【作者】 牛龙刚；

【导师】 张明升；

【作者基本信息】 山西医科大学 ， 生理学， 2014， 博士

【摘要】 研究背景：生理性事件或病理性疾病都可导致机体重要脏器如心脏和脑对能量的需求增加,高耗能组织短期或长期内对能量的过多需求,可导致有氧的三羧酸循环不能提供足够的能量,进而无氧的糖酵解过程增强,其提供能量的同时产生大量的酸性代谢产物如丙酮酸、乳酸等释放入血引起酸中毒。在心肌梗塞发生时,局部的缺血缺氧导致局部较严重的酸中毒,由于循环障碍时不利于代谢产物排出,酸中毒对局部组织的影响会较大,尤其是对CA ASMCs和心肌细胞。研究表明,在猪CA、家兔CA、大鼠肠系膜动脉和主动脉酸中毒可引起动脉平滑肌张力减弱,在大鼠肺动脉和CA酸中毒能够引起动脉平滑肌张力增强。尤其是对大鼠CA的酸中毒研究存在争议,相关研究认为酸中毒舒张大鼠CA,引起冠脉流量增大。导致酸中毒对不同种属动物动脉以及同一种属如大鼠不同动脉作用差异的机制至今仍未阐明。更重要的是,目前已有研究表明酸中毒可造成心肌细胞受损,适当的碱化治疗对心肌细胞有保护作用,那么心梗时CA再通以及酸中毒的及时纠正对心梗后心肌损伤的恢复可能是非常重要的。因此,阐明酸中毒对CA张力的影响如何及其机制,以及其对不同动脉张力差异性影响的原因将有利于给临床缺血缺氧性疾病的治疗提供参考依据。研究目的：1.研究酸中毒对大鼠CA张力的影响,包括其对CA基础张力的影响,以及酸中毒条件下,CA对血管活性物质肌源性反应的影响如何。2.研究相同酸中毒条件下,大鼠CA, RIA和MA肌源性反应的异同点及其机制。3. ASMCs的L-型钙通道和Kv通道是调节动脉张力的重要通道,这些通道受酸中毒影响时其电流大小有何变化。4.酸中毒发生时极易影响到ASMCs pHin值,而pHin值的稳态受到细胞膜上酸碱转运体的调节,本文进一步研究酸碱转运体抑制剂对酸中毒所致的动脉张力变化、ASMCs [Ca2+]in和pHin值变化的影响。5.阐明酸碱转运体与pHin值、[Ca2+]in和CA张力之间的关系。研究内容及方法：采用体重为200～220克成年♂Wistar大鼠,从器官、组织和细胞三个水平研究酸中毒对冠脉张力的影响及其机制。第一部分采用离体心脏恒压Langendorff灌流的方法观察酸中毒对CPF的影响。同时,为了排除心肌收缩力和心率变化对CPF的影响,采用离体CA插管恒压灌流的方式观察酸中毒时CA直径的变化。第二部分采用离体动脉环张力记录法,观察酸中毒时CA、RIA和MA基础张力的变化,以及其对CA由缩血管剂引起的收缩反应曲线的影响。观察外钙内流和内钙释放在酸中毒引起CA收缩过程中的作用。观察酸碱转运体抑制剂如：1μM州或3μM Lans、10μM Amil、10μM IDIS和1μM KB R7943对酸中毒引起CA收缩的影响。第三部分观察酸中毒对CA、RIA和MA的ASMCs形态的影响。同时利用全细胞膜片钳技术,观察pHex值和pHin值变化对CA ASMCs LTCC通道的影响以及pHex值变化对Kv通道的影响。为了探索pHex值变化对pHin值影响以及酸碱转运体在此过程中的作用,观察酸碱转运体抑制剂如：1μM或3μM Lans、10μM Amil、10μM DIDS和1μM KB R7943对酸中毒引起的ASMCs [Ca2+]in和pHin值变化的影响。研究结果：1.酸中毒可使离体灌流心脏的LVP降低和CPF减小。在离体插管恒压灌流的CA可观察到CA直径随酸中毒程度增加而逐渐变小。2.在离体动脉环实验中,结果显示酸中毒仅仅引起CA基础张力升高,而对RIA和MA的张力没有影响。在相同酸中毒的条件下,由KCl或U46619引起的CA收缩反应明显增强。3.在酸中毒引起CA收缩的过程中,外Ca2+内流所起作用比内Ca2+释放要大。4.酸碱转运体抑制剂尤其是质子泵和Na+/HCO3-转运体的抑制剂明显抑制酸中毒引起的CA收缩,其中DIDS的作用较强。Na+/Ca2+交换体抑制剂KB R7943的作用也较明显,但远不及DIDS。5.急性分离单细胞实验结果表明,酸中毒特异性地使CA ASMCs的长度缩短和面积变小。6. pHex值降低可使CA ASMCs的Ba2+电流增强,相反可使MA ASMCs的Ba2+电流减弱,而对RIA ASMCs的Ba2+电流幅度没有显著影响。而pHin值降低时对Ba2+电流抑制作用和其升高对Ba2+电流增强作用,在CA和MA ASMCs是一致的。7. pHex值降低可抑制CAASMCs的Kv通道电流,而对RIA和MA的Kv通道电流没有显著影响。8. pHex值降低也可特异性地使CAASMCs [Ca2+]in升高,而对RIA和MA ASMCs [Ca2+]in没有显著影响。酸碱转运体抑制剂如质子泵抑制剂Lans和Na+/HCO3-转运体抑制剂DIDS可抑制酸中毒所致的[Ca2+]in的升高,其中DIDS的作用最强,而Amil对[Ca2+]in的变化几乎没有影响。9. pHex值降低可使CA ASMCs pHin值升高,相反使MAASMCs的pHin值降低。酸碱转运体抑制剂DIDS可使pHex值降低引起的CA ASMCs pHin值的升高作用受到抑制,Lans的此作用次之,而Amil的作用不显著。结论：酸中毒可引起或促进RCA的收缩,而对RIA和MA的静息张力没有显著影响。细胞外酸中毒增强RCAASMCs的LTCC电流和抑制其Kv电流,细胞外酸化或细胞内碱化可增强RCAASMCs的LTCC电流。细胞外酸化引起CAASMCs细胞内碱化,其碱化程度受到酸碱转运体抑制剂尤其是Na+/HCO3转运体和质子泵抑制剂的影响。据此我们推测,酸中毒引起RCA收缩、RCA ASMCs pHin值升高和电生理学的改变与细胞膜上的Na+/HCO3-转运体和质子泵有关。更多还原

【Abstract】 Background:Physiological events or pathophysiological diseases result in the increasing need to energy in critical organs, such as brain and heart. The excessive requirement to energy of the high energy-intensive tissues in the long or short term leads to likely that tricarboxylic acid cycle cannot afford the require of overmuch energy, and that the process of glycolysis is enhanced to increase the output of acidic metabolite, such as pyruvate and lactate. Acidosis induced by the release of acidic metabolite affects the arterial tone of different regions of vascular tree. Ischemia/hypoxia of local tissue in myocardial infarction induces the severe acidosis which produces the harmful injury to the local tissue, such as CA ASMCs and myocardial cells. The present studies indicate that acidosis can induce vasodilation in the CA of rabbit and pig. Conversely, acidosis can induce the increase of arterial tone in rat aorta and pulmonary artery. Controversially, acidosis dilated rat CA to enhance coronary arterial flow. So far it is elusive that acidosis develops the inconsistently vascular function in different species and different regions of the vascular tree. What’s more, the present studies also verify that acidosis jeopardizes myocardium and proper alkalinization of extracellular fluid may be cardioprotective. The above opinions indicate that the recovery of CA reperfusion in myocardial infarction and the therapy of acidosis are likely of importance to recover the injured myocyte. Therefore, it is vital to clarify the effects of acidosis to CA tone, the mechanisms of acidosis-induced CA contraction, and the different vascular functions of acidosis that would contribute to provide the interesting guideline for doctors to remedy the ischemia/hypoxia diseases. Objectives:we established the current study to demonstrate:1. Effects of acidosis on the resting tone of CA and the dose-response curve of CA constriction induced by vasoconstrictors.2. To explore the likely differences among vasoconstrictions induced by acidosis in CA, RIA and MA.3. To observe the effects of acidosis on the current amplitude of LTCC and Kv in CA, RIA and MA owing to they are the vital regulators of vascular function.4. Acidosis rapidly affects SMC pHin that is adjusted by the acid-base transporters in cell membrane. Therefore, it is essential that we focus on the effects of the inhibitors of acid-base transporters on the arterial force, ASMCs [Ca2+]in and pHin.5. To clarify the relationship of among pHin, pHex and CA tone.Research contents and methodsMale Wistar rats weighing200-220g were used. To study the effects of acidosis on CAtone and related mechanisms study.The first section To observe the effect of acidosis on CPF of the isolated heart with Langendorff method. Meanwhile, the direct effect of acidosis on CA diameter was observed by cannulated and pressurized artery in order to remove the effects of heart rate and myocardial contractility.The second section Effects of acidosis on the basic force of CA, RIA and MA and vasoconstriction induced by vasoconstrictors in CA were observed by wire myograph method. The releasing of organelles Ca2+and influx of extracellular Ca2+were focused during pHex6.8-induced contraction in CA. Effects of acid-base transporter inhibitors, such as1μM or3μM Lans,10μM Amil,10μM DIDS, and1μM KB R7943(Na+/Ca2+exchanger inhibitor), simultaneously, on acidosis-induced CA contraction were the focal points.The third section To observe the morphological changes of SMC in CA, RIA and MA during acidosis in order to demonstrate the its direct action on ASMCs. In the whole-cell patch clamp method, we observed the action of changes of pHex and pHin on LTCC of ASMCs and effect of pHex changes on KV. To clarify the effects of pHex on pHin and acid-base transporters during vasoconstriction of CA in acidosis, we observed the likely actions of acid-base transporter inhibitors, such as1μM or3uM Lans,10μM Amil and10μM DIDS, in the process of changes of SMC [Ca2+]in and pHin due to the acidic pHex.Results:1. Acidosis attenuated the LVP and CPF in the isolated Langendorff rat hearts, and narrowed the cannulated CA.2. Acidosis augmented singly the basic force of CA, but there was no effect on the resting tone of RIA and MA. It was logically consistent that acidosis contributed to vasoconstriction triggered by KCl and U46619in CA.3. Vasoconstriction induced by acidosis was mainly dependent on influx of extracellular Ca2+in CA.4. Inhibitors of proton pump and Na+/HCO3-transporter attenuated both pHex6.8-induced vasoconstriction of CA. Especially, the decreasing action of DIDS was stronger than that of Lans. There was no effect of Amil on acidosis-triggered CA contraction. Na+/Ca2+exchanger inhibitors, KB-R7943, slightly weakened the above vasoconstriction.5. Acidosis only decreased the length and area of CA SMC.6. Lowing pHex augmented ASMCs Ba2+current in CA, attenuated in MA, and did not affect in RIA. There was no difference in the current amplitude change induced by pHin variation in CA and MA SMC.7. Acidic pHex diminished SMC Kv current of CA specifically, and did not alter the amplitude of KV current in RIA and MA ASMCs:8. Acidic pHex increased ASMCs [Ca2+]in in CA specifically, and did not alter [Ca2+]in in RIA and MA. The increasing [Ca2+]in of CA inhibited by Lans and DIDS. Function of DIDS is bigger than Lans. Another inhibitor, Amil, did not vary the [Ca2+]in increasing in CA.9. Acidic pHex led to the increasing pHin in CA and the decreasing pHin in MA. The increasing pHin was reversed by DIDS, and became lowing pHin. Acidosis did not affect the physiological pHin owing to pretreatment of Lans. However, Amil did not alter the increasing pHin induce by acidosis in CA ASMCs. Conclusion:Acidosis induced and contributed to RCA constriction, while did not affect significantly the basic force of RIA and MA. Acidic pHex enhanced LTCC current and decreased KV current of RCA ASMCs. Lower pHex and alkalized pHin increased both its LTCC current amplitude. The cytoplasmic alkalization induced by acidic pHex was attenuated by Na+/HCO3-transporter inhibitor (DIDS) and proton pump inhibitor (Lans) in RCA ASMCs. According to the above results, we thought that vasoconstriction, pHin increasing and the changes of electrophysiological characteristics were involved in Na+/HCO3-transporter and proton pump of cell membrane in RCA ASMCs.更多还原