OBJECTIVE: To test the expression of autoantibody against the M2-muscarinic receptor in patients with severe preeclampsia.
 METHODS: A case-control study, including 78 patients with severe preeclampsia and 78 women with normal pregnancy in Department of Gynaecology and Obstetrics of Capital Medical University affiliated Beijing Chao-Yang Hospital from Jan, 2013 to Jan, 2015, were conducted, and 60 non-pregnant women were served as a control group. ELISA protocol was used to test serum autoantibody against the M2-muscarinic receptor. The clinical significance of the autoantibody against the M2-muscarinic receptor among women with severe preeclampsia was estimated.
 RESULTS: Autoantibody against the M2-muscarinic receptor were positive in 32.1% (25/78) of patients with severe preeclampsia, in 10.3% (8/78, P<0.05) of normal pregnant women and in 8.3% (5/60, P<0.05) of non-pregnant controls. The concentration of creatine kinase in patients with severe preeclampsia and normal pregnant women were (101.49±142.75) and (57.94±31.64) U/L,
respectively. Mask difference exist between the severe preeclampsia group and the normal pregnancy group (P<0.05).
 CONCLUSION The expression of autoantibody against the M2-muscarinic receptor in patients with severe preeclampsia is elevated significantly, which is associated with severe preeclampsia. However, its etiological role needs further to be investigated.
Autoantibodies ; Case-Control Studies ; Enzyme-Linked Immunosorbent Assay ; Female ; Humans ; Pre-Eclampsia ; Pregnancy ; Receptor, Muscarinic M2 ; Severity of Illness Index

The present study was aimed to investigate the positive inotropic mechanism of carbachol (CCh) on rat ventricular myocytes. The effects of CCh on L-type calcium current (I(Ca,L)) and Na(+)/Ca(2+) exchange current (I(Na/Ca)) were investigated in isolated rat ventricular myocytes. After loading myocytes with Fura-2/AM, electrically triggered Ca(2+) transient and cell shortening in single myocyte were measured simultaneously using ion imaging system with charge-coupled device (CCD) camera. CCh (100 mumol/L) increased I(Na/Ca) in forward mode from (1.18 +/- 0.57) pA/pF in the control group to (1.65 +/- 0.52) pA/pF (P<0.01) and that in reverse mode from (1.11 +/- 0.49) pA/pF in the control group to (1.53 +/- 0.52) pA/pF (P<0.01), respectively. CCh had no effect on I(Ca,L). The stimulatory effect of CCh on I(Na/Ca) was blocked by application of atropine, a non-selective M muscarinic receptor antagonist, and methoctramine, a selective M(2) muscarinic receptor antagonist. CCh (100 mumol/L) increased cell shortening from (3.00 +/- 0.67) mum in the control group to (3.55 +/- 1.21) mum. Ca(2+) transient was also increased from 203.8 +/- 50.0 in the control group to 234.8 +/- 64.3 in 100 mumol/L CCh group. KB-R7943, a selective inhibitor of reverse mode Na(+)/Ca(2+) exchange, did not change the baseline level of cell shortening and Ca(2+) transient, while completely abolished CCh-induced increments of both Ca(2+) transient and cell shortening. CCh increased cell shortening and Ca(2+) transient in the presence of nicardipine, indicating that the positive inotropic effect of CCh was through activation of Na(+)/Ca(2+) exchange. Calcium sensitivity was not changed by CCh. Both atropine and methoctramine abolished the positive inotropic effects of CCh, demonstrating that CCh induced positive inotropism via the M(2) muscarinic receptor. The results suggest that CCh increases cell contraction and Ca(2+) transient in rat ventricular myocytes. This positive inotropic effect of CCh is through activation of reverse mode Na(+)/Ca(2+) exchange, and M(2) receptors are involved in mediating CCh-induced contraction.
Animals ; Calcium ; Carbachol ; pharmacology ; Heart Ventricles ; Male ; Myocardial Contraction ; Myocytes, Cardiac ; drug effects ; Rats ; Receptor, Muscarinic M2 ; Receptors, Muscarinic ; drug effects ; Sodium ; Sodium-Calcium Exchanger ; Thiourea ; analogs & derivatives

OBJECTIVETo explore the effect of organophosphorus insecticides (OPs) on G protein-coupled receptor kinase 2 mediated phosphorylation of M2 muscarinic receptors in vitro and to understand an alternative target of the OPs for human and other animals.

METHODSThe acetylcholine M2 muscarinic receptors (mAChR2) were purified from rat brain by single step affinity chromatography. In vitro experiments, the purified mAChR2, G-protein coupled receptor kinase 2 (GRK2) and the (gamma-p32) labeled ATP were incubated with paraoxon (PO), chlorpyrifos oxon (CPO) or chlorpyrifos (CPF) of varying concentrations. The proteins were separated by the polyacrylamide gel electrophoresis. The gels were dried and the phosphorylation of mAChR2 was detected with autoradiograms. Bands containing M2 receptor were excised and counted by liquid scintillation.

RESULTSCPO inhibited phosphorylation of M2 muscarinic receptors by GRK2 with a median inhibition concentration (IC(50)) at 70 micromol/L. CPF also inhibited M2 receptors phosphorylation, but was less potent and less efficacious than that of CPO. PO and parathion (PT) had little effect on the receptor phosphorylation under the same conditions. CPO and CPF didn't inhibit the beta2 Adrenalin (beta2-AR) receptor phosphorylation also mediated by GRK2.

CONCLUSIONCPO and CPF can selectively inhibit the GRK2 mediated mAChR2 phosphorylation while PO and PT have no this effect.

Animals ; Chlorpyrifos ; analogs & derivatives ; toxicity ; Cholinesterase Inhibitors ; toxicity ; G-Protein-Coupled Receptor Kinase 2 ; Paraoxon ; toxicity ; Phosphorylation ; Rats ; Receptor, Muscarinic M2 ; metabolism ; beta-Adrenergic Receptor Kinases ; metabolism ; physiology

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.
Acetylcholine ; Atropine ; Contracts ; Guanidines ; Indazoles ; Nerve Fibers ; Neurons ; NG-Nitroarginine Methyl Ester ; Nitrergic Neurons ; Nitric Oxide ; Nitric Oxide Synthase ; Nitric Oxide Synthase Type I ; Phenylephrine ; Prostate ; Receptor, Muscarinic M2 ; Receptor, Muscarinic M3 ; Receptors, Muscarinic ; Relaxation

OBJECTIVETo investigate the general pattern of cholinergic nerve distribution and M(2) receptors in adult rat heart.

METHODSKarnovsky-Roots histochemical staining combining point counting method and immunochemical SABC method with image analysis were used to identify the cholinergic nerves and M(2) receptors, respectively, in adult rat heart.

RESULTSPositive staining of cholinergic nerves and M(2) receptors was found in all regions of the rat heart, and the point count of cholinergic nerves in the atria was 4.6 times as much as that in ventricles, and the area of immunoreactive substance for M(2) receptors two-fold higher in the atria than in the ventricles. The point counts of the cholinergic nerves in the medial-layer myocardium were fewer than that in subepicardial and endocardial tissues of the left ventricular free wall. However, M(2) receptors were comparable among the 3 layers of the left free ventricular wall.

CONCLUSIONCholinergic nerves and M(2) receptors are located in both rat atria and ventricles, but their density is much higher in the atria than in the ventricles. Transmural heterogeneity characterizes cholinergic nerve innervation in the left ventricular free wall without significant differences in M(2) receptor density.

Animals ; Cholinergic Fibers ; metabolism ; Female ; Heart ; innervation ; Heart Atria ; innervation ; metabolism ; Heart Ventricles ; innervation ; metabolism ; Immunohistochemistry ; Male ; Myocardium ; metabolism ; Rats ; Rats, Sprague-Dawley ; Receptor, Muscarinic M2 ; analysis

OBJECTIVETo explore the role of the autoantibodies against M(2)-muscarinic receptor (M(2)-receptor), beta(1)-adrenergic receptor (beta(1)-receptor) in the development of diabetic with refractory hypertension.

METHODSSerum autoantibodies against M(2) and beta(1) were detected by ELISA using synthesized epitopes of the second extracellular loop of M(2) receptor (169 - 193) and beta(1) receptor (197 - 222) in healthy controls (n = 40), diabetic patients (n = 62), diabetic patients with non-refractory hypertension (n = 55) and diabetic patients with refractory hypertension (n = 81).

RESULTSThe positive rates of the autoantibodies against M(2) receptor and beta(1) receptor were similar among healthy controls (15.0% and 17. 5%), diabetes mellitus patients (17.7% and 14.5%) and diabetic patients with non-refractory hypertension (16.4% and 12.7%) but are significantly higher in diabetic patients with refractory hypertension (64.2% and 55.6%, P < 0.01 vs. other 3 groups).

CONCLUSIONThis finding suggests that autoimmune mechanisms might play a role in the pathogenesis of diabetic patients with refractory hypertension.

Adult ; Autoantibodies ; blood ; Diabetes Mellitus, Type 2 ; blood ; complications ; Female ; Humans ; Hypertension ; blood ; complications ; Male ; Middle Aged ; Receptor, Muscarinic M2 ; immunology ; Receptors, Adrenergic, beta-1 ; immunology

OBJECTIVETo study the acute effects of dimethoate on the muscarinic-receptors(M1, M2) in the brain of rats.

METHODS24 Sprague-Dawley rats were divided into 4 groups randomly. They were administered subcutaneously with 0, 25, 50, 100 mg/kg dimethoate, respectively. Brains were removed after 48 hours of administration. Radioligand binding assay was used to determine the density and affinity of M1 and M2 receptors.

RESULTSRats in the treated group showed low density of M1 and M2 receptors compared with the control rats. The brain M1 receptor density of the rats in the highest dosage group was significantly lower than that in the control group while brain M2 receptors density had a decrease trend with increasing dosage, but the difference showed no significance. However, there were no differences of the affinity of both M1 and M2 among different treated groups. Correlation analysis showed there is positive relationship between cholinesterase activity and density of M1 receptors(r = 0.583, P < 0.01).

CONCLUSIONM1 and M2 receptors density decreased with the increasing dosage of dimethoate. It is suggested that the alleviating of cholinergic symptoms may be due to the decrease of M1 and M2 receptors in rat brain.

Animals ; Brain ; drug effects ; metabolism ; Cholinesterases ; metabolism ; Dimethoate ; pharmacology ; Dose-Response Relationship, Drug ; Radioligand Assay ; Rats ; Rats, Sprague-Dawley ; Receptor, Muscarinic M1 ; analysis ; drug effects ; Receptor, Muscarinic M2 ; analysis ; drug effects

BACKGROUND: Non-depolarizing muscle relaxants have their muscle relaxing effect by competing with acetylcholine (ACh) at the nicotinic receptor level. What are the effects of such muscle relaxants on the tracheal smooth muscle? This present study was set up to address the question as to how vecuronium and pancuronium influence the tracheal smooth muscle. METHODS: Sixty male Sprague-Dawley rat tracheal smooth muscles were isolated at optimal length for isometric force. The preparations were set up in an organ bath containing Tyrode's solution. And isometric force displacement transducer and physiograph were used to record the change in force. After the equilibration period the preparations were contracted with ACh 10(-5) M and carbachol 3x10(-7)M seperately. The preparations were washed with fresh tyrode's solution and allowed to return passively to resting tone. Then the cumulartive effect of ACh (from 3 10(-7) M through 10(-5) M) and carbachol (CCh, from 10(-8) M through 3 10(-6) M) were produced before and after pretreating the preparation with vecuronium (10(-5) M and 10(-6) M) and pancuronium (10(-5) M and 10(-6) M) respectively. Also, we studied the changes of contraction produced by neostigmine before and after pretreatment with vecuronium (10(-5) M and 3 10(-5) M) and pancuronium (3 10(-6) M and 3 10(-5) M). RESULTS: Vecuronium shifted the ACh dose-response curve of the tracheal contraction to the left (p<0.0001) and pancuronium shifted the curve to the right side (p<0.0001). However vecuronium shifted the carbachol dose-response curve to the right side as did pancuronium. Also the contraction effects of neostigmine after pretreatment with muscle relaxants decreased in the group pretreated with vecuronium (p<0.05) and a higher concentration of pancuronium (p<0.01), but with a low concentration of pancuronium the effect increased (p>0.05). CONCLUSIONS: Vecuronium inhibits the ACh hydrolyzing enzyme, especially acetylcholinesterase. Therefore it potentiates ACh contraction in the tracheal smooth muscle, but not the CCh contraction, while pancuronium has a different effect in comparison with vecuronium. That is, at a low concentration it reveals an antagonistic effect on the muscarinic M2 receptor and at a higher concentration it has an antagonistic effect on the muscarinic M3 receptor in the tracheal smooth muscle.
Acetylcholine ; Acetylcholinesterase ; Animals ; Baths ; Carbachol ; Humans ; Male ; Muscle, Smooth* ; Neostigmine ; Neuromuscular Nondepolarizing Agents ; Pancuronium* ; Rats* ; Rats, Sprague-Dawley ; Receptor, Muscarinic M2 ; Receptor, Muscarinic M3 ; Receptors, Nicotinic ; Transducers ; Vecuronium Bromide*

OBJECTIVETo determine whether autoantibodies against beta(1)-adrenergic and M(2)-muscarinic receptors are related to patients with congestive heart failure (CHF).

METHODSBoth synthetic peptides corresponding to amino acids sequence 197-222 and 169-173 of the second extracellular loops of the beta(1) and M(2) receptors were used as antigens to screen sera from 265 patients.188 were congestive heart failure ( CHF) patients with different heart diseases, among them 42 were ischemic cardiomyopathy (ICD) and 52 were idiopathic dilated cardiomyopathy (IDCM) 44 were hypertensive heart disease (HHD) 50 were rheumatic valvular heart disease (RVHD); 77 were controls, among them 36 were simple hypertension and 41 were healthy donors (NC).

RESULTSPositive sera for beta(1)-adrenergic receptor was found in 45.73% (86/188) of CHF patients, while in the controls it was 10.4% (8/77) (P < 0.01); positive sera for M(2)-muscarinic receptor in CHF patients was found in 49.5% (99/188), while in the control it was 11.7% (9/77) (P < 0.01). The positive ratio of autoantibodies against beta(1)-adrenergic and M(2)-muscarinic receptors in CHF patients with cardiac function class II-III (NYHA) were significantly higher than cardiac function class IV. The average titer of autoantibodies against beta(1)-adrenergic and M(2)-muscarinic receptors of the former was significantly higher than the latter; 56.1% of patients with autoantibodies against beta(1)-adrenergic receptor had autoantibodies against M(2)-muscarinic receptor.

CONCLUSIONSAutoantibodies against beta(1)-adrenergic receptor and M(2)-muscarinic receptor were found in sera from heart failure patients with different cardiac diseases. We propose that autoantibodies against beta(1) and M(2) receptors are not only related to the IDCM, but also to cardiac structural and functional changes.

Adult ; Aged ; Amino Acid Sequence ; Autoantibodies ; blood ; Female ; Heart Failure ; immunology ; physiopathology ; Humans ; Male ; Middle Aged ; Molecular Sequence Data ; Receptor, Muscarinic M2 ; Receptors, Adrenergic, beta-1 ; immunology ; Receptors, Muscarinic ; immunology

PURPOSE: The aim of this study was to analyze patterns of sensory protein expression and urothelial dysfunction in ketamine-related cystitis (KC) in humans. METHODS: Biopsies of bladder mucosa were performed in 29 KC patients during cystoscopy. Then specimens were analyzed for tryptase, zonula occludens-1 (ZO-1), E-cadherin, and terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) with immunofluorescence staining and quantification. In addition, 10 healthy control bladder specimens were analyzed and compared with the KC specimens. Another 16 whole bladder specimens obtained from partial cystectomy were also analyzed for the muscarinic receptors M2 and M3, endothelial nitric oxide synthase (eNOS), inducible nitric oxide synthase (iNOS), β-3 adrenergic receptors (β3-ARs), and the P2X₃ receptor by western blotting. In addition, 3 normal control bladder specimens were analyzed and compared with the KC specimens. RESULTS: The KC bladder mucosa revealed significantly less expression of ZO-1 and E-cadherin, and greater expression of TUNEL and tryptase activity than the control samples. The expression of M3 and β3-AR in the KC specimens was significantly greater than in the controls. The expression of iNOS, eNOS, M2, and P2X3 was not significantly different between the KC and control specimens. CONCLUSIONS: The bladder tissue of KC patients revealed significant urothelial dysfunction, which was associated with mast-cell mediated inflammation, increased urothelial cell apoptosis, and increased expression of the M3 and β3-AR.
Apoptosis ; Biopsy ; Blotting, Western ; Cadherins ; Cystectomy ; Cystitis* ; Cystoscopy ; Fluorescent Antibody Technique ; Humans* ; In Situ Nick-End Labeling ; Inflammation ; Ketamine ; Mucous Membrane ; Nitric Oxide Synthase Type II ; Nitric Oxide Synthase Type III ; Receptor, Muscarinic M2 ; Receptors, Adrenergic ; Tryptases ; Urinary Bladder ; Urothelium