AIMTo establish the whole-cell recording techniques of the neuronal alpha4beta2, alpha4beta4, and alpha7-nicotinic acetylcholine receptors heterologously expressed in SH-EP1 cell line and discuss the electrophysiological characteristics of their open states.

METHODSThe cells were cultured with DEME medium(high glucose) and suitable for electrophysiological experiments three days after passage. The receptors were induced from resting states into open states by rapid application of nicotine (alpha4beta2, alpha4beta4) or choline (alpha7).

RESULTSThe SH-EP1 cells cultured by this method were in good conditions and expressed plenty of receptors. Alpha4beta2, alph4beta4 and alpha7 inward currents could be induced by rapid application of agonists but had different dynamic processes against time. All the three types of currents were dose and voltage-dependent and had inward rectification property.

CONCLUSIONThe open states of neuronal alpha4beta2, alpha4beta4, and alpha7-nicotinic acetylcholine receptors and their transitions have distinct characteristics and the inward currents of all this three types of receptors are dose and voltage-dependent and have inward rectification property.

Brain ; cytology ; metabolism ; Cell Line ; Epithelial Cells ; cytology ; Humans ; Membrane Potentials ; physiology ; Neurons ; cytology ; metabolism ; Patch-Clamp Techniques ; Receptors, Nicotinic ; physiology ; Transfection ; alpha7 Nicotinic Acetylcholine Receptor

Recent studies showed that Eph/Ephrin tyrosine kinase family plays an important role in the development and functional maintenance of the nervous system, but its function in the sympathetic nervous system is still obscure. In the present study, we examined the effect of Eph/Ephrin-B1 signaling on the whole-cell currents mediated by either alpha7 or alpha3-nicotinic acetylcholine receptors (nAChRs) in acutly dissociated ciliary ganglion (CG) neurons. Firstly, we detected the effect of Ephrin-B1 on nAChRs currents. The neurons were randomly divided into control group, Ephrin-B1Fc-treated group that was stimulated by recombinant Ephrin-B1Fc, IgG-treated group, and Ephrin-B1-treated group. Secondly, we studied the regulatory mechanism of Ephrin-B1Fc on nAChRs currents. The neurons were randomly divided into control group, Ephrin-B1Fc-treated group, PP2 (inhibitor of Src tyrosine kinase) or PD98095 (antagonist of mitogen-activated protein kinase)-treated group, Ephrin-B1Fc + PP2 or PD98095-treated group. The results showed that there was no significant difference between the currents in control group, IgG-treated group and Ephrin-B1-treated group, but Ephrin-B1Fc significantly suppressed both alpha3-nAChRs and alpha7-nAChRs-mediated currents (P=0.002, P=0.003). Pretreatment with PP2 or PD98095 could partially rescue the Ephrin-B1Fc-induced suppression of currents mediated by alpha3-nAChRs or alpha7-nAChRs respectively. These results suggest that the Eph/Ephrin-B1 signaling may inhibit alpha3-nAChRs and alpha7-nAChRs-mediated currents on CG neurons, involving Src tyrosine kinase and mitogen-activated protein kinase signaling in the regulation of sympathetic nervous system.
Ephrin-B1 ; metabolism ; Ganglia, Parasympathetic ; enzymology ; Mitogen-Activated Protein Kinases ; metabolism ; Neurons ; enzymology ; Receptors, Nicotinic ; metabolism ; Signal Transduction ; alpha7 Nicotinic Acetylcholine Receptor ; metabolism ; src-Family Kinases ; metabolism

The effects of presynaptic nicotinic acetylcholine receptors (nAChRs) on excitatory synaptic transmission in CA1 pyramidal neurons of the rat hippocampus were examined by blind whole-cell patch clamp recording from hippocampal slice preparations. Local application of the nAChRs agonist dimethylphenyl-piperazinium iodide (DMPP) did not induce a postsynaptic current response in CA1 pyramidal cells. However, DMPP enhanced the frequency and amplitude of spontaneous excitatory postsynaptic current (sEPSC) in these cells in a dose-dependent manner. This enhancement was blocked by the selective nicotinic alpha-7 receptor antagonist alpha-bungarotoxin, but not by the antagonist mecamylamine, hexamethonium or dihydro-beta-erythroidine. The frequency of miniature excitatory postsynaptic current (mEPSC) in CA1 pyramidal neurons was also increased by application of DMPP, indicating a presynaptic site of action of the agonist. Taken together, these results suggest that activation of presynaptic nAChRs in CA1 pyramidal neurons, which contain alpha-7 subunits, potentiates presynaptic glutamate release and consequently modulate excitatory synaptic transmission in the hippocampus.
Animals ; Bungarotoxins ; physiology ; Dimethylphenylpiperazinium Iodide ; pharmacology ; Glutamic Acid ; pharmacology ; Hippocampus ; physiology ; Male ; Neurons ; physiology ; Nicotinic Agonists ; pharmacology ; Pacemaker, Artificial ; Patch-Clamp Techniques ; Rats ; Rats, Wistar ; Receptors, Nicotinic ; physiology ; Receptors, Presynaptic ; physiology ; Synapses ; physiology ; Synaptic Transmission ; alpha7 Nicotinic Acetylcholine Receptor

BACKGROUNDInflammation is one of important mechanisms for myocardial ischemia reperfusion injury (IRI). Ischemia postconditioning (IPOC) can protect the heart against IRI by inhibiting inflammation, but its cardioprotection is weaker than that of ischemia preconditioning. Recently, the α7 subunit-containing nicotinic acetylcholine receptor (α7nAChR) agonist has shown anti-inflammatory effects in many diseases related to inflammation. This randomized controlled experiment was designed to evaluate whether combined postconditioning with IPOC and the α7nAChR agonist could produce an enhanced cardioprotection in a rat in vivo model of acute myocardial IRI.

METHODSFifty Sprague-Dawley rats were randomly divided into five equal groups: sham group, control group, IPOC group, α7nAChR agonist postconditioning group (APOC group) and combined postconditioning with IPOC and α7nAChR agonist group (combined group). Hemodynamic parameters were recorded during the periods of ischemia and reperfusion. Serum concentrations of troponin I (TnI), tumor necrosis factor α (TNF-α) and high-mobility group box 1 (HMGB-1) at 180 minutes after reperfusion were assayed in all groups. At the end of the experiment, the infarct size was assessed from excised hearts by Evans blue and triphenyl tetrazolium chloride staining.

RESULTSAs compared to the sham group, the infarct size in the other four groups was significantly increased, serum levels of TnI, TNF-α and HMGB1 in the control group and TNF-α, HMGB1 in the IPOC group were significantly increased. The infarct size and serum concentrations of TNF-α, HMGB1 and TnI in the IPOC, APOC and combined groups were significantly lower than those in the control group. As compared to the IPOC group, the infarct size in the combined group was significantly decreased, serum concentrations of TnI, TNF-α and HMGB1 in the APOC and combined groups were significantly reduced. Although the infarct size was significantly smaller in the combined group than in the APOC group, serum levels of TNF-α and HMGB1 were significantly higher in the combined group than in the APOC group.

CONCLUSIONSIn a rat in vivo model of acute myocardial IRI, combined postconditioning with IPOC and the α7nAChR agonist can produce enhanced protection against myocardial IRI by increasing the anti-inflammatory effect.

Animals ; Heart ; drug effects ; Ischemic Preconditioning, Myocardial ; methods ; Male ; Myocardial Reperfusion Injury ; prevention & control ; Myocardium ; pathology ; Nicotinic Agonists ; therapeutic use ; Rats ; Rats, Sprague-Dawley ; Receptors, Nicotinic ; metabolism ; Tumor Necrosis Factor-alpha ; blood ; alpha7 Nicotinic Acetylcholine Receptor

The cholinergic anti-inflammatory pathway (CAP) is a neurophysiological mechanism that regulates the immune system. The CAP inhibits inflammation by suppressing cytokine synthesis via release of acetylcholine in organs of the reticuloendothelial system, including the lungs, spleen, liver, kidneys and gastrointestinal tract. Acetylcholine can interact with alpha7 nicotinic acetylcholine receptors (alpha7 nAchR) expressed by macrophages and other cytokine producing cells, down-regulate pro-inflammatory cytokine synthesis and prevent tissue damage. Herein is a review of the neurophysiological mechanism in which the CAP regulates inflammatory response, as well as its potential interventional strategy for inflammatory diseases.
Acetylcholine ; pharmacology ; Animals ; Humans ; Inflammation ; immunology ; prevention & control ; Myocardial Infarction ; immunology ; Pancreatitis ; immunology ; Receptors, Muscarinic ; physiology ; Receptors, Nicotinic ; physiology ; Reperfusion Injury ; immunology ; Sepsis ; immunology ; Shock, Hemorrhagic ; immunology ; Spleen ; immunology ; innervation ; Vagus Nerve ; physiology ; alpha7 Nicotinic Acetylcholine Receptor

PURPOSE: To investigate the role of alpha3 and alpha7 nicotinic acetylcholine receptor subunits (nAChRs) in the bladder, using a rat model with detrusor overactivity induced by partial bladder outlet obstruction (BOO). METHODS: Forty Sprague-Dawley rats were used: 10 were sham-operated (control group) and 30 were observed for 3 weeks after partial BOO. BOO-induced rats were further divided into 3 groups: Two groups of 10 rats each received intravesicular infusions with hexamethonium (HM group; n=10) or methyllycaconitine (MLC group; n=10), which are antagonists for alpha3 and alpha7 nAChRs, respectively. The remaining BOO-induced rats received only saline infusion (BOO group; n=10). Based on the contraction interval measurements using cystometrogram, the contraction pressure and nonvoiding bladder contractions were compared between the control and the three BOO-induced groups. Immunofluorescent staining and Western blotting were used to analyze alpha3 and alpha7 nAChRs levels. RESULTS: The contraction interval of the MLC group was higher than that of the BOO group (P<0.05). Nonvoiding bladder contraction almost disappeared in the HM and MLC groups. Contraction pressure increased in the BOO group (P<0.05) compared with the control group and decreased in the HM and MLC groups compared with the BOO group (P<0.05). Immunofluorescence staining showed that the alpha3 nAChR signals increased in the urothelium, and the alpha7 nAChR signals increased in the urothelium and detrusor muscle of the BOO group compared with the control group. Western blot analysis showed that both alpha3 and alpha7 nAChR levels increased in the BOO group (P<0.05). CONCLUSIONS: Alpha3 and alpha7 nAChRs are associated with detrusor overactivity induced by BOO. Furthermore, nAChR antagonists could help in clinically improving detrusor overactivity.
alpha7 Nicotinic Acetylcholine Receptor ; Animals ; Blotting, Western ; Fluorescent Antibody Technique ; Hexamethonium ; Models, Animal ; Rats* ; Rats, Sprague-Dawley ; Receptors, Nicotinic* ; Urinary Bladder ; Urinary Bladder Neck Obstruction* ; Urinary Bladder, Overactive ; Urothelium

OBJECTIVETo investigate the expression of alpha7-nAChR on rat hippocampal astrocytes in vivo and in vitro.

METHODSRat hippocampus was isolated and cut into 30-microm cryosections with anti-GFAP antibody staining followed by staining for alpha7-nAChR. The cultured astrocytes obtained from newborn rat (1-2 days old) hippocampus were identified with GFAP, and the expression of alpha7-nAChR was measured by fluorescein isothcyanate-tagged alpha-bungarotoxin staining analysis and double immunolabeling.

RESULTSThe localization of alpha7-nAChR was visualized in green with fluorescein isothiocyanate labeled IgG, whereas that of GFAP in red with Texas red-labeled IgG in the hippocampal slices, and the yellow spots indicating colocalization of the two fluorescent probes was shown in a merging image. alpha-bungarotoxin-binding nicotinic receptors were clustered, and the colocalization of alpha7-nAChR and GFAP on the cultured hippocampal astrocytes was visualized with the two fluorescent probes.

CONCLUSIONThe expression of alpha7-nAChR is identified on hippocampal astrocytes in vivo and in vitro.

Animals ; Animals, Newborn ; Astrocytes ; cytology ; metabolism ; Cells, Cultured ; Glial Fibrillary Acidic Protein ; analysis ; Hippocampus ; cytology ; metabolism ; Immunohistochemistry ; Microscopy, Fluorescence ; Rats ; Rats, Sprague-Dawley ; Receptors, Nicotinic ; biosynthesis ; alpha7 Nicotinic Acetylcholine Receptor

OBJECTIVETo understand the mechanism of effect of conditioned immune response in curing bronchial asthma.

METHODSAn experimental asthma modal was produced on healthy BALB/C mice (female, 4 - 6 weeks old) by sensitization and stimulation with ovalbumin (OV A). Totally 105 mice were divided into 7 groups randomly with 15 in each and treated differently: in group CIR(1), noise was used as conditioned stimulus (CS) and budesonide and salbutamol as unconditioned stimulus (UCS) respectively, a conditioned immune response model of mice with asthma was established by the combination of CS and UCS 7 times (7 days), then the mice were given CS only, and the combination were given once a week for 20 weeks. In group CIR(2) saccharin (SAC) was taken as CS, and the other treatments were the same as the group CIR(1). In the group of conventional therapy, the mice were given inhalation of nebulized budesonide and salbutamol only for 20 weeks. In the group of lower dose conventional therapy, the mice were given nebulized inhalation of budesonide and salbutamol for the first 7 days, then once a week for 20 weeks. In the noise group the mice were given noise only everyday for 20 weeks. In SAC group the mice were treated with SAC only everyday for 20 weeks. In the blank control group the mice were treated with placebo for 20 weeks. The mice in all the groups were stimulated with OVA once a day. The mice in the healthy control group were given PBS inhalation for 20 weeks. After 20 weeks therapy, the bronchoalveolar lavage fluid (BALF) was taken for eosinophils (EOS) counting. The spleens were taken to obtain CD4(+)T lymphocytes and the expression of neuronal acetylcholine receptor alpha 7 (nAChRalpha7), IL-4, IFN-gamma and IL-17 were detected by flow cytometry.

RESULTS(1) The percent of EOS of groups CIR(1), CIR(2), conventional therapy and healthy control was much lower than that of blank control (P < 0.01), and there was no significant difference among groups CIR(1), CIR(2) and conventional therapy (P > 0.05). (2) The expression of nAChRalpha7, IL-4 and IL-17 of groups CIR(1), CIR(2), conventional therapy and healthy control was much lower than that in blank control group, IFN-gamma was much higher (P < 0.01), and no significant difference was found among groups CIR(1), CIR(2) and conventional therapy (P > 0.05). There was a positive correlation between nAChRalpha7 and IL-4 (r = 0.76, P < 0.01), nAChRalpha7 and IL-17 (r = 0.46, P < 0.01). There was a negative correlation between nAChRalpha7 and IFN-gamma (r = 0.69, P < 0.01). (3) In the groups treated with lower dose of conventional therapy, noise, SAC and blank control, the epithelial tissue of airway were much thicker, the lumens were much narrower, and inflammatory cells and collagen fibers were much more than in the healthy control group, and after therapy, the inflammation in groups CIR(1), CIR(2) and conventional therapy was significantly improved.

CONCLUSIONThe conditioned immune response models established by both noise and SAC as CS and budesonide and salbutamol as UCS can downregulate nAChRalpha7 on CD4(+)T lymphocytes, regulate the function of CD4(+)T lymphocytes, and achieve the same therapeutic efficacy in treatment of asthma.

Administration, Inhalation ; Animals ; Asthma ; drug therapy ; immunology ; Budesonide ; therapeutic use ; CD4-Positive T-Lymphocytes ; immunology ; metabolism ; Female ; Gene Expression Regulation ; Mice ; Mice, Inbred BALB C ; Receptors, Nicotinic ; metabolism ; alpha7 Nicotinic Acetylcholine Receptor

OBJECTIVETo investigate the association between neuronal nicotinic acetylcholine receptor alpha 7 subunit (CHRNA7) gene and schizophrenia.

METHODSThe three polymorphisms rs2337980, rs1909884, rs883473 in CHRNA7 gene were detected based on PCR and polyacrylamide gel microarray in 129 schizophrenic trios. The results of genotyping were analyzed by haplotype relative risk analysis based on haplotype(HHRR), transmission disequilibrium test(TDT) and hyplotype analysis.

RESULTS(1)The HHRR analysis suggested that there was significant differences in rs2337980 allele frequencies between schizophrenia group and dummy control group(P= 0.017); (2)In TDT test, there may be transmission disequilibrium between rs2337980 and schizophrenia, the heterozygous parents excessively transferred the C allele to patients (P= 0.021); (3)The haplotype between rs2337980 and rs1909884 as well as the hyplotype among rs2337980, rs1909884 and rs883473 may have significant association with schizophrenia (global P= 0.034; global P= 0.027), the T-C and T-C-T hyplotype may have transmission disequilibrium with schizophrenia.

CONCLUSIONThere may be association between CHRNA7 gene polymorphisms and schizophrenia, the variant allele T in rs2337980 may have a protective effect to schizophrenia.

Adolescent ; Adult ; Female ; Gene Frequency ; Genetic Predisposition to Disease ; genetics ; Haplotypes ; Humans ; Linkage Disequilibrium ; genetics ; Male ; Middle Aged ; Polymorphism, Single Nucleotide ; genetics ; Receptors, Nicotinic ; genetics ; Schizophrenia ; genetics ; Young Adult ; alpha7 Nicotinic Acetylcholine Receptor

OBJECTIVESTo investigate the neuroprotective function of alpha7 nicotinic receptor (nAChR) and its roles in the pathogenesis of Alzheimer's disease (AD).

METHODSpecific RNA interference to alpha7 nAChR mRNA expression was performed by gene specific small interference RNA (siRNA). SH-SY5Y cells were transfected with the siRNA or treated with 20 micromol/L 3-[2, 4-dimethoxybenzylidene] anabaseine (DMXB), an alpha7 nAChR agonist. After 48 hrs culture, levels of alpha7 nAChR mRNA and protein were monitored by RT-PCR and Western blotting, respectively. In the second experiment, SH-SYSY cells treated with siRNA or DMXB were exposed to 1 micromol/L Abeta(25-35), followed by protein analysis of alpha-form of secreted beta-amyloid precursor peptide (alphaAPPs), and total APP was assayed by Western blotting. In addition, lipid peroxidation and MTT [3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide] reduction were measured by spectrophotometry.

RESULTIn RNA interference group, as compared with controls, alpha7 nAChR mRNA and protein levels were decreased with inhibitory efficiency by 80% and 69%, respectively, along with a decrease in protein levels of alphaAPP and reduction of MTT. However the product of lipid peroxidation was increased. There was an enhanced gene inhibition of alpha7 nAChR by Abeta. While cells treated with DMXB, the alpha7 nAChR protein was increased by 23% as compared with that of the control, along with decrease of alphaAPP and ERK 1/2 at the protein level. The enhanced expression of alpha7 nAChR reduced the neurotoxic effects resulted from Abeta.

CONCLUSIONThe findings indicate that alpha7 nAChR may play a significant neuroprotective role by enhancing cleavage of APP, improving antioxidant defenses and limiting the toxicity of Abeta, which has been implied in the pathogenesis of AD.

Acetylcholine ; pharmacology ; Alzheimer Disease ; pathology ; physiopathology ; Amyloid beta-Peptides ; metabolism ; toxicity ; Amyloid beta-Protein Precursor ; pharmacology ; Cells, Cultured ; Humans ; Lipid Peroxidation ; Neurons ; drug effects ; pathology ; Neuroprotective Agents ; pharmacology ; Nicotinic Agonists ; pharmacology ; Protease Nexins ; RNA Interference ; RNA, Messenger ; drug effects ; metabolism ; RNA, Small Interfering ; pharmacology ; Receptors, Cell Surface ; Receptors, Nicotinic ; metabolism ; physiology ; alpha7 Nicotinic Acetylcholine Receptor