二十二碳六烯酸对大鼠肺动脉平滑肌细胞大电导钙激活性钾通道的影响

 目的： 探讨二十二碳六烯酸（DHA）对大鼠肺动脉平滑肌细胞（PASMCs）大电导钙激活性钾离子通道（BKCa）的作用。方法：采用酶解法获得单个活性良好的PASMC,PASMCs的BKCa电流变化采用全细胞膜片钳技术记录,PASMCs胞浆内游离钙离子浓度(［Ca2+］i)采用激光共聚焦显微镜技术测定。结果：DHA 0.01 μmol/L对BKCa无显著激活作用;0.1、1、10 μmol/L DHA可显著激活BKCa。不同浓度的DHA（0、0.1、1、10 μmol/L）在指令电压为+60 mV时,BKCa电流密度分别为(30.5±6.5) pA/pF、(59.4±5.8) pA/pF、(87.2±4.3) pA/pF和(117.3±7.1) pA/pF (P<0.01)。急性缺氧在指令电压为+60 mV时,BKCa电流密度从(32.7±8.5) pA/pF降低至(11.9±5.8) pA/pF (P<0.01)。DHA (10 μmol/L) 能明显逆转急性缺氧对BKCa的抑制作用（P<0.01）,同时DHA触发PASMCs内［Ca2+］i的增加,最大增加速率为（71.9±4.1）%（P<0.01）。结论：DHA通过增加PASMCs［Ca2+］i激活BKCa,逆转急性缺氧对BKCa的抑制作用,具有舒张肺血管的作用。     

Stretch-inactivated cationic channels in single smooth muscle cells

Stretch-inactivated channels (SICs) were identified in single smooth muscle cells freshly dissociated from the stomach of the toad, Bufo marinus. In both cell-attached and excised inside-out patches, negative pressure applied to the extracellular surface of the membrane patch suppressed the activity of SICs. These channels were permeable to cations and were not significantly permeable to Cl^–. The current-voltage relationship showed outward rectification in cell-attached patches with high NaCl in the pipette solution (2 mM MgCl₂), and the slope conductance at negative potentials was approximately 8 pS under these conditions. When divalent cations were eliminated from the pipette solution, the slope conductance at negative potentials increased to 30 pS. No significant voltage dependence of SIC gating could be observed between –100 mV and 60 mV.     

Calcium channel currents in isolated smooth muscle cells from the basilar artery of the guinea pig

Ca²⁺ channel currents were studied in smooth muscle cells from the basilar artery of the guinea pig using the whole-cell patch-clamp technique, 10 mM Ba²⁺ as the charge carrier and strong buffering of intracellular Ca²⁺ with EGTA (pCa_i=8). Cell capacitance was 18.8 ± 6.6 pF (n= 96) and maximum current density at +10 to +20 mV (holding potential < –55 mV), measured early in dialysis, was –14.8 ± 4.9 pA/pF (n= 83). Currents reversed at approximately +95 mV and, at more positive potentials, outward Cs⁺ currents were recorded that were blocked by either external Cd²⁺ or Ca²⁺. One component of current was identified that had properties consistent with L-type channels. On the basis of measurements of tail currents, its threshold for activation was –15 mV, its voltage dependence of activation was steep and it was half-activated at +8.5 mV. It inactivated very slowly at +15 mV (2787 ± 511 ms) and it deactivated rapidly (251 ± 55 s) at –55 mV. It was quickly lost during dialysis and was largely blocked by 1 nM nifedipine (1-s pulses, holding potential = –55 mV). A second component, termed B-type current, was identified that had properties inconsistent with those of T-type channels. On the basis of tail currents, its threshold for activation was –30 mV, its voltage dependence of activation was less steep and it was half-activated at +33.7 mV. It was half-inactivated at –32.1 mV, it inactivated with a time constant of 162 ± 13 ms at +15 mV and it deactivated relatively slowly (3.8 ± 0.8 ms) at –55 mV. It was less sensitive than L-type current to dialysis and to block by nifedipine.     

Membrane rectification in single smooth muscle cells from the rat tail artery

Membrane rectification to depolarization was studied by voltage recording with patch electrodes in freshly isolated cells from the rat tail artery. Injection of depolarizing currents elicited electrotonic potentials that developed with a single-exponential time course (time constant of 94.8 ms). When the cell was depolarized beyond –30 mV, delayed rectification was observed. A second type of rectification, characterized by oscillations, was observed when the cell was depolarized positive to + 30 mV. The threshold of this rectification and the oscillations were sensitive to changes in intracellular Ca²⁺. Delayed rectification was more sensitive to 4-aminopyridine but more resistant to tetraethylammonium and charybdotoxin than the Ca²⁺-sensitive rectification. A 4-aminopyridine-sensitive outward current (I _K,dr) with a threshold of around –30 mV and a second Ca²⁺-sensitive outward current (I _K,Ca) activated at around + 30 mV were observed from whole-cell voltage clamp recordings. I _K,Ca was blocked by tetraethylammonium and charybdotoxin. An 11-pS and a 122-pS channel, having characteristics similar to I _K,dr and I _K,Ca respectively, were identified from single-channel recordings. These observations showed how membrane depolarization of vascular smooth-muscle cells was regulated by these two populations of K⁺ channels under various conditions.     

Aluminofluoride activates hyperpolarization- and stretch-activated cationic channels in single smooth muscle cells

Aluminofluoride (AF) has a variety of biological actions such as activation of GTP binding proteins and inhibition of phosphatases. In the present study, the effects of AF on hyper-polarization- and stretch-activated cationic channels (HA-SACs) were investigated in isolated gastric smooth muscle cells from the toad, Bufo marinus, using the patch-clamp technique. In cell-attached patches extracellular application of AF (20 mM KF plus 20 M AlCl₃) reversibly increased HA-SAC activity without changing its voltage sensitivity. The single channel current amplitude of HA-SACs was not affected during this procedure. The mechanism of AF-induced activation of HA-SACs remains unclear. However, this activation may play a role in contraction of smooth muscle induced by AF.     

Protein kinase A increases availability of calcium channels in smooth muscle cells from guinea pig basilar artery

We studied single Ca²⁺ channels in smooth muscle cells from the basilar artery of the guinea pig using conventional patch-clamp techniques. With 40 mM or 90 mM Ba²⁺ as the charge carrier, a 23-pS inward current channel was observed in 46/187 cell-attached patches studied without the dihydropyridine, BAY K8644, in the pipette solution. At 0 mV, this channel exhibited short and long openings with time constants of 1.03 and 3.65 ms, respectively. The probability of channel opening was voltage dependent with half-activation occurring at +9.9 mV. In 14/26 patches tested, addition of 8-bromo-cyclic adenosine monophosphate (8-Br-cAMP) to the bath increased the probability of opening at -10 mV by a factor of 2.6, from 0.0272±0.0429 to 0.0695±0.0788 (P <0.01, paired t-test). Mean data from five patches fit to a Boltzmann function indicated that at positive potentials, the probability of opening increased by a factor of 1.7, from 0.352 to 0.600, whereas the voltage dependence, the number of channels, the number of open states, the time constants of the open states, and the proportion of time spent in each open state were unchanged. When BAY K8644 was added to the pipette solution, the 23-pS channel was observed in nearly all patches (62/66), but the voltage dependence of activation was shifted –15.3 mV compared to control. In some patches studied with 90 mM Ba²⁺, a 9-pS inward current channel also was observed and its activity also was increased significantly by 8-Br-cAMP. When membrane patches were excised from the cell and studied in an inside-out configuration, single-channel activity due to the 23-pS channel lasted 1–3 min before being irreversibly lost, regardless of the presence of BAY K8644 in the pipette or of 8-Br-cAMP plus Mg · ATP and leupeptin in the bath. Subsequent addition of the catalytic subunit of protein kinase A (PKA_CS) did not restore Ca²⁺ channel activity. Conversely, when patches were excised into a solution already containing 8-Br-cAMP plus Mg · ATP, leupeptin and PKA_CS, channel activity was prominent and generally lasted until the seal was lost, or until the experiment was terminated at 30–45 min, unless protein kinase inhibitor also was present, in which case channel lifetime was short. Our findings indicate that availability of the L-type Ca²⁺ channel in basilar artery smooth muscle cells is increased by activation of cAMP-dependent protein kinase A, and that the (or one of the) phosphoprotein(s) involved may not be membrane bound.     

在inside-out膜片上caffeine对猪冠状动脉平滑肌细胞钙激活钾通道的调节作用及ryanodine的影响

目的:利用膜片钳技术,研究咖啡因对钙激活钾通道(calcium-activated potassium channel,KCa)的作用机制,及咖啡因存在时兰尼定(ryanodine)对KCa的影响以阐明冠状动脉平滑肌的调控机制。方法:采用急性酶分离方法,应用膜片钳单通道电流记录技术记录猪冠状动脉平滑肌钿胞上KCa电流活动。电流信号经放大、滤波及A/D、D/A转换后输入微机进行采样和储存。应用PCLAMP9.0软件系统进行数据采集及分析。结果:在内面向外式(inside-out)膜片下,咖啡因(0.1、0.5、1.0、5.0mmol/L)可浓度依赖性地增加通道开放概率,而对电流幅值无明显影响,开放概率增加是通过明显缩短平均关闭时间实现的(n=8,P0.01);洗去药物后通道活性可以恢复到对照水平;5.0mmol/L咖啡因对KCa激活作用最大(P0.01)。在细胞贴附式(cell-attached)膜片上,咖啡因激活KCa后,ryanodine(10-40μmol/L)浓度依赖性地抑制通道开放概率,开放时间缩短,关闭时间延长,对电流幅度无明显影响。结论:在inside-out膜片下,咖啡因能够直接激活猪冠状动脉平滑肌细胞KCa通道。在cell-atta-ched构型上,ryanodine可通过胞内一定的信号通路浓度依赖性间接抑制咖啡因对KCa激活。     

慢性缺氧对大鼠肺动脉平滑肌细胞胞内钙的影响及其机制研究

目的:研究慢性缺氧对大鼠肺动脉平滑肌细胞(PASMCs)胞内钙浓度(［Ca²⁺］_i)的影响及L-型钙通道和胞内钙库的作用,为缺氧性肺动脉高压(HPH)发病机制的进一步研究提供理论依据。 方法:复制大鼠缺氧性肺动脉高压动物模型，利用Fura－2/AM钙离子成像方法测定PASMCs在不同钙离子浓度细胞外液及L-型钙通道阻滞剂nifedipine和IP3R钙通道抑制剂肝素干预前后 ［Ca²⁺］_i变化。 结果:(1)缺氧＋含钙外液组PASMCs ［Ca²⁺］_i 显著高于对照＋含钙外液组（P<0.05）。缺氧＋含钙外液组PASMCs ［Ca²⁺］_i显著高于缺氧＋无钙外液组（P<0.05）。(2)缺氧nifedipine组PASMCs［Ca²⁺］_i在加药前后无显著差异（P>0.05）。（3）缺氧未干预组与缺氧肝素组PASMCs ［Ca²⁺］_i无明显差异（P>0.05）。 结论:慢性缺氧可使PASMCs的［Ca²⁺］_i增加。慢性缺氧引起［Ca²⁺］_i增加可能与细胞外钙内流有关，L-型钙通道和IP3R钙通道在调节［Ca²⁺］_i的过程中可能不独立发挥作用。     

丹参素对猪冠脉平滑肌细胞钙激活钾通道的作用

目的观察丹参素(DS-182)对原代培养猪冠脉平滑肌细胞钙激活钾通道(KCa)的作用,从单个钾通道水平揭示其扩冠机制。方法采用膜片钳单通道电流记录技术。结果①猪冠脉平滑肌细胞KCa的单通道电导值高,为(291.74±4.89)pS;对膜电位变化及细胞内钙离子浓度变化敏感,能被5~20 mmol/L膜内侧四乙基铵(TEA)所阻断。②在细胞贴附式膜片方式下,细胞外应用10~20μmol/L的DS-182对通道具明显的激活效应;而在内面向外式膜片方式下,细胞内应用5~30μmol/L的DS-182对通道均无明显作用。结论DS-182对通道的激活作用是非直接的,需要一系列的胞内过程。     

Multiple types of voltage-dependent Ca channels in mammalian intestinal smooth muscle cells

(1) Whole-cell and single channel recording techniques have been applied to smooth muscle cells isolated from guinea-pig taenia coli to examine whether multiple types of Ca channels exist. (2) Whole-cell recordings under physiological Ca concentration (1.8 mM) revealed two current components with fast and slow inactivating kinetics. The fast inactivating component was present when cells were held at very negative potentials (–80 mV). It was insensitive to the dihydropyridine (DHP) derivative, nifedipine. In contrast, the slow inactivating component was present at less negative holding potentials. It was blocked by nifedipine. (3) The two current components were found to have closely similar voltage dependencies for activation. (4) These results suggest that the fast inactivating decay of the Ca current was mediated not only by the entry of Ca into the cell but also by a voltage-dependent process via a different type of Ca channel with fast inactivating kinetics. (5) Recordings from cell-attached membrane patches with 100 mM external Ba clearly showed the existence of multiple types of Ca channels with different conductances. (6) The large conductance channels (30 pS) activated at more positive potentials (0 mV) and their averaged current decayed much more slowly. The DHP Ca antagonist, nifedipine, inhibited the large conductance channels increasing the proportion of blank sweeps and reducing the averaged current. On the other had, the DHP Ca-agonist, BayK 8644, increased the averaged current by increasing the mean open-times of the large conductance channels. The presence of micromolar Cd in the patch pipettes produced a flickering block of the large conductance channels. (7) The small conductance channels (7 pS) activated at more negative potentials (–40 mV 30 mV) and the averaged current decayed rapidly within 100 ms. They showed no sensitivity to nifedipine and Cd ions. (8) In summary, we have identified at least two distinct types of Ca channels with different conductances, different pharmacological sensitivities, and different voltage- and time-dependent kinetics in smooth muscle cells isolated from guinea-pig taenia coli. The large and small conductance channels could be classified asl- andt-type Ca channels, respectively, which have been described in neuronal and heart cells. The contribution of those channels to the macroscopic component of whole-cell Ca current is also discussed.     

Cytoplasmic sequestration phenomena in smooth muscle cells of kidney resistance vessels and epithelioid cells of the juxtaglomerular apparatus

Summary The occurrence of vacuoles in cells of contractile tissues and especially in media cells of resistance vessels has been known for quite some time. Recently, it has been widely accepted that these vacuoles, characteristically lined by a double membrane, result from herniation of one vascular smooth muscle cell into the other as a result of vasoconstriction. In our electronmicroscopic investigations we found double membrane-bounded vacuoles not only in kidney resistance vessels of rats and mice under conditions of vasoconstriction, but also in control animals and animals with maximal renal vasodilation. Part of our observations are compatible with the assumption that such vacuoles arise from a damage of club-shaped, musculo-muscular contacts due to shape changes of media cells during maximal vasoconstriction or vasodilation. However, serial thin sectioning revealed that some of the cytoplasmic vacuoles have no connections with neighbouring cells. This finding and various parallels to the generation of autophagic vacuoles indicate that the so-called herniations may also represent demarcations of large cytoplasmic areas within an individual cell. Irrespective of the origin of these vacuoles, their contents show different stages of deterioration. At later stages, the vacuoles appear to be adjacent, with only one membrane, to the extracellular space, into which they are believed to discharge finally. Cytoplasmic vacuolization has not only been observed in smooth muscle cells, but also in juxtaglomerular epithelioid cells of the afferent arteriole. Here the vacuoles - besides other organelles - also contain secretory granules; it is therefore proposed that autophagic phenomena with final extrusion of cytoplasmic material may be involved in the programmed down-regulation of the granular renin store following inhibition of renin synthesis and secretion.These studies were supported by the German Research Foundation within the Forschergruppe Niere, Heidelberg     

钾通道对大鼠肺动脉平滑肌细胞[Ca2+]i的调节

目的:探讨在常氧、低氧条件下钾通道对大鼠肺动脉平滑肌细胞(PASMCs)[Ca²⁺]_i的调节。方法:采用钙荧光探针(Fura-2/AM)负载培养的大鼠PASMCs,观察常氧、低氧培养后3种钾通道抑制剂(4AP,TEA、Glib)对PASMCs[Ca²⁺]_i的调节,同时用四唑盐(MTT)比色法比较4AP、TEA、Glib对大鼠PASMCs增殖的影响。结果:(1)常氧状态下,PASMCs[Ca²⁺]_i为(156.91±8.60)nmol/L,低氧时为(294.01±16.81)nmol/L(P＜0.01)。(2)常氧状态下,4AP可引起PASMCs[Ca²⁺]_i升高,达(280.52±23.21)nmol/L(P＜0.01),而TEA、Glib无此作用。(3)低氧时,4AP和TEA都可引起PASMCs[Ca²⁺]_i的升高,分别为(422.41±24.28)nmol/L、(380.84±11.02)nmol/L(P<0.01),Glib无作用。(4)MTT比色法中,常氧和低氧状态下4AP均引起吸光度(A)值升高,分别是0.582±0.062,0.873±0.043(P＜0.01)。TEA仅在低氧时A值升高(0.729±0.041,P＜0.05),而Glib无论常氧还是低氧均无影响。结论:无论常氧还是低氧条件下,电压依赖性钾通道(K_V)对PASMCs[Ca²⁺]_i及其增殖起主要作用。钙激活的钾通道(K_Ca)在常氧条件下对[Ca²⁺]_i不起调节作用,而在低氧下使[Ca²⁺]_i降低,反应性地调节PASMCs增殖。ATP敏感性钾通道(K_ATP)无论在常氧还是低氧情况下对[Ca²⁺]_i的调节不起作用。     

自发性高血压鼠脑基底动脉ET-1的表达及其意义

为探讨高血压时期内皮素-1(endothelin-1,ET-1)在脑血管神经源性调节中的作用,本研究应用反转录-聚合酶链式反应(RT-PCR)技术,检测了自发性高血压鼠脑基底动脉ET-1mRNA的表达变化;应用免疫印迹(Western blotting)方法,观察了自发性高血压鼠脑基底动脉ET-1的表达。结果显示:自发性高血压鼠脑基底动脉ET-1mRNA的表达较正常血压鼠明显增加;自发性高血压鼠脑基底动脉ET-1表达亦较正常血压鼠明显增加(P<0.05)。此结果提示自发性高血压鼠脑基底动脉高表达的ET-1和ET-1mRNA在自发性高血压鼠脑血管的神经源性调节中可能发挥重要作用。     

UTP对猪冠状动脉平滑肌细胞BK_(Ca)通道作用机制的研究

目的:利用膜片钳技术,研究UTP对急性酶分离的猪冠状动脉平滑肌细胞上BKCa通道的作用,探讨生理条件下UTP调节BKCa通道的机制。方法:应用Cell-attached技术记录急性酶分离的猪冠状动脉平滑肌细胞上BKCa电流。用pCLAMP9.0软件系统进行数据采集及分析。结果:在Cell-attached膜片上,80μM UTP可明显激活BKCa通道(P0.05,n=21)。PLC抑制剂U73122可抑制UTP对BKCa的激活作用(P0.05,n=5)。PKC激动剂PMA(10nM),可使BKCa通道开放概率明显降低(P0.05,n=9)。UTP(80μM)预处理细胞之后,IP3抑制剂2APB(80μM),可使BKCa通道开放受抑(P0.05,n=6)。结论:在Cell-attached膜片上,UTP能激活猪冠脉平滑肌细胞BKCa通道。PLC信使转导通路中的IP3介导的胞内Ca2+释放是UTP激活BKCa通道的重要途径。     

神经调节蛋白1对冠状动脉平滑肌细胞表达血管生成因子的影响

目的:探讨神经调节蛋白1(neuregulin-1,NRG-1)对人冠状动脉平滑肌细胞(HCASMCs)表达血管生成因子的影响。方法:培养HCASMCs,实验使用第3代细胞。用Western blot法检测细胞Erb B的表达和磷酸化。在正常、缺氧缺血清或NRG-1(100μg/L)处理条件下,用Western blot法检测血管内皮生长因子(VEGF)、血管生成素1(Ang-1)和血管生成素2(Ang-2)表达的改变。结果:Erb B2、Erb B3和Erb B4均能在HCASMCs中表达,加入NRG-1后,这3种Erb B的磷酸化水平均增加。与对照组比较,在缺氧缺血清组HCASMCs中VEGF和Ang-1的表达明显增加(P0.05),而Ang-2的表达差异无统计学显著性。与缺氧缺血清组比较,NRG-1处理组的HCASMCs表达VEGF和Ang-1进一步显著增加(P0.05),而Ang-2的表达差异无统计学显著性。结论:HCASMCs能表达Erb B2、Erb B3和Erb B4,加入NRG-1增强Erb B2、Erb B3和Erb B4的磷酸化。缺氧缺血清和NRG-1处理均能增加VEGF和Ang-1在HCASMCs中的表达。     

平滑肌细胞对共培养体系滋养细胞侵袭力与MMP-2及MMP-9表达的影响

目的模拟体内环境,建立可保持平滑肌细胞与绒毛外细胞滋养层细胞(EVCT)生物学特性的共培养细胞模型,应用于研究平滑肌细胞与滋养细胞理化特性与滋养细胞的侵袭行为。方法利用组织块培养法培养脐动脉平滑肌细胞,组织块培养、胰酶消化和Percoll梯度沉降,收集纯化人早孕绒毛组织的滋养细胞,免疫组化检测细胞的纯度。将滋养细胞与平滑肌细胞分别放入Transwell的上下小室,观察该模型下滋养细胞形态变化、细胞活力、侵袭力改变与分泌功能等特性。结果免疫组化显示EVCT的细胞角蛋白7阳性表达的细胞数占95%以上,SMC a-actin阳性表达的细胞数也超过95%,证实共培养系统中EVCT和SMC纯度均在95%以上,且生物学特性得以维持。上室中的EVCT保持了其侵袭能力,且平滑肌细胞能促进滋养细胞增殖活性、侵蚀能力及MMP2、MMP9的表达。结论成功地建立了平滑肌细胞与滋养细胞原代共培养系统模型,便于研究滋养细胞侵袭和子宫螺旋动脉重铸障碍的分子机制。     

低氧抑制猪肺动脉平滑肌细胞MMP-2和MMP-9的表达

目的探讨低氧对猪肺动脉平滑肌细胞（PASMC）分泌基质金属蛋白酶（MMPs）的影响。方法采用酶谱法测定PASMC培养基中MMP-2和MMP-9的酶活性，免疫印迹法检测培养基中MMP-2和MMP-9的蛋白表达，免疫组化法测定细胞原位MMP-2和MMP-9的蛋白表达，RT-PCR法检测mRNA的表达。结果低氧后PASMC分泌的MMP-2酶活性、细胞内外蛋白表达量、mRNA表达量均下降；MMP-9酶活性、细胞外蛋白表达量下降，而细胞内蛋白表达无变化。结论低氧可抑制PASMC分泌MMP-2和MMP-9的酶活性，其机制可能是低氧影响PASMC中MMP-2基因的转录、影响MMP-9蛋白表达后的分泌与活化，导致MMP-2和MMP-9酶活性的改变。     

蛋白激酶C活性下调对钙库操纵的钙通道活性和气道平滑肌细胞增殖的作用

目的:研究蛋白激酶C(PKC)活性下调对大鼠气道平滑肌细胞(ASMCs)的Ca2+库操纵的Ca2+通道(SOC)和ASMCs增殖的影响。方法:分离培养大鼠支气管平滑肌细胞;利用激光共聚焦显微镜测定ASMCs的fluo-3/AM的荧光信号;利用长时间暴露于PKC活化剂诱导PKC活性下调,观察PKC活性下调对ASMCs的SOC通道和增殖的影响;Alamar blue还原率测定法测定ASMCs的增殖。结果:PKC激活剂PMA(10μmol/L)和PDBu(1μmol/L)作用24h下调PKC活性后可以抑制ASMCs的增殖,PKC抑制剂chelerythrine同样抑制ASMCs的增殖;PKC活性下调和chelerythrine均可以抑制ASMCs的SOC通道活性;小剂量PKC激活剂PMA(100nmol/L)可以促进ASMCs的增殖,这种作用被SOC通道抑制剂SKF-96365抑制。结论:PKC活性下调或抑制导致SOC通道的活性下降,表明PKC可能参与了SOC通道功能调控;SOC通道开放可能参与了PKC促进ASMCs增殖的作用。