大黄素对家兔离体主动脉平滑肌细胞增殖影响的可能途径

目的:探讨大黄素干预对家兔离体血管平滑肌细胞增殖的作用,并观察其浓度效应剂量。方法:实验于2004-06/12在第三军医大学西南医院药学部完成。选择雄性家兔1只作为动脉平滑肌细胞的动物来源,采用组织贴块法进行主动脉平滑肌细胞的培养。细胞培养成功后分2组:对照组和大黄素组。对照组不加药,大黄素组按剂量分为5个亚组即1.25,2.5,5,10,20mg/L组,每组6孔。选用生长良好的第三四代细胞,加3H-标记的胸腺脱氧核苷酸行液闪计数以表征细胞增殖的程度,细胞增殖测定采用酶标仪上比色(波长为570nm)用吸光度值表示。超过氧化物歧化酶测定采用邻苯三酚自氧化抑制法,直接以每毫升培养液中超过氧化物歧化酶kat表示。脂质过氧化物测定采用改良TBA微量法,以脂质过氧化物的稳定终产物丙二醛浓度(nmol/L)表示。实验结果采用单因素方差分析(方差齐性检验),用SNK法进行均数之间的显著性检验。结果:①大黄素对细胞增殖的影响:随着大黄素剂量的增加,吸光度值逐渐减少,抑制率逐渐增高。大黄素5,10,20mg/L剂量组的吸光度值明显低于对照组(0.594±0.037,0.410±0.014,0.301±0.024,0.730±0.041,P<0.05)。②大黄素对胸腺嘧啶脱氧核苷酸掺入量的影响:随着大黄素剂量的增加,胸腺嘧啶脱氧核苷酸掺入量逐渐减少,抑制率逐渐增高。大黄素5,10,20mg/L剂量组每分液闪计数值明显低于对照组大黄素(33537±3713,29304±4336,21155±1938,73958±2276,t=5.862,7.330,18.688,P<0.05)。③大黄素对超过氧化物歧化酶的影响:大黄素剂量1.25,2.5mg/L剂量组,超过氧化物歧化酶高于对照组,但差异不显著(P>0.05)。5,10,20mg/L剂量组明显高于对照组犤(118.19±3.56),(127.48±4.59),(140.23±4.96),(100.16±9.83)kat/L,t=4.224,6.170,8.914,P<0.05犦。④对丙二醛含量变化的影响:随着大黄素剂量的增加,其水平逐渐减少,当大黄素剂量为5,10,20mg/L时与对照组相比显著减低犤(2.336±0.113),(1.945±0.135),(1.381±0.193),(3.110±0.256)nmol/L,t=6.774,9.857,13.187,P<0.05犦。结论:随着培养液中大黄素浓度的增加,超过氧化物歧化酶活性逐渐增强,同时丙二醛水平逐渐降低,从而抑制血管平滑肌细胞的增殖此,此作用均从5mg/L剂量组开始,并具有浓度依赖性。     

恒磁场对离体兔动脉平滑肌细胞的抑制效应

目的 探讨恒磁场对离体兔主动脉平滑肌细胞增殖的抑制全盘和。为研究冠脉内磁化支架对经皮腔内冠脉成形术（ＰＴＣＡ）及支架术后冠脉再狭窄的防治作用提供理论基础。方法 用含１０％小牛血清的ＲＰＭＩ－１６４０培养液体外培养新西兰白兔的主动脉平滑肌细胞至３～７代,将含５％小牛血清的ＲＰＭＩ－１６４０培养液主动脉平滑肌细胞县液１００ｕｌ加入９６孔培养板中培养,对照组持续培养７２ｈ,实验组２４ｈ后将培养板置于０．     

衰老对离体大鼠主动脉内皮依赖性血管舒张反应的影响

目的：研究增龄对离体大鼠主动脉内皮依赖性舒张的影响。方法：成年及老年大鼠各20只，利用组织器官水浴系统，通过乙酰胆碱、N-硝基左旋精氨酸，测量离体主动脉环的舒张反应变化(％)。同时利用免疫组织化学方法观察诱导型一氧化氮合酶(inducible nitrleoxide synthase，iNOS)和内皮型一氧化氮合酶(endothelial nitricoxide svnthase，eNOS)在不同年龄大鼠主动脉中的表达。结果：①老年组大鼠主动脉环对1&;#215;10^-8，1&;#215;10^-7，1&;#215;10^-6，1&;#215;10^-5,1&;#215;10^-4mol／L乙酰胆碱诱发的内皮依赖舒张反应[(3．13&;#177;0．85)％，(12．60&;#177;2．18)％，(25．23&;#177;4．63)％，(37．73&;#177;6．95)％，(50．78&;#177;3．58)％]均较成年组明显减弱，两组之间有显著差异(P&;lt;0．01)。②离体大鼠主动脉环环经1&;#215;10^-4mol／LNNIA预处理20min，阻断一氧化氮合酶(nitricoxide synthase，NOS)后，同样剂量的乙酰胆碱不再诱发上述内皮依赖性舒血管反应。③eNOS组：在老年组血管内膜层可见棕褐色的阳性反应颗粒120．524&;#177;2．037，但较成年组相比，可发现阳性反应明显减弱。iNOS组：成年组内膜及中膜层可见较弱的棕褐色阳性反应颗粒沉在老年组(112．371&;#177;1．64)，阳性反应明显增强。结论：随增龄，血管内皮分泌的一氧化氮逐渐减少，导致血管内皮依赖性舒张减弱；NOS的异常表达对一氧化氮减少起着重要的作用，eNOS随增龄表达减弱，而iNOS随增龄表达则明显增强。     

家兔离体主动脉血管对天麻水煎剂干预的反应

背景:现代医学研究发现天麻(Gastrodiaelata B1)具有明显地心肌保护和血管扩张作用,可用于冠心病、高血压的治疗,但其作用机制仍需明确.目的:观察天麻水煎剂对去甲肾上腺素、KCl预收缩血管反应的舒张作用,并分析其作用途径.设计:分组设计、离体动物组织对照实验.单位:川北医学院生理学教研室.材料:选用健康家兔12只,兔龄七、八个月,雌雄不拘,由川北医学院实验动物中心提供.实验所用药品天麻水煎剂由慧仁堂药店鉴定提取,将其配制成10%,20%.40%和80%浓度备用.β肾上腺素能受体阻断剂普萘洛尔为北京第二制药厂产品:一氧化氮合酶抑制剂左旋硝基精氨酸为Sigma公司产品:甲烯蓝、环氧酶抑制剂吲哚美辛为江苏太仓制药厂产品.方法:实验于2006-01/2007-01在川北医学院药物研究所完成.采用家兔离体主动脉平滑肌标本,以去甲肾上腺素(1×10-6mol/L)预收缩主动脉后给予1.0,2.0,4.0,8.0和16.0g/L天麻水煎剂观察其张力变化,并观察去除血管内皮、给予1×10-4mol/L左旋硝基精氨酸、1×10-5mol/L甲烯蓝、1×10-5mol/L吲哚美辛和1×10-5mol/L普萘洛尔对血管张力的影响:血管张力经张力换能器输入BL-410智能型生物信号处理系统进行处理.另外观察8g/L天麻水煎剂对无内皮细胞血管环NA和KCl的量效收缩反应.主要观察指标:离体血管张力.结果:天麻对血管环静息张力无明显影响.但不同剂量的天麻可使1×10-6mol/L去甲肾上腺素预收缩血管产生明显舒张(r=0.85,t=18.45,P＜0.01).去除血管内皮、1×10-4mol/L左旋硝基精氨酸或1×10-5mol/L甲烯蓝可减弱天麻的舒张作用,但吲哚美辛和普萘洛尔对其无影响.另外,8g/L天麻水煎剂对无内皮细胞血管环去甲肾上腺素和KCI的量效收缩反应明显降低,且去甲肾上腺素和KCl的PD2(-log游离药物半数有效浓度)分别由温育前(6.90±0.93)mol/L和(1.53±0.55)mmol/L,降低为(5.61±0.70)mol/L和(1.10±0.25)mmol/L,(t=2.41,3.82,P＜0.05).结论:天麻水煎剂对主动脉的舒张是内皮依赖性的,并与内皮一氧化氮的释放有关;也可通过拮抗ROC和PDC通道,抑制Ca2 的内流和释放等机制有关.     

干扰素-γ对血管紧张素Ⅱ诱导的大鼠胸主动脉血管平滑肌细胞增殖的影响

目的 观察血管紧张素Ⅱ（AngⅡ）对培养大鼠胸主动脉血管平滑肌细胞（VSMCs)增殖的作用及干扰素-γ对其增殖的影响。方法 幼龄Wistar大鼠（4周龄）12只，分成对照组，血管紧张素Ⅱ（AngⅡ）组，干扰素-γ组和干扰素-γ AngⅡ组，采用组织块贴壁法培养胸主动脉平滑肌细胞，分别测定^3H-胸腺嘧啶（^3H-TdR),^3H-亮氨酸（^3H-Leu)的掺入量和细胞周期中各期细胞构成比。结果 AngⅡ（0.1μmol/L）作用24h能使VSMCs的^3H-TdR与^3H-TdR与^3H-Leu的掺入量比对照组增多。且S期和G2/M期细胞增多，细胞增殖指数增大；干扰素-γ组无以上作用，与AngⅡ组相比，干扰素-γ（500U/mL) AngⅡ组^3H-TdR,^3H-Leu的掺入量明显减少，S期细胞构成比和细胞增殖指数亦明显减少。结论 AngⅡ对血管平滑肌细胞有促增殖作用。这种促增殖作用能被干扰素-γ所拮抗。     

大黄素对IL-1β诱导血管平滑肌细胞增殖的影响及机制研究

[目的]观察大黄素对IL-1诱导下血管平滑肌细胞增殖的影响并探讨其作用机制.[方法]将IL-1β作用于平滑肌细胞,并用大黄素进行干预,实验设置对照组、IL-1β组、大黄素干预组.MTT法检测各组细胞的增殖能力;流式细胞术检测各组细胞的周期变化;RT-PCR检测各组细胞PCNA、CyclinD1 mRNA的表达;Western blot检测各组细胞PCNA、CyclinD1蛋白的表达以及JAK2、STAT 3的磷酸化情况.[结果]与对照组相比IL-1β组细胞的增殖能力显著增加(P＜0.01),与IL-1β组相比大黄素干预组细胞的增殖能力显著降低(P＜0.01).IL-1β组S期细胞所占比例(50.71±4.17)％,显著高于对照组(30.91±2.04)％(P＜0.01).大黄素干预组S期细胞所占比例(32.34±4.35)％,显著低于IL-1β组(P＜0.05),同时其G0～G1期细胞所占比例(48.83±4.55)％显著高于IL-1β组的(33.88±1.67)％(P ＜0.01).与对照组相比IL-1β组PCNA、CyclinD1 mRNA以及蛋白的表达都显著升高(P ＜0.01),而大黄素干预组PCNA、CyclinD1 mRNA以及蛋白的表达显著低于IL-1β组(P ＜0.05).与对照组相比IL-1β组JAK2、STAT3蛋白磷酸化程度显著增加(P＜0.05),与IL-1β组相比大黄素干预组JAK2、STAT 3蛋白磷酸化程度显著降低(P＜0.05).[结论]大黄素能够抑制IL-1β诱导的血管平滑肌细胞增殖,并且其抑制作用与JAK2/STAT 3信号通路的阻断有关.     

足阳明经穴针刺对家兔离体胃窦平滑肌细胞舒缩活动与胞内三磷酸肌醇含量的影响

目的：探讨针刺血清足三里、天枢、梁门等足阳明经穴针刺血清对家兔离体胃窦平滑肌细胞舒缩活动及胞内三磷酸肌醇含量的影响.方法：实验于2003-07/12在广州中医药大学和广州中医药大学附属第二医院完成.将45只家兔随机分为5组,每组9只.生理盐水组：耳缘静脉滴注0.9%氯化钠溶液剂量为10 mL/kg;阿托品模型组：耳缘静脉滴注以生理盐水配制的2.5 mg/100 mL的阿托品;足阳明经穴组、足少阳经穴组、足太阳经穴组均先静滴阿托品（滴注方法同阿托品模型组）,在滴完阿托品总量的一半时,开始分别电针双侧足阳明经穴（足三里、天枢、梁门、四白）、足少阳经穴（阳陵泉、带脉、日月、阳白）、足太阳经穴（承筋、背下点、背上点、攒竹）30 min,待电针完毕后,颈动脉采血6 mL离心15 min,取上清,制备成“针刺血清”.另取家兔10只,手术取胃,分离胃窦平滑肌,经Ⅱ型胶原酶消化后制备胃窦平滑肌细胞悬液,以“针刺血清”与家兔胃窦平滑肌细胞作用,用戊二醛固定后测定细胞长度和细胞内三磷酸肌醇含量.结果：进入细胞长度分析的家兔为43只,进入三磷酸肌醇含量分析家兔为45只.[1]细胞长度比较：足阳明经穴组明显低于足少阳经穴组[（42.05&;#177;7.85,77.33&;#177;9.80）μm,（P＜0.01）];足太阳经穴组明显高于生理盐水组[（75.88&;#177;9.14,64.58&;#177;6.59）μm,（P＜0.05）].[2]三磷酸肌醇含量比较：足少阳经穴组明显低于组阳明经穴组[（33.15&;#177;7.45,48.96&;#177;8.89）pmol/10^6,（P＜0.01）];阿托品模型组明显低于生理盐水组[（31.65&;#177;7.69,40.84&;#177;9.82）pmol/10^6,（P＜0.05）].结论：足阳明经穴针刺血清能使离体胃窦平滑肌细胞产生明显的收缩效应,其收缩效应的产生与升高细胞内的三磷酸肌醇含量有关.     

超声联合微泡对不同细胞周期血管平滑肌细胞增殖和凋亡的影响

目的 探讨超声联合微泡对不同细胞周期血管平滑肌细胞(VSMCs)增殖和凋亡的影响.方法 组织贴块法培养大鼠胸主动脉VSMCs,采用血清饥饿法和胸苷双阻断法使细胞同步化, 并用流式细胞仪分析同步化结果.以频率1 MHz、声强0.3 W/cm2的连续波超声联合脂质微泡辐照不同细胞周期的VSMCs 120 s,分别用噻唑蓝(MTT)比色法、流式细胞仪AnnexinV/PI染色、免疫细胞化学技术检测VSMCs的增殖、凋亡和增殖细胞核抗原(PCNA)表达.结果 成功获得同步化的G0/G1期和S期VSMCs,其同步化率分别为89.53%和66.87%.超声联合微泡可显著抑制S期细胞的增殖并下调PCNA蛋白表达,对G0/G1期细胞的增殖和PCNA表达无明显影响;G0/G1期和S期细胞的凋亡率均较辐照前增高,且对于S期细胞超声联合微泡诱导凋亡的作用更为显著.结论 超声联合微泡对处于增殖状态的VSMCs具有显著的抑制增殖和诱导凋亡的作用.     

足阳明经穴针刺对家兔离体胃窦平滑肌细胞舒缩活动与胞内三磷酸肌醇含量的影响

目的探讨针刺血清足三里、天枢、梁门等足阳明经穴针刺血清对家兔离体胃窦平滑肌细胞舒缩活动及胞内三磷酸肌醇含量的影响.方法实验于2003-07/12在广州中医药大学和广州中医药大学附属第二医院完成.将45只家兔随机分为5组,每组9只.生理盐水组耳缘静脉滴注0.9%氯化钠溶液剂量为10 mL/kg;阿托品模型组耳缘静脉滴注以生理盐水配制的2.5 mg/100 mL的阿托品;足阳明经穴组、足少阳经穴组、足太阳经穴组均先静滴阿托品(滴注方法同阿托品模型组),在滴完阿托品总量的一半时,开始分别电针双侧足阳明经穴(足三里、天枢、梁门、四白)、足少阳经穴(阳陵泉、带脉、日月、阳白)、足太阳经穴(承筋、背下点、背上点、攒竹)30 min,待电针完毕后,颈动脉采血6 mL离心15 min,取上清,制备成"针刺血清".另取家兔10只,手术取胃,分离胃窦平滑肌,经Ⅱ型胶原酶消化后制备胃窦平滑肌细胞悬液,以"针刺血清"与家兔胃窦平滑肌细胞作用,用戊二醛固定后测定细胞长度和细胞内三磷酸肌醇含量.结果进入细胞长度分析的家兔为43只,进入三磷酸肌醇含量分析家兔为45只.[1]细胞长度比较足阳明经穴组明显低于足少阳经穴组[(42.05±7.85,77.33±9.80)μm,(P＜0.01)];足太阳经穴组明显高于生理盐水组[(75.88±9.14,64.58±6.59)μm,(P＜0.05)].[2]三磷酸肌醇含量比较足少阳经穴组明显低于组阳明经穴组[(33.15±7.45,48.96±8.89)pmol/106,(P＜0.01)];阿托品模型组明显低于生理盐水组[(31.65±7.69,40.84±9.82)pmol/106,(P＜0.05)].结论足阳明经穴针刺血清能使离体胃窦平滑肌细胞产生明显的收缩效应,其收缩效应的产生与升高细胞内的三磷酸肌醇含量有关.     

BCL-6对血管平滑肌细胞增殖和氧化应激的影响

目的探讨原癌基因BCL-6对血管平滑肌细胞增殖和氧化应激的影响。方法人主动脉平滑肌细胞株(HA-VSMCs)随机分为三组:对照组、空载组和BCL-6组。对照组不进行转染,空载组转染空载体pcDNA3. 1 2μg,BCL-6组转染pcDNA3. 1-BCL-6 2μg。MTT法检测细胞增殖,二氯荧光素双醋酸盐法检测活性氧(ROS)水平,流式细胞仪检测细胞周期,Western blot检测BCL-6及α-SMA蛋白表达。结果转染后24 h与48 h,BCL-6组的细胞增殖指数显著高于对照组与空载组(P 0. 05),BCL-6组的ROS水平均显著对照组与空载组(P 0. 05)。转染后48h,BCL-6组的GO/G1期比率、α-SMA蛋白相对表达量显著低于对照组与空载组(P 0. 05),S期比率、BCL-6蛋白相对表达量显著高于对照组与空载组(P 0. 05)。对照组与空载组对比差异无统计学意义(P 0. 05)。结论 BCL-6可抑制血管平滑肌细胞α-SMA蛋白的表达,抑制细胞周期由G2期向M期转变,激活氧化应激反应,从而促进细胞增殖。     

Preparation of functional smooth muscle cells from the rabbit aorta

A procedure for dissociating the rabbit aorta into single, functional smooth muscle cells is described. After removal of adventitia and intima, slices of media were incubated with purified collagenase, elastase, and soybean trypsin inhibitor in a Krebs-Ringer buffer modified with Hepes, amino acids, and a [Ca2+] of 0.2 mM. After enzymatic digestion and mechanical shear, the yield of dispersed cells was approximately 25% based on DNA recovered. Greater than 95% of the cells excluded trypan blue and approximately 80-90% adhered to tissue culture dishes. By phase contrast microscopy, most of the cells were elongate and approximately 10 micron X 30 micron in size. The remainder were either spherical or highly crenated and contracted. Electron microscopy of the cells showed that immediately after dissociation greater than 95% could be identified as smooth muscle, though most had undergone some degree of structural change compared to cells in situ. Depending on the preparation, from 5 to 50% of these cells contracted in response to agonists. Cells shortened by 10-15% and developed numerous evaginations when stimulated by angiotensin II norepinephrine, or carbamylcholine. Cells relaxed after washout of agonists and could subsequently be restimulated. Specific inhibitors of each of the agonists blocked the contractile response. Dispersed cells cultured for 1-5 days contracted in even higher numbers than the freshly prepared cells, suggesting restoration of hormone binding and/or contractile function in culture. This preparation provides a system in which the physiology of individual vascular smooth muscle cells may be studied.     

Thrombin stimulates proliferation of cultured rat aortic smooth muscle cells by a proteolytically activated receptor.

Thrombin has been implicated in the stimulation of smooth muscle cell (SMC) proliferation that contributes to post angioplasty restenosis. The present studies demonstrated that human alpha-thrombin was a potent and efficacious mitogen for cultured rat aortic SMC, stimulating an increase in 3H-thymidine incorporation, as well as an increase in cell number at 1 to 10 nM concentration. gamma-Thrombin, which is enzymatically active but lacks fibrinogen clotting activity, stimulated SMC mitogenesis but was approximately 10-fold less potent than alpha-thrombin. In contrast, D-phenylalanyl-L-propyl-L-arginyl-chloromethyl ketone-alpha-thrombin, which lacked enzymatic activity, had no mitogenic effect. Diisopropylfluorophosphate-alpha-thrombin failed to stimulate mitogenesis except at concentrations having equivalent enzymatic activity as that of alpha-thrombin at its threshold for mitogenesis. Thus, thrombin-induced proliferation was dependent on enzymatic activity. A 14-residue peptide (SFLLRNPNDKYEPF) corresponding to amino acids 42 through 55 of the human thrombin receptor (Vu, T. K., D. T. Hung, V. I. Wheaton, and S. R. Coughlin, 1991. Cell. 64:1057-1068) had full efficacy in stimulating SMC proliferation. Reversing the first two amino acids of this peptide abolished mitogenic activity. Northern analysis demonstrated that SMC expressed a single mRNA species that hybridized to a labeled thrombin receptor cDNA probe. These findings indicate that alpha-thrombin stimulates SMC proliferation via the proteolytic activation of a receptor very similar or identical to that previously identified.     

Human macrophages modulate the phenotype of cultured rabbit aortic smooth muscle cells through secretion of platelet-derived growth factor

Phenotypic change of aortic smooth muscle cells (SMC) is a key step for their abnormal proliferation in atheromatous lesions. In this study modulation of the growth properties of SMC by macrophages was investigated to clarify the mechanism regulating the SMC phenotype. Cultured rabbit SMC preincubated with either macrophages derived from human peripheral monocytes, or conditioned medium from macrophages grew faster than control SMC in the absence of either macrophages or conditioned medium. SMC preincubated with purified platelet-derived growth factor (PDGF) also grew faster than control SMC in the absence of PDGF, and their rapid growth was maintained for at least two passages. SMC preincubated with conditioned medium of macrophages plus anti-PDGF antibody did not grow faster than control SMC. Furthermore SMC preincubated with PDGF acquired the ability to secrete some mitogen, which differed from PDGF. These results suggest that macrophages modulate the phenotype of SMC by a mechanism mediated by PDGF. As a result the SMC grow faster and at the same time secrete some mitogen probably distinct from PDGF in an autocrine manner.     

同型半胱氨酸刺激动脉平滑肌细胞的初步研究

目的观察不同分型及不同浓度的同型半胱氨酸(Hcy)刺激动脉平滑肌细胞的结果,揭示高同型半胱氨酸血症致动脉粥样硬化的作用机制。方法对大鼠动脉平滑肌细胞进行体外原代培养,在培养中加入不同型及不同浓度的同型半胱氨酸进行对平滑肌细胞刺激,然后用MTT比色方法,观察平滑肌细胞的增殖情况。结果经单因素方差分析,DHcy促进平滑细胞的增殖与对照组之间没有显著性差异。D,LHcy与LHcy在一定浓度时,与对照组之间存在明显差异(P<001)。DHcy、D,LHcy、LHcy在促进平滑肌细胞增殖方面,经双因素方差分析,只有D,LHcy及LHcy之间存在显著差异(P<005)。结论大鼠动脉平滑肌细胞的增殖程度与D,LHcy及LHcy的水平呈正相关,而DHcy促进平滑肌细胞增殖作用不明显。LHcy促进平滑肌细胞增殖的程度与D,LHcy之间差异没有显著性,与DHcy之间存在显著性差异。     

Alterations in 28Mg distribution and movements in rabbit aortic smooth muscle

The distribution and transmembrane fluxes of 28Mg were examined in the isolated media-intimal layer of rabbit aorta. Accumulation of 28Mg was slow and not complete after a 3-hr incubation. The major portion of the cellular Mg++ is not exchangeable. The 28Mg efflux rate was increased by 1.5 mM nonradioactive Mg++ after a time lag of 5 to 10 min; this increase was blocked reversibly by decreasing bathing solution temperature to 4 degrees C. A rapid and sustained increase in 28Mg efflux rate was elicited with added EDTA. Accumulation of 28Mg by rabbit aorta was increased more than 5-fold by substituting sucrose for NaCl in the bathing solution. Q10 values obtained for Mg++ accumulation in rabbit aorta incubated at different temperatures either in normal solution or low-Na+ solution ranged from 1.3 to 2.0. Uptake of 28Mg was inhibited substantially by 60 mM added K+, 1.5 or 15.0 mM La , 7 mM neomycin or 1 microgram/ml of antimycin A. These ions and drugs did not significantly increase 28Mg efflux when added during the slow component phase of the washout. Thus, the major portion of slowly accumulated Mg++ appears to be stored and exchanged intracellularly. The transmembrane movements of Mg++ depend upon simple diffusion, Mg++-Mg++ exchange and a transport process that is increased when Na+ is decreased. The EDTA-induced increase in 28Mg efflux rate may result from nonspecific membrane permeability increases, whereas ions and drugs decrease cellular Mg++ content by reducing uptake rather than increasing loss.     

Effects of nifedipine on smooth muscle cells of the rabbit mesenteric artery

The effects of nifedipine on electrical and mechanical responses of smooth muscle cells of the rabbit mesenteric artery were investigated using microelectrode and isometric tension recording methods for intact cells. The effects of nifedipine on the mechanical response on saponin-treated skinned muscles were also studied. Nifedipine inhibited the Ca spike evoked by outward current pulses in the presence of tetraethylammonium and that by perivascular nerve stimulation without affecting the amplitude of excitatory junction potentials. Nifedipine (less than 3 X 10(-7) M) modified neither the amplitude of excitatory junction potentials nor the facilitation process. This drug inhibited the contractions evoked by direct muscle stimulation under conditions of treatment with guanethidine and tetrodotoxin, excess concentrations of [ K ]O, exogenously applied norepinephrine (NE) and perivascular nerve stimulation. The K-induced contraction was markedly inhibited by nifedipine (greater than 3 X 10(-9) M) and the potency of the inhibitory action of nifedipine appeared in the following order: direct muscle stimulation greater than perivascular nerve stimulation greater than exogenously applied NE. Nifedipine inhibited the NE-induced oscillatory contractions more than the NE-induced tonic and phasic contractions. In Na-free solution, the tissue generated a small tonic contraction after 20 to 30 min superfusion. This contraction ceased with application of nifedipine. In the saponin-treated skinned muscles (50 micrograms/ml for 20 min), Ca accumulation into and Ca release from the store sites, as well as the contractile proteins including calmodulin, were not affected by nifedipine (1 X 10(-7) M). These results indicate that nifedipine only acts on the myoplasmic membrane of smooth muscles of the mesenteric artery. The nifedipine-induced relaxation appears to be due to inhibition of the voltage-dependent Ca channel.     

Studies on cholesterol esterase in cultured rabbit smooth muscle cells

Acid and neutral cholesterol esterases (CEases) in cultured rabbit smooth muscle cells (SMC) were examined. The optimal pH of acid CEase was 4.5 and its apparent Km was 83 microM. The optimal pH of neutral CEase was 7.5 and its apparent Km was 38 microM. CEases were most active with substrate vesicles prepared with phosphatidylcholine. Acid CEase hydrolyzed cholesteryl [1-14C]oleate labeled LDL (LDL-CE), but neutral CEase did not. Incubation with LDL increased the acid and neutral CEase activities of SMC, but incubation with acetylated-LDL did not increase their activities.