大黄素对豚鼠结肠收缩性的影响及其机制的研究

目的研究大黄素对豚鼠离体远端结肠平滑肌收缩活动的影响及其可能机制。方法急性分离豚鼠远端结肠平滑肌,采用生理记录仪记录不同浓度大黄素对远端结肠收缩的影响;木瓜蛋白酶消化分离豚鼠近端结肠平滑肌细胞,采用全细胞膜片钳技术记录钾电流。结果不同浓度的大黄素（1～50μmol.L-1）对豚鼠远端结肠具有双向调节作用,且其效应呈剂量依赖的趋势1～10μmol.L-1大黄素可浓度依赖性地增强豚鼠远端结肠平滑肌的收缩10μmol.L-1浓度时,大黄素的作用达到平台期。当大黄素的浓度大于10μmol.L-1时,逐渐呈现明显的抑制作用,且其抑制作用与浓度正相关10μmol.L-1硝苯地平孵育肌条后,大黄素不能增强肌条的收缩。大黄素（1～30μmol.L-1）浓度依赖性地抑制豚鼠近端结肠平滑肌细胞钾电流。结论大黄素增强豚鼠结肠的收缩作用可能与L-型钙通道及钾通道有关。     

大黄素增强结肠近端平滑肌细胞钙离子依赖氯离子通道的表达

目的:研究大黄素对结肠近端平滑肌细胞钙离子依赖氯离子通道(ClCa channel)的作用。方法:采用RM6200四道仪记录黄体酮对平滑肌的等长收缩活动作用,相对定量的单细胞RT-PCR法检测平滑肌细胞ClCa通道mRNA的表达。结果:大黄素显著增强离体结肠平滑肌肌条和单个平滑肌细胞的收缩,并可使ClCa电流显著增强,作用与剂量呈正相关。DIDS和NFA可阻滞大黄素的作用。豚鼠近端结肠平滑肌细胞有ClCa1和ClCa2基因的mRNA表达,ClCa1基因的mRNA表达强度是ClCa2基因的mR-NA的5.3±0.71(n=5,P<0.01)倍。50μM大黄素孵育可以使ClCa1基因的mRNA表达增强(n=5,P<0.01)。结论:大黄素可通过兴奋氯离子通道电流引起结肠平滑肌收缩,大黄素对氯离子通道兴奋作用可能与增强结肠平滑肌细胞ClCa1基因表达有关。     

黄体酮、雌二醇对豚鼠离体胆囊肌条收缩的活动影响

目的：观察不同阻断剂对黄体酮、雌二醇效应的影响。方法：将豚鼠胆囊制备成平滑肌条标本,固定在浴槽中,连接张力换能器。用8个灌流肌槽同时记录胆囊肌条的自发收缩。结果：黄体酮和雌二醇均抑制胆囊肌条的收缩活动;雷尼替丁可阻断雌二醇对豚鼠胆囊肌条的抑制作用,而六烃季胺、消炎痛、心得安和L－NNA无此作用;实验中所用的阻断剂均未阻断黄体酮对豚鼠胆囊肌条的抑制作用。结论：雌二醇对胆囊肌条的抑制作用可被雷尼替丁阻断。黄体酮对胆囊肌条的作用可能是直接作用于平滑肌。     

染料木黄酮对豚鼠结肠平滑肌细胞L型钙通道的影响(英文)

目的研究酪氨酸激酶抑制剂染料木黄酮(GST)对豚鼠结肠平滑肌细胞L型钙通道电流的作用。方法木瓜蛋白酶法分离单个豚鼠结肠平滑肌细胞,应用全细胞式膜片钳技术记录L型钙通道电流。结果 GST(10 ~100μmo·lL-1)浓度依赖性地阻断L型钙通道电流,其作用可被洗脱,半数有效抑制浓度为(39.9±3.6)μmol·L-1。GST可使L型钙通道的稳态失活曲线向超极化方向左移约10 mV(P<0.01),对其斜率没有影响。GST的无活性拟似物大豆异黄酮对L型钙通道电流的作用明显小于GST。酪氨酸磷酸酶抑制剂原钒酸钠可阻断GST对钙通道电流的抑制作用。结论 GST可通过酪氨酸激酶途径抑制豚鼠结肠平滑肌L型钙通道。     

糖尿病豚鼠胆囊收缩功能的变化

目的探讨糖尿病豚鼠胆囊运动功能的变化。方法 30只雄性豚鼠随机均分为成实验组和对照组。实验组腹腔注射链脲菌素复制糖尿病模型。造模成功4周后处死,观察禁食情况下胆囊体积、胆囊胆汁的外观以及胆汁中胆固醇结晶发生率。制作胆囊平滑肌条,在恒温、氧浴的条件下体外观察胆囊肌条自发性收缩的频率和收缩张力;观察胆囊肌条对不同浓度的乙酰胆碱(ACh)、胆囊收缩素(CCK)和氯化钾(KCl)的反应。结果实验组胆囊体积为对照组的1.6倍;实验组胆囊胆汁浑浊,而对照组胆囊胆汁清亮;实验组胆囊胆汁的胆固醇结晶率显著高于对照组(66.7%vs.13.3%)(P<0.01)。与对照组相比,实验组胆囊肌条对ACh(10-4 mol/L)和CCK(10-7 mol/L)的反应低于对照组(P<0.05和P<0.01)。结论糖尿病胆囊平滑肌对兴奋性神经递质ACh和胃肠激素CCK反应减弱。该异常反应可能导致胆囊运动减弱、胆汁淤滞和胆囊体积增加。     

黄体酮对豚鼠结肠平滑肌及细胞膜钙依赖的钾通道电流的影响

目的　观察黄体酮 (Progesterone)对豚鼠结肠平滑肌肌条和细胞的作用 ,借此探讨肠易激综合征 (IBS)的病理生理机制。方法　急性分离豚鼠结肠平滑肌肌条和单个平滑肌细胞。采用TD 112S型等张传感器测量肌条收缩与舒张的幅值、频率 ,并用Axopatch 1 D膜片钳放大器测全细胞模式下的单个平滑肌细胞大电导的钙离子依赖钾通道电流(BKCa)。结果　 6 4 8μmol·L-1黄体酮可抑制结肠带肌条的收缩 (0 1792± 0 0 873) g(n =6 ,P <0 0 5 ) ,而低浓度黄体酮仅抑制结肠环行平滑肌肌条的收缩 (16 2pmol·L-10 2 36 0g± 0 15 78g ,n =6 ,P <0 0 5 ;1 6 2nmol·l-10 4 332 g±0 2 111g ,n =6 ,P <0 0 1;3 2 4nmol·l-1部分肌条完全抑制P <0 0 1) ,其效应呈剂量依赖的趋势。细胞实验中 12 96μmol·L-1的黄体酮可抑制BKCa的幅值约 6 0 %± 17% (n =10 ,P <0 0 1) ,而灌流液含 5 μmol·L-1尼卡地平 (nicardip ine)时抑制BKCa的作用不明显。结论　高浓度黄体酮对纵行和环行平滑肌均有抑制作用 ,而低浓度主要抑制环行平滑肌的收缩 ,作用机制与减少细胞外钙离子内流及抑制BKCa有关 ,这种作用可部分解释在临床上IBS妇女患病更普遍的现象 ,对女性IBS患者的激素治疗有一定的提示作用     

白杨素对大鼠离体肾动脉的舒张作用

目的研究白杨素对大鼠离体肾动脉的舒张作用及其机制是否涉及抑制肾动脉血管平滑肌细胞L-型电压依赖性钙通道。方法利用微血管张力记录仪(DMT)观察白杨素对预收缩大鼠肾动脉血管环的肌源性反应;利用全细胞膜片钳电生理学实验方法,观察白杨素对大鼠离体肾动脉血管平滑肌细胞L-型电压依赖性钙电流的作用。结果 1白杨素浓度依赖性的舒张60 mmol/L KCl或10-5 mol/L去氧肾上腺素(PE)预收缩的大鼠肾动脉血管环,其最大舒张幅度分别为88.99%和67.47%,RC50值分别为26.25μmol/L和51.68μmol/L。2白杨素(浓度为RC50值)可抑制大鼠肾动脉血管平滑肌细胞L-型电压依赖性钙电流,使其I-V曲线非平行上移;给予0 mV单电压刺激时,白杨素(浓度为RC50值)使大鼠肾动脉血管平滑肌细胞L-型电压依赖性钙电流值降低45.43%。结论 1白杨素浓度依赖性舒张60 mmol/L KCl或10-5 mol/L PE预收缩的大鼠肾动脉血管环;2白杨素抑制大鼠肾动脉血管平滑肌细胞L-型电压依赖性钙电流,使其I-V曲线非平行上移。     

蛋白激酶C 参与内源性大麻素花生四烯乙醇胺对L型钙电流的抑制作用

目的：研究内源性大麻素物质花生四烯乙醇胺是否通过改变蛋白激酶C（ PKC）活性从而抑制心肌L型钙电流,并进一步探讨可能改变PKC活性的信号途径。方法应用全细胞膜片钳技术记录单个心肌细胞的L型钙电流（P ＜0 n．05）;应用PepTag非放射性蛋白激酶C检测系统（ Promega）检测PKC活性;Elisa试剂盒测定细胞中二脂酰甘油（ DAG）的含量;western blot 技术测定磷脂酶Cβ（ PLCβ）和磷酸化磷脂酶C β（ p-PLCβ）表达。结果应用花生四烯乙醇胺灌流心肌细胞后显著抑制心肌L型钙电流（ P ＜0．05）,预先应用大麻素1型受体（ CB1）阻断剂AM251或PKC非特异性激动剂佛波醇酯（PMA）可以完全阻断此抑制效应,而大麻素2型受体（CB2）阻断剂AM630没有阻断花生四烯乙醇胺抑制L型钙电流的作用。检测心肌细胞PKC活性发现,花生四烯乙醇胺明显抑制PKC活性（ P ＜0．05）,同样预先应用AM251或PMA完全阻断花生四烯乙醇胺对PKC活性的抑制效应,而AM630无此效应。应用花生四烯乙醇胺没有影响心肌细胞DAG含量和PLCβ的磷酸化。结论本实验首次证明内源性大麻素花生四烯乙醇胺激活心肌细胞CB1受体后抑制细胞PKC的活性,从而抑制L型钙电流,此过程没有PLCβ-DAG途径参与。     

银杏酮酯对缺血豚鼠心室肌细胞Ⅰ_(Ca-L)和游离钙的影响

目的观察银杏酮酯GBE50对模拟缺血游离豚鼠心室肌细胞L型钙电流和游离钙浓度的影响,探讨GBE50抗心肌缺血的作用机制。方法应用单酶酶解法分离游离单个豚鼠心室肌细胞,采用膜片钳全细胞记录心室肌细胞L型钙电流,通过激光共聚焦显微镜扫描测定细胞内游离钙浓度的动态变化。结果缺血抑制豚鼠心室肌细胞的ICa-L(n=9,P<0.01),使豚鼠心室肌细胞内游离钙增加(n=10,P<0.01),而50mg·mL-1GBE50减轻缺血对ICa-L的抑制效应(n=6,P>0.05),减少缺血后心室肌细胞内游离钙浓度的增加(n=10,P>0.05)。结论GBE50可减轻心肌缺血区域与非缺血区域电生理的异质性,维持缺血后豚鼠心肌细胞电生理的稳定性,并减轻缺血后心肌细胞内钙超载介导的心肌损伤。     

大黄素对豚鼠单个心室肌细胞胞浆游离钙浓度及L-型钙电流的影响

目的研究大黄素(emodin)对豚鼠心室肌细胞钙信号的影响。方法酶解法分离豚鼠单个心室肌细胞,应用激光扫描共聚焦显微镜联合全细胞膜片钳技术测量豚鼠心室肌细胞钙信号的变化。结果在静息状态下,1～100 μmol·L^-1大黄素对［Ca²⁺］i均无影响;对60 mmol·L^-1 KCl诱导的外钙内流引起的胞浆钙升高有不同的影响,1 μmol·L^-1表现为促进作用;10 μmol·L^-1无作用;100 μmol·L^-1则表现为抑制作用。膜片钳研究结果表明,1 μmol·L^-1大黄素可明显促进L-型钙电流,10 μmol·L^-1对L-型钙电流无影响;100 μmol·L^-1明显抑制L-型钙电流。结论大黄素对心肌细胞内钙及L-型钙电流具有双向调节作用。     

Emodin increases Ca(2+) influx through L-type Ca(2+) channel in guinea pig gallbladder smooth muscle

Emodin is known to be used in the treatment of cholesterol stones and cholecystitis. This study sought to investigate the effects of emodin on the contraction of gallbladder smooth muscle (GBSM), intracellular Ca(2+) concentration and L-type calcium current in GBSM cells. Gallbladder muscle strips were obtained from adult guinea pigs and the resting tension was recorded. Gallbladder smooth muscle cells were isolated by enzymatic digestion. Cells were loaded with fluo-3/AM and [Ca(2+)](i) was determined by a laser confocal microscope. Calcium current was recorded by the whole-cell patch clamp method. Emodin increased the resting tension of GBSM strips in a dose-dependent manner. Emodin elevated [Ca(2+)](i) in GBSM cells, and this effect was attenuated by pretreatment with nifedipine. In addition, Emodin increased L-type calcium current at concentrations of 1 to 30 microM (at +10 mV, 10 microM, 45.1+/-5.2% compared to control, EC(50) =3.11 microM). In the presence of protein kinase C (PKC) inhibitor, Staurosporine, emodin did not significantly affect the calcium current. However, phorbol 12, 13-dibutyrate mimicked emodin in enhancement of the calcium current. These results suggest that emodin promotes gallbladder contraction by increasing Ca(2+) influx through L-type calcium channel via PKC pathway.     

Emodin Inhibits Voltage‐Dependent Potassium Current in Guinea Pig Gallbladder Smooth Muscle

Abstract: Emodin is known to prompt bile secretion in gallbladder and to be used in the treatment of cholesterol stones. We studied the effects of emodin on the contraction of gallbladder smooth muscle and voltage‐dependent K⁺ current in gallbladder smooth muscle cells. Gallbladder muscle strips were obtained from adult guinea pigs and the resting tension was recorded. Gallbladder smooth muscle cells were isolated by enzymatic digestion, and K⁺ current was recorded by the whole‐cell patch clamp method. Emodin increased the resting tension of gallbladder smooth muscle strips and inhibited voltage‐dependent K⁺ current in a dose‐dependent manner. When 10 µM emodin was applied to gallbladder smooth muscle cells for 3–6 min., the amplitude of voltage‐dependent K⁺ current was decreased by 31.5 ± 0.5% at +40 mV, and this inhibitory effect mostly recovered after washout. The steady‐state inactivation curves were shifted in a hyperpolarizing direction by emodin. In the presence of the protein kinase C inhibitors staurosporine and chelerythrine, the effect of emodin on voltage‐dependent K⁺ current was significantly attenuated. In conclusion, emodin promotes gallbladder contraction, mainly by inhibiting voltage‐dependent K⁺ current via the protein kinase C pathway. These findings provide theoretical foundation for the application of emodin in gallbladder motility disorders.     

大黄素对豚鼠结肠带平滑肌细胞钾通道活性的影响

用检测平滑肌细胞电活动和张力技术,研究了大黄素对豚鼠结肠带平滑肌细胞电和收缩活动的影响并与 cromakalim, glybenclamide,四乙胺及BaCl₂的作用进行比较。结果表明,大黄素加强平滑肌细胞电和收缩活动,作用效果与剂量有关;大黄素与cromakalim的作用相互抑制。其促进平滑肌细胞电和收缩活动的作用与glybenclamide相似,而与四乙胺和BaCl₂有明显区别。提示大黄素的作用机制与抑制细胞膜K_ATP等钾通道的活性相关。     

Mechanism of inhibitory effects of ethanol on gallbladder contraction: differences in cases of histamine- and acetylcholine-induced contraction]

We investigated the effects of different concentrations of ethanol on contraction of gallbladder isolated from guinea pig. Ethanol at 25 mM significantly inhibited the contraction induced by histamine, but not those by KCl and acetylcholine. A higher concentration (100 mM) of ethanol inhibited both histamine-, acetylcholine- and KCl- induced contractions. The inhibitory effect of the lower concentration (25 mM) of ethanol was not observed in the presence of verapamil, an antagonist of L-type Ca2+ channel or staurosporine, a selective inhibitor of protein kinase C. Verapamil and staurosporine significantly inhibited both histamine- and acetylcholine-induced contractile responses: the inhibitory effect was more potent for the histamine contraction. Our recent study has demonstrated that contraction caused by protein kinase C activation is completely dependent on Ca2+ influx through the L-type Ca2+ channel in gallbladder smooth muscle of guinea pig. Therefore, the difference in 25 mM ethanol effect on histamine- and acetylcholine-induced contractions may be due to different degree of involvement of protein kinase C activation in the agonist-induced contraction. On the other hands, the higher concentration (100 mM) of ethanol inhibits the common pathway of contraction in gallbladder smooth muscle cells.     

Marrubenol interacts with the phenylalkylamine binding site of the L-type calcium channel

Marrubenol inhibits contraction of rat arteries by blocking L-type calcium (Ca(2+)) channels in smooth muscle cells, but its interaction with binding sites for calcium antagonists had never been investigated. Competition binding studies indicated that marrubenol was a weak inhibitor of 1,4-dihydropyridine binding in membranes isolated from rat intestinal smooth muscle but completely displaced specifically bound (-)-[(3)H]desmethoxyverapamil ([(3)H]D888) with an apparent K(i) value of 16 microM (95% confidence interval: 6.5-39.5 microM). As marrubenol inhibited the contraction evoked by KCl depolarization of intestinal smooth muscle half-maximally at a concentration of approximately 12 microM, interaction with the phenylalkylamine binding site seems to account for the inhibition of L-type Ca(2+) channels by marrubenol.     

Neurokinin A-induced guinea pig gallbladder contraction: Potential mechanism of action

The present study was performed to examine the mechanism of action of neurokinin A (NKA) on guinea pig gallbaldder smooth muscle. Muscle strips were prepared and mounted in 10 mL tissue bath containing Krebs' solution under 1 g tension. NKA induced a concentration-dependent increase in gallbladder muscle tension and reached a maximal response at 1 μM. The EC₅₀ value was approximately 30nM. Preincubation of the muscle strips with neurotoxins, tetrodotoxin (1 μM), or omega-conotoxin (0.1 μM) had no effect on the NKA contractile response. NKA-induced gallbladder contractions were insensitive to cyclooxygenase inhibitors (5 μM) piroxicam and indomethacin. In contrast, the calcium channel blockers verapamil and diltiazem (0.1–1 μM) significantly blocked the contractile response to NKA. The intracellular calcium chelator BAPTA/AM had no significant effect on NKA activity. The removal of extracellular calcium, however, completely abolished the contractile response of NKA. These data suggest that NKA has a direct contractile effect on guinea pig gallbladder smooth muscle, which is independent of prostaglandin release. The primary source of calcium involved in mediating the NKA contractile response is the extracellular pool, suggesting that NKA might act via activation of L-type voltage-operated calcium channels to mediate its action. © 1992 Wiley-Liss, Inc.     

Conserved smooth muscle contractility and blood pressure increase in response to high-salt diet in mice lacking the beta3 subunit of the voltage-dependent calcium channel

Voltage-dependent calcium channels are crucially important for calcium influx and the following smooth muscle contraction. Beta subunits of these channels are known to modify calcium currents through pore-forming alpha subunits. Among the four reported independent beta subunits, the beta3 subunit is expressed in smooth muscle cells and thought to compose L-type calcium channels in the tissue. To determine the role of the beta3 subunit in the cardiovascular system, we have analyzed beta3-null mice. Electrophysiological examinations proved the existence of dihydropyridine (DHP)-sensitive. L-type calcium channels in the smooth muscle cells. Beta3-null mice show no apparent changes in smooth muscle contraction and sensitivity to DHP, and normal blood pressure when they are raised on a normal diet, but the 13 subunit deficient mice show elevated blood pressure in response to a high-salt diet, with significant reductions in plasma catecholamine concentrations. Our finding strongly suggests a close relationship between voltage-dependent channels and high blood pressure.     

大黄素对大鼠近端结肠平滑肌细胞电压依赖性钾通道的影响

目的研究大黄素对大鼠近端结肠电压依赖性钾离子通道的影响，以探讨其增强结肠运动的机制。方法 采用全细胞膜片钳技术测定电压依赖性钾离子通道快速激活型钾电流及延迟整流型钾电流。结果大黄素(1～30 μmol·L^-1)浓度依赖性地阻断延迟整流性钾通道，加快电流失活，其阻断作用不需要钾通道的开放。30 μmol·L^-1大黄素可抑制快速激活型钾电流。5 μmol·L^-1大黄素对钾通道的激活动力学及失活动力学没有影响，但30 μmol·L^-1大黄素使其激活动力学曲线明显右移，斜率常数由(13.0±0.6)上升至(19.6±2.5) mV，同时也使失活动力学曲线明显右移。结论大黄素可阻断延迟整流型钾通道及快速激活型钾通道，其阻断作用不是开放阻断。