This experiment, using cultured bovine pulmonary artery endothelial cells (BPAEC), was undertaken to investigate roles of endogenous ONOO- in lipopolysaccharide (LPS)-caused injury to endothelial cells. The fluorescent intensity of nitrotyrosine (NT), a marker of ONOO- generation, in BPAEC represented content of endogenous ONOO- generation. The fluorescent intensity of NT and number of NT positive cells were detected with flow cytometry, and the percentage of NT positive cells was calculated. Results were as follows. (1) LPS (1 mg/L, 5 mg/L and 10 mg/L) caused marked increase in fluorescent intensity of NT in a dose dependent manner. The number and percentage of NT positive cells were markedly increased (P＜0.05). Aminoguanidine (AG), a selective inhibitor of inducible nitric oxide synthase, inhibited the increase in fluorescent intensity of NT in BPAEC induced by LPS. However, the number and percentage of NT positive cells had tendency to reduce. (2) LPS caused the enhancement of MDA content and activity of LDH in cultured supernatant (P＜0.01). AG reversed the enhancement of MDA content induced by LPS (P＜0.01). In contrast, AG had marginal effect on activity of LDH. (3) LPS induced the increase in apoptotic rate in BPAEC in a dose dependent manner. Some BPAEC stained with fluorescent probe ethidium bromide showed morphological features of apoptosis with chromatin condensation and nuclear fragmentation. AG reduced the apoptotic rate and number of apoptotic cells, both of which were still higher than those of vehicle group (P＜0.05). (4) LPS inhibited mitochondrial respiration. Effect of LPS on mitochondrial membrane potential (ΔΨ) depended on the doses of LPS. 1 mg/L LPS led to a little increase in ΔΨ, while 5 mg/L and 10 mg/L LPS significantly reduced ΔΨ. In conclusion, LPS caused injury to cultured BPAEC and increased production of ONOO-. Cytotoxicity of LPS may be mediated by endogenous ONOO-.

Our previous experiments confirmed that endothelial derived ONOO- mediated injury to cultured BPAEC induced by LPS, and that CCK protected endothelial functions against the detrimental effect of LPS in vitro. In the present study, using cultured BPAEC, we investigated effect of CCK on LPS-induced generation of ONOO- in BPAEC and on injury to BPAEC induced by LPS. Results were as follows.(1)CCK inhibited increase in endothelial generation of ONOO- induced by LPS, for fluorescent intensity of NT in BPAEC reduced from (6.55±0.30) AU of LPS group to (4.37±0.08) AU (P＜0.01), the latter being still higher than (3.27±0.15) AU of vehicle group (P＜0.01). In contrast, the number and percentage of NT positive cells reduced a little. Proglumide, a nonspecific inhibitor of CCK receptors, might in part reverse the effect of CCK. (2)CCK markedly reduced MDA content in supernatant in LPS group (P＜0.01), which was completely reversed by proglumide(P＜0.01). Activities of LDH in supernatant in LPS, LPS+CCK group were (85.30±8.66) U/L and (71.33±4.07) U/L, respectively. Proglumide elicited increase in activity of LDH ［(81.00±6.35) U/L］. However, effect of CCK or proglumide was not significant in the alterations of activity of LDH. (3)CCK significantly inhibited increase in apoptotic rate in BPAEC induced by LPS, from 13.50%±0.60% of LPS group to 5.35%±0.25%(P＜0.001), the latter being completely reversed by proglumide with apoptotic rate of 11.45%±0.65%(P＜0.05).These results further confirmed that CCK afforded the cyoprotection for the mitigation lipoperoxide damage and inhibition of apoptosis in BPAEC induced by LPS. The cytoprotection of CCK may be mediated by CCK receptors and related to the reductive ability of endothelia to generate ONOO- induced by LPS.

In this study we found: 1\, There was endogenous ONOO- formation in lungs in the early stage of endotoxic shock. Exogenous ONOO- led to increase in microvascular permeability, severe lung pathological changes and enhanced MDA content. 2\, It was, for the first time, found that responses of isolated pulmonary artery preincubated with ONOO- showed abnormal manifestations. (1) Low dose of ONOO- let to the inhibition of endothelial dependent relaxation, but enhacement of contractile response, both of which were similar to changes of reactivity in isolated pulmonary artery induced by LPS. (2) High dose of ONOO- reduced contractile response to PE and relaxation to SNP. 3\, ONOO- had direct effect for relaxation of precontracted isolated pulmonary artery. The relaxing action of ONOO- was weak and was negtively regulated by endothelial cells, supporting the notion that ONOO- may be involved in pulmonary hypertension in the early stage of endotoxic shock. 4\, It was, for the first time, found that LPS-induced increase in endogenous ONOO- generation in BPAEC and that endogenous ONOO- mediated injury to BPAEC induced by LPS, which may be a novel mechanism for endotoxin-elicited damage to endothelial cells. 5\, Exposure of pulmonary artery to LPS led to reduction in endothelial dependent relaxation but enhancement in contractile response, both of which were reversed by concomitant exposure to CCK and LPS. 6\, CCK protected cultured BPAEC against the detrimental effects of LPS such as lipoperoxide damages and cellular apoptosis as well as LPS-induced endogenous ONOO- formation. The underlying mechanism of CCK for cytoprotection may be mediated by its receptors and related to its reduced ability of endothelia to generate ONOO- induced by LPS.

In practice of forensic medicine, potential disease can be associated with fatal asphyxia in re-straint position. Research has demonstrated that nitric oxide (NO) and nitric oxide synthase (NOS) are plentifully distributed in skeletal muscle, contributing to the regulation of contractile and relaxation. In the current study, respiratory functions, indices of diaphragmatic biomechanical functions ex vivo, as well as NO levels in serum, the expressions of diaphragmatic inducible NOS (iNOS) mRNA, and the effects of L-NNA on contractility of the diaphragm were observed in sepsis induced by cecal ligation and punc-ture (CLP) under the condition of restraint position. The results showed that in the CLP12-18 h rats, respiratory dysfunctions; indices of diaphragmatic biomechanical functions (Pt, +dT/dtmax, -dT/dtmax, CT, Po, force over the full range of the force-frequency relationship and fatigue resistance ) declined progressive-ly; the NO level in serum, and iNOS mRNA expression in the diaphragm increased progressively; force increased significantly at all stimulation frequencies after L-NNA pre-incubation. Restraint position 1 h in CLP12 h rats resulted in severe respiratory dysfunctions after relative stable respiratory functions, almost all the indices of diaphragmatic biomechanical functions declined further, whereas little change took place in NO level in serum and diaphragmatic iNOS mRNA expression; and the effects of L-NNA were lack of statistical significance compared with those of CLP12 h, but differed from CLP18 h group. These results suggest that restraint position and sepsis act together in a synergistic manner to aggravate the great reduction of diaphragmatic contractility via, at least in part, the negative modulation of NO, which may contribute to the pathogenesis of positional asphyxia.

AIM: To study the changes of iNOS mRNA ,protein and the production of nitric oxide(NO) as well as whether puerarin regulates the expression of iNOS mRNA during the formation of diabetic rat cataract. METHODS: One hundred and eight Sprague-Dawley(SD) rats were randomly divided into three groups, thirty-six rats were taken as control group, seventy two rats were injected peritoneally with streptozotocin(STZ,45mg/kg) to establish animal model of diabetic cataract, and then divided into STZ (36) and puerarin(36) treatment groups. Morphologic changes of lens were observered with slit lamp and light microscope; Samples were taken at 20th, 40th, 60th day and the changes in iNOS mRNA and protein expression of lens epithelium cells(LEC)as well as production of NO and NOS activity were determined with reverse transcription -polymerase chain reaction(RT-PCR), western blot, and biochemical method ,respectively. RESULTS: Morphologyic changes of LEC, up-regulation of iNOS mRNA and iNOS protein as well as increase in NO production and NOS activity in the LEC were observered during the formation of rat diabetic cataract. Compared with TZ group, puerarin treatment group showed distinctly down-regulation of iNOS mRNA and iNOS protein and decrease in NO production and NOS activity as well as attenuation of morphologic changes. CONCLUSIONS: There are morphologic changes of LEC and up-regulation of iNOS mRNA and as well as increase in NO production and NOS activity in the LEC during the formation of diabetic rat cataract , and treatment with puerarin can reverse the above changes.

In order to investigate the possible role of ONOO- in regulatory disorder of pulmonary arterial tension in endotoxic shock, the responses of rabbit pulmonary arterial rings (PARs) preincubated with ONOO- to endothelial dependent and receptor dependent relaxants acetylcholine (ACh) and adenosine diphosphate (ADP), endothelial dependent and receptor independent relaxant A23187, endothelial independent relaxant sodium nitroprusside (SNP) and α1-adrenoceptor agonist phenylephrine (PE) were observed in vitro in accumulative manner. Results were as follow: (1) Relaxations of PARs to ACh, A23187 and ADP were markedly impaired with shift of accumulative dose response curve of each agonist to the right. Inhibition of endothelial dependent and receptor dependent or independent relaxation by ONOO- was dose dependent. (2) ONOO- incubation inhibited SNP-induced relaxation in a dose dependent manner. Accumulative dose response curve of SNP was right shift to some degree depending on the doses of ONOO-. (3) Contractile response of PARs to PE varied with the different doses of ONOO-. In PARs preincubated with 0.5 mmol/L ONOO-, contractile reponse was significantly enhanced with shift of PE accumulative dose response curve to the left, while in PARs preincubated with 1.0 mmol/L or 2.0 mmol/L ONOO-, it was markedly reduced with right shift of PE accumulative dose response curve. (4) Vehicle of ONOO- had no effect on responses to every agonist, whereas decomposed ONOO- had minimal effect on the response to PE and ADP. In contrast, relaxation of PARs to ACh, A23187 and SNP were enhanced. These results suggested that direct effect of ONOO- on pulmonary artery may be a key factor contributing to regulatory disorder of pulmonary arterial tension induced by LPS and pulmonary hypertension in the early stage of endotoxic shock.

AIM:To study the effect of ONOO- on the airway epithelial injury. METHODS: The mitochondrial respiration, the amount of lactate dedydrogenase (LDH) release into the cell culture medium, the levels of 8-hydroxy-2′-deoxyguanosine (8-OHdG), and the cellular apoptosis were examined after exposure of cultured rat tracheal epithelial (RTE) cells to ONOO-. RESULTS: Exposure of RTE cells to 0.25, 0.5 and 1 mmol/L ONOO- caused a dose-dependent suppression of the mitochondrial respiration . ONOO- also caused a dose-dependent increase in the percentage of LDH release. Exposure of RTE cells to ONOO- resulted in an increased generation of 8-OHdG in a dose-dependent manner. ONOO- caused an increase in apoptotic percentage in RTE cells in a time-dependent manner at different concentrations. CONCLUSION: ONOO- could cause necrosis and apoptosis in cultured RTE cells. Low concentration of ONOO- caused apoptosis in a time-dependent manner. Whereas exposure to high concentration of ONOO- resulted in cell necrosis, ONOO- caused a dose-dependent increase in the percentage of LDH release. Suppression of mitochondrial respiration and oxidative DNA damage by ONOO- may be the major cause of cellular injury induced by ONOO-.

This study was designed to invesigate vasodilatory action of exogenous peroxynitrite (ONOO-), and effect of endothelial cells on ONOO- -induced relaxation in isolated rabbit pulmonary arterial rings (PARs). Results were as follows: (1) In precontracted PARs, ONOO- could give rise to vasodilation in a dose-dependent manner. Relaxations of PARs to ONOO- at doses of 10-5 mol/L, 5×10-5 mmol/L and 10-4 mol/L were 11.09%±1.84%, 31.10%±3.53% and 64.35%±3.83%, respectively, all of which were significantly higher than those of decomposed ONOO- with 5.88%±1.27%、16.15%±1.82% and 34.44%±3.26% at same concentrations, respectively. (2) Compared with SNP and ACh, ONOO- had weak relaxant action. (3) ONOO- induced more significantly enhanced relaxation in denuded endothelial PARs than in intact endothelial PARs. (4) In this experimental condition, the relaxation of PARs to 10-6 mol/L ACh remained unchanged before and after observation of relaxation to ONOO-. (5) The relaxations of PARs to 5×10-5 mol/L ONOO- in repetitively administered manner appeared progressively decreased. These results suggested that ONOO- might be implicated in pulmonary hypertension in the early stage of endotoxic shock.

AIM and METHODS: to elucidater the effect of poly(ADP-ribose) polymerase(PARP) on tracheal hyperreactivity of guinea - pig induced by peroxynitrite, the responses of guinea pig tracheas to histamine af- ter incubation with peroxynitrite in the absence and presence of 3 - aminobenzamide(3 - AB), a highly selective inhibitor for PARP, were observed in vitro. RESULTS: The exposure of tracheal strips to peroxynitrite led to epithelial damage and hyperreacitivity to histamine, both of which were reversed by 3 - AB(l mmol/L or 5mmol/L), whereas incubation of tracheal strips with 3 - AB(5 mmol/L) had no effect on the reponses. CONCLUSION:PARP is involved in the epithelial damage and hyperreactivity of guinea - pig tracheas induced by peroxynitrite. The results suggested that inhibition of excessive activation of PARP may represent a novel strategy for the prevention and therapy of airway hyperreactivity in asthma.

AIM: To explore the effect of peroxynitrite (ONOO -) on pulmonary microvascular endothelial barrier and roles of ONOO - in the pathogenesis of acute lung injury in vivo. METHODS: SD rats in different groups were insufflated with various concentrations of ONOO -, decomposed ONOO - or vehicle (alkaline normal saline), respectively. Then permeability changes in pulmonary microvascular walls were detected and the pathological alterations of pulmonary tissue were examined under light microscope. Malondialdehyde(MDA) contents were measured in normal lung homogenate pretreated with various concentration of ONOO -. RESULTS: Intratracheal insufflation of ONOO - resulted in dose-dependent increase in lung coefficient, lung wet/dry ratio, lung water contents and Evans blue contents, together with significant pulmonary pathological changes such as diffuse alveolar collapse, capillary congestion, focal hemorrhage, and endothelial swollen. In addition, ONOO - can also elicit increase in MDA contents in normal lung homogenate. CONCLUSION: ONOO - may induce dysfunctions of pulmonary microvascular endothelial barriers, it is suggested that enhanced endogenous ONOO - generation may take part in the pathogenesis of acute lung injury.