Kv和BK通道在大鼠小动脉平滑肌细胞的分布及牛磺酸对钾通道电流的影响
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摘要
研究背景与方法:
血管的反应性差异很大程度上与其血管平滑肌的多种受体及离子通道的分布类型和分布密度差异有密切关系。平滑肌细胞膜离子通道的活动正常是血管平滑肌兴奋收缩过程起始的关键,也是细胞的兴奋性与膜的稳定性得以维持的保证。K~+通道分布广泛,是调节血管平滑肌的收缩性与舒张性的主要离子通道。K~+通道电流微小的改变即可导致膜电位的很大变化,K~+经K~+通道跨细胞膜外流,细胞内电位降低,具有细胞膜稳定作用;细胞内电位降低还使电压依赖性钙通道的开放几率降低。K~+通道也因此成为治疗药物作用的重要靶标之一。K~+通道是目前已知的亚型(包括功能分型和结构分型)最多的一类细胞膜离子通道。一些通过影响K~+通道而起作用的药物已逐渐应用于临床。但目前临床上所用的K~+通道开放药对组织的选择性不够高,在组织特异性和功能多样性方面的一系列问题导致了药物在临床应用过程中的疗效不理想,甚至治疗失败。因此,了解各组织细胞K~+通道的分布情况和功能状态对研发新药、临床针对性地合理选择药物治疗极其重要。
大电导钙激活钾通道(high-conductance Ca~(2+)-activated K~+ channel, BK通道)和电压依赖性钾通道(voltage-dependent K~+ channel, Kv通道)是血管平滑肌细胞(vascular smooth muscle cell, VSMC)中普遍存在而且发挥优势作用的两类负载外向型钾电流的K~+通道。它们不同的通道亚型对细胞功能的调节和影响有所不同,保障了VSMC能对各种不同的刺激做出适当的反应。尽管Kv通道和BK通道在某些血管上的情况已经得到一些研究,但不同部位的血管上的各种K~+通道的表达和功能还远不清楚。心脏和脑的血液供应受心脏冠状动脉和脑血管平滑肌张力的影响。明确冠状动脉和大脑中动脉平滑肌细胞(SMC)的Kv通道和BK通道的分布差异有助于更好地掌握其生理作用,进一步了解其在不同生理和病理状态的动态调整;为明确Kv通道和BK通道在冠状动脉和大脑中动脉SMC介导多种血管活性物质的调节作用机制以及在多种病理过程中的作用奠定基础;也为研究开发以K~+通道为靶点的选择性高、副作用少的药物提供新思路、新线索。
牛磺酸是人和哺乳动物体内广泛存在的含硫β氨基酸,在心血管组织中含量最丰富。国内外大量研究表明,牛磺酸具有广泛生物学效应,是调节机体正常生理功能的重要物质,对多种情况下病理损伤具有积极的保护作用。在心血管功能异常的病理状态下补充牛磺酸可以对疾病的发生发展起积极防治作用。作为天然内源性物质,与一般化学合成药物不同的是,牛磺酸作用靶点十分广泛,可以通过多环节、多途径调节心血管系统功能,在防治心脑血管疾病方面展示出良好应用前景。许多研究表明,牛磺酸能够通过作用于包括K~+通道在内的多种离子通道而产生心血管效应,但到目前为止,国内外有关牛磺酸的研究多集中于心肌和骨骼肌。我室前期研究表明,牛磺酸对血管平滑肌收缩和舒张功能的影响与多种K~+通道有关。
本实验中,急性酶解分离成年雄性SD大鼠冠状动脉和大脑中动脉SMC,利用全细胞膜片钳技术记录Kv通道和BK通道的电流,应用单细胞反转录聚合酶链反应(RT-PCR)方法观察Kv通道和BK通道亚型在转录水平的基因表达情况;记录给予牛磺酸后Kv通道和BK通道电流的变化,了解牛磺酸对此两类通道的影响。
主要实验结果:
一、大鼠冠状动脉和大脑中动脉SMC的Kv通道
大鼠冠状动脉和大脑中动脉SMC的静息电位分别为-28.91±3.93 mV(n = 15)和-30.82±2.79 mV(n = 19)(P>0.05);膜电容分别为17.66±0.95 pF(n =46)和14.28±1.01 pF(n =42)(P>0.05)。
大鼠冠状动脉和大脑中动脉SMC的Kv电流约在膜电位正于-40mV开始呈电压依赖性的增加和外向整流特性;电流在除极后约10~20ms达到最大,500 ms内无明显失活,呈延迟整流特性。Boltzmann方程拟合得到大鼠冠状动脉和大脑中动脉SMC的Kv通道电流半激活电位(V1/2)分别为-4.39±1.44mV和-9.89±1.72 mV(P<0.05),斜率因子(K)分别为12.42±1.19和13.03±1.62(P>0.05);半失活电位(V1/2)分别为-11.78±1.47 mV和-25.77±2.29 mV(P<0.05),斜率因子(K)分别为14.73±1.54和16.37±2.33(P>0.05)。给予Kv通道阻断药4-氨基吡啶(4-aminopyridine, 4-AP, 3mM)后,通道电流分别被抑制61.2%(n=11)和62.7%(n=12)。
国外研究资料表明,Kv通道的Kv1.1、Kv1.2、Kv1.5、Kv1.6和Kv2.1亚型具有延迟整流特性,因此,我们采用单细胞RT-PCR方法检测了这些亚型在大鼠冠状动脉和大脑中动脉SMC转录水平的表达。结果表明,Kv1.2和Kv1.5亚型在大鼠冠状动脉和大脑中动脉SMC均有表达。取甘油醛-3-磷酸脱氢酶(GAPDH)作为内参照,计算各目的基因相对表达量。Kv1.2亚型在此二动脉VSMC的表达分别为94.4±7.59%和98.1±5.08%(P>0.05, n=7)。Kv1.5亚型在此二动脉VSMC的表达分别为87.3±4.20%和80.2±6.06%(P>0.05, n=7)。Kv1.1亚型在大脑中动脉有表达,其与GAPDH的相对值为54.7±3.05%,表达率100%;在冠状动脉偶有弱的表达,其与GAPDH的相对值为37.6%,表达率14.3%。Kv1.1亚型在此二动脉VSMC的表达有显著性差异(P<0.01, n=7)。Kv1.6和Kv2.1亚型在此二动脉VSMC均无表达。
二、大鼠冠状动脉和大脑中动脉SMC的BK通道
大鼠冠状动脉和大脑中动脉SMC的BK电流呈电压依赖性,在除极大于+10mV开始呈明显外向整流特性;浴槽液中给予BK通道阻断药四乙胺(tetraethylammonium, TEA,1mM)后通道电流明显降低,峰值电流分别较给药前减小63.4%(n=9)和72.7%(n=8)。采用EGTA作胞内钙的螯合剂,控制胞内钙螯合状态及游离钙浓度,可见大鼠冠状动脉和大脑中动脉SMC的BK电流随细胞内游离Ca~(2+)浓度增减而升降,具有钙敏感性。BK通道α亚单位在大鼠冠状动脉SMC上的表达为48±8.80%(与GAPDH的相对值),而在大脑中动脉SMC上的表达为80.9±8.37%(P<0.01, n=7);BK通道β1亚单位在此二动脉VSMC上的表达相当,与GAPDH的相对值分别为23.5±5.33%和22.7±5.27%(P>0.05, n=7)。
三、牛磺酸对大鼠冠状动脉和大脑中动脉Kv通道和BK通道电流的影响
1.牛磺酸对大鼠冠状动脉SMC的Kv通道电流的影响牛磺酸在较低浓度(1mM、3mM、10mM)时表现出对大鼠冠状动脉SMC的Kv通道电流的抑制作用,而在较高浓度(40mM、60mM)时表现为增强作用。
1mM、3mM和10mM牛磺酸对大鼠冠状动脉SMC上的Kv通道电流有显著抑制作用。在+20mV膜电位,1mM(n=11)、3mM(n=12)和10mM(n=11)牛磺酸使大鼠冠状动脉SMC的Kv通道电流分别降到5.28±0.23 pA/pF、4.47±0.26 pA/pF和4.19±0.28 pA/pF,与给药前(6.29±0.2 pA/pF)相比差异均显著(P<0.05)。在+20mV膜电位,3mM和10mM牛磺酸分别与1mM牛磺酸相比对电流的抑制具有显著性差异(P<0.05)。在+10mV膜电位,3mM和10mM牛磺酸使Kv通道电流从给药前的5.18±0.28 pA/pF分别降到3.71±0.29 pA/pF(P<0.05)和3.72±0.14 pA/pF(P<0.05)。在0mV膜电位,3mM和10mM牛磺酸使Kv通道电流从给药前的4.06±0.24 pA/pF分别降到2.96±0.18 pA/pF(P<0.05)和2.69±0.13 pA/pF(P<0.05)。
20mM(n=9)牛磺酸对各膜电位水平的Kv电流均与给药前无显著性差异(P>0.05)。
40mM(n=11)和60mM(n=11)牛磺酸对Kv电流具有增强作用。60mM牛磺酸在+10mV和+20mV膜电位的电流幅度分别为6.34±0.54 pA/pF和7.27±0.35 pA/pF,与给药前(5.18±0.28 pA/pF和6.29±0.2 pA/pF)相比均有显著性差异(P<0.05)。
2.牛磺酸对大鼠大脑中动脉SMC的Kv通道电流的影响
在大鼠大脑中动脉SMC,1mM(n=11)和3mM(n=11)牛磺酸降低Kv电流,且1mM牛磺酸在+10mV(4.18±0.21 pA/pF)和+20mV(4.29±0.18 pA/pF)膜电位对电流的抑制作用与给药前(4.99±0.31 pA/pF和5.94±0.26 pA/pF)相比差异显著(P<0.05)。10mM(n=12)牛磺酸与给药前相比对电流影响不明显(P>0.05)。20mM(n=11)、40mM(n=11)和60mM(n=11)牛磺酸浓度依赖性地增强Kv电流。与给药前(4.99±0.31 pA/pF和5.94±0.26 pA/pF)相比,40mM和60mM牛磺酸在+10mV(5.91±0.25 pA/pF和6.89±0.43 pA/pF)和+20mV(6.87±0.25 pA/pF和7.74±0.28 pA/pF)膜电位的电流幅度差异均具有显著性(P<0.05)。40mM和60mM牛磺酸之间对Kv电流的增强作用在+10mV和+20mV膜电位均具有显著性差异(P<0.05)。
3.1mM牛磺酸对大鼠VSMC的Kv通道作用的时间依赖性
大鼠冠状动脉和大脑中动脉SMC均显示,给予1mM牛磺酸后1min即可见对通道的抑制作用,给药后5min其作用明显,给药后10min与给药后5min水平相当。而洗脱后5min记录,通道电流可恢复到给药前的90%以上。
4.60mM牛磺酸对大鼠VSMC的BK通道电流的影响
在大鼠冠状动脉SMC,60mM牛磺酸(n=12)在低膜电位水平(-30mV ~ +10mV)表现为对BK通道电流的增强作用,在-20mV、-10mV和0mV膜电位时BK电流分别为:4.9±0.46 pA/pF、5.35±0.49 pA/pF和6.47±0.6 pA/pF,与给药前(3.29±0.31 pA/pF、3.82±0.35 pA/pF和4.42±0.38 pA/pF)相比差异均显著(P< 0.05);在高膜电位水平(+40mV-+80mV)表现为对BK通道电流的抑制作用,+60mV、+70mV和+80mV膜电位时BK电流分别为:16.26±1.51 pA/pF、19.81±1.84 pA/pF和24.04±2.24 pA/pF,与给药前(24.26±1.55 pA/pF、29.31±1.86 pA/pF和32.89±2.02 pA/pF)相比差异均显著(P< 0.05)。
在大鼠大脑中动脉SMC,60mM牛磺酸(n=9)表现为从-40mV到+40mV膜电位对BK通道电流的增强作用,而膜电位高于+40mV时对BK电流影响不明显,但未产生如冠状动脉平滑肌一样的抑制作用。60mM牛磺酸对BK通道电流的增强作用从-40mV到+10mV膜电位(4.65±1.35 pA/pF,5.18±1.32 pA/pF,5.98±1.4 pA/pF,6.48±1.51 pA/pF,8.2±1.5 pA/pF和10.05±1.43 pA/pF)均与给药前(1.33±0.25 pA/pF,1.74±0.37 pA/pF,1.94±0.46 pA/pF,2.34±0.5 pA/pF,2.84±0.59 pA/pF和3.54±0.52 pA/pF)有显著差异(P< 0.05)。
综合以上研究结果,Kv通道和BK通道在大鼠冠状动脉和大脑中动脉SMC上的功能和表达有一定的差异。Kv通道在大鼠冠状动脉SMC的激活与失活阈值均较大脑中动脉SMC高。大鼠冠状动脉SMC的Kv通道亚型主要是Kv1.2和Kv1.5亚型,而大脑中动脉SMC主要是Kv1.2、Kv1.5和Kv1.1亚型。BK通道α亚单位在大鼠大脑中动脉表达高于冠状动脉。对于其他通道亚型的表达,以及各通道亚型在蛋白水平的表达情况仍需进一步的研究,从而明确通道四聚体的分子构成,以证实这些通道亚型在生理及病理条件下在VSMC中确切的功能。
高浓度牛磺酸增强大鼠冠状动脉和大脑中动脉SMC的Kv通道电流;低浓度牛磺酸抑制大鼠冠状动脉和大脑中动脉SMC的Kv通道电流,且具有时间依赖性和可逆性。牛磺酸对大鼠冠状动脉和大脑中动脉SMC的BK通道的作用表现不一致。60mM牛磺酸在大鼠冠状动脉SMC表现为负膜电位增强而正膜电位抑制BK电流的双向作用;在大鼠大脑中动脉SMC则仅表现为低于+40mV膜电位增强BK电流作用,高于+40mV膜电位对BK电流无明显影响。牛磺酸对大鼠冠状动脉和大脑中动脉SMC的Kv通道和BK通道的作用具有一定的差异性和多样性,有待于深入研究。
Diversity of vascular smooth muscle cell (VSMC) function may be important in physiology and pathophysiology, allowing responses to vasodilators and vasoconstrictors to vary within or between vascular beds. Heterogeneity of VSMC is related to many kinds of recepts and ion channels distributed in their membrane. The normal of ion channels in VSMC plays pivotal role not only in the regulation of vascular smooth muscle contraction, but also in the maintenance of cellular excitation and membrane stability. Potassium channel, a family of widely expressed in VSMC, is required for maintenance of vascular tone by providing a repolarizing current to counteract and balance vasoconstrictive in?uences. Relatively a few potassium current may vary membrane potential severely. Potassium efflux from VSMC results in membrane repolarization and, by promoting the close for voltaged-dependent calcium channel, reduces intracellular free calcium concentration. As an important therapeutic target, some potassium channel regulators have been used clinically. However, as the most complicated classification (including functional and structural classification) among the membrane ion channels, various subfamilies of potassium channels result in the low selectivity and satisfaction of potassium channel regulators which had been used in clinical therapy at present.
Four main classes of K~+ channels have been described in VSMC. Two types of K~+ channels that are known to be prevalent and contribute significantly to the repolarization of VSMC membranes are the high-conductance Ca~(2+)-activated K~+ (BK) channels and the voltage-gated K~+ (Kv) channels. While most VSMC identified to date contain both BK and Kv channels, the expression of individual channel occurs in a tissue-specific manner, thereby providing functional specificity. The heterogeneity of channel subunits in VSMC ensures the appropriate responses of VSMC to diverse stimuli. Although BK channel and Kv channel had been studied in some arteries, the function and expression of them in the VSMC of different circulatory beds remain largely unknown. Vascular tone of coronary and cerebral artery is thought to regulate the blood flow of heart and brain. It is essential to establish the precise diversity of VSMC Kv channel and BK channel in coronary artery and middle cerebral artery in physiology, pathophysiology and pharmacology.
In the last few years, several laboratories have focused their attentions on the investigation of the cellular mechanisms underlying the effects of taurine, a well known sulfonic amino acid distributed widely in different tissues and abundantly in cardiovascular tissues. These concerns come from the multiple and surprising effects exerted by taurine involved in many physiological processes and the positive effects induced by supplement of taurine in some cardiovascular pathologic state.
The targets of taurine are complicated. The cellular mechanism of action of taurine is under investigation and appears to involve the interaction with several ion channels including potassium channels. Previous findings from our laboratory have demonstrated that taurine changes vasomotion by interaction with VSMC potassium channels.
In this study, VSMCs were freshly isolated from rat coronary arteries and middle cerebral arteries. Whole cell patch clamp and multicell RT-PCR techniques were used in the experiments to investigate the function and expression diversities of Kv channel and BK channel between coronary artery and middle cerebral artery. The changes of Kv currents and BK currents after taurine perfusion were recorded as well.
The results were as follows:
1. VSMC Kv channel of rat coronary artery and middle cerebral artery
The VSMC resting membrane potential of coronary artery and middle cerebral artery was -28.91±3.93 mV (n = 15) and -30.82±2.79 mV (n = 19), respectively. The capacitance was 17.66±0.95pF (n = 46) and 14.28±1.01pF (n = 42), respectively. Both of them had insignificant difference between coronary artery and middle cerebral artery (P> 0.05).
In rat coronary artery and middle cerebral artery, the VSMC Kv currents were voltage dependent, delayed rectifying and outward rectifying. The currents were activated about -40 mV and slowly inactivated during the 500ms recording period. 4-aminopyridine (4-AP; 3 mM), the classic Kv channel blocker, inhibited Kv currents significantly. Fitted to a conventional Boltzmann distribution equation, membrane potential producing half-maximal activation and the slope were -4.39±1.44 mV and 12.42±1.19 in coronary artery SMC, whereas -9.89±1.72 mV and 13.03±1.62 in middle cerebral artery SMC. Membrane potential producing half-maximal inactivation and the slope were -11.78±1.47 mV and 14.73±1.54 in coronary artery SMC, whereas -25.77±2.29 mV and 16.37±2.33 in middle cerebral artery SMC. The test potentials at which VSMC outward current was half-activated or half-inactivated were significantly shifted toward more positive membrane potentials in coronary artery compared with middle cerebral artery (P< 0.05, n=11), whereas slope was not different.
To identify the tetramer composition of Kv channels in rat VSMCs, multicell RT-PCR was used to screen for mRNAs encoding each of the five Kv subfamilies (Kv1.1, Kv1.2, Kv1.5, Kv1.6 and Kv2.1) which have delayed rectifier properties. Kv1.2 and Kv1.5 subfamilies were detected outstandingly both in rat coronary artery and middle cerebral artery SMC. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) served as a housekeeping gene. The expression of VSMC Kv1.2 relative to GAPDH was 94.4±7.59% and 98.1±5.08% (P>0.05, n=7) in coronary artery and middle cerebral artery, respectively. The amounts of VSMC Kv1.5 transcripts relative to GAPDH were 87.3±4.20% and 80.2±6.06% (P>0.05, n=7) in coronary artery and middle cerebral artery, respectively. VSMC Kv1.1 subfamily was detected moderately in rat middle cerebral artery and the amounts of Kv1.1 transcripts relative to GAPDH were 54.7±3.05%. However, in rat middle cerebral artery, a faint band of VSMC Kv1.1 expression was detected in one group and its amount of transcripts relative to GAPDH was 37.6%. The positive expression rate of VSMC Kv1.1 was 100% in middle cerebral artery and was 14.3% in coronary artery. VSMC Kv1.6 and Kv2.1 were silent both in rat coronary artery and middle cerebral artery.
2. VSMC BK channel of rat coronary artery and middle cerebral artery
The BK current recorded was voltage dependent, outward rectifying and intracellular calcium sensitive. The rectifying component of the outward current was inhibited by tetraethylammonium (TEA, 1 mM), a BK channel blocker.
The expression of VSMC BKαsubunit in rat coronary artery was less than which in middle cerebral artery. The amounts of VSMC BKαsubunit transcripts relative to GAPDH were 48±8.80% in coronary artery whereas it was 80.9±8.37% in middle cerebral artery (P<0.01, n=7). The level of VSMC BKβ1 transcripts in coronary artery was comparable to that in middle cerebral artery. The amounts of VSMC BKβ1 transcripts relative to GAPDH were 23.5±5.33% and 22.7±5.27% (P>0.05, n=7) in coronary artery and middle cerebral artery, respectively.
3. Effect of taurine on VSMC Kv current and BK current of rat coronary artery and middle cerebral artery
1) The effect of taurine on VSMC Kv current of rat coronary artery
Kv current was attenuated after perfusing 1mM, 3mM or 10mM taurine whereas it was increased by 40mM or 60mM taurine.
Kv current was decreased significantly by 1mM (n=11) taurine at +20 mV test potential (5.28±0.23 pA/pF) (P<0.05, compared with absence of taurine, 6.29±0.2 pA/pF).
Kv current was decreased significantly by 3mM (n=12), 10mM (n=11) taurine at +20 mV, +10 mV and 0 mV test potential (P<0.05, compared with absence of taurine). At +20 mV test potential, Kv current was 6.29±0.2pA/pF, 4.47±0.26 pA/pF and 4.19±0.28 pA/pF in absence of taurine or perfused with 3mM and 10mM taurine, respectively. At +10 mV test potential, Kv current was 5.18±0.28 pA/pF, 3.71±0.29 pA/pF and 3.72±0.14 pA/pF in absence of taurine or perfused with 3mM and 10mM taurine. At 0 mV test potential, Kv current was 6.29±0.2 pA/pF, 4.47±0.26 pA/pF and 4.19±0.28 pA/pF in absence of taurine or perfused with 3mM and 10mM taurine, respectively.
Kv current was not altered by 20mM taurine (P>0.05).
Kv current was increased by 40mM (n=11) and 60mM taurine (n=11). In the presence of 60mM taurine, Kv current was 6.34±0.54pA/pF and 7.27±0.35 pA/pF at +10 mV and +20 mV test potential that was augmented significantly compared with absence of taurine (5.18±0.28 pA/pF and 6.29±0.2 pA/pF, P<0.05).
2) The effect of taurine on VSMC Kv current of rat middle cerebral artery
Kv current was decreased by 1mM and 3mM taurine (n=11). At +10 mV and +20 mV test potential, Kv current was attenuated significantly by 1mM taurine (4.18±0.21 pA/pF and 4.29±0.18 pA/pF) (P<0.05, compared with absence of taurine, 4.99±0.31 pA/pF and 5.94±0.26 pA/pF).
Kv current was not altered by 10mM but was significantly increased by 20mM (n=11), 40mM (n=11) and 60mM (n=11) taurine concentration-dependently.
At +20 mV test potential, Kv current was 5.94±0.26 pA/pF, 6.87±0.25 pA/pF and 7.74±0.28 pA/pF in absence of taurine or perfusing 40mM and 60mM taurine, respectively. At +10 mV test potential, Kv current was 4.99±0.31 pA/pF, 5.91±0.25pA/pF and 6.89±0.43pA/pF in absence of taurine or perfused with 40mM and 60mM taurine, respectively. Campared with absence of taurine, the increase of Kv current in presence of taurine was significant (P<0.05). Campared with presence of 40mM taurine, the increase of Kv current in presence of 60mM taurine was significant at +20 mV and +10 mV test potential (P<0.05).
3) Time-dependent and reversible effect of taurine (1mM) on VSMC Kv current
Both in rat coronary artery and middle cerebral artery SMC, Kv current decreased after 1min taurine perfusion and attenuated significantly at 5min perfusion. The current recovered to about 90% level of pretaurine after washout taurine for 5min.
4) The effect of taurine (60mM) on VSMC BK current of rat coronary artery and middle cerebral artery
In rat coronary artery SMC, BK current was increased at negative membrane potential whereas it was attenuated at positive membrane potential after perfusion of taurine (60mM, n=12). Compared with absence of taurine (3.29±0.31pA/pf, 3.82±0.35pA/pF and 4.42±0.38pA/pF), BK current was augmented significantly to 4.9±0.46pA/pF, 5.35±0.49pA/pF and 6.47±0.6pA/pF at -20 mV, -10 mV and 0 mV test potential, respectively (P< 0.05). Compared with absence of taurine (24.26±1.55 pA/pF, 29.31±1.86 pA/pF and 32.89±2.02 pA/pF), BK current was attenuated significantly to 16.26±1.51pA/pF,19.81±1.84pA/pF and 24.04±2.24 pA/pF at +60 mV, +70 mV and +80 mV test potential, respectively (P< 0.05).
In rat middle cerebral artery SMC, BK current was increased significantly by taurine (60mM, n=9) from -40 mV to +10 mV test potential (P< 0.05, compared with absence of taurine) whereas was not altered above +40 mV test potential. From -40 mV to +10 mV test potential, BK current was 4.65±1.35 pA/pF, 5.18±1.32 pA/pF, 5.98±1.4pA/pF, 6.48±1.51 pA/pF, 8.2±1.5 pA/pF and 10.05±1.43 pA/pF in the presence of taurine. Absence of taurine, BK current was 1.33±0.25 pA/pF, 1.74±0.37 pA/pF, 1.94±0.46 pA/pF, 2.34±0.5 pA/pF, 2.84±0.59 pA/pF and 3.54±0.52 pA/pF from -40 mV to +10 mV test potential, respectively.
In summary, the function and expression of both VSMC Kv channel and BK channel are disparity between rat coronary artery and middle cerebral artery. The VSMC Kv channel thresholds of both activation and inactivation potential were significantly higher in coronary artery than in middle cerebral artery. Among the detected five Kv channel subfamilies, the member was Kv 1.2 and Kv1.5 subfamily in rat coronary artery SMC and the member was Kv 1.2, Kv1.5 and Kv 1.1 subfamily in rat middle cerebral artery SMC. The expression level of VSMC BKαsubunit was lower in coronary artery than in middle cerebral artery. The precise functions of these members under physiological and pathological conditions should be determined by further studies.
In the presence of taurine, Kv currents of rat coronary artery and middle cerebral artery SMC were augmented in higher concentration whereas inhibited in low concentration time-dependently and reversibly. As for the effects of taurine on BK channel, it was different between rat coronary artery and middle cerebral artery SMC. In coronary artery, VSMC BK currents were increased at negative membrane potential and decreased at positive membrane potential after taurine perfusion. In middle cerebral artery, taurine inhibited VSMC BK currents when the membrane potential was lower than +40 mV test potential and had no effect above +40 mV test potential. The effect of taurine on VSMC Kv current and BK current seems to be different and complicated in rat coronary artery and middle cerebral artery.
血管的反应性差异很大程度上与其血管平滑肌的多种受体及离子通道的分布类型和分布密度差异有密切关系。平滑肌细胞膜离子通道的活动正常是血管平滑肌兴奋收缩过程起始的关键,也是细胞的兴奋性与膜的稳定性得以维持的保证。K~+通道分布广泛,是调节血管平滑肌的收缩性与舒张性的主要离子通道。K~+通道电流微小的改变即可导致膜电位的很大变化,K~+经K~+通道跨细胞膜外流,细胞内电位降低,具有细胞膜稳定作用;细胞内电位降低还使电压依赖性钙通道的开放几率降低。K~+通道也因此成为治疗药物作用的重要靶标之一。K~+通道是目前已知的亚型(包括功能分型和结构分型)最多的一类细胞膜离子通道。一些通过影响K~+通道而起作用的药物已逐渐应用于临床。但目前临床上所用的K~+通道开放药对组织的选择性不够高,在组织特异性和功能多样性方面的一系列问题导致了药物在临床应用过程中的疗效不理想,甚至治疗失败。因此,了解各组织细胞K~+通道的分布情况和功能状态对研发新药、临床针对性地合理选择药物治疗极其重要。
大电导钙激活钾通道(high-conductance Ca~(2+)-activated K~+ channel, BK通道)和电压依赖性钾通道(voltage-dependent K~+ channel, Kv通道)是血管平滑肌细胞(vascular smooth muscle cell, VSMC)中普遍存在而且发挥优势作用的两类负载外向型钾电流的K~+通道。它们不同的通道亚型对细胞功能的调节和影响有所不同,保障了VSMC能对各种不同的刺激做出适当的反应。尽管Kv通道和BK通道在某些血管上的情况已经得到一些研究,但不同部位的血管上的各种K~+通道的表达和功能还远不清楚。心脏和脑的血液供应受心脏冠状动脉和脑血管平滑肌张力的影响。明确冠状动脉和大脑中动脉平滑肌细胞(SMC)的Kv通道和BK通道的分布差异有助于更好地掌握其生理作用,进一步了解其在不同生理和病理状态的动态调整;为明确Kv通道和BK通道在冠状动脉和大脑中动脉SMC介导多种血管活性物质的调节作用机制以及在多种病理过程中的作用奠定基础;也为研究开发以K~+通道为靶点的选择性高、副作用少的药物提供新思路、新线索。
牛磺酸是人和哺乳动物体内广泛存在的含硫β氨基酸,在心血管组织中含量最丰富。国内外大量研究表明,牛磺酸具有广泛生物学效应,是调节机体正常生理功能的重要物质,对多种情况下病理损伤具有积极的保护作用。在心血管功能异常的病理状态下补充牛磺酸可以对疾病的发生发展起积极防治作用。作为天然内源性物质,与一般化学合成药物不同的是,牛磺酸作用靶点十分广泛,可以通过多环节、多途径调节心血管系统功能,在防治心脑血管疾病方面展示出良好应用前景。许多研究表明,牛磺酸能够通过作用于包括K~+通道在内的多种离子通道而产生心血管效应,但到目前为止,国内外有关牛磺酸的研究多集中于心肌和骨骼肌。我室前期研究表明,牛磺酸对血管平滑肌收缩和舒张功能的影响与多种K~+通道有关。
本实验中,急性酶解分离成年雄性SD大鼠冠状动脉和大脑中动脉SMC,利用全细胞膜片钳技术记录Kv通道和BK通道的电流,应用单细胞反转录聚合酶链反应(RT-PCR)方法观察Kv通道和BK通道亚型在转录水平的基因表达情况;记录给予牛磺酸后Kv通道和BK通道电流的变化,了解牛磺酸对此两类通道的影响。
主要实验结果:
一、大鼠冠状动脉和大脑中动脉SMC的Kv通道
大鼠冠状动脉和大脑中动脉SMC的静息电位分别为-28.91±3.93 mV(n = 15)和-30.82±2.79 mV(n = 19)(P>0.05);膜电容分别为17.66±0.95 pF(n =46)和14.28±1.01 pF(n =42)(P>0.05)。
大鼠冠状动脉和大脑中动脉SMC的Kv电流约在膜电位正于-40mV开始呈电压依赖性的增加和外向整流特性;电流在除极后约10~20ms达到最大,500 ms内无明显失活,呈延迟整流特性。Boltzmann方程拟合得到大鼠冠状动脉和大脑中动脉SMC的Kv通道电流半激活电位(V1/2)分别为-4.39±1.44mV和-9.89±1.72 mV(P<0.05),斜率因子(K)分别为12.42±1.19和13.03±1.62(P>0.05);半失活电位(V1/2)分别为-11.78±1.47 mV和-25.77±2.29 mV(P<0.05),斜率因子(K)分别为14.73±1.54和16.37±2.33(P>0.05)。给予Kv通道阻断药4-氨基吡啶(4-aminopyridine, 4-AP, 3mM)后,通道电流分别被抑制61.2%(n=11)和62.7%(n=12)。
国外研究资料表明,Kv通道的Kv1.1、Kv1.2、Kv1.5、Kv1.6和Kv2.1亚型具有延迟整流特性,因此,我们采用单细胞RT-PCR方法检测了这些亚型在大鼠冠状动脉和大脑中动脉SMC转录水平的表达。结果表明,Kv1.2和Kv1.5亚型在大鼠冠状动脉和大脑中动脉SMC均有表达。取甘油醛-3-磷酸脱氢酶(GAPDH)作为内参照,计算各目的基因相对表达量。Kv1.2亚型在此二动脉VSMC的表达分别为94.4±7.59%和98.1±5.08%(P>0.05, n=7)。Kv1.5亚型在此二动脉VSMC的表达分别为87.3±4.20%和80.2±6.06%(P>0.05, n=7)。Kv1.1亚型在大脑中动脉有表达,其与GAPDH的相对值为54.7±3.05%,表达率100%;在冠状动脉偶有弱的表达,其与GAPDH的相对值为37.6%,表达率14.3%。Kv1.1亚型在此二动脉VSMC的表达有显著性差异(P<0.01, n=7)。Kv1.6和Kv2.1亚型在此二动脉VSMC均无表达。
二、大鼠冠状动脉和大脑中动脉SMC的BK通道
大鼠冠状动脉和大脑中动脉SMC的BK电流呈电压依赖性,在除极大于+10mV开始呈明显外向整流特性;浴槽液中给予BK通道阻断药四乙胺(tetraethylammonium, TEA,1mM)后通道电流明显降低,峰值电流分别较给药前减小63.4%(n=9)和72.7%(n=8)。采用EGTA作胞内钙的螯合剂,控制胞内钙螯合状态及游离钙浓度,可见大鼠冠状动脉和大脑中动脉SMC的BK电流随细胞内游离Ca~(2+)浓度增减而升降,具有钙敏感性。BK通道α亚单位在大鼠冠状动脉SMC上的表达为48±8.80%(与GAPDH的相对值),而在大脑中动脉SMC上的表达为80.9±8.37%(P<0.01, n=7);BK通道β1亚单位在此二动脉VSMC上的表达相当,与GAPDH的相对值分别为23.5±5.33%和22.7±5.27%(P>0.05, n=7)。
三、牛磺酸对大鼠冠状动脉和大脑中动脉Kv通道和BK通道电流的影响
1.牛磺酸对大鼠冠状动脉SMC的Kv通道电流的影响牛磺酸在较低浓度(1mM、3mM、10mM)时表现出对大鼠冠状动脉SMC的Kv通道电流的抑制作用,而在较高浓度(40mM、60mM)时表现为增强作用。
1mM、3mM和10mM牛磺酸对大鼠冠状动脉SMC上的Kv通道电流有显著抑制作用。在+20mV膜电位,1mM(n=11)、3mM(n=12)和10mM(n=11)牛磺酸使大鼠冠状动脉SMC的Kv通道电流分别降到5.28±0.23 pA/pF、4.47±0.26 pA/pF和4.19±0.28 pA/pF,与给药前(6.29±0.2 pA/pF)相比差异均显著(P<0.05)。在+20mV膜电位,3mM和10mM牛磺酸分别与1mM牛磺酸相比对电流的抑制具有显著性差异(P<0.05)。在+10mV膜电位,3mM和10mM牛磺酸使Kv通道电流从给药前的5.18±0.28 pA/pF分别降到3.71±0.29 pA/pF(P<0.05)和3.72±0.14 pA/pF(P<0.05)。在0mV膜电位,3mM和10mM牛磺酸使Kv通道电流从给药前的4.06±0.24 pA/pF分别降到2.96±0.18 pA/pF(P<0.05)和2.69±0.13 pA/pF(P<0.05)。
20mM(n=9)牛磺酸对各膜电位水平的Kv电流均与给药前无显著性差异(P>0.05)。
40mM(n=11)和60mM(n=11)牛磺酸对Kv电流具有增强作用。60mM牛磺酸在+10mV和+20mV膜电位的电流幅度分别为6.34±0.54 pA/pF和7.27±0.35 pA/pF,与给药前(5.18±0.28 pA/pF和6.29±0.2 pA/pF)相比均有显著性差异(P<0.05)。
2.牛磺酸对大鼠大脑中动脉SMC的Kv通道电流的影响
在大鼠大脑中动脉SMC,1mM(n=11)和3mM(n=11)牛磺酸降低Kv电流,且1mM牛磺酸在+10mV(4.18±0.21 pA/pF)和+20mV(4.29±0.18 pA/pF)膜电位对电流的抑制作用与给药前(4.99±0.31 pA/pF和5.94±0.26 pA/pF)相比差异显著(P<0.05)。10mM(n=12)牛磺酸与给药前相比对电流影响不明显(P>0.05)。20mM(n=11)、40mM(n=11)和60mM(n=11)牛磺酸浓度依赖性地增强Kv电流。与给药前(4.99±0.31 pA/pF和5.94±0.26 pA/pF)相比,40mM和60mM牛磺酸在+10mV(5.91±0.25 pA/pF和6.89±0.43 pA/pF)和+20mV(6.87±0.25 pA/pF和7.74±0.28 pA/pF)膜电位的电流幅度差异均具有显著性(P<0.05)。40mM和60mM牛磺酸之间对Kv电流的增强作用在+10mV和+20mV膜电位均具有显著性差异(P<0.05)。
3.1mM牛磺酸对大鼠VSMC的Kv通道作用的时间依赖性
大鼠冠状动脉和大脑中动脉SMC均显示,给予1mM牛磺酸后1min即可见对通道的抑制作用,给药后5min其作用明显,给药后10min与给药后5min水平相当。而洗脱后5min记录,通道电流可恢复到给药前的90%以上。
4.60mM牛磺酸对大鼠VSMC的BK通道电流的影响
在大鼠冠状动脉SMC,60mM牛磺酸(n=12)在低膜电位水平(-30mV ~ +10mV)表现为对BK通道电流的增强作用,在-20mV、-10mV和0mV膜电位时BK电流分别为:4.9±0.46 pA/pF、5.35±0.49 pA/pF和6.47±0.6 pA/pF,与给药前(3.29±0.31 pA/pF、3.82±0.35 pA/pF和4.42±0.38 pA/pF)相比差异均显著(P< 0.05);在高膜电位水平(+40mV-+80mV)表现为对BK通道电流的抑制作用,+60mV、+70mV和+80mV膜电位时BK电流分别为:16.26±1.51 pA/pF、19.81±1.84 pA/pF和24.04±2.24 pA/pF,与给药前(24.26±1.55 pA/pF、29.31±1.86 pA/pF和32.89±2.02 pA/pF)相比差异均显著(P< 0.05)。
在大鼠大脑中动脉SMC,60mM牛磺酸(n=9)表现为从-40mV到+40mV膜电位对BK通道电流的增强作用,而膜电位高于+40mV时对BK电流影响不明显,但未产生如冠状动脉平滑肌一样的抑制作用。60mM牛磺酸对BK通道电流的增强作用从-40mV到+10mV膜电位(4.65±1.35 pA/pF,5.18±1.32 pA/pF,5.98±1.4 pA/pF,6.48±1.51 pA/pF,8.2±1.5 pA/pF和10.05±1.43 pA/pF)均与给药前(1.33±0.25 pA/pF,1.74±0.37 pA/pF,1.94±0.46 pA/pF,2.34±0.5 pA/pF,2.84±0.59 pA/pF和3.54±0.52 pA/pF)有显著差异(P< 0.05)。
综合以上研究结果,Kv通道和BK通道在大鼠冠状动脉和大脑中动脉SMC上的功能和表达有一定的差异。Kv通道在大鼠冠状动脉SMC的激活与失活阈值均较大脑中动脉SMC高。大鼠冠状动脉SMC的Kv通道亚型主要是Kv1.2和Kv1.5亚型,而大脑中动脉SMC主要是Kv1.2、Kv1.5和Kv1.1亚型。BK通道α亚单位在大鼠大脑中动脉表达高于冠状动脉。对于其他通道亚型的表达,以及各通道亚型在蛋白水平的表达情况仍需进一步的研究,从而明确通道四聚体的分子构成,以证实这些通道亚型在生理及病理条件下在VSMC中确切的功能。
高浓度牛磺酸增强大鼠冠状动脉和大脑中动脉SMC的Kv通道电流;低浓度牛磺酸抑制大鼠冠状动脉和大脑中动脉SMC的Kv通道电流,且具有时间依赖性和可逆性。牛磺酸对大鼠冠状动脉和大脑中动脉SMC的BK通道的作用表现不一致。60mM牛磺酸在大鼠冠状动脉SMC表现为负膜电位增强而正膜电位抑制BK电流的双向作用;在大鼠大脑中动脉SMC则仅表现为低于+40mV膜电位增强BK电流作用,高于+40mV膜电位对BK电流无明显影响。牛磺酸对大鼠冠状动脉和大脑中动脉SMC的Kv通道和BK通道的作用具有一定的差异性和多样性,有待于深入研究。
Diversity of vascular smooth muscle cell (VSMC) function may be important in physiology and pathophysiology, allowing responses to vasodilators and vasoconstrictors to vary within or between vascular beds. Heterogeneity of VSMC is related to many kinds of recepts and ion channels distributed in their membrane. The normal of ion channels in VSMC plays pivotal role not only in the regulation of vascular smooth muscle contraction, but also in the maintenance of cellular excitation and membrane stability. Potassium channel, a family of widely expressed in VSMC, is required for maintenance of vascular tone by providing a repolarizing current to counteract and balance vasoconstrictive in?uences. Relatively a few potassium current may vary membrane potential severely. Potassium efflux from VSMC results in membrane repolarization and, by promoting the close for voltaged-dependent calcium channel, reduces intracellular free calcium concentration. As an important therapeutic target, some potassium channel regulators have been used clinically. However, as the most complicated classification (including functional and structural classification) among the membrane ion channels, various subfamilies of potassium channels result in the low selectivity and satisfaction of potassium channel regulators which had been used in clinical therapy at present.
Four main classes of K~+ channels have been described in VSMC. Two types of K~+ channels that are known to be prevalent and contribute significantly to the repolarization of VSMC membranes are the high-conductance Ca~(2+)-activated K~+ (BK) channels and the voltage-gated K~+ (Kv) channels. While most VSMC identified to date contain both BK and Kv channels, the expression of individual channel occurs in a tissue-specific manner, thereby providing functional specificity. The heterogeneity of channel subunits in VSMC ensures the appropriate responses of VSMC to diverse stimuli. Although BK channel and Kv channel had been studied in some arteries, the function and expression of them in the VSMC of different circulatory beds remain largely unknown. Vascular tone of coronary and cerebral artery is thought to regulate the blood flow of heart and brain. It is essential to establish the precise diversity of VSMC Kv channel and BK channel in coronary artery and middle cerebral artery in physiology, pathophysiology and pharmacology.
In the last few years, several laboratories have focused their attentions on the investigation of the cellular mechanisms underlying the effects of taurine, a well known sulfonic amino acid distributed widely in different tissues and abundantly in cardiovascular tissues. These concerns come from the multiple and surprising effects exerted by taurine involved in many physiological processes and the positive effects induced by supplement of taurine in some cardiovascular pathologic state.
The targets of taurine are complicated. The cellular mechanism of action of taurine is under investigation and appears to involve the interaction with several ion channels including potassium channels. Previous findings from our laboratory have demonstrated that taurine changes vasomotion by interaction with VSMC potassium channels.
In this study, VSMCs were freshly isolated from rat coronary arteries and middle cerebral arteries. Whole cell patch clamp and multicell RT-PCR techniques were used in the experiments to investigate the function and expression diversities of Kv channel and BK channel between coronary artery and middle cerebral artery. The changes of Kv currents and BK currents after taurine perfusion were recorded as well.
The results were as follows:
1. VSMC Kv channel of rat coronary artery and middle cerebral artery
The VSMC resting membrane potential of coronary artery and middle cerebral artery was -28.91±3.93 mV (n = 15) and -30.82±2.79 mV (n = 19), respectively. The capacitance was 17.66±0.95pF (n = 46) and 14.28±1.01pF (n = 42), respectively. Both of them had insignificant difference between coronary artery and middle cerebral artery (P> 0.05).
In rat coronary artery and middle cerebral artery, the VSMC Kv currents were voltage dependent, delayed rectifying and outward rectifying. The currents were activated about -40 mV and slowly inactivated during the 500ms recording period. 4-aminopyridine (4-AP; 3 mM), the classic Kv channel blocker, inhibited Kv currents significantly. Fitted to a conventional Boltzmann distribution equation, membrane potential producing half-maximal activation and the slope were -4.39±1.44 mV and 12.42±1.19 in coronary artery SMC, whereas -9.89±1.72 mV and 13.03±1.62 in middle cerebral artery SMC. Membrane potential producing half-maximal inactivation and the slope were -11.78±1.47 mV and 14.73±1.54 in coronary artery SMC, whereas -25.77±2.29 mV and 16.37±2.33 in middle cerebral artery SMC. The test potentials at which VSMC outward current was half-activated or half-inactivated were significantly shifted toward more positive membrane potentials in coronary artery compared with middle cerebral artery (P< 0.05, n=11), whereas slope was not different.
To identify the tetramer composition of Kv channels in rat VSMCs, multicell RT-PCR was used to screen for mRNAs encoding each of the five Kv subfamilies (Kv1.1, Kv1.2, Kv1.5, Kv1.6 and Kv2.1) which have delayed rectifier properties. Kv1.2 and Kv1.5 subfamilies were detected outstandingly both in rat coronary artery and middle cerebral artery SMC. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) served as a housekeeping gene. The expression of VSMC Kv1.2 relative to GAPDH was 94.4±7.59% and 98.1±5.08% (P>0.05, n=7) in coronary artery and middle cerebral artery, respectively. The amounts of VSMC Kv1.5 transcripts relative to GAPDH were 87.3±4.20% and 80.2±6.06% (P>0.05, n=7) in coronary artery and middle cerebral artery, respectively. VSMC Kv1.1 subfamily was detected moderately in rat middle cerebral artery and the amounts of Kv1.1 transcripts relative to GAPDH were 54.7±3.05%. However, in rat middle cerebral artery, a faint band of VSMC Kv1.1 expression was detected in one group and its amount of transcripts relative to GAPDH was 37.6%. The positive expression rate of VSMC Kv1.1 was 100% in middle cerebral artery and was 14.3% in coronary artery. VSMC Kv1.6 and Kv2.1 were silent both in rat coronary artery and middle cerebral artery.
2. VSMC BK channel of rat coronary artery and middle cerebral artery
The BK current recorded was voltage dependent, outward rectifying and intracellular calcium sensitive. The rectifying component of the outward current was inhibited by tetraethylammonium (TEA, 1 mM), a BK channel blocker.
The expression of VSMC BKαsubunit in rat coronary artery was less than which in middle cerebral artery. The amounts of VSMC BKαsubunit transcripts relative to GAPDH were 48±8.80% in coronary artery whereas it was 80.9±8.37% in middle cerebral artery (P<0.01, n=7). The level of VSMC BKβ1 transcripts in coronary artery was comparable to that in middle cerebral artery. The amounts of VSMC BKβ1 transcripts relative to GAPDH were 23.5±5.33% and 22.7±5.27% (P>0.05, n=7) in coronary artery and middle cerebral artery, respectively.
3. Effect of taurine on VSMC Kv current and BK current of rat coronary artery and middle cerebral artery
1) The effect of taurine on VSMC Kv current of rat coronary artery
Kv current was attenuated after perfusing 1mM, 3mM or 10mM taurine whereas it was increased by 40mM or 60mM taurine.
Kv current was decreased significantly by 1mM (n=11) taurine at +20 mV test potential (5.28±0.23 pA/pF) (P<0.05, compared with absence of taurine, 6.29±0.2 pA/pF).
Kv current was decreased significantly by 3mM (n=12), 10mM (n=11) taurine at +20 mV, +10 mV and 0 mV test potential (P<0.05, compared with absence of taurine). At +20 mV test potential, Kv current was 6.29±0.2pA/pF, 4.47±0.26 pA/pF and 4.19±0.28 pA/pF in absence of taurine or perfused with 3mM and 10mM taurine, respectively. At +10 mV test potential, Kv current was 5.18±0.28 pA/pF, 3.71±0.29 pA/pF and 3.72±0.14 pA/pF in absence of taurine or perfused with 3mM and 10mM taurine. At 0 mV test potential, Kv current was 6.29±0.2 pA/pF, 4.47±0.26 pA/pF and 4.19±0.28 pA/pF in absence of taurine or perfused with 3mM and 10mM taurine, respectively.
Kv current was not altered by 20mM taurine (P>0.05).
Kv current was increased by 40mM (n=11) and 60mM taurine (n=11). In the presence of 60mM taurine, Kv current was 6.34±0.54pA/pF and 7.27±0.35 pA/pF at +10 mV and +20 mV test potential that was augmented significantly compared with absence of taurine (5.18±0.28 pA/pF and 6.29±0.2 pA/pF, P<0.05).
2) The effect of taurine on VSMC Kv current of rat middle cerebral artery
Kv current was decreased by 1mM and 3mM taurine (n=11). At +10 mV and +20 mV test potential, Kv current was attenuated significantly by 1mM taurine (4.18±0.21 pA/pF and 4.29±0.18 pA/pF) (P<0.05, compared with absence of taurine, 4.99±0.31 pA/pF and 5.94±0.26 pA/pF).
Kv current was not altered by 10mM but was significantly increased by 20mM (n=11), 40mM (n=11) and 60mM (n=11) taurine concentration-dependently.
At +20 mV test potential, Kv current was 5.94±0.26 pA/pF, 6.87±0.25 pA/pF and 7.74±0.28 pA/pF in absence of taurine or perfusing 40mM and 60mM taurine, respectively. At +10 mV test potential, Kv current was 4.99±0.31 pA/pF, 5.91±0.25pA/pF and 6.89±0.43pA/pF in absence of taurine or perfused with 40mM and 60mM taurine, respectively. Campared with absence of taurine, the increase of Kv current in presence of taurine was significant (P<0.05). Campared with presence of 40mM taurine, the increase of Kv current in presence of 60mM taurine was significant at +20 mV and +10 mV test potential (P<0.05).
3) Time-dependent and reversible effect of taurine (1mM) on VSMC Kv current
Both in rat coronary artery and middle cerebral artery SMC, Kv current decreased after 1min taurine perfusion and attenuated significantly at 5min perfusion. The current recovered to about 90% level of pretaurine after washout taurine for 5min.
4) The effect of taurine (60mM) on VSMC BK current of rat coronary artery and middle cerebral artery
In rat coronary artery SMC, BK current was increased at negative membrane potential whereas it was attenuated at positive membrane potential after perfusion of taurine (60mM, n=12). Compared with absence of taurine (3.29±0.31pA/pf, 3.82±0.35pA/pF and 4.42±0.38pA/pF), BK current was augmented significantly to 4.9±0.46pA/pF, 5.35±0.49pA/pF and 6.47±0.6pA/pF at -20 mV, -10 mV and 0 mV test potential, respectively (P< 0.05). Compared with absence of taurine (24.26±1.55 pA/pF, 29.31±1.86 pA/pF and 32.89±2.02 pA/pF), BK current was attenuated significantly to 16.26±1.51pA/pF,19.81±1.84pA/pF and 24.04±2.24 pA/pF at +60 mV, +70 mV and +80 mV test potential, respectively (P< 0.05).
In rat middle cerebral artery SMC, BK current was increased significantly by taurine (60mM, n=9) from -40 mV to +10 mV test potential (P< 0.05, compared with absence of taurine) whereas was not altered above +40 mV test potential. From -40 mV to +10 mV test potential, BK current was 4.65±1.35 pA/pF, 5.18±1.32 pA/pF, 5.98±1.4pA/pF, 6.48±1.51 pA/pF, 8.2±1.5 pA/pF and 10.05±1.43 pA/pF in the presence of taurine. Absence of taurine, BK current was 1.33±0.25 pA/pF, 1.74±0.37 pA/pF, 1.94±0.46 pA/pF, 2.34±0.5 pA/pF, 2.84±0.59 pA/pF and 3.54±0.52 pA/pF from -40 mV to +10 mV test potential, respectively.
In summary, the function and expression of both VSMC Kv channel and BK channel are disparity between rat coronary artery and middle cerebral artery. The VSMC Kv channel thresholds of both activation and inactivation potential were significantly higher in coronary artery than in middle cerebral artery. Among the detected five Kv channel subfamilies, the member was Kv 1.2 and Kv1.5 subfamily in rat coronary artery SMC and the member was Kv 1.2, Kv1.5 and Kv 1.1 subfamily in rat middle cerebral artery SMC. The expression level of VSMC BKαsubunit was lower in coronary artery than in middle cerebral artery. The precise functions of these members under physiological and pathological conditions should be determined by further studies.
In the presence of taurine, Kv currents of rat coronary artery and middle cerebral artery SMC were augmented in higher concentration whereas inhibited in low concentration time-dependently and reversibly. As for the effects of taurine on BK channel, it was different between rat coronary artery and middle cerebral artery SMC. In coronary artery, VSMC BK currents were increased at negative membrane potential and decreased at positive membrane potential after taurine perfusion. In middle cerebral artery, taurine inhibited VSMC BK currents when the membrane potential was lower than +40 mV test potential and had no effect above +40 mV test potential. The effect of taurine on VSMC Kv current and BK current seems to be different and complicated in rat coronary artery and middle cerebral artery.
引文
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