Nicotinic cholinergic receptors in esophagus:Early alteration during carcinogenesis and prognostic value

AIM: To compare expression of nicotinic cholinergic receptors(CHRNs) in healthy and squamous cell carcinoma-affected esophagus and determine the prognostic value.METHODS: We performed RT-q PCR to measure the expression of CHRNs in 44 esophageal samples from healthy individuals and in matched normal surrounding mucosa, and in tumors from 28 patientsdiagnosed with esophageal squamous cell carcinoma(ESCC). Next, we performed correlation analysis for the detected expression of these receptors with the habits and clinico-pathological characteristics of all study participants. In order to investigate the possible correlations between the expression of the different CHRN subunits in both healthy esophagus and tissues from ESCC patients, correlation matrices were generated. Subsequently, we evaluated whether the detected alterations in expression of the various CHRNs could precede histopathological modifications during the esophageal carcinogenic processes by using receiver operating characteristic curve analysis. Finally, we evaluated the impact of CHRNA5 and CHRNA7 expression on overall survival by using multivariate analysis.RESULTS: CHRNA3, CHRNA5, CHRNA7 and CHRNB4, but not CHRNA1, CHRNA4, CHRNA9 or CHRNA10, were found to be expressed in normal(healthy) esophageal mucosa. In ESCC, CHRNA5 and CHRNA7 were overexpressed as compared with patient-matched surrounding non-tumor mucosa(ESCC-adjacent mucosa; P < 0.0001 and P = 0.0091, respectively). Positive correlations were observed between CHRNA3 and CHRNB4 expression in all samples analyzed. Additionally, CHRNB4 was found to be differentially expressed in the healthy esophagus and the normalappearing ESCC-adjacent mucosa, allowing for distinguishment between these tissues with a sensitivity of 75.86% and a specificity of 78.95%(P = 0.0002). Finally, CHRNA5 expression was identified as an independent prognostic factor in ESCC; patients with high CHRNA5 expression showed an increased overall survival, in comparison with those with low expression. The corresponding age- and tumor stage-adjusted hazard ratio was 0.2684(95%CI: 0.075-0.97, P = 0.0448).CONCLUSION: Expression of CHRNs is homogeneous along healthy esophagus and deregulated in ESCC, suggesting a pathogenic role for these receptors in ESCC development and progression.

Involvement of M3 Cholinergic Receptor Signal Transduction Pathway in Regulation of the Expression of Chemokine MOB-1, MCP-1 Genes in Pancreatic Acinar Cells

Whether M3 cholinergic receptor signal transduction pathway is involved in regulation of the activation of NF-κB and the expression of chemokine MOB-1, MCP-1genes in pancreatic acinar cells was investigated. Rat pancreatic acinar cells were isolated, cultured and treated with carbachol, atropine and PDTC in vitro. The MOB-1 and MCP-1 mRNA expression was detected by using RT-PCR. The activation of NF-κB was monitored by using electrophoretic mobility shift assay. The results showed that as compared with control group, M3 cholinergic receptor agonist (10 -3 mol/L, 10 -4 mol/L carbachol) could induce a concentration-dependent and time-dependent increase in the expression of MOB-1, MCP-1 mRNA in pancreatic acinar cells. After treatment with 10 -3 mol/L carbachol for 2 h, the expression of MOB-1, MCP-1 mRNA was strongest. The activity of NF-κB in pancreatic acinar cells was significantly increased (P<0.01) after treated with M3 cholinergic receptor agonist (10 -3 mol/L carbachol) in vitro for 30 min. Either M3 cholinergic receptor antagonist (10 -5 mol/L atropine) or NF-κB inhibitor (10 -2 mol/L PDTC) could obviously inhibit the activation of NF-κB and the chemokine MOB-1, MCP-1 mRNA expression induced by carbachol (P<0.05). This inhibitory effect was significantly increased by atropine plus PDTC (P<0.01). The results of these studies indicated that M3 cholinergic receptor signal transduction pathway was likely involved in regulation of the expression of chemokine MOB-1 and MCP-1genes in pancreatic acinar cells in vitro through the activation of NF-κB.

Cholinergic receptor, nicotinic, alpha 7 as a target molecule of Arctic mutant amyloid β

Alzheimer’s disease（AD）is a progressive cognitive disorder that develops predominantly in elderly patients and is characterized by cognitive impairments affecting memory,learning,and attention（Selkoe,2002）.

Research on Autoantibodies Against Myocardialβ_1-adrenergic and M_2 Cholinergic Receptors in Patients With Chronic Keshan Disease

Objectives To explore the relationship between serum autoantibodies against myocardial β1-adrenergic, M2-cholinergic receptors and chronic Keshan disease （CKD）. Methods The second extracellular loops of β1 and M2 receptors on human cardiomyocytes were used as the antigens. Enzyme linked immunosorbent assay （ELISA） was applied to determine serum autoantibodies against myocardial β1 and ME receptors in 32 CKD patients. 31 healthy subjects from endemic area were selected as the control. Results Positive rate of autoantibodies against myocardial β1 adrenergic （51.3%, 17/32） and M2 cholinergic （56.3% , 18/32） receptors were significantly higher than those in the control （9.7%, 3/ 31; 12.9%, 4/31） （both P〈 0.01）. Both positive rate and titers of above autoantibodies in NYHA Ⅱ - Ⅲ CKD patients were significantly higher than those in NYHA Ⅳ, demonstrating an apparently positive correlation between serum antibodies against myocardial β1 and M2 receptors （r=0.95）. Conclusions Autoantibodies against myocardial β1 and M2 receptors were found in sera of CKD patients; distribution of positive rate and titers of the autoantibodies in CKD patients in various NYHA are significantly different. classes of cardiac function

An increase in intracelluar free calcium ions modulated by cholinergic receptors in rat facial nucleus

Background Ca^2＋ in the central nervous system plays important roles in brain physiology, including neuronal survival and regeneration in rats with injured facial motoneurons. The present research was to study the modulations of intracellular free Ca^2＋ concentrations by cholinergic receptors in rat facial nucleus, and the mechanisms of the modulations. Methods The fluorescence intensity of facial nucleus in Fluo-3 AM loaded acute brainstem slices was detected by applying intracellular free Ca^2＋ measurement technique via confocal laser scanning microscope. The changes of fluorescence intensity of facial nucleus indicate the average changes of intracellular free Ca^2＋ levels of the neurons. Results Acetylcholine was effective at increasing the fluorescence intensity of facial nucleus. Muscarine chloride induced a marked increase of fluorescence intensity in a concentration dependent fashion. The enhancement of fluorescence intensity by muscarine chloride was significantly reduced by thapsigargin （depletor of intracellular Ca^2＋ store; P 〈0.01）, rather than Ca^2＋ free artifical cerebrospinal fluid or EGTA （free Ca^2＋ chelator; P〉0.05）. And the increase of fluorescence intensity was also significantly inhibited by pirenzepine （M1 subtype selective antagonist; P 〈0.01） and 4-DAMP （M3 subtype selective antagonist; P 〈0.01）. In addition, fluorescence intensity was markedly increased by nicotine. The enhancement of fluorescence intensity by nicotine was significantly reduced by EGTA, nifedipine （L-type voltage-gated Ca^2＋ channel blocker）, dihydro-β-erythroidine （α4β2 subtype selective antagonist）, and in Ca^2＋ free artificial cerebrospinal fluid （P 〈0.01）, but not in the presence of mibefradil （M-type voltage-gated Ca^2＋ channel blocker） or thapsigargin （P〉0.05）. Conclusions The data provide the evidence that muscarinic receptors may induce the increase of intracellular free Ca^2＋ levels through the Ca^2＋ release of intracellular Ca^2＋ stores, in a manner related to M1 and M3 subtypes of muscarinic receptors in rat facial nucleus. Nicotine may increase intracellular free Ca^2＋ concentrations via the influx of extracellular Ca^2＋ mainly across L-type voltacle-clated Ca^2＋ channels, in a manner related to the α4β2 subtype of nicotinic receptors.

Effects of Isoflurane on the Actions of Neuromuscular Blockers on the Muscle Nicotine Acetylcholine Receptors

Summary: In this study, we tested the hypothesis that volatile anesthetic enhancement of muscle relaxation is the result of combined drug effects on the nicotinic acetylcholine receptors. The poly A m RNA from muscle by isolation were microinjected into Xenopus oocytes for receptor expression. Concentration-effect curves for the inhibition of Ach-induced currents were established for vecuronium, rocuranium, and isoflurane. Subsequently, inhibitory effects of NDMRs were studied in the presence of the isoflurane at a concentration equivalent to half the concentration producing a 50 % inhibition alone. All tested drugs produced rapid and readily reversible concentration-dependent inhibition. The 50 % inhibitory concentration values were 889 μmol/L (95 % CI: 711-1214 μmol), 33.4 μmol (95 % CI: 27.1-41.7 nmol) and 9.2 nmol (95 % CI: 7.9-12.3 nmol) for isoflurane, rocuranium and vecuronium, respectively. Coapplication of isoflurane significantly enhanced the inhibitory effects of rocuranium and vecuronium, and it was especially so at low concentration of NMDRs. Isoflurane increases the potency of NDMRs, possibly by enhancing antagonist affinity at the receptor site.

Berberine Relieves Insulin Resistance via the Cholinergic Anti-inflammatory Pathway in HepG2 Cells

Berberine（BBR） is an isoquinoline alkaloid extracted from Rhizoma coptidis and has been used for treating type 2 diabetes mellitus（T2DM） in China. The development of T2 DM is often associated with insulin resistance and impaired glucose uptake in peripheral tissues. In this study, we examined whether BBR attenuated glucose uptake dysfunction through the cholinergic anti-inflammatory pathway in Hep G2 cells. Cellular glucose uptake, quantified by the 2-[N-（7-Nitrobenz-2-oxa-1,3-diazol-4-yl）-amino]-2-deoxy-D-glucose（2-NBDG）, was inhibited by 21% after Hep G2 cells were incubated with insulin（10-6 mol/L） for 36 h. Meanwhile, the expression of alpha7 nicotinic acetylcholine receptor（α7n ACh R） protein was reduced without the change of acetylcholinesterase（ACh E） activity. The level of interleukin-6（IL-6） in the culture supernatant, the ratio of phosphorylated I-kappa-B kinase-β（IKKβ） Ser181/IKKβ and the expression of nuclear factor-kappa B（NF-κB） p65 protein were also increased. However, the treatment with BBR enhanced the glucose uptake, increased the expression of α7n ACh R protein and inhibited ACh E activity. These changes were also accompanied with the decrease of the ratio of p IKKβ Ser181/IKKβ, NF-κB p65 expression and IL-6 level. Taken together, these results suggest that BBR could enhance glucose uptake, and relieve insulin resistance and inflammation in Hep G2 cells. The mechanism may be related to the cholinergic anti-inflammatory pathway and the inhibition of ACh E activity.

Effect of Varenicline on Detrusor Overactivity in Rat Model of Parkinson’s Disease Induced by Intranigral 6-Hydroxydopamine

Background: Parkinson disease (PD) is a progressive neurodegenerative disorder characterized by both motor and non-motor symptoms. Bladder dysfunction is the common non-motor symptom of PD, most often presenting with detrusor overactivity (DO). Treatment of DO is currently limited, poorly tolerated and sometimes ineffective. Bladder responses are not only mediated by muscarinic cholinergic receptors (mAChR) but also by nicotinic cholinergic receptors (nAChR). However, nicotinic receptor subtypes and functions in the bladder are not clearly identified. Purpose: This study aimed at investigating the effect of varenicline, an alpha7 full agonist and alpha4beta2/alpha3 partial agonist, on detrusor strips in rat PD model induced by substantia nigra injection of 6-hydroxydopamine. Method: The detrusor activity was studied in an isolated organ bath system. Results: In PD group, the detrusor activity was increased, whereas varenicline decreased the DO. Conclusion: Alpha7 nAChR agonists may have therapeutic potential in treatment of bladder overactivity in PD.

Effects of rhubarb on isolated gastric muscle strips of guinea pigs

AIM:To study the effects of rhubarb (dried root of Rheum officinale Baill.) on contractile activity of isolated gastric muscle strips of guinea pigs and its possible mechanism. METHODS: A total of 48 guinea pigs were killed to remove the whole stomach. Then, the stomach was opened and the mucosal layer was removed. Parallel to the circular fibers, muscle strips were cut from the body. Each isolated gastric muscle strip was suspended in a tissue chamber containing 5 mL Krebs solution, constantly warmed by water jacket at 37℃ and bubbled continuously with a mixed gas of 950 mL/L O2 and 50 mL/L CO2. After being incubated for 1 h with 1 g tension, rhubarb of varied concentrations (1%, 2%, 7%, 20% and 70%) was added cumulatively into the tissue chamber at intervals of 2 min. Atropine (10-6 mol/L) or isoptin (5x10-8 mol/L) or hexamethonium (10-5 mol/L) was given 2 min before the administration of rhubarb. The isometrical response was measured with an ink-writing recorder. RESULTS: Rhubarb dose dependently increased the resting tension of gastric body circular muscle (CM) (r = 0.726, P<0.05). Atropine (r= 0.829, A:0.05), isoptin (r= 0.764, A;0.05) and hexamethonium (r = 0.797, P<0.05) did not affect its action in a dose-related manner. Atropine apparently reduced the increasing action of 1%, 3%, 10%, 30% and 100% rhubarb on the resting tension of gastric body CM. Isoptin inhibited the effect of 10%, 30% and 100% rhubarb on the resting tension of gastric body CM. Hexamethonium reduced the increasing action of 1%, 10%, 30% and 100% rhubarb on the resting tension of gastric body CM. Rhubarb increased the contractile frequency of CM of body. While atropine, isoptin and hexamethonium did not inhibit the contractile frequency of gastric body CM in comparison with rhubarb at the same concentration, rhubarb at the highest concentration (100%) decreased the mean contractile amplitude of gastric body CM. Atropine, isoptin and hexamethonium did not affect the mean contractile amplitude of gastric body CM compared to rhubarb at the same concentration. CONCLUSION: Rhubarb has exciting actions on isolated gastric smooth muscle strips of guinea pig. The exciting action of rhubarb is partly mediated via cholinergic M receptor, cholinergic N receptor and L-type calcium channel.

肾上腺素β受体和胆碱能M受体对边缘细胞钾离子转运的调控

耳蜗血管纹边缘细胞向内淋巴转运钾离子对耳蜗内淋巴的生成,电化学特征的维持起到直接作用。钾离子经边缘细胞转运的过程除了受细胞内外离子浓度、膜电压的调控外还受细胞膜受体及细胞内信号转导系统调节。目前研究发现肾上腺素β受体促进边缘细胞的钾离子转运;胆碱能M受体则抑制边缘细胞的钾离子转运。肾上腺素β受体和胆碱能M受体对边缘细胞钾离子转运的调控作用具有重要的临床意义。

Cholinergic agonists increase intracellular calcium concentration in guinea pig vestibular hair cells

Abstract:Objective To better understand the cholinergic receptors in vestibular hair cells (VHC) and their subtypes, and to investigate the effects of cholinergic agonists on intracellular calcium concentration ([Ca2+]i ) in guinea pig VHCs.Methods VHCs were isolated from guinea pig crista ampullaris by enzymatic and mechanical methods. The effect of cholinergic agonists on [Ca2+]i was examined using laser scanning confocal microscopy and the Ca2+ sensitive dye Fluo-3.Results The results showed that the addition of acetylcholine (ACh) and carbachol (CCh), muscarnic and nicotinic agonists, induced [Ca2+]i increases in all the VHCs, whereas acetylcholine bromide (ACh-Br), a nicotinic agonist, induced the [Ca2+]i increase in only a small percentage of VHCs. The ACh or CCh-induced Ca2+ response could be partially suppressed by atropine. In the presence of 0.1?mmol/L atropine, the amplitudes of ACh or CCh-induced [Ca2+]i responses became significantly smaller than those in atropine free medium (P<0.01). Conclusions The results suggest the existence of cholinergic receptors in guinea pig VHCs. It is the muscarnic agonists rather than nicontic receptors that dominate [Ca2+]i variation. Atropine can suppress muscarnic agonist-induced Ca2+ responses.

Autoantibodies against the myocardial β_1-adrenergic and M_2-muscarinic receptors in patients with congestive heart failure

Objective To determine whether autoantibodies against β 1-adrenergic and M 2-muscarinic receptors are related to patients with congestive heart failure (CHF).Methods Both synthetic peptides corresponding to amino acids sequence 197-222 and 169-173 of the second extracellular loops of the β 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). Results Positive sera for β 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 β 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 β 1-adrenergic and M 2-muscarinic receptors of the former was significantly higher than the latter; 56.1% of patients with autoantibodies against β 1-adrenergic receptor had autoantibodies against M 2-muscarinic receptor. Conclusions Autoantibodies against β 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 β 1 and M 2 receptors are not only related to the IDCM, but also to cardiac structural and functional changes.

Autoantibodies against the myocardial β1-adrenergic and M2-muscarinic receptors in patients with congestive heart failure

Objective To determine whether autoantibodies against β 1-adrenergic and M 2-muscarinic receptors are related to patients with congestive heart failure (CHF).Methods Both synthetic peptides corresponding to amino acids sequence 197-222 and 169-173 of the second extracellular loops of the β 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). Results Positive sera for β 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 β 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 β 1-adrenergic and M 2-muscarinic receptors of the former was significantly higher than the latter; 56.1% of patients with autoantibodies against β 1-adrenergic receptor had autoantibodies against M 2-muscarinic receptor. Conclusions Autoantibodies against β 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 β 1 and M 2 receptors are not only related to the IDCM, but also to cardiac structural and functional changes.

Effect of Jianpiyiqi granule on ocular myasthenia gravis in children prepared with a formula from Traditional Chinese Medicine

OBJECTIVE: To investigate the effect of Jianpiyiqi granule, prepared according to a Traditional Chinese Medicine(TCM) formula, on the ocular type of myasthenia gravis(MG) caused by a defect in synaptic transmission at the neuromuscular junction.METHODS: A total of 155 children with ocular MG were recruited from January 2008 to January 2015.All individuals received ineffective glucocorticoid treatment prior to admission, and were given Jianpiyiqi granules(two doses per day) for 12 months.Plasma levels of acetylcholine receptor autoantibodies(ACh R-Ab), cytokines, immune parameters and clinical score were analyzed.RESULTS: After intervention with Jianpiyiqi granule for 12 months, the levels of cytokines [including interleukin(IL)-6, IL-12, IL-17], immune parameters[including immunoglobulin G(Ig G), Ig M, free triiodothyronine, free thyroxine] and ACh R-Ab were sig-nificantly decreased(P = 0.042, P = 0.049, P = 0.011,P = 0.02, P = 0.04, P = 0.03, P = 0.01; F = 21.60, P =0.000). In contrast, IL-4 levels were significantly increased(P = 0.049). The absolute clinical score after treatment declined significantly compared with before treatment(F = 33.24, P = 0.000). The effective cure rate and the total effective rate gradually increased to a maximum of 149(96.2%) and 151(97.4%) in MG patients after 12 months of treatment, respectively.CONCLUSION: The Jianpiyiqi granule treatment lowered ACh R-Ab levels and improved cytokine and other immune parameter levels, which suggests that the granule could be an ancillary treatment for ocular MG in children caused by a defect in synaptic transmission at the neuromuscular junction.

Effects of Electroacupuncture on the Ability of Learning and Memory in Rats with Ischemia-reperfusion Injury

Objective： To observe the effects of pretreatment and treatment with electroacupuncture on the ability of learning and memory in rats with ischemia-reperfusion injury, and to provide the research basis for preventing and treating the disorder of learning and memory in patients with apoplectic sequelae. Methods： Seventy-two SD rats, clean class, were randomly divided into sham operation group, model group, pretreatment group, and treatment group. The step-through latency and number of errors, nerve injury score, and the number of cholinoceptor in the affected hippocampus were recorded to analyze the effects of pretreatment and treatment with electroacupuncture on the ability of learning and memory in rats with ischemia-reperfusion injury. Results： 1） The rats with ischemia had various signs of nerve injury. Compared with that in the sham operation rats, the nerve injury score in the model rats was lower, the step-through latency and number of errors were increased, and the content of special mAchR was decreased （P〈0.05）; 2） Electroacupuncture could decrease the nerve injury score of the model rats. Compared with those in the model group, the step-through latency and number of errors all had significant difference in the pretreatment and treatment groups （P〈0.05）; 3） Compared with that in the model group, the content of mAchR was increased in the pretreatment group （P〈0.05）, and increased a little in the treatment group. Conclusion： Pretreatment and treatment with electroacupuncture can prolong step-through latency, and