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.

Background and purpose: The a 1L -adrenoceptor has pharmacological properties that distinguish it from three classical a 1 -adrenoceptors (a 1A , a 1B and a 1D ). The purpose of this was to identify a 1L -adrenoceptors in mice and to examine their relationship to classical a 1 -adrenoceptors. Experimental approach: Radioligand binding and functional bioassay experiments were performed on the cerebral cortex, vas deferens and prostate of wild-type (WT) and a 1A -, a 1B -and a 1D -adrenoceptor gene knockout (AKO, BKO and DKO) mice. Key results: The radioligand [3 H]-silodosin bound to intact segments of the cerebral cortex, vas deferens and prostate of WT, BKO and DKO but not of AKO mice. The binding sites were composed of two components with high and low affinities for prazosin or RS-17053, indicating the pharmacological profiles of a 1A -adrenoceptors and a 1L -adrenoceptors. In membrane preparations of WT mouse cortex, however, [3 H]-silodosin bound to a single population of prazosin high-affinity sites, suggesting the presence of a 1A -adrenoceptors alone. In contrast, [3 H]-prazosin bound to two components having a 1A -adrenoceptor and a 1B -adrenoceptor profiles in intact segments of WT and DKO mouse cortices, but AKO mice lacked a 1A -adrenoceptor profiles and BKO mice lacked a 1B -adrenoceptor profiles. Noradrenaline produced contractions through a 1L -adrenoceptors with low affinity for prazosin in the vas deferens and prostate of WT, BKO and DKO mice. However, the contractions were abolished or markedly attenuated in AKO mice. Conclusions and implications: a 1L -Adrenoceptors were identified as binding and functional entities in WT, BKO and DKO mice but not in AKO mice, suggesting that the a 1L -adrenoceptor is one phenotype derived from the a 1A -adrenoceptor gene.

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.

Background and purpose: In addition to a 1A , a 1B and a 1D -adrenoceptors (ARs), putative a 1L -ARs with a low affinity for prazosin have been proposed. The purpose of the present study was to identify the a 1A -AR and clarify its pharmacological profile using a radioligand binding assay. Conclusions and implications:The present study clearly reveals the presence of a 1L -ARs as a pharmacologically distinct entity from a 1A and a 1B -ARs in intact tissue segments of rat cerebral cortex but not tail artery. However, the a 1L -ARs disappeared after tissue homogenization, suggesting their decomposition and/or their pharmacological profile changes to that of a 1A -ARs.