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BACKGROUND AND PURPOSEMuscarinic acetylcholine receptors (mAChRs) and b-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. EXPERIMENTAL APPROACHThe regional distribution and relative proportion of mAChR and b-adrenoceptor subtypes were evaluated in human bronchus and lung parenchyma by a tissue segment binding method with [ KEY RESULTSThe M3 subtype predominantly occurred in the bronchus, but the density decreased from the segmental to subsegmental bronchus, and was absent in lung parenchyma. On the other hand, the M1 subtype occurred in the lung only, and the M2 subtype was distributed ubiquitously in the bronchus and lungs. b2-adrenoceptors were increased along the airways, and their densities in the subsegmental bronchus and lung parenchyma were approximately twofold higher than those of mAChRs in the same region. b1-adrenoceptors were also detected in lung parenchyma but not in the bronchus. The muscarinic contractions and adrenoceptor relaxations in both bronchial regions were mediated through M3-mAChRs and b2-adrenoceptors, respectively. CONCLUSIONS AND IMPLICATIONSFrom the present radioligand binding approach with intact tissue segments, we constructed a distribution map of mAChRs and b-adrenoceptors in human bronchus and lung parenchyma for the first time, providing important evidence for future pharmacotherapy and new drug development for respiratory disorders.

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. Radioligandbinding 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. Keywords: cell surface and intracellular GPCR, ERK1/2, LTP, M1-muscarinic receptor, synaptic plasticity. Among the five muscarinic acetylcholine receptor (mAChR) subtypes, M1-mAChRs predominantly exist in the CNS and are involved in cognitive enhancement (Wess 2004;

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, [ H]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 [ H]ACh in rat cerebral cortex was temperature-dependent, and the uptake capacity was comparable to that of [ H]choline. However, [ H]ACh uptake was inhibited by lower concentrations of ACh, carbachol, tetraethylammonium (TEA), compared with uptake of [ H]choline. Uptake of [ H]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 Alzheimer's disease, such as donepezil, galantamine, and rivastigmine, also suppressed the uptake of [ H]ACh, but not [ H]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.

J. Neurochem. (2011) 118, 958–967. Abstract Signaling by muscarinic agonists is thought to result from the activation of cell surface acetylcholine receptors (mAChRs) that transmit extracellular signals to intracellular systems. In N1E‐115 neuroblastoma cells, we detected both plasma membrane and intracellular M1‐mAChRs using both biochemical and pharmacological methods. In intact cells, both plasma membrane and intracellular M1‐mAChRs were detected by the hydrophobic ligand probe, 1‐quinuclidinyl‐[phenyl‐4‐3H]‐benzilate ([3H]‐QNB) whereas the hydrophilic probe, 1‐[N‐methyl‐3H] scopolamine ([3H]‐NMS), detected only cell surface receptors. These probes detected comparable numbers of receptors in isolated membrane preparations. Immunohistochemical studies with M1‐mAChR antibody also detected both cell‐surface and intracellular M1‐mAChRs. Carbachol‐stimulated phosphatidylinositol hydrolysis and Ca2+ mobilization were completely inhibited by a cell‐impermeable M1 antagonist, muscarinic toxin ‐7 and the Gq/11 inhibitor YM‐254890. However, carbachol‐stimulated extracellular‐regulated kinase 1/2 activation was unaffected by muscarinic toxin‐7, but was blocked by the cell‐permeable antagonist, pirenzepine. extracellular regulated kinase 1/2 phosphorylation was resistant to blockade of Gq/11 (YM‐254890) and protein kinase C (bisindolylmaleimide I). Our data suggest that the geographically distinct M1‐mAChRs (cell surface versus intracellular) can signal via unique signaling pathways that are differentially sensitive to cell‐impermeable versus cell‐permeable antagonists. Our data are of potential physiological relevance to signaling that affects both cognitive and neurodegenerative processes.