Role of K+ channels in hypoxic pulmonary vasoconstriction in normal and chronic hypoxic rats

AIM:To investigate the role of K⁺ channels in the decreased hypoxic pulmonary vasoconstriction(HPV) in chronic hypoxic rats. METHODS:Blockers of three kinds of K⁺ channels, 4-AP(voltage dependent K⁺ channel blocker), TEA(Ca²⁺ activated K⁺ channel blocker), GLIB(ATP sensitive K⁺ channel blocker) were used in isolated perfused rat lungs to detect the role of K⁺ channels in HPV. RESULTS:In normal rats, 4-AP and TEA, but not GLIB, both elicited a significant increase in pulmonary artery baseline pressure, and also potentiated the acute hypoxic pulmonary vasoconstriction. In chronic hypoxic rats, the HPV is significantly decreased, while 4-AP, TEA, GLIB all elicited a significant but smaller increase in pulmonary artery baseline pressure. Additionally, all these three blockers potentiated the HPV stronger in chronic hypoxic rats than in control rats. CONCLUSION:The opening of Kv, K_Ca, K_ATP might modulate the hypoxic pulmonary vasoconstriction in isolated rat lungs, and the increase in this modulation by potassium channel in chronic hypoxic rats might play a role in its decrease in HPV.     

Role of K+ channels in attenuation of pulmonary vasoreactivity following chronic hypobaric hypoxia in rats

AIM and METHODS: To determine the role of different K⁺-channels in attenuation of vasoreactivity of intrapulminary artery rings induced by chronic hypoxia. RESULTS: ①Acute hypoxia-induced pulmonary vasoconstriction (HPV) could be significantly attenuated by chronic hypobaric hypoxia for 15 days and for 30 days. ②HPV could be significantly potentiated by ATP-sensitive K⁺-channel (K_ATP) blocker or Ca²⁺-activated K⁺-channel (K_Ca) blocker, and the potentiated scope in chronic hypoxic group was much higher than that observed in control group. ③Delayed rectifier K⁺-channel (K_DR) blocker showed no effect on HPV in both control group and chronic hypoxic group. CONCLUSION: Both K_ATP and K_Ca play an important modulating role in HPV and its potentiation may be a critical mechanism for the attenuated vasoreactivity to acute hypoxia following chronic hypobaric hypoxia.     

Effects of 15-HETE and ERK1/2 on pulmonary artery constriction in chronic hypoxic rats

AIM:The aim of the present study was to investigate whether the extracellular signal regulated kinase-1/2 (ERK1/2) pathway was involved in 15-hydroxyeicosatetraenoic acid (15-HETE)-induced chronic hypoxic pulmonary artery (PA) constriction and whether ERK1/2 activity was influenced by 15-HETE, for clarifying the mechanism of hypoxic pulmonary vasoconstriction (HPV). METHODS:Rats were placed in hypoxic box with fractional inspired oxygen (FiO2) 0.12 for 9 days to make hypoxic models, while those lived in FiO₂ 0.21 served as normal controls. Heart and lungs were taken out from chest and PA in diameter of 1-1.5 mm was isolated and cut into rings with 3 mm long for tension studies in organ baths. The ring tensions before and after adding 15-HETE were compared. Influences of ERK1/2 upstream kinase inhibitor U0126 as well as endothelium integrity on 15-HETE-induced HPV were observed. Expression and activity of ERK1/2 in cultured rat pulmonary artery smooth muscle cells (PASMCs) treated with 15-HETE for different times and concentrations were examined by Western blotting. RESULTS:15-HETE significantly constricted PA rings from hypoxic rats, and the response of the hypoxic rings were significantly greater than that of normoxic ones (P<0.05). U0126 significantly reduced vasoconstriction induced by 15-HETE both in endothelium-intact and -denuded rings (both were P<0.05). Western blotting results showed 15-HETE enhanced activity of ERK1/2 in PASMCs, increasing with concentration and decreasing with time. CONCLUSION:15-HETE upregulates activity of ERK1/2 in PASMCs of rats. The activation of ERK1/2 is an important step in 15-HETE- induced HPV in rats.     

Roles of the Kv1.2, Kv1.5, Kv2.1 potassium channels in hypoxia pulmonary vasoconstriction

AIM: To determine the role of Kv1.2, Kv1.5, Kv2.1 in the hypoxia pulmonary vasoconstriction (HPV). METHODS: Male Wistar rats were divided into two groups: normoxic group and hypoxic group. The single smooth muscle cell was obtained from pulmonary artery of Wistar rats with acute enzymatic digestion method. The conventional whole-cell patch clamp technique was used to record the resting membrane potential (Em) and the potassium currents of voltage-gated potassium channel (IKv) in rat pulmonary arterial smooth muscle cells (PASMC). Intracellular application of Kv1.2/Kv1.5/Kv2.1 antibodies (1∶125) was conducted through the whole-cell patch clamp system. RESULTS: ① Em of PASMC was depolarized after 24 h hypoxia compared with that of control cells . IKv of PASMC was decreased after 24 h hypoxia, . ② The mixture of Kv1.2/Kv1.5/Kv2.1 antibodies depolarized Em and inhibited IKv in PASMC from normoxic rat, whereas the mixture of Kir2.1/Kir2.3/Kir4.1 antibodies had no effects on them. ③ The mixture of Kv1.2/Kv1.5/Kv2.1 antibodies and the mixture of Kir2.1/Kir2.3/Kir4.1 antibodies had no effects on IKv and Em from rats hypoxic for 24 h. CONCLUSION: Kv1.2, Kv1.5, Kv2.1 might be oxygen sensitive potassium channels which mediated HPV.     

Protective effect of ambroxol against the lung damage in chronically hypoxic rats

AIM: To investigate the effect of ambroxol on pulmonary and vascular injury in chronically hypoxic rats. METHODS: 36 male Wistar rats were randomly divided into 3 groups: normal control,chronically intermittent hypoxia(CIH) and ambroxol precaution group(AP).The CIH and AP groups were made into the chronically hypoxic models.The mean pulmonary artery pressure(PAPM) and the levels of plasma superoxide dismutase(SOD) and plasma nitric oxide(NO),lipid peroxide(LPO) were determined. The levels of the lung homogenates SOD, LPO, NO and the changes in pulmonary vascular structure were also examined. RESULTS: The levels of plasma and lung homogenates SOD,NO in CIH group were respectively significantly lower than that of normal control and AP group( P <0.01),but the levels of plasma and lung homogenates LPO were significantly higher( P< 0.01). PAPM in AP group is significantly lower than that of CIH group( P< 0.01);The damage of pulmonary artery smooth muscle cells and extra cell matrix of AP group is much slighter than that of CIH group. CONCLUSION: Ambroxol might be an effective protector in chronically hypoxic rats.     

Role of cyclic nucleotides in the acute hypoxic responses of hypoxic subcultured porcine pulmonary arterial smooth muscle and endothelial cells

AIM: To detect the role of cyclic nucleotides in the alleviation of hypoxic pulmonary vasoconstriction (HPV) in chronic hypoxic animals. METHODS: The intracellular cAMP and cGMP of the cultured porcine pulmonary arterial smooth muscle cells (PASMC) and endothelial cells (PAEC) were assayed by RIA. The length of single PASMC during acute hypoxia was measured by imaging analysis system. RESULTS: The basal levels of cAMP and cGMP in PASMC and cGMP in PAEC of Chronic hypoxic groups decreased remarkably compared with normoxic groups (P＜0.01). Under acute hypoxia, the contents of cAMP and cGMP in PASMC of chronic hypoxic groups increased significantly (P＜0.01). Meanwhile, the percentage of PASMC with weak constrictive response in chronic hypoxic group was higher than that of control group. CONCLUSION:It's suggested that the changes of cAMP and cGMP in chronic hypoxic PASMC and PAEC might contribute to the increase in the basic tension of pulmonary artery and the alleviation of HPV in chronic hypoxic animals.     

Role of SB203580 in enhancement of hypoxia hypercapnia-induced pulmonary vasoconstriction by voltage-dependent K+ channel blocker 4-aminopyridione in rats

AIM:To investigate the roles of voltage-dependent K+ channel (KV) and p38 mitogen-activated protein kinase (MAPK) signal pathway in the pathological process of hypoxia hypercapnia-induced pulmonary vasoconstriction (HHPV) in rats. METHODS:The second-order pulmonary artery rings isolated from SD rats in vitro was prepared, and randomly divided into control group (N group), hypoxia hypercapnia group (H group), hypoxia hypercapnia+DMSO incubation group (HD group), hypoxia hypercapnia+4-aminopyridione (4-AP) group (4-AP group), hypoxia hypercapnia+SB203580 incubation group (SB group) and hypoxia hypercapnia+4-AP+SB203580 incubation group (4-AP+SB group). Under acute hypoxia hypercapnia condition, the changes of the 3 stages of HHPV incubated by 4-AP or the combined application of 4-AP and SB203580 were observed. At the same time, the tension values of the rings were recorded. RESULTS:Under hypoxia hypercapnia condition, a biphasic pulmonary artery contractile response was observed. The phase II persistent vasoconstriction of the pulmonary artery rings incubated with 4-AP was enhanced. Under hypoxia hypercapnia condition, SB203580 significantly relieved the phase II persistent vasoconstriction induced by 4-AP. CONCLUSION: The KV blocker 4-AP enhances HHPV. SB203580 significantly relieves the phase II persistent vasoconstriction induced by 4-AP, indicating that the participation of p38 MAPK may be one of the important mechanisms for the regulation of HHPV by KV in the rats.     

Effect of cilazapril on pulmonary vascular endothelial dysfunction in hypoxic rats

AIM:To study the effect of cilazapril on pulmonary vascular endothelial dysfunction in hypoxic rats. METHODS:The structure and function of endothelium in hypoxic rats were studied by biochemical analysis, radioimmunoassay, transmission electron microscope and correlated with hemodynamic. RESULTS:1) The change and damage of ultrastructure in endothelial cell (EC) were obsevered in hypoxic rats. 2) The contents of plasma nitric oxide (NO) and superoxide dismutase (SOD) activity in blood as well as endothelial nitric oxide synthase (eNOS) activity in the lung tissue were significantly lower in the hypoxic rat than those in contral animals. The concentrations of plasma endothelin-1(ET-1) and angiotensin converting enzyme(ACE) as well as malondialdehyde(MDA) were significantly higher in the hypoxic rat than these in contral animals. The relaxing and contracting factors had a significant positive/negative correlation with mean pulmonary artery pressure (mPAP). 3) Cilazapril significantly decreased the level of ET-1 and ACE and significantly increased the level of NO and activity of eNOS and SOD. At the same time, cilazapril extenuated hypoxia-induced injuries of EC. CONCLUSION:The results indicate that damaging structure and dysfunction of EC existes in hypoxic rats. The cilazapril effectively preventes and treates the chronic hypoxic PH by relieving the injury and improving secretion in EC.     

Expression of NO and H2S in hypoxic pulmonary hypertension and intervention of sodium nitrite in rats

AIM: To observe the change of nitric oxide(NO)and hydrogen sulfide(H2S)in blood and lung homogenate of hypoxic pulmonary hypertension(HPH)rat model, and to discuss the meaning of inhalation sodium nitrite and these factors in the treatment of HPH. METHODS: Fifty healthy male Wistar rats were assigned randomly into 5 groups(10 rats each): normoxia control group(NC), normoxia sodium nitrite group(NNI), hypoxic control group(HC), hypoxic normal saline group(HNS)and hypoxic sodium nitrite group(HNI). The mean pulmonary arterial pressure(mPAP), weight of right ventricle, weight of left ventricle plus septum, and the ratio of the weight of right ventricle to that of left ventricle plus septum(right ventricle hypertrophy index, RVHI)were also determined. The serum level of NO and plasma level of H2S were measured, and at the same time the levels of NO in the lung homogenate were detected. The structures in pulmonary arteries were examined using optical microscope. RESULTS: After model established, compared to that in the normoxia groups, the body weight decreased significantly in hypoxia groups(P<0.05), although no difference of body weight in five groups before producing model was observed. Compared to that in normoxia groups, the levels of mPAP and RVHI increased significantly in hypoxia groups, and compared to that in hypoxia control groups and hypoxia normal saline group, mPAP and RVHI levels decreased significantly in hypoxia sodium nitrite group(P<0.05). Compared to that in normoxia groups, the serum level of NO decreased significantly in hypoxia groups(P<0.05). NO level in lung homogenate decreased significantly in hypoxia control group and hypoxia normal saline group as compared to that in normoxia groups(P<0.05), and no obvious difference between hypoxic sodium nitrite group and normoxia groups was found. The plasma level of H2S was decreased significantly in hypoxia groups(P<0.05)as compared to that in normoxia groups. H2S level increased significantly in hypoxia sodium nitrite group as compared to that in hypoxia control groups and hypoxia normal saline group(P<0.05). Observation under optical microscope, the lumen structure of lung in normoxia control group was normal. No significant change in normoxia sodium nitrite group was found. The proliferation of smooth muscle cells(SMCs), the collagen fiber deposition in the vessel wall and every caliber thickening was observed in hypoxic control group. The same changes were also observed in hypoxic normal saline group. The thickened caliber was relieved significantly in hypoxic nitrite group. CONCLUSION: Pulmonary hypertension and right ventricle reconstitution can be relieved by inhalation of sodium nitrite, and can be regulated by the level of NO and H2S in rats. Above all, inhalation of sodium nitrite may degrade HPH directly or by affecting the externalization and synthesizing of gas signaling molecule indirectly.     

Effect of endogenous H2S on pulmonary hypertension during acute lung injury induced by LPS and its interaction with NO

AIM: To investigate the effect of H2S on pulmonary artery hypertension during acute lung injury induced by LPS and the interaction between the systems of hydrogen sulfide (H2S)/cystathionine-β-lyase (CSE) and nitric oxide (NO)/nitric oxide synthase (NOS) in this process. METHODS: Seventy-two adult male rats were randomly divided into four groups: control group, LPS group, LPS+L-NAME group and LPS+propargylglycine (PPG) group. Mean pulmonary artery pressure (mPAP) of each rat was examined at 2 h, 4 h, 6 h and 8 h after treatment. H2S and NO contents in plasma, NO content, iNOS, cNOS and CSE activity in lung were measured at 4 h or 8 h after treatment, respectively. Expression of iNOS in lung tissue was also detected by immunohistochemistry technique, and the injury of lung was evaluated with morphological changes under microscope. RESULTS: LPS could induce severe lung injury, and mPAP, NO content, iNOS activity and its protein expression in LPS group significantly increased, but cNOS activity, H2S content and CSE activity decreased compared with those of control group. Administration of L-NAME before LPS could attenuate the changes induced by LPS. Pre-administration of PPG, a CSE inhibitor, exacerbated the injury by LPS, but there was no prominent variation in cNOS activity. CONCLUSION: Reduced endogenous H2S could increase pulmonary artery hypertension during acute lung injury induced by LPS. There is a negative effect between H2S/CSE system and NO/NOS system in this process.     

Changes of nitric oxide synthase and nitric oxide in lung of smoking rats

AIM:To observe the changes of iNOS and eNOS in lung tissue and NO in bronchoalveolar lavage fluid (BALF) in smoking rats.METHODS:80 Wistar rats were divided into control, smoking group, L-NIL group and L-NAME group (rats were exposed to smoke and injected (i.p.) with selective iNOS inhibitor L-NIL or NOS inhibitor L-NAME). iNOS and eNOS protein levels in whole lung were detected by immunohistochemical staining, and NOS mRNA was quantified using RT-PCR. In addition, NO₂^-/NO₃^- was determined using Griess assay.RESULTS:The expression of iNOS mRNA and protein in smoking rats increased, the expression of eNOS mRNA and eNOS protein decreased, and the total cell count and the level of NO₂^-/NO₃^-in BALF increased(P＜0.05). In vivo, L-NIL reduced the total cell count and NO₂^-/NO₃^- in BALF (P＜0.05), while L-NAME had no effect on them.CONCLUSION:Cigarette smoke increased expression of iNOS mRNA and protein and decreased expression of eNOS mRNA and protein. The large amount of NO generated by iNOS may amplify inflammation in lung tissue.     

The regulation of nitric-oxide synthase/nitric-oxide system by endogenous carbon monoxide in rats with pulmonary hypertension

AIM:To study the role and the mechanism of heme oxygenas/endogenous carbon monoxide on nitric oxide synthase/nitric oxide system in rats with pulmonary hypertension induced by hypoxic hypercapnia.METHODS:Sprague-Dawley rats were randomly divided into three groups: control group (A group), hypoxic hypercapnic group (B group), hypoxic hypercapnia+hemin group (C group). Blood CO concentration (COHb%), NO concentration, HO-1 activity, iNOS, cNOS in blood serum and lung homogenate were measured, respectively. RESULTS:① mPAP and RV/(LV+S) of B group were significantly higher than those of A and C group(P＜0.01).② Blood CO concentration, activity of HO-1in blood serum and lung homogenate in rats of B group were significantly higher than those of A group, but were significantly lower than those of C group (P＜0.01). ③ NO concentration in blood serum and lung homogenate in rats of B group were significantly lower than those of A group, those of C group were significantly higher than those of B group (P＜0.01).④The activity of iNOS in blood serum and lung homogenate in rats of B group were significantly higher than those of A group, but were significantly lower than those of C group (P＜0.01). Activity of cNOS in blood serum and lung homogenate of B group were significantly lower than those of A group (P＜0.01), and there was no significant difference between cNOS in B and C group.CONCLUSION:Endogenous carbon monoxide upregulated iNOS/NO system in rats with chronic pulmonary hypertension induced by hypoxic hypercapnia.     

Insulin influences the vasoconstriction caused by endothelin-1 in the hyperglycemic rat

AIM: To investigate the effects of insulin on aortic constriction caused by endothelin-1(ET-1) in normal and hyperglycemic rats. METHODS: Hyperglycemic rat models were prepared. Two aortic rings were immersed in the Krebs-Hensleit fluid with and without insulin (40 mIU/L) and the responses of rings to 10^-9 mol/L ET-1 were observed. RESULTS:ET-1 induced more obvious aortic ring constriction in normal rats than those in hyperglycemic rats ( P< 0.01). Rings constriction caused by ET-1 were attenuated in normal rats if the rings were immersed in the KH fluid with insulin ( P< 0.01), but no obvious changes were found in hyperglycemic rats.CONCLUSION: (1) the vasoconstriction of aortic rings caused by ET-1 can be reduced by hyperglycemia. (2) Insulin can attenuate the aortic response to ET-1. (3) Insulin can not attenuate the vasoconstriction caused by ET-1 in hyperglycemic rats. These results suggest that insulin may affect vasoconstriction caused by ET-1, which may be involved in the pathogenesis of vascular complication in diabetic or insulin resistance condition.     

Changes of NO- and H2O2-dependent soluble guanylate cyclase pathway in the hypoxic hypercapnic pulmonary hypertension rats

AIM: To study the effect of hypoxia and hypercapnia on nitric oxide (NO) in plasma and superoxide dismutase (SOD), catalase (CAT), soluble guanylate cyclase (sGC), cyclic guanosine monophospholate (cGMP) in lung tissue in rats, and to explore the effect of NO- and H2O2-sGC pathway on the development of the pulmonary hypertension. METHODS: The model of hypoxic and hypercapnic 1, 2, 4-week group (HH 1 week, HH 2 weeks, HH 4 weeks) and control group was set up. NO content in plasma, CAT and SOD in rat lung were determined by spectrophotometry. The sGC activity in lung tissue was detected by enzyme kinetic analysis. cGMP content in lung tissue was examined with ［125I］-radioimmunoassay. RESULTS: The mean pulmonary artery pressure (mPAP) showed significantly higher in HH 1 week, HH 2 weeks and HH 4 weeks groups compared with control group (all P<0.05). NO concentration in plasma, CAT, SOD, basal or nitroprusside-or H2O2- stimulated sGC activity and cGMP concentration in lung homogenates were significantly lower (P<0.05, P<0.01, P<0.01, respectively) in HH 1 week, HH 2 weeks and HH 4 weeks groups compared with control group. CONCLUSION: The inhibition of NO- and H2O2-sGC pathway by hypoxia and hypercapnia plays an important role in the development of pulmonary hypertension.     

Role of potassium channels in the regulation of intracellular free Ca2+ concentration of pulmonary artery smooth muscle cells in rats

AIM: To investigate the role of potassium channels in the regulation of intracellular free calcium concentration ( [Ca²⁺]_i) of pulmonary artery smooth muscle cells (PASMCs) in rats. METHODS: The fluorescence Ca²⁺ indicator Fura-2/AM was used to observe [Ca²⁺]_i of rat PASMCs in normal and chronic hypoxic condition. The influences of potassium channels on PASMCs proliferation were assessed by MTT assay. RESULTS: 1. In normoxic condition, [Ca²⁺]_i was (156.91±8.60) nmol/L, and in hypoxic condition, [Ca²⁺]_i was (294.01±16.81) nmol/L. 2. In normoxic condition, the voltage-dependent K⁺-channel antagonist 4-aminopyridine (4AP), but not the Ca²⁺-activated K⁺-channel antagonist tetraethylammonium (TEA) and the ATP-sensitive K⁺-channel antagonist glibenclamide (Glib) increased [Ca²⁺]_i. 3. In hypoxic condition, 4AP and TEA caused the rise in [Ca²⁺]_i , but Glib had no effect on [Ca²⁺]_i. 4. MTT assay showed that 4AP increased the value of absorbing light degree (A value) in normoxic and hypoxic condition (0.582±0.062,0.873±0.043,respectively, P＜0.01), TEA increased A value only in hypoxic condition, and Glib had no effect on the proliferation of PASMCs. CONCLUSIONS: K_V plays an important role in the regulation of [Ca²⁺]_i and proliferation of PASMCs. K_Ca serves as distinct responsive roles in the regulation of proliferation of PASMCs in hypoxic condition. K_ATP has no effect on [Ca²⁺]_i and proliferation of PASMCs in normoxic and hypoxic conditions.     

Changes in endogenous nitric oxide and effects of inhaled nitric oxide on pulmonary artery hypertension in chronically hypoxic rats

AIM: To investigate the role of nitric oxide (NO)in the development of chronically hypoxic pulmonary artery hypertension (PAH) and the hemodynamic effects of inhaled NO on pulmonary circulation. METHODS: 67 male adult SD rats were randomly divided into 7 groups: (1) control (n=9);(2) chronically intermitent hypoxia (CIH, 6 h/d, 7 d/w) 1 week(n=7); (3) CIH 2 weeks (n=11); (4) CIH 3 weeks (n=11); (5) CIH 1 week+L-NAME (NO synthase inhibitor, 30 mg/kg, by gavage, n=10); (6)CIH 3 weeks+L-Arg (NO precursor, 10 mg/kg, by gavage, n=9); (7) CIH 3 weeks+inhaled NO (0.0004% for 20 min, n=10) to determine the mean pulmonary artery pressure (MPAP), weigh the right ventricle (R) and ventricular segment plus left ventricle (S+L), and calculate R/(S+L) (g/g) and R/Wt (Wt: body weight, g/kg). RESULTS: 1.MPAP increased compared with control when CIH 1 week, reaching the highest when CIH 2 weeks; R/(S+L) and R/Wt also increased notably when CIH 1 week (P＜0.01); 2. The level of plasma NO₂^-/NO₃^- elevated significantly when CIH 2 weeks, but fell when CIH 3 weeks; the content of plasma ET-1(endothelin-1) also increased significantly. The level of plasma ET-1 correlated with R/(S+L) and R/Wt, r=0.43 and 0.46, respectively, both P＜0.01; 3. The level of plasma NO₂^-/NO₃^- droped 33.2 % (P＜0.01) after treatment with L-NAME, with R/(S+L) increasing 15.2 % (P＜0.05); 4. L-Arg decreased the MPAP 17.8 %(P＜0.01). CONCLUSION: The endogenous NO release increases at early stage (1-2 weeks) of chronic hypoxia, but falls at the prolonged stage; the elevated level of plasma ET-1 possibly plays an important role in remodeling of chronically hypoxic pulmonary vessels and ventricle; inhaled NO significantly decreases the chronically hypoxic PAH.     

Effect of nitric oxide on apoptosis in lung injury following ischemia-reperfusion of hind limbs

AIM: To investigate the injury of lung and the role of cell apoptosis in the pathogenesis of acute lung injury following ischemia-reperfusion of hind limbs and the influence of nitric oxide (NO) to apoptosis.METHODS: Referring to our laboratory normal method,the model rats,which underwent 4 hours ischemia and 4 hours reperfusion of hind limbs were made.L-arginine (L-Arg) and N-nitro-L-arginine methyl ester (L-NAME) was administrated respectively to these rats before the experiment.Apoptosis was detected by TdT-mediated dUTP nick end labeling (TUNEL),respectively.The radioimmunoassay (RIA) was used to detect level in the expression of TNF-α.The immunohistochemistry (IHC) method was used to detect the level in the expression of Bcl-2,Bax,caspase-3 and TNF-α.The morphologic changes were observed under microscope,respectively.The results of the RIA and the IHC were analyzed quantitatively by relative computer analytical system.RESULTS: After rats’s hind limbs suffered from ischemia-reperfusion,the apoptosis in alveolar epithelial cells and pulmonary vascular endothelial cells was found.The expression of TNF-α,caspase-3 and Bax increased.Compared with IR rats,the expressions of TNF-α,caspase-3 and Bax were not obvious in the L-Arg administrated group,but the expression of Bcl-2 was obvious in that group.Compared with IR rats,the expressions of TNF-α,caspase-3 and Bax were obvious in the L-NAME administrated group,but the expression of Bcl-2 was not obvious in that group even weaker than normal ones.CONCLUSION: Apoptosis participated in acute lung injury following ischemia-reperfusion of hind limbs.The excess expression of TNF-α related with apoptosis of alveolar epithelial cells and pulmonary vascular endothelial cells.NO may reduce the occurrence of apoptosis and other lung injury through down-regulating the level in the expression of TNF-α.     

Effect of 15(S)-hydroxyeicosate traenoic acid (15-HETE) on eNOS Thr495 phosphorylation in pulmonary artery endothelial cells

AIM: To examine the effect of 15(S)-hydroxyeicosate traenoic acid (15-HETE) on eNOS activity in pulmonary artery endothelial cells (PAECs).METHODS: 1.Rat pulmonary artery (PA) rings of intrapulmonary arteries were suspended under isometric tension in oxygenated Krebs buffer in order to observe the differences on constriction induced by 15-HETE using nitro-L-arginine methyl ester (L-NAME, 10-4 mol/L, an inhibitor of eNOS) and removal of endothelium.2.The bovine PAECs was cultured for NO assay by Greiss reaction.3.Immunoprecipitation (IP) and Western blotting (WB) were used to analyze the cell extracts from bovine PAECs treated with 2×10-6 mol/L 15-HETE for 30 and 60 min, using phosphatase-eNOS (Thr495) antibody for IP and eNOS antibody for WB.RESULTS: 1.Inhibition of eNOS with L-NAME and removal of endothelium significantly enhanced 15-HETE induced contractions in rat PA rings in a concentration-dependent manner (P＜0.05, P＜0.01).2.15-HETE increased phospho-eNOS (Thr495) levels (P＜0.01) in cultured bovine PAECs.3.10-6 mol/L 15-HETE (P＜0.05) did significantly reduce nitrite production, whereas 10-5 mol/L cinnamy l 3, 4-dihydroxy-［alpha］-cyanocinnamate (CDC, P＜0.05), 10-4 mol/L nordihydroguiairetic acid (NDGA, P＜0.01) significantly increased nitrite production, as measured by a spectrophotometric assay.CONCLUSION: 15-HETE inhibits eNOS activity and decreases production of NO (NO-2/NO-3).The pathway of eNOS/NO is involved in 15-HETE-induced contraction in the rat PA.