Synthesis, activity determination of tropane compounds as muscarinic M3 receptor antagonists and quantitative relationship between their molecular structure and activity / 上海交通大学学报（医学版）

Objective • To provide theoretical guidance for the design of molecules with high activity by building the quantitative structure-activity relationship (QSAR) model for tropane compounds as muscarinic M3 receptor antagonists. Methods • Six compounds (J1-J6) were prepared with 3α-hydroxy-tropane (J0) as the starting material by modifying the structure in C-3α position of the tropane skeleton. The antagonistic activity of new tropane compounds to muscarinic M3 receptors on tracheal rings of guinea pigs was evaluated by functional assays in vitro. The antagonistic parameters (pA2) of new tropane compounds prepared in this paper and former studies were applied to construct the QSAR model. The information about structural optimization was acquired by analyzing the effect of steric field and electrostatic field of model on activity of tropane compounds. Results • The six new tropane compounds showed obvious antagonistic activity against M3 receptors. Among them, J4 had the greatest activity (pA2=7.992). The cross-validation correlation coefficient squared (q2) and the non-cross-validated correlation coefficient squared (r2) of the QSAR model were 0.585 and 0.993, respectively. Conclusion • The antagonistic activity to muscarinic M3 receptors can be obviously improved, when the conjugating extent of π-bonds is large and O or N atoms participate in conjugation on the rings in the R-substituting group at C-3α position of the compounds.

Synthesis, activity determination of tropane compounds as muscarinic M3 receptor antagonists and quantitative relationship between their molecular structure and activity / 上海交通大学学报（医学版）

Objective · To provide theoretical guidance for the design of molecules with high activity by building the quantitative structure-activity relationship (QSAR) model for tropane compounds as muscarinic M3 receptor antagonists. Methods · Six compounds (J1-J6) were prepared with3α-hydroxy-tropane (J0) as the starting material by modifying the structure in C-3α position of the tropane skeleton. The antagonistic activity of new tropane compounds to muscarinic M3 receptors on tracheal rings of guinea pigs was evaluated by functional assays in vitro. The antagonistic parameters (pA2) of new tropane compounds prepared in this paper and former studies were applied to construct the QSAR model. The information about structural optimization was acquired by analyzing the effect of steric field and electrostatic field of model on activity of tropane compounds. Results · The six new tropane compounds showed obvious antagonistic activity against M3 receptors. Among them, J4 had the greatest activity (pA2=7.992). The cross-validation correlation coefficient squared (q2) and the non-cross-validated correlation coefficient squared (r2) of the QSAR model were 0.585 and 0.993, respectively.Conclusion · The antagonistic activity to muscarinic M3 receptors can be obviously improved, when the conjugating extent of π-bonds is large and O or N atoms participate in conjugation on the rings in the R-substituting group at C-3α position of the compounds.

Role of M3 receptor in the effect of penehyclidine hydrochloride up-regulating β-arrestin-1 expression / 重庆医学

Objective To investigate the role of M3 receptor in the effect of penehyclidine hydrochloride(PHC) upregulating β-arrestin-1 expression in lipopolysaccharide(LPS)-induced human pulmonary microvascular endothelial cell(HPMVEC) injury.Methods.M3 shRNA transfected HPMVEC and normal HPMVEC cells were randomly divided into LPS group(A),LPS+pHC group(B),LPS+ M3 shRNA transfection group(C) and PHC+ LPS+ M3 shRNA transfection group(D).The cytoskeleton change was observed by laser scanning confocal.The LDH level in cellular supernate was detected.The VCAM 1 protein expression was examined by immunofluorescence chemistry.β-arrestin-1 protein expression was determined by Western blot and β-arrestin-1mRNA expression was measured by real-time PCR.Results Compared with the group A or C,F-actin cytoskeleton arrangement in the group B or D was neat,the LDH level and VCAM-1 protein expression were decreased,and β-arrestin-1 expression was increased;compared with group A or B,F-actin cytoskeleton arrangement in the group C or D was neat,the LDH level and VCAM-1 protein expression were decreased,while the β-arrestin-1 expression had no obvious change.Conclusion Silence M3 receptor is conducive to reduce LPS-induced HPMVEC injury.But the role of PHC up-regulating β-arrestin-1 expression has no necessary connection with M3 receptor.

Study of antagonistic activity against M3 receptor and inhibition activity to neutrophil elastase of tropane compounds / 上海交通大学学报（医学版）

Objective · To design and synthesize five new tropane compounds, and test their antagonistic activity against M3 receptor and inhibition activity to neutrophil elastase (NE), of which the structure-activity relationship were preliminarily investigated. Methods · The five compounds, A1-A3,B1 and C1, were prepared with 3α-hydroxy-tropane (A0) as the starting material by modifying the structure in C-3α position and N atom on the tropane skeleton. The antagonistic activity of the compounds to muscarinic M3 receptors on tracheal rings of guinea pigs was evaluated by functional assays in vitro. The hydrolysis of PGlu-Pro-Val-PNA as substrate was catalyzed by NE to get colorful nitroaniline (PNA). The NE inhibition activity of the tropane compounds was obtained by determining the absorbance [(D(405 nm)] of PNA. Results · The five new tropane compounds generated strong antagonistic activity against M3 receptors. Among them, A2 had the greatest activity [antagonistic parameter pA2(M3)=9.004], and elicited obvious inhibitory effect to NE (inhibition ratio YA2=20.29%). Conclusion · Introducing strong electron-attraction group, such as sulfuryl and hydrophobic group with large volume into C-3α position on the tropane skeleton can improve the M3 receptor antagonistic activity as well as the NE inhibition activity.

Impacts on urodynamic parameters and the protein expressions of M2 and M3 receptors of detrusor in the rats of detrusor hyperreflexia treated with ginger-salt-isolated moxibustion at "Shenque" (CV 8) / 中国针灸

<p><b>OBJECTIVE</b>To discuss the effects on detrusor hyperreflexia treated with ginger-salt-isolated moxibustion at "Shenque" (CV 8) and its mechanism.</p><p><b>METHODS</b>Thirty female adult SD rats were selected. The model of detrusor hyperreflexia was prepared with complete spinal transection at T, of which, 20 rats were randomized into a model group (10 rats) and a moxibustion group (10 rats). A sham-operation group (10 rats) was set up for sham-spinal transection. In the moxibustion group, when urine incontinence occurred (about in 2 weeks of modeling), the ginger-salt-isolated moxibustion at "Shenque" (CV 8) was given, 3 moxa cones each time, once a day, continuously for 7 days. After treatment, in each group, the urodynamic parameters were determined, after which, the bladder detrusor was collected. Western blot was used to determine the protein expressions of M2 and M3 receptors.</p><p><b>RESULTS</b>Compared with the sham-operation group, the micturition interval was shortened apparently (<0.01); the maximal bladder pressure was increased apparently (<0.01); the protein expression of M2 receptor in the detrusor was increased significantly (<0.05) and that of M3 receptor had no apparent change (>0.05) in the rats of the model group. Compared with the model group, the micturition interval was longer apparently (<0.01), the maximal bladder pressure was reduced apparently (<0.01), the protein expression of M2 receptor in the detrusor was reduced significantly (<0.05) and that of M3 receptor had no apparent change (>0.05) in the rats of the moxibustion group.Compared with the sham-operation group, the results of the above indicators were not different significantly in the moxibustion group (all>0.05).</p><p><b>CONCLUSIONS</b>The ginger-salt-isolated moxibustion at "Shenque" (CV 8) suppresses the overactive bladder in the rat with spinal transection and its effect mechanism is possibly relevant with reducing the protein expression of detrusor M2 and inhibiting the excessive contraction of the detrusor.</p>

Relaxing Effect of Acetylcholine on Phenylephrine-Induced Contraction of Isolated Rabbit Prostate Strips Is Mediated by Neuronal Nitric Oxide Synthase

PURPOSE: The location of acetylcholinesterase-containing nerve fibers suggests a role for acetylcholine in both contractility and secretion in the prostate gland. The colocalization of nitrergic nerves with cholinergic nerves, and the cotransmission of nitric oxide with acetylcholine in cholinergic nerves, has been demonstrated in the prostate glands of various species. Thus, we investigated the effects of acetylcholine on phenylephrine-induced contraction and the correlation between cholinergic transmission and nitric oxide synthase by using isolated prostate strips of rabbits. MATERIALS AND METHODS: Isolated prostate strips were contracted with phenylephrine and then treated with cumulative concentrations of acetylcholine. Changes in acetylcholine-induced relaxation after preincubation with NG-nitroarginine methyl ester, 7-nitroindazole, and aminoguanidine were measured. The effects of selective muscarinic receptor antagonists were also evaluated. RESULTS: In the longitudinal phenylephrine-contracted strip, the cumulative application of acetylcholine (10(-9) to 10(-4) M) elicited a concentration-dependent relaxation effect. Acetylcholine-induced relaxation was inhibited not only by nitric oxide synthase inhibitors (10 microM L-NAME or 10 microM 7-nitroindazole) but also by 10 microM atropine and some selective muscarinic receptor antagonists (10(-6) M 11-([2-[(diethylamino)methyl]-1-piperdinyl]acetyl)-5,11-dihydro-6H-pyrido[2,3-b][1,4]benzodiazepine-6-one and 10(-6) M 4-diphenylacetoxy-N-methyl-piperidine). In contrast, relaxation was significantly increased by pretreatment of the strips with 10 mM L-arginine. CONCLUSIONS: Acetylcholine relaxed phenylephrine-induced contractions of isolated rabbit prostate strips. This relaxation may be mediated via both cholinergic and constitutive nitric oxide synthase with both the M2 and M3 receptors possibly playing key roles.

Advances in the study of arrhythmogenic mechanisms / 国际药学研究杂志

Arrhythmia is a common complication of cardiovascular diseases and a risk factor for human health. Especially, ventricular tachycardia and ventricular fibrillation may not only exacerbate original heart diseases, but also cause cardiac sudden death which has been an mportant death reason in China. However, anti-arrhythmic drugs nowadays cannot effectively treat these arrhythmias, with an efficiency of only 30%-60%, which indicates that our knowledge about arrhythmias is limited. Hence, to explore the potential mechanism, look for novel targets, and develop drugs with multiple-channel action are the focus of the research direction. Recent studies displayed that the atrial-specific potassium channels such as IKur and IKAch were involved in atrial fibrillation, which provided a prospective target for atrial fibrillation treatment. Calcium leak, gap junction protein and autoantibody against ICaL channel were shown to participate in arrhythmogenesis. These findings provided a theoretical basis for the development of more effective anti-arrhythmic drugs. Remarkably, as a kind of mportant RNA regulating gene expression, microRNA (miRNA) was shown to possess anti-arrhythmic activities which may prevent cardiac sudden death. miR-1, miR-133 and miR-590 regulated the arrhythmia in various types of animal models. Because of the multiple-gene regulation actions of miRNA, it has the potential to be developed as novel anti-arrhythmic target.

The value of anti-M3 receptor polypeptide antibody in the diagnosis of sj(o)gren's syndrome / 中华风湿病学杂志

Objective To establish the enzyme-linked immunosorbent assay (ELISA)method in detecting the anti-M3RP antibody in Sj(o)gren's syndrome (SS) patients.and to explore the significance of this autoantibody in the diagnosis of SS.Methods The synthesized M3 receptor polypeptide was used as antigen in EUSA to detect the anti-M3RP antibody in sera of patients with SS.other CTDS and healthy controls.and the association between the clinical features of SS and anti-M3RP antibody was analyzed.Results Antibodies against M3RP were detected in 84.6%patients with pSS and 81-3% with sSS.8.8%with other CTD and 1%in heahhy controls.The positive rates of antibodies in pSS and sSS were higher than those in other CTDs and healthy controls.The presence of anti-M3RP antibodies had no significant correlation with clinical manifesta-tions and internal organ involvement.Furthermore.the positive rates of anti-M3RP antibodies in anti-α-fodrin.SSA,SSB,and ANA antibodies negative SS patients were 85%.89-3%,88.9%and 95.2%,respectively.body is a complementary parameter in the diagnosis of antibody-negative SS.

The application of combined detection of autoantibodies in primary Sj(o)gren's syndrome / 中华内科杂志

Objective To compare the specificity and sensitivity of antibodies against SSA and SSB,anti-M3 receptor polypeptide antibodies,anti-α-fodrin(IgG)antibodies in primary SjSgren's syndrome (pSS).Methods One hundred and ten pSS patients(mean age was 49.2±14.8.mean disease duration was 5.6±4.6),80 systemic lupus erythmatosis(SLE)patients(mean age was 25.5 4-4.6,mean disease duration was 2.5±1.2)and 80 rheumatoid arthritis(RA)patients(mean age was 44.6±3.5.mean disease duration was 4.2±1.1)were studied.Enzyme-linked immunosorbent assay was used to measure these antibodies.Results The seropositive rates of anti-SSA,anti-SSB antibodies,anti-M3 receptor polypeptide antibodies and anti-α-fodrin(IgG)antibodies were 45.5%,30.9%,78.2%and 77.3%,respectivelv in pSS.They were much higher than those in RA and SLE patients(P<0.05).Specificities of SSA、SSB antibodies,anti-M3 receptor antibodies and anti-α-fodrin IgG were 83.8%,97.7%.92.0%and 90.O% respectively.With the combination of these antibodies in the diagnosis of pSS.the Sellsitivity can be increased at least to 88.2%and the specificity was not decreased significantly.Conclusion Combination of these antibodies can significantly improve the sensitivity of these antibodies in the diagnosis of pSS.Anti-SSA and SSB antibodies,anti-M3 receptor antibodies and anti-α-fodrin(IgG)antibodies are specific antibodies for the diagnosis of pSS.

The clinical significance of autoantibodies against acetylcholine muscarinic 3 receptor in primary Sj(o)gren's Syndrome / 中华内科杂志

Objective To detect anti-M3 receptor antibodies in primary Sj(o)gren's syndrome(pSS)patients and to explore its association with clinical manifestations.Methods Anti-M3 antibodies were tested in 70 patients with pSS,50 patients with systemic lupus erythematosus(SLE)and 76 normal controls with ELISA and Western blot.The correlation between anti-M3 antibodies and other clinical manifestations was analyzed.Results (1)The positive rate of anti-M3 antibodies in pSS Was 47.14% using ELISA and 60.00% using Western blot in SLE 4.00% using ELISA and 12.00% using Western blot and in normal controls 14.47% using ELISA and 15.79% using Western blot.(2)The incidence of sehirmer test,tear break-up time(BUT),whole saliva flow rate,punctate epithelial erosions(PEE)on the corneas and external eye examination were not significantly different between the anti-M3 positive and negative groups.(3)The incidences of IgG,rheumatoid factor,SSB and fluorescence index(FI)＞3 were higher in the positive group than in the negative group using EUSA.Conclusion The positive rate of anti-M3 antibodies is higher in pSS than in SLE and normal controls and in some degree it has correlation with the lesion in salivary gland.

Protective effects of M_3 receptor agonists on the arrhythmias of rats induced by barium chloride / 中国药理学通报

Aim To observe the protective effects of choline and pilocarpine,the M3 receptor agonists,on the arrhythmias of Wistar rats induced by barium chloride.Methods Barium chloride was used to induce the experimental arrhythmias of Wistar rats.Choline,pilocarpine,and 4-diphenylacetoxy-N-methylpiperidine-methiodide (4-DAMP),the selective antagonist of M3 receptor,were used to explore the effects of M3 receptor on the arrhythmias induced by barium chloride.The occurence and the severity of arrhythmias were observed.Results Choline 10 mg?kg-1 and pilocarpine 0.2 mg?kg-1 inhibited the occurence of arrhythmias,shortened the duration of arrhythmias (P