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 expressio...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.展开更多
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...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展开更多
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 mo...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.展开更多
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 ...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.展开更多
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 panc...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.展开更多
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 resistan...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.展开更多
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).
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 ...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.展开更多
基金Supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico(CNPq,Brazil),Fundacao de AmparoàPesquisa do Estado do Rio de Janeiro(FAPERJ),Ministério da Saúde(INCA) and Swiss Bridge Foundation
文摘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.
文摘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
基金This work was supported by a grant from the Youth Science Foundation of Qingdao University (No. 2007).
文摘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.
文摘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.
文摘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.
基金supported by the National Natural Science Foundation of China(No.81373872)
文摘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.
基金supported by a grant KAKENHI 15K06786the Center of Innovation Science and Technology based Radical Innovation and Entrepreneurship Program(COI STREAM)of the Ministry of Education,Culture,Sports,Science and Technology(MEXT),Japan
文摘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).
文摘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.