Inducible nitric-oxide synthase is regulated by the proteasome degradation pathway

Inducible nitric-oxide synthase (iNOS) is responsible for nitric oxide (NO) synthesis from l-arginine in response to inflammatory mediators. To determine the degradation pathway of iNOS, human epithelial kidney HEK293 cells with stable expression of human iNOS were incubated in the presence of various degradation pathway inhibitors. Treatment with the proteasomal inhibitors lactacystin, MG132, and N-acetyl-l-leucinyl-l-leucinyl-l-norleucinal resulted in the accumulation of iNOS, indicating that these inhibitors blocked its degradation. Moreover, proteasomal inhibition blocked iNOS degradation in a dose- and time-dependent manner as well as when NO synthesis was inhibited by N(omega)-nitro-l-arginine methyl ester. Furthermore, proteasomal inhibition blocked the degradation of an iNOS splice variant that lacked the capacity to dimerize and of an iNOS mutant that lacks l-arginine binding ability, suggesting that iNOS is targeted by proteasomes, notwithstanding its capacity to produce NO, dimerize, or bind the substrate. In contrast to proteasomal inhibitors, the calpain inhibitor calpastatin and the lysosomal inhibitors trans-epoxysuccinyl-l-leucylamido-4-guanidino butane, leupeptin, pepstatin-A, chloroquine, and NH(4)Cl did not lead to significant accumulation of iNOS. Interestingly, when cytokines were used to induce iNOS in RT4 human epithelial cells, the effect of proteasomal inhibition was dichotomous. Lactacystin added prior to cytokine stimulation prevented iNOS induction by blocking the degradation of the NF-kappaB inhibitor IkappaB-alpha, thus preventing activation of NF-kappaB. In contrast, lactacystin added 48 h after iNOS induction led to the accumulation of iNOS. Similarly, in murine macrophage cell line RAW 264.7, lactacystin blocked iNOS degradation when added 48 h after iNOS induction by lipopolysaccharide. These data identify the proteasome as the primary degradation pathway for iNOS.     

缺血预处理对大鼠肺缺血/再灌注损伤的保护作用

目的 :观察缺血预处理 (IPC)对大鼠肺缺血 /再灌注 (I/R)损伤的保护作用 ,并初步探讨其作用机制。方法 :建立离体大鼠肺灌流模型 ,36只wistar大鼠随机分为对照组、I/R组和IPC组 ,处理完毕后分别测定平均肺动脉压(MPAP)、肺组织湿 /干重比、支气管肺泡灌洗液中肺表面活性物质磷脂及表面张力改变 ,肺组织标本送电镜检查。结果 :①电镜下观察IPC组肺损伤明显减轻。②肺组织湿 /干重比值IPC组为 4.41± 0 .2 4,显著低于I/R组 ,但仍高于缺血前 (P <0 .0 1) ;③IPC组大鼠缺血 1h后MPAP为 ( 1.88± 0 .2 9)kPa ,明显低于I/R组 (P <0 .0 1) ;④IPC组支气管肺泡灌洗液中总磷脂为 ( 2 33 .42± 14.0 5 ) μg/kg ,大聚体为 ( 10 5 .39± 6 .17) μg/kg ,与I/R组相比显著增高 ,但低于对照组 (P <0 .0 1) ,三组之间小聚体含量没有显著差异 ;⑤IPC组表面张力为 ( 36 .88± 3.49)mN/m ,显著低于I/R组 ,与对照组相比则无显著性差异 (P >0 .0 5 )。结论 :缺血预处理对大鼠肺I/R损伤有保护作用 ,保护机制可能与促进肺表面活性物质 (PS)磷脂分泌、改善PS组成 ,从而提高PS功能有关。     

缺血预处理对肢体缺血／再灌注肾损伤保护作用的机制初探

目的：探讨缺血预处理对肢体缺血／再灌注时肾损伤的保护作用。方法：复制家兔肢体缺血／再灌注（I／R）损伤模型,观察肢体缺血4h再灌注4h后以及应用缺血预处理干预对肾损伤的影响。分别从右颈外静脉、肾动脉和肾静脉取血,代表外周血以及入、出肾血,观察外周血超氧化物歧化酶（SOD）、丙二醛（MDA）及尿素氮（BUN）;同时测定入肾血和出肾血NO、SOD、MDA和肾组织SOD、MDA、诱导型一氧化氮合酶（iNOS）以及缺血预处理对上述指标的影响。结果：与对照组比较,缺血再灌组松夹后4h外周血、入、出肾血及肾组织SOD活性明显降低,MDA含量增高（P〈0．01）;外周血BUN以及入、出肾血NO和肾组织iNOS含量升高（P〈0．01）;在缺血前给予缺血预处理组．SOD活性升高,而MDA、BUN、NO、iNOS含量降低（P〈0．01）。相关分析显示MDA与SOD间存在明显负相关（P〈0．01）．而MDA与NO、BUN间呈显著正相关（P〈0．01）。结论：肢体缺血／再灌注时伴有肾脏氧自由基代谢紊乱,缺血预处理可以增强肾组织的抗氧化能力,对肢体缺血再灌注肾损伤具有保护作用。     

Inducible nitric oxide synthase in innate immune cells is important for restricting cyst formation of Toxoplasma gondii in the brain but not required for the protective immune process to remove the cysts

Significantly larger numbers of Toxoplasma gondii cysts were detected in the brains of RAG1^?/?NOS2^?/? than RAG1^?/? mice following infection. In contrast, the cyst numbers markedly decreased in a same manner in both strains of mice after receiving CD8⁺ immune T cells. Thus, NOS2-mediated innate immunity is important for inhibiting formation of cysts in the brain but not required for the T cell-initiated cyst removal, which is associated with phagocyte accumulation. Treatment with chloroquine, an inhibitor of endolysosomal acidification, partially but significantly inhibited the T cell-mediated cyst removal, suggesting that phagosome–lysosome fusion could be involved in the T. gondii cyst elimination.     

Biphasic response of cardiac NO synthase isoforms to ischemic preconditioning in conscious rabbits

In conscious rabbits, a sequence of six 4-min coronary occlusion/4-min reperfusion cycles, which elicits late preconditioning (PC), caused rapid activation of calcium-dependent nitric oxide (NO) synthase (NOS) [cNOS; endothelial NOS (eNOS) and/or neuronal NOS (nNOS)], whereas calcium-independent NOS [inducible NOS (iNOS)] activity remained unchanged. The enhanced cNOS activity was associated with increased myocardial levels of NO(2) and/or NO(3) (NO(x)). Twenty-four hours after ischemic PC was induced, the opposite pattern was observed, i.e., there was a pronounced increase in cytosolic iNOS activity but no change in cNOS activity. The initial burst of ischemia-induced cNOS activity was not affected by pretreatment with the antioxidant N-2-mercaptopropionyl glycine (MPG), the protein kinase C (PKC) inhibitor chelerythrine, or the tyrosine kinase inhibitor lavendustin A, indicating that it is independent of the generation of oxidant species and the activation of PKC and tyrosine kinases. In contrast, the delayed upregulation of iNOS 24 h after PC was prevented by pretreatment with N(omega)-nitro-L-arginine, MPG, or chelerythrine before the PC ischemia, indicating that it is triggered by a signaling mechanism that involves the generation of NO, the formation of oxidant species, and the activation of PKC. Taken together, these results demonstrate that, in conscious animals, ischemic PC elicits a biphasic response in cardiac NOS activity, i. e., an immediate activation of cNOS (most likely eNOS) followed 24 h later by a delayed upregulation of iNOS. To our knowledge, this is the first study to directly measure NOS activity after brief myocardial ischemia in vivo. In conjunction with previous functional studies, the data support a distinctive role of NOS isoforms in late PC, with eNOS serving as the trigger on day 1 and iNOS as the mediator on day 2.     

Activation of constitutive nitric-oxide synthase activity is an early signaling event induced by ionizing radiation

Ionizing radiation at clinical dose levels activates both pro- and anti-proliferative signal transduction pathways, the balance of which determines cell fate. The initiating and amplifying mechanisms involved in the activation are poorly understood. We demonstrate that one mechanism involves stimulation of constitutive nitric-oxide synthase (NOS) activity. NOS activity of Chinese hamster ovary cells was measured by the arginine --> citrulline conversion assay. Irradiation stimulated a transient activation of NOS with maximal activity at 5 min of post-irradiation. Western blot analysis and genetic manipulation by overexpression of wild type or dominant negative NOS mutant identify the radiation-induced isoform as NOS-1. Further evidence that NOS-1 is activated by radiation was the demonstration of radiation-induced cGMP formation in cells transiently transfected with the NO-dependent soluble guanylate cyclase. Protein Tyr nitration, a footprint of peroxynitrite formation, followed radiation exposure and was inhibited by expression of a dominant negative NOS-1 mutant. Radiation-induced ERK1/2 kinase activity, a cytoprotective response to radiation, was also blocked by inhibiting NOS activity. These experiments establish NO-dependent signal transduction pathways as being radio-responsive. Given the lipophilic and relatively stable properties of NO, these results also suggest a possible mechanism by which ionization events in one cell may activate signaling processes in adjacent cells.     

MnSOD in mouse heart: acute responses to ischemic preconditioning and ischemia-reperfusion injury

Manganese superoxide dismutase (MnSOD) is one of the main antioxidant enzymes that protects the heart against ischemia-reperfusion (I/R) injury. Ischemic preconditioning (IPC) is a short period of ischemia-reperfusion that reduces subsequent prolonged I/R injury. Although MnSOD localizes in mitochondria, the immediate subcellular distribution of MnSOD in heart after IPC and I/R has not been studied. In a Langendorff mouse heart model, IPC significantly improved cardiac function and reduced the infarction size induced by I/R. Immunoblotting and double immunostaining in fresh preparations revealed that I/R resulted in an increase in cytosolic MnSOD content accompanied by the release of cytochrome c. In contrast, IPC increased mitochondrial MnSOD and reduced cytosolic MnSOD and cytochrome c release induced by I/R. We found that compared with freshly prepared fractions, the freeze-thaw approach results in mitochondrial integrity disruption and release of large amounts of MnSOD into the cytosol along with mitochondrial markers even in the absence of I/R. In contrast, fresh preparations exhibit early MnSOD release into the cytosol after I/R that is prevented by IPC and cyclosporin A administration.     

Decreased capacity of immune cells to cause tissue injury mediates kidney ischemic preconditioning

Ischemic preconditioning (IP) is a well-established phenomenon, and the underlying mechanisms of IP are thought to involve adaptive changes within the injured tissue. Because one of the main functions of immune cells is to harbor memory, we hypothesized that circulating immune cells could mediate IP by responding to an initial ischemia reperfusion injury (IRI) and then mediate decreased injury after a second IRI event. C57BL/6 mice underwent 30 min of bilateral renal clamping or sham operation. At 5 days after ischemia, purified leukocytes from spleen were adoptively transferred into T cell-deficient (nu/nu) mice. After 1 wk, these mice underwent 30 min of renal IRI. The nu/nu mice receiving leukocytes from ischemic wild-type mice had significantly reduced renal injury compared with nu/nu mice receiving leukocytes from sham-operated, wild-type mice. Infiltration of neutrophil and macrophage in postischemic kidney did not correlate with the protection. No difference in kidney C3d or IgG deposition was detected between groups. Given that inducible NO synthase (iNOS) has been implicated in IP, leukocytes from ischemic or sham-operated, iNOS-deficient mice were transferred into nu/nu mice. Effects similar to those of wild-type transfer of ischemic leukocytes were demonstrated; thus, iNOS was not mediating the IP effect of leukocytes. This is the first evidence that immune cells are primed after renal IRI and thereby lose the capacity to cause kidney injury during a second episode of IRI. This finding may also be relevant for elucidating the mechanisms underlying cross-talk between injured kidney and distant organs.     

Tyrosine as important contributor to the antioxidant capacity of seminal plasma

A novel post-addition method, based on the trapping of ABTS-radicals, is applied for studying the total antioxidant capacity of seminal plasma. A remarkable profile is observed, in which seminal plasma quenches radicals in a continuous, relatively slow fashion. Five putative antioxidants present in seminal plasma were studied using the same assay. Some of the compounds such as ascorbic acid, alpha-tocopherol and uric acid exert immediate, fast radical trapping, whereas hypotaurine and tyrosine give rise to the same slow radical trapping curve as seminal plasma. Due to this slow, continuous radical trapping, quantification of the total antioxidant capacity (expressed as trolox equivalent antioxidant capacity, TEAC) strongly depends on the chosen time point after onset of radical trapping. When determined during the slow antioxidant trapping phase, tyrosine has a powerful antioxidant capacity, which in combination with its relatively high plasma concentration makes it an important contributor to the total antioxidant capacity of seminal plasma.     

How important is endogenous muscle glycogen to fatigue in prolonged exercise?

Endogenous muscle glycogen represents a primary fuel source during large muscle group activity in the human. The depletion of this fuel source during submaximal exercise at intensities ranging between 60 and 85% of maximal aerobic power (Vo2max) is widely believed to be the cause of an inability to sustain exercise. Alterations of preexercise muscle glycogen reserves by dietary and exercise manipulations and changing the degree of dependency on endogenous glycogen during exercise by modifying the availability of other fuel sources have in general served to establish a close relationship between muscle glycogen and fatigue resistance. However, in spite of the evidence implicating glycogen depletion to fatigue, the mechanism remains elusive. The most popular theory is that glycogen is an essential substrate, the depletion of which results in a reduction in the rate of ATP regeneration and an inability to maintain energy supply to one or more of the processes involved in excitation and contraction in the muscle. As a consequence, the muscle is unable to translate the motor drive into an expected force and fatigue develops. However, there is little experimental evidence to support this theory. Most studies report no or only minimal changes in ATP concentration at fatigue with low glycogen and no further change in the by-products of ATP hydrolysis. These findings suggest that fatigue might be caused by other nonmetabolic factors. This review examines these other nonmetabolic factors and analyzes their potential role in fatigue during prolonged exercise with depletion of muscle glycogen reserves.     

Involvement of src-kinase activation in ischemic preconditioning induced protection of mouse brain

AimsTo investigate the role of src-kinase in ischemic preconditioning induced reversal of ischemia and reperfusion induced cerebral injury in mice.Main methodsBilateral carotid artery occlusion of 17 min followed by reperfusion for 24 h was employed to produce ischemia and reperfusion induced cerebral injury in mice. Cerebral infarct size was measured using triphenyltetrazolium chloride staining using both by volume and by weight methods differently. Memory was evaluated using elevated plus maze test. Rota rod test was employed to assess motor incoordination.Key findingsBilateral carotid artery occlusion followed by reperfusion produced cerebral infarction and impaired memory and motor co-ordination. Three preceding episodes of bilateral carotid artery occlusion for 1 min and reperfusion of 1 min (ischemic preconditioning) prevented markedly ischemia–reperfusion-induced cerebral injury measured in terms of infarct size (38.5 ± 1.3% and 38.5 ± 2.9% mean infarct of control animals was reduced to 24.3 ± 1.2% and 23.5 ± 1.8% of the preconditioning groups respectively), loss of memory (72.2 ± 3.6 mean transfer latency time of control animals was reduced to 25.6 ± 5.2 of the preconditioning group respectively) and motor coordination (78.3 ± 17.6 s mean falling down latency time of control animals was increased to a mean value of 180.9 ± 6.5 s of the preconditioning groups respectively). SU6656 (2 mg/kg, ip) and PP1 (0.1 mg/kg, ip), highly selective src-kinase inhibitors, attenuated this neuroprotective effect of ischemic preconditioning.SignificanceTherefore, neuroprotective effect of ischemic preconditioning may be due to src-kinase linked mechanism.     

Glucosyceramide synthase of mouse kidney: further characterization with an improved assay method

The synthesis of glucosylceramide from ceramide and UDP-[3H]glucose by mouse kidney homogenates is very sensitive to the concentration of tissue. This was shown to be due to the presence of a UDP-glc pyrophosphatase, which could be blocked by adding NAD to the medium. A new solvent partitioning system is described, containing t-butyl methyl ether, isopropyl alcohol, and aqueous sodium sulfate, which separates the original substrate (UDP-[3H]glc) from the enzyme product, [3H]cerebroside. A particular advantage of the solvent system is that only a single partitioning step is needed, without backwashes, and the enzyme product appears in the upper phase, making transfer to a counting vial more reliable. A novel incubation device, a thermostatically controlled ultrasonic bath, is used to produce highly uniform enzyme reaction rates. Ca2+, as well as Mg2+ and Mn2+, was found to be a good stimulator of the glucosyltransferase. The enzyme activity in kidney of 22-day old mice, approximately 240 pmol/h/mg tissue, is significantly greater than previously demonstrated. The enzyme was stable in intact kidneys stored at -70 degrees C but unstable at 4 degrees C. The enzyme, when acting on endogenous ceramides, showed no demonstrable glucosylation of the C24 family of ceramides although this family is the predominant one in kidney.     

The C terminus of mouse macrophage inducible nitric-oxide synthase attenuates electron flow through the flavin domain

The sequences of nitric-oxide synthase (NOS) flavin domains closely resemble that of NADPH-cytochrome P450 reductase (CPR), with the exception of a few regions. One such region is the C terminus; all NOS isoforms are 20-40 amino acids longer than CPR, forming a "tail" that is absent in CPR. To investigate its function, we removed the 21-amino acid C-terminal tail from murine macrophage inducible NOS (iNOS) holoenzyme and from a flavin domain construct. Both the truncated holoenzyme and reductase domain exhibited cytochrome c reductase activities that were 7-10-fold higher than the nontruncated forms. The truncated holoenzyme catalyzed NO formation approximately 20% faster than the intact form. Using stopped-flow spectrophotometry, we demonstrated that electron transfer into and between the two flavins and from the flavin to the heme domain is 2-5-fold faster in the absence of the C-terminal tail. The heme-nitrosyl complex, formed in all NOS isoforms during NO catalysis, is 5-fold less stable in truncated iNOS. Although both CPR and intact NOS can exist in a stable, one electron-reduced semiquinone form, neither the truncated holoenzyme nor the truncated flavin domain demonstrate such a form. We propose that this C-terminal tail curls back to interact with the flavin domain in such a way as to modulate the interaction between the two flavin moieties.     

Inhibition of p38 MAPK alpha/beta reduces ischemic injury and does not block protective effects of preconditioning

We examined the effect of inhibition of p38 mitogen-activated protein kinase (MAPK) alpha/beta during ischemia and preconditioning by using the inhibitor SB-202190. Isolated rat hearts were perfused with Krebs-Henseleit buffer, while left ventricular developed pressure (LVDP) and (31)P nuclear magnetic resonance spectra were acquired continuously. After 20 min of ischemia and 25 min of reperfusion, recovery of LVDP in untreated hearts was 32 +/- 4%, whereas hearts treated with SB-202190 5 min before ischemia recovered 59 +/- 7% of their pretreatment LVDP. Preconditioning improved functional recovery to 65 +/- 5%, which was unaffected by SB-202190 treatment, added either throughout the preconditioning protocol (56 +/- 5% recovery) or during the final reperfusion period of preconditioning (71 +/- 11% recovery). Necrosis was assessed after 40 min of ischemia and 2 h of reperfusion using 2,3,5-triphenyltetrazolium chloride (TTC) staining and creatine kinase release. The untreated group had 54 +/- 8% necrotic myocardium, whereas the SB-202190-treated group had 32 +/- 7% and the preconditioned group had 21 +/- 4% necrotic tissue by TTC staining.     

Cardioprotective effect of ischemic preconditioning is preserved in food-restricted senescent rats

Ischemic preconditioning (PC) has been proposed as an endogenous form of protection against-ischemia reperfusion injury. We have shown that PC does not prevent postischemic dysfunction in the aging heart. This phenomenon could be due to the reduction of cardiac norepinephrine release, and it has also been previously demonstrated that age-related decrease of norepinephrine release from cardiac adrenergic nerves may be restored by caloric restriction. We investigated the effects on mechanical parameters of PC against 20 min of global ischemia followed by 40 min of reperfusion in isolated hearts from adult (6 mo) and "ad libitum"-fed and food-restricted senescent (24 mo) rats. Norepinephrine release in coronary effluent was determined by high-performance liquid chromatography. Final recovery of percent developed pressure was significantly improved after PC in adult hearts versus unconditioned controls (85.2 +/- 19% vs. 51.5 +/- 10%, P < 0.01). The effect of PC on developed pressure recovery was absent in ad libitum-fed rats, but it was restored in food-restricted senescent hearts (66.6 +/- 13% vs. 38.3 +/- 11%, P < 0.05). Accordingly, norepinephrine release significantly increased after PC in both adult and in food-restricted senescent hearts, and depletion of myocardial norepinephrine stores by reserpine abolished the PC effect in both adult and in food-restricted senescent hearts. We conclude that PC reduces postischemic dysfunction in the hearts from adult and food-restricted but not in ad libitum-fed senescent rats. Despite the possibility of multiple age-related mechanisms, the protection afforded by PC was correlated with increased norepinephrine release, and it was blocked by reserpine in both adult and food-restricted senescent hearts. Thus caloric restriction may restore PC in the aging heart probably via increased norepinephrine release.