Molecular and metabolic evidence for the restricted expression of inducible nitric oxide synthase in healing wounds

Tissue injury initiates a temporally ordered sequence of local cellular and metabolic responses presumably necessary for successful repair. Previous investigations demonstrated that metabolic evidence for nitric oxide synthase (NOS) activity is detectable in wounds only during the initial 48 to 72 hours of the repair process. Present results identify the cell types contributing inducible NOS (iNOS) to experimental wounds in rats. iNOS antigen was expressed in most macrophages present in wounds 6 to 24 hours after injury, and these cells exhibited NAPDH diaphorase and NOS activity. Polymorphonuclear leukocytes contained little iNOS antigen and no NADPH diaphorase activity and were minimally able to convert L-arginine to L-citrulline. The frequency of iNOS-positive macrophages declined on days 3 and 5 after wounding. By day 10, most macrophages in the wound were negative for iNOS. These cells, however, acquired iNOS antigen and activity in culture. Wound fluids, but not normal rat serum, suppressed the induction of iNOS during culture. Findings indicate that the expression of iNOS in healing wounds is restricted to macrophages present during the early phases of repair and that components of wound fluid suppress the induction of iNOS in macrophages in late wounds. Polymorphonuclear leukocytes contribute little iNOS activity to the healing wound.     

The role of inducible nitric oxide synthase following spinal cord injury in rat

Acute spinal cord injury (SCI) is two-step process that first involves the primary mechanical injury and then the secondary injury is induced by various biochemical reactions. Apoptosis is one of secondary SCI mechanisms and it is thought to play an important role for the delayed neuronal injury. The enhanced formation of nitric oxide (NO) via inducible nitric oxide synthase (iNOS) has been implicated in the pathogenesis of apoptosis in SCI. The level of .iNOS mRNA peaked at 6 hr after SCI and it declined until 72 hr after SCI in a rat model. Double-immunofluorescence staining revealed that iNOS positive cells were stained for ED-1, synaptophysin, GFAP, and oligodendrocyte marker. The terminal deoxynucleotidyl-transferase-mediated dUDP-biotin nick end-labeling (TUNEL) positive cell count was higher for the 72 hr post-SCI group than for the 24 hr post-SCI group. This cell count was also higher going in the caudal direction than in the rostral direction from the epicenter, and especially for the 72 hr group. Treatment with a selective iNOS inhibitor resulted in the reduction of TUNEL-positive cells at the lesion site. These findings suggest that nitric oxide generated by the iNOS of macrophages, neurons, oligodentrocytes, and astrocytes plays an important role for the acute secondary SCI that results from apoptotic cell death.     

急性缺氧小鼠海马CAl区NOS活力和nNOS蛋白表达的时程变化

目的:观察急性缺氧小鼠海马CAl区一氧化氮合酶(NOS)和神经元型一氧化氮合酶(nNOS) 阳性神经元的时程变化,探讨NO在脑缺氧中的作用并为抗脑缺氧提供依据。方法:复制小鼠急性缺氧模型,采用NADPH-d组织化学和nNOS免疫组织化学方法,研究急性缺氧后不同时程点小鼠海马CAl区NADPH-d 和nNOS阳性神经元数量的变化。结果:与正常对照组相比较,急性缺氧后0.5h组小鼠海马CAl区NADPH-d 和nNOS阳性神经元的数量无明显变化,差异无显著性(P>0.05),3h、6h和12h组逐渐增多并于12h升高达到最高峰,差异有显著性(P<0.05),而于24h后开始降低,48h恢复正常。结论:急性缺氧后早期海马CAl区NOS和nNOS水平明显增多,NO在缺氧所致早期脑损伤中起重要作用。     

Protective effect of growth hormone on neuronal apoptosis after hypoxia-ischemia in the neonatal rat brain

Recent studies have shown that growth hormone (GH) can reduce neuronal loss after hypoxic-ischemic injury (HI) in neonatal and juvenile rat brains. Here, we investigated whether GH exerts its neuroprotective role through an anti-apoptotic effect in neonatal rat brains damaged by severe HI. Gross and histological observations showed that the extent of brain damage was found to be reduced in GH-treated brain at E7 after injury. In a terminal transferase-mediated dUTP nick-end-labeling (TUNEL) study, TUNEL-positive apoptotic cells were localized only at the damaged region in animals treated with saline, which was confirmed by an electron microscopy. In an immunohistochemical study with anti-bcl-2, -bax, -bad, -neuronal nitric oxide synthase (nNOS), -inducible NOS (iNOS) and -endothelial NOS (eNOS) antibodies, we observed that bax, bad, iNOS and eNOS were elevated in the saline-treated group. This study thus suggests that the protective role of GH against HI injury is mediated thorough an anti-apoptotic effect, which offers the possibility of a GH application for the treatment of neonatal HI encephalopathy.     

The effect of N-nitro-L-arginine and aminoguanidine treatment on changes in constitutive and inducible nitric oxide synthases in the spinal cord after sciatic nerve transection

Ca2+-dependent and Ca2+-independent nitric oxide synthase (NOS) activity, and neuronal and inducible NOS immunoreactivity (nNOS-IR and iNOS-IR), were investigated in the rabbit lower lumbar spinal cord after i) sciatic nerve transection and survival of experimental animals for 2 weeks, ii) treatment of animals with N-nitro-L-arginine (NNLA), an inhibitor of nNOS dosed at 20 mg/b.w. for 12 days, and iii) after treatment of animals with the inducible NOS (iNOS) inhibitor, aminoguanidine, dosed at 100 mg/b.w. for 4 and 12 days. Our attention was focused on the dorsal part of L4-L6 segments receiving sensory inputs from the sciatic nerve, and on the ventral part consisting of sciatic nerve motor neurons. Sciatic nerve transection increased Ca2+-dependent NOS activity and the density of nNOS in the dorsal part of the spinal cord on the ipsilateral side. NNLA treatment effectively reduced nNOS-IR in both the dorsal horn and the dorsal column, and decreased Ca2+-dependent NOS activity in the lower lumbar segments. Immunocytochemical analysis disclosed the up-regulation of iNOS immunoreactive staining after peripheral axotomy in alpha-motoneurons. The changes in iNOS expression and Ca2+-independent NOS activity were not significantly corrected by aminoguanidine treatment for 4 days. Long-lasting iNOS inhibition decreased Ca2+-independent NOS activity, but caused motor neuron degeneration and mediated small necrotic foci in the ventrolateral portion of the ventral horn. The results of the present study provide evidence that constitutive NOS inhibition by NNLA is more effective than specific long-lasting inhibition of iNOS by aminoguanidine treatment.     

Age-related changes in nitric oxide synthase and arginase in the rat prefrontal cortex

Increasing evidence suggests that nitric oxide (NO), generated by nitric oxide synthase (NOS) from l-arginine, plays an important role in the ageing process. The present study, for the first time, investigates age-related changes in NOS and arginase, an enzyme that shares a common substrate with NOS, in the prefrontal cortex of rats assessed with and without prior behavioural testing. A significant increase in total NOS activity was found in the prefrontal cortex in aged (24-month-old) as compared with young (4-month-old) rats. Western blotting revealed that there were no significant differences between young and aged rats in neuronal NOS (nNOS) and endothelial NOS (eNOS) protein expression. Inducible isoform of NOS (iNOS), in terms of activity and protein expression, was not detected in either group. Total arginase activity and arginase I and II protein expression did not differ between the young and aged groups. The present findings support the contribution of NOS/NO to ageing but question the importance of iNOS in the normal ageing process.     

mRNA and protein expression and activities of nitric oxide synthases in the lumbar spinal cord of neonatal rats after sciatic nerve transection and melatonin administration

Sciatic axotomy in 2-day-old rats (P2) causes lumbar motoneuron loss, which could be associated with nitric oxide (NO) production. NO may be produced by three isoforms of synthase (NOS): neuronal (nNOS), endothelial (eNOS) and inducible (iNOS). We investigated NOS expression and NO synthesis in the lumbar enlargement of rats after sciatic nerve transection at P2 and treatment with the antioxidant melatonin (sc; 1 mg/kg). At time points ranging from P2 to P7, expression of each isoform was assessed by RT-PCR and immunohistochemistry; catalytic rates of calcium-dependent (nNOS, eNOS) and independent (iNOS) NOS were measured by the conversion of [3H]L-arginine to [3H]L-citrulline. All NOS isoforms were expressed and active in unlesioned animals. nNOS and iNOS were detected in some small cells in the parenchyma. Only endothelial cells were positive for eNOS. No NOS isoform was detected in motoneurons. Axotomy did not change these immunohistochemical findings, nNOS and iNOS mRNA expression and calcium-independent activity at all survival times. However, sciatic nerve transection reduced eNOS mRNA levels at P7 and increased calcium-dependent activity at 1 and 6 h. Melatonin did not alter NOS expression. Despite having no action on NOS activity in unlesioned controls the neurohormone enhanced calcium-dependent activity at 1 and 72 h and reduced calcium-independent catalysis at 72 h in lesioned rats. These results suggest that NOS isoforms are constitutive in the neonatal lumbar enlargement and are not overexpressed after sciatic axotomy. Changes in NO synthesis induced by axotomy and melatonin administration in the current model are discussed considering some beneficial and deleterious effects that NO may have.     

一氧化氮合酶在坐骨神经夹伤后腓肠肌中的表达变化

目的:探讨外周神经损伤后同侧腓肠肌中3种一氧化氮合酶(nitric oxide synthase,NOS)的表达变化及定位。方法:采用H-E染色及Masson三色染色法分析大鼠坐骨神经夹伤后同侧腓肠肌的病理变化,NADPH-黄递酶组织化学研究其总NOS的改变,并利用Western印迹法、免疫荧光双标法,对3种NOS表达变化及定位进行分析。结果:神经夹伤后相应腓肠肌发生了明显的病理变化且总NOS发生改变,3种NOS变化不尽相同,其表达高峰均约在4周左右。nNOS与神经丝标记物NF-200有共定位,iNOS、eNOS则分别在巨噬细胞、血管内皮细胞中有表达。结论:3种NOS在坐骨神经夹伤后相应腓肠肌中表达变化不同,可能对肌肉损伤及再生修复发挥不同作用。     

大鼠严重烧伤后体内一氧化氮水平变化与预后的关系

目的:研究一氧化氮(NO)及一氧化氮合酶(NOS)在严重烧伤早期大鼠体内的变化规律及其与预后的可能联系。方法:检测严重烧伤前后大鼠血液中NO代谢产物NO^-₂/NO^-₃及脑、肺脏和十二指肠组织中神经型(nNOS)和诱生型一氧化氮合酶(iNOS)蛋白的水平,同时统计各组大鼠的存活率。结果:烧伤后大鼠血液中NO^-₂/NO^-₃水平显著增高,非选择性NOS抑制剂L-NAME和选择性iNOS抑制剂氨基胍(AG)对其均有抑制作用,以L-NAME为甚;nNOS蛋白在伤后部分升高,L-NAME和AG均轻度上调nNOS水平;iNOS在正常组织中不表达,烧伤后表达异常增高,L-NAME和AG对此均无影响;与对照组比较,AG组大鼠存活时间延长,L-NAME组存活时间缩短。结论:严重烧伤后的血管扩张、血压降低和血管反应性低下与iNOS蛋白水平过度增高及其释放的大量NO关系密切。     

Constitutively synthesized nitric oxide is a physiological negative regulator of mammalian angiogenesis mediated by basic fibroblast growth factor

We recently reported that the systemically administered nitric oxide synthase (NOS) inhibitor Nw-nitro-L-arginine methyl ester, L-NAME, administered before, during and after the angiogenic treatment stimulated angiogenesis induced by basic fibroblast growth factor, bFGF, in the rat. This suggests that suppression of constitutively expressed NOS, cNOS, plus inducible NOS, iNOS, and thus reduced production of nitric oxide, NO, was the stimulating factor. In those studies, the rat mesenteric-window angiogenesis assay was used. Moreover, the systemic administration of a NO releaser inhibited bFGF-mediated angiogenesis. Using the same experimental system, we have now studied whether the inhibition of cNOS alone in adult animals under physiological conditions, i.e. prior to the administration of the angiogenic stimulation with bFGF, affected the subsequent angiogenic response. cNOS constitute endothelial cell NOS (ecNOS) and neuronal NOS (nNOS). L-NAME or its inactive enantiomer Nw-nitro-D-arginine methyl ester, D-NAME, were given continuously in the drinking water (1.0 g/L) during 14 days prior to the start of the treatment with bFGF. The treatment with L-NAME significantly enhanced the subsequent angiogenic response. NO synthesized under physiological conditions by ecNOS in endothelial cells and platelets or nNOS in platelets may thus act as a first constitutional angiostatic factor in bFGF-mediated mammalian angiogenesis.     

Pseudorabies virus-induced expression of nitric oxide synthase isoforms

In addition to its role as a neurotransmitter, studies have postulated both neuroprotective and neurotoxic roles for nitric oxide (NO) generated in response to infections with neurotropic viruses. This study examined the expression of neuronal nitric oxide synthase (nNOS) and inducible nitric oxide synthase (iNOS) isoforms of NOS induced by neuronal infection with virulent and attenuated strains of pseudorabies virus (PRV). Caudal brainstem neurons infected by peripheral inoculation of the viscera served as the model system. Neuronal infection induced the expression of nNOS and iNOS, but the timing and the apparent magnitude of NOS expression varied according to the virulence of the infecting strain of virus. Expression of nNOS was observed in infected neurons that did not express this enzyme in control animals, and the onset of expression was earlier in animals infected with virulent PRV. Expression of iNOS was largely restricted to monocytes and macrophages that invaded the brain in response to PRV infection. These iNOS-expressing cells were observed earlier in animals infected with the virulent virus, and were differentially concentrated in areas exhibiting virus-induced neuropathology. Collectively, these data suggest functionally diverse roles for NO in the brain response to PRV neuronal infection.     

Changes in mRNA of protein inhibitor of neuronal nitric oxide synthase following facial nerve transection

Protein inhibitor of neuronal nitric oxide synthase (PIN) is reported as the protein inhibiting neuronal nitric oxide synthase (nNOS) activity by preventing dimerization of nNOS. It was also reported that PIN inhibits the activity of all nitric oxide synthase (NOS) isozymes. We examined the effects of facial nerve transection on PIN mRNA and NOS expression by in situ hybridization for PIN mRNA and nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d) staining. PIN mRNA was initially expressed and transiently increased from 3 to 5 days and returned to the basal level at 7 days after axotomy in the motoneurons of the facial nucleus. NADPH-d-positive motoneurons were found from 7 days post-operation in the facial nucleus. These results suggest that PIN may interact with NOS from 7 days post-operation.     

内源性H2S对LPS所致大鼠急性肺损伤时肺动脉高压的影响及其与一氧化氮的相互作用

目的： 探讨硫化氢（H2S）对脂多糖（LPS）所致急性肺损伤时肺动脉高压（PAH）的影响及H2S/胱硫醚-γ-裂解酶（CSE）体系和一氧化氮（NO）/一氧化氮合酶（NOS）体系在其发生机制中的相互作用。 方法： 将72只大鼠随机分为生理盐水（NS）对照组、LPS组、LPS+L-NAME组、LPS+PPG组，检测给药后2、4、6、8 h的平均肺动脉压（mPAP），以及4、8 h血浆H2S、NO含量和iNOS、cNOS活性、肺组织NO含量和iNOS、cNOS、CSE活性，免疫组化法测定肺组织iNOS蛋白表达，并结合肺光镜形态等指标综合评价肺损伤程度。 结果： LPS组各时点的mPAP显著高于对照组，给药后4、8 h，NO含量、iNOS活性和蛋白表达升高，cNOS活性及H2S含量、CSE活性降低，肺组织损伤较重。预先给予L-NAME可减轻LPS所致上述指标的改变。而预先给予PPG可加重LPS所致肺损伤，但对cNOS活性无明显影响。 结论： LPS使内源性H2S减少导致mPAP升高； H2S/CSE体系与NO/NOS体系共同参与LPS所致急性肺损伤时PAH形成的调控机制，在其中呈相互的负性调节作用。     

Intra- and inter-hemispheric coupling of electroencephalographic 8-13 Hz rhythm in humans and force of static finger extension

Information on equipment and subcellular distribution of nitric oxide synthase (NOS) isoforms in myenteric neurons and pacemaker cells (ICC) might help to identify nitric oxide (NO) pathway(s) acting on gastrointestinal motility. In sections of mouse colon labelled with neuronal (n)NOS, endothelial (e)NOS and inducible (i)NOS antibodies, all myenteric neurons co-expressed eNOS and iNOS and a subpopulation of them co-expressed nNOS. ICC co-expressed nNOS and eNOS. In the neurons, nNOS-labeling was intracytoplasmatic, in the ICC at cell periphery. In both cell types, eNOS-labeling was on intracytoplasmatic granules, likely mitochondria. In conclusion, myenteric neurons and ICC co-express several NOS isoforms with specific subcellular distribution. Different nNOS splice variants are presumably present: intracytoplasmatic nNOSbeta and nNOSalpha producing neurogenic NO, plasma membrane-bound nNOSalpha producing ICCgenic NO. eNOS might be implicated in mitochondrial respiration and, in ICC, also in pacemaker activity. Neurons express iNOS also in basal condition.     

iNOS expression in rat aorta is increased after spinal cord transection: a possible cause of orthostatic hypotension in man

Orthostatic hypotension commonly occurs in persons with spinal cord injury (SCI), limiting rehabilitation and independence. Findings of increased production of nitric oxide (NO) by inducible nitric oxide synthase (iNOS) after exposure to simulated microgravity suggest that increased iNOS expression contributes to OH in persons with SCI. To test this possibility, male Wistar rats underwent surgical transection of the spinal cord (T10) or sham-SCI surgery followed by euthanasia 3, 7 or 14 days later. Expression in thoracic aortic of inducible (iNOS), endothelial (eNOS) and neuronal (nNOS) NOS was then determined. In SCI rats, expression of iNOS mRNA was decreased at 3 days, had returned to normal levels of expression at 7 days and was increased at 14 days post-SCI (1.8-fold). In contrast, levels of eNOS mRNA were increased at 3 days (1.4-fold), then declined over time reaching levels by day 14 that were reduced compared to sham-SCI (0.23-fold). There were no significant effects of SCI on nNOS expression. These findings suggest a possible role for increased iNOS expression in the pathogenesis of OH in persons with SCI.     

Changes in nitric oxide and inducible nitric oxide synthase following stretch-induced injury to the tibialis anterior muscle of rabbit

This study investigated the changes in nitric oxide (NO) together with inducible nitric oxide synthase (iNOS) content and enzyme activity at 0, 4, 12, 24, and 48 h following acute muscle stretch injury. A single stretch injury was induced to the tibialis anterior muscle of 30 male New Zealand white rabbits (n = 6 at each time point). Injured and uninjured contralateral sham-operated muscles were harvested and analyzed for NO levels, iNOS content, and iNOS activity at each time point. Furthermore, three animals were used to estimate baseline NO levels and iNOS activity. There was a progressive reduction in NO content in the injured and the sham-operated muscles up to 24 h postoperation and stretch injury (p < 0.05). At 48 h postinjury, however, NO levels were 146% higher in injured muscles than in sham-operated muscles (p < 0.05). iNOS protein content was higher at 4 h and 48 h in injured versus shamoperated muscles (p < 0.05). Similarly, iNOS activity was higher at 4 h (p < 0.05) and at 48 h (p < 0.01) in injured versus sham-operated muscles. These results suggest that NO may play an active role during the postinjury recovery of skeletal muscle modulated by iNOS expression.     

Effects of nitric oxide on dentate gyrus cell proliferation after seizures induced by pentylenetrazol in the adult rat brain

Epileptic seizures have been shown to increase the proliferation of granule cell precursors in the adult brain, but the underlying mechanisms remain largely unknown. This study examined the effect of nitric oxide (NO) on the proliferation of granule cell precursors in adult rats after pentylenetrazol (PTZ)-induced generalized clonic seizures. Using systemic bromodeoxyuridine (BrdU) to label dividing cells, we found that injection of the neuronal nitric oxide synthase (nNOS) inhibitor 7-nitroindazole (50 mg/kg i.p.) 10 min before PTZ significantly reduced the number of BrdU labeled cells in the dentate gyrus 3, 7, and 14 days after seizures (P < 0.05). Administration of the inducible NOS (iNOS) inhibitor aminoguanidine (100 mg/kg i.p.) also significantly inhibited the proliferation rate of neural precursor cells in the dentate gyrus at various time points after PTZ-induced seizures. Our findings suggest that epileptic seizures lead to increased cell proliferation in the adult rat dentate gyrus through NO-dependent mechanisms. Both the NO originating from nNOS and iNOS may be involved in brain repair after seizures.     

八肽胆囊收缩素对内毒素休克时大脑皮质损伤的影响及其机制初探

目的：观察八肽胆囊收缩素（CCK-8）对内毒素休克（ES）时大脑皮质损伤的影响，并探讨其可能的机制。 方法： 将日本大耳白兔经静脉注入脂多糖（LPS，8 mg/kg）复制ES模型。32只家兔随机分为对照组、LPS组、CCK-8+LPS组和非特异性CCK受体拮抗剂丙谷胺（Pro）+LPS组，每组8只。监测平均动脉压（MAP）变化，光、电镜观察大脑皮质的组织形态学改变，比色法检测大脑皮质NOS和SOD活性、NO和MDA含量的改变。用SD大鼠 （12只，同上复制模型及分组） 以免疫组织化学染色法观察大脑皮质iNOS和nNOS表达的变化。 结果： 注入LPS后，MAP明显持续低于对照组（P<0.01），大脑皮质组织水肿，iNOS和nNOS表达增强，NOS活性、NO和MDA含量显著高于对照组（P<0.05、P<0.01和P<0.01），SOD活性显著低于对照组（P<0.01）。预先注入CCK-8可明显减轻上述变化，预先注入丙谷胺Pro则加剧以上变化。 结论： CCK-8可减轻ES时的大脑皮质损伤，其机制可能与其抗氧化作用和抑制NO的过量生成有关。     

Regional variations and age-related changes in nitric oxide synthase and arginase in the sub-regions of the hippocampus

L-arginine can be metabolised by nitric oxide synthase (NOS) with the formation of L-citrulline and nitric oxide (NO), or arginase with the production of L-ornithine and urea. In contrast to studies showing a potential involvement of NOS/NO in the aging process, the role of arginase has not been well documented. The present study investigates for the first time the regional variations and age-related changes in both NOS and arginase in sub-regions of the hippocampus. In young adult rats, although the total NOS activity was not significantly different across the hippocampal CA1, CA2/3 and the dentate gyrus (DG) sub-regions, the total arginase activity showed a clear regional variation with the highest level in DG. Western blotting revealed that the highest levels of neuronal NOS (nNOS) and endothelial NOS (eNOS) proteins were located in CA1. Arginase I is expressed at a very low level in the brain (the whole hippocampus) as compared with the liver. By contrast, arginase II protein shows an extremely high expression in the brain with little or no expression in the liver. There was no regional variation in arginase I or arginase II protein expression across the sub-regions of the hippocampus. When a comparison was made between young (4-month-old) and aged (24-month-old) rats, a significant increase in total NOS activity was found in DG and significant decreases in arginase activity were observed in the CA1 and CA2/3 regions in the aged animals. Western blotting further revealed a dramatic decrease in eNOS protein expression in aged CA2/3 with no age-associated changes in nNOS, arginase I and II protein expression in any region examined. Interestingly, evidence of activity or protein expression of the inducible isoform of NOS (iNOS) was not detected in any tissue from either group. The present results, in conjunction with previous findings, support the contribution of NOS/NO to aging but question the involvement of iNOS in the normal aging process. Region-specific changes in arginase suggest that this enzyme may also contribute to aging.