Search

Inflammatory bowel disease (IBD), which includes Crohn’s disease and ulcerative colitis, is an intestinal disorder that causes prolonged inflammation of the gastrointestinal tract. Currently, the etiology of IBD is not fully understood and treatments are insufficient to completely cure the disease. In addition to absorbing essential nutrients, intestinal epithelial cells prevent the entry of foreign antigens (micro-organisms and undigested food) through mucus secretion and epithelial barrier formation. Disruption of the intestinal epithelial homeostasis exacerbates inflammation. Thus, the maintenance and reinforcement of epithelial function may have therapeutic benefits in the treatment of IBD. Muscarinic acetylcholine receptors (mAChRs) are G protein-coupled receptors for acetylcholine that are expressed in intestinal epithelial cells. Recent studies have revealed the role of mAChRs in the maintenance of intestinal epithelial homeostasis. The importance of non-neuronal acetylcholine in mAChR activation in epithelial cells has also been recognized. This review aimed to summarize recent advances in research on mAChRs for intestinal epithelial homeostasis and the involvement of non-neuronal acetylcholine systems, and highlight their potential as targets for IBD therapy.

Cholinergic transmission underlies higher brain functions such as cognition and movement. To elucidate the process whereby acetylcholine (ACh) release is maintained and regulated in the central nervous system, uptake of [

Regulation of neurotransmitter release in the central nervous system is complex. Here, we investigated regulatory mechanisms for acetylcholine (ACh) release from cholinergic neurons by performing superfusion experiments with rat striatal segments after labelling the cellular ACh pool with [3 H]choline. Electrical stimulation-evoked pronounced [3 H]ACh release from cholinergic neurons. The estimated quantity of [3 H]ACh release per pulse of electrical stimulation was reduced by an increase in stimulus frequency, showing an inverse correlation between release probability of ACh and neuronal excitation. ACh release was also negatively regulated by pre-synaptic muscarinic ACh receptors (mAChRs). The autoinhibition induced by released ACh was predominantly suppressed by the M2 -selective antagonist AF-DX 116, partially inhibited by M3 -selective darifenacin, and minimally by M4 -selective PD 102807. Other subtype-selective antagonists had no effect at subtype-selective concentrations. ACh esterase (AChE) inhibitors (diisopropylfluorophosphate, donepezil and galantamine) at concentrations that mostly inhibit esterase activity reduced [3 H]ACh release, and the reduction was abolished by treatment with atropine. This implies that pre-synaptic autoreceptors are activated more after blockade of ACh hydrolysis, leading to autoinhibition of ACh release and consequent reduction in synaptic ACh concentrations. [3 H]efflux was also enhanced by ACh uptake inhibitors (100 μM hemicholinium-3 and physostigmine), regardless of ACh hydrolysis. This study shows that synaptic ACh concentrations in striatal cholinergic neurons are regulated in a complex manner by many factors such as release probability, pre-synaptic M2 /M3 /M4 mAChRs, AChE and post-synaptic ACh uptake, and provides important information about cholinergic neurotransmission for future exploration of therapeutic strategies for Alzheimer's and other central nervous system diseases. OPEN SCIENCE BADGES: This article has received a badge for *Open Materials* because it provided all relevant information to reproduce the study in the manuscript. The complete Open Science Disclosure form for this article can be found at the end of the article. More information about the Open Practices badges can be found at https://cos.io/our-services/openscience-badges/.

PNU-120596 is a classical positive allosteric modulator (PAM) of α7 nicotinic acetylcholine receptor (α7 nAChR) and widely used to investigate the effect of α7 nAChR activation on several inflammation-associated diseases including rheumatoid arthritis, inflammatory bowel disease and cerebral ischemia. In this study, we report that PNU-120596 directly inhibits p38 mitogen-activated protein kinase (MAPK) activity. In 293A cells, p38 MAPK phosphorylation by several factors (oxidative stress, osmotic stress, TNF-α, or muscarinic stimulation) was inhibited by PNU-120596 as well as p38 MAPK inhibitor BIRB-796. Inhibition of p38 MAPK phosphorylation by PNU-120596 was not affected by α7 nAChR antagonist, methyllycaconitine (MLA). In vitro kinase assay revealed that PNU-120596 directly inhibits p38α MAPK-induced activating transcription factor 2 (ATF2) phosphorylation. MKK6-induced phosphorylation of p38α MAPK was also inhibited by PNU-120596. Real-time monitoring of binding to p38α MAPK using fluoroprobe SKF-86002 showed quite rapid binding of PNU-120596 compared to BIRB-796 which is known as a slow binder. Finally, we showed that PNU-120596 suppressed LPS-induced phosphorylation of p38 MAPK and expression of inflammatory factors including TNF-α, IL-6 and COX-2, independent on α7 nAChR activity in microglial cell BV-2. Thus, PNU-120596 might exert an anti-inflammatory effect through not only α7 nAChR potentiation but also direct inhibition of p38 MAPK.

In addition to hydrolysis by acetylcholine esterase (AChE), acetylcholine (ACh) is also directly taken up into brain tissues. In this study, we examined whether the uptake of ACh is involved in the regulation of synaptic ACh concentrations. Superfusion experiments with rat striatal segments pre-incubated with [3 H]choline were performed using an ultra-mini superfusion vessel, which was developed to minimize superfusate retention within the vessel. Hemicholinium-3 (HC-3) at concentrations less than 1 μM, selectively inhibited the uptake of [3 H]choline by the high affinity-choline transporter 1 and had no effect on basal and electrically evoked [3 H]efflux in superfusion experiments. In contrast, HC-3 at higher concentrations, as well as tetraethylammonium (>10 μM), which inhibited the uptake of both [3 H]choline and [3 H]ACh, increased basal [3 H]overflow and potentiated electrically evoked [3 H]efflux. These effects of HC-3 and tetraethylammonium were also observed under conditions where tissue AChE was irreversibly inactivated by diisopropylfluorophosphate. Specifically, the potentiation of evoked [3 H]efflux was significantly higher in AChE-inactivated preparations and was attenuated by atropine. On the other hand, striatal segments pre-incubated with [3 H]ACh failed to increase [3 H]overflow in response to electrical stimulation. These results show that synaptic ACh concentrations are significantly regulated by the postsynaptic uptake of ACh, as well as by AChE hydrolysis and modulation of ACh release mediated through presynaptic muscarinic ACh receptors. In addition, these data suggest that the recycling of ACh-derived choline may be minor in cholinergic terminals. This study reveals a new mechanism of cholinergic transmission in the central nervous system.

Aims Endothelial dysfunction is one of the earliest symptoms in septic patients and plays an important role in the cardiovascular alterations. However, the endothelial mechanisms involved in the impaired sympathetic regulation of the cardiovascular system are not clear. This study aimed to determine the role of the endocardial endothelium (EE) in the cardiac β-adrenergic (β-AR) remodeling at the early phase of endotoxemic shock. Main methods Rats received either lipopolysaccharide (LPS) or saline (control) intravenously. Three hours later, β-AR cardiac contractility was evaluated on papillary muscles with or without a functional EE. Key findings Isoproterenol-induced contractility was strongly increased in papillary muscles from LPS rats. A similar increase was observed with a β1-AR stimulation, whereas β2-AR and β3-AR produced similar contractility in control and LPS treatments. The removal of the EE did not modify β1-AR-induced contractility in controls, whereas it abolished the increased β1-AR response in LPS-treated muscles. In LPS-treated papillary muscle, the increased β1-AR-induced contractility was not modified by pretreatment with a NOS inhibitor or an endothelin receptor antagonist. Conversely, the increased β1-AR-induced contractility was abolished by indomethacin, a non-selective cyclooxygenase (COX) inhibitor, as well as by selective inhibitors of COX1 and COX2. An early treatment with indomethacin improved the survival of LPS rat. Significance Our results suggest that the EE is involved in the increased cardiac β1-AR contractility in the early phase of endotoxemic shock. This effect is mediated through the activation of COX1 and COX2 and suggests these may be novel putative therapeutic targets during endotoxemic shock.

Endothelial dysfunction is one of the earliest symptoms in septic patients and plays an important role in the cardiovascular alterations. However, the endothelial mechanisms involved in the impaired sympathetic regulation of the cardiovascular system are not clear. This study aimed to determine the role of the endocardial endothelium (EE) in the cardiac β-adrenergic (β-AR) remodeling at the early phase of endotoxemic shock.Rats received either lipopolysaccharide (LPS) or saline (control) intravenously. Three hours later, β-AR cardiac contractility was evaluated on papillary muscles with or without a functional EE.Isoproterenol-induced contractility was strongly increased in papillary muscles from LPS rats. A similar increase was observed with a βOur results suggest that the EE is involved in the increased cardiac β

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, [3 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 [3 H]ACh in rat cerebral cortex was temperature-dependent, and the uptake capacity was comparable to that of [3 H]choline. However, [3 H]ACh uptake was inhibited by lower concentrations of ACh, carbachol, tetraethylammonium (TEA), compared with uptake of [3 H]choline. Uptake of [3 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 [3 H]ACh, but not [3 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.

Hippocampal cholinergic activity enhances long-term potentiation (LTP) of synaptic transmission in intrahippocampal circuits and regulates cognitive function. We recently demonstrated intracellular distribution of functional M1-muscarinic acetylcholine receptors (mAChRs) and neuronal uptake of acetylcholine (ACh) in the central nervous system. Here we examined whether endogenous ACh acts on intracellular M1-mAChRs following its uptake and causes cholinergic facilitation of hippocampal LTP. ACh esterase (AChE) activities and [3H]ACh uptake was measured in rat hippocampal segments. LTP of evoked field excitatory postsynaptic potentials at CA1 synapses was induced by high frequency stimulation in hippocampal slices. Pretreatment with diisopropylfluorophosphate (DFP) irreversibly inhibited AChE, augmented ACh uptake, and significantly enhanced the LTP. This cholinergic facilitation was inhibited by pirenzepine, a membrane-permeable M1 antagonist, while only the early stage of cholinergic facilitation was inhibited by a membrane-impermeable M1 antagonist, muscarinic toxin 7. Tetraethylammonium (TEA) inhibited ACh uptake in hippocampal segments and selectively suppressed late stage cholinergic facilitation without changing the early stage. In contrast, LTP in DFP-untreated slices was not affected by the muscarinic antagonists and TEA. Carbachol (CCh; an AChE-resistant muscarinic agonist) competed with ACh for its uptake and produced cholinergic facilitation of LTP in DFP-untreated slices. The late stage of CCh-induced facilitation was also selectively inhibited by TEA. Our results suggest that when AChE is inactivated by inhibitors, LTP in hippocampal slices is significantly enhanced by endogenous ACh and that cholinergic facilitation is caused by direct activation of cell-surface M1-mAChRs and subsequent activation of intracellular M1-mAChRs after ACh uptake.

The calcitonin (CT)/CT gene-related peptide (CGRP) family is conserved in vertebrates. The activities of this peptide family are regulated by a combination of two receptors, namely the calcitonin receptor (CTR) and the CTR-like receptor (CLR), and three receptor activity-modifying proteins (RAMPs). Furthermore, RAMPs act as escort proteins by translocating CLR to the cell membrane. Recently, CT/CGRP family peptides have been identified or inferred in several invertebrates. However, the molecular characteristics and relevant functions of the CTR/CLR and RAMPs in invertebrates remain unclear. In this study, we identified three CT/CGRP family peptides (Bf-CTFPs), one CTR/CLR-like receptor (Bf-CTFP-R), and three RAMP-like proteins (Bf-RAMP-LPs) in the basal chordate amphioxus (Branchiostoma floridae). The Bf-CTFPs were shown to possess an N-terminal circular region typical of the CT/CGRP family and a C-terminal Pro-NH2. The Bf-CTFP genes were expressed in the central nervous system and in endocrine cells of the midgut, indicating that Bf-CTFPs serve as brain and/or gut peptides. Cell surface expression of the Bf-CTFP-R was enhanced by co-expression with each Bf-RAMP-LP. Furthermore, Bf-CTFPs activated Bf-CTFP-R·Bf-RAMP-LP complexes, resulting in cAMP accumulation. These results confirmed that Bf-RAMP-LPs, like vertebrate RAMPs, are prerequisites for the function and translocation of the Bf-CTFP-R. The relative potencies of the three peptides at each receptor were similar. Bf-CTFP2 was a potent ligand at all receptors in cAMP assays. Bf-RAMP-LP effects on ligand potency order were distinct to vertebrate CGRP/adrenomedullin/amylin receptors. To the best of our knowledge, this is the first molecular and functional characterization of an authentic invertebrate CT/CGRP family receptor and RAMPs.

Impaired intestinal barrier function is one of the critical issues in inflammatory bowel diseases. The aim of this study is to investigate muscarinic cholinoceptor (mAChR)-mediated signaling for the amelioration of cytokine-induced barrier dysfunction in intestinal epithelium. Rat colon challenged with TNF-α and interferon γ reduced transepithelial electrical resistance (TER). This barrier injury was attenuated by muscarinic stimulation. In HT-29/B6 intestinal epithelial cells, muscarinic stimulation suppressed TNF-α-induced activation of NF-κB signaling and barrier disruption. Finally, muscarinic stimulation promoted the shedding of TNFR1, which would be a mechanism for the attenuation of TNF-α/NF-κB signaling and barrier disruption via mAChR.

In the central nervous system, acetylcholine (ACh) is an important neurotransmitter related to higher brain functions and some neurodegenerative diseases. It is released from cholinergic nerve terminals and acts on presynaptic and postsynaptic ACh receptors (AChRs). Following release, ACh is rapidly hydrolyzed and the resultant choline is recycled as a substrate for new ACh synthesis. However, this classical concept of cholinergic transmission is currently reevaluated due to new evidence. In the cholinergic synapse, ACh may be itself taken up into postsynaptic neurons by a specific transport system and may act on AChRs at intracellular organelles (Golgi apparatus and mitochondria). Choline for ACh synthesis in cholinergic nerve terminals may be mainly supplied from choline at relevant concentration levels present in the extracellular space, rather than recycled from ACh-derived choline. Recent evidence has reopened the issue of classical cholinergic transmission and cognition, and may provide a novel approach to rational drug development for the treatment of neurodegenerative disorders such as Alzheimer’s disease.

The transient receptor potential vanilloid 1 (TRPV1) channel is highly expressed in a subset of sensory neurons in the dorsal root ganglia (DRG) and trigeminal ganglia of experimental animals, responsible for nociception. Many researches have revealed that some TRPV1-positive neurons co-express the transient receptor potential ankyrin 1 (TRPA1) channel whose activities are closely modulated by TRPV1 channel. However, it is less investigated whether the activities of TRPV1 channel are modulated by the presence of TRPA1 channel in primary sensory neurons. This study clarified the difference in electrophysiological responses induced by TRPV1 channel activation between TRPA1-positive and TRPA1-negative DRG. TRPV1 and TRPA1 channel activations were evoked by capsaicin (1 μM), a TRPV1 agonist, and allyl isothiocyanate (AITC; 500 μM), a TRPA1 agonist, respectively. Capsaicin perfusion for 15 s caused a large inward current without a desensitization phase at a membrane potential of −70 mV in AITC-insensitive DRG (current density; 29.6 ± 5.6 pA/pF, time constant of decay; 12.8 ± 1.8 s). The capsaicin-induced currents in AITC-sensitive DRG had a small current density (12.7 ± 2.9 pA/pF) with a large time constant of decay (24.3 ± 5.4 s). In calcium imaging with Fura-2, the peak response by capsaicin was small and duration reaching the peak response was long in AITC-sensitive neurons. These electrophysiological differences were completely eliminated by HC-030031, a TRPA1 antagonist, in an extracellular solution or 10 mM EGTA, a Ca2+ chelator, in an internal solution. Capsaicin perfusion for 120 s desensitized the inward currents after a transient peak. The decay during capsaicin perfusion was notably slow in AITC-sensitive DRG; ratio of capsaicin-induced current 60 s after the treatment per the peak current in AITC-sensitive neurons (78 ± 9%) was larger than that in AITC-insensitive neurons (48 ± 5%). The capsaicin-induced current in the desensitization phase was attenuated by HC-030031 in AITC-insensitive DRG. These results indicate that (1) TRPV1-mediated currents in TRPA1-positive neurons characterize small current densities with slow decay, which is caused by TRPA1 channel activities and intracellular Ca2+ mobilization and (2) desensitization of TRPV1-mediated current in TRPA1-positive neurons is apparently slow, due to appending TRPA1-mediated current.

Background and Purpose The pharmacological properties of particular receptors have recently been suggested to vary under different conditions. We compared the pharmacological properties of the α1B-adrenoceptor subtype in various tissue preparations and under various conditions. Experimental Approach [3H]-prazosin binding to α1B-adrenoceptors in rat liver (segments, dispersed hepatocytes and homogenates) was assessed and the pharmacological profiles were compared with the functional and binding profiles in rat carotid artery and recombinant α1B-adrenoceptors. Key Results In association and saturation-binding experiments with rat liver, binding affinity for [3H]-prazosin varied significantly between preparations (KD value approximately ten times higher in segments than in homogenates). The binding profile for various drugs in liver segments also deviated from the representative α1B-adrenoceptor profile observed in liver homogenates and recombinant receptors. L-765,314 and ALS-77, selective antagonists of α1B-adrenoceptors, showed high binding and antagonist affinities in liver homogenates and recombinant α1B-adrenoceptors. However, binding affinities for both ligands in the segments of rat liver and carotid artery were 10 times lower, and the antagonist potencies in α1B-adrenoceptor-mediated contractions of carotid artery were more than 100 times lower than the representative α1B-adrenoceptor profile. Conclusions and Implications In contrast to the consistent profile of recombinant α1B-adrenoceptors, the pharmacological profile of native α1B-adrenoceptors of rat liver and carotid artery varied markedly under various receptor environments, showing significantly different binding properties between intact tissues and homogenates, and dissociation between functional and binding affinities. In addition to conventional ‘subtype’ characterization, ‘phenotype’ pharmacology must be considered in native receptor evaluations in vivo and in future pharmacotherapy.

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.

Calcium influx via store-operated calcium entry (SOCE) has an important role for regulation of vast majority of cellular physiological events. MAPK signalling is also another pivotal modulator of many cellular functions. However, the relationship between SOCE and MAPK is not well understood. In this study, we elucidated the involvement of SOCE in Gαq/11 protein-mediated activation of p38 MAPK in an intestinal epithelial cell line HT-29/B6. In this cell line, we previously showed that the stimulation of M3 muscarinic acetylcholine receptor (M3-mAChR) but not histamine H1 receptor (H1R) led to phosphorylation of p38 MAPK which suppressed tumor necrosis factor-α (TNF-α)-induced NF-κB signalling through ADAM17 protease-mediated shedding of TNF receptor-1 (TNFR1). First, we found that stimulation of M3-mAChR and protease-activated receptor-2 (PAR-2) but not H1R induced persistent upregulation of cytosolic Ca2+ concentration through SOCE. Activation of M3-mAChR or PAR-2 also suppressed TNF-α-induced NF-κB phosphorylation, which was dependent on the p38 MAPK activity. Time course experiments revealed that M3-mAChR stimulation evoked intracellular Ca2+-dependent early phase p38 MAPK phosphorylation and extracellular Ca2+-dependent later phase p38 MAPK phosphorylation. This later phase p38 MAPK phosphorylation, evoked by M3-mAChRs or PAR-2, was abolished by inhibition of SOCE. Thapsigargin or ionomycin also phosphorylate p38 MAPK by Ca2+ influx through SOCE, leading to suppression of TNF-α-induced NF-κB phosphorylation. Finally, we showed that p38 MAPK was essential for thapsigargin-induced cleavage of TNFR1 and suppression of TNF-α-induced NF-κB phosphorylation. In conclusion, SOCE is important for p38 MAPK phosphorylation and is involved in TNF-α signalling suppression.

Background and Purpose Two distinct α1-adrenoceptor phenotypes (α1A and α1L) have recently been demonstrated to originate from a single α1A-adrenoceptor gene. Here, we examined the agonist profiles of recombinant α1A and α1L phenotypes and of lower urinary tract (LUT) α1-adrenoceptors. Experimental Approach A series of drugs (A61603, Ro 115–1240, NS-49 , MK017 and ESR1150) originally developed for stress urinary incontinence (SUI) therapy were used to stimulate recombinant α1A- and α1L-adrenoceptor phenotypes, and their potencies and intrinsic activity estimated from Ca2+ responses. Agonist-induced contractions were also examined in LUT tissues of rats and humans and in human mesenteric artery and rat tail artery. Key Results All the drugs were potent agonists of the α1A-adrenoceptor compared with the α1L-adrenoceptor phenotype. Among them, Ro 115–1240 was shown to be an α1A-specific partial agonist that produced partial contractions through α1A-adrenoceptors in rat prostate and tail artery, but not in the other LUT tissues and human mesenteric artery. In contrast, P-come 102 showed full agonist activity at α1A- and α1L-adrenoceptors, but was less selective than noradrenaline for α1A-adrenoceptors. Like noradrenaline, P-come 102 was highly potent at inducing contractions in all of the LUT tissues tested. However, the potency and intrinsic activity of P-come 102 were significantly lower than those of noradrenaline in human mesenteric artery. Conclusions and Implications The α1A- and α1L-adrenoceptor phenotypes and LUT α1-adrenoceptors were demonstrated to have distinct agonist profiles. As adrenergic contractions in LUT are predominantly mediated through α1L-adrenoceptors, the development of α1L-selective agonists may provide clinically useful drugs for SUI therapy.

Aims Recombinant systems have been used for evaluating the properties of G-protein-coupled receptors (GPCRs) on the assumption of cell surface expression. However, many GPCRs, including muscarinic acetylcholine receptors (mAChRs), have also been reported to be distributed in intracellular organelles in native tissues and cell lines. In this study, we compared the pharmacological profiles of exogenously and endogenously expressed M1-mAChRs, and evaluated the functional properties of these receptors. Main methods Recombinant M1-mAChRs were expressed exogenously in Chinese hamster ovary cells (CHO-M1 cells) and compared with endogenously expressed M1-mAChRs in N1E-115 neuroblastoma cells. The pharmacological and functional profiles were evaluated using cell-permeable antagonists (1-quinuclidinyl-benzilate (QNB), pirenzepine and atropine) and cell-impermeable antagonists (N-methylscopolamine (NMS) or MT-7). Key findings M1-mAChRs were seen at the cell surface and intracellular sites in both cell lines. Under whole cell conditions, intracellular M1-mAChRs were mainly recognized by cell-permeable ligands, but scarcely by cell-impermeable ligands (at less than 100 nM). In CHO-M1 cells, M1-mAChR activation by carbachol resulted in Ca 2 + mobilization, ERK1/2 phosphorylation and a reduction in thymidine incorporation, all of which were completely inhibited by MT-7, indicating the involvement of surface M1-mAChRs. In N1E-115 cells, Ca 2 + mobilization occurred through surface M1-mAChRs, whereas ERK1/2 phosphorylation and acceleration of thymidine incorporation were mediated through intracellular M1-mAChRs. Significance Exogenous and endogenous M1-mAChRs are present at both the cell surface and the intracellular organelles, and the pharmacological properties of geographically distinct M1-mAChRs are different, and may depend on cell background and/or exogenous or endogenous origin.

CC-BY-NC-ND, 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.

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. EXPERIMENTAL APPROACH The regional distribution and relative proportion of mAChR and β-adrenoceptor subtypes were evaluated in human bronchus and lung parenchyma by a tissue segment binding method with [3H]-N-methylscopolamine ([3H]-NMS) for mAChRs and [3H]-CGP-12,177 for β-adrenoceptors. Functional responses to carbachol and isoprenaline were also analysed in the bronchus. KEY RESULTS The 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. β2-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. β1-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 β2-adrenoceptors, respectively. CONCLUSIONS AND IMPLICATIONS From the present radioligand binding approach with intact tissue segments, we constructed a distribution map of mAChRs and β-adrenoceptors in human bronchus and lung parenchyma for the first time, providing important evidence for future pharmacotherapy and new drug development for respiratory disorders.

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. LINKED ARTICLE This article is commented on by Ventura, pp. 1223–1225 of this issue. To view this commentary visit http://dx.doi.org/10.1111/j.1476-5381.2011.01663.x

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.

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 [(3)H]N-methylscopolamine ([(3)H]NMS). Recombinant M(1) and M(3) mAChRs were also examined. The density of mAChRs detected by [(3)H]NMS binding to rat cerebral cortex segments and homogenates was the same (approximately 1400 fmol/mg tissue protein), but the dissociation constant of [(3)H]NMS was significantly different (1400-1700 pM in segments and 260 pM in homogenates). A wide variation in [(3)H]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 M(1), M(2), M(3), and M(4) subtypes, which showed typical subtype pharmacology in the homogenates. However, in the cortex segments the M(3) subtype showed a low selectivity for M(3) antagonists (darifenacin, solifenacin) and was not distinguished by the M(3) antagonists from the other subtypes. Recombinant M(1) and M(3) mAChRs showed high affinity for [(3)H]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 [(3)H]NMS binding affinity among mAChRs of many rat tissues and the presence of an atypical M(3) 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.

Background and purpose: The expression of multiple pharmacological phenotypes including α1L-adrenoceptor has recently been reported for α1-adrenoceptors. The purpose of the present study was to identify α1-adrenoceptor phenotypes in the irises of pigmented and albino rabbits. Experimental approach: Radioligand binding and functional bioassay experiments were performed in segments or strips of iris of pigmented and albino rabbits, and their pharmacological profiles were compared. Key results: [3H]-silodosin at subnanomolar concentrations bound to intact segments of iris of pigmented and albino rabbits at similar densities (approximately 240 fmol·mg−1 protein). The binding sites in the iris of a pigmented rabbit were composed of a single component showing extremely low affinities for prazosin, hydrochloride [N-[2-(2-cyclopropylmethoxyphenoxy)ethyl]-5-chloro-α,α-dimethyl-1H-indole-3-ethamine hydrochloride (RS-17053)] and 5-methylurapidil, while two components with high and low affinities for prazosin, RS-17053 and 5-methylurapidil were identified in irises from albino rabbits. In contrast, specific binding sites for [3H]-prazosin were not clearly detected because a high proportion of non-specific binding and/or low affinity for prazosin occurred. Contractile responses of iris dilator muscle to noradrenaline were antagonized by the above ligands, and their antagonist affinities were consistent with the binding estimates at low-affinity sites identified in both strains of rabbits. Conclusions and implications: A typical α1L phenotype with extremely low affinity for prazosin is exclusively expressed in the iris of pigmented rabbits, while two distinct phenotypes (α1A and α1L) with high and moderate affinities for prazosin are co-expressed in the iris of albino rabbits. This suggests that a significant difference in the expression of phenotypes of the α1-adrenoceptor occurs in the irises between the two strains of rabbits.

Alpha(1)-adrenoceptors are involved in physiological functions such as urinary excretion and ejaculation in the lower urinary tract (LUT). Several alpha(1) antagonists are clinically used for the treatment of urinary obstruction in patients with benign prostatic hyperplasia. At present, three classical alpha(1)-adrenoceptor subtypes (alpha(1A), alpha(1B), and alpha(1D)) have been identified, among which the alpha(1A) and alpha(1D)-adrenoceptor subtypes have been regarded as the main targets of alpha(1) antagonist therapy for LUT symptoms. Prazosin has been used as a prototypic, classical antagonist, to characterize alpha(1)-adrenoceptors pharmacologically, (i.e. all classical alpha(1)-adrenoceptor subtypes show high-affinity for the drug). However, we found that alpha(1)-adrenoceptors in the LUT show atypical low-affinity for prazosin. Therefore, the concept alpha(1L)-receptor, which indicates alpha(1)-adrenoceptor(s) showing low-affinity for prazosin has been introduced. A recent study demonstrated that the alpha(1L)-adrenoceptor is a specific phenotype present in the many intact tissues including human LUT, and that it originates from the ADRA1A gene. Therefore, the alpha(1L)-adrenoceptor in the LUT is now re-defined as alpha(1A(L))-adrenoceptor. The physiological and pharmacological difference between classical alpha(1A(H),) and alpha(1A(L)) which is the native receptor expressed in the LUT is of special interest as it provides fundamental bases for urological alpha(1A)-adrenoceptor blocking pharmacotherapy. Here, we briefly review the alpha(1)-adrenoceptors in the LUT with special reference to phenotype-based (pharmacome) analysis.

This study evaluates ocular (iris, ciliary body and ciliary process) and nonocular (atria and lung) beta-adrenoceptors in rabbit to characterize the plasma membrane beta-adrenoceptors and binding affinities of beta-adrenoceptor antagonists.The tissue segment binding method with a hydrophilic radioligand (-)-4-[3-t-butylamino-2-hydroxypropoxy]-[5,7-(3)H]benzimidazol-2-one ([(3)H]-CGP12177) was employed.Specific and saturable binding of [(3)H]-CGP12177 to intact tissue segments was detected by using (+/-)-propranolol to define nonspecific binding, showing a single population of plasma membrane binding sites with high affinity. Competition experiments with selective beta(1)- and beta(2)-adrenoceptor antagonists revealed a single population of beta(2)-adrenoceptors in ocular tissues and of beta(1)-adrenoceptors in atria, but mixed populations of beta(1)- and beta(2)-adrenoceptors in 70% and 30%, respectively, in lung. A competition curve for timolol was biphasic in lung and its binding affinity for beta(2)-adrenoceptors was approximately 158-fold higher than for beta(1)-adrenoceptors, indicating the beta(2)-selectivity of timolol. In contrast, competition curves for stereoisomers of befunolol, carteolol, and propranolol were monophasic in all tissues. The (-)-enantiomers of these antagonists were more potent than corresponding (+)-enantiomers in displacing from [(3)H]-CGP12177 binding, and the isomeric potency ratios of befunolol and carteolol were less than those of propranolol.This study with tissue segment binding method suggests that the binding affinity of (-)-enantiomers of beta-adrenoceptor antagonists for plasma membrane beta-adrenoceptors (beta(1)-adrenoceptors of atria, beta(2)-adrenoceptors of ocular tissues, and mixed beta(1)-/beta(2)-adrenoceptors of lung) is higher than that of corresponding (+)-enantiomers and their stereoselectivity is different between beta-adrenoceptor antagonists.

This study examines the influence of receptor expression level on signaling pathways activated via endothelin type A receptor (ETAR) expressed in Chinese hamster ovary cells at 32,100 (ETAR-high-CHO) and 893 (ETAR-low-CHO) fmol·mg protein−1. Endothelin-1 (ET-1) elicited a sustained increase in intracellular Ca2+ concentration ([Ca2+]i), which was dependent on Gq/11 protein, phospholipase C (PLC), Na+/H+ exchanger (NHE), and p38 mitogen–activated protein kinase (p38MAPK) in ETAR-high-CHO, whereas the sustained [Ca2+]i increase was negligible in ETAR-low-CHO. Functional study with CytosensorTM microphysiometer showed that ET-1 evoked an NHE1-mediated increase in extracellular acidification rate (ECAR) in ETAR-high-CHO and ETAR-low-CHO. In ETAR-high-CHO, the ECAR response at 30 min after ET-1 stimulation was insensitive to Gq/11 and PLC inhibitors, but sensitive to the p38MAPK inhibitor. In ETAR-low-CHO, the ECAR response at 30 min was sensitive to these inhibitors. Western blot analysis demonstrated that ET-1–induced p38MAPK phosphorylation in ETAR-low-CHO but not in ETAR-high-CHO was mediated via Gq/11 and PLC. The Gq/11/PLC-independent p38MAPK phosphorylation in ETAR-high-CHO was suppressed by expression of the C terminus of Gα12 protein to disrupt receptor-G12 protein coupling. These results provide evidence for multiple signaling pathways of ETAR that were activated via at least the Gq/11/PLC/NHE, G12/p38MAPK/NHE, and Gq/11/PLC/p38MAPK/NHE cascades in an expression level–dependent manner. Keywords:: endothelin type A receptor, receptor expression level, intracellular free Ca2+ concentration, Na+/H+ exchanger, p38 mitogen–activated protein kinase

Distinct muscarinic acetylcholine receptor subtypes widely distribute in stomach tissues and are involved in many physiological functions. Although mRNA of M 1 subtype was found in gastric mucosa, the M 1 subtype has not been detected by conventional membrane binding assays. In the present study, muscarinic receptor subtypes in the rat stomach were reevaluated by using the tissue segment binding technique recently developed to recognize the inherent/native profiles of receptors without receptor environment perturbation. [ 3 H]-N-methylscopolamine (NMS) bound to muscarinic receptors in the intact segments of rat gastric mucosa and muscle layers. The muscarinic receptors in the mucosal segments were composed of M 1 , M 2 and M 3 subtypes, among which the M 1 subtype selectively showed high affinity for pirenzepine. However, in the membrane preparations, binding sites with high affinity for pirenzepine could not be detected. In the muscle layer, M 2 and M 3 subtypes, but not M 1 , were identified in tissue segment and conventional membrane binding assays. Western blotting analysis recognized the M 1 subtype in the membrane preparations of mucosal but not muscle layers. Chronic immobilization stress increased the M 3 subtype in mucosal and muscle layers and decreased the M 2 subtype in the muscle layer, whereas M 1 and M 2 subtypes in mucosal layer did not change after the stress. The current study shows that M 1 subtype occurs as a pirenzepine-high affinity entity in intact segments of rat gastric mucosa, but that it loses the affinity for pirenzepine upon homogenization. Careful identification of native in vivo muscarinic receptors may further elucidate their functions in stomach.

Background and purpose: The α1L-adrenoceptor has pharmacological properties that distinguish it from three classical α1-adrenoceptors (α1A, α1B and α1D). The purpose of this was to identify α1L-adrenoceptors in mice and to examine their relationship to classical α1-adrenoceptors. Experimental approach: Radioligand binding and functional bioassay experiments were performed on the cerebral cortex, vas deferens and prostate of wild-type (WT) and α1A-, α1B- and α1D-adrenoceptor gene knockout (AKO, BKO and DKO) mice. Key results: The radioligand [3H]-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 α1A-adrenoceptors and α1L-adrenoceptors. In membrane preparations of WT mouse cortex, however, [3H]-silodosin bound to a single population of prazosin high-affinity sites, suggesting the presence of α1A-adrenoceptors alone. In contrast, [3H]-prazosin bound to two components having α1A-adrenoceptor and α1B-adrenoceptor profiles in intact segments of WT and DKO mouse cortices, but AKO mice lacked α1A-adrenoceptor profiles and BKO mice lacked α1B-adrenoceptor profiles. Noradrenaline produced contractions through α1L-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: α1L-Adrenoceptors were identified as binding and functional entities in WT, BKO and DKO mice but not in AKO mice, suggesting that the α1L-adrenoceptor is one phenotype derived from the α1A-adrenoceptor gene. British Journal of Pharmacology (2008) 155, 1224–1234; doi:10.1038/bjp.2008.360; published online 22 September 2008

Background and purpose: a1-Adrenoceptors in the rabbit prostate have been studied because of their controversial pharmacological profiles in functional and radioligand binding studies. The purpose of the present study is to determine the native profiles of a1-adrenoceptor phenotypes and to clarify their relationship. Experimental approach: Binding experiments with [ 3 H]-silodosin and [ 3 H]-prazosin were performed using intact tissue segments and crude membrane preparations of rabbit prostate and the results were compared with a1-adrenoceptor-mediated prostate contraction. Key results: [ 3 H]-Silodosin at subnanomolar concentrations bound specifically to intact tissue segments of rabbit prostate. However, [ 3 H]-prazosin at the same range of concentrations failed to bind to a1-adrenoceptors of intact segments. Binding sites of [ 3 H]-silodosin in intact segments were composed of a1L phenotype with low affinities for prazosin (pKi ¼ 7.1), 5-methyurapidil and N-[2-(2-cyclopropylmethoxyphenoxy)ethyl]-5-chloro-a,a-dimethyl-1H-indole-3-ethamine hydrochloride (RS-17053), and a1A-like phenotype with moderate affinity for prazosin (pKi ¼ 8.8) but high affinity for 5-methyurapidil and RS17053. In contrast, both radioligands bound to a single population of a1-adrenoceptors in the membrane preparations at the same density with a subnanomolar affinity, showing a typical profile of ‘classical’ a1A-adrenoceptors (pKi for prazosin ¼ 9.8). The pharmacological profile of a1-adrenoceptor-mediated prostate contraction was in accord with the a1L phenotype observed by intact segment binding approach. Conclusions and implications: Three distinct phenotypes (a1L and a1A-like phenotypes in the intact segments and a classical a1A phenotype in the membranes) with different affinities for prazosin were detected in rabbit prostate. It appears that the three phenotypes are phenotypic subtypes of a1A-adrenoceptors, but are not genetically different subtypes. British Journal of Pharmacology (2008) 155, 906–912; doi:10.1038/bjp.2008.318; published online 11 August 2008

The pharmacological profiles of alpha1-adrenoceptors for ketanserin, prazosin, silodosin, and BMY 7378 (8-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-8-azaspiro[4,5]decane-7,9-dione dihydrochloride) were examined under different assay conditions. Among the tested antagonists and alpha1-adrenoceptors subtypes, ketanserin showed significantly lower affinity for the alpha1B-adrenoceptor subtype in intact tissue sampled from the rat tail artery, thoracic aorta, and cerebral cortex (functional pKB and binding pKi were approximately 6), than in cerebral cortex membrane preparations or whole cell and membrane preparations of alpha1B-adrenoceptor transfected human embryonic kidney 293T (HEK 293T) cells (pKi was approximately 8). In these tissues and cells, however, ketanserin showed a similar affinity (pKi = approximately 8) for alpha1A- and alpha1D-adrenoceptors even though the assays were conducted under different conditions. In contrast, the affinities of alpha1A-, alpha1B-, and alpha1D-adrenoceptors for prazosin, silodosin, and BMY 7378 did not significantly change under different assay conditions and in different tissues. The present study reveals that the pharmacological profiles of native alpha 1B-adrenoceptors for ketanserin is strongly influenced by the assay conditions and suggest that antagonist affinity is not necessarily constant.

Background and purpose: In addition to α1A, α1B and α1D-adrenoceptors (ARs), putative α1L-ARs with a low affinity for prazosin have been proposed. The purpose of the present study was to identify the α1A-AR and clarify its pharmacological profile using a radioligand binding assay.

Recently, tissue segment binding method with a hydrophilic radioligand [(3)H]-CGP12177 was developed to detect plasma membrane beta-adrenoceptors in rat heart (Horinouchi et al., 2006). In the present study, propranolol (40 mg kg(-1) day(-1)), atenolol (40 mg kg(-1) day(-1)) and bevantolol (200 mg kg(-1) day(-1)) were administered to rats for 6 weeks, and the changes of plasma membrane beta-adrenoceptors and their mRNA expression in rat ventricle were examined. Chronic administration of propranolol increased the beta(1)-adrenoceptors but decreased the beta(2)-adrenoceptors without changing total amount of plasma membrane beta-adrenoceptors. Atenolol increased both plasma membrane beta(1)- and beta(2)-adrenoceptors, whereas bevantolol had no effect on the beta-adrenoceptor density and their subtype proportions. In contrast, the density of beta-adrenoceptors detected in conventional homogenate binding study was extremely low (approximately 60% of plasma membrane beta-adrenoceptors detected with the tissue segment binding method) and the effects of chronic administration of beta-adrenoceptor antagonists were not necessarily in accord with those at the plasma membrane beta-adrenoceptors. The mRNA levels of beta(1)- and beta(2)-adrenoceptors were not altered by propranolol treatment, while beta(1)-adrenoceptor mRNA significantly decreased after administration of atenolol or bevantolol without changing the level of beta(2)-adrenoceptor mRNA. The present binding study with intact tissue segments clearly shows that the plasma membrane beta(1)- and beta(2)-adrenoceptors of rat heart, in contrast to the homogenate binding sites and the mRNA levels, are differently affected by chronic treatment with three beta-adrenoceptor antagonists; up- and down-regulations of beta(1)- and beta(2)-adrenoceptors, respectively, by propranolol, and up-regulation of both the subtypes by atenolol, but no significant change in both the subtypes by bevantolol.

The calcitonin (CT)/CT gene-related peptide (CGRP) family is conserved in vertebrates. The activities of this peptide family are regulated by a combination of two receptors, namely the calcitonin receptor (CTR) and the CTR-like receptor (CLR), and three receptor activity-modifying proteins (RAMPs). Furthermore, RAMPs act as escort proteins by translocating CLR to the cell membrane. Recently, CT/CGRP family peptides have been identified or inferred in several invertebrates. However, the molecular characteristics and relevant functions of the CTR/CLR and RAMPs in invertebrates remain unclear. In this study, we identified three CT/CGRP family peptides (Bf-CTFPs), one CTR/CLR-like receptor (Bf-CTFP-R), and three RAMP-like proteins (Bf-RAMP-LPs) in the basal chordate amphioxus (Branchiostoma floridae). The Bf-CTFPs were shown to possess an N-terminal circular region typical of the CT/CGRP family and a C-terminal Pro-NH2. The Bf-CTFP genes were expressed in the central nervous system and in endocrine cells of the midgut, indicating that Bf-CTFPs serve as brain and/or gut peptides. Cell surface expression of the Bf-CTFP-R was enhanced by co-expression with each Bf-RAMP-LP. Furthermore, Bf-CTFPs activated Bf-CTFP-R·Bf-RAMP-LP complexes, resulting in cAMP accumulation. These results confirmed that Bf-RAMP-LPs, like vertebrate RAMPs, are prerequisites for the function and translocation of the Bf-CTFP-R. The relative potencies of the three peptides at each receptor were similar. Bf-CTFP2 was a potent ligand at all receptors in cAMP assays. Bf-RAMP-LP effects on ligand potency order were distinct to vertebrate CGRP/adrenomedullin/amylin receptors. To the best of our knowledge, this is the first molecular and functional characterization of an authentic invertebrate CT/CGRP family receptor and RAMPs.

Alpha(1)-adrenoceptors are widely distributed in the human body and play important physiologic roles. Three alpha(1)-adrenoceptor subtypes (alpha(1A), alpha(1B) and alpha(1D)) have been cloned and show different pharmacologic profiles. In addition, a putative alpha(1)-adrenoceptor (alpha(1L) subtype) has also been proposed. Recently, three drugs (tamsulosin, naftopidil, and silodosin) have been developed in Japan for the treatment of urinary obstruction in patients with benign prostatic hyperplasia. In this review, we describe recent alpha(1)-adrenoceptor subclassifications and the pharmacologic characteristics (subtype selectivity and clinical relevance) of alpha(1)-adrenoceptor antagonists.

We obtained a new mutant of the β1-adrenergic receptor (β1-AR) by point mutations that can constitutively activate β1-AR. Aspartate104 of the β1-AR in the 2nd transmembrane was replaced with alanine. The β1-AR mutant expressed in human embryonic kidney (HEK)-293 cells displayed high level of constitutive activity with respect to wild-type (P