ß2-Adrenoceptor signaling in airway epithelial cells promotes eosinophilic inflammation, mucous metaplasia, and airway contractility.

The mostly widely used bronchodilators in asthma therapy are ß2-adrenoreceptor (ß2AR) agonists, but their chronic use causes paradoxical adverse effects. We have previously determined that ß2AR activation is required for expression of the asthma phenotype in mice, but the cell types involved are unknown. We now demonstrate that ß2AR signaling in the airway epithelium is sufficient to mediate key features of the asthmatic responses to IL-13 in murine models. Our data show that inhibition of ß2AR signaling with an aerosolized antagonist attenuates airway hyperresponsiveness (AHR), eosinophilic inflammation, and mucus-production responses to IL-13, whereas treatment with an aerosolized agonist worsens these phenotypes, suggesting that ß2AR signaling on resident lung cells modulates the asthma phenotype. Labeling with a fluorescent ß2AR ligand shows the receptors are highly expressed in airway epithelium. In ß2AR-/- mice, transgenic expression of ß2ARs only in airway epithelium is sufficient to rescue IL-13-induced AHR, inflammation, and mucus production, and transgenic overexpression in WT mice exacerbates these phenotypes. Knockout of ß-arrestin-2 (ßarr-2-/-) attenuates the asthma phenotype as in ß2AR-/- mice. In contrast to eosinophilic inflammation, neutrophilic inflammation was not promoted by ß2AR signaling. Together, these results suggest ß2ARs on airway epithelial cells promote the asthma phenotype and that the proinflammatory pathway downstream of the ß2AR involves ßarr-2. These results identify ß2AR signaling in the airway epithelium as capable of controlling integrated responses to IL-13 and affecting the function of other cell types such as airway smooth muscle cells.

The IUPHAR/BPS Guide to PHARMACOLOGY in 2016: towards curated quantitative interactions between 1300 protein targets and 6000 ligands.

The IUPHAR/BPS Guide to PHARMACOLOGY (GtoPdb, http://www.guidetopharmacology.org) provides expert-curated molecular interactions between successful and potential drugs and their targets in the human genome. Developed by the International Union of Basic and Clinical Pharmacology (IUPHAR) and the British Pharmacological Society (BPS), this resource, and its earlier incarnation as IUPHAR-DB, is described in our 2014 publication. This update incorporates changes over the intervening seven database releases. The unique model of content capture is based on established and new target class subcommittees collaborating with in-house curators. Most information comes from journal articles, but we now also index kinase cross-screening panels. Targets are specified by UniProtKB IDs. Small molecules are defined by PubChem Compound Identifiers (CIDs); ligand capture also includes peptides and clinical antibodies. We have extended the capture of ligands and targets linked via published quantitative binding data (e.g. Ki, IC50 or Kd). The resulting pharmacological relationship network now defines a data-supported druggable genome encompassing 7% of human proteins. The database also provides an expanded substrate for the biennially published compendium, the Concise Guide to PHARMACOLOGY. This article covers content increase, entity analysis, revised curation strategies, new website features and expanded download options.

The IUPHAR/BPS Guide to PHARMACOLOGY: an expert-driven knowledgebase of drug targets and their ligands.

The International Union of Basic and Clinical Pharmacology/British Pharmacological Society (IUPHAR/BPS) Guide to PHARMACOLOGY (http://www.guidetopharmacology.org) is a new open access resource providing pharmacological, chemical, genetic, functional and pathophysiological data on the targets of approved and experimental drugs. Created under the auspices of the IUPHAR and the BPS, the portal provides concise, peer-reviewed overviews of the key properties of a wide range of established and potential drug targets, with in-depth information for a subset of important targets. The resource is the result of curation and integration of data from the IUPHAR Database (IUPHAR-DB) and the published BPS 'Guide to Receptors and Channels' (GRAC) compendium. The data are derived from a global network of expert contributors, and the information is extensively linked to relevant databases, including ChEMBL, DrugBank, Ensembl, PubChem, UniProt and PubMed. Each of the ∼6000 small molecule and peptide ligands is annotated with manually curated 2D chemical structures or amino acid sequences, nomenclature and database links. Future expansion of the resource will complete the coverage of all the targets of currently approved drugs and future candidate targets, alongside educational resources to guide scientists and students in pharmacological principles and techniques.

Confocal myography for the study of hypertensive vascular remodelling.

Hypertension is associated with vascular structural alterations known as "vascular remodelling", which initially are adaptive but in the long run, lead to vascular damage and loss of function. Despite decades of study, there is still modest information on the 3-dimensional (3D) arrangement of vascular cells and extracellular matrix (ECM) and how they change under pathological situations. To address this problem we developed a technique which combines fluorescence confocal microscopy, pressure myography and image analysis, "confocal myography", which permits the study of intact resistance-sized vessels at cellular level and at physiological pressure. With the aid of this method, we have identified, in arteries from hypertensive rats, abnormal orientation of endothelial, smooth muscle cells (SMC) and elastic fibres; elongation and denudation of endothelial cells, and adventitial hypercellularity. Confocal myography offers a new approach to the study of vascular remodelling in intact small arteries from a 3D point of view.

Alterations in rabbit aorta induced by types I and II pyrethroids.

Since pyrethroids are involved in reactive oxygen species production and no investigations have yet been performed on smooth muscle cell integrity, we studied the influence of permethrin- and cypermethrin-treatment on rabbit aorta using confocal laser scanning fluorescence microscopy, which allows cell viability to be assessed within the wall of living rabbit aorta. The data obtained show that the pyrethroid-treatment (10-100µM) impairs the smooth muscle cell viability. A double-labeling protocol allowed us to distinguish cytotoxic effects of permethrin- and cypermethrin-treatment in aortic rings. In conclusion, permethrin seems to induce more oxidative stress on the aorta wall than that cypermethrin does.

Localization of the mouse alpha1A-adrenergic receptor (AR) in the brain: alpha1AAR is expressed in neurons, GABAergic interneurons, and NG2 oligodendrocyte progenitors.

alpha(1)-Adrenergic receptors (ARs) are not well defined in the central nervous system. The particular cell types and areas that express these receptors are uncertain because of the lack of high avidity antibodies and selective ligands. We have developed transgenic mice that either systemically overexpress the human alpha(1A)-AR subtype fused with the enhanced green fluorescent protein (EGFP) or express the EGFP protein alone under the control of the mouse alpha(1A)-AR promoter. We confirm our transgenic model against the alpha(1A)-AR knockout mouse, which expresses the LacZ gene in place of the coding region for the alpha(1A)-AR. By using these models, we have now determined cellular localization of the alpha(1A)-AR in the brain, at the protein level. The alpha(1A)-AR or the EGFP protein is expressed prominently in neuronal cells in the cerebral cortex, hippocampus, hypothalamus, midbrain, pontine olivary nuclei, trigeminal nuclei, cerebellum, and spinal cord. The types of neurons were diverse, and the alpha(1A)-AR colocalized with markers for glutamic acid decarboxylase (GAD), gamma-aminobutyric acid (GABA), and N-methyl-D-aspartate (NMDA) receptors. Recordings from alpha(1A)-AR EGFP-expressing cells in the stratum oriens of the hippocampal CA1 region confirmed that these cells were interneurons. We could not detect expression of the alpha(1A)-AR in mature astrocytes, oligodendrocytes, or cerebral blood vessels, but we could detect the alpha(1A)-AR in oligodendrocyte progenitors. We conclude that the alpha(1A)-AR is abundant in the brain, expressed in various types of neurons, and may regulate the function of oligodendrocyte progenitors, interneurons, GABA, and NMDA receptor containing neurons.

Phosphorylation-independent internalisation and desensitisation of the human sphingosine-1-phosphate receptor S1P3.

Here we demonstrate that phosphorylation of the sphingosine-1-phosphate (S1P) receptor S1P(3) is increased specifically in response to S1P. Truncation of the receptor's carboxyl-terminal domain revealed that the presence of a serine-rich stretch of residues between Leu332 and Val352 was essential to observe this effect. Although agonist-occupied wild-type (WT) S1P(3) could be phosphorylated in vitro by G-protein-coupled receptor kinase 2 (GRK2), a role of S1P(3) phosphorylation in controlling S1P(3)-G(q/11) coupling was excluded since A) a phosphorylation-resistant S1P(3) mutant desensitised in a manner indistinguishable from the WT receptor and was phosphorylated to a greater extent than the WT receptor by GRK2 in vitro, and B) co-expression with GRK2 or GRK3 failed to potentiate S1P(3) phosphorylation. S1P(3) phosphorylation was also not required for receptor sequestration away from the cell surface. Together, these data suggest that S1P(3) function is not subject to conventional regulation by GRK phosphorylation and that novel aspects of S1P(3) function distinct from classical G-protein coupling and receptor internalisation may be controlled its carboxyl-terminal domain.

Fluorescent ligands, antibodies, and proteins for the study of receptors.

Fluorescent molecules bound to receptors can show their location and, if binding is reversible, can provide pharmacological information such as affinity and proximity between interacting molecules. The spatial precision offered by visualisation transcends the diverse localisation and low molecular concentration of receptor molecules. Consequently, the relationships between receptor location and function and life cycles of receptors have become better understood as a result of fluorescent labeling. Each of these aspects contributes new insights to drug action and potential new targets. The relationships between spatial distribution of receptor and function are largely unknown. This is particularly apparent for native receptors expressed in their normal host tissues where communication between heterogeneous cell types influences receptor distribution and function. In cultured cell systems, particularly for G-protein-coupled receptors (GPCR), fluorescence-based methods have enabled the visualisation of the cycle of agonist-stimulated receptor clustering, endocytic internalisation to the perinuclear region, degradation of the receptor-ligand complex, and recycling back to the surface membrane. Using variant forms of green fluorescent protein (GFP), antibodies, or fluorescent ligands, it is possible to detect or visualise the formation of oligomeric receptor complexes. Careful selection of fluorescent molecules based on their spectral properties enables resonance energy transfer and multilabel visualisation with colocalisation studies. Fluorescent agonist and antagonist ligands are now being used in parallel with GFP to study receptor cycling in live cells. This review covers how labeling and visualisation technologies have been applied to the study of major pharmacologically important receptors and illustrates this by giving examples of recent techniques that have relied on GFP, antibodies, or fluorescent ligands alone or in combination for the purpose of studying GPCR.

Evidence for involvement of alpha1D-adrenoceptors in contraction of femoral resistance arteries using knockout mice.

The role of alpha(1D)-adrenoceptors in vasoconstrictor responses to noradrenaline in mouse femoral resistance arteries was investigated using wire myography in alpha(1D)-adrenoceptor knockout (alpha(1D)-KO) and wild-type (WT) mice of the same genetic background.alpha(1D)-KO mice were 2.5-fold less sensitive than WTs to exogenous noradrenaline and BMY 7378 was significantly less potent against noradrenaline in alpha(1D)-KO mice than in WTs, showing a minor contribution of alpha(1D)-adrenoceptors in response to noradrenaline. Prazosin and 5-methyl-urapidil were equally effective against noradrenaline in alpha(1D)-KO and WT mice. Chloroethylclonidine produced a significantly greater attenuation of the response to noradrenaline in alpha(1D)-KO mice than in WTs. Responses to electrical field stimulation (EFS), at 2-20 Hz for 10 s and 0.09 ms pulse width were significantly smaller overall in alpha(1D)-KOs than in WTs although no significant differences were seen at the different frequencies.BMY 7378 produced significantly greater inhibition of responses at 2 and 5 Hz than at higher frequencies in WTs. In alpha(1D)-KOs, this greater sensitivity to BMY 7378 at lower frequencies was not apparent, confirming that the effect of BMY 7378 was due to blockade of alpha(1D)-adrenoceptors. Prazosin and 5-methyl-urapidil had similar inhibitory effects on responses to EFS in alpha(1D)-KO and WT mice. Chloroethylclonidine inhibited responses to EFS to a significantly greater extent in alpha(1D)-KO mice. The present study with alpha(1D)-KO mice shows that alpha(1D)-adrenoceptors contribute to vasoconstrictor responses to exogenous and neurally released noradrenaline in femoral resistance arteries.

Direct demonstration of beta1- and evidence against beta2- and beta3-adrenoceptors, in smooth muscle cells of rat small mesenteric arteries.

1 Recent evidence supports additional subtypes of vasodilator beta-adrenoceptor (beta-AR) besides the 'classical' beta(2). The aim of this study was to investigate the distribution of beta-ARs in the wall of rat mesenteric resistance artery (MRA), to establish the relative roles of beta-ARs in smooth muscle and other cell types in mediating vasodilatation and to analyse this in relation to the functional pharmacology. 2 We first examined the vasodilator beta-AR subtype using 'subtype-selective' agonists against the, commonly employed, phenylephrine-induced tone. Concentration-related relaxation was produced by isoprenaline (pEC(50): 7.70+/-0.1) (beta(1) and beta(2)). Salbutamol (beta(2)), BRL 37344 (beta(3)) and CGP 12177 (atypical beta) caused relaxation but were 144, 100 and 263 times less potent than isoprenaline; the 'beta(3)-adrenoceptor agonist' CL 316243 was ineffective. 3 In arteries precontracted with 5-HT or U 46619, isoprenaline produced concentration-related relaxation but salbutamol, BRL 37344, CGP 12177 and CL 316243 did not. SR 59230A, CGP 12177 and BRL 37344 caused a parallel rightward shift in the concentration-response curve to phenylephrine indicating competitive alpha(1)-AR antagonism, explaining the false-positive 'vasodilator' action against phenylephrine-induced tone. Endothelial denudation but not L-NAME slightly attenuated isoprenaline-mediated vasodilatation in phenylephrine and U 46619 precontracted MRA. 4 The beta-AR fluorescent ligand BODIPY TMR-CGP 12177 behaved as an irreversible beta(1)-AR antagonist in MRA and bound to the surface and inside vascular smooth muscle cells in intact vascular wall. Beta-ARs in smooth muscle cells were observed in a perinuclear location, consistent with the location of Golgi and endoplasmic reticulum. 5 Binding of BODIPY TMR-CGP 12177 was inhibited by BAAM (1 microM) in all three vascular tunics, confirming the presence of beta-ARs in adventitia, media and intima. Binding in adventitia was observed in both neuronal and non-neuronal cell types. Lack of co-localisation with a fluorescent ligand for alpha-ARs confirms the selectivity of BODIPY TMR-CGP 12177 for beta-ARs over alpha-ARs. 6 Our results support the presence of functional vasodilator beta(1)-ARs and show that they are mainly located in smooth muscle cells. Furthermore, we have demonstrated, for the first time, the usefulness of BODIPY TMR-CGP 12177 for identifying beta-AR distribution in the 'living' vascular wall.

Insights into the functional roles of alpha(1)-adrenoceptor subtypes in mouse carotid arteries using knockout mice.

1. alpha(1)-Adrenoceptor (AR) subtypes in mouse carotid arteries were characterised using a combination of agonist/antagonist pharmacology and knockout (KO) mice. 2. Phenylephrine (PE) was most potent in the alpha(1B)-KO (pEC(50)=6.9+/-0.2) followed by control (pEC(50)=6.3+/-0.06) and alpha(1D)-KO (pEC(50)=5.5+/-0.07). Both N-[5-(4,5-dihydro-1H-imidazol-2yl)-2-hydroxy-5,6,7,8-tetrahydronaphthalen-1-yl] methanesulphonamide hydrobromide (A-61603) and 5-hydroxytryptamine (5-HT) were more potent in the alpha(1D)-KO (pEC(50)=7.4+/-0.27 and 7.4+/-0.05, respectively) than the control (pEC(50)=6.9+/-0.09 and 6.9+/-0.08, respectively) and equipotent with the control in the alpha(1B)-KO (pEC(50)=6.7+/-0.07 and 6.8+/-0.04). Maximum responses to PE and A-61603 were reduced in the alpha(1D)-KO compared to control; there was no difference in maximum responses to 5-HT. 3. In control arteries, prazosin and 5-methylurapidil acted competitively with pA(2) of 9.6 and 7.5, respectively. BMY7378 produced antagonism only at the highest concentration used (100 nM; pK(B) 8.3). 4. Prazosin, 5-methylurapidil and BMY7378 acted competitively in alpha(1B)-KO carotid arteries with pA(2) of 10.3, 7.6 and 9.6, respectively. 5. In the alpha(1D)-KO, against PE, 5-methylurapidil produced a pA(2) of 8.1. pK(B) values were calculated for prazosin (10.6) and BMY7378 (7.0). Against A-61603, 5-methylurapidil had a pA(2) of 8.5, prazosin 8.6, while BMY7378 had no effect. 6. In conclusion, the alpha(1B)-KO mediates contraction solely through alpha(1D)-ARs and the alpha(1D)-KO through alpha(1A)-ARs. Extrapolating back to the control from the knockout data suggests that all three subtypes could be involved in the responses, but we propose that the alpha(1D)-AR causes the contractile response and that the role of the alpha(1B)-AR is mainly regulatory.

The alpha 1-adrenoceptor profile in human skeletal muscle resistance arteries in critical limb ischaemia.

OBJECTIVE: We recently reported the hyper-responsiveness of human skeletal muscle resistance arteries (SkMRAs) to noradrenaline in critical limb ischaemia (CLI). In this study we investigated the characteristics of alpha(1)-adrenoceptor subtypes and evaluated the agonist affinity and adrenoceptor reserve in the ischaemic arteries. METHODS: Human SkMRAs were isolated from non-ischaemic and ischaemic areas of limbs amputated for CLI. Subcutaneous resistance arteries were isolated from inguinal biopsies from healthy subjects. Arterial segments were mounted on a small vessel wire myograph. RESULTS: Contractile responses to agonists, adrenaline and A-61603 (alpha(1A)-selective) were significantly increased in ischaemic arteries compared to those in non-ischaemic arteries. Receptor inactivation studies indicated an increase in the alpha-adrenoceptor reserve in the ischaemic arteries but the affinity of noradrenaline was unaffected. Healthy subcutaneous arteries had a similar noradrenaline affinity but a higher receptor reserve than skeletal muscle arteries. In the ischaemic arteries, the antagonists prazosin (alpha(1)-selective), 5-methyl-urapidil (alpha(1A)-selective) and BMY 7378 (alpha(1D)-selective) produced rightward shifts in the concentration response curves (CRCs) of noradrenaline giving pK(B)s of 9.6+/-0.3, 8.4+/-0.2 and 7.1+/-0.4, respectively. Pretreatment with 10 microM chloroethylclonidine decreased the contractile responses to noradrenaline and A-61603 to 57+/-7 and 72+/-4% of their respective controls. CONCLUSIONS: These results demonstrate that the ischaemic SkMRAs have an increased alpha-adrenoceptor reserve with no change in the predominant alpha(1A)-adrenoceptor profile.

Localization of α-adrenoceptors: JR Vane Medal Lecture.

UNLABELLED: This review is based on the JR Vane Medal Lecture presented at the BPS Winter Meeting in December 2011 by J.C. McGrath. A recording of the lecture is included as supporting information. It covers his laboratory's work from 1990 to 2010 on the localization of vascular α1 -adrenoceptors in native tissues, mainly arteries. MAIN POINTS: (i) α1 -adrenoceptors are present on several cell types in arteries, not only on medial smooth muscle, but also on adventitial, endothelial and nerve cells; (ii) all three receptor subtypes (α1 A , α1 B , α1 D ) are capable of binding ligands at the cell surface, strongly indicating that they are capable of function and not merely expressed. (iii) all of these cell types can take up an antagonist ligand into the intracellular compartments to which endocytosing receptors move; (iv) each individual subtype can exist at the cell surface and intracellularly in the absence of the other subtypes. As functional pharmacological experiments show variations in the involvement of the different subtypes in contractions of different arteries, it is concluded that the presence and disposition of α1 -adrenoceptors in arteries is not a simple guide to their involvement in function. Similar locations of the subtypes, even in different cell types, suggest that differences between the distribution of subtypes in model systems do not directly correlate with those in native tissues. This review includes a historical summary of the alternative terms used for adrenoceptors (adrenergic receptors, adrenoreceptors) and the author's views on the use of colours to illustrate different items, given his partial colour-blindness.