The Effects of Etimicin and Gentamicin on Renal Endoplasmic Reticulum ^(45)Ca^(2+)-Uptake and Ca^(2+)-Mg^(2+)-ATPase Activity

Objective: To study the effects of etimicin (EM) and gentamicin (GM) on renal endoplasmic reticulum 45 Ca 2+ uptake and Ca 2+ Mg 2+ ATPase activity. Methods: Using 45 Ca 2+ incorporation technique and peacock blue spectrophotometry respectively. Results: EM and GM (≥3.4×10 4 mol·L 1 ) inhibited endoplasmic reticulum 45 Ca 2+ uptake (the rate of inhibition: ≥17.4% and ≥25.5%, respectively); EM and GM (3.4×10 2 mol·L 1 ) inhibited Ca 2+ Mg 2+ ATPase activity (the rate of inhibition: 24.2% and 29.2%, respectively). Conclusion: At high concentration, EM and GM elevated intracellular calcium which may be related to their nephrotoxicity.

心力衰竭大鼠心肌β_3肾上腺素受体表达及其对钙离子相关调节蛋白的作用

目的研究心力衰竭(HF)大鼠心肌β3肾上腺素受体(beta3-adrenergic receptors,β3-AR)表达情况及对钙离子相关调节蛋白的作用。方法以正常大鼠作对照组,皮下注射生理盐水;实验组大鼠皮下注射异丙肾上腺素(Isoproterenol,ISO),常规饲养8w。造模成功后实验组随机分为3组,激动剂组给予尾静脉注射β3-AR激动剂BRL37344,抑制剂组给予尾静脉注射β3-AR抑制剂SR59230A,HF组给予生理盐水。用药8w后行心脏彩超、RT-PCR法检测心肌组织钙调蛋白(CaM)、钙调蛋白激酶Ⅱ(CaMKⅡ)、肌内质网Ca2+-ATP酶异构体2a(SERCA2a)的mRNA表达。结果①HF组较对照组β3-ARmRNA表达水平增高,CaM、CaMKⅡ、SERCA2amRNA表达水平减低(均P<0.05)。②与HF组相比较,激动剂组β3-ARmRNA水平表达增加,CaM、CaMKⅡ、SERCA2a表达进一步减少(均P<0.05)。③抑制剂组β3-ARmRNA表达水平较对照组减少,CaM、CaMKⅡ、SERCA2a较对照组减低,但较HF组增加(均P<0.05)。结论β3-AR激动剂减低衰竭心肌CaM、CaMKⅡ、SERCA2a的mRNA表达,导致心功恶化,β3-AR抑制剂则相反,可延缓HF进展,预示β3-AR抑制剂在药物治疗HF中的应用前景。

长时间运动对大鼠骨骼肌红、白肌纤维肌浆网功能的影响

大鼠以18m／min的速度运动100min后，测定股四头肌和腓肠肌的浅层、深层的白肌、红肌肌浆网Ca2＋--Mg2＋ATPase活性和Ca2＋转运。结果发现，大鼠长时间运动后骨骼肌肌浆网功能下降，红肌比白肌变化明显。这一结果提示运动性骨骼肌疲劳可能与肌浆网功能下降有关。

Shen-Fu Injection(参附注射液)Alleviates Post-resuscitation Myocardial Dysfunction by Up-regulating Expression of Sarcoplasmic Reticulum Ca2+-ATPase

Objective： To compare the effect of Shen-Fu Injection （SFI） and epinephrine on the expression of sarcoplasmic reticulum Ca2. ATPase 2a （SERCA2a） in a pig model with post-resuscitation myocardial dysfunction. Methods： Ventricular fibrillation （VF） was electrically induced in Wu-zhi-shan miniature pigs. After 8 min of untreated VF and 2 min of cardiopulmonary resuscitation （CPR）, all animals were randomly administered a bolus injection of saline placebo （SA group, n=10）, SFI （0.8 mg/kg, SFI group, n=10） or epinephrine （20 t~ g/kg, EPI group, n=10）. After 4 min of CPR, a 100-J shock was delivered. If the defibrillation attempt failed to attain restoration of spontaneous circulation （ROSC）, manual chest compressions were rapidly resumed for a further 2 rain followed by a second defibrillation attempt. Hemodynamic variables were recorded, and plasma concentrations of catecholamines were measured. Adenylate cyclase （AC）, cyclic adenosine monophosphate （cAMP） and the expressions of 13 1-adrenoceptor （AR） and SERCA 2a were determined. Results： Cardiac output, left ventricular dp/dtr,~x and negative dp/dtm^x were significantly higher in the SFI group than in the SA and EPI groups at 4 and 6 h after ROSC. The expression of 13 1-AR and SERCA2a at 24 h after ROSC were significantly higher in the SFI group than in the SA and EPI groups （P〈0.05 or P〈0.01）. Conclusions： The administration of epinephrine during CPR decreased the expression of SERCA2a and aggravated postresuscitation myocardial function （P〈0.01）. SFI attenuated post-resuscitation myocardial dysfunction, and the mechanism might be related to the up-regulation of SERCA2a expression.

Study on the effect of doxorubicin on expressions of genes encoding myocardial sarcoplasmic reticulum Ca^2＋ transport proteins and the effect of taurine on myocardial protection in rabbits

To investigate the effect of doxorubicin(DOX) on gene expression of the myocardial sarcoplasmic reticulum (SR)Ca 2+ transport proteins and the mechanism of taurine(Tau) protecting cardiac muscle cells, 9 rabbits were injected with DOX , 8 rabbits with DOX and Tau, and 9 rabbits with normal saline. Cardiac function , concentration of calcium in cardiomyocytes (Myo[Ca 2+ ] \%i\%), activity of SR Ca 2+ ATPase(SERCA2a), level of SERCA2a mRNA and Ca 2+ released channels(RYR2)mRNA were detected. The left ventricle tissues were observed by electron microscopy. The results showed that cardiac index, left ventricular systolic pressure, activity of SR Ca 2+ ATPase and level of SERCA2a mRNA decreased , while Myo[Ca 2+ ] \%i\% increased in DOX treated rabbits. DOX could not affect the level of RYR2 mRNA. Tau intervention could alleviate the increase of left ventricular diastolic pressure, Myo[Ca 2+ ] \%i\% and the decrease of SERCA2a mRNA induced by doxorubicin. The results suggested that downregulation of SERCA2a gene expression was an important mechanism of DOX induced cardiomyopathy and that Tau could partially improve the heart function by reducing calcium overload and alleviating downregulation of SERCA2a mRNA.

Total Glycosides of Ranunculus Japonius Prevent Hypertrophy in Cardiomyocytes via Alleviating Chronic Ca^(2+) Overload

Objective To evaluate the in vitro anti-hypertrophic effect of total Glycosides of Ranunculus Japonius （TGRJ）. Methods Neonatal rat cardiomyocytes were cultured and hypertrophy was induced by adminis- trating isoproterenol （ISO, 10 gmol/L） or angiotensin Ⅱ （AngⅡ, 1 gmol/L） for 48 hours. In the treatment groups, cells were pretreated with TGRJ （0.3 g/L） for 30 minutes prior to hypertrophic stimuli. The anti-hypertrophic effects of TGRJ were examined by measuring cell size, total protein content, and protein synthesis. Intracellular free Ca2＋ concentration （[Ca2＋]i） was evaluated using fluorescence dye Fura-2/AM. Sacroplasmic/endoplasmic reticulum Ca2＋ ATPase 2a （SERCA2a）, atrial natriuretic peptide （ANP）, B-type natriuretic peptide （BNP）, and beta-myosin heavy chain （β-MHC） protein expression levels were measured by Western blotting. SERCA2a activity was assayed by p-nitrophenal phosphate disodium salt hexahydrate method. Results Increased cell size, total protein content, and protein synthesis following ISO or Ang II stimulation were significantly inhibited by pretreatment with TGRJ （all P〈0.05）. This anti-hypertrophic effect of TGRJ was confirmed by its suppressing effect on elevated expression of the three hypertrophic related genetic markers, ANP, BNP, and ^-MHC. In addition, TGRJ inhibited ISO or Ang Ⅱ induced up-regulation of [Ca2＋] under chronic but not acute conditions. And ISO or Ang Ⅱ induced down-regulation of SERCA2a expression and activity was also effectively rectified byTGRJ pretreatment. Conclusions The results of present study suggested that TGRJ could prevent ISO or Ang Ⅱ induced cardiac hypertrophy through improving chronic [Ca2＋]i disorder, might via normalizing SERCA2a expression and activity.

Switching-on of serotonergic calcium signaling in activated hepatic stellate cells

AIM:To investigate serotonergic Ca 2+ signaling and the expression of 5-hydroxytryptamine(5-HT) receptors,as well as Ca 2+ transporting proteins,in hepatic stellate cells(HSCs) . METHODS:The intracellular Ca 2+ concentration([Ca 2+ ]i) of isolated rat HSCs was measured with a fluorescence microscopic imaging system.Quantitative PCR was per-formed to determine the transcriptional levels of 5-HT receptors and endoplasmic reticulum(ER) proteins involved in Ca 2+ storage and release in cultured rat HSCs. RESULTS:Distinct from quiescent cells,activated HSCs exhibited[Ca 2+ ]i transients following treatment with 5-HT,which was abolished by U-73122,a phospholipase C inhibitor.Upregulation of 5-HT2A and 5-HT2B receptors,but not 5-HT3,was prominent during trans-differentiation of HSCs.Pretreatment with ritanserin,a 5-HT2 antagonist,inhibited[Ca 2+ ]i changes upon application of 5-HT.Expression of type 1 inositol-5'-triphosphate receptor and type 2 sarcoplasmic/endoplasmic reticulum Ca 2+ ATPase were also increased during activation of HSCs and serve as the major isotypes for ER Ca 2+ storage and release in activated HSCs.Ca 2+ binding chaperone proteins of the ER,including calreticulin,calnexin and calsequestrin,were up-regulated following activation of HSCs. CONCLUSION:The appearance of 5-HT-induced[Ca 2+ ]i response accompanied by upregulation of metabotropic 5-HT2 receptors and Ca 2+ transporting/chaperone ER proteins may participate in the activating process of HSCs.

基于家族分析的苜蓿逆境应答Ca^(2+) ATPase家族基因筛选与鉴定

Ca^(2+) ATPase属于P型ATPase超家族,在植物对外界逆境的应答中发挥重要作用,但迄今尚未有苜蓿Ca^(2+) ATPase基因研究的相关报道。为筛选逆境胁迫应答Ca^(2+) ATPases,本研究鉴定获得了16个苜蓿Ca^(2+) ATPase家族成员,分布在7条染色体上,且存在一对旁系同源基因。系统进化分析表明苜蓿Ca^(2+) ATPases分为ACA和ECA亚家族,ACA亚家族分为4组,且组内成员具有相似的内含子-外显子组成模式。此外,苜蓿Ca^(2+) ATPase家族具有高度保守的蛋白序列组成,包括4个功能结构域、10个跨膜结构域和11个保守motif。转录组数据分析发现该家族具有不同的逆境诱导表达模式,且多数苜蓿Ca^(2+) ATPases家族基因受低温和冷冻胁迫诱导表达,其中Mt ACA9和Mt ACA8上调表达最明显。q RT-PCR结果证实Mt ACA9和Mt ACA8表达在低温和冷冻胁迫下显著上调。本研究结果为揭示苜蓿Ca^(2+) ATPase基因在低温冷害应答中的功能提供了重要参考。

Changes in Ca^(2+) -ATPase in a guinea pig endolymphatic hydrops model

OBJECTIVE: To investigate the localization of Ca(2+)-ATPase (Ca(2+) pump) in the cochlear and its change after endolymphatic hydrops, and to study the relationship between compound action potential (CAP) threshold and the Ca(2+)-ATPase activety. METHODS: The left endolymphatic sac was ablated to induce endolymphatic hydrops in fourteen healthy guinea pigs with normal action potential thresholds measured after a sliver ball electrode placed on the round window. Ca(2+)-ATPase activity was studied cytochemically using a lead citrate reaction in control and hydropic ears. The reaction product was lead phosphate particles as an expression of Ca(2+)-ATPase activity, observed with an eletron microscope. RESULTS: Ca(2+)-ATPase activity is mainly found on the endolymphatic surface of Reis sner's membrane, the stereocilia and cuticular plate of inner and outer hair cells, and along the infolded plasma membrane of strial marginal cells. CAP thresholds of filtered click are increased and Ca(2+)-ATPase activity significantly decreased after endolymphatic hydrops in the mentioned locations. CONCLUSIONS: CAP thresholds are increased and Ca(2+)-ATPase activity are significantly decreased in the cochlea after endolymphatic hydrops. These results suggest that there is a negative correlation between them.

Changes of sarcoplamic reticular Ca^(2+)-ATPase and IP_3-I receptor mRNA expression in patients with atrial fibrillation

OBJECTIVE: To investigate changes in the expression of sarcoplamic reticular Ca(2+)-ATPase (SERCA) and IP(3)-I receptors (IP(3)R(1)) mRNA in patients with atrial fibrillation. METHODS: Thirty-eight patients with mitral stenosis undergoing open heart surgery were studied. 100 mg of atrial tissue was obtained during surgery from the right appendage and the right atrium. The amount of messenger ribonucleic acid (mRNA) amount of SERCA and IP(3)R(1) was measured by reverse transcription-polymerase chain reaction (RT-PCR) and normalized to the mRNA levels of glyceraldehyde 3-phosphate dehydrogenase (GAPDH). RESULTS: Levels of mRNA expression of SERCA in patients with AF, as compared with subjects in sinus rhythm, was lower and that of IP(3)R(1) was higher. The longer AF was sustained, the higher the levels of mRNA. There was no significant difference between right atrial free wall and right appendage. CONCLUSIONS: The expression changes of SERCA and IP3R mRNA may correlate with the initiation or maintenance of AF.

Changes of sarcoplamic reticular Ca2+-ATPase and IP3-I receptor mRNA expression in patients with atrial fibrillation

Objective To investigate changes in the expression of sarcoplamic reticular Ca 2+ ATPase (SERCA) and IP 3 I receptors (IP 3R 1) mRNA in patients with atrial fibrillation Methods Thirty eight patients with mitral stenosis undergoing open heart surgery were studied 100 mg of atrial tissue was obtained during surgery from the right appendage and the right atrium The amount of messenger ribonucleic acid (mRNA) amount of SERCA and IP 3R 1 was measured by reverse transcription polymerase chain reaction (RT PCR) and normalized to the mRNA levels of glyceraldehyde 3 phosphate dehydrogenase (GAPDH) Results Levels of mRNA expression of SERCA in patients with AF, as compared with subjects in sinus rhythm, was lower and that of IP 3R 1 was higher The longer AF was sustained, the higher the levels of mRNA There was no significant difference between right atrial free wall and right appendage Conclusions The expression changes of SERCA and IP3R mRNA may correlate with the initiation or maintenance of AF

Effect of tetrandrine on cellular electrophysiology and calcium uptake of myocardium in guinea pigs and dogs

Objective To study the effects of tetrandrine, a Chinese herbal medicine, on the action potential (AP),contraction as well as sarcoplasmic reticulum (SR) calcium uptake of myocardium in guinea-pigs and dogs.Methods Changes in AP, dV/dt, peak tension (PT) and dT/dt of myocardial cells were studied using the technique of glass electrode. Changes of the calcium uptake rate by sarcoplasmic reticulum and release of inorganic phosphate from sarcoplasmic reticulum were assessed with biochemical techniques.Results Tetrandrine exerts a concentration-dependent and frequency-dependent negative inotropic effect and shortens action potential duration. Tetrandrine depresses both dT(E)/dt and dT(L)/dt as well as the tension of myocardium, and reduces dV/dt and amplitude only in the slow action potential, thus implying that tetrandrine blocks the slow calcium channel. In addition, compared with thapsigargin, a specific inhibitor of Ca2+-ATPase on SR, tetrandrine more apparently suppresses the contraction of the myocardium.Conclusions Tetrandrine is a wide-range calcium antagonist of plant origin. Not only it blocks the voltageoperated calcium channels as other authors reported, but also may play an important role in affecting the function of Ca2+ -ATPase and calcium release channels on SR. From this study, we also suggest that the calcium channel appears to be more critical than SR for the contraction of my ocardium.

A Comparative Study on the Therapeutic Effect of Astragaloside (黄芪总皂甙) and Perindopril in Treating CVB_3-Infected Cardiomyocytes in Rats

Objective: To compare the therapeutic effects of Astragaloside and Perindopril on myocardial sarco/endoplasmic reticulum Ca 2+ ATPase (SERCA) activity and the SERCA type 2 mRNA level in Coxsackievirus B 3 (CVB 3) infected cardiomyocytes. Methods: Cultured cardiomyocytes of rats were divided into normal, model, Astragaloside and Perindopril groups. The model, Astragaloside and Perindopril groups were infected with CVB 3. Meanwhile, the Astragaloside and the Perindopril groups were treated with Astragaloside (10 μg/ml) and Perindopril (1.3 μg/ml) respectively. Cytopathic effect (CPE), cardiac troponin I ( cTnI) , the SERCA activity and mRNA level of the SERCA type 2 were observed after 96 hours. Results: The CPE and cTnI of model group were significantly higher than those of normal, Astragaloside and Perindopril groups ( P <0.01). The activity and the mRNA expression of myocardial SERCA of model group were significantly lower than those of normal, Astragaloside and Perindopril groups ( P <0.01-0.05). Compared with Astragaloside group, the CPE, cTnI of Perindopril group were higher and the activity and mRNA level of Perindopril group were lower. But there were no significant difference between the two groups ( P >0.05). Conclusion: Astragaloside and Perindopril were able to reverse the down regulations of cardiac SERCA activity and mRNA expression caused by virus infection to alleviate the cardiomyocyte injury.

Changes of neuronal calcium channel following brain damage induced by injection of pertussis bacilli in rats

Objective: To explore changes of neuronal calcium channel following brain damage induced by injection of pertussis bacilli in rats, and to investigate the relationship between cytosolic free calcium concentration ([Ca 2+ ] i) in the synaptosome and Ca 2+ ATPase activities of mitochondria. Methods: The level of [Ca 2+ ] i in the synaptosome and Ca 2+ ATPase activities of mitochondria in the acute brain damage induced by injection of pertussis bacilli (PB) in rat was determined and nimodipine was administrated to show its effects on [Ca 2+ ] i in the synaptosome and on alteration of Ca 2+ ATPase activity in the mitochondria. Seventy three rats were randomly divided into four groups, ie, normal control group (Group A), sham operation control group (Group B), PB group (Group C) and nimodipine treatment group (Group D). Results: The level of [Ca 2+ ] i was significantly increased in the PB injected cerebral hemisphere in the Group C as compared with that in the Group A and the Group B at 30 minutes after injection of PB. The level of [Ca 2+ ] i was kept higher in the 4 hours and 24 hours subgroups after the injection in the Group C (P< 0.05 ). In contrast, the Ca 2+ ATPase activities were decreased remarkably among all of the subgroups in the Group C. Nimodipine, which was administered after injection of PB, could significantly decrease the [Ca 2+ ] i and increase the activity of Ca 2+ ATPase (P< 0.05 ). Conclusions: The neuronal calcium channel is opened after injection of PB. There is a negative correlation between activities of Ca 2+ ATPase and [Ca 2+ ] i. Nimodipine can reduce brain damage through stimulating the activities of Ca 2+ ATPase in the mitochondria, and decrease the level of [Ca 2+ ] i in the synaptosome. Treatment with nimodipine dramatically reduces the effects of brain damage induced by injection of PB.