SHR心肌细胞离子泵活性与血压及左心室肥厚的相关性研究

目的:研究心肌细胞膜离子泵活性与血压及左心室肥厚之间的关系。方法:将12只自发性高血压大鼠(SHR)分成两组,一组灌喂缬沙坦(24mg/kg),另一组和6只正常大鼠(WKY)灌喂生理盐水共4周。测量实验前后血压及实验后心肌细胞膜的Na⁺-K⁺-ATP酶、Ca²⁺-ATP酶和Mg²⁺-ATP酶活性,同时测量心肌细胞横径(TDM)和心脏重量/体重(HW/BW)。结果:SHR生理盐水组的血压和TDM及HW/BW显著高于WKY和SHR用药组(P＜0.01),Na⁺-K⁺-ATP酶和Ca²⁺-ATP酶活性显著低于WKY和SHR用药组(P＜0.01)。Ca²⁺-ATP酶活性与血压、HW/BW和TDM呈显著负相关(r=-0.5945,-0.7077和-0.5026,P＜0.01和P＜0.05);与Na⁺-K⁺-ATP酶呈显著正相关(r=0.7543,P＜0.01)。Na⁺-K⁺-ATP酶与血压呈显著负相关(r=-0.6338,P＜0.01)。结论:SHR心肌细胞膜Na⁺-K⁺-ATP酶和Ca²⁺-ATP酶活性的改变与血压和左心室肥厚之间有密切的内在关系,心肌细胞膜离子泵活性的缺陷不仅在高血压同时可在左心室肥厚的形成和发展过程中起重要作用。     

β-淀粉样蛋白对D-半乳糖致衰大鼠海马线粒体膜流动性的影响

目的:研究β-淀粉样蛋白(β-AP)对衰老大鼠海马线粒体膜流动性的影响。方法:用D-半乳糖(D-gal)建立衰老动物模型,并且海马内微注射β-AP;检测各组大鼠学习记忆行为;以DPH为荧光探针,测定大鼠海马线粒体膜粘滞系数,同时测定海马Na⁺-K⁺ATP酶活性的变化。结果:D+A组大鼠Na⁺-K⁺ATP酶活性明显低于D组和A组(P＜0.05),与N组比较有显著差异(P＜0.01);海马线粒体膜流动性显著低于N组(P＜0.01),与D组和A组比较也有显著差异(P＜0.05)。结论:β-AP与D-gal联合可导致脑海马自由基损伤效应加重,海马线粒体膜流动性显著降低,Na⁺-K⁺ATP酶活性明显降低。     

创伤后应激障碍大鼠海马Ca2+/钙调蛋白的改变

目的 观察创伤后应激障碍（PTSD）大鼠海马神经元Ca²⁺信号及钙调蛋白（CaM）的表达变化,探讨PTSD行为异常的神经生物学机制。 方法 采用国际认定的无连续单一应激(SPS)方法刺激建立大鼠PTSD模型。成年健康雄性Wistar大鼠60只,随机分为SPS模型的12h、1d、4d、7d组及正常对照组,采用荧光探针标记法、免疫组织化学、免疫印迹和RT-PCR法检测Ca²⁺含量和CaM的表达变化。结果 SPS刺激后大鼠海马神经元内游离Ca²⁺浓度（nmol/L）于12h内升高,24h增至顶峰,7d恢复正常。CaM的表达于SPS刺激后1d表达最多,之后渐趋下降。 结论 海马Ca²⁺信号调控与CaM的表达变化可能是PTSD大鼠情感行为异常的重要病理生理基础之一。     

金属硫蛋白在培养乳鼠心肌细胞缺氧预处理中的作用

目的:探讨金属硫蛋白(MT)在培养乳鼠心肌细胞缺氧预处理中的作用机制。方法:建立培养的乳鼠心肌细胞缺氧/复氧模型,检测心肌细胞预缺氧24 h后MT及丙二醛(MDA)含量,Na⁺-K⁺ATP酶、Ca²⁺-Mg²⁺ATP酶活性的变化以及用MT抗体阻断后的相应变化。结果:缺氧预处理组MT含量及Na⁺-K⁺ATP酶、Ca²⁺-Mg²⁺ATP酶活性显著高于对照组及缺氧/复氧组(P＜0.05),MDA含量显著低于缺氧/复氧组(P＜0.01);使用MT抗体后,酶活性则显著降低,而MDA含量显著高于未加抗体和对照组(P＜0.01)。结论:缺氧预处理产生大量MT,后者可能通过减少MDA及促进Na⁺-K⁺ATP酶、Ca²⁺-Mg²⁺ATP酶的活性升高起到保护心肌的作用。     

参麦注射液对大鼠急性心肌缺血再灌注损伤的影响

目的:观察参麦注射液对大鼠急性心肌缺血再灌注损伤的影响,并探讨其作用机制。方法:结扎冠状动脉左前降支10min再灌15min复制大鼠急性心肌缺血再灌注损伤模型,描记标准肢体Ⅱ导联心电图,测定心肌组织匀浆中超氧化物歧化酶(SOD)、Na⁺,K⁺-ATP酶和Ca²⁺-ATP酶活性及丙二醛(MDA)含量,电镜观察心肌线粒体改变。结果:参麦注射液使再灌注性心律失常的发生率低于模型组、持续时间较短,心肌组织匀浆中SOD、Na⁺,K⁺-ATP酶和Ca²⁺-ATP酶的活性高于模型组,MDA的含量低于模型组,心肌线粒体损伤轻于模型组。结论:参麦注射液对大鼠急性心肌缺血再灌注损伤有明显的防治作用,其机制与减轻氧自由基及钙超载损伤有关。     

败血症休克肝细胞线粒体损伤机制的实验研究

目的:探讨败血症休克肝细胞线粒体损伤的机制。方法:30只SD大鼠随机分为3组,假手术组,12h手术组,16h手术组。采用盲肠结扎穿孔术(cecalligationandpuncture)制作败血症休克模型,比较手术前后肝细胞线粒体呼吸功能和氧化磷酸化功能的变化及线粒体膜ATP酶的活性改变与大鼠死亡率和血压变化的关系。结果:12h手术组平均动脉压(9.54±1.26)kPa明显低于假手术组(14.58±1.32)kPa(P<0.05),死亡率明显高于假手术组(P<0.05),12h手术组肝细胞线粒体Ⅲ态(1.28±0.25)、P/O比值(1.67±0.34)、呼吸控制率(1.27±0.25)、线粒体膜Ca²⁺-ATP酶(58.00±2.43)、Na⁺-K⁺-ATP酶(40.80±3.45)、Mg²⁺-ATP酶(78.30±4.16)、Ca²⁺-Mg²⁺-ATP酶(2.70±2.25)活性与假手术组相比较,具有显著差异(P<0.05)。术后16h组更低于12h组,与大鼠死亡率增加呈显著正相关。结论:肝细胞线粒体摄氧功能和氧化磷酸化功能减弱,膜流动性降低,能量代谢功能障碍,线粒体内钙镁平衡紊乱是败血症休克肝细胞线粒体损伤的主要机制。     

一氧化碳对缺血脑组织神经细胞胞内Ca2+浓度的影响

目的:研究一氧化碳对局灶性缺血脑组织神经细胞胞内Ca²⁺浓度的影响,试图从离子水平阐明CO对脑组织保护作用的机制。方法:将SD大鼠随机分为3组(n=6), 使用HO诱导剂、HO抑制剂腹腔注射为实验组,等量生理盐水腹腔注射为对照组,12 h后制成MCAO模型。栓塞后24 h检测血浆CO浓度、神经细胞胞内Ca²⁺浓度。结果: HO诱导剂组CO浓度明显高于生理盐水组,而胞内Ca²⁺浓度低于生理盐水组(P<0.05);HO抑制剂组CO浓度明显低于生理盐水组, 胞内Ca²⁺浓度高于生理盐水组(P<0.05)。HO诱导剂、HO抑制剂对非栓塞侧神经细胞胞内Ca²⁺浓度没有影响(P>0.05)。结论: CO通过作用于细胞膜上的Ca²⁺-K⁺通道,引起缺血脑组织神经细胞胞内Ca²⁺浓度的变化可能是CO脑保护作用的机制之一。     

泡沫细胞形成与胞浆Ca2+水平变化

目的:探讨巨噬细胞在泡沫化过程中,胞浆Ca²⁺水平的变化规律及其病理机制。方法:选择动脉粥样硬化易感性C57BL/6J小鼠,取其腹膜巨噬细胞,在10mg·L^-1氧化低密度脂蛋白中孵育96h,制备了富含脂质成分的泡沫样细胞。在此基础上,应用Ca²⁺荧光指示剂技术和NADH氧化偶联差光谱变化的分析方法,检测了前述泡沫样细胞的胞浆Ca²⁺水平及膜成分Ca²⁺-ATP酶活性。结果:泡沫样细胞的胞浆Ca²⁺水平是对照巨噬细胞的2.7倍,膜成分Ca²⁺-ATP酶活性为后者的24%。结论:在巨噬细胞源性泡沫细胞的形成过程中,伴随着缓慢的膜外Ca²⁺内流或肌质网Ca²⁺释放,这可能与初期膜上Ca²⁺通道的持续性开放及后期膜上Ca²⁺泵功能的不可逆性钝化有关。     

严重烧伤早期心肌[Ca2+]m变化及其机制研究

目的:探讨严重烧伤早期心肌线粒体Ca²⁺浓度([Ca²⁺]_m)的动态变化规律及其发生机制。方法:复制30%Ⅲ°烫伤大鼠模型,测定伤后1、3、6、12、24h大鼠心肌[Ca²⁺]_m,同时检测影响[Ca²⁺]_m的相关指标—胞浆Ca^2＋浓度(_c)及线粒体Ca^2＋转运速率。结果:烧伤后1、3、6h[Ca²⁺]_m依次升高,12、24h较6h虽有所下降,但仍高于正常对照组;_c除伤后1h无明显变化外,其余各时相点变化趋势与[Ca²⁺]_m相同,且伤后[Ca²⁺]_m与_c呈显著正相关,相关系数为0.9177(P＜0.01)。伤后1h心肌线粒体Ca^2＋摄取速率明显升高,而Ca^2＋释放速率无明显改变,但3、6、12、24h心肌线粒体Ca^2＋摄取速率与Ca^2＋释放速率均显著降低,且烧伤后3、6、12、24h[Ca²⁺]_m分别与线粒体Ca^2＋释放速率呈明显负相关。结论:烧伤后心肌线粒体存在明显的Ca^2＋超载和转运紊乱。     

应激状态下大鼠胃壁细胞H+-K+-ATP酶活性测定及电镜酶细胞化学研究

目的：探讨应激状态下大鼠胃壁细胞H⁺-K⁺-ATP酶活性及电镜酶细胞化学染色的变化。方法:将24只SD大鼠随机分为对照组、应激组和应激+奥美拉唑（OM）组，采用水浸-束缚应激（WRS）动物模型，检测胃粘膜溃疡指数（UI）和壁细胞H⁺-K⁺-ATP酶活性，观察壁细胞超微结构变化及H⁺-K⁺-ATP酶细胞化学染色结果。结果:应激组胃粘膜UI和壁细胞H⁺-K⁺-ATP酶活性明显高于对照组（P<0.01和P<0.05），而应激+OM组UI和H⁺-K⁺-ATP酶活性明显低于应激组（P<0.01）；电镜下对照组壁细胞呈静息状态，应激组壁细胞内分泌小管密集呈激活状态，而应激+OM组分泌小管明显扩张，绒毛稀少；酶细胞化学染色显示对照组壁细胞的分泌小管和顶部质膜有少量黑色点状酶反应产物沉积，应激组壁细胞的分泌小管可见多量的、密集分布的黑色点状酶反应产物，而应激+OM组分泌小管上几乎无反应产物沉积。结论:应激状态下大鼠胃壁细胞H⁺-K⁺-ATP酶活性升高，且与壁细胞超微结构改变相一致，提示胃酸是应激性溃疡发生的重要因素之一。     

Cation pumps in skeletal muscle: potential role in muscle fatigue.

Two membrane bound pumps in skeletal muscle, the sarcolemma Na⁺-K⁺ adenosine triphosphatase (ATPase) and the sarcoplasmic reticulum Ca²⁺-ATPase, provide for the maintenance of transmembrane ionic gradients necessary for excitation and activation of the myofibrillar apparatus. The rate at which the pumps are capable of establishing ionic homeostasis depends on the maximal activity of the enzyme and the potential of the metabolic pathways for supplying adenosine triphosphate (ATP). The activity of the Ca²⁺-ATPase appears to be expressed in a fibre type specific manner with both the amount of the enzyme and the isoform type related to the speed of contraction. In contrast, only minimal differences exist between slow-twitch and fast-twitch fibres in Na⁺-K⁺ ATPase activity. Evidence is accumulating that both active transport of Na⁺ and K⁺ across the sarcolemma and Ca²⁺-uptake by the sarcoplasmic reticulum may be impaired in vivo in a task specific manner resulting in loss of contractile function. In contrast to the Ca²⁺-ATPase, the Na⁺-K⁺ ATPase can be rapidly upregulated soon after the onset of a sustained pattern of activity. Similar programmes of activity result in a downregulation of Ca²⁺-ATPase but at a much later time point. The manner in which the metabolic pathways reorganize following chronic activity to meet the changes in ATP demand by the cation pumps and the degree to which these adaptations are compartmentalized is uncertain.     

Time course of changes in in vitro sarcoplasmic reticulum Ca2+-handling and Na+-K+-ATPase activity during repetitive contractions

Time-dependent changes in sarcoplasmic reticulum (SR) Ca²⁺-handling and Na⁺-K⁺-ATPase activity, as assessed in vitro, were investigated in the superficial (GS) and deep regions (GD) of rat gastrocnemius muscles undergoing short-term (up to 30 min) electrical stimulation. There was a rapid and progressive loss of force output during the first 5 min of stimulation. For GS, significant depressions (P < 0.05) in SR Ca²⁺-uptake rate and Ca²⁺-ATPase activity were observed during only the first 1 min. No further reductions occurred with stimulation time. SR Ca²⁺-release rate was significantly (P < 0.05) decreased at 3 min. For GD, significant reductions (P < 0.05) in Ca²⁺-uptake rate, Ca²⁺-release rate and Ca²⁺-ATPase activity were manifested after 3, 5, and 5 min, respectively. A decay in Na⁺-K⁺-ATPase activity was found only in 1-min stimulated GD and 30-min stimulated GS. After 30 min, the depressed functions reverted to resting levels in GD but not in GS. The alterations in any variables examined were not parallel with changes in force output. These results suggest that, at least under the conditions used in this study, in vivo disruptions in cation regulation mediated by vigorous contractile activity would be attributable primarily to events other than structural alterations to the respective proteins.     

Stimulation of Na+, K+-pump activity in skeletal muscle by methylxanthines: evidence and proposed mechanisms

Evidence is presented to support the hypothesis that submillimolar concentrations of methylxanthines stimulate Na⁺, K⁺-ATPase activity in skeletal muscle. Administration of methylxanthines to skeletal muscle results in plasma membrane hyperpolarization and increased rates of K⁺ uptake and Na⁺ efflux. These effects are both dose- and time-dependent and inhibited by blockers of the Na⁺, K⁺ ATPase. The mechanisms for stimulation of Na⁺, K⁺-ATPase activity and the signal transduction pathways are not known. The methylxanthine concentrations required for stimulation of Na⁺, K⁺-ATPase activity are less than those required to cause a 50% inhibition of phosphodiesterase activity, and therefore increases in cyclic AMP due to inhibition of the enzyme are not involved. Possible mechanisms by which methylxanthines may increase Na⁺, K⁺-ATPase activity include: (1) a role for increased intracellular [Ca²⁺]; (2) Ca²⁺ or adenosine-receptor-mediated increases in intracellular cyclic AMP; and (3) a direct action of methylxanthines on the Na⁺, K⁺ ATPase.     

Unchanged sarcoplasmic reticulum Ca2+-ATPase activity, reduced Ca2+ sensitivity, and negative force-frequency relationship in transgenic rats overexpressing the mouse renin gene

Transgenic rats overexpressing the mouse Ren-2 gene [TG(mREN2)27 rats, TGR] were used to characterize alterations in force generation and relaxation following cardiac hypertrophy. Age-matched Sprague-Dawley rats were used as the control group. The β-adrenoceptor dependent increase in force of contraction was reduced in the transgenic animals but not the Ca²⁺-dependent increase in force generation. Additionally, force of contraction decreased after increasing stimulation frequencies (up to 7 Hz), but the frequency-dependent decrease in force of contraction was significantly more pronounced in the transgenic group. The Ca²⁺ sensitivity in chemically skinned fiber preparations of TGR was reduced than that in Sprague-Dawley rats while maximum effectiveness was the same. Unexpectedly, the sarcoplasmic reticulum Ca²⁺-ATPase activity measured in crude membrane preparations from TGR did not differ from that in Sprague-Dawley rats; however, the activity of the Na⁺/K⁺-ATPase was less while the Na⁺/Ca²⁺-exchanger activity was significantly greater. In the same preparations the protein expression of SERCA2 was reduced in TGR while expression of phospholamban and calsequestrin remained the same. Thus in the model of cardiac hypertrophy harboring the mouse Ren-2 gene the hypothesized correlation between SERCA2 function and force-frequency relationship was not observed. Possible reasons for the more negative force-frequency relationship in TGR included changes at the level of the myofilaments and altered intracellular Na⁺ homeostasis which may result from the reciprocal changes in the Na⁺/K⁺-ATPase and the Na⁺/Ca²⁺-exchanger activity. Received: 15 September 1997 / Accepted: 7 April 1998     

Intracellular Na+ measurements in smooth muscle using SBFI – changes in [Na+], Ca2+ and force in normal and Na+-loaded ureter

 Our understanding of the control and effects of intracellular [Na⁺] ([Na⁺]_i) in intact smooth muscle is limited by the lack of data concerning [Na⁺]_i. The initial aim of this work was therefore to investigate the suitability of using the Na⁺-sensitive fluorophore SBFI in intact smooth muscle. We find this to be a good method for measuring [Na⁺]_i in ureteric smooth muscle. Resting [Na⁺]_i was found to be around 10 mM and rose to 25 mM when the Na⁺-K⁺-ATPase was inhibited by ouabain. This relatively low [Na⁺]_i in the absence of Na⁺-K⁺-ATPase suggests that other cellular processes, such as Na⁺-Ca²⁺ exchange, play a role in maintaining [Na⁺]_i under these conditions. Simultaneous measurements of [Na⁺]_i or [Ca²⁺] _i and force showed that Na⁺-Ca²⁺ exchange can play a functional role in ureteric smooth muscle. We found that the greater the driving force for Na⁺ exit and hence Ca²⁺ entry, the larger the contraction. In addition the Na⁺-Ca²⁺ exchanger activity under these conditions was found to be pH sensitive: acidification reduced the contraction and concomitant changes in [Ca²⁺] and [Na⁺]_i. We conclude that SBFI is a useful method for monitoring [Na] in smooth muscle and that Na⁺-Ca²⁺ exchange may play a functional role in the ureter. Received: 26 August 1997 / Received after revision: 27 October 1997 / Accepted: 28 October 1997     

Regulatory volume increase after secretory volume decrease in colonic epithelial cells under muscarinic stimulation

To address the question of whether colonic secretory cells change their volume in response to carbachol (CCh) stimulation and, if so, the mechanisms involved therein, we used two-photon laser scanning microscopy to measure the volume of individual epithelial cells in the fundus region of crypts isolated from the guinea-pig distal colon. We also measured the volume of human colonic epithelial T84 cells using an electronic sizing technique. Both types of colonocytes responded to stimulation by CCh with shrinkage and then underwent a regulatory volume increase (RVI), even during continued stimulation by CCh. The secretory volume decrease (SVD) induced by CCh was antagonized by atropine, BAPTA loading and niflumic acid, a blocker of Ca²⁺-activated Cl^– channels. An increase in the intracellular free [Ca²⁺] was observed with fura-2 during these volume responses to CCh. Removal of all Na⁺ or K⁺ or of most of the Cl^– from the extracellular solution abolished the RVI, but not the preceding SVD. The RVI, but not the preceding SVD, was abolished by bumetanide, a blocker of the Na⁺-K⁺-2Cl^– cotransporter. We conclude that guinea-pig crypt colonocytes and human T84 cells exhibit a cytosolic Ca²⁺-dependent SVD and undergo a subsequent RVI that is dependent on the operation of Na⁺-K⁺-2Cl^– cotransporters.     

Protective effects of Chlorpromazine and Verapamil against cadmium-induced kidney damage in vivo

Overexposure to cadmium (Cd) can induce kidney damage, which was related to the oxidative damage and disturb intracellular Ca²⁺ homeostasis. Chlorpromazine (CPZ), targeting calmodulin (CaM), and the Ca²⁺ channel blocker Verapamil (Ver) are involved in intracellular Ca²⁺ homeostasis processes. The aim of the study was to investigate the kidney damage caused by Cd administrated for 6 weeks and to evaluate the effects of pre-treatment with either chlorpromazine or verapamil on Cd-induced kidney damage. Thirty-two Wistar rats were divided randomly into 4 groups by weight, i.e., control group, Cd-treated group, and CPZ or Ver pre-treated group. The Cd-treated group rats were subcutaneously (s.c.) injected with 7 μmol CdCl₂/kg body weight/day. The CPZ and Ver pre-treated group rats were intraperitoneally (i.p.) injected with 5 mg CPZ/kg body weight/day, 4 mg Ver/kg body weight/day, respectively, 1 h before the s.c. administration of 7 μmol CdCl₂/kg body weight/day. The control group rats were injected s.c. with saline at the same time. The volume of injection was 2 ml/kg body weight, 5 times per week, for up to 6 weeks. After 6 weeks, Cd concentrations in the renal cortex and urine were significantly higher in Cd-treated group than that in controls. Cd concentrations of the urine in CPZ and Ver pre-treated groups were significantly lower than that in Cd-treated group, but there were no significant changes in the renal cortex. Compared with the controls, urinary NAG, ALP activities, and the levels of GSH, MDA, and the activities of PKC, Na⁺–K⁺-ATPase, and Ca²⁺-ATPase in rats from the Cd-treated group were significantly increased. SOD activity was suppressed by Cd. Urinary NAG activity and the level of GSH and the activities of PKC and Ca²⁺-ATPase in both CPZ and Ver pre-treated groups were significantly lower than that in Cd-treated rats. The present results showed that Cd-induced kidney damage was related to the oxidative damage and disturb intracellular Ca²⁺ homeostasis. Both CPZ and Ver possess some ability to prevent cadmium-induced kidney damage via antioxidative action and by maintaining calcium homeostasis.     

Changes in K+, Na+ and calcium contents during in vivo stimulation of rat skeletal muscle

The effects of in vivo stimulation via the sciatic nerve on Na⁺ K⁺ and calcium contents in slow-twitch and fast-twitch muscles were compared. Whereas intermittent stimulation for 24 h at 20 Hz caused only minor changes in soleus (SOL), a considerable loss of K⁺ (around 24%) and gain of Na⁺ (around 84%) was observed in extensor digitorum longus (EDL) and tibialis anterior (TA) muscles. These changes could be detected within 0.5 h and a plateau was maintained from 2 to 24 h. Total calcium content increased progressively, reaching values 245 and 382% above the control level in EDL and TA muscle, respectively, after 24 h of 20 Hz stimulation. Whereas the Na⁺ and K⁺ content recovered within a few hours, calcium content did not return towards control level until after 48 h of rest. In a pilot study performed with continuous stimulation at 10 Hz, the changes in Na⁺ and K⁺ contents in SOL, EDL and TA muscle were comparable to those at 20 Hz. The concentration of the Na⁺-K⁺ pumps was highest in the fast-twitch EDL and TA muscles and was unaffected by 10 Hz stimulation. It is concluded that a stimulation pattern leading to a rise in intracellular Na⁺ and a loss of K⁺ may cause a marked accumulation of calcium. These events seem to be related to insufficient activation of the Na-K⁺ pump rather than to variations in the total Na⁺-K' pump capacity.