Julia K. L. Walker

Muscarinic Supersensitivity and Impaired Receptor Desensitization in G Protein-Coupled Receptor Kinase 5-Deficient Mice

G protein-coupled receptor kinase 5 (GRK5) is a member of a family of enzymes that phosphorylate activated G protein-coupled receptors (GPCR). To address the physiological importance of GRK5-mediated regulation of GPCRs, mice bearing targeted deletion of the GRK5 gene (GRK5-KO) were generated. GRK5-KO mice exhibited mild spontaneous hypothermia as well as pronounced behavioral supersensitivity upon challenge with the nonselective muscarinic agonist oxotremorine. Classical cholinergic responses such as hypothermia, hypoactivity, tremor, and salivation were enhanced in GRK5-KO animals. The antinociceptive effect of oxotremorine was also potentiated and prolonged. Muscarinic receptors in brains from GRK5-KO mice resisted oxotremorine-induced desensitization, as assessed by oxotremorine-stimulated [5S]GTPgammaS binding. These data demonstrate that elimination of GRK5 results in cholinergic supersensitivity and impaired muscarinic receptor desensitization and suggest that a deficit of GPCR desensitization may be an underlying cause of behavioral supersensitivity.

β-Arrestin-2 regulates the development of allergic asthma

Asthma is a chronic inflammatory disorder of the airways that is coordinated by Th2 cells in both human asthmatics and animal models of allergic asthma. Migration of Th2 cells to the lung is key to their inflammatory function and is regulated in large part by chemokine receptors, members of the seven-membrane-spanning receptor family. It has been reported recently that T cells lacking beta-arrestin-2, a G protein-coupled receptor regulatory protein, demonstrate impaired migration in vitro. Here we show that allergen-sensitized mice having a targeted deletion of the beta-arrestin-2 gene do not accumulate T lymphocytes in their airways, nor do they demonstrate other physiological and inflammatory features characteristic of asthma. In contrast, the airway inflammatory response to LPS, an event not coordinated by Th2 cells, is fully functional in mice lacking beta-arrestin-2. beta-arrestin-2-deficient mice demonstrate OVA-specific IgE responses, but have defective macrophage-derived chemokine-mediated CD4+ T cell migration to the lung. This report provides the first evidence that beta-arrestin-2 is required for the manifestation of allergic asthma. Because beta-arrestin-2 regulates the development of allergic inflammation at a proximal step in the inflammatory cascade, novel therapies focused on this protein may prove useful in the treatment of asthma.

PROPERTIES OF SECRETIN RECEPTOR INTERNALIZATION DIFFER FROM THOSE OF THE BETA 2-ADRENERGIC RECEPTOR

The endocytic pathway of the secretin receptor, a class II GPCR, is unknown. Some class I G protein-coupled receptors (GPCRs), such as the β2-adrenergic receptor (β2-AR), internalize in clathrin-coated vesicles and this process is mediated by G protein-coupled receptor kinases (GRKs), β-arrestin, and dynamin. However, other class I GPCRs, for example, the angiotensin II type 1A receptor (AT1AR), exhibit different internalization properties than the β2-AR. The secretin receptor, a class II GPCR, is a GRK substrate, suggesting that like the β2-AR, it may internalize via a β-arrestin and dynamin directed process. In this paper we characterize the internalization of a wild-type and carboxyl-terminal (COOH-terminal) truncated secretin receptor using flow cytometry and fluorescence imaging, and compare the properties of secretin receptor internalization to that of the β2-AR. In HEK 293 cells, sequestration of both the wild-type and COOH-terminal truncated secretin receptors was unaffected by GRK phosphorylation, whereas inhibition of cAMP-dependent protein kinase mediated phosphorylation markedly decreased sequestration. Addition of secretin to cells resulted in a rapid translocation of β-arrestin to plasma membrane localized receptors; however, secretin receptor internalization was not reduced by expression of dominant negative β-arrestin. Thus, like the AT1AR, secretin receptor internalization is not inhibited by reagents that interfere with clathrin-coated vesicle-mediated internalization and in accordance with these results, we show that secretin and AT1A receptors colocalize in endocytic vesicles. This study demonstrates that the ability of secretin receptor to undergo GRK phosphorylation and β-arrestin binding is not sufficient to facilitate or mediate its internalization. These results suggest that other receptors may undergo endocytosis by mechanisms used by the secretin and AT1Areceptors and that kinases other than GRKs may play a greater role in GPCR endocytosis than previously appreciated.

New perspectives regarding β2‐adrenoceptor ligands in the treatment of asthma

In the last two decades several significant changes have been proposed in the receptor theory that describes how ligands can interact with G protein‐coupled receptors (GPCRs). Here we briefly summarize the evolution of receptor theory and detail recent prominent advances. These include: (i) the existence of spontaneously active GPCRs that are capable of signalling even though they are unoccupied by any ligand; (ii) the discovery of ligands that can inactivate these spontaneously active receptors; (iii) the notion that a ligand may simultaneously activate more than one GPCR signalling pathway; and (iv) the notion that certain ligands may be able to preferentially direct receptor signalling to a specific pathway. Because the data supporting these receptor theory ideas are derived primarily from studies using artificial expression systems, the physiological relevance of these new paradigms remains in question. As a potential example of how these new perspectives in receptor theory relate to drug actions and clinical outcomes, we discuss their relevance to the recent controversy regarding the chronic use of β2‐adrenoceptor agonists in the treatment of asthma.

Altered airway and cardiac responses in mice lacking G protein-coupled receptor kinase 3

Contraction and relaxation of airway smooth muscles is mediated, in part, by G protein-coupled receptors (GPCRs) and dysfunction of these receptors has been implicated in asthma. Phosphorylation of GPCRs, by G protein-coupled receptor kinase (GRK), is an important mechanism involved in the dampening of GPCR signaling. To determine whether this mechanism might play a role in airway smooth muscle physiology, we examined the airway pressure time index and heart rate (HR) responses to intravenous administration of the cholinergic agonist methacholine (MCh) in genetically altered mice lacking one copy of GRK2 (GRK2 +/-), homozygous GRK3 knockout (GRK3 -/-), and wild-type littermates. (GRK2 -/- mice die in utero.) GRK3 -/- mice demonstrated a significant enhancement in the airway response to 100 and 250 μg/kg doses of MCh compared with wild-type and GRK2 +/- mice. GRK3 -/- mice also displayed an enhanced sensitivity of the airway smooth muscle response to MCh. In addition, GRK3 -/- mice displayed an altered HR recovery from MCh-induced bradycardia. Although direct stimulation of cardiac muscarinic receptors measured as vagal stimulation-induced bradycardia was similar in GRK3 -/- and wild-type mice, the baroreflex increase in HR associated with sodium nitroprusside-induced hypotension was significantly greater in GRK3 -/- than wild-type mice. Therefore, these data demonstrate that in the mouse, GRK3 may be involved in modulating the cholinergic response of airway smooth muscle and in regulating the chronotropic component of the baroreceptor reflex.Contraction and relaxation of airway smooth muscles is mediated, in part, by G protein-coupled receptors (GPCRs) and dysfunction of these receptors has been implicated in asthma. Phosphorylation of GPCRs, by G protein-coupled receptor kinase (GRK), is an important mechanism involved in the dampening of GPCR signaling. To determine whether this mechanism might play a role in airway smooth muscle physiology, we examined the airway pressure time index and heart rate (HR) responses to intravenous administration of the cholinergic agonist methacholine (MCh) in genetically altered mice lacking one copy of GRK2 (GRK2 +/-), homozygous GRK3 knockout (GRK3 -/-), and wild-type littermates. (GRK2 -/- mice die in utero.) GRK3 -/- mice demonstrated a significant enhancement in the airway response to 100 and 250 microgram/kg doses of MCh compared with wild-type and GRK2 +/- mice. GRK3 -/- mice also displayed an enhanced sensitivity of the airway smooth muscle response to MCh. In addition, GRK3 -/- mice displayed an altered HR recovery from MCh-induced bradycardia. Although direct stimulation of cardiac muscarinic receptors measured as vagal stimulation-induced bradycardia was similar in GRK3 -/- and wild-type mice, the baroreflex increase in HR associated with sodium nitroprusside-induced hypotension was significantly greater in GRK3 -/- than wild-type mice. Therefore, these data demonstrate that in the mouse, GRK3 may be involved in modulating the cholinergic response of airway smooth muscle and in regulating the chronotropic component of the baroreceptor reflex.

β-Arrestins specifically constrain β2-adrenergic receptor signaling and function in airway smooth muscle

Chronic use of inhaled beta‐agonists by asthmatics is associated with a loss of bronchoprotective effect and deterioration of asthma control. Beta‐agonist‐promoted desensitization of airway smooth muscle beta‐2‐adrenergic receptors, mediated by G protein‐coupled receptor kinases and arrestins, is presumed to underlie these effects, but such a mechanism has never been demonstrated. Using in vitro, ex vivo, and in vivo murine models, we demonstrate that beta‐arrestin‐2 gene ablation augments beta‐agonist‐mediated airway smooth muscle relaxation, while augmenting beta‐agonist‐stimulated cyclic adenosine monophosphate production. In cultures of human airway smooth muscle, small interfering RNA‐mediated knockdown of arrestins also augments beta‐agonist‐stimulated cyclic adenosine monophosphate production. Interestingly, signaling and function mediated by m2/m3 muscarinic acetylcholine receptors or prostaglandin E2 receptors were not affected by either beta‐arrestin‐2 knockout or arrestin knockdown. Thus, arrestins are selective regulators of beta‐2‐adrenergic receptor signaling and function in airway smooth muscle. These results and our previous findings, which demonstrate a role for arrestins in the development of allergic inflammation in the lung, identify arrestins as potentially important therapeutic targets for obstructive airway diseases.—Deshpande, D. A., Theriot, B. S., Penn, R. B., Walker, J. K. L. β‐Arrestins specifically constrain β2‐adrenergic receptor signaling and function in airway smooth muscle. FASEB J. 22, 2134–2141 (2008)

3He MRI in mouse models of asthma

In the study of asthma, a vital role is played by mouse models, because knockout or transgenic methods can be used to alter disease pathways and identify therapeutic targets that affect lung function. Assessment of lung function in rodents by available methods is insensitive because these techniques lack regional specificity. A more sensitive method for evaluating lung function in human asthma patients uses hyperpolarized (HP) 3He MRI before and after bronchoconstriction induced by methacholine (MCh). We now report the ability to perform such 3He imaging of MCh response in mice, where voxels must be ∼3000 times smaller than in humans and 3He diffusion becomes an impediment to resolving the airways. We show three‐dimensional (3D) images that reveal airway structure down to the fifth branching and visualize ventilation at a resolution of 125 × 125 × 1000 μm3. Images of ovalbumin (OVA)‐sensitized mice acquired after MCh show both airway closure and ventilation loss. To also observe the MCh response in naive mice, we developed a non‐slice‐selective 2D protocol with 187 × 187 μm2 resolution that was fast enough to record the MCh response and recovery with 12‐s temporal resolution. The extension of 3He MRI to mouse models should make it a valuable translational tool in asthma research. Magn Reson Med 58:893–900, 2007.

β-Arrestin-2 mediates the proinflammatory effects of proteinase-activated receptor-2 in the airway

Proteinase-Activated rreceptor-2 (PAR2), a G-protein–coupled Receptor, activated by serine proteinases, is reported to have both protective and proinflammatory effects in the airway. Given these opposing actions, both inhibitors and activators of PAR2 have been proposed for treating asthma. PAR2 can signal through two independent pathways: a β-arrestin–dependent one that promotes leukocyte migration, and a G-protein/Ca2+ one that is required for prostaglandin E2 (PGE2) production and bronchiolar smooth muscle relaxation. We hypothesized that the proinflammatory responses to PAR2 activation are mediated by β-arrestins, whereas the protective effects are not. Using a mouse ovalbumin model for PAR2-modulated airway inflammation, we observed decreased leukocyte recruitment, cytokine production, and mucin production in β-arrestin-2−/− mice. In contrast, PAR2-mediated PGE2 production, smooth muscle relaxation, and decreased baseline airway resistance (measures of putative PAR2 “protective” effects) were independent of β-arrestin-2. Flow cytometry and cytospins reveal that lung eosinophil and CD4 T-cell infiltration, and production of IL-4, IL-6, IL-13, and TNFα, were enhanced in wild-type but not β-arrestin-2−/− mice. Using the forced oscillation technique to measure airway resistance reveals that PAR2 activation protects against airway hyperresponsiveness by an unknown mechanism, possibly involving smooth muscle relaxation. Our data suggest that the PAR2-enhanced inflammatory process is β-arrestin-2 dependent, whereas the protective anticonstrictor effect of bronchial epithelial PAR2 may be β-arrestin independent.

Breath timing, volume and drive to breathe in conscious rats: comparative aspects.

In conscious animals, respiratory frequency (f) and tidal volume (VT) vary breath to breath. Examining the average value of variables associated with specific bins of another variable, such as breath f, provides a unique tool to examine respiratory behaviour. In conscious Sprague-Dawley rats respiratory breath timing, tidal volume (VT) and drive (VT/TI) were characterized using a plethysmograph. In the majority of rats at low breath f, expiratory time (TE) exceeded inspiratory time (TI) and these times became equal as f exceeded 150 breaths/min; there was no evidence for TI greater than TE at higher f, as observed in cats and dogs. When VT is normalized per kg, rat breath VT and VT/TI, binned by breath f, are continuous with those for the cat and non-panting dog at the lowest breath f. Relative to breath f, breath VT and VT/TI in rats are greater than in normothermic panting dogs (20 degrees C), but only slightly greater than those variables in panting dogs in the heat (30 degrees C). Lower values of breath VT/TI, binned by breath f or V, in cats and dogs are compensated for by a greater TI relative to the duration of a given breath. This comparative analysis suggests continuities of respiratory pattern generation among species.

β-Adrenoceptor inverse agonists in asthma

Beta(2)-adrenoceptor (beta(2)-AR) agonists are very effective bronchodilators and play a major role in every stage of asthma management. However, their chronic, regular use is associated with detrimental effects including an increase in asthma-related deaths. Conversely, recent data suggest that certain beta-blockers, specifically beta-adrenoceptor (beta-AR) inverse agonists, may be useful in the chronic treatment of asthma. Here we review the data for this observation and the signaling pathways that may be involved. The data suggest that beta(2)-AR signaling is required to produce maximal airway inflammation and hyperresponsiveness, and the signaling pathway responsible for these effects is likely the non-canonical beta-arrestin-2 pathway. Therefore, beta-AR inverse agonists may produce their beneficial chronic effects by inhibiting constitutive or ligand-induced activation of this pathway. Both lung parenchymal and hematopoietic cells appear to be involved in mediating the beneficial effects of beta-AR inverse agonists.