Deepak A. Deshpande

Bitter taste receptors on airway smooth muscle bronchodilate by localized calcium signaling and reverse obstruction

Bitter taste receptors (TAS2Rs) on the tongue probably evolved to evoke signals for avoiding ingestion of plant toxins. We found expression of TAS2Rs on human airway smooth muscle (ASM) and considered these to be avoidance receptors for inhalants that, when activated, lead to ASM contraction and bronchospasm. TAS2R agonists such as saccharin, chloroquine and denatonium evoked increased intracellular calcium ([Ca2+]i) in ASM in a Gβγ–, phospholipase Cβ (PLCβ)- and inositol trisphosphate (IP3) receptor–dependent manner, which would be expected to evoke contraction. Paradoxically, bitter tastants caused relaxation of isolated ASM and dilation of airways that was threefold greater than that elicited by β-adrenergic receptor agonists. The relaxation induced by TAS2Rs is associated with a localized [Ca2+]i response at the cell membrane, which opens large-conductance Ca2+-activated K+ (BKCa) channels, leading to ASM membrane hyperpolarization. Inhaled bitter tastants decreased airway obstruction in a mouse model of asthma. Given the need for efficacious bronchodilators for treating obstructive lung diseases, this pathway can be exploited for therapy with the thousands of known synthetic and naturally occurring bitter tastants.

CD38/cyclic ADP-ribose-mediated Ca2+ signaling contributes to airway smooth muscle hyper-responsiveness

We previously demonstrated that cyclic ADP‐ribose (cADPR) elicits Ca2+ release in airway smooth muscle (ASM) cells through ryanodine receptor channels. CD38 is a cell surface protein that catalyzes the synthesis and degradation of cADPR. In inflammatory diseases such as asthma, augmented Ca2+ responses and Ca2+ sensitivity contribute to increased ASM contractility in response to agonists. In this study, we investigated the regulation of CD38 expression and the role of cADPR‐mediated Ca2+ release in airway inflammation. Human ASM cells in culture between the second and fifth passages were exposed to tumor necrosis factor α (TNF‐α), interleukin 1β, or interferon γ, or bovine serum albumin (controls). CD38 expression was measured by reverse transcriptase‐polymerase chain reaction (RT‐PCR), real‐time PCR, and Western blot analysis, and ADP‐ribosyl cyclase activity was assayed with nicotinamide guanine dinucleotide as the substrate. Ca2+ responses to acetylcholine, bradykinin, and thrombin were measured in fura‐2AM‐loaded cells by fluorescence microscopy. Cytokines caused significant augmentation of CD38 expression, ADP‐ribosyl cyclase activity, and Ca2+ responses to the agonists, compared with the control. TNF‐α effects were greater than those of the other two cytokines. The cADPR antagonist 8‐bromo‐cADPR attenuated the Ca2+ responses to the agonists in control and cytokine‐treated cells, with the magnitude of inhibition correlating with the level of CD38. This study provides the first demonstration of a role for CD38‐cADPR signaling in a model of inflammatory airway disease.

IL-13 enhances agonist-evoked calcium signals and contractile responses in airway smooth muscle.

Growing evidence suggests that interleukin (IL)‐13, a Th2‐type cytokine, plays a critical role in the development of bronchial hyper‐responsiveness (BHR), an essential feature of asthma, although the underlying mechanisms remain unknown. In the present study, we investigated whether IL‐13 directly affects airway smooth muscle (ASM) function. In murine tracheal rings, IL‐13 (100 ng ml−1, 24 h) significantly increased both the carbachol‐ and KCl‐induced maximal force generation without affecting ASM sensitivity. In cultured human ASM cells, IL‐13 (50 ng ml−1, 24 h) also augmented cytosolic calcium levels to bradykinin, histamine and carbachol by 60, 35 and 26%, respectively. The present study demonstrates that IL‐13 may promote BHR by directly modulating ASM contractility, an effect that may be due to enhanced G protein‐coupled receptor (GPCR)‐associated calcium signaling.

Allosteric ligands for the pharmacologically dark receptors GPR68 and GPR65

At least 120 non-olfactory G-protein-coupled receptors in the human genome are ‘orphans’ for which endogenous ligands are unknown, and many have no selective ligands, hindering the determination of their biological functions and clinical relevance. Among these is GPR68, a proton receptor that lacks small molecule modulators for probing its biology. Using yeast-based screens against GPR68, here we identify the benzodiazepine drug lorazepam as a non-selective GPR68 positive allosteric modulator. More than 3,000 GPR68 homology models were refined to recognize lorazepam in a putative allosteric site. Docking 3.1 million molecules predicted new GPR68 modulators, many of which were confirmed in functional assays. One potent GPR68 modulator, ogerin, suppressed recall in fear conditioning in wild-type but not in GPR68-knockout mice. The same approach led to the discovery of allosteric agonists and negative allosteric modulators for GPR65. Combining physical and structure-based screening may be broadly useful for ligand discovery for understudied and orphan GPCRs.

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)

The GPCR OGR1 (GPR68) mediates diverse signalling and contraction of airway smooth muscle in response to small reductions in extracellular pH

Previous studies have linked a reduction in pH in airway, caused by either environmental factors, microaspiration of gastric acid or inflammation, with airway smooth muscle (ASM) contraction and increased airway resistance. Neural mechanisms have been shown to mediate airway contraction in response to reductions in airway pH to < 6.5; whether reduced extracellular pH (pHo) has direct effects on ASM is unknown.

Transcriptional regulation of CD38 expression by tumor necrosis factor-α in human airway smooth muscle cells: role of NF-κB and sensitivity to glucocorticoids

The transmembrane glycoprotein CD38 catalyzes the synthesis of the calcium mobilizing molecule cyclic ADP‐ribose from NAD. In human airway smooth muscle (HASM) cells, the expression and function of CD38 are augmented by the inflammatory cytokine tumor necrosis factor‐alpha (TNF‐α), leading to increased intracellular calcium response to agonists. A glucocorticoid response element in the CD38 gene has been computationally described, providing evidence for transcriptional regulation of its expression. In the present study, we investigated the effects of dexamethasone, a glucocorticoid, on CD38 expression and ADP‐ribosyl cyclase activity in HASM cells stimulated with TNF‐α. In HASM cells, TNF‐α augmented CD38 expression and ADP‐ribosyl cyclase activity, which were attenuated by dexamethasone. TNF‐α increased NF‐κB expression and its activation, and dexamethasone partially reversed these effects. TNF‐α increased the expression of IκBα, and dexamethasone increased it further. An inhibitor of NF‐κB activation or transfection of cells with IκB mutants decreased TNF‐α‐induced CD38 expression. The results indicate that TNF‐α‐induced CD38 expression involves NF‐κB expression and its activation and dexamethasone inhibits CD38 expression through NF‐κB‐dependent and ‐independent mechanisms.— Kang, B.‐N., Tirumurugaan, K. G., Deshpande, D. A., Amrani, Y., Panettieri, R. A., Walseth, T. F., Kannan, M. S. Transcriptional regulation of CD38 expression by tumor necrosis factor‐α in human airway smooth muscle cells: role of NF‐κB and sensitivity to glucocorticoids. FASEB J. 20, E170–E179 (2006)

Anti-mitogenic effects of β-agonists and PGE2 on airway smooth muscle are PKA dependent

Inhaled β‐agonists are effective airway smooth muscle (ASM)‐relaxing agents that help reverse bronchoconstriction in asthma, but their ability to affect the aberrant ASM growth that also occurs with asthma is poorly understood. β‐Agonists exhibit PKA‐dependent antimitogenic effects in several cell types. However, recent studies suggest that Epac, and not PKA, mediates the antimitogenic effect of cAMP in both ASM and fibroblasts. This study aims to clarify the roleof PKA in mediating the effect of GS‐coupled receptors on human ASM growth. Pretreatmentof ASM cultures with β‐agonists albuterol, isoproterenol, or salmeterol (100 nM to 10 µM) caused a significant (‐25–30%) inhibition of EGF‐stimulated ASM thymi‐dine incorporation and cell proliferation, whereas a much greater inhibition was observed from pretreatment with PGE2 (75–80%). However, all agents were ineffective in cells expressing GFP chimeras of either PKI (a PKA inhibitor) or a mutant PKA regulatory subunit relative to the control cells expressing GFP. The antimitogenic efficacy of PGE2 in inhibiting control cultures was associated with greater ability to stimulate sustained PKA activation and greater inhibition of late‐phase promitogenic p42/p44 and PI3K activities. These findings suggest that therapeutic approaches enabling superior PKA activation in ASM will be most efficacious in deterring ASM growth.—Yan, H., Deshpande, D. A., Misior, A. M., Miles, M. C., Saxena, H., Riemer, E. C., Pascual, R. M., Panettieri, R. A., Penn. R. B. Anti‐mitogenic effects of β‐agonists and PGE2 on airway smooth muscle are PKA dependent. FASEB J. 25, 389–397 (2011). www.fasebj.org

Agonist-Promoted Homologous Desensitization of Human Airway Smooth Muscle Bitter Taste Receptors

Bitter taste receptors (TAS2Rs) were shown to be expressed in human airway smooth muscle (ASM). They couple to specialized [Ca(2+)](i) release, leading to membrane hyperpolarization, the relaxation of ASM, and marked bronchodilation. TAS2Rs are G-protein-coupled receptors, known to undergo rapid agonist-promoted desensitization that can limit therapeutic efficacy. Because TAS2Rs represent a new drug target for treating obstructive lung disease, we investigated their capacity for rapid desensitization, and assessed their potential mechanisms. The pretreatment of human ASM cells with the prototypic TAS2R agonist quinine resulted in a 31% ± 5.1% desensitization of the [Ca(2+)](i) response from a subsequent exposure to quinine. No significant change in the endothelin-stimulated [Ca(2+)](i) response was attributed to the short-term use of quinine, indicating a homologous form of desensitization. The TAS2R agonist saccharin also evoked desensitization, and cross-compound desensitization with quinine was evident. Desensitization of the [Ca(2+)](i) response was attenuated by a dynamin inhibitor, suggesting that receptor internalization (a G-protein coupled receptor kinase [GRK]-mediated, β-arrestin-mediated process) plays an integral role in the desensitization of TAS2R. Desensitization was insensitive to antagonists of the second messenger kinases protein kinase A and protein kinase C. Using intact airways, short-term, agonist-promoted TAS2R desensitization of the relaxation response was also observed. Thus these receptors, which represent a potential novel target for direct bronchodilators, undergo a modest degree of agonist-promoted desensitization that may affect clinical efficacy. Collectively, the results of these mechanistic studies, along with the multiple serines and threonines in intracellular loop 3 and the cytoplasmic tail of TAS2Rs, suggest a GRK-mediated mode of desensitization.

β-Agonist-mediated Relaxation of Airway Smooth Muscle Is Protein Kinase A-dependent

Background: Mechanisms by which β-2-adrenoreceptor agonists effect bronchorelaxation remain unestablished. Results: Direct inhibition of PKA via molecular approaches reversed β-agonist-mediated antagonism of procontractile signaling and relaxation of contracted airway smooth muscle (ASM) despite augmenting intracellular cAMP. Conclusion: PKA is the primary mechanism by which β-agonists relax ASM. Significance: PKA-dependent signaling and functions should guide the development of bronchodilator drugs. Inhaled β-agonists are effective at reversing bronchoconstriction in asthma, but the mechanism by which they exert this effect is unclear and controversial. PKA is the historically accepted effector, although this assumption is made on the basis of associative and not direct evidence. Recent studies have asserted that exchange protein activated by cAMP (Epac), not PKA, mediates the relaxation of airway smooth muscle (ASM) observed with β-agonist treatment. This study aims to clarify the role of PKA in the prorelaxant effects of β-agonists on ASM. Inhibition of PKA activity via expression of the PKI and RevAB peptides results in increased β-agonist-mediated cAMP release, abolishes the inhibitory effect of isoproterenol on histamine-induced intracellular calcium flux, and significantly attenuates histamine-stimulated MLC-20 phosphorylation. Analyses of ASM cell and tissue contraction demonstrate that PKA inhibition eliminates most, if not all, β-agonist-mediated relaxation of contracted smooth muscle. Conversely, Epac knockdown had no effect on the regulation of contraction or procontractile signaling by isoproterenol. These findings suggest that PKA, not Epac, is the predominant and physiologically relevant effector through which β-agonists exert their relaxant effects.