that the nonspecific inhibition of nitric oxide synthase (NOS) enhances endothelin-1 (ET-1) effects, which can play a role in ET-A receptor-dependent myocardial contractile responses. The myocardial contractility was estimated from the slope of the left ventricular end-systolic pressure–diameter relationship in closed-chest, pentobarbital-anesthetized dogs. Group 1 (n = 7) was the saline-treated control, while in groups 2 (n = 7) and 3 (n = 7) N-nitro-l-arginine (NNA, 4 mg kg^?1), a nonselective NOS blocker, was administered with or without pretreatment with the ET-A receptor antagonist ETR-P1/fl peptide (100 nmol kg^?1 iv). Plasma ET-1, nitrite/nitrate (NO_x) and blood superoxide levels were measured, and myocardial ET-1 content and xanthine oxidoreductase (XOR) activity were determined from myocardial biopsies. The infusion of NNA over 120 min decreased the plasma NO_x, significantly elevated the plasma ET-1 and blood superoxide levels, and in parallel greatly increased the left ventricular contractility as compared with the untreated controls [47.5 vs 30 mm Hg mm^?1]. The myocardial ET-1 content decreased simultaneously, while the XOR activity and blood superoxide level were significantly elevated. These effects, including NNA-induced positive inotropy, were significantly suppressed by pretreatment with ETR-P1/fl peptide. These results demonstrate that a diminished NO synthesis leads to a preponderant ET-1 effect, which increases myocardial contractility through an ET-A receptor-dependent mechanism.     

Extracellular alkalinization induces endothelium-derived nitric oxide dependent relaxation in rat thoracic aorta

AimTo investigate the mechanism through which the extracellular alkalinization promotes relaxation in rat thoracic aorta.MethodsThe relaxation response to NaOH-induced extracellular alkalinization (7.4–8.5) was measured in aortic rings pre-contracted with phenylephrine (Phe, 10^?6 M). The vascular reactivity experiments were performed in endothelium-intact and -denuded rings, in the presence or and absence of indomethacin (10^?5 M), NG-nitro-l-arginine methyl ester (L-NAME, 10^?4 M), N-(6-Aminohexyl)-5-chloro-1-naphthalenesulfonamide/HCl (W-7, 10^?7 M), 2,5-dimethylbenzimidazole (DMB, 2 × 10^?5 M) and methyl-β-cyclodextrin (10^?2 M). In addition, the effects of NaOH-induced extracellular alkalinization (pH 8.0 and 8.5) on the intracellular nitric oxide (NO) concentration was evaluated in isolated endothelial cells loaded with diaminofluorescein-FM diacetate (DAF-FM DA, 5 μM), in the presence and absence of DMB (2 × 10^?5 M).ResultsThe extracellular alkalinization failed to induce any change in vascular tone in aortic rings pre-contracted with KCl. In rings pre-contracted with Phe, the extracellular alkalinization caused relaxation in the endothelium-intact rings only, and this relaxation was maintained after cyclooxygenase inhibition; completely abolished by the inhibition of nitric oxide synthase (NOS), Ca²⁺/calmodulin and Na⁺/Ca²⁺ exchanger (NCX), and partially blunted by the caveolae disassembly.ConclusionsThese results suggest that, in rat thoracic aorta, that extracellular alkalinization with NaOH activates the NCX reverse mode of endothelial cells in rat thoracic aorta, thereby the intracellular Ca²⁺ concentration and activating the Ca²⁺/calmodulin-dependent NOS. In turn, NO is released promoting relaxation.     

Nitric oxide synthase activation and oxidative stress,but not intracellular zinc dyshomeostasis,regulate ultraviolet B light-induced apoptosis

AimsTo investigate the role of nitric oxide synthase (NOS) and intracellular free zinc ion (Zn²⁺) in regulation of ultraviolet B light (UVB)-induced cell damage and apoptosis.Main methodsReal-time confocal microscopy measurement was used to determine the changes of intracellular free zinc concentration under different conditions. Cell apoptotic death was determined using fluorescein isothiocyanate (FITC) conjugated-annexin V (ANX5)/PI labeling followed by flow cytometry. Western analysis was used to determine cell apoptosis and eNOS uncoupling.Key findingsUVB induced an elevation of Zn²⁺ within 2 min of exposure. The UVB-induced intracellular Zn²⁺ elevation was dependent on the increase of constitutive nitric oxide synthase (cNOS) activity and production of superoxide. Removal of Zn²⁺ with a lower concentration (< 25 μM) of N,N,N′,N′-tetrakis (2-pyridylmethyl) ethylenediamine (TPEN), a Zn²⁺-specific chelator, did not induce cell death or prevent cells from UVB-induced apoptosis. However, a higher [TPEN] (> 50 μM) was cytotoxic to cells, but prevented cells from further UVB-induced apoptosis. The higher [TPEN] also induced cNOS uncoupling. Furthermore, treating the cells with a membrane permeable superoxide dismutase (PEG-SOD) inhibited Zn²⁺ release and reduced apoptotic cell death after UVB treatment. The results demonstrated a complex and dynamic regulation of UVB-induced cell damage.SignificanceOur findings not only advance our understanding of the correlations between cNOS activation and Zn elevation, but also elucidated the role of cNOS in regulation of oxidative stress and apoptosis upon UVB-irradiation.     

Unusual hepatic mitochondrial arginase in an Indian air-breathing teleost,Heteropneustes fossilis: Purification and characterization

A functional urea cycle with both cytosolic (ARG I) and mitochondrial (ARG II) arginase activity is present in the liver of an ureogenic air-breathing teleost, Heteropneustes fossilis. Antibodies against mammalian ARG II showed no cross-reactivity with the H. fossilis ARG II. ARG II was purified to homogeneity from H. fossilis liver. Purified ARG II showed a native molecular mass of 96 kDa. SDS–PAGE showed a major band at 48 kDa. The native enzyme, therefore, appears to be a homodimer. The pI value of the enzyme was 7.5. The purified enzyme showed maximum activity at pH 10.5 and 55 °C. The K_m of purified ARG II for l-arginine was 5.25 ± 1.12 mM. l-Ornithine and N^ω-hydroxy-l-arginine showed mixed inhibition with K_i values 2.16 ± 0.08 and 0.02 ± 0.004 mM respectively. Mn^+ 2 and Co^+ 2 were effective activators of arginase activity. Antibody raised against purified H. fossilis ARG II did not cross-react with fish ARG I, and mammalian ARG I and ARG II. Western blot with the antibodies against purified H. fossilis hepatic ARG II showed cross reactivity with a 96 kDa band on native PAGE and a 48 kDa band on SDS–PAGE. The molecular, immunological and kinetic properties suggest uniqueness of the hepatic mitochondrial ARG II in H. fossilis.