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.

Role of Cholinergic Receptors in Colorectal Cancer: Potential Therapeutic Implications of Vagus Nerve Stimulation?

Inflammatory Bowel Disease (IBD) patients, such as Crohn’s disease or ulcerative colitis suffer from chronic and relapsing intestinal inflammation that favours the development of colitis associated cancer (CAC). This inflammation is initiated by aberrant activations of the innate immune responses associated to intestinal barrier defects. The conventional medical therapies consist to decrease the inflammatory response, which also decrease the risk of colon carcinoma but lead to severe side-effects. Recently, a number of animal studies have demonstrated that innate immune responses are attenuated by stimulation of the efferent arm of vagus nerve (VN) through its neurotransmitter acetylcholine (ACh), that acts on resident macrophages α7 nicotinic receptor (α7 nAChR). ACh also acts as a signalling molecule in epithetlial cells through cholinergic receptors such as nAChR or muscarinic (mAChR) receptors. In the current study, we aimed to extend these findings to CAC prevention by treating human adenocarcinoma cell lines through targeting cholinergic receptors with nicotine (which binds nAChR) and ACh (which binds both cholinergic receptors). Using HT-29 and Caco-2 cell lines, we demonstrated that ACh-induced activation of mAChR results in cell dissociation together with changes in expression and localization of intestinal tight and adherens junction proteins. ACh-induced modulation of cell adhesion proprieties correlates with the acquisition of invasive potential. By contrast, nicotine-mediated activation of nAChR maintains epithelial cell organisation. ACh-released by VN stimulation (VNS) could effectively preserve epithelium integrity thus limiting inflammatory response and tumor development. However, attention should be paid on the nature of the cholinergic receptor solicited. Indeed, regarding to the protective effects of nAChR signalling on epithelial cells, activation of mAChR would worsen the disease and led to increase inflammation. These data have important repercussions on the therapeutic potential of VNS in IBD and CAC, which may represent “the yin and yang” of the intestinal homeostasis.

METABOLIC KINETICS OF BRAIN MUSCARINIC CHOLINERGIC RECEPTORS IN NORMAL AND HYPOTHYROID MICE

A technique for studying in vivo the production rate and turnover rate constant of mouse brain M-receptors was established. A single injection of 25 mg / kg of Benzilylcholine Mustard to living mice resulted in 90 % irreversible block of brain M-receptors. The time course of the receptor density was then monitored by 3H-QNB binding assay and the production rate and turnover rate constant were calculated from the time course curve with a computer program. It was found that in normal mice the turnover rate constant was about 0.035 h-1 (half-life was about 20 h) and the production rate was 30-42 fmol / (h ·mg protein). Parallel experiments revealed a significant slow down of the turnover of brain M-receptors in hypothyroid mice (turnover rate constant was 0.0257±0.0012 h-1 in hypothyroid vs. 0.0356±0.0021 h-1 in normal) while the production rate was not changed significantly. The results suggest that thyroid hormones have a regulatory action on the turnover of brain M-receptors and the elevation of brain M-receptor density together with slow down of the turnover of brain M- receptors is probably one of the important mechanisms relevant to the brain dysfunction in hypothyroidism.

THE EFFECT OF ACUPUNCTURE ON PREVENTION AND TREATMENT OF ISCHEMIC ARRHYTHMIA AND THE RELATION BETWEEN EFFECT OF ACUPUNCTURE AND CHOLINERGIC MUSCARINE-LIKE RECEPTORS

For investigating the effect of acupuncture on ischemic arrhythmia and its mech-anism, adult albino rats with ligated anterior descending branch of coronary artery as experimentalmodel were treated with or without acupuncture, and others with imitative operation but without bothcoronary artery ligation and acupuncture treatment were used as control. It was found in acupuncturegroup that the fibrillation-liability of ischemic myocardium was efficiently decreased, the affinity ofAch-M receptors on membranes of ischemic myocardium was markedly increased, and the tolerance ofischemic myocardium to atropine was elevated in the experiment of atropine inducing fibrillation.These results indicate that acupuncture may play a therapeutic role on ischemic arrhythmia throughactivating the activity of muscarine-like receptors of cholinergic nervous system.

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

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.

Activities of nicotinic acetylcholine receptors modulate neurotransmission and synaptic architecture

The cholinergic system is involved in a broad spectrum of brain function, and its failure has been implicated in Alzheimer＇s disease. Acetylcholine transduces signals through muscarinic and nicotinic acetylcholine receptors, both of which influence synaptic plasticity and cognition. However, the mechanisms that relate the rapid gating of nicotinic acetylcholine receptors to persistent changes in brain function have remained elusive. Recent evidence indicates that nicotinic acetylcholine receptors activities affect synaptic morphology and density, which result in persistent rearrangements of neural connectivity. Further investigations of the relationships between nicotinic acetylcholine receptors and rearrangements of neural circuitry in the central nervous system may help understand the pathogenesis of Alzheimer＇s disease.

Effect of soman on the neuro-muscle junctional and extrajunctional nicotinic acetylcholine receptors

Autoradiography of nicotinic acetytcholine receptors(N-ACHR)with the application ofhistochemical staining location of cholinesterase was used to observe the effect of soman onjunctional and extrajunctional N-AChR.Testing with the diaphragms and extensor digitorum longusmuscles of mice and rats,we found that soman mainly increased the number of extrajunctionalN-AChR.It did not alter the number of junctional N-AChR significantly,nor did it have any pro-nouneed effects on the gtycoprotein property and isoelectfic point(pI)of junctional andextrajunctional N-AChR.The change of extrajunctional N-AChR number caused by somanis similar to the phenomenon of increased extrajunctional N-AChR number and sensitivity resultingfrom denervation,but the mechanism of action is different from the latter.The increase ofN-AChR number is one of the important characteristics of soman poisoning which make it differ-ent from other nerve agents.To maintain the metabofic balance of N-AChR may be an importantnew approach to the treatment of soman poisoning.

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.

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.

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.

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）.

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.

Puerarin partly counteracts the inflammatory response after cerebral ischemia/reperfusion via activating the cholinergic anti-inflammatory pathway

Puerarin, a major isoflavonoid derived from the Chinese medical herb radix puerariae （Gegen）, has been reported to inhibit neuronal apoptosis and play an anti-inflammatory role in focal cerebral ischemia model rats. Recent findings regarding stroke pathophysiology have recognized that anti-inflammation is an important target for the treatment of ischemic stroke. The cholinergic anti-inflammatory pathway is a highly robust neural-immune mechanism for inflammation control. This study was to investigate whether activating the cholinergic anti-inflammatory pathway can be involved in the mechanism of inhibiting the inflammatory response during puerarin-induced cerebral ischemia/reperfusion in rats. Results showed that puerarin pretreatment （intravenous injection） re- duced the ischemic infarct volume, improved neurological deficit after cerebral ischemia/reperfusion and decreased the levels of interleukin-1β, interleukin-6 and tumor necrosis factor-a in brain tissue. Pretreatment with puerarin （intravenous injection） attenuated the inflammatory response in rats, which was accompanied by janus-activated kinase 2 （JAK2） and signal transducers and activators of transcription 3 （STAT3） activation and nuclear factor kappa B （NF-KB） inhibition. These observa- tions were inhibited by the alpha7 nicotinic acetylcholine receptor （a7nAchR） antagonist a-bungarotoxin （a-BGT）. In addition, puerarin pretreatment increased the expression of a7nAchR mRNA in ischemic cerebral tissue. These data demonstrate that puerarin pretreatment strongly protects the brain against cerebral ischemia/reperfusion injury and inhibits the inflammatory re- sponse. Our results also indicated that the anti-inflammatory effect of puerarin may partly be medi- ated through the activation of the cholinergic anti-inflammatory pathway.

The Acoustic Sensorimotor Gating Predicts the Efficiency of Hypoxic Preconditioning. Participation of the Cholinergic System in This Phenomenon

Moderate one-off hypobaric hypoxia (HBH) provokes preconditioning and prolongs the resistance (T, the time before apnoea) to severe hypobaric hypoxia (SHBH). Hypoxic preconditioning has therapeutic potential;however, the efficiency of hypoxic preconditioning varies greatly and the methods for its preliminary evaluation are absent in both animals and humans. This rodent study evaluates the dependence of SHBH resistance, initiated by HBH, on the rate of sensorimotor gating estimated in the model of the acoustic startle prepulse inhibition (PPI). A stable negative correlation was found between PPI and T. Low doses of the α7 nicotinic receptor agonist, PNU-282987 (PNU), and more pronouncedly dimethyl sulfoxide (DMSO) (a PNU solvent), inverted the correlation between PPI and T from negative to positive. The DMSO and PNU effects were reversed at PPIs of 0.36 - 0.40 (36% - 40%). DMSO increased T values by 52.2% ± 9.7% in the region of lower HBH efficiency (PPI ≥ 0.40) and reduced it by 35.2% ± 9.3% in the region of higher HBH efficiency (PPI < 0.40). PNU reduced both DMSO effects. The involvement of the central cholinergic mechanisms was substantiated in both DMSO and PNU influences on HBH. In conclusion, 1) PPI can be used to predict the efficiency of hypoxic preconditioning and to study its mechanisms, 2) two opposite cholinergic PPI-related mechanisms participate in the preconditioning effects of HBH, 3) the sensitivity of rats to DMSO and PNU diverges when the PPI is 0.36 - 0.40, and 4) DMSO can enhance resistance to severe hypoxia in the region of the lower preconditioning efficiency of HBH at PPI ≥ 0.4.

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.

Mechanisms mediating cholinergic antral circular smooth muscle contraction in rats

AIM:To investigate the pathway(s)mediating rat antral circular smooth muscle contractile responses to the cholinomimetic agent,bethanechol and the subtypes of muscarinic receptors mediating the cholinergic contraction. METHODS:Circular smooth muscle strips from the antrum of Sprague-Dawley rats were mounted in muscle baths in Krebs buffer.Isometric tension was recorded.Cumulative concentration-response curves were obtained for(+)-cis- dioxolane(cD),a nonspecific muscarinic agonist,at 10^(-8)- 10^(-4)mol/L,in the presence of tetrodotoxin(TTX,10^(-7)mol/L). Results were normalized to cross sectional area.A repeat concentration-response curve was obtained after incubation of the muscle for 90 min with antagonists for M1(pirenzepine), M2(methoctramine)and M3(darifenadn)muscarinic receptor subtypes.The sensitivity to PTX was tested by the ip injection of 100 mg/kg of PTX 5 d before the experiment.The antral circular smooth muscles were removed from PTX-treated and non-treated rats as strips and dispersed smooth muscle cells to identify whether PTX-linked pathway mediated the contractility to bethanechol. RESULTS:A dose-dependent contractile response observed with bethanechol,was not affected by TTx.The pretreatment of rats with pertussis toxin decreased the contraction induced by bethanechol.Lack of calcium as well as the presence of the L-type calcium channel blocker,nifedipine,also inhibited the cholinergic contraction,with a reduction in response from 2.5±0.4 g/mm^2 to 1.2±0.4 g/mm^2(P<0.05).The dose- response curves were shifted to the right by muscarinic antagonists in the following order of affinity:darifenacin (M_3)>methocramine(M_2)>pirenzepine(M_1). CONCLUSION:The muscarinic receptors-dependent contraction of rat antral circular smooth muscles was linked to the signal transduction pathway(s)involving pertussis-toxin sensitive GTP-binding proteins and to extracellular calcium via L-type voltage gated calcium channels.The presence of the residual contractile response after the treatment with nifedipine,suggests that an additional pathway could mediate the cholinergic contraction.The involvement of more than one muscarinic receptor(functionally predominant type 3 over type 2)also suggests more than one pathway mediating the cholinergic contraction in rat antrum.

Accelerating Alzheimer’s pathogenesis by GRK5 deficiency via cholinergic dysfunction

G protein-coupled receptors (GPCRs) mediate a wide variety of physiological function. GPCR signaling is negatively regulated by the receptor desensitization, a procedure initiated by a group of kinases, including GPCR kinases (GRKs). Studies using genetargeted mice revealed that deficiency of a particular GRK member led to dysfunction of a highly selective group of GPCRs. In particular, for example, GRK5 deficiency specifically disrupts M2/M4-mediated muscarinic cholinergic function. Emerging evidence indicates that ?-amyloid accumulation may lead to GRK5 deficiency, while the latter impairs desensitization of M2/M4 receptors. Within memory circuits, M2 is primarily presynaptic autoreceptor serving as a negative feedback to inhibit acetylcholine release. The impaired desensitization of M2 receptor by GRK5 deficiency leads to hyperactive M2, which eventually suppresses acetylcholine release and results in an overall cholinergic hypofunctioning. Since the cholinergic hypofunctioning is known to cause ?-amyloid accumulation, the GRK5 deficiency appears to connect the cholinergic hypofunctioning and ?-amyloid accumulation together into a self-amplifying cycle, which accelerates both changes. Given that the ? -amyloid accumulation and the cholinergic hypofucntioning are the hallmark changes in the ?-amyloid hypothesis and the cholinergic hypothesis, respectively, the GRK5 deficiency appears to bring the two major hypotheses in Alzheimer’s disease together, whereas the GRK5 deficiency is the pivotal link. Therefore, any strategies that can break this cycle would be therapeutically beneficial for Alzheimer’s patients.

Anal metastasis from recurrent breast lobular carcinoma:A case report

We report a case of isolated gastrointestinal metastasis from breast Iobular carcinoma, which mimicked primary anal cancer. In July 2000, an 88-year-old woman presented with infiltrating Iobular cancer （pT1/G2/N2）. The patient received postoperative radiotherapy and hormonal therapy. Four years later, she presented with an anal polypoid lesion. The mass was removed for biopsy. Immunohistochemical staining suggested a breast origin. Radiotherapy was chosen for this patient, which resulted in complete regression of the lesion. The patient died 3 years after the first manifestation of gastrointestinal metastasis. According to the current literature, we consider the immunohistochemistry features that are essential to support the suspicion of gastrointestinal breast metastasis, and since we consider the gastrointestinal involvement as a sign of systemic disease, the therapy should be less aggressive and systemic.

Therapeutic potential of α7 nicotinic receptor agonists to regulate neuroinflammation in neurodegenerative diseases

Neurodegenerative diseases, such as Alzheimer＇s, Parkinson＇s and Huntington＇s diseases, are all character- ized by a component of innate immunity called neuroinflammation. Neuronal loss and neuroinflammation are two phenomena closely linked. Hence, the neuroinflammation is a relevant target for the management of the neurodegenerative diseases given that, to date, there is no treatment to stop neuronal loss. Several studies have investigated the potential effects of activators of alpha 7 nicotinic acetylcholine receptors in animal models of neurodegenerative diseases. These receptors are widely distributed in the central nervous system. After activation, they seem to mediate the cholinergic anti-inflammatory pathway in the brain. This anti-inflammatory pathway, first described in periphery, regulates activation of microglial cells considered as the resident macrophage population of the central nervous system. In this article, we shortly review the agonists of the alpha 7 nicotinic acetylcholine receptors that have been evaluated in vivo and we focused on the selective positive allosteric modulators of these receptors. These compounds represent a key element to enhance receptor activity only in the presence of the endogenous agonist.