大鼠心室肌M3受体与间隙连接蛋白43作为抗心律失常药物靶点的综合研究

目的在蛋白质分子水平研究心律失常相关蛋白质M₃受体与间隙连接蛋白43之间的结构相互作用，并为其作为筛选药物靶点提供依据。方法通过免疫组化结合激光共聚焦显微镜，及免疫沉淀与免疫印迹技术，研究M受体与间隙连接蛋白43的结构性共定位关系。结果证实了大鼠心室肌细胞膜蛋白中M₁～M₅等5个亚型的存在；观察到大鼠单个心肌细胞膜上M₃受体与间隙连接蛋白43的结构性共定位；发现M受体各亚型与间隙连接蛋白43均存在结构整合，且一定浓度离子型去垢剂可破坏M₃受体与间隙连接蛋白43的结构整合关系，并进一步发现参与M₃受体结构整合的是间隙连接蛋白43的磷酸化形式。结论大鼠心室肌M受体亚型与间隙连接蛋白43的磷酸化形式存在结构性共定位关系，且可被一定浓度离子型去垢剂破坏。     

Clinical pharmacology of β3-adrenoceptors

1An atypical non β₁/β₂-adrenoceptor (AR) subtype (β₃-AR) has been identified which is selectively stimulated by a group of ligands which mediate lipolytic and thermic responses in brown and white adipose tissue. 2Molecular studies have shown that β₃-AR in man are mainly expressed in visceral adipocytes, and to a lesser extent in gall-bladder and colon. In vitro studies with β₃-AR agonists have shown activity at other sites including skeletal muscle and myocardium. 3Regulation of β₃-AR may differ from β₁/β₂-AR subtypes in that continuous agonist exposure does not result in receptor down-regulation. 4A polymorphism of the human β₃-AR gene (Trp64Arg) has been identified which is associated with obesity, insulin resistance and an earlier onset of non-insulin-dependent diabetes mellitus (NIDDM). Studies are required to establish whether expression of the mutant gene results in altered metabolic responses to β₃-AR stimulation in man. 5There is accumulating evidence to support a therapeutic role of β₃-AR agonists in NIDDM because of anti-obesity and anti-diabetic activity, as a consequence of thermogenic effects as well as increased insulin sensitivity and glucose tolerance. 6Selectivity studies with BRL35135 and isoprenaline in humans have demonstrated a β₃-AR mediated component to thermogenesis which is dissociated from β₁/β₂-mediated effects on carbohydrate and fat metabolism. Similar studies have suggested a functional β₃-AR mediating cardiac but not airway responses in humans. An evaluation of β₃-AR agonists in irritable bowel syndrome may be warranted in view of colonic antimotility properties in vitro.     

SPECIES DIFFERENCE IN THE β1/β2-ADRENOCEPTOR SELECTIVITY OF SM220CL IN THE CAT AND GUINEA-PIG

1. The β-receptor stimulant effects of Sm220Cl, dl-N- (1,1-dimethyl-3-phenylpropyl)-2-hydroxy-2(3,4-dihydroxy-2-methoxyphenyl)ethylamine, and (-)-isoprenaline have been compared in isolated atrial (β₁) and tracheal (β₂) preparations from guinea-pigs and cats. 2. The compounds were also tested for their ability to increase the heart rate (β₁), reduce serotonin-induced increases in pulmonary resistance (β₂), and decrease soleus muscle contractility (β₂) in vivo in the two species. 3. In all experiments cumulative concentration or dose-effect curves were established, EC₅₀ or ED₅₀ values obtained and molar activity-ratios (Sm220Cl: (-)-isoprenaline) calculated. 4. Calculated selectivity ratios [activity-ratio (heart):activity-ratio (bronchial smooth muscle)] from the in vitro experiments showed that Sm220Cl possessed β₂-receptor selectivity. This was more marked in guinea-pig than in cat preparations. 5. In the anaesthetized animals this species difference was more apparent; in cats Sm220Cl was non-selective in its actions for β₁- and β₂-receptor mediated responses, while marked β₂-receptor selectivity was obtained in the guinea-pig. 6. Since in both species the activity-ratios for β₂-receptor mediated actions are similar, the differences in the β₁/β₂-receptor selectivity of Sm220Cl are caused by the divergent cardiac effects produced by the drug.     

REDUCED INHIBITORY ACTIONS OF ADENOSINE A1 AND K1-OPIOID RECEPTOR AGONISTS ON β-ADRENOCEPTORS IN SPONTANEOUSLY HYPERTENSIVE RAT HEART

1. The modulatory actions of both adenosine A₁ and K₁-opioid receptor agonists on β-adrenoceptor stimulation in the heart of both spontaneously hypertensive rats (SHR) and nor-motensive Wistar-Kyoto (WKY) rats were compared. 2. In both types of rats, bot. R(-)-N⁶-(2-phenyliso-propyl)adenosine (R-PIA), an adenosine A₁ receptor agonist, and U50 488H, a K₁-opioid receptor agonist, inhibited the stimulatory effects of β-adrenoceptor activation on electrically induced [Ca²⁺]i transients measured by a spectrofluorometric method with fura-2/AM as the calcium indicator. The effects of these two agonists were blocked by their respective antagonists, namely 8-cyclopentyl-l,3-diprolxanthine and norbinaltorphimine. 3. The inhibitory actions of both R-PIA and U50488H on β-adrenoceptor augmentation of electrically induce. [Ca²⁺]i transients in the heart were more significantly reduced in SHR than in WKY rats, suggesting the negative modulatory actions of endogenous substances on 3-adrenoceptors were impaired in SHR, which may contribute to hypertension.     

β2-Adrenoceptors in Rat Liver Microcirculation

1. Rat liver sinusoids were observed by a microscopic in vivo transillumination method. The diameter of liver sinusoids and intrasinusoid erythrocyte flow velocities were measured quantitatively by a close-circuit television technique. 2. Both isoprenaline and the selective β₂-adrenoceptor agonist terbutaline produced a concentration-dependent dilatation of liver sinusoids and slowed erythrocyte flow velocity. The effects of isoprenaline and terbutaline were antagonized by propranolol but not by the selective β₁-adrenoceptor antagonist atenolol. Propranolol alone produced constriction of the liver sinusoids and increased erythrocyte flow velocity; these effects were not produced by atenolol. 3. The percentage dilatations produced by isoprenaline alone, isoprenaline in the presence of phenoxybenzamine and isoprenaline in the presence of phenylephrine-induced constriction were similar. 4. It is proposed that the β-adrenoceptors in liver sinusoids are of the β₂-type, and their physiological role was to counteract constrictor responses produced by a-adrenoceptor activity.     

M3受体与大鼠缺血性心肌细胞凋亡关系的研究M3受体与大鼠缺血性心肌细胞凋亡关系的研究

目的观察M₃受体对大鼠缺血性心肌细胞凋亡的作用及其机制。方法结扎大鼠左冠状动脉前降支建立急性心肌缺血模型,给予M₃受体激动剂胆碱或阻断剂4DAMP进行干预,观察M₃受体对其的影响。结果缺血前15 min iv胆碱10 mg·kg^-1可提高血清超氧化物歧化酶(SOD)活力,降低丙二醛(MDA)含量,减少凋亡细胞的数量(P<0.01),并可增加Bcl-2表达,减少Fas表达。预先5 min iv 4DAMP 0.12 mg·kg^-1阻断心肌M₃受体可逆转胆碱的作用。结论激动M₃受体对结扎大鼠冠状动脉诱导的心肌损伤有保护作用,其机制可能与调节Bcl-2和Fas表达从而抑制心肌细胞凋亡有关。     

In vitro functional interactions of acetylcholine esterase inhibitors and muscarinic receptor antagonists in the urinary bladder of the rat

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心脏M3受体作为抗心律失常药物新靶点研究的进展

心脏在交感神经和副交感神经的精细控制下完成正常的生理功能，心脏迷走神经通过刺激M型乙酰胆碱受体（muscarinic acetylcholine receptor．mAchR）调节心脏收缩力、兴奋性和传导。目前，通过分子生物学技术已经克隆出哺乳动物M受体的5个亚型，即m1，m2，m3，m4和m5。由于尚无m5受体相对特异性阻滞剂和激动剂，通过药理学的方法，将M受体分为M1，M2，M3及M4等4个亚型。     

STIMULATION OF β1-ADRENOCEPTORS ENHANCES ELECTRICALLY EVOKED [3H]-ACETYLCHOLINE RELEASE FROM RAT PHRENIC NERVE

1. The effects of isoprenaline, noradrenaline and fenoterol on the electrically evoked release of [3H]-acetylcholine from the rat phrenic nerve were investigated. 2. Isoprenaline (0.1 mumol/L) and noradrenaline (1 mumol/L) enhanced evoked [3H]-acetylcholine release by about 90%, an effect which was abolished by CGP 20712A (0.1 mumol/L), a specific antagonist at beta 1-adrenoceptors. Noradrenaline still enhanced [3H]-acetylcholine release in the presence of phentolamine (1 mumol/L). 3. The enhancing effect of both isoprenaline and noradrenaline decreased at prolonged exposure times (24-32 min). A pre-exposure of the tissue to a low concentration (0.01 mumol/L) of isoprenaline prevented the enhancing effect of 0.1 mumol/L isoprenaline. 4. Fenoterol, a specific agonist at beta 2-adrenoceptors, did not modify evoked [3H]-acetylcholine release. 5. The present results indicate the existence of facilitatory beta 1-adrenoceptors on the motor nerve. These receptors appear to be desensitized either by a high concentration of, or by a long exposure to, agonists. Under the present conditions noradrenaline enhances the release of newly synthesized transmitter mainly by stimulation of beta-adrenoceptors.     

Mechanisms of synergy of autoantibodies to M3 muscarinic acetylcholine receptor and secretory pathway Ca2+/Mn2+-ATPase isoform 1 in patients with non-desmoglein pemphigus vulgaris

Pemphigus vulgaris (PV) is a potentially lethal mucocutaneous blistering disease characterized by IgG autoantibodies (AuAbs) binding to epidermal keratinocytes and inducing a devastating blistering disease affecting oral and/or esophageal surfaces and, sometimes, also the skin. Anti-keratinocyte AuAbs developed by the desmoglein (Dsg) 1/3 AuAb-negative acute PV patients are pathogenic, as they induced acantholysis and epidermal split in the experimental models of PV in vitro and in vivo. These PV patients have various combinations of AuAbs to keratinocyte muscarinic acetylcholine receptor subtype M3 (M3AR), the secretory pathway Ca²⁺/Mn²⁺-ATPase isoform 1 (SPCA1), and desmocollin 3 whose relative concentrations correlate with the disease activity. In this study, we identified new molecular mechanisms of the synergistic cooperation of AuAbs to M3AR and SPCA1 in inducing acantholysis in the anti-Dsg 1/3 AuAb-negative PV patients. Anti-M3AR AuAb was found to play an important role in determining the level of intraepidermal split just above the basal cells, caspase to mediate early pro-apoptotic events triggered by anti-SPCA1 AuAb, and the neonatal Fc receptor (FcRn) to contribute to the pathobiological actions of both anti-M3AR and anti-SPCA1 AuAbs. Altogether, these novel results support our original hypothesis that pemphigus acantholysis is a complex disease process (also known as apoptolysis) initiated by AuAbs directed against different keratinocyte proteins that play important roles in supporting cell viability and regulating vital cell functions.     

Effects of oral and inhaled corticosteroid on lymphocyte β2-adrenoceptor function in asthmatic patients

Aims We have previously demonstrated that a single dose of oral prednisolone but not single doses of inhaled fluticasone had facilitatory effects on lymphocyte β₂-adrenoceptor (AR) function. To address possible differences in steady-state time-course, the aim of this study was to determine if repeated dosing with inhaled fluticasone would have facilitatory effects on lymphocyte β₂-AR. Plasma cortisol was also evaluated as a measure of systemic bioactivity. Methods Ten asthmatic subjects, mean (s.e.mean) age 29 (3) years, FEV₁ 89 (5) % predicted, were randomised in a double-blind crossover study to receive inhaled placebo (PL), inhaled fluticasone 1000 μg day⁻¹ (F1000) and inhaled fluticasone 2000 μg day⁻¹, each for 4 days and also a single dose of oral prednisolone 50 mg (PRED). Prednisolone was given as open medication. The last dose of study drug was taken at 22.00 h and subjects attended the laboratory at 08.00 h the following day. Results β₂-AR density (B_max; fmol/10⁶ cells) was significantly increased after PRED compared with PL and inhaled fluticasone. B_max (geometric mean) after each treatment were: PL 1.51, F1000 1.20, F2000 1.20 and PRED 2.14 (a 1.4 fold difference PRED vs PL; 95% CI 1.05 to 1.95; P<0.001). There was significant (P<0.001) suppression of plasma cortisol (nmol l⁻¹ ) following F2000 and PRED compared with PL: 393.8, F1000 302.1, F2000 205.0 (95% CI F2000 vs PL 58.1 to 319.4) and PRED 87.0 (95% CI PRED vs PL 176.2 to 437.5). The estimated milligram equivalence ratio for adrenal suppression was calculated at 1:11 for fluticasone vs prednisolone. Conclusions Repeated dosing with high-dose inhaled fluticasone did not up-regulate lymphocyte β₂-AR as compared with a single dose of oral prednisolone, despite having significantly suppressed early morning plasma cortisol. This study confirms our previous finding of a dissociation in sensitivity between effects of inhaled corticosteroid on adrenal suppression and lymphocyte β₂-AR regulation, at least for doses up to 2 mg day⁻¹ of fluticasone.     

白藜芦醇能够修复离体缺血再灌注损伤大鼠心脏M3受体与间隙连接蛋白43间结构及功能性整合

为了探讨白藜芦醇是否能通过影响M₃受体和间隙连接蛋白43(Cx43)间结构及功能性整合发挥其抗心肌缺血再灌注损伤作用，应用免疫共沉淀、免疫印迹及免疫荧光技术研究白藜芦醇对M₃受体与Cx43间结构及功能性整合的影响。结合大鼠离体II导联心电图及心肌超氧化物歧化酶(SOD)、丙二醛(MDA)的检测观察白藜芦醇是否能恢复心肌缺血再灌注损伤。白藜芦醇能修复心肌缺血再灌注损伤所致的M₃受体与Cx43间结构及功能性整合的破坏及纠正Cx43表达异常。同时QRS波时限﹑SOD及MDA的改变也得到相应恢复。白藜芦醇能修复M₃受体与Cx43间结构及功能性整合而发挥抗缺血再灌注损伤作用。     

Rosiglitazone‐induced myocardial protection against ischaemia–reperfusion injury is mediated via a phosphatidylinositol 3‐kinase/Akt‐dependent pathway

1. Rosiglitazone is widely used in the treatment of Type 2 diabetes. However, in recent years it has become evident that the therapeutic effects of peroxisome proliferator‐activated receptor γ ligands reach far beyond their use as insulin sensitizers. Recently, the ability of rosiglitazone pretreatment to induce cardioprotection following ischaemia–reperfusion (I/R) has been well documented; however, the protective mechanisms have not been elucidated. In the present study, examined the role of the phosphatidylinositol 3‐kinase (PI3‐K)/Akt signalling pathway in rosiglitazone cardioprotection following I/R injury. 2. Mice were pretreated with 3 mg/kg per day rosiglitazone for 14 days before hearts were subjected to ischaemia (30 min) and reperfusion (2 h). Wortmannin (1.4 mg/kg, i.p.), an inhibitor of PI3‐K, was administered 10 min prior to myocardial I/R. Then, activation of the PI3‐K/Akt/glycogen synthase kinase (GSK)‐3α signalling pathway was examined. The effects of PI3‐K inhibition on rosiglitazone‐induced cardioprotection were also evaluated. 3. Compared with control rats, the ratio of infarct size to ischaemic area (area at risk) and the occurrence of sustained ventricular fibrillation in rosiglitazone‐pretreated rats was significantly reduced (P < 0.05). Rosiglitazone pretreatment attenuated cardiac apoptosis, as assessed by ELISA to determine cardiomyocyte DNA fragmentation. Rosiglitazone pretreatment significantly increased levels of phosphorylated (p‐) Akt and p‐GSK‐3α in the rat myocardium. Pharmacological inhibition of PI3‐K by wortmannin markedly abolished the cardioprotection induced by rosiglitazone. 4. These results indicate that rosiglitazone‐induced cardioprotection in I/R injury is mediated via a PI3‐K/Akt/GSK‐3α‐dependent pathway. The data also suggest that modulation of PI3‐K/Akt/GSK‐3α‐dependent signalling pathways may be a viable strategy to reduce myocardial I/R injury.     

M3受体在脑心综合征心律失常中的作用

观察M₃受体(M₃R)在脑心综合征(CCS)模型大鼠心室肌中的作用及其表达，以探讨其与CCS心律失常的关系。线栓法建立CCS模型，监测心电图，激光共聚焦扫描显微镜记录激动M₃R后心肌细胞内钙的变化，Western blotting检测心室肌中M₃R表达水平的改变。同对照组相比，模型组心电图QRS、QT间期显著延长(P<0.01)；Western blotting结果显示模型组心室肌中M₃R的表达水平较对照组显著降低(P<0.05)；胆碱激动M₃R可以抑制KCl除极诱导的模型组心肌细胞内钙的升高幅度(P<0.01)，其特异阻断剂4-DAMP可以阻断这一作用。M₃R表达降低可能是CCS心律失常的重要原因之一。激动M₃R可以降低心肌细胞内钙，M₃R可能是治疗CCS心律失常新的靶点。     

5-HT3 receptor antagonists inhibit the response of κ oploid receptors in the morphine-reduced straub tail

We used the morphine-induced Straub tail to examine the actions of a 5-HT₃ receptor antagonist and κ opioid receptor agonist. The κ opioid receptor agonist, U-50,488H (4–16 mg/kg i.p.), produced a dose-related inhibition of the tail elevation induced by morphine (10 mg/kg s.c.) in ICR male mice. ICS-205-930 (3 and 10 mg/kg i.p.) and zacopride (0.3 and 1 mg/kg i.p.), 5-HT₃ receptor antagonists, attenuated the inhibitory effect of U-50,488H in a dose-dependent manner. ICS-205-930 and zacopride did not inhibit the morphine-induced Straub tail. These observations suggest that the actions of κ opioid receptors may be modulated by 5-HT³ receptors in the morphine-induced Straub tail.     

PODOCYTE INJURY IS SUPPRESSED BY 1,25-DIHYDROXYVITAMIN D3 VIA MODULATION OF TRANSFORMING GROWTH FACTOR-β1/BONE MORPHOGENETIC PROTEIN-7 SIGNALLING IN PUROMYCIN AMINONUCLEOSIDE NEPHROPATHY RATS

• 1 Accumulating evidence suggests that vitamin D and its analogues are renoprotective. However, the precise mechanisms and the molecular targets by which active vitamin D exerts its beneficial effects remain obscure. The objective of the present study was to evaluate the effect of active vitamin D on rats with puromycin aminonucleoside (PAN) nephropathy, a model that is characterized by predominant podocyte injury.
• 2 The PAN nephropathy rats were created by a single intravenous injection of 100 mg/kg PAN. Changes in renal pathology and podocyte numbers were observed. Real‐time polymerase chain reaction (PCR) was performed to examine mRNA expression of nephrin, transforming growth factor (TGF)‐β1 and bone morphogenetic protein (BMP)‐7. Protein expression of nephrin, TGF‐β1, BMP‐7 and p‐Smad2/3 and p‐Smad1/5/8 was examined by immunofluorescence, immunohistochemistry and western blotting, respectively. Rats were treated with 1,25(OH)₂D₃ by gastric gavage at a dose of 2.5 µg/kg per day, starting 2 days before PAN injection and continuing throughout the experiment.
• 3 A single injection of PAN induced massive proteinuria and elevated serum creatinine on Day 7, both of which were significantly suppressed by 1,25‐dihydroxyvitamin D₃ (1,25(OH)₂D₃). Immunofluorescence and real‐time PCR of the podocyte‐associated protein nephrin revealed reduced and discontinuous staining and this change was reversed by 1,25(OH)₂D₃. In PAN nephropathy rats, TGF‐β1 and p‐Smad2/3 expression was upregulated, whereas that of BMP‐7 and p‐Smad1/5/8 was downregulated. Treatment with 1,25(OH)₂D₃ significantly restored BMP‐7/Smad signalling while suppressing TGF‐β1/Smad signalling.
• 4 In conclusion, 1,25(OH)₂D₃ can ameliorate podocyte damage and proteinuria induced by PAN. The beneficial effects of 1,25(OH)₂D₃ on podocytes may be attributable, in part, to direct modulation of TGF‐β1/BMP‐7 signalling.