Expression Of Acetylcholine Receptor mRNA Research Articles

Role of non-neuronal cholinergic system in the early stage response of epithelial-mesenchymal transformation related markers in A549 cells induced by coal particles

ObjectiveThis study was aimed to investigate the role of non-neuronal cholinergic system (NNCS) in the early stage response of epithelial-mesenchymal transformation (EMT) related markers in human lung adenocarcinoma A549 cells induced by coal particles. MethodsA549 cells were exposed to different concentrations of GBW11110K, GBW11126D and exogenous acetylcholinesterase (AChE) (the exposure doses were determined according to the results of CCK-8 experiment, and the doses that had no significant effects on cell viability were selected) for 24 h. After exposure, the indexes of oxidative stress (SOD and MDA), inflammatory factors (IL-6 and TNF-α), EMT marker proteins (E-cadherin and vimentin), AChE enzymatic activity and mRNA expression levels of different types of acetylcholine receptors (CHRM3, CHRM5, CHRNA5, CHRNA7, CHRNA9 and CHRNB2) were determined. ResultsGBW11110K and GBW11126D exposure could lead to the following injury effects: the levels of oxidative stress and inflammatory factors changed to a certain extent (SOD decreased gradually, while MDA, IL-6 and TNF-α increased). The protein level of E-cadherin decreased while the vimentin level increased (P < 0.05), suggesting the occurrence of EMT. The AChE enzymatic activity decreased gradually. The expression of acetylcholine receptor mRNA changed as follows (GBW11110K/GBW11126D: CHRM3 (↑↑), CHRM5 (↓↓), CHRNA5 (↓↓), CHRNA7 (↓↓), CHRNA9 (– ↑), CHRNB2 (– –). The addition of exogenous AChE recombinant protein could antagonize the damage effects caused by the coal particles to a certain extent. ConclusionThe coal particle exposure could induce the change of oxidative stress response, inflammatory response and EMT related markers, down-regulate the AChE enzymatic activity, and interfere the mRNA expression levels of AChRs in A549 cells. The addition of exogenous AChE recombinant protein could reverse the above effects to a certain extent.

The M1 muscarinic acetylcholine receptor in the crypt stem cell compartment mediates intestinal mucosal growth.

Localization of a specific subtype of the muscarinic acetylcholine receptor in the crypt stem cell compartment suggests a critical role in intestinal mucosal homeostasis. Here we demonstrate the localization of the M1 muscarinic acetylcholine receptor to the stem cell compartment and demonstrate increase morphometric and proliferative parameters when this is stimulated in vivo. These data provide novel information about this complex signaling microenvironment and offer potential future therapeutic targets for future study.

Melatonin attenuated mediators of neuroinflammation and alpha-7 nicotinic acetylcholine receptor mRNA expression in lipopolysaccharide (LPS) stimulated rat astrocytoma cells, C6

Melatonin has been known to affect a variety of astrocytes functions in many neurological disorders but its mechanism of action on neuroinflammatory cascade and alpha-7 nicotinic acetylcholine receptor (α7-nAChR) expression are still not properly understood. Present study demonstrated that treatment of C6 cells with melatonin for 24 hours significantly decreased lipopolysaccharide (LPS) induced nitrative and oxidative stress, expressions of cyclooxigenase-2 (COX-2), inducible nitric-oxide synthase (iNOS) and glial fibrillary acidic protein (GFAP). Melatonin also modulated LPS-induced mRNA expressions of α7-nAChR and inflammatory cytokine genes. Furthermore, melatonin reversed LPS-induced changes in C/EBP homologous protein 10 (CHOP), microsomal prostaglandin E synthase-1(mPGES-1) and phosphorylated p38 mitogen activated protein kinase (P-p38). Treatment with pyrrolidine dithiocarbamate (PDTC) inhibited α7-nAChR mRNA expression in LPS-induced C6 cells. Our findings explored anti-neuroinflammatory action of melatonin, which may suggests its beneficial roles in the neuroinflammation associated disorders.

Immunohistochemical Expression of Muscarinic Receptors in the Urothelium and Suburothelium of Neurogenic and Idiopathic Overactive Human Bladders, and Changes With Botulinum Neurotoxin Administration

To investigate the possible associations of urothelial and suburothelial muscarinic receptors with human bladder pathophysiology we examined the immunohistochemical expression of muscarinic receptors types 1, 2 and 3 in the bladder urothelium and suburothelium of patients with neurogenic or idiopathic detrusor overactivity compared with that in controls. We also examined associations with patient quantified symptoms and the effect of intradetrusor botulinum neurotoxin type A treatment. We obtained bladder biopsies from 36 patients with detrusor overactivity before, and 4 and 16 weeks after treatment with intradetrusor botulinum neurotoxin type A via flexible cystoscopy. Patients with neurogenic detrusor overactivity were injected with 300 U botulinum neurotoxin type A and those with idiopathic detrusor overactivity received 200 U. Control biopsies were taken from 7 patients during investigation for asymptomatic microscopic hematuria. We studied muscarinic receptor immunohistochemical expression using commercial antibodies to muscarinic receptors 1, 2 and 3 with results quantified by image analysis. We noted decreased suburothelial muscarinic receptor immunoreactivity in detrusor overactivity biopsies vs controls, which were significant for muscarinic receptors 1 and 3. After successful botulinum neurotoxin treatment we noted only increased muscarinic receptor 1 and 2 immunoreactivity. Urothelial muscarinic receptor 1 and 3 immunoreactivity was increased after treatment. We identified no substantial urothelial muscarinic receptor 2 immunoreactivity. Receptor levels showed inverse correlations with patient urgency and frequency. Decreased muscarinic receptor levels in the urothelium and suburothelium of patients with detrusor overactivity were largely restored to control levels after successful treatment with botulinum neurotoxin type A. Correlations of receptor levels with patient symptoms further support a role for urothelial and suburothelial muscarinic receptors in detrusor overactivity in humans.

α7 nicotinic acetylcholine receptor mRNA expression and binding in postmortem human brain are associated with genetic variation in neuregulin 1

Studies in cell culture and in animals suggest that neuregulin 1 (NRG1), a probable schizophrenia susceptibility gene, regulates the expression of the alpha7 nicotinic acetylcholine receptors (nAChRs). We hypothesized that schizophrenia-associated allelic variations within the NRG1 gene, via their effects on NRG1 isoform expression, would be associated with alterations in nAChR alpha7 receptor levels. We examined the effects of four disease-associated single-nucleotide polymorphisms (SNPs) in the 5' region of the NRG1 gene on nAChR alpha7 mRNA transcript expression in both the dorsolateral prefrontal cortex (DLPFC) and hippocampus of normal controls and patients with schizophrenia using quantitative real-time PCR. NRG1 risk alleles at SNPs SNP8NRG221132 and rs6994992 predicted significantly lower nAChR alpha7 mRNA expression in the DLPFC. Haplotypes containing the risk alleles at the above SNPs were also associated with lower expression of nAChR alpha7 in the DLPFC. The genotype effect for rs6994992 and the haplotype effect were more pronounced within the schizophrenic patient group. To determine whether receptor levels follow that of mRNA expression, we performed receptor binding and autoradiography using [(125)I] alpha-bungarotoxin in the DLPFC. Consistent with the mRNA findings, we found a decrease in binding in risk allele carriers of SNP8NRG221132 as compared with heterozygous individuals. Together, these results suggest that the molecular mechanism of the association between NRG1 risk alleles and schizophrenia may include down-regulation of nAChR alpha7 expression.

α7‐Type Nicotinic Acetylcholine Receptor and Prodynorphin mRNA Expression after Administration of (−)‐Nicotine and U‐50,488H in β‐Amyloid Peptide (25‐35)‐Treated Mice

We previously reported that (-)-nicotine and kappa-opioid receptor agonists lessened impairment of learning and/or memory in several animal models. Furthermore, these drugs prevented neurodegenerative damage induced by ischemia or beta-amyloid peptide (25-35). In the present study, we tested whether (-)-nicotine and U-50,488H prevent delayed-memory impairment induced by beta-amyloid peptide (25-35), and changes of expression of alpha7-type nicotinic acetylcholine receptor mRNA and prodynorphin mRNA. Seven days after treatment with beta-amyloid peptide (25-35) (9 nmol/mouse, i.c.v.), memory impairment was observed in the Y-maze test. Memory impairment was prevented when (-)-nicotine (6.16 micromol/kg, s.c.) or U-50,488H (21 micromol/kg, s.c.) was administered 1 h before, but not 1 h after, beta-amyloid peptide (25-35) treatment. There was no change in prodynorphin mRNA or alpha7-type nicotinic acetylcholine receptor mRNA expression in the hippocampus 10 days after beta-amyloid peptide (25-35) treatment alone. Of interest, mRNA expression of not only prodynorphin, but also the alpha7-type nicotinic acetylcholine receptor, was significantly decreased when U-50,488H was administered 1 h before, but not 1 h after, treatment with beta-amyloid peptide (25-35). However, these changes were not observed after the administration of (-)-nicotine. These results suggest that activation of the kappa-opioid system, but not beta7-type nicotinic receptors has a neuroprotective effect on beta-amyloid peptide (25-35)-induced memory impairment, and may be involved in the long-lasting changes in the expression of these mRNAs.

Muscle nicotinic acetylcholine receptor mRNA expression in hyperplastic and neoplastic myasthenia gravis thymus.

Muscle nicotinic acetylcholine receptor mRNA expression in hyperplastic and neoplastic myasthenia gravis thymus.

Developmental expression of ?9 acetylcholine receptor mRNA in the rat cochlea and vestibular inner ear

Expression of alpha9 acetylcholine receptor (AChR) mRNA was studied by in situ hybridization in the rat adult and developing cochlea and vestibular inner ear. Alpha9 AChR mRNA was first observed in cochlear hair cells (HCs) at embryonic day 18 (E18), increased markedly after birth, stayed high until postnatal day 10 (P10), and decreased to substantially lower adult levels by P14. High levels of alpha9 AChR mRNA expression were also noted in the developing nonneuronal structures of the inner sulcus, chondrocytes, and/or osteoblasts in the cochlear capsule and interscalar laminae. Both developing and adult bone marrow cells also expressed intense alpha9 AChR mRNA. In the vestibular system, alpha9 AChR mRNA was first observed in HCs at E16 in all sensory epithelia, increased to its highest levels by P0-P4, then decreased slightly to reach adult levels by P10. The results are consistent with the alpha9 AChR subserving efferent neurotransmission to both cochlear and vestibular HCs. The observation of alpha9 AChR mRNA in cochlear HCs 2 weeks prior to functional onset in the cochlea further suggests that expression of this gene is not related to HC activity. The observation of substantial nonneuronal expression of alpha9 AChR mRNA suggests that this receptor also has functions separate from its role in neurotransmission.

Nuclear clustering in myotubes: a proposed role in acetylcholine receptor mRNA expression

We investigated the functional relationship between nuclear topology, as expressed by degree and type of nuclear aggregation, and appearance of acetylcholine receptor (AChR) subunit mRNAs. Embryonic chick muscle cell cultures treated with the muscle activity blocking agents decamethonium (DCM), d-tubocurare (TBC), and tetrodotoxin (TTX) or co-cultured with cholinergic neurons were examined for the influence of muscle activity on nuclear aggregation and its effects on AChR α-, γ-, and δ-subunit message expression. mRNA was measured by in situ hybridization and nuclei were visualized by bis-benzimide DNA staining. DCM and TBC treatments, as well as neuronal co-culture, resulted in increased nuclear clustering within myotubes and a per nucleus upregulation in mRNA expression relative to control for each subunit. The pattern of nuclear aggregation was treatment dependent, with more and larger aggregates found when myotubes were co-cultured with neurons. Moreover, as nuclear aggregates became larger: (1) nearly all nuclei within active aggregates expressed mRNA and (2) local accumulation (mRNA per unit area) was elevated relative to single nuclei, while per nucleus mRNA production decreased. To determine whether mRNA expression was transient and did not result in steady-state upregulation of AChR receptor protein, we performed a double labeling of surface AChRs with 125 I -α-bungarotoxin ( 125 I -α-BTX) concomitant to the in situ hybridization for mRNA quantification on TTX treated muscle cells. Surface receptor expression tracked mRNA expression for all types of nuclear topology observed, indicating that message levels are in fact reliable indicators of receptor population on the plasma membrane surface in myotubes. We propose that nuclear clustering is an organelle-level, accessory mechanism whereby cells concentrate relatively large amounts of AChR mRNA/protein in specific myotube regions.

Agonist-induced down-regulation of the m4 muscarinic acetylcholine receptor occurs without changes in receptor mRNA steady-state levels

The regulation of m4 muscarinic acetylcholine receptor mRNA expression by receptor activation was studied in N1E-115 neuroblastoma and AtT-20 pituitary cells that endogeneously express the m4 muscarinic receptor. Receptor concentration was measured by binding of the muscarinic receptor radioligand [3H]quinuclidinyl benzilate, and RNA-RNA solution hybridization/RNase protection assay with a m4 receptor-specific [32P]-cRNA probe was used to evaluate the levels of receptor mRNA. Treatment of both cell lines with a receptor-saturating concentration of the agonist carbachol decreased receptor number. However, there was no change in steady-state levels of m4 mAChR mRNA in both cell lines. Determination of mRNA stability in the presence of the transcription blocker actinomycin D revealed that carbachol treatment increased half-life of receptor mRNA in N1E-115 cells, but not in AtT-20 cells, suggesting that receptor activation can regulate m4 receptor mRNA stability dependently on cell type. Analysis of receptor degradation kinetics in the presence of the protein synthesis inhibitor cycloheximide showed that receptor down-regulation in N1E-115 and AtT-20 cells is sufficiently accounted for by increased receptor degradation. These results indicate than m4 muscarinic receptor down-regulation is substantially different from that of the muscarinic receptor subtypes m2 and m3 which is reported to be associated with agonist-induced reduction in receptor mRNA.

Regulation of muscarinic acetylcholine receptor mRNA expression by activation of homologous and heterologous receptors.

Muscarinic acetylcholine receptors (mAChR) in the embryonic chicken heart undergo agonist-induced internalization and a subsequent decrease in receptor number (downregulation). Cloning studies have identified two subtypes of mAChR expressed in the embryonic chicken heart, the cm2 and cm4 receptors. We report here that persistent activation of the mAChR in cultured chicken heart cells with the cholinergic agonist carbachol causes significant decreases in the levels of both cm2 and cm4 mRNA, as measured by solution hybridization analyses. The half-lives of the cm2 and cm4 mRNAs are not altered by agonist treatment, indicating that agonist most likely regulates mRNA levels by regulating the rate of gene transcription. Activation of mAChR in chicken heart causes both inhibition of adenylate cyclase activity and stimulation of phospholipase C activity. To test whether changes in the levels of intracellular second messengers were involved in the changes in mAChR mRNAs observed following agonist exposure, we determined the effects of incubation with agonists for the A1 adenosine receptors (which inhibit adenylate cyclase in chicken heart) and angiotensin II receptors (which stimulate phospholipase C) on mAChR receptor number and mRNA levels. Activation of these pathways together through heterologous receptors resulted in decreased mAChR number and mRNA levels, although these changes were not as large as those seen with direct activation of the mAChR. These results suggest that regulation of adenylate cyclase and phospholipase C activities may be involved in the regulation of mAChR gene expression.

Factors released by ciliary neurons and spinal cord explants induce acetylcholine receptor mRNA expression in cultured muscle cells

The nuclei of cultured noninnervated muscle cells are heterogeneous with respect to production of mRNA for the nicotinic acetylcholine receptor (AChR). Some nuclei actively express AChR mRNA while others have a low level of activity or are inactive. To determine if innervation, or a factor released by neurons, influences nuclear expression of AChR mRNA, we examined mRNA at a single cell level via in situ hybridization and autoradiography with an alpha-subunit AChR genomic probe. Four days after plating, we co-cultured chicken primary muscle cells with spinal cord explants, ciliary neurons, or dorsal root ganglia (DRG) cells. In situ hybridization of the spinal-cord and muscle-cell co-cultures with the AChR alpha-subunit probe revealed a high density of silver grains on muscle cells, which were within two explant diameters of the spinal cord explant, and a graded decrease in silver grain density as the distance from the explant increased, as well as the appearance of a strikingly nonhomogenous distribution of active and inactive muscle cell nuclei. When ciliary neurons were uniformly distributed over the muscle cells, a high level of AChR mRNA was induced, but no gradients appeared. Neither an increased mRNA level nor a gradient was observed when DRG cells were co-cultured with muscle cells. When ciliary neurons are cultured within Costar permeable inserts, which prevent any contact between the neurons and the underlying muscle cells, AChR messenger RNA is still induced, showing that diffusible factors are responsible. Our results indicate that molecules released by cholinergic neurons regulate the expression of AChR mRNA in the myotubes and raise the possibility that AChR expression depends on both neuronal signals and on intracellular information from the muscle cell.

Differential expression of acetylcholine receptor mRNA in nuclei of cultured muscle cells.

Muscle cells in vitro and in vivo are multinucleated and express acetylcholine receptors (AcChoRs). On innervated cells, the AcChoRs form clusters which lie under the nerve terminals. However, noninnervated cells in culture also express clusters of AcChoR. Both in vivo and in vitro the AcChoR clusters appear to be associated with clusters of nuclei. We have used in situ hybridization to determine whether all the nuclei in cultured chicken embryo myotubes are equally active in expressing the AcChoR alpha subunit message. Cells were hybridized with 35S-labeled probes that contained either both an exon and an intron region or only exon sequences. Control cultures were hybridized with a labeled actin DNA probe or poly(U). The hybrids were detected by emulsion autoradiography; simultaneously, the nuclei were visualized with bisbenzamide. Cells hybridized with the intron/exon probe showed a striking preferential silver grain localization in and around some of the myotube nuclei, whereas those hybridized with the exon probe gave a rather homogeneous grain distribution in the cytoplasm. These results show that myotube nuclei possess differential activation capacities for the expression of AcChoR alpha subunit mRNA and that this difference is due to differential rates of transcription.