杜鹃素通过激活Kv通道舒张C57BL/6J小鼠离体肺动脉

Farrerol relaxes isolated pulmonary arteries in C57BL/6J mice by activating Kv channel

摘要:

目的]研究杜鹃素对C57BL/6J小鼠离体肺动脉的舒张作用及机制。 [方法]麻醉小鼠,迅速取出肺组织置于4 ℃ K-H缓冲液中,显微镜下分离得到肺动脉,并剪成长约2 mm的血管环备用。(1)杜鹃素对小鼠离体肺动脉静息张力的影响:选取具有活性的小鼠肺动脉环,在静息状态下加入不同浓度的杜鹃素(10－6、3×10－6、10－5、3×10－5及10－4 mol/L)。(2)杜鹃素舒张小鼠肺动脉实验:苯肾上腺素(PE,1 μmol/L)或KCl(60 mmol/L)诱导小鼠肺动脉收缩达到平台期后,加入不同浓度的杜鹃素(10－6、3×10－6、10－5、3×10－5及10－4 mol/L)。(3)杜鹃素抑制肺动脉收缩实验:在加入或未加入杜鹃素的情况下,加入PE(10－9、3×10－9、10－8、3×10－8、10－7、3×10－7及10－6 mol/L)或KCl(20、30、40、60、80及120 mmol/L)诱导小鼠离体肺动脉收缩,并记录血管张力的变化。(4)无钙或复钙实验:在加入或未加入杜鹃素的情况下,记录小鼠离体肺动脉在无钙或复钙{2.5 mmol/L[Ca²＋]ex}状态下血管张力的变化。(5)杜鹃素舒张小鼠离体肺动脉与钾离子通道的关系:首先用60 mmol/L KCl溶液收缩小鼠肺动脉至平台期,随后分别加入3 mmol/L 4-氨基吡啶(4-AP)、2 mmol/L四乙胺(TEA)、30 μmol/L BaCl₂和10 μmol/L格列本脲(Gli)处理15 min,最后通过加入浓度梯度杜鹃素对肺动脉进行舒张。 [结果]杜鹃素对静息状态下小鼠肺动脉张力无明显影响,但对PE和KCl诱导的小鼠肺动脉收缩具有浓度依赖性舒张作用；3×10－5 mol/L杜鹃素预处理显著降低PE和KCl诱导的小鼠肺动脉最大收缩(P＜0.01),以及显著降低在无钙或复钙条件下KCl诱导的小鼠肺动脉收缩(P＜0.01)。加入电压依赖性钾离子通道阻滞剂4-AP显著降低杜鹃素对小鼠肺动脉的最大舒张率(P＜0.01),而加入大电导钙激活钾离子通道阻滞剂TEA、内向整流钾离子通道阻滞剂BaCl₂或ATP敏感钾离子通道阻滞剂Gli对杜鹃素的舒张血管作用均无显著影响(P＞0.05)。 [结论]杜鹃素对小鼠离体肺动脉具有舒张作用,其机制可能与开放电压依赖性钾离子通道相关。

Abstract:

Aim To study the diastolic effect and mechanism of farrerol on isolated pulmonary arteries of C57BL/6J mice. Methods After anesthesia, mouse lung tissue was quickly removed and placed into the 4 ℃ K-H buffer, pulmonary arteries were isolated under the microscope and cut into 2 mm long vascular rings for spare use. (1)The effect of farrerol on the resting tension of isolated mouse pulmonary arteries:in the resting state, the active mouse pulmonary artery rings were treated with different concentrations of farrerol (10－6,3×10－6,0－5,3×10－5 and 10－4 mol/L). (2)Farrerol relaxed mouse pulmonary artery experiment:pulmonary arteries were contracted using phenylephrine (PE, 1 μmol/L) or KCl (60 mmol/L), and when the contraction reached the platform, different concentrations of farrerol (10－6,3×10－6,0－5,3×10－5 and 10－4 mol/L) was added. (3) Farrerol inhibited pulmonary artery contraction experiment:under conditions with or without the addition of farrerol, pulmonary arteries were contracted using different concentrations of PE (10－9,3×10－9,0－8,3×10－8,0－7,3×10－7 and 10－6 mol/L) or KCl (0,0, 0,0, 80 and 120 mmol/L), and the pulmonary artery muscle tension was recorded. (4)Calcium free and recalcification experiments:under conditions with or without the addition of farrerol, the changes of isolated mouse pulmonary artery tension were measured in the state of calcium free or recalcification {2.5 mmol/L [Ca²＋]ex}. (5)The relationship between farrerol induced relaxation of isolated mouse pulmonary arteries and potassium ion channels:firstly, 60 mmol/L KCl solution was used to contract the mouse pulmonary arteries until the platform. Then, 3 mmol/L aminopyridine (4-AP), 2 mmol/L tetraethylammonium (TEA), 30 μmol/L BaCl₂, and 10 μmol/L glibenclamide (Gli) were added and treated for 15 min. Subsequently, the pulmonary arteries were relaxed using a concentration gradient of farrerol. Results Farrerol had no significant effect on the mouse pulmonary arteries in the resting state, but had a concentration-dependent relaxing effect on the mouse pulmonary arteries pre-contracted with PE and KCl. While the pretreatment of 3×10－5 mol/L farrerol could significantly reduce the maximum contraction of mouse pulmonary arteries induced by PE and KCl (P＜0.01), as well as significantly reduce the contraction of mouse pulmonary arteries induced by KCl under calcium free or recalcification conditions (P＜0.01). Addition of the voltage-dependent potassium ion channel blocker 4-AP significantly reduced the maximum diastolic rate of mouse pulmonary arteries induced by farrerol (P＜0.01), while addition of the high conductivity calcium activated potassium ion channel blocker TEA, inward rectifying potassium ion channel blocker BaCl₂, or ATP sensitive potassium ion channel blocker Gli had no significant effect on the vasodilation effect of farrerol (P＞0.05). ConclusionFarrerol has a relaxing effect on isolated mouse pulmonary arteries, and its mechanism may be related to open voltage-dependent potassium ion channels.