In order to compare the potential selectivity of R-（-）-DM-phencynonate hydrochloride with its racemate （±）-DM- phencynonate hydrochloride on acetylcholine muscarinic receptor subtypes, the five human acetylch...In order to compare the potential selectivity of R-（-）-DM-phencynonate hydrochloride with its racemate （±）-DM- phencynonate hydrochloride on acetylcholine muscarinic receptor subtypes, the five human acetylcholine muscarinic receptor subtypes （M1- M5） （CHO-hml-5R） were cloned and expressed in Chinese hamster ovary （CHO-K1） cell line. The specific mRNAs of the five acetylcholine muscarinic receptor subtypes were detected by the reverse transcription-polymerase chain reaction （RT-PCR） method, demonstrating the definite expression of muscarinic receptor subtype genes （CHO-hml-5R）. The affinity and saturability of different muscarinic receptor subtypes to [^3H] N-methylscopolamine （[^3H]-NMS） were obtained by radioligand binding assay. Equilibrium binding assay revealed that the maximum binding capacity of [^3H]-NMS （Bmax value） to CHO-hml-5R were 40.22±3.23, 24.53±4.11, 29.65±2.65, 25.41±2.46, 32.78±4.81 pmol/mg·protein, respectively. Kd values of [^3H]-NMS to muscarinic receptors M1 to M5 were 0.97±0.22, 1.16±0.14, 0.99±0.06, 0.56±0.08, 1.12±0.06 nM, respectively. R-（-）-DM- phencynonate hydrochloride was found to block the M4 receptor with a much higher potency （pD2 = 7.48） than those displayed on M1 （pD2 = 6.20）, M2 （pD2 = 5.99）, M3 （pD2 = 5.99） and M5 （pD2 = 6.70） subtypes. However, for （±）-DM-phencynonate hydrochloride, no significant subtype receptor selectivity was found. Both （±）-DM- and R-（-）-DM-phencynonate hydrochloride showed allosteric effects on muscarinic receptors, the Hill coefficient （nH） of five receptor subtypes was less than 1, respectively. The results revealed that R-（-）-DM-phencynonate hydrochloride showed selectivity torwards M4 subtype, and there were allosteric effects for both R-（-）-DM-phencynonate hydrochloride and （±）-DM-phencynonate hydrochloride on muscarinic receptors.展开更多
BACKGROUND:Cholangiocarcinoma,a type of malignant tumor,originates from epithelial cells of the bile duct.Perineural invasion is common path for cholangiocarcinoma metastasis,and it is highly correlated with postopera...BACKGROUND:Cholangiocarcinoma,a type of malignant tumor,originates from epithelial cells of the bile duct.Perineural invasion is common path for cholangiocarcinoma metastasis,and it is highly correlated with postoperative recurrence and poor prognosis.It has been reported that muscarinic acetylcholine receptor M3(mAChR M3) is widely expressed in digestive tract cancer,and may play an important role in the proliferation,differentiation,transformation and carcinogenesis of tumors.This study was to explore the effect of mAChR M3 on the growth of cholangiocarcinoma cells in vitro and provide a new approach to the pathogenesis and treatment of cholangiocarcinoma.METHODS:Streptavidin-biotin complex immunohistochemistry was carried out to assess the expression of mAChR M3 in surgical specimens of cholangiocarcinomas(40 cases) and normal bile duct tissues(9),as well as to investigate nerve infiltration.The cholangiocarcinoma cells were treated with different concentrations of selective M-receptor agonist pilocarpine and M-receptor blocker atropine sulfate to induce changes in cell proliferation.The experimental data were analyzed by the Chi-square test.RESULTS:The strongly-positive expression rate of mAChR M3 was much higher in poorly-differentiated(69%,9/13) than in well-and moderately-differentiated cholangiocarcinomas(30%,8/27)(χ 2 =5.631,P<0.05).The strongly-positive mAChR M3 expression rate in hilar cholangiocarcinoma(50%,14/28) was higher than that in cholangiocarcinomas from the middle and lower common bile duct(25%,3/12)(χ 2 =2.148,P<0.05).Cholangiocarcinomas with distant metastasis had a stronglypositive expression rate(75%,9/12),which was much higher than those without distant metastasis(29%,8/28)(χ 2 =7.410,P<0.01).The absorbance value in the pilocarpine+atropine group was significantly higher than the corresponding value in the pilocarpine group.CONCLUSIONS:The expression of mAChR M3 is influenced by the extent of differentiation,distant metastasis and the site of cholangiocarcinoma.It also plays a key role in the proliferation and metastasis of cholangiocarcinoma.展开更多
Background: Evidences have shown that anti-M3 muscarinic acetylcholine receptor IgG (anti-M3 mAChR IgG) are clinically useful autoantibody that exert a cholinergic pharmacologic effect binding and interacting with M3 ...Background: Evidences have shown that anti-M3 muscarinic acetylcholine receptor IgG (anti-M3 mAChR IgG) are clinically useful autoantibody that exert a cholinergic pharmacologic effect binding and interacting with M3 mAChR at the level of exocrine gland (salivary and ocular). Aims: The aim of this study was to determine the associations between serum level of anti-M3 mAChR IgG in patients with systemic lupus erythematosus (SLE) and other autoantibodies, serum prostaglandin E2 (PGE2), and clinical manifestations. Methods: Serum autoantibodies against M3 mAChR synthetic peptide were measured by enzyme-linked immuno absorbent assay (ELISA) using, as an antigen, a 25-mer peptide K-R-T-V-P-D-N-Q-C-F-I-Q-F-L-S-N-P-A-V-T-F-G-T-A-I corresponding to the amino acid sequence of the second extracellular loop of the human M3 mAChR. Serum levels of antinuclear antibodies (ANA), anti-Smith (Sm) antibodies, anti-phospholipid (APL) antibodies, and PGE2 were determined by ELISA in patients with SLE. Results: We found significantly enhanced titers of anti-M3 mAChR IgG in sera from SLE patients compared with healthy individuals (control). In addition, serum levels of PGE2 were significantly higher in SLE patients than in control patients and were significantly higher in active than in non-active SLE. No correlation was found with other autoantibodies present in SLE. By contrast, a positive correlation was found between anti-M3 mAChR IgG and PGE2 serum levels in SLE. Conclusions: As anti-M3 mAChR antibodies present in the sera of SLE patients may be another factor in the pathogenesis of this disease, and the increment of PGE2 in the sera of SLE has a modulatory action on the inflammatory process, suggesting that the presence of these autoantibodies against M3 mAChR may contribute to sustained immune deregulation and the strong inflammatory component observed in SLE.展开更多
Aims: This paper investigates the presence of M3 muscarinic acetylcholine receptor autoantibody present in the serum of patients with primary Sj?gren syndrome (pSS). Main methods: We detected the levels of M3mAChR pep...Aims: This paper investigates the presence of M3 muscarinic acetylcholine receptor autoantibody present in the serum of patients with primary Sj?gren syndrome (pSS). Main methods: We detected the levels of M3mAChR peptide IgG, PGE2, IL-1β in serum of SS patients using the enzyme-linked immune sorbent assay (ELISA). To measure the quantity of nitrite/nitrate, we used Griess reagent system. Key findings: Titres of M3mAChR antibody in sera from SS patients are significantly enhanced compared to healthy subjects (control). The enhancement of these autoantibodies is accompanied by the increase of the levels of PGE2, IL-1β and nitrite/nitrate in serum. Under in vitro conditions, the synthetic human M3 peptide impaires the increment of M3mAChR antibody but not that of nati-Ro/SSA antibody. In positive anti-Ro/SSA antibody patients, the increment of M3mAChR peptide IgG and the measured pro-inflammatory substances is related. Significance: On this basis, anti M3mAChR peptide IgG can be said to act as a modulator of the immune system and to play a role in the host-chronic increment of proinflammatory substances in SS patients with positive Ro/SSA antibody. This association between the antibody and the pathogenesis of SS disease may result in useful predicting SS.展开更多
Acetylcholine (ACh), as an important neuro-transmitter in animals, also plays a significant role in various kinds of physiological functions in plants. But relatively little is known about its receptors in plants. A g...Acetylcholine (ACh), as an important neuro-transmitter in animals, also plays a significant role in various kinds of physiological functions in plants. But relatively little is known about its receptors in plants. A green fluorescence BODIPY FL-labeled ABT, which is a high affinity ligand of muscarinic acetylcholine receptor (mAChR), was used to localize mAChR in plant guard cells. In Vicia faba L. and Pisum sativum L., mAChR was found both on the plasma membrane of guard cells. mAChR may also be distributed on guard cell chloroplast membrane of Vicia faba L. The evidence that mAChR localizes in the guard cells provides a new possible signal transduction pathway in ACh mediated stomata movement.展开更多
Acetyicholine (ACh) is an important neuro-chemical transmitter in animals; it also exists in plants and plays a significant role in various kinds of physiological functions in plants. ACh has been known to induce the ...Acetyicholine (ACh) is an important neuro-chemical transmitter in animals; it also exists in plants and plays a significant role in various kinds of physiological functions in plants. ACh has been known to induce the stomatal opening. By monitoring the changes of cytusolic Ca^2+ with fluorescent probe Fiuo-3 AM under the confocal microscopy, we found that exogenous ACh increased cytosolic Ca^2+ concentration of guard cells of Vicia faba L. Muscarlne, an agonist of muscarine acetyicholine receptor (mAChR), could do so as well. In contrast, atropine, the antagonist of mAChR abolished the ability of ACh to increase Ca^2+ in guard cells. This mechanism is similar to mAChR in animals. When EGTA was used to chelate Ca^2+ or ruthenium red to block Ca^2+ released from vacuole respectively, the results showed that the increased cytosolic Ca^2+ mainly come from intracellular Ca^2+ store. The evidence supports that Ca^2+ is involved in guard-cell response to ACh and that Ca^2+ sigual is coupled to mAChRs in ACh signal transduction in guard cells.展开更多
基金National Natural Science Foundation of China (Grant No. 30672445)
文摘In order to compare the potential selectivity of R-（-）-DM-phencynonate hydrochloride with its racemate （±）-DM- phencynonate hydrochloride on acetylcholine muscarinic receptor subtypes, the five human acetylcholine muscarinic receptor subtypes （M1- M5） （CHO-hml-5R） were cloned and expressed in Chinese hamster ovary （CHO-K1） cell line. The specific mRNAs of the five acetylcholine muscarinic receptor subtypes were detected by the reverse transcription-polymerase chain reaction （RT-PCR） method, demonstrating the definite expression of muscarinic receptor subtype genes （CHO-hml-5R）. The affinity and saturability of different muscarinic receptor subtypes to [^3H] N-methylscopolamine （[^3H]-NMS） were obtained by radioligand binding assay. Equilibrium binding assay revealed that the maximum binding capacity of [^3H]-NMS （Bmax value） to CHO-hml-5R were 40.22±3.23, 24.53±4.11, 29.65±2.65, 25.41±2.46, 32.78±4.81 pmol/mg·protein, respectively. Kd values of [^3H]-NMS to muscarinic receptors M1 to M5 were 0.97±0.22, 1.16±0.14, 0.99±0.06, 0.56±0.08, 1.12±0.06 nM, respectively. R-（-）-DM- phencynonate hydrochloride was found to block the M4 receptor with a much higher potency （pD2 = 7.48） than those displayed on M1 （pD2 = 6.20）, M2 （pD2 = 5.99）, M3 （pD2 = 5.99） and M5 （pD2 = 6.70） subtypes. However, for （±）-DM-phencynonate hydrochloride, no significant subtype receptor selectivity was found. Both （±）-DM- and R-（-）-DM-phencynonate hydrochloride showed allosteric effects on muscarinic receptors, the Hill coefficient （nH） of five receptor subtypes was less than 1, respectively. The results revealed that R-（-）-DM-phencynonate hydrochloride showed selectivity torwards M4 subtype, and there were allosteric effects for both R-（-）-DM-phencynonate hydrochloride and （±）-DM-phencynonate hydrochloride on muscarinic receptors.
文摘BACKGROUND:Cholangiocarcinoma,a type of malignant tumor,originates from epithelial cells of the bile duct.Perineural invasion is common path for cholangiocarcinoma metastasis,and it is highly correlated with postoperative recurrence and poor prognosis.It has been reported that muscarinic acetylcholine receptor M3(mAChR M3) is widely expressed in digestive tract cancer,and may play an important role in the proliferation,differentiation,transformation and carcinogenesis of tumors.This study was to explore the effect of mAChR M3 on the growth of cholangiocarcinoma cells in vitro and provide a new approach to the pathogenesis and treatment of cholangiocarcinoma.METHODS:Streptavidin-biotin complex immunohistochemistry was carried out to assess the expression of mAChR M3 in surgical specimens of cholangiocarcinomas(40 cases) and normal bile duct tissues(9),as well as to investigate nerve infiltration.The cholangiocarcinoma cells were treated with different concentrations of selective M-receptor agonist pilocarpine and M-receptor blocker atropine sulfate to induce changes in cell proliferation.The experimental data were analyzed by the Chi-square test.RESULTS:The strongly-positive expression rate of mAChR M3 was much higher in poorly-differentiated(69%,9/13) than in well-and moderately-differentiated cholangiocarcinomas(30%,8/27)(χ 2 =5.631,P<0.05).The strongly-positive mAChR M3 expression rate in hilar cholangiocarcinoma(50%,14/28) was higher than that in cholangiocarcinomas from the middle and lower common bile duct(25%,3/12)(χ 2 =2.148,P<0.05).Cholangiocarcinomas with distant metastasis had a stronglypositive expression rate(75%,9/12),which was much higher than those without distant metastasis(29%,8/28)(χ 2 =7.410,P<0.01).The absorbance value in the pilocarpine+atropine group was significantly higher than the corresponding value in the pilocarpine group.CONCLUSIONS:The expression of mAChR M3 is influenced by the extent of differentiation,distant metastasis and the site of cholangiocarcinoma.It also plays a key role in the proliferation and metastasis of cholangiocarcinoma.
文摘Background: Evidences have shown that anti-M3 muscarinic acetylcholine receptor IgG (anti-M3 mAChR IgG) are clinically useful autoantibody that exert a cholinergic pharmacologic effect binding and interacting with M3 mAChR at the level of exocrine gland (salivary and ocular). Aims: The aim of this study was to determine the associations between serum level of anti-M3 mAChR IgG in patients with systemic lupus erythematosus (SLE) and other autoantibodies, serum prostaglandin E2 (PGE2), and clinical manifestations. Methods: Serum autoantibodies against M3 mAChR synthetic peptide were measured by enzyme-linked immuno absorbent assay (ELISA) using, as an antigen, a 25-mer peptide K-R-T-V-P-D-N-Q-C-F-I-Q-F-L-S-N-P-A-V-T-F-G-T-A-I corresponding to the amino acid sequence of the second extracellular loop of the human M3 mAChR. Serum levels of antinuclear antibodies (ANA), anti-Smith (Sm) antibodies, anti-phospholipid (APL) antibodies, and PGE2 were determined by ELISA in patients with SLE. Results: We found significantly enhanced titers of anti-M3 mAChR IgG in sera from SLE patients compared with healthy individuals (control). In addition, serum levels of PGE2 were significantly higher in SLE patients than in control patients and were significantly higher in active than in non-active SLE. No correlation was found with other autoantibodies present in SLE. By contrast, a positive correlation was found between anti-M3 mAChR IgG and PGE2 serum levels in SLE. Conclusions: As anti-M3 mAChR antibodies present in the sera of SLE patients may be another factor in the pathogenesis of this disease, and the increment of PGE2 in the sera of SLE has a modulatory action on the inflammatory process, suggesting that the presence of these autoantibodies against M3 mAChR may contribute to sustained immune deregulation and the strong inflammatory component observed in SLE.
文摘Aims: This paper investigates the presence of M3 muscarinic acetylcholine receptor autoantibody present in the serum of patients with primary Sj?gren syndrome (pSS). Main methods: We detected the levels of M3mAChR peptide IgG, PGE2, IL-1β in serum of SS patients using the enzyme-linked immune sorbent assay (ELISA). To measure the quantity of nitrite/nitrate, we used Griess reagent system. Key findings: Titres of M3mAChR antibody in sera from SS patients are significantly enhanced compared to healthy subjects (control). The enhancement of these autoantibodies is accompanied by the increase of the levels of PGE2, IL-1β and nitrite/nitrate in serum. Under in vitro conditions, the synthetic human M3 peptide impaires the increment of M3mAChR antibody but not that of nati-Ro/SSA antibody. In positive anti-Ro/SSA antibody patients, the increment of M3mAChR peptide IgG and the measured pro-inflammatory substances is related. Significance: On this basis, anti M3mAChR peptide IgG can be said to act as a modulator of the immune system and to play a role in the host-chronic increment of proinflammatory substances in SS patients with positive Ro/SSA antibody. This association between the antibody and the pathogenesis of SS disease may result in useful predicting SS.
基金supported by the National Natural Science Foundation of China(No.31300915)the Scientific Research Project Fund for Youth Teachers from Central China Normal University(No.CCNU15A05035)
基金the National NaturalScience Foundation of China (Grant No. 39870076) and the State Key Basic Research Development Program of China (Grant No. G19990117).
文摘Acetylcholine (ACh), as an important neuro-transmitter in animals, also plays a significant role in various kinds of physiological functions in plants. But relatively little is known about its receptors in plants. A green fluorescence BODIPY FL-labeled ABT, which is a high affinity ligand of muscarinic acetylcholine receptor (mAChR), was used to localize mAChR in plant guard cells. In Vicia faba L. and Pisum sativum L., mAChR was found both on the plasma membrane of guard cells. mAChR may also be distributed on guard cell chloroplast membrane of Vicia faba L. The evidence that mAChR localizes in the guard cells provides a new possible signal transduction pathway in ACh mediated stomata movement.
文摘Acetyicholine (ACh) is an important neuro-chemical transmitter in animals; it also exists in plants and plays a significant role in various kinds of physiological functions in plants. ACh has been known to induce the stomatal opening. By monitoring the changes of cytusolic Ca^2+ with fluorescent probe Fiuo-3 AM under the confocal microscopy, we found that exogenous ACh increased cytosolic Ca^2+ concentration of guard cells of Vicia faba L. Muscarlne, an agonist of muscarine acetyicholine receptor (mAChR), could do so as well. In contrast, atropine, the antagonist of mAChR abolished the ability of ACh to increase Ca^2+ in guard cells. This mechanism is similar to mAChR in animals. When EGTA was used to chelate Ca^2+ or ruthenium red to block Ca^2+ released from vacuole respectively, the results showed that the increased cytosolic Ca^2+ mainly come from intracellular Ca^2+ store. The evidence supports that Ca^2+ is involved in guard-cell response to ACh and that Ca^2+ sigual is coupled to mAChRs in ACh signal transduction in guard cells.
