Junsuke Uwada

Regional quantification of muscarinic acetylcholine receptors and β‐adrenoceptors in human airways

BACKGROUND AND PURPOSE Muscarinic acetylcholine receptors (mAChRs) and β‐adrenoceptors in the airways and lungs are clinically important in chronic obstructive pulmonary disease (COPD) and asthma. However, the quantitative and qualitative estimation of these receptors by radioligand binding approaches in human airways has not yet been reported because of tissue limitations.

Novel contribution of cell surface and intracellular M1-muscarinic acetylcholine receptors to synaptic plasticity in hippocampus.

Muscarinic acetylcholine receptors (mAChRs) are well known to transmit extracellular cholinergic signals into the cytoplasm from their position on the cell surface. However, we show here that M1‐mAChRs are also highly expressed on intracellular membranes in neurons of the telencephalon and activate signaling cascades distinct from those of cell surface receptors, contributing uniquely to synaptic plasticity. Radioligand‐binding experiments with cell‐permeable and ‐impermeable ligands and immunohistochemical observations revealed intracellular and surface distributions of M1‐mAChRs in the hippocampus and cortex of rats, mice, and humans, in contrast to the selective occurrence on the cell surface in other tissues. All intracellular muscarinic‐binding sites were abolished in M1‐mAChR‐gene‐knockout mice. Activation of cell surface M1‐mAChRs in rat hippocampal neurons evoked phosphatidylinositol hydrolysis and network oscillations at theta rhythm, and transiently enhanced long‐term potentiation. On the other hand, activation of intracellular M1‐mAChRs phosphorylated extracellular‐regulated kinase 1/2 and gradually enhanced long‐term potentiation. Our data thus demonstrate that M1‐mAChRs function at both surface and intracellular sites in telencephalon neurons including the hippocampus, suggesting a new mode of cholinergic transmission in the central nervous system.

Intracellular distribution of functional M1‐muscarinic acetylcholine receptors in N1E‐115 neuroblastoma cells

J. Neurochem. (2011) 118, 958–967.

Regulation of Steroidogenesis, Development, and Cell Differentiation by Steroidogenic Factor-1 and Liver Receptor Homolog-1

Steroidogenic factor-1 (SF-1) and liver receptor homolog-1 (LRH-1) belong to the nuclear receptor superfamily and are categorized as orphan receptors. In addition to other nuclear receptors, these play roles in various physiological phenomena by regulating the transcription of target genes. Both factors share very similar structures and exhibit common functions. Of these, the roles of SF-1 and LRH-1 in steroidogenesis are the most important, especially that of SF-1, which was originally discovered and named to reflect such roles. SF-1 and LRH-1 are essential for steroid hormone production in gonads and adrenal glands through the regulation of various steroidogenesis-related genes. As SF-1 is also necessary for the development of gonads and adrenal glands, it is also considered a master regulator of steroidogenesis. Recent studies have clearly demonstrated that LRH-1 also represents another master regulator of steroidogenesis, which similarly to SF-1, can induce differentiation of non-steroidogenic stem cells into steroidogenic cells. Here, we review the functions of both factors in these steroidogenesis-related phenomena.

Intracellular localization of the M1 muscarinic acetylcholine receptor through clathrin-dependent constitutive internalization is mediated by a C-terminal tryptophan-based motif.

ABSTRACT The M1 muscarinic acetylcholine receptor (M1-mAChR, encoded by CHRM1) is a G-protein-coupled membrane receptor that is activated by extracellular cholinergic stimuli. Recent investigations have revealed the intracellular localization of M1-mAChR. In this study, we observed constitutive internalization of M1-mAChR in mouse neuroblastoma N1E-115 cells without agonist stimulation. Constitutive internalization depended on dynamin, clathrin and the adaptor protein-2 (AP-2) complex. A WxxI motif in the M1-mAChR C-terminus is essential for its constitutive internalization, given that replacement of W442 or I445 with alanine residues abolished constitutive internalization. This WxxI motif resembles Yxx&PHgr;, which is the canonical binding motif for the &mgr;2 subunit of the AP-2 complex. The M1-mAChR C-terminal WxxI motif interacted with AP-2 &mgr;2. W442A and I445A mutants of the M1-mAChR C-terminal sequence lost AP-2-&mgr;2-binding activity, whereas the W442Y mutant bound more effectively than wild type. Consistent with these results, W442A and I445A M1-mAChR mutants selectively localized to the cell surface. By contrast, the W442Y receptor mutant was found only at intracellular sites. Our data indicate that the cellular distribution of M1-mAChR is governed by the C-terminal tryptophan-based motif, which mediates constitutive internalization.

Phenotype pharmacology of lower urinary tract α1-adrenoceptors

α1‐Adrenoceptors are involved in numerous physiological functions, including micturition. However, the pharmacological profile of the α1‐adrenoceptor subtypes remains controversial. Here, we review the literature regarding α1‐adrenoceptors in the lower urinary tract from the standpoint of α1L phenotype pharmacology. Among three α1‐adrenoceptor subtypes (α1A, α1B and α1D), α1a‐adrenoceptor mRNA is the most abundantly transcribed in the prostate, urethra and bladder neck of many species, including humans. In prostate homogenates or membrane preparations, α1A‐adrenoceptors with high affinity for prazosin have been detected as radioligand binding sites. Functional α1‐adrenoceptors in the prostate, urethra and bladder neck have low affinity for prazosin, suggesting the presence of an atypical α1‐adrenoceptor phenotype (designated as α1L). The α1L‐adrenoceptor occurs as a distinct binding entity from the α1A‐adrenoceptor in intact segments of variety of tissues including prostate. Both the α1L‐ and α1A‐adrenoceptors are specifically absent from Adra1A (α1a) gene‐knockout mice. Transfection of α1a‐adrenoceptor cDNA predominantly expresses α1A‐phenotype in several cultured cell lines. However, in CHO cells, such transfection expresses α1L‐ and α1A‐phenotypes. Under intact cell conditions, the α1L‐phenotype is predominant when co‐expressed with the receptor interacting protein, CRELD1α. In summary, recent pharmacological studies reveal that two distinct α1‐adrenoceptor phenotypes (α1A and α1L) originate from a single Adra1A (α1a‐adrenoceptor) gene, but adrenergic contractions in the lower urinary tract are predominantly mediated via the α1L‐adrenoceptor. From the standpoint of phenotype pharmacology, it is likely that phenotype‐based subtypes such as the α1L‐adrenoceptor will become new targets for drug development and pharmacotherapy.

Influence of tissue integrity on pharmacological phenotypes of muscarinic acetylcholine receptors in the rat cerebral cortex

Distinct pharmacological phenotypes of muscarinic acetylcholine receptors (mAChRs) have been proposed. We compared the pharmacological profiles of mAChRs in intact segments and homogenates of rat cerebral cortex and other tissues by using radioligand binding assays with [3H]N-methylscopolamine ([3H]NMS). Recombinant M1 and M3 mAChRs were also examined. The density of mAChRs detected by [3H]NMS binding to rat cerebral cortex segments and homogenates was the same (approximately 1400 fmol/mg tissue protein), but the dissociation constant of [3H]NMS was significantly different (1400–1700 pM in segments and 260 pM in homogenates). A wide variation in [3H]NMS binding affinity was also observed among the segments of other tissues (ranging from 139 pM in urinary bladder muscle to 1130 pM in the hippocampus). The mAChRs of cerebral cortex were composed of M1, M2, M3, and M4 subtypes, which showed typical subtype pharmacology in the homogenates. However, in the cortex segments the M3 subtype showed a low selectivity for M3 antagonists (darifenacin, solifenacin) and was not distinguished by the M3 antagonists from the other subtypes. Recombinant M1 and M3 mAChRs showed high affinity for [3H]NMS and subtype-specific pharmacology for each tested ligand. The present binding study under conditions where tissue integrity was kept demonstrates a wide variation in [3H]NMS binding affinity among mAChRs of many rat tissues and the presence of an atypical M3 phenotype in the cerebral cortex, suggesting that the pharmacological properties of mAChRs are not necessarily constant, rather they may be significantly modified by tissue integrity and tissue type.

Activation of muscarinic receptors prevents TNF-α-mediated intestinal epithelial barrier disruption through p38 MAPK

Intestinal epithelial cells form a tight barrier to act as selective physical barriers, repelling hostile substances. Tumor necrosis factor-α (TNF-α) is a well characterized pro-inflammatory cytokine which can compromise intestinal barrier function and the suppression of TNF-α function is important for treatment of inflammatory bowel disease (IBD). In this study, we investigated the contribution of G-protein-coupled receptor (GPCR)-induced signalling pathways to the maintenance of epithelial barrier function. We first demonstrated the existence of functional muscarinic M3 and histamine H1 receptors in colonic epithelial cell HT-29/B6. As we previously reported, muscarinic M3 receptor prevented TNF-α-induced barrier disruption through acceleration of TNF receptor (TNFR) shedding which is carried out by TNF-α converting enzyme (TACE). M3 receptor-mediated suppression of TNF-α function depends on Gαq/11 protein, however, histamine H1 receptor could not ameliorate TNF-α function, while which could induce Gαq/11 dependent intracellular Ca2+ mobilization. We found that p38 MAPK was predominantly phosphorylated by M3 receptor through Gαq/11 protein, whereas H1 receptor barely upregulated the phosphorylation. Inhibition of p38 MAPK abolished M3 receptor-mediated TNFR shedding and suppression of TNF-α-induced NF-κB signalling. The p38 MAPK was also involved in TACE- mediated EGFR transactivation followed by ERK1/2 phosphorylation. These results indicate that not H1 but M3 receptor-induced activation of p38 MAPK might contribute to the maintenance of epithelial barrier function through down-regulation of TNF-α signalling and activation of EGFR.

Activation of focal adhesion kinase via M1 muscarinic acetylcholine receptor is required in restitution of intestinal barrier function after epithelial injury

Impairment of epithelial barrier is observed in various intestinal disorders including inflammatory bowel diseases (IBD). Numerous factors may cause temporary damage of the intestinal epithelium. A complex network of highly divergent factors regulates healing of the epithelium to prevent inflammatory response. However, the exact repair mechanisms involved in maintaining homeostatic intestinal barrier integrity remain to be clarified. In this study, we demonstrate that activation of M1 muscarinic acetylcholine receptor (mAChR) augments the restitution of epithelial barrier function in T84 cell monolayers after ethanol-induced epithelial injury, via ERK-dependent phosphorylation of focal adhesion kinase (FAK). We have shown that ethanol injury decreased the transepithelial electrical resistance (TER) along with the reduction of ERK and FAK phosphorylation. Carbachol (CCh) increased ERK and FAK phosphorylation with enhanced TER recovery, which was completely blocked by either MT-7 (M1 antagonist) or atropine. The CCh-induced enhancement of TER recovery was also blocked by either U0126 (ERK pathway inhibitor) or PF-228 (FAK inhibitor). Treatment of T84 cell monolayers with interferon-γ (IFN-γ) impaired the barrier function with the reduction of FAK phosphorylation. The CCh-induced ERK and FAK phosphorylation were also attenuated by the IFN-γ treatment. Immunological and binding experiments exhibited a significant reduction of M1 mAChR after IFN-γ treatment. The reduction of M1 mAChR in inflammatory area was also observed in surgical specimens from IBD patients, using immunohistochemical analysis. These findings provide important clues regarding mechanisms by which M1 mAChR participates in the maintenance of intestinal barrier function under not only physiological but also pathological conditions.

Pharmacological evidence of specific acetylcholine transport in rat cerebral cortex and other brain regions

Functional acetylcholine receptors (AChRs) were recently demonstrated to exist not only in the plasma membrane but also intracellularly in brain tissues. In order to activate intracellular AChRs, endogenous hydrophilic ACh must cross the plasma membrane. Here, we examined the pharmacological characteristics of this process, including whether it is mediated by active ACh uptake. When ACh esterase (AChE) was suppressed by diisopropylfluorophosphate, [3H]ACh was effectively taken up into segments of rat cerebral cortex and other brain regions, in contrast to peripheral tissues such as liver and kidney. The uptake of [3H]ACh in rat cerebral cortex was temperature‐dependent, and the uptake capacity was comparable to that of [3H]choline. However, [3H]ACh uptake was inhibited by lower concentrations of ACh, carbachol, tetraethylammonium (TEA), compared with uptake of [3H]choline. Uptake of [3H]ACh was also inhibited by several organic cations, including choline, hemicholinium‐3 (HC‐3), quinidine, decynium 22, clonidine, diphenhydramine, but was little affected by some amino acids and biogenic amines, corticosterone, spermine, atropine, and tetrodotoxin. Unlike diisopropylfluorophosphate, several ACh esterase inhibitors, including drugs for Alzheimers disease, such as donepezil, galantamine, and rivastigmine, also suppressed the uptake of [3H]ACh, but not [3H]choline. These results indicate that in the brain, ACh is specifically taken up through a unique transport system with different pharmacological properties from known organic cation transporters (OCTs), and suggest that this mechanism may be involved in intracellular cholinergic transmission in the brain.