成纤维细胞内肌动蛋白、钙离子与瘢痕挛缩的相关研究

目的　探讨增生性瘢痕、瘢痕疙瘩成纤维细胞中肌动蛋白 (actin)和钙离子 (Ca2 + )含量与瘢痕挛缩的关系。方法　取两类瘢痕各 10例 ,蛋白质电泳及凝胶扫描测定成纤维细胞内总肌动蛋白 ,聚合 (F肌动蛋白 )、球 (G肌动蛋白 )肌动蛋白及F/G肌动蛋白比值。Fura 2荧光探剂标记、图像分析胞内Ca2 + 浓度。结果　增生性瘢痕总肌动蛋白 [(2 .3 8± 0 .3 5 )ng/10 4细胞 ]、F肌动蛋白 [(0 .98± 0 .19)ng/10 4细胞 ]、F/G肌动蛋白比值 (0 .68± 0 .0 7)、Ca2 + 浓度 (82 .64± 10 .0 0 )均明显高于瘢痕疙瘩总肌动蛋白 [(1.68± 0 .2 5 )ng/10 4细胞 ]、F肌动蛋白 [(0 .46± 0 .10 )ng/10 4细胞 ]、F/G肌动蛋白比值 (0 .3 6± 0 .0 5 )及Ca2 + 浓度 (73 .2 6± 2 .5 5 ) (P <0 .0 1)。G 肌动蛋白含量差异无显著性 (P >0 .0 5 )。结论　成纤维细胞内肌动蛋白与钙离子浓度的改变 ,是造成两类瘢痕不同挛缩特性的重要原因。     

川芎嗪对家兔阴茎海绵体平滑肌细胞游离钙浓度的影响

目的 :研究中药川芎嗪 (TMP)对体外培养家兔阴茎海绵体平滑肌细胞 (PCSMC)胞质内游离钙浓度([Ca2 + ] i)的影响。　方法 :用新型Ca2 + 荧光染色剂Fluo 3/AM负载家兔PCSMC ,细胞分为氯化钾 (KCl)作用组和去甲肾上腺素 (NE)作用组 ,应用激光扫描共聚焦显微镜 (LSCM)实时测定胞质内 [Ca2 + ] i的变化 ,分别观察不同浓度的TMP对高钾和NE诱导胞质内 [Ca2 + ] i升高的影响 ,并与经典钙拮抗剂维拉帕米 (Ver)的作用相对比。　结果 :静息状态下 ,TMP对家兔PCSMC胞质内 [Ca2 + ] i无明显影响。 1、1 0、1 0 0 μmol/LTMP能显著抑制高钾诱发的细胞内 [Ca2 + ] i升高 ,抑制率分别为 (38.6± 3 .0 ) %、(44.1± 2 .4) %和 (53 .7± 4 .1 ) % ;也能抑制 1 μmol/LNE诱发钙库释放所致的细胞内 [Ca2 + ] i升高 ,抑制率分别为 (1 3 .9± 2 .7) %、(2 1 .2± 1 .9) %和 (2 9.5± 3 .6) %。　结论 :TMP通过对家兔PCSMC电压依赖性钙通道和细胞内钙库释放的双重抑制作用 ,降低PCSMC胞质内 [Ca2 + ] i水平 ,其作用效果与Ver相似 ,这是TMP治疗阴茎勃起功能障碍的重要机制。     

聚集素抗LNCaP细胞凋亡作用的研究

目的研究聚集素抗LNCaP细胞凋亡的作用及聚集素反义寡核苷酸(AS-ODN)对肿瘤坏死因子-α(TNF-α)细胞毒性作用的影响。方法培养转染全长聚集素序列的LNCaP细胞(A)为实验组,野生型LNCaP细胞(L)及转染PIRES2-EGFP空载体的LNCaP细胞(M)为对照组,以20 ng/ml TNF-α进行诱导,MTT及ELISA法检测3组细胞增殖活性与凋亡程度；转染聚集素AS-ODN后A细胞分4组,①对照组：常规培养；②AS-ODN组：转染AS-ODN,培养液不含TNF-α；③TNF-α组：未转染AS-ODN,培养液含20ng/ml TNF-α；④TNF-α+AS-ODN组：转染AS-ODN,培养液含20ng/ml TNF-α,观察4组细胞增殖活性与凋亡程度的变化。结果L、M、A3组细胞增殖活性分别为0.84±0.03、0.85±0.04、0.95±0.03,L与M间差异无统计学意义(P＞0.05),L、M与A相比增殖活性显著降低(P值均＜0.01)。3组细胞ELISA吸光度值分别为0.59±0.04、0.62±0.03、0.33±0.04,L与M间差异无统计学意义(P＞0.05),但L、M与A相比凋亡程度显著增高(P值均＜0.01)。A细胞①～④组细胞增殖活性吸光度值分别为1.30±0.03,1.25±0.03,0.99±0.03,0.80±0.03,两两比较组间差异均有统计学意义(P值均＜0.05)；4组细胞凋亡程度吸光度值分别为0.02±0.00,0.21±0.02,0.63±0.07,1.16±0.04,两两比较组间差异均有统计学意义(P值均＜0.01)。结论转染并永久表达全长序列聚集素后,TNF-α对LNCaP细胞的细胞毒性作用显著降低,转染聚集素AS-ODN显著增强TNF-α的细胞毒性作用,聚集素具有抗LNCaP细胞凋亡的作用。     

胰岛素对体外培养兔骨骼肌肌管蛋白降解的调节作用

目的观察胰岛素对体外培养兔骨骼肌肌管蛋白降解的调节作用。方法无菌分离幼兔下肢骨骼肌肌肉,采用组织块法分离、培养成肌细胞,待其融合形成肌管,采用L-[3,5-3H]-酪氨酸标记肌管内蛋白后,随机分为对照组(用含体积分数10％胎牛血清的DMEM培养液培养)、胰岛素组(用含100 nmol／L胰岛素+体积分数10％胎牛血清的DMEM培养液培养)、地塞米松组(用含100 nmol／L地塞米松+体积分数10％胎牛血清的DMEM培养液培养)和胰岛素+地塞米松组(用含100 nmoL／L胰岛素+100 nmol／L地塞米松+体积分数10％胎牛血清的DMEM培养液培养),每组含24孔肌管。培养24 h后．应用液体闪烁计数仪测定培养液和肌管内L-[3,5-3H]-酪氨酸的含量,计算肌管内蛋白的降解率。RNA印迹法测定肌管内泛素-蛋白酶体C2亚基mRNA的表达水平,以其与内参照甘油醛-3-磷酸脱氢酶的灰度值之比表示。结果肌管内蛋白的降解比:地塞米松组为0．50±0．03,明显高于对照组(0．38±0．04,P<0．01);胰岛素组为0．35±0．03,明显低于对照组(P< 0．05);胰岛素+地塞米松组为0．41±0．03,明显低于地塞米松组(P<0．01),但仍高于对照组(P< 0．05)。肌管内泛素-蛋白酶体C2亚基的mRNA表达水平:与对照组(泛素2．4 kb条带为0．82±0．15、1．2 kb条带为0．60±0．10,C2亚基为0．75±0．16)比较,地塞米松组(泛素2．4 kb条带为2．15±0．23、1．2 kb条带为1．50±0．14,C2亚基为1．50±0．13)明显升高(P<0．01);胰岛素+地塞米松组(泛素2．4 kb条带为1．25±0．17、1．2 kb条带为0．85±0．09,C2亚基为0．90±0．15)明显低于地塞米松组(P<0．01);胰岛素组(泛素2．4 kb条带为0．85±0．07、1．2 kb条带为0．65±0．12,C2亚基为0．76±0．09)与对照组相近(P>0．05)。结论胰岛素对兔骨骼肌肌管内泛素-蛋白酶体途径的活性和蛋白代谢分别有较弱的促进与抑制作用,但能有效拮抗地塞米松诱导的该途径活性的增强和蛋白的高降解,其机制尚有待进一步探讨。     

肺癌淋巴结细胞培养及体内外的杀伤作用

目的探讨肺癌淋巴结细胞的培养方法及体内外杀伤作用。方法将肺癌引流淋巴结剪碎小于1 mm~3,悬于含有白细胞介素2(IL-2)的RPMI 1640培养基,置于37℃,5％CO_2培养箱培养,并行显微镜下观察和细胞计数。细胞扩增后用流式细胞仪测定细胞的免疫表型,测定其对自体肿瘤细胞的杀伤活性。将肺癌组织接种子裸鼠皮下,观察TDLN对移植瘤的抑制效果。结果淋巴组织在低浓度的IL-2培养1周,开始释放细胞群落,持续产生1～2月。1．0 g淋巴组织中在 7、14、21 d分别获得1．0×10~8、1．4×10~8和1．2×10~8细胞。表型分析显示TDLN细胞主要由CD3~+ T细胞(84．22％),和CD83~+成熟树突细胞(20．40％)组成,TDLN细胞对自体肿瘤细胞的杀伤率为 69．21％,明显低于外周血LAK细胞的杀伤率(30．17％,P<0．05)。TDLN处理后4周,移植瘤体积为(388．75±33．64)mm~3,而生理盐水组、IL-2组和LAK组分别为(742．56±160．14)mm~3、 (672．24±86．17)mm~3、(544．79±30．88)mm~3(P<0．01)。结论肺癌淋巴结细胞可在肺癌免疫治疗中发挥重要作用。     

生理浓度睾酮对前列腺素F_(2α)诱导SD大鼠血管平滑肌细胞中Ca~(2+)升高的影响

目的:观察生理浓度睾酮短时作用对前列腺素F2α(PGF2α)诱导血管平滑肌细胞(VSMCs)中Ca2+升高的影响。方法:贴块法培养雄性SD大鼠胸主动脉VSMCs,钙敏荧光指示剂Fura-2负载细胞,Nikon TE-2000E活细胞工作站对VSMCs内钙信号进行测定。结果:VSMCs给以单纯PGF2α(10μmol/L)刺激,[Ca2+]i在数秒钟内由基线水平100nmol/L左右迅速升至约500nmol/L的峰值,随后呈缓慢下降,约150s降至基线水平。在PGF2α作用的峰值给以生理浓度(40nmol/L)睾酮或乙醇溶媒干预,前者能够明显缩短[Ca2+]i由峰值回落至基线水平的时间[与乙醇溶媒相比,(104.9±27.0)svs(153.5±40.4)s,P<0.01]。在PGF2α作用前预先给以睾酮或乙醇溶媒处理2min,前者对PGF2α刺激的[Ca2+]i峰值水平无明显影响[与乙醇溶媒相比,(390.0±126.0)nmol/Lvs(403.4±160.7)nmol/L,P>0.05],但使[Ca2+]i由峰值降至基线水平的时间显著缩短[与乙醇溶媒相比,(120.6±32.0)svs(151.4±27.4)s,P<0.01]。结论:睾酮对PGF2α诱导的VSMCs中[Ca2+]i升高具有快速抑制作用,提示睾酮通过与VSMCs细胞膜作用抑制受体门控钙通道介导的Ca2+内流。     

低温保存-再灌注肝脏糖原含量与肝细胞凋亡的关系

目的　研究肝脏低温保存 再灌注期间肝糖原含量与肝细胞凋亡之间的关系及其相关机制。方法　制备糖原含量分别为 (1 5 .42± 2 .60 )mg/ g(A组 )、(51 .83± 6 .61 )mg/ g(B组 )及(63 .2 2± 5 .84)mg/ g(C组 )的 3组兔肝脏模型 ,检测各组肝脏低温保存 再灌注过程中肝细胞凋亡及肝细胞内游离Ca2 + 浓度变化情况。结果　各组肝脏于低温保存 9h后再灌注 60min时 ,组织内可见明显的肝实质细胞凋亡现象 ;其凋亡细胞数量分别为 (× 1 0 - 2 个 / μm2 ) :A组 :1 7.58± 3 .64 ,B组 :1 2 .61± 2 .95 ,C组 :8.2 7± 2 .60 ,3组间差异均有显著性 (P <0 .0 1 ) ;并且此时 ,3组肝实质细胞内游离Ca2 + 浓度 (nmol/L)依次为 :A组 :379.1 8± 50 .2 6 ,B组 :331 .59± 43 .56 ,C组 :2 81 .47±40 .1 2 ,各组间差异均有显著性 (P <0 .0 1 )。结论　肝脏低温保存 再灌注过程中 ,肝细胞内高糖原含量能显著拮抗再灌注期间肝实质细胞凋亡的发生 ;其可能与高糖原含量的肝实质细胞内ATP充裕 ,细胞膜上Ca2 + ATP酶功能完善 ,细胞内外Ca2 + 平衡相对稳定有关     

纤维连接蛋白调理的人表皮角质细胞粘附、迁移功能

目的观察纤维连接蛋白(FN)对人表皮角质细胞(EKC)在体外培养过程中有无粘附、迁移作用.方法利用不同剂量的FN,对新鲜EKC及体外培养7～10d的EKC进行粘附、迁移功能测定.应用间接免疫荧光染色法,检测α5β1受体在细胞培养前后的表达.结果(1)新鲜EKC在FN调理下的粘附、迁移值明显低于体外培养7～10d的EKC,粘附值分别为(0.9±0.5)%、(37.26±8.34)%(P＜0.01),迁移指数分别为17.5±2.5、48.7±3.2(P＜0.01).(2)培养1d的EKC及组织块体外培养后增生的表皮细胞,α5β1受体的表达染色阳性;培养7d的EKC及组织块增生表皮外周边缘染色呈强阳性;新鲜EKC染色阴性或弱阳性.结论(1)新鲜EKC与培养7～10d的EKC生物学功能间差异有显著性意义.(2)EKC生物学功能活跃的阶段(培养7d)和部位(组织块边缘增生的表皮细胞)α5β1受体表达最强.(3)体外培养能有效地诱导和激活EKC的粘附、迁移功能,使生物学功能相对静止的EKC变成了功能活跃的EKC细胞.     

氟桂利嗪对小鼠生精细胞T型Ca~(2+)通道的作用

目的:研究二苯烷基氨类衍生物氟桂利嗪(F lu)对小鼠生精细胞T型钙电流(ICaT)的影响。方法:采用急性机械分离的小鼠生精细胞,应用膜片钳全细胞记录技术,观察F lu对ICaT的影响。结果:钳制电位为-90 mV、刺激电压为-30 mV时,F lu(0.1、1、10、100μmol/L)可抑制小鼠生精细胞Ca2+电流,抑制率分别为(23.34±2.76)%、(46.04±3.52)%、(62.52±3.70)%、(73.52±3.12)%(P<0.05);F lu对T型Ca2+通道的半数最大抑制浓度为0.29μmol/L。结论:F lu对生精细胞T型Ca2+通道有阻断作用,呈浓度依赖性;Cav3.2是生精细胞T型Ca2+电流的主要贡献者。     

对不同月龄雄性大鼠成骨细胞内钙和钙通道电流的实验研究

目的研究不同月龄的雄性大鼠成骨细胞内钙浓度及钙通道电流是否存在差异.方法采用二次酶消化法分离不同月龄(分别为1、2、3、5、7、9、11、13、15)雄性大鼠的原代成骨细胞,通过激光扫描共聚焦技术测定细胞内游离钙浓度([Ca2+]i)(以平均荧光强度表示),同时应用全细胞膜片钳技术记录成骨细胞膜钙电流(ICa)的变化.结果共聚焦结果显示随月龄增加,成骨细胞内[Ca2+]i逐渐降低,但相邻2月龄组之间细胞内[Ca2+]i无显著性差异(P＞0.05);1、2、3月龄组与11、13、15月龄组成骨细胞内[Ca2+]i有显著性差异(P＜0.05),5、7、9月龄组与各组相比均无差异(P＞0.05).应用全细胞膜片钳技术发现刺激电压为+10 mV时,2、7、13月龄组鼠ICa分别为(-392.77±97.07)pA、(-330.33±33.86)pA和(-287.68±71.01)pA,13月龄组鼠与2月龄组鼠相比,ICa明显降低(P＜0.05),而7月龄组鼠与2月龄组鼠和13月龄组鼠相比,ICa均没有明显差异(P＞0.05).结论不同月龄大鼠成骨细胞内[Ca2+]i存在差异,其机制可能与细胞膜上钙通道活性改变相关.     

Intracellular free calcium accumulation in ferret vascular smooth muscle during crystalloid and blood cardioplegic infusions.

OBJECTIVE: The effects of magnesium- and potassium-based crystalloid and blood-containing cardioplegic solutions on coronary smooth muscle intracellular free calcium ([Ca2+]i) accumulation and microvascular contractile function were examined. METHODS: Isolated ferret hearts were subjected to hyperkalemic (25 mmol/L K+) blood cardioplegic infusion, hypermagnesemic (25 mmol/L Mg2+, K+-free) crystalloid cardioplegic infusion, or hyperkalemic crystalloid cardioplegic infusion for 1 hour. Coronary arterioles were isolated, cannulated, and loaded with fura 2. Reactivity and [Ca2+]i were assessed with videomicroscopy. [Ca2+]i was measured at baseline and after application of 50 mmol/L KCl. In addition, [Ca2+]i and vascular contraction were measured during exposure to Mg2+ and K+ cardioplegic solution at both 4 degrees C and 37 degrees C. RESULTS: From a baseline [Ca2+]i of 177 +/- 52 nmol/L, K+ cardioplegic infusion (302 +/- 80 nmol/L potassium) markedly increased [Ca2+]i, whereas blood cardioplegic infusion (214 +/- 53 nmol/L) and Mg2+ cardioplegic infusion (180 +/- 42 nmol/L) did not alter [Ca2+]i. Although a difference between groups in percentage contraction after application of 50 mmol/L KCl was not observed, [Ca2+]i increased significantly more in vessels in the control group (764 +/- 327 nmol/L) and the K+ crystalloid cardioplegic infusion group (698 +/- 215 nmol/L) than in vessels in the blood cardioplegic infusion group (402 +/- 45 nmol/L) and the Mg2+ cardioplegic infusion group (389 +/- 80 nmol/L). Mg2+ cardioplegic solution induced no microvascular contraction at either 4 degrees C or 37 degrees C, nor was an increase in [Ca2+]i observed. K+ cardioplegic solution induced microvascular contraction at 37 degrees C but not at 4 degrees C; it increased [Ca2+]i at both 4 degrees C and 37 degrees C. CONCLUSION: An Mg2+-based cardioplegic solution, or appropriate Mg2+ or blood supplementation of a K+ crystalloid cardioplegic solution, may decrease the accumulation of [Ca2+]i in the vascular smooth muscle during ischemic arrest.     

Experimental conditions are important determinants of cardiac inotropic effects of propofol

BACKGROUND: The rationale for this study is that the depressant effect of propofol on cardiac function in vitro is highly variable but may be explained by differences in the temperature and stimulation frequency used for the study. Both temperature and stimulation frequency are known to modulate cellular mechanisms that regulate intracellular free Ca2+ concentration ([Ca2+]i) and myofilament Ca2+ sensitivity in cardiac muscle. The authors hypothesized that temperature and stimulation frequency play a major role in determining propofol-induced alterations in [Ca2+]i and contraction in individual, electrically stimulated cardiomyocytes and the function of isolated perfused hearts. METHODS: Freshly isolated myocytes were obtained from adult rat hearts, loaded with fura-2, and placed on the stage of an inverted fluorescence microscope in a temperature-regulated bath. [Ca2+]i and myocyte shortening were simultaneously measured in individual cells at 28 degrees or 37 degrees C at various stimulation frequencies (0.3, 0.5, 1, 2, and 3 Hz) with and without propofol. Langendorff perfused hearts paced at 180 or 330 beats/min were used to assess the effects of propofol on overall cardiac function. RESULTS: At 28 degrees C (hypothermic) and, to a lesser extent, at 37 degrees C (normothermic), increasing stimulation frequency increased peak shortening and [Ca2+]i. Times to peak shortening and rate of relengthening were more prolonged at 28 degrees C compared with 37 degrees C at low stimulation frequencies (0.3 Hz), whereas the same conditions for [Ca2+]i were not altered by temperature. At 0.3 Hz and 28 degrees C, propofol caused a dose-dependent decrease in peak shortening and peak [Ca2+]i. These changes were greater at 28 degrees C compared with 37 degrees C and involved activation of protein kinase C. At a frequency of 2 Hz, there was a rightward shift in the dose-response relation for propofol on [Ca2+]i and shortening at both 37 degrees and 28 degrees C compared with that observed at 0.3 Hz. In Langendorff perfused hearts paced at 330 beats/min, clinically relevant concentrations of propofol decreased left ventricular developed pressure, with the effect being less at 28 degrees C compared with 37 degrees C. In contrast, only a supraclinical concentration of propofol decreased left ventricular developed pressure at 28 degrees C at either stimulation frequency. CONCLUSION: These results demonstrate that temperature and stimulation frequency alter the inhibitory effect of propofol on cardiomyocyte [Ca2+]i and contraction. In isolated cardiomyocytes, the inhibitory effects of propofol are more pronounced during hypothermia and at higher stimulation frequencies and involve activation of protein kinase C. In Langendorff perfused hearts at constant heart rate, the inhibitory effects of propofol at clinically relevant concentrations are more pronounced during normothermic conditions.     

Experimental Conditions Are Important Determinants of Cardiac Inotropic Effects of Propofol

Background: The rationale for this study is that the depressant effect of propofol on cardiac function in vitro is highly variable but may be explained by differences in the temperature and stimulation frequency used for the study. Both temperature and stimulation frequency are known to modulate cellular mechanisms that regulate intracellular free Ca2+ concentration ([Ca2+]i) and myofilament Ca2+ sensitivity in cardiac muscle. The authors hypothesized that temperature and stimulation frequency play a major role in determining propofol-induced alterations in [Ca2+]i and contraction in individual, electrically stimulated cardiomyocytes and the function of isolated perfused hearts.

Methods: Freshly isolated myocytes were obtained from adult rat hearts, loaded with fura-2, and placed on the stage of an inverted fluorescence microscope in a temperature-regulated bath. [Ca2+]i and myocyte shortening were simultaneously measured in individual cells at 28[degrees] or 37[degrees]C at various stimulation frequencies (0.3, 0.5, 1, 2, and 3 Hz) with and without propofol. Langendorff perfused hearts paced at 180 or 330 beats/min were used to assess the effects of propofol on overall cardiac function.

Results: At 28[degrees]C (hypothermic) and, to a lesser extent, at 37[degrees]C (normothermic), increasing stimulation frequency increased peak shortening and [Ca2+]i. Times to peak shortening and rate of relengthening were more prolonged at 28[degrees]C compared with 37[degrees]C at low stimulation frequencies (0.3 Hz), whereas the same conditions for [Ca2+]i were not altered by temperature. At 0.3 Hz and 28[degrees]C, propofol caused a dose-dependent decrease in peak shortening and peak [Ca2+]i. These changes were greater at 28[degrees]C compared with 37[degrees]C and involved activation of protein kinase C. At a frequency of 2 Hz, there was a rightward shift in the dose-response relation for propofol on [Ca2+]i and shortening at both 37[degrees] and 28[degrees]C compared with that observed at 0.3 Hz. In Langendorff perfused hearts paced at 330 beats/min, clinically relevant concentrations of propofol decreased left ventricular developed pressure, with the effect being less at 28[degrees]C compared with 37[degrees]C. In contrast, only a supraclinical concentration of propofol decreased left ventricular developed pressure at 28[degrees]C at either stimulation frequency.     

Nephron segment and calcium as determinants of anoxic cell death in renal cultures

Proximal tubules of the S1, S2 and S3 segments, medullary thick ascending limbs of Henle's loop (MAL) and cortical collecting tubules (CCT) were individually microdissected from rabbit kidneys and cultured for seven days in hormonally defined media. Anoxia was induced by incubation of cultures in normal medium for 45 min at 25 degrees C in an atmosphere of nitrogen (N2), and cell death was measured by nigrosine dye uptake. Immediately after anoxia, cell death was highest in S3 and MAL segments greater than S2 greater than S1 = CCT. The combined effects of anoxia and substrate (glucose, vitamins, amino acid) omission determined after incubation of cultures in phosphate buffered saline containing Ca2+ and Mg2+ (PBS) for 45 min in N2 also showed differential killing dependent on segment of origin: MAL greater than S3 greater than S2 CCT greater than S1. The effects of in vitro "reflow" were tested by returning cells to their normal oxygenated culture media at 37 degrees C. After the 45 min of anoxia and four to six hr of reflow in normal calcium-containing media, all cells from each segment were dead. Reflow in media lacking calcium for two hr immediately after anoxia then followed by return to normal calcium-containing media was associated with the survival of a significant percentage of cells for 48 hr: S1 (35.3 +/- 2.0%), S2 (30.0 +/- 2.0%), S3 (46.2 +/- 3.0%), MAL (38.7 +/- 3.0%), CCT (28.2 +/- 2.0%).(ABSTRACT TRUNCATED AT 250 WORDS)     

Role of physiological state 'normothermia' in internal thoracic artery spasm after harvesting.

OBJECTIVE: Vasospasm is often faced after the operative preparation of internal thoracic artery. Different vasodilating pharmacological agents are being used to eliminate this problem. During the preparation of internal thoracic artery, normal, local, and systemic temperatures are lost. We aimed to find out the effect of this decrease in temperature on the free flow of internal thoracic artery. METHODS: We investigated the effects of normal saline solution at 20 degrees C (group I), papaverine at 20 degrees C (group II) and normal saline solution at 37 degrees C (group III). Each group contained 20 patients undergoing coronary bypass. Free flow of the left internal thoracic artery was measured after mobilization. After approximately 18 min the graft had been sprayed with one of the agents, and the second free flow was measured (t-interval was 18.2+/-2.1 for group I, 18.3+/-1.8 for group II, and 17.5+/-1.9 for group III). RESULTS: Normal saline solution at 20 degrees C did not cause a significant change. Topical papaverine at 20 degrees C increased the flow from 42.1+/-10.7 ml/min to 77.5+/-17.9 ml/min (p < 0.0001). A significant increase also occurred with normal saline solution at 37 degrees C from 41+/-11.9 ml/min to 75.3+/-18.9 ml/min (p < 0.0001). CONCLUSIONS: After harvesting the graft, regaining physiological normothermic state is enough for vasodilatation. Even using one of the most potent vasodilating agent papaverine at 20 degrees C is not superior to normothermia.     

pH paradox and neonatal heart.

BACKGROUND: To evaluate the functional status of the Na+/H+ exchanger in the neonatal heart. METHODS: On the Langendorff system, isolated neonatal rabbit hearts were arrested by using cardioplegia with or without a specific Na+/H+ exchanger blocker, 5-(N,N dimethyl) amiloride (DMA) (20 microM). Ischemic period was 40 minutes at 37 degrees C or 120 minutes at 20 degrees C before 30 minutes of reperfusion at 37 degrees C. When DMA was added to the cardioplegia solution, it was also added to the reperfusate for the first 5 minutes of reperfusion (20 microM). RESULTS: Postischemic developed pressure was 50.3+/-7.1 mmHg in the DMA group versus 25.9+/-6 mmHg in the control group (p<0.05) at 37 degrees C and 74.8+/-14.6 mmHg in the DMA group versus 60.6+/-11.5 mmHg in the control group (p<0.05) at 20 degrees C. Postischeimic diastolic pressure was 40.4+/-3.3 mmHg in the DMA group versus 28.4+/-7 mmHg in the control group (p<0.05) at 37 degrees C and 9.6+/-3.1 mmHg in the DMA group versus 15+/-3.7 in the control group (p<0.05) at 20 degrees C. Creatine kinase washout was 296+/-97 IU/L in the DMA group versus 1253+/-537 IU/L in the control group (p<0.05) at 37 degrees C and 370+/-156 IU/L in the DMA group versus 524+/-104 IU/L in the control group (p<0.05) at 20 degrees C. CONCLUSIONS: 1) The Na+/H+ exchanger is active in the neonatal heart. 2) The Na+/H+ exchanger plays a key-role in the pathogenesis of reperfusion injury of the neonatal myocardium. 3) This exchanger is sensitive even for low H+ transmembrane gradients and even under hypothermic conditions.     

Possible involvement of Na(+)-H+ exchange in the early phase of reperfusion in myocardial stunning.

Calcium overload during reperfusion after prolonged ischemia has been associated with the Na(+)-Ca2+ exchange system. It has been proposed that the promotion of Na(+)-Ca2+ exchange at reperfusion may be mediated by Na(+)-H+ exchange. To evaluate whether this hypothesis is applicable for stunned myocardium, we examined the influence of temporary suppression of Na(+)-H+ and/or Na(+)-Ca2+ exchange during early reperfusion in isolated rat hearts. Myocardial stunning was produced by global ischemia for 15 min at 37 degrees C. The initial reperfusate was given during the subsequent 10 min after ischemia, and followed by reperfusion with normal Krebs-Henseleit buffer solution for 40 min. Hemodynamic indices, creatine kinase in coronary effluent, and myocardial water content were measured during reperfusion. The functional recovery of stunned myocardium was improved with higher extracellular Na+ concentration and/or lower Ca2+ concentration of the initial reperfusate. Aortic flow recovery of group II (135 mM Na(+)-0.5 mM Ca2+) was 77.0 +/- 3.4%, which was substantially greater (P < 0.05) than that of other groups: group I (control, 135 mM Na(+)-1.5 mM Ca2+), 68.2 +/- 2.4%; group III (25 mM Na(+)-0.5 mM Ca2+), 48.7 +/- 2.9%; group IV (25 mM Na(+)-1.5 mM Ca2+), 21.6 +/- 1.5%. Administration of amiloride, an inhibitor of Na(+)-H+ exchange, in the initial reperfusate ameliorates cardiac damage and improved aortic flow recovery in a dose-dependent manner (10(-6) M, 70.1 +/- 3.7%; 10(-5) M, 77.3 +/- 1.7%; 10(-4) M, 82.0 +/- 2.1% vs control 68.2 +/- 2.4%).(ABSTRACT TRUNCATED AT 250 WORDS)     

Divalent cations modulate human colon cancer cell adhesion

BACKGROUND: Iatrogenic tumor implantation within surgical sites can compromise curative cancer surgery. Cancer cell adhesion to extracellular matrix proteins is mediated by diverse matrix receptors, most notably integrins. Divalent cations may modulate integrin-ligand interactions in some cells. MATERIALS AND METHODS: We studied adhesion of SW620 and Caco-2 human colon cancer cells to collagen I, the dominant collagen of the interstitial matrix, and confirmed our results in primary human colon cancer cells from surgical specimens. Single cell suspensions in either HEPES/NaCl buffer or media supplemented with 0-1 mM Mn2+ or Mg2+, and 0-10 mM Zn2+ or Ca2+ were plated onto collagen-I-precoated dishes for 30 min. RESULTS: Supplementation of the HEPES/NaCl/BSA buffer with 1 mM Mn2+, Mg2+, Zn2+, or Ca2+ affected adhesion differently. Mn2+ (1 mM) markedly promoted SW620 adhesion vs control (21.17 +/- 0.08-fold). Mg2+ (1 mM) had a similar but lesser effect (14.71 +/- 0.02-fold). However, 1-10 mM Ca2+ inhibited basal cell adhesion by 22.0 +/- 3.1 to 88.0 +/- 7.3 % inhibition. Ca2+ (2.5-10 mM) also inhibited Mn2+-induced adhesion. Zn2+ stimulated basal adhesion slightly at lower concentrations but inhibited Mn2+-stimulated adhesion similarly to Ca2+ at higher concentrations. Results were duplicated in conventional serum containing culture medium supplemented with these cations. Caco-2 cells and primary cancer cells yielded similar results. All results are significant to P < 0.01. DISCUSSION: Integrin-mediated colon cancer cell adhesion is affected by extracellular divalent cation concentrations. Washing the surgical site with dilute calcium or zinc solutions might diminish perioperative tumor implantation.     

Harmful effects of inotropic agents on myocardial protection.

Using an isolated working rat heart model, the pretreatment effects of positive inotropic agents on ischemia-reperfusion injury were investigated. The experiment consisted of (1) working control perfusion; (2) working perfusion with isoproterenol (I), milrinone (M), a combination of these drugs (I + M) and dibutyl-cyclic adenosine monophosphate (DB) followed by ischemic arrest for 33 minutes at 37 degrees C or 150 minutes at 20 degrees C and Langendorff reperfusion; and (3) working perfusion. Under conditions of normothermic ischemia, percent recoveries of postischemic cardiac output (mean +/- standard error of the mean) in the I, M, I + M, and DB groups were 37.8% +/- 12.7%, 61.3% +/- 3.1%, 0%, and 53.1% +/- 5.2%, respectively. Under conditions of hypothermic ischemia, the percent recoveries in I + M and DB groups were 10.9% +/- 7.9% and 29.8% +/- 9.5%; they were all significantly lower than that in the control group. The addition of diltiazem or ryanodine at several concentrations and lowering of the Ca2+ concentration in the St. Thomas' cardioplegic solution did not prevent I + M-induced injury. Our data suggest that pretreatment by I + M aggravated ischemia-reperfusion injury, and adjustments in Ca2+ concentration were not sufficient to prevent that injury.     

Propofol Attenuates Capacitative Calcium Entry in Pulmonary Artery Smooth Muscle Cells

Background: Depletion of intracellular Ca2+ stores results in capacitative Ca2+ entry (CCE) in pulmonary artery smooth muscle cells (PASMCs). The authors aimed to investigate the effects of propofol on CCE and to assess the extent to which protein kinase C (PKC) and tyrosine kinases mediate propofol-induced changes in CCE.

Methods: Pulmonary artery smooth muscle cells were cultured from explants of canine intrapulmonary artery. Fura 2-loaded PASMCs were placed in a dish (37[degrees]C) on an inverted fluorescence microscope. Intracellular Ca2+ concentration was measured using fura 2 in PASMCs using a dual-wavelength spectrofluorometer. Thapsigargin (1 [mu]m), a sarcoplasmic reticulum Ca2+-adenosine triphosphatase inhibitor, was used to deplete intracellular Ca2+ stores after removing extracellular Ca2+. CCE was activated when extracellular Ca2+ (2.2 mm) was restored.

Results: Thapsigargin caused a transient increase in intracellular Ca2+ concentration (182 +/- 11%). Restoring extracellular calcium (to induce CCE) resulted in a peak (246 +/- 12% of baseline) and a sustained (187 +/- 7% of baseline) increase in intracellular Ca2+ concentration. Propofol (1, 10, 100 [mu]m) attenuated CCE in a dose-dependent manner (peak: 85 +/- 3, 70 +/- 4, 62 +/- 4%; sustained: 94 +/- 5, 80 +/- 5, 72 +/- 5% of control, respectively). Tyrosine kinase inhibition (tyrphostin 23) attenuated CCE (peak: 67 +/- 4%; sustained: 74 +/- 5% of control), but the propofol-induced decrease in CCE was still apparent after tyrosine kinases inhibition. PKC activation (phorbol 12-myristate 13-acetate) attenuated CCE (peak: 48 +/- 1%; sustained: 53 +/- 3% of control), whereas PKC inhibition (bisindolylmaleimide) potentiated CCE (peak: 132 +/- 11%; sustained: 120 +/- 4% of control). Moreover, PKC inhibition abolished the propofol-induced attenuation of CCE.