小檗胺对高钾,去甲肾上腺素及咖啡因引起大鼠心肌细胞内钙动拮？…

AIM: To study the effects of berbamine (Ber) on intracellular calcium concentration ([Ca2+]i) mobilized by KCl depolarization, norepinephrine (NE), and caffeine. METHODS: [Ca2+]i was measured with fluorescent intensity (FI) by confocal microscope in single cultured cardiomyocytes of newborn rats loaded with Fluo 3-AM 2 mumol.L-1. RESULTS: FI value of [Ca2+]i in control level was 248 +/- 70 in the presence of extracellular calcium 1.5 mmol.L-1 and was not changed by Ber 3-30 mumol.L-1. KCl (60 mmol.L-1)- and NE (30 mumol.L-1)-induced [Ca2+]i mobilizations were inhibited (P < 0.01) by Ber 30 mumol.L-1, similar to that of verapamil (Ver). The inhibitory effect of Ber on [Ca2+]i induced by KCl was further increased (P < 0.05) in the presence of egtazic acid 3 mmol.L-1, but that on [Ca2+]i induced by NE was not changed. The [Ca2+]i mobilized by caffeine 80 and 160 mumol.L-1 in D-Hanks' solution was not affected (P > 0.05) by Ber and Ver. CONCLUSION: Ber possessed the antagonistic effects on [Ca2+]i increases via voltage-dependent Ca2+ channel and receptor-operated Ca2+ channel in newborn rat cardiomyocytes, but without effect on intracellular Ca2+ release.     

脱水长春胺乙酯对离体犬基底动脉的作用

本文采用离体犬基底动脉实验。结果表明脱水长春胺乙酯能对抗5-HT和高钾所致基底动脉收缩,其对5-HT具二重拮抗作用,用Brink作图法求出pA₂为6.06,pD′₂为4.96。     

小檗胺对血管平滑肌细胞钙动力学影响

本文以Fluo-3/AM 荧光技术和激光扫描共聚焦显微镜检查法, 研究小檗胺 (Ber) 对培养家兔胸主动脉血管平滑肌细胞(VSMC)内游离钙 ([Ca 2 ]i ) 的影响结果表明：在胞外钙 ([Ca 2 ]o ) 为?.0 mmol·L -1 条件下，Ber 抑制60 mmol·L -1 KCL1 mmol·L -1 哇巴因Ouabain 30 mmol·L -1 去甲肾上腺素 ( NE ) 1 mmol·L -1 5-羟色胺5-HT 和30 mmol·L -1 三磷酸腺苷 (ATP)引起的 [Ca 2 ]i 升高，而且荧光强度达峰时间亦延长。在无胞外钙条件下，Ber 对咖啡因 (Caffeine)引起的[Ca 2 ]i 升高没有作用。结论：Ber 抑制电压依赖性钙通道 (VDCC) 和受体调控性钙通道 (ROCC) 激活引起的外钙内流，对Caffeine 引起的内钙释放没有影响。Ber 可抑制Ouabain 诱导的细胞内钙升高。     

过氧化氢酶和内皮素对缺氧引起的猪基底动脉收缩的体外影响

AIM: To study whether anoxia-induced vasoconstriction was related to the release of endothelin (ET). METHODS: Acute anoxia was induced by gassing the organ chamber with 95% N2 + 5% CO2. Changes in tension of porcine basilar arterial ring was recorded. RESULTS: Anoxia-induced increases in tension were 0.21 g +/- 0.08 g and 0.24 g +/- 0.09 g under basal tension and during ET 3 nmol.L-1-induced contractions, respectively. In the rings tension did not further augment following the increase of ET from 100 to 300 nmol.L-1, acute anoxia did cause further increase in tension of 0.16 g +/- 0.10 g (n = 4). Catalase 800 and 2400 kU.L-1 decreased the anoxia-induced contraction, with inhibitory rate of 33% +/- 7% and 47% +/- 9%, respectively. CONCLUSION: Anoxia-induced vasoconstriction was related to release of hydrogen peroxide from endothelial cells.     

小檗胺对小鼠迟发型超敏反应的影响

本文研究了小檗胺对小鼠由绵羊红细胞，二硝基氯苯及同种异型脾细胞所诱导的迟发型超敏反应的影响。实验结果显示小檗胺能明显抑制小鼠的迟发型超敏反应，表明小檗胺可能对小鼠的细胞免疫功能具有抑制作用。     

双苯氟嗪,氟桂利嗪,桂利嗪对5—羟色胺所致离体猪基底动脉收缩…

目的：探索双苯氟嗪（Ｄｉｐ，一种我国自行合成的桂利嗪衍生物）对５－羟色胺所致的脑动脉收缩的影响。方法：比较双苯氟嗪，氟桂利嗪（Ｆｌｕ），桂利嗪（Ｃｉｎ）对５－羟色胺所致离体猪基底动脉收缩的抑制及两种收缩成分的影响。结果：三者的拮抗作用强度顺序（ＩＣ５０）为Ｄｉｐ４．０μｍｏｌ．Ｌ＾－１〉Ｆｌｕ１５．６μｍｏｌ．Ｌ＾－１〉Ｃｉｎ２５．２ｕｍｏ．Ｌ＾－１，这三种药对５－羟色胺所致离体猪基底动脉的两种收     

小檗胺对糖尿病肾小管上皮细胞损伤的影响

目的 研究小檗胺对体外糖尿病肾小管上皮细胞(RTEC)损伤的影响。方法 将RTEC分成对照组、模型组(高糖)、低剂量实验组(2 mg·L^-1小檗胺和高糖)、中剂量实验组(4 mg·L^-1小檗胺和高糖)、高剂量实验组(8 mg·L^-1小檗胺和高糖)、BBM-H+miR-NC组(转染mimics control, 8 mg·L^-1小檗胺和高糖)、BBM-H+miR-135b组(转染miR-135b mimics, 8 mg·L^-1小檗胺和高糖)。以噻唑蓝(MTT)法检测细胞增殖活性,以流式细胞术检测细胞凋亡率,以蛋白质印迹法检测细胞B淋巴细胞瘤-2(Bcl-2)、Bcl-2相关X(Bax)蛋白表达,以化学荧光法检测活性氧(ROS)水平,以硫代巴比妥酸法检测丙二醛(MDA)水平,以黄嘌呤氧化法检测超氧化物歧化酶(SOD)水平,以酶联免疫吸附试验(ELISA)法检测肿瘤坏死因子-α(TNF-α)、白细胞介素-8(IL-8)和白细胞介素-1β(IL-1β)水平。结果 对照组、模型组和低、...     

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。     

Mechanism of asynchronous Ca2+ waves underlying agonist-induced contraction in the rat basilar artery

Background and purpose:

Uridine 5''-triphosphate (UTP) is a potent vasoconstrictor of cerebral arteries and induces Ca²⁺ waves in vascular smooth muscle cells (VSMCs). This study aimed to determine the mechanisms underlying UTP-induced Ca²⁺ waves in VSMCs of the rat basilar artery.

Experimental approach:

Isometric force and intracellular Ca²⁺ ([Ca²⁺]_i) were measured in endothelium-denuded rat basilar artery using wire myography and confocal microscopy respectively.

Key results:

Uridine 5''-triphosphate (0.1–1000 µmol·L⁻¹) concentration-dependently induced tonic contraction (pEC₅₀ = 4.34 ± 0.13), associated with sustained repetitive oscillations in [Ca²⁺]_i propagating along the length of the VSMCs as asynchronized Ca²⁺ waves. Inhibition of Ca²⁺ reuptake in sarcoplasmic reticulum (SR) by cyclopiazonic acid abolished the Ca²⁺ waves and resulted in a dramatic drop in tonic contraction. Nifedipine reduced the frequency of Ca²⁺ waves by 40% and tonic contraction by 52%, and the nifedipine-insensitive component was abolished by SKF-96365, an inhibitor of receptor- and store-operated channels, and KB-R7943, an inhibitor of reverse-mode Na⁺/Ca²⁺ exchange. Ongoing Ca²⁺ waves and tonic contraction were also abolished after blockade of inositol-1,4,5-triphosphate-sensitive receptors by 2-aminoethoxydiphenylborate, but not by high concentrations of ryanodine or tetracaine. However, depletion of ryanodine-sensitive SR Ca²⁺ stores prior to UTP stimulation prevented Ca²⁺ waves.

Conclusions and implications:

Uridine 5''-triphosphate-induced Ca²⁺ waves may underlie tonic contraction and appear to be produced by repetitive cycles of regenerative Ca²⁺ release from the SR through inositol-1,4,5-triphosphate-sensitive receptors. Maintenance of Ca²⁺ waves requires SR Ca²⁺ reuptake from Ca²⁺ entry across the plasma membrane via L-type Ca²⁺ channels, receptor- and store-operated channels, and reverse-mode Na⁺/Ca²⁺ exchange.     

Cl~-通道阻断剂对5-HT和CPA引起的兔脑椎基底动脉平滑肌细胞Ca~(2+)内流的影响

目的　在培养的兔脑椎基底动脉平滑肌细胞上观察5 HT和CPA诱导的Ca2 + 内流的特性 ,电压依赖性Ca2 + 通道 (VDC)抑制药尼莫地平 ,非电压依赖性Ca2 + 通道抑制药SK&F963 65及Cl-通道阻断剂DIDS、NPPB对两种激动剂引起 [Ca2 + ]i 反应的影响 ,以探讨脑血管平滑肌细胞中 5 HT引起Ca2 + 内流的特性、Cl-通道与Ca2 + 内流的关系。方法　采用生物荧光双波长影像分析系统瞬即测定单细胞胞质[Ca2 + ]i 技术。结果　① 5 HT和CPA均能诱导平滑肌细胞[Ca2 + ]i 呈双相升高 ,并且 5 HT诱导的Ca2 + 释放是环匹阿尼酸 (CPA)敏感Ca2 + 池的一部分 ;②尼莫地平对 5 HT和CPA触发的Ca2 + 内流无明显影响 ,而SK&F963 65可阻止二者触发的Ca2 + 内流 ;③Cl-通道阻断剂DIDS、NPPB呈浓度依赖性抑制Ca2 + 内流 ,在SK&F963 65最大限度抑制Ca2 + 内流后 ,DIDS、NPPB可进一步抑制Ca2 + 内流 ;而Ca2 +内流被DIDS、NPPB分别最大抑制后 ,SK&F963 65也可进一步抑制Ca2 + 内流。结论　 5 HT引起的Ca2 + 内流是经SK&F963 65敏感的非VDC ,其中包含Ca2 + 释放引起的Ca2 + 内流 (CRAC)成分与非CRAC成分 ,并且这两部分Ca2 +内流均与DIDS、NPPB敏感的Cl-通道开放有关     

Characterization of Ca2+ channels involved in endothelin-1-induced contraction of rabbit basilar artery

This study attempted to characterize Ca2+ channels involved in endothelin-1-induced contraction of rabbit basilar artery using whole-cell patch-clamp and measurement of intracellular free Ca2+ concentration. Endothelin-1 activates two types of Ca2+-permeable nonselective cation channels (NSCC-1 and NSCC-2) and a store-operated Ca2+ channel (SOCC) in addition to the voltage-operated Ca2+ channel (VOCC). These channels can be discriminated using Ca2+ channel blockers, SK&F 96365 and LOE 908. Tension study was conducted to clarify the Ca2+ channels involved in endothelin-1-induced contraction of basilar artery. Endothelin-1-induced basilar artery contraction is fully dependent on extracellular Ca2+ influx. Based on sensitivity to nifedipine, an L-type VOCC blocker, VOCCs have a minor role in endothelin-1-induced contraction. Both LOE 908 and SK&F 96365 inhibit endothelin-1-induced contraction in a concentration-dependent manner, and their combination abolished it. The median inhibitory concentrations of these blockers for endothelin-1-induced contraction correlated well with those of the endothelin-1-induced [Ca2+]i responses. Thus, the inhibitory action of these blockers on endothelin-1-induced contraction may be mediated by blockade of NSCC-1, NSCC-2, and the SOCC. Extracellular Ca2+ influx through NSCC-1, NSCC-2, and SOCC may be essential for endothelin-1-induced basilar artery contraction.     

Effects of isosorbide dinitrate and diltiazem on Ca2+ flux and contraction in artery

We studied the effects of isosorbide dinitrate and diltiazem on histamine-stimulated 45Ca fluxes and contractions of isolated porcine coronary artery. Isosorbide dinitrate was slightly more potent as an inhibitor of intracellular compared to extracellular calcium-dependent contraction. Isosorbide dinitrate inhibited histamine-stimulated calcium efflux and intracellular calcium-dependent contraction over similar concentration ranges. Isosorbide dinitrate partially inhibited histamine-stimulated calcium influx, but this effect was significant only at high concentration and correlated weakly with inhibition of contraction that was dependent on extracellular calcium. Diltiazem more potently inhibited extracellular vs. intracellular calcium-dependent contraction. Diltiazem partially inhibited histamine-stimulated calcium efflux and intracellular calcium-dependent contraction to similar extents (55-60%) and produced similar concentration-response relationships for inhibition of histamine-stimulated calcium influx and extracellular calcium-dependent contraction. The data suggest that alterations of cellular calcium metabolism are major mechanisms of vascular smooth muscle relaxation by isosorbide dinitrate and diltiazem, but that the specific alterations differ for the two drugs. Isosorbide dinitrate may inhibit contraction primarily by enhancing intracellular calcium sequestration, but possibly also by inhibiting agonist-stimulated calcium influx at high isosorbide dinitrate concentrations. Diltiazem primarily inhibits stimulated calcium influx, but may also inhibit intracellular calcium release.     

Contribution of Ca2+ influx to carbachol-induced detrusor contraction is different in human urinary bladder compared to pig and mouse

Carbachol-induced detrusor contractions are mainly mediated via M3 receptor subtype and depend not only on Ca2+ release from the intracellular calcium stores but also on Ca2+ influx via L-type Ca2+ channels. The purpose of this study was to examine the different contributions of Ca2+ influx and Ca2+ release underlying muscarinic receptor-mediated contractions in human, porcine and murine urinary bladder. Detrusor contractions were measured in urothelium-denuded detrusor strips as responses to cumulatively increasing carbachol concentrations, release of intracellular Ca2+ was determined in Chinese hamster ovary cells stably transfected with human muscarinic M3 (hM3) receptors. In human tissue, 1 microM of the L-type Ca2+-channel blocker nifedipine reduced carbachol contractions to 74%, in pig to 18% and in mouse to 27% of pre-drug controls. 2-aminoethoxyphenyl borate (2-APB, 300 microM), which impairs inositol trisphosphate (IP3)-induced release of Ca2+, reduced carbachol responses in human detrusor to 60%, in pig to 35% and in mouse to 20%, whereas block of the Ca2+-induced Ca2+ release with ryanodine had no significant effect on carbachol contractions in all three species. Carbachol-induced release of intracellular Ca2+ in Chinese hamster ovary cells expressing muscarinic hM3 receptors was completely prevented by 100 microM 2-APB. The direct intracellular IP3 receptor antagonist xestospongin C (10 microM) reduced carbachol-stimulated intracellular Ca2+ to 41% of the control value. Blockade of ATP-dependent Ca2+ uptake into intracellular stores with thapsigargin was associated with a concentration-dependent increase of detrusor contraction, but limited on-top contractions with carbachol. In conclusion, carbachol-induced contractions in human, porcine and mouse detrusor depend differently on Ca2+ influx, since potency of nifedipine reducing muscarinic receptor-mediated detrusor contraction is lower in human bladder. On the other hand, slight species differences are also found when inhibiting IP3-induced Ca2+ release and Ca2+ reuptake into intracellular stores. Taken together, our data show considerable species differences between human, porcine and murine detrusor regarding the relative contributions of Ca2+ influx and maybe also carbachol-induced Ca2+ release that could be of relevance when using different animal models.     

Maturation attenuates the effects of cGMP on contraction, [Ca2+]i and Ca2+ sensitivity in ovine basilar arteries.

The present study explores the hypothesis that age-related variations in cerebrovascular responses to vasodilators reflect corresponding age-dependent differences in the mechanisms coupling changes in cytosolic cGMP to vasorelaxation. The experiments focused on cGMP's ability to decrease either [Ca2+]i or myofilament Ca2+ sensitivity, because both effects can contribute to cGMP-induced vasodilation. Use of the cGMP analog 8-pCPT-cGMP minimized problems associated with limited cell permeation or cGMP hydrolysis. In fetal basilars contracted with 10 microM serotonin, the EC30 for 8-pCPT-cGMP-induced relaxation was 6 microM. In fura-2 loaded fetal basilars, pretreatment with 6 microM 8-pCPT-cGMP significantly depressed the sensitivity of [Ca2+]i to 5HT, and also myofilament sensitivity to calcium, but only in fetal arteries. In fetal basilar arteries contracted with 120 mM potassium, the EC30 for 8-pCPT-cGMP-induced relaxation was 25 microM. In fura-2 loaded ovine arteries, pretreatment with 25 microM 8-pCPT-cGMP had no effect on the ability of graded concentrations of potassium to elevate [Ca2+]i but reduced potassium's ability to induce contraction and attenuated myofilament calcium sensitivity; these latter effects were significant only in fetal arteries. In alpha-toxin permeabilized preparations, 25 microM 8-pCPT-cGMP significantly depressed both basal- and agonist-stimulated myofilament calcium sensitivity, only in fetal but not in adult basilars. Together, these results demonstrate that: (1) sensitivity to cGMP is greater in fetal than adult sheep arteries independent of method of contraction; (2) cGMP can reduce [Ca2+]i but only in agonist-contracted and not in potassium-contracted arteries; (3) and cGMP attenuates myofilament calcium sensitivity regardless of method of contraction. Overall, the data demonstrate that variations in the ability of cGMP to produce vasodilatation reflect age-, artery-, and agonist-dependent differences in the combination of mechanisms mediating responses to cGMP.     

蛋白酪氨酸激酶抑制剂对脑血管平滑肌细胞Ca~(2+)池操纵性Ca~(2+)内流的影响

目的　研究蛋白酪氨酸激酶和蛋白酪氨酸磷酸酶抑制剂对牛脑血管平滑肌细胞 (CSMC)Ca2 + 池操纵性Ca2 + 内流的影响。方法　采用培养的CSMC ,在生物荧光双波长影像分析系统用Fura 2 /Am荧光探针测定单个细胞内游离Ca2 + 浓度。结果　 (1)蛋白酪氨酸激酶抑制剂 (genistein ,2 5 ,5 ,10 μmol·L-1)能浓度依赖性降低内皮素 1(ET 1,10 -7mol·L-1)刺激引起的CSMCCa2 + 内流 ,抑制率分别为5 6%± 2 .9%、2 5 6%± 3 9%、48 9%± 3 7% ;蛋白酪氨酸磷酸酶抑制剂 (vanadate ,2 ,4,8μmol·L-1)能浓度依赖性升高CPA刺激引起的CSMCCa2 + 内流 ,增加比率分别为8 2 %± 3 9%、18 8%± 4 9%、46 6%± 6 9% ;(2 ) genistein(2 5 ,5 ,10 μmol·L-1)能浓度依赖性降低ATP(10 μmol·L-1)刺激引起的CSMCCa2 + 内流 ,抑制率分别为 6 7%±2 6%、2 4 6%± 6 5 %、5 1 3 %± 6 9% ;vanadate (2 ,4,8μmol·L-1)能浓度依赖性升高ATP刺激引起的CSMCCa2 +内流 ,增加比率分别为 4 8%± 2 0 %、2 8 5 %± 4 6%、49 6%± 3 3 % ;(3 ) genistein (2 5 ,5 ,10 μmol·L-1)能浓度依赖性降低环匹阿尼酸 (Cyclopiazonicacid ,CPA ,10 μmol·L-1)刺激引起的CSMCCa2 + 内流 ,抑制率分别为 6 5 %± 3 0 %、2 2 5 %± 5 2 %、     

双苯氟嗪、氟桂利嗪、桂利嗪对5-羟色胺所致离体猪基底动脉收缩的拮抗作用(英文)

目的:探索双苯氟嗪(Dip,一种我国自行合成的桂利嗪衍生物),对5-羟色胺所致的脑动脉收缩的影响。方法:比较双苯氟嗪、氟桂利嗪(Flu)、桂利嗪(Cin)对5-羟色胺所致离体猪基底动脉收缩的抑制及两种收缩成分的影响。结果:三者的拮抗作用强度顺序(IC_(50))为Dip 4.0μmol·L~(-1)>Flu15.6μmol·L~(-1)>Cin 25.2umo·L~(-1)。这三种药对5-羟色胺所致离体猪基底动脉的两种收缩成分均有拮抗。Dip和Cin抑制收缩的快速相强于持续相,而Flu对二者的作用无显著差异。结论:在Dip,Flu和Cin三种药之中,Dip对脑血管的扩张作用最强,其原因主要与抑制内钙的释放有关。