| The mechanism of pulmonary vasoconstricition induced by chronic hypoxia is extremely complex, and remains unknown till now. Hypoxic pulmonary vasoconstriction (HPV) helps match perfusion with ventilation, thus diverting blood flow from poorly ventilated portions of the lung to maximize arterial saturation. But that pulmonary arterioles continuously constrict, lead to hypertrophia,fibrosis and pulmonary artery hypertension (PAH), ultimately ventricular dilatation and congestive heart failure to death. Since the mechanism of PAH induced by the hypoxia is elusive, the potently etiotropic curatives are deficient. The existing therapeutic tools are symptomatic treatment, which can not achieve satisfactory therapeutic effect. It is essential to explore the mechanism of HPV.Our previous study indicated that chronic hypoxia increased the activity of 15-lipoxygenase (15-LO), and transfered 15-LO from cytoplasm to plasma membrane, which catalyzed archidonic acid (AA) to produce the metabolisms of 15-HETE,15-KETE. Therefore, these metabolisms of 15-LO from AA have their correspond intracellular distribution and location. That 15-HETE constricts PA rings in dose-dependent manner is more obvious in the hypoxic group. But 15-HETE is instability in animal in vivo and can further metabolism. The roles of its products in HPV have not been reported. In the present study, we studied the effect of 15-HETE and its products on rat pulmonary vasoconstriction in the hypoxic and normoxic group by using organ bath technique. The results demonstrated that 15-KETE constricted rat pulmonary arteries more strongly than that of 15-HETE, which explained that 15-KETE may be a new mediator in HPV and had synergistic effect with 15-HETE. At the same time, by using the specific blocker of K+ channel and L-type Ca2+ channel,whole cell patch-clamp technique,laser scanning confocal microscope(LSCM)technique,Western blot and RT-PCR technique, we revealed the mechanism of 15-KETE on rat pulmonary vasoconstriction ion channel from vascular function,electrophysiology,protein and mRNA levels, and provided a theory foundation for HPV.In the isolated pulmonary arterial bath experiments, thirty-two male SD rats were randomly divided into two groups (n=16): normal group breathing fresh air (FiO2=21 %) and hypoxic group breathing hypoxic air (N2 = 90 %, O2 = 10 %). After 9 days , the rats were anesthetized with pentobarbital sodium (40 mg/kg ) , and the chests were opened for removal of the heart and lungs en bloc and PA rings (0.3～0.7 mm in diameter and 3 mm in length) were prepared. By using organ bath technique, one half in each group were used to observe the effect of 15-HETE,15-KETE and 8(S),15 (S) -DiHETE on hypoxic and normoxic rat isolated pulmonary arterial rings, the others in each group were used to investigate the effect of 15-KETE and its mechanism through ion channels on hypoxic and normoxic rat isolated pulmonary arterial rings by using the specific K+ channel blockers,L-type Ca2+ channel blocker and Ca2+-free Krebs solution. The results demonstrated that 15-HETE , 15- KETE and 8 (S) ,15 (S)–DiHETE gradually increased PA rings tension in a dose- dependent manner in the normal and hypoxic group, respectively; Constriction effect of 15-HETE , 15- KETE on rat PA rings in hypoxic group was significantly greater than that in normal group , but the constriction effect of 8(S) ,15(S)-DiHETE on rat PA rings between normal and hypoxic groups had no statistical difference. Contractility effect of 15-KETE , 8(S) ,15(S)-DiHETE on normoxic rat PA rings was greater than that of 15-HETE; 4-aminopyridine (2×10-3 mol/L) , a Kv channel blocker significantly decreased constriction of rat isolated PA rings induced by 15-KETE. The results were similar in two groups; The KATP channel blocker glibenclamide (10-6 mol/L) and the BKCa channel blocker tetraethylammonium (1×10-2 mmol/L) did not affect constriction of rat isolated PA rings induced by 15-KETE; The L-type Ca2+ channel blocker nifedipine (10-6 mol/L) and Ca2+-free Krebs solution significantly decreased constriction of rat isolated PA rings induced by 15-KETE.In whole cell patch-clamp experiments, single PASMC of the normoxic and hypoxic group were obtained with acute enzyme (collagenase I plus elastase) dispersing method. Using whole cell patch-clamp technique, the effect of 15-KETE on Kv current was recorded. The results demonstrated that 15-KETE( 1×10-7 mol/L,1×10-6 mol/L ) caused membrane depolarization. The effect of 15-KETE on membrane potential persisted in cells in which intracellular Ca2+ was buffered with 1 ,2-bis (2-aminophenoxy) ethane-N , N , N′,N′-tetraacetic acid (BAPTA).15-KETE did not affect the membrane capacitance of PASMCs.15-KETE (1×10-8 to 1×10-6 mol/L) caused a concentration-dependent and reversible inhibition of Kv current. The inhibition of 15-KETE (1×10-6 mol/L) on peak Kv current was greater when containing intracellular Ca2+ at physiological level ([Ca2+]i =75 nmol/L) than that of intracellular free of Ca2+. Chronic hypoxia did not change the effect of 15-KETE on the passive electrical properties of concentration-dependent inhibition of voltage-gated K+ current, but might alter the sensitivity of PASMCs to 15-KETE. The inhibition of K+ current from chronic hypoxic rats was greater than that from normoxic rats at high concentration (1×10-7 mol/L, 1×10-6 mol/L ). In the laser scanning confocal microscope (LSCM) experiments, PASMCs obtained with enzyme (collagenase I plus elastase) dispersing method were cultured in the first passage in vitro. Cells with concentration of 2×10-5 cells/ml were seeded onto a glass coverslip in 6 well plate, and cultured for 24h in the incubator at 37℃. After cell adherence, the glass coverslip was taken out from 6 well plate, placed in special-made chamber, and washed with D-Hanks solution for 3 times. Cells were loaded with Fluo-3/AM (final concentration 10μmol/L), followed by 30min in the incubator at 37℃, then washed with D-Hanks solution for 3 times, extracellular dye was washed out, the Fluo-3/AM loaded cells were available for [Ca2+]i measurement within 2h. Using LSCM technique, the effect of 15-KETE on cytosolic [Ca2+]i in rat PASMCs was measured. The results demonstrated that 15-KETE (1×10-81×10-6 mol/L) increased cytosolic [Ca2+]i of rat PASMCs in a concentration-dependent manner. The L-type Ca2+ channel blocker verapamil (10-6 mol/L) and Ca2+ -free Krebs solution significantly inhibited the increase of cytosolic [Ca2+]i induced by 15-KETE.Western blot and RT-PCR were applied to determine the effect of 15-KETE (1×10-6 mol/L) in the cell cultured media on the protein and mRNA expression level of Kv1.2,Kv1.5,Kv2.1 and Kv3.4 at different time point (0h,0.5h,1h,2h,4h,8h,24h). The results demonstrated that 15-KETE (1×10-6 mol/L) decreased the protein expression level of Kv1.2,Kv1.5,Kv2.1 and Kv3.4; simultaneously decreased mRNA expression level of them.In brief, 15-KETE strongly constricted rat PA rings in the concentration-dependent manner, which was more significant in the hypoxic group. 15-KETE inhibited voltage-gated K+ current in rat PASMCs and depolarized rat PASMCs so that promoted extracellular Ca2+ to inflow and increased cytosolic [Ca2+]i in rat PASMCs. Simultaneously, 15-KETE decreased the protein and mRNA expression level of Kv1.2,Kv1.5,Kv2.1 and Kv3.4. Therefore, 15-KETE may be a new mediator in the hypoxic pulmonary vasoconstriction. |
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