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

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

This paper is aimed to study the effect of ADL on expression of β1-AR and M2-AchR in myocardial cells of rats exposed to microwave radiation. Immunohistochemistry, Western blot and image analysis were used to detect the expression of β1-AR and M2-AchR in myocardial cells at 7 and 14 d after microwave exposure. The results show that the expression level was higher in microwave exposure group and 0.75 g/(kg•d) ADL group than in sham operation group and significantly lower in 1.5 and 3.0 g/(kg•d) ADL groups than in microwave group. So we have a conclusion that the expression of β1-AR and M2-AchR is down-regulated in myocardial cells of rats exposed to microwave radiation. ADL can protect rats against microwave-induced heart tissue injury.
Animals ; Down-Regulation ; drug effects ; Drugs, Chinese Herbal ; pharmacology ; Heart ; drug effects ; Male ; Microwaves ; adverse effects ; Myocardium ; cytology ; metabolism ; Protective Agents ; pharmacology ; Rats, Wistar ; Receptor, Muscarinic M2 ; metabolism ; Receptors, Adrenergic, beta-1 ; metabolism

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

AIMTo study the relation between Respiratory Syncytial Virus infection and asthma development by measuring airway responsiveness (AR) and M2R function.

METHODSGuinea pigs (n = 34) were randomly divided into 4 groups: Hep-2/NS group (group A, n = 9), RSV/NS group (group B, n =9), Hep-2/OVA group (group C, n = 8) and RSV/OVA group(group D, n = 8). On day 21 after infection we tested AR and M2R. Then counted eosinophils in BALF and observed pathological change.

RESULTSIntraairway pressure(IP mmH20) of group B had no significant difference with group A(P > 0.01), and the extent of IP decrease also had no difference between groups A and B (P > 0. 05), but IP of C group were much higher than group A (P<0.05), with extent of IP decrease lower than group A (P < 0.05). And IP of group D were higher than group C (P < 0.01), with the extent of IP decrease much lower than group C (P < 0.05).

CONCLUSIONRSV infection could enhance OVA-induced M2R dysfunction, then develop AHR.

Animals ; Asthma ; immunology ; physiopathology ; virology ; Bronchial Hyperreactivity ; immunology ; physiopathology ; virology ; Female ; Guinea Pigs ; Male ; Ovalbumin ; immunology ; Random Allocation ; Receptor, Muscarinic M2 ; physiology ; Respiratory Syncytial Virus Infections ; immunology ; Respiratory Syncytial Viruses ; immunology

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 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 investigate the effect of serum autoantibodies against the human M(2) muscarinic acetylcholine receptors (M(2)-receptors, Abs) from patients with congestive heart failure on L-Type Ca(2+) channel activity in guinea pig cardiac myocytes.

METHODUsing whole cell patch-clamp technique, we quantitatively measured the ionic intensity and density of L-Type Ca(2+) channel (I(Ca-L)).

RESULTSThe M(2)-receptors agonist (carbachol) could decrease the I(Ca-L) peak intensity and density stimulated by isoprenaline from (2111.65 +/- 203.13) pA and (18.83 +/- 1.14) pA/pF to (1230.87 +/- 208.14) pA (P < 0.01) and (10.72 +/- 1.06) pA/pF (P < 0.01). The serum Abs could also decrease I(Ca-L) peak intensity and density [from (1995.21 +/- 195.13) pA and (18.13 +/- 1.03) pA/pF to (636.42 +/- 110.07) pA (P < 0.01) and (5.54 +/- 0.81) pA/pF, P < 0.01]. The M(2)-receptors antagonist, atropine was able to block these effects of carbachol and Abs.

CONCLUSIONSThe circulating serum autoantibodies against the M(2)-receptors has similar effect as M(2)-receptors agonist on decreasing the isoprenaline stimulated I(Ca-L) in guinea pig cardiac myocytes and possess negative inotropic effect. These results further suggest that serum autoantibodies against the human M(2) muscarinic acetylcholine receptors may participate in the pathophysiological processes in patients with heart failure.

Adult ; Aged ; Animals ; Autoantibodies ; pharmacology ; Calcium Channels, L-Type ; drug effects ; Female ; Guinea Pigs ; Heart Failure ; immunology ; Humans ; Male ; Middle Aged ; Myocytes, Cardiac ; drug effects ; metabolism ; Patch-Clamp Techniques ; Receptor, Muscarinic M2 ; immunology ; Serum ; immunology

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 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