黄连素对结肠平滑肌细胞膜钙激活钾通道和延迟整流钾通道的影响

目的　研究黄连素对结肠平滑肌细胞膜钙离子激活钾通道 (IK(Ca) )和延迟整流钾通道 (IK(V) )的影响以初步探讨其治疗运动性腹泻的机制。方法　酶解法急性分离单个豚鼠结肠平滑肌细胞 ,运用膜片钳方法检测 10、5 0、10 0 μmol·L-1的黄连素对结肠平滑肌细胞膜IK(Ca) 和IK(V) 的影响。结果　 10、5 0、10 0 μmol·L-1的黄连素可抑制豚鼠单个结肠平滑肌细胞膜IK(Ca) (P <0 0 1) ,当阶跃刺激为 +80mV时 ,其IK(Ca) 分别为生理盐水对照组的 (6 8 2 0± 5 17) % ,(5 5 89± 1 6 1) % ,(4 8 0 8± 2 4 5 ) % (P <0 0 1) ;10、5 0、10 0 μmol·L-1的黄连素可抑制豚鼠单个结肠平滑肌细胞膜IK(V) (P <0 0 1) ,当阶跃刺激为 +80mV时 ,其IK(V) 分别为生理盐水对照组的 (77 0 6± 6 4 2 ) % ,(6 8 6 7± 6 79) % ,(6 1 0 7±7 72 ) % (P <0 0 1)。结论　Ber能抑制豚鼠结肠平滑肌钙离子激活钾通道和延迟整流钾通道的开放 ,这可能是其治疗运动性腹泻的机制之一。     

大电导钙激活钾通道在平滑肌细胞中的调节机制

大电导钙激活钾通道由形成孔道的α亚基及具有调节作用的β亚基组成,因其电导大,对调节平滑肌细胞功能起重要作用,从而成为近年来研究的热点。大电导钙激活钾通道的调节机制多种多样,现对其近年来调节机制的研究进展作一综述。     

葛根素对大鼠肾动脉血管平滑肌细胞大电导钙激活钾通道电流的影响

<正>葛根素是从豆科植物野葛或甘葛根的干燥根中提取的单体,其化学名为8-β-D-葡萄吡喃糖-4,7-二羟基异黄酮。据研究报道,葛根素具有多种药理学活性,包括降低血压[1]、舒张大鼠冠状动脉[2]、改善急性心肌缺血[3]、清除氧自由基[4]等,但关于葛根素对大鼠肾动脉的作用却未见报道。本实验主要研究葛根素对大鼠离体肾动脉的肌源性作用,葛根素对大鼠肾动脉血管平滑肌细胞大电导钙激活钾通道(BKCa)电流的作     

阿托伐他汀对自发性高血压大鼠肠系膜动脉平滑肌细胞大电导钙激活钾通道的影响

目的:探讨阿托伐他汀对自发性高血压大鼠(SHR)动脉血压及肠系膜动脉平滑肌细胞大电导钙激活钾通道(large-conductance calcium-activated potassium channel,BKCa,MaxiK)的影响。方法:选取雄性9周龄SHR12只,随机分为阿托伐他汀组(AVT组)、蒸馏水组(DW组),每组6只,以Wistar-Kyoto大鼠为对照组(WKY组)(n=6)。AVT组以AVT加适量蒸馏水灌胃10周(50 mg.kg-1.d-1)。观察给药前后大鼠腹主动脉血压的变化,测定大鼠血清TC、TG及高密度脂蛋白胆固醇(HDL-C)含量。利用膜片钳全细胞模式记录肠系膜动脉平滑肌细胞钾电流、用四乙胺阻断大电导钙激活钾通道后的电流、膜电容,以计算BKCa电流值、电流密度。结果:AVT组腹主动脉血压明显低于DW组[(171±8)mmHgvs(190±10)mmHg,P<0.01](1 mmHg=0.133 kPa);与DW组相比,AVT组肠系膜动脉平滑肌细胞BKCa电容减低[(23.8±2.6)pFvs(30.0±2.5)pF,P<0.01]而电流密度则显著高于DW组[(13.2±1.2)pA...     

糖尿病大鼠冠状动脉平滑肌细胞中大电导钙离子激活钾通道电流和钙离子浓度变化

目的探讨糖尿病大鼠冠状动脉平滑肌细胞中大电导钙离子激活钾通道(BK通道)电流及钙离子浓度的变化。方法 40只SD大鼠随机均分为正常对照组(A组)和糖尿病组(B组)。采用链脲霉素腹腔内注射建立糖尿病大鼠模型,酶消化法分离冠状动脉平滑肌细胞,全细胞膜片钳实验和荧光测定方法分别检测冠状动脉平滑肌细胞BK通道电流和钙离子浓度。结果与A组相比,当刺激电压大于60mV时,B组冠状动脉平滑肌细胞BK通道电流密度明显下降(P<0.05);A组冠状动脉平滑肌细胞内钙离子浓度明显低于B组[(103±23)nmol/L vs.(193±22)nmol/L](P<0.05)。结论冠状动脉平滑肌细胞中BK通道电流下降及细胞内钙离子浓度升高可能是糖尿病冠状动脉功能损伤的重要原因。     

动脉粥样硬化兔血管平滑肌细胞大电导钙激活钾通道NO直接激活研究

目的:研究NO是否可以直接激活VSMC BKCa以及AS时血管平滑肌细胞BKCa对NO的反应性是否发生改变,为治疗AS提供新思路.方法:3月龄新西兰兔20只,雌雄各半,随机分为实验组(AS组)和对照组(正常组),每组10只.实验组兔喂高脂饲料8周以建立AS模型,对照组兔喂普通饲料8周.以ISMN为外源性NO供体,应用单通道膜片钳技术检测NO对兔VSMC BKCa的直接激活作用及两组兔对NO的反应性差别.结果:在inside-out patches(膜电位 40 mV)下,ISMN显著增加BKCa通道开放事件数,缩短Tc,增加Po,且具有浓度依赖性.10-6 mol·L-1的ISMN(膜电位 40 mV)可分别使AS组和对照组BKCa的Po增加4.917±1.475倍和9.616±3.227倍(P＜0.01).结论:NO对VSMCBKCa有直接激活作用,但在AS时VSMC BKCa对NO的激活敏感性是降低的.     

大电导钙激活钾通道对平滑肌的调控作用研究进展

钾通道是组织器官中的一种重要通道,几乎所有的组织中都有该通道的分布,并且它在调节细胞功能方面起着极其重要的作用,例如动作电位的形成和信号传导等。大电导钙激活钾通道(BKca/Maxi K)以其广泛的分布,以及参与调节多种细胞功能吸引了更多研究者的关注。BKca/Maxi K的激活可导致细胞膜的超极化,从而抑制电压依赖性钙通道的激活,抑制钙离子内流,引起平滑肌舒张。近年来研究发现,BKca/Maxi K的激活、失活和变异与多种疾病的发病有关,BKca/Maxi K对心血管平滑肌、子宫平滑肌、呼吸道平滑肌和阴茎勃起等具有调控作用,尤其是其基因疗法对阴茎勃起障碍的治疗逐渐显现出较大的优势。     

大电导钙激活钾通道对血管平滑肌的作用

大电导钙激活钾通道(BKCa)是血管平滑肌细胞膜电位的主要离子通道,在血管的舒缩及调节细胞功能方面发挥着重要的作用。BKCa通道的激活可使细胞膜发生超极化,从而抑制电压依赖性钙通道的激活及抑制钙离子内流,导致平滑肌舒张。近年来研究发现,BKCa通道的激活、失活和变异与多种疾病的发病有关,如BKCa对糖尿病、失血性休克、高血压、妊娠期高血压疾病等具有调控作用。     

N-甲基小檗胺对大鼠肠系膜动脉平滑肌细胞钙激活钾电流的作用

目的研究N-甲基小檗胺对大鼠肠系膜阻力血管平滑肌细胞钙激活钾电流(IK·Ca)的作用,以阐明其降血压的离子机制。方法膜片钳技术全细胞记录模式记录IK·Ca。结果指令电位为+60mV时,N-甲基小檗胺0·1,1,10μmol·L-1使IK·Ca幅值分别由给药前的(33·6±2·0)pA·pF-1增至给药后的(35·7±1·9),(42·9±2·7)和(59·4±1·4)pA·pF-1(n=6,P<0·01)。结论N-甲基小檗胺可以增加大鼠肠系膜阻力血管平滑肌细胞钙激活钾电流,这可能是其降压机制之一。     

粉防己碱对大鼠肺动脉平滑肌细胞小电导钙激活钾通道的作用

目的　研究粉防己碱 (Tet)对大鼠肺动脉平滑肌细胞小电导钙激活钾通道 (SKCa)的作用。方法　内面朝外膜片箝单通道记录法。结果　Tet 7 5 μmol·L-1对电导值为 10pS的SKCa无明显影响 ,15 μmol·L-1可增加SKCa开放的概率 ,改变通道的开放和关闭模式 ,30 μmol·L-1降低通道的开放概率 ,通道开放以短暂簇状为主。结论　Tet对肺动脉平滑肌SKCa的作用与Tet的浓度有关 ,合适浓度下可增加通道的开放 ,K+ 外流增多 ,与Tet降低肺动脉张力有关。     

The Na(+)/Ca(2+) exchanger inhibitor KB-R7943 activates large-conductance Ca(2+)-activated K(+) channels in endothelial and vascular smooth muscle cells

The effect of the selective inhibitor of Na(+)/Ca(2+) exchanger (NCX), KB-R7943, on large-conductance Ca(2+)-activated K(+) (BK(Ca)) channels was examined in cultured human umbilical vein endothelial cells (HUVECs) and freshly isolated mouse aortic smooth muscle cells (MASMCs). In voltage-clamped cells, KB-R7943 reversibly activated BK(Ca) currents in HUVECs and MASMCs. The EC(50) of KB-R7943 for BK(Ca) current activation in HUVECs was determined to be 6.78+/-0.7 microM. In inside-out and outside-out patches, KB-R7943 markedly increased BK(Ca) channel activity and slightly decreased single channel current amplitudes. In inside-out patches, KB-R7943 shifted the relationship between [Ca(2+)](i) and open probability (P(o)) to the left; the [Ca(2+)](i) required to evoke half-maximal activation changed from 1220+/-68 nM (in the absence of KB-R7943) to 620+/-199 nM (in the presence of 10 microM KB-R7943). In addition, KB-R7943 shifted the relationship between membrane potential and P(o) to the left; the membrane potential to evoke half-maximal activation changed from 76.86+/-1.09 mV (in the absence of KB-R7943) to 49.62+/-2.55 mV (in the presence of 10 microM KB-R7943). In conclusion, KB-R7943 was found to act as a potent BK(Ca) channel activator, which increases the sensitivity of BK(Ca) channels to cytosolic free Ca(2+) and membrane potential, and thereby BK(Ca) channel activity. These results should be considered when KB-R7943 is used as NCX blocker.     

二十二碳六稀酸对大鼠冠状动脉平滑肌细胞BKCa单通道的作用

目的 探讨二十二碳六烯酸(DHA)对大鼠冠状动脉平滑肌细胞(CASMCs)大电导钙激活性钾通道(BKca)的影响.方法 采用酶消化法获得大鼠CASMCs,用膜片钳技术以单通道内膜向外模式记录在跨膜电位60 mV下,加入0、10、20、30、40、50、60、70和80μmol/L DHA后,大鼠CASNXs的BKca单通道开放概率(Po)、电流幅度值(Am)、平均开放时间(To)、平均关闭时间(Tc)的变化.结果 在跨膜电位60mV时,当DHA浓度＞10/μmol/L时,BKca通道的Po增大,且呈浓度依赖性,Po从(0.072±0.003)(0 μmol/L DHA,n=6)增至(0.606±0.089)(80μmol/L DHA,μ=10)(P＜0.05),EC50是(36.11±0.08)μmol/L,当DHA浓度＜10μmol/L时,Po增加不明显;加入不同浓度的DHA时,对通道Am及To的作用不明显,通道Tc明显缩短,Tc由(492.91±42.12)ms(0μmol/L DHA,n=6)缩短至(72.39±6.97)ms(80μmol/L DHA,n=10)(P＜0.05).结论 DHA能直接激活BKca通道而产生舒张血管、增加血流量的效应.     

Effects of docosahexaenoic acid on large-conductance Ca2+-activated K+ channels and voltage-dependent K+ channels in rat coronary artery smooth muscle cells

Aim:

To investigate the effects of docosahexaenoic acid (DHA) on large-conductance Ca²⁺-activated K⁺(BK_Ca) channels and voltage-dependent K⁺ (K_V) channels in rat coronary artery smooth muscle cells (CASMCs).

Methods:

Rat CASMCs were isolated by an enzyme digestion method. BK_Ca and K_V currents in individual CASMCs were recorded by the patch-clamp technique in a whole-cell configuration at room temperature. Effects of DHA on BK_Ca and K_V channels were observed when it was applied at 10, 20, 30, 40, 50, 60, 70, and 80 μmol/L.

Results:

When DHA concentrations were greater than 10 μmol/L, BK_Ca currents increased in a dose-dependent manner. At a testing potential of +80 mV, 6.1%±0.3%, 76.5%±3.8%, 120.6%±5.5%, 248.0%±12.3%, 348.7%±17.3%, 374.2%±18.7%, 432.2%±21.6%, and 443.1%±22.1% of BK_Ca currents were increased at the above concentrations, respectively. The half-effective concentration (EC₅₀) of DHA on BK_Ca currents was 37.53±1.65 μmol/L. When DHA concentrations were greater than 20 μmol/L, K_V currents were gradually blocked by increasing concentrations of DHA. At a testing potential of +50 mV, 0.40%±0.02%, 1.37%±0.06%, 11.80%±0.59%, 26.50%±1.75%, 56.50%±2.89%, 73.30%±3.66%, 79.70%±3.94%, and 78.1%±3.91% of K_V currents were blocked at the different concentrations listed above, respectively. The EC₅₀ of DHA on K_V currents was 44.20±0.63 μmol/L.

Conclusion:

DHA can activate BK_Ca channels and block K_V channels in rat CASMCs, and the EC₅₀ of DHA for BK_Ca channels is lower than that for K_V channels; these findings indicate that the vasorelaxation effects of DHA on vascular smooth muscle cells are mainly due to its activation of BK_Ca channels.     

Acetic acid opens large-conductance Ca2+-activated K+ channels in guinea pig detrusor smooth muscle cells

Acetic acid was found to have actions on urinary bladder smooth muscle in our routine ion channel screening assays. Numerous studies have examined the mechanisms of bladder irritation by acetic acid; however, the direct effect of acetic acid on ion channels in detrusor smooth muscle cells has not been evaluated. We used whole-cell patch-clamp techniques to examine the effect of acetic acid on large-conductance Ca2+-activated K+ channels (BKCa) from guinea pig detrusor smooth muscle cells and CHO cells expressing recombinant human BKCaalphabeta1 (CHO BKCaalphabeta1) and human BKCaalpha (CHO BKCaalpha). Acetic acid activated BKCa currents in a concentration-dependent (0.01% to 0.05% v/v) manner in all the cell systems studied. Acetic acid (0.05%) increased BKCa current at +30 mV by 2764+/-918% (n=8) in guinea pig detrusor smooth muscle cells. Acetic acid (0.03%) shifted the V1/2 of conductance-voltage curve by 64+/-14 (n=5), 128+/-14 (n=5), and 126+/-12 mV (n=4) in CHO BKCaalpha, CHO BKCaalphabeta1 and detrusor smooth muscle cells, respectively. This effect of acetic acid was found to be independent of pH and was also not produced by its salt form, sodium acetate. Automated patch-clamp experiments also showed similar activation of CHO BKCaalphabeta1 by acetic acid. In conclusion, acetic acid directly activates BKCa channels in detrusor smooth muscle cells. This novel study necessitates caution while interpreting the results from acetic acid bladder irritation model.     

Cyclopiazonic acid增加SHR和WKY血管平滑肌细胞Ca~(2+)-依赖性外向K~+-电流

采用全细胞及细胞贴附式斑片钳技术记录自发性高血压大鼠(SHR)和Wistar-Kyoto对照鼠(WKY)培养主动脉平滑肌细胞的Ca~(2+)-依赖性外向K~+电流[I_(k(Ca))],测定肌浆网Ca~(2+)泵抑制剂CPA对其影响.CPA能增加I_K(Ca))单通道开放时间,缩短关闭时间,增加全细胞I_(K(Ca))幅度,这些作用与Ca~(2+)相关并可被K~+通道阻断药glybenclamide阻断。CPA作用在SHR和WKY之间无明显差异。结果提示高血压状态下血管平滑肌的功能改变可能与I_(K(Ca))无关。     

异丙肾上腺素及氨茶碱通过cAMP诱导的高电导Ca^2＋激活钾通道活化舒张支气管平滑肌

目的：探讨异丙肾上腺素(Iso)和氨茶碱(Ami)是否通过蛋白激酶A(PKA)通道激活高电导Ca^2 活化钾通道(BKCa)来舒张支气管平滑肌．方法：运用等长张力记录和穿孔膜片箝技术,观察Iso和Ami对大鼠离体支气管平滑肌细胞BKCa的作用以及PKA抑制剂Rp—cAMP对该效应的影响．结果：(1)Iso和Ami均可诱发甲酰胆碱预收缩的离体支气管产生浓度依赖性舒张反应,BKCa阻断剂四乙胺(TEA)5mmol/L使两者的量效曲线向右显著移位;(2)Iso 1 μmol/L显著增加方波刺激时(从-60mV到 50mV)平滑肌细胞BKCa电流,该效应可被Rp—cAMP l00μmol/L显著抑制,Iso使BKCa电流电压关系曲线向上移位．在斜坡刺激时(从-100mV到 100mV)亦获得类似结果;(3)Ami 1 mmol/L显著增加支气管平滑肌细胞在方波刺激时的BKCa电流,该效应被Rp—cAMP l00μmol/L显著抑制,Ami使BKCa电流电压关系曲线向上移位．在斜坡刺激时亦获得类似结果．结论：cAMP依赖性的气道舒张剂Iso和Ami对大鼠气道的舒张效应至少部分通过PKA的活化激活BKCa通道而实现的．     

Protein kinase C modulates Ca2+-activated K+ channels in cultured rat mesenteric artery smooth muscle cells

The electrical and pharmacological properties of protein kinase C (PKC) and its effect on the single Ca2+-activated K+ channel (Kca-channel) in the cultured smooth muscle cells of rat mesenteric artery were studied using a patch-clamp technique. The Kca-channel had a slope conductance of 151+/-7 pS (mean+/-S.E.) in symmetrical 142 mm K solutions. The high conductance K+ channel, applied to the outer side of membrane patches, was potently inhibited by charybdotoxin (0.1 microM) and tetraethylammonium (0.5 microM), but not by apamin (0.4 microM). In cell-attached patches, bath application of phorbol 12-myristate 13-acetate (PMA, 2 microM), a PKC activator, inhibited the activity of the Kca-channel in the presence of the Ca2+ ionophore, A 23187 (10 microM). This inhibition was reversed by subsequent application of staurosporine (1 nM), a PKC inhibitor. Application of 1-oleoyl-2-acetylglycerol (OAG, 30 microM), another PKC activator, also inhibited the A 23187-induced activation of the K+ channel, and this inhibition was reversed by staurosporine. In inside-out patches, bath application of PKC (0.2 munits), in the presence of ATP (1 mM) and PMA (1 microM), inhibited the K+ channel. These results indicate that protein kinase C inhibits the Ca2+-activated K+ channel of mesenteric artery smooth muscle cells in the rat.     

The pure anti-oestrogen ICI 182,780 (Faslodex) activates large conductance Ca(2+)-activated K(+) channels in smooth muscle

Oestrogen and tamoxifen activate large conductance Ca(2+)-activated K(+) (BK(Ca)) channels in smooth muscle through a non-genomic mechanism that depends on the regulatory beta1 subunit and an extracellular binding site. It is unknown whether a "pure" anti-oestrogen such as ICI 182,780 (Faslodex), that has no known oestrogenic properties, would have any effect on BK(Ca) channels. Using single channel patch clamp techniques on canine colonic myocytes, the hypothesis that ICI 182,780 would activate BK(Ca) channels was tested. ICI 182,780 increased the open probability of BK(Ca) channels in inside-out patches with an EC(50) of 1 microM. These data suggest that molecules with the ability to bind nuclear oestrogen receptors, regardless of oestrogenic or anti-oestrogenic nature, activate BK(Ca) channels through this nongenomic, membrane-delimited mechanism. The identity and characteristics of this putative binding site remain unclear; however, it has pharmacological similarity to oestrogen receptors alpha and beta, as ICI 182,780 interacts with it.     

The amiodarone derivative 2-methyl-3-(3,5-diiodo-4-carboxymethoxybenzyl)benzofuran (KB130015) opens large-conductance Ca2+-activated K+ channels and relaxes vascular smooth muscle

2-methyl-3-(3,5-diiodo-4-carboxymethoxybenzyl)benzofuran (KB130015) has been developed to retain the antiarrhythmic properties of the parent molecule amiodarone but to eliminate its undesired side effects. In patch-clamp experiments, KB130015 activated large-conductance, Ca2+-activated BK(Ca) channels formed by hSlo1 (alpha) subunits in HEK 293 cells. Channels were reversibly activated by shifting the open-probability/voltage (P(o)/V) relationship by about -60 mV in 3 muM intracellular free Ca2+ ([Ca2+]in). No effect on the single-channel conductance was observed. KB130015-mediated activation of BK(Ca) channels was half-maximal at 20 microM with a Hill coefficient of 2.8. BK(Ca) activation by KB130015 did not require the presence of Ca2+ and still occurred with saturating (100 microM) [Ca2+]in. Effects of the prototypic BK(Ca) activator NS1619 (1,3-dihydro-1-[2-hydroxy-5-(trifluoromethyl)phenyl]-5-(trifluoromethyl)-2H-benzimidazol-2-one) and those of KB130015 were not additive suggesting that both activators may at least partially share a common mechanism of action. KB130015-mediated activation was observed also for BK(Ca) channels from insects and for human BK(Ca) channels with already profoundly left-shifted voltage-dependence. In contrast, human intermediate conductance Ca2+-activated channels were inhibited by KB130015. Using segments of porcine pulmonary arteries, KB130015 induced endothelium-independent vasorelaxation, half-maximal at 43 microM KB130015. Relaxation was inhibited by 1 mM tetraethylammonium, suggesting that KB130015 can activate vascular smooth muscle type BK(Ca) channels under physiological conditions. Interestingly, the shift in the P(o)/V relationship was considerably stronger (-90 mV in 3 microM [Ca2+]in) for BK(Ca) channels containing Slo-beta1 subunits. Thus, KB130015 belongs to a novel class of BK(Ca) channel openers that exert an effect depending on the subunit composition of the channel complex.