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.

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.

AIM and METHODS: To investigate the effect of endotoxin on the celluar activity and secretion of endothelin-1 by radioimmunoassay and MTT methods in cultured human umbilical vein endothelial cells stimulated by E coli endotoxin (E coli O55:B5, Sigma) of various concentrations (1 g/L, 100 mg/L， 10 mg/L，1 mg/L，100 μg/L，10 μg/L, 1 μg/L) and at the same time interval (HUVEC stimulated by endotoxin for 6 hours) in vitro.RESULTS:Endotoxin showed a slightly inhibitory effect on the viability of endothelial cells at low doses (1 μg/L， 10 μg/L, 100 μg/L, 1 mg/L). The viabilities were 92.00%±1.45%， 91.81%±2.03%, 89.52%±1.49%, 88.35%±1.88%, respectively, versus control group, P＜0.01. The cells were impaired significantly at the higher dose of LPS (100 mg/L), the viability was 80.49%±8.76%, versus control group, P＜0.01. The cells were killed evidently at the concentration of LPS (1 g/L), the viability was 73%±8%, versus control group, P＜0.01. The secretion of ET-1 increased gradually with the concentration of endotoxin manifolding. The concentration of ET-1 reached its peak at the dose of 100 μg/L, and it was (324.384±17.023) ng/L, versus control group (251.636±17.023) ng/L, P＜0.01. Endotoxin was effective in stimulating the endothelial cells to secret ET-1 in a dose dependent manner. CONCLUSION: These findings suggested ET-1 may be one of the important factors in endotoxic shock, and the increase in plasma ET-1 level in endotoxemia may be associated with increase in ET-1 secretion.

AIM：The mechanism of tumor necrosis factor-alpha (TNF-α) induced pulmonary artery hypertension(PAH) in endotoxic shock (ES) is not clear. Cholecystokinin-octapeptide (CCK-8) had anti-ES and anti-PAH effects. The impact of CCK-8 on changes in vascular reactivity and endothelial ultrastructure induced by TNF-α was studied. The role of nitric oxide (NO) was preliminarily studied. METHODS：Rabbit pulmonary artery rings were divided into four groups: TNF-α, TNF-α+CCK-8, CCK-8 and Vehicle. The rings were incubated for 2 h, 7 h or 14 h. Relaxative responses to ACh(10-8-10-5 mol/L)， SNP (10-9-10-6 mol/L) and contractile responses to PE(10-8-10-5 mol/L) were generated seperately. The NOS activity of rings was detected and the ultrastructure of endothelium was observed in the groups that incubated for 7 h.RESULTS:The relaxative responses to ACh were not affected by TNF-α and CCK-8 after incubation for 2 h. TNF-α(7 h,14 h) significantly reduced ACh-induced endothelium-dependent relaxation response of pulmonary artery. CCK-8 reversed the effect. CCK-8 itself had no effect on responses of normal pulmonary artery. Contraction to PE and relaxation to SNP were unaffected by TNF-α, CCK-8. The NOS activity increased in the TNF-α and the TNF-α+CCK-8 groups. While no significant difference was obseved between the Vehicle and the CCK-8 groups. Endothelial injury in TNF-α group and alleviated changes in TNF-α+CCK-8 group were observed. CONCLUSION:TNF-α significantly inhibits endothelium-dependent relaxation, which be one of the mechanisms of PAH induced by TNF-α during ES. It was found for the first time that CCK-8 reversed TNF-α induced impairment of endothelium-dependent relaxation and alleviated structural injury of endothelium, which might be one of the mechanisms of anti-PAH effect by CCK-8 during ES. The effects of TNF-α and CCK-8 might be related to NO.

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.