Pharmacogenetic characterization of indacaterol, a novel beta 2-adrenoceptor agonist.

BACKGROUND AND PURPOSE: Indacaterol is a novel beta(2)-adrenoceptor agonist in development for the treatment of chronic obstructive pulmonary disease. The aim of this study was to investigate the comparative pharmacology of indacaterol in recombinant cells expressing the common polymorphic variants of the human beta(2)-adrenoceptor and in human primary airway smooth muscle (ASM) cells. EXPERIMENTAL APPROACH: Chinese hamster ovarian-K1 cell lines expressing high and low levels of the common human beta(2)-adrenoceptor variants were generated [Gly16-Glu27-Val34-Thr164(GEVT), RQVT, GQVT] and also the rare GQVI variant. Human primary ASM cells were isolated from explants of trachealis muscle. Adenosine-3',5'-cyclic-monophosphate production was used as an outcome measure. KEY RESULTS: In both the low- and high-expression recombinant GEVT 'wild type' cell lines indacaterol is a high-efficacy agonist. Salmeterol and formoterol were identified as low- and high-efficacy agonists, respectively, and showed similar potencies to indacaterol irrespective of the beta(2)-adrenoceptor genotype. The I164 variant cell line was associated with a reduced capacity to generate adenosine-3',5'-cyclic-monophosphate in response to beta(2)-adrenoceptor agonist. In the human primary ASM cells indacaterol gave a maximal response intermediate between that of salmeterol and formoterol. CONCLUSIONS AND IMPLICATIONS: These data demonstrate that indacaterol is a high-efficacy agonist in recombinant cell systems but acts with lower efficacy in human primary ASM cells. No marked genotype-dependent effects were observed for common variants; however, changes in I164 receptor activity were identified, which were dependent on the level of expression of beta(2)-adrenoceptors.

Mechanisms of cytokine effects on G protein-coupled receptor-mediated signaling in airway smooth muscle.

Numerous in vitro and in vivo studies have implicated the cytokines interleukin-1 beta (IL-1 beta) and tumor necrosis factor-alpha (TNF-alpha) as mediators of airway inflammation and therefore potentially important substances in the pathogenesis of asthma. In this study, we examined the mechanisms by which IL-1 beta and TNF-alpha affect inhibition of cell growth, G protein-coupled receptor (GPCR) desensitization, and the recently reported adenylyl cyclase sensitization in human airway smooth muscle (HASM) cultures. Our findings demonstrate that adenylyl cyclase sensitization is independent of cytokine-mediated cyclooxygenase type 2 (COX-2) and prostaglandin E(2) (PGE(2)) induction, whereas COX-2 induction appears to be required for both growth inhibition and GPCR desensitization. However, GPCR desensitization was highly dependent on the presence of EGF during chronic treatment with cytokines, which could be explained by a synergistic effect of EGF on cytokine-mediated COX-2 and PGE(2) induction. Interestingly, various agents (including inhibitors of p42/p44 and p38 mitogen-activated protein kinase signaling) were significantly more effective in inhibiting cytokine-mediated PGE(2) induction, GPCR desensitization, and cell growth inhibition than in inhibiting COX-2 induction. These data demonstrate disparity in the requirement and sufficiency of COX-2 induction in promoting different functional effects of IL-1 beta and TNF-alpha in HASM.

Interleukin-1beta and rhinovirus sensitize adenylyl cyclase in human airway smooth-muscle cells.

Rhinovirus (RV) is a major cause of wheezing in asthmatics and has been reported to cause beta2 adrenergic receptor hyporesponsiveness in human airway smooth muscle (HASM) via cellular secretion of interleukin (IL)-1beta. We studied the effects of IL-1beta and RV on cyclic adenosine monophosphate (cAMP) production in HASM cells. Chronic incubation with IL-1beta or RV caused a significant increase (approximately 3- and approximately 2-fold, respectively) in forskolin (FSK)-stimulated cAMP production, suggesting a sensitization of adenylyl cyclase (AC). The observed augmentation of FSK-stimulated cAMP formation by IL-1beta was completely abrogated by pretreatment with an IL-1 receptor antagonist or cycloheximide, demonstrating that the effect is mediated via the IL-1 receptor 1 (IL-1R1) and that de novo protein synthesis is required. In contrast, RV-induced AC sensitization was not mediated via the IL-1R1 but was observed to be protein kinase C-dependent. We suggest that the sensitization of AC observed after exposure to IL-1beta or RV infection is a cellular defense mechanism to promote pathways that induce relaxation in the inflamed airway.

Inflammatory and contractile agents sensitize specific adenylyl cyclase isoforms in human airway smooth muscle.

Beta-agonists, through activation of the beta(2)-adrenergic receptor (beta(2)AR)-G(s)-adenylyl cyclase (AC) pathway, promote bronchodilation via functional antagonism of airway smooth muscle (ASM) spasmogens associated with the asthmatic state. Although previous studies have demonstrated that beta(2)AR signaling in ASM is subject to homologous (beta-agonist-induced) beta(2)AR desensitization, the potential for inflammatory and contractile agents to impact beta(2)AR signaling in ASM through heterologous mechanisms has not been defined. Here we report that chronic exposure of human ASM (HASM) to carbachol, serotonin, the thromboxane analogue U46619, or histamine induced little change or a small increase in isoproterenol-stimulated cyclic adenosine monophosphate (cAMP) formation, but significantly increased cAMP formation elicited by stimulation with forskolin. This latter increase in intrinsic AC activity was largely reversed by pertussis toxin pretreatment, and was unaffected by protein kinase C inhibition. Analysis of both AC function and isoform expression supports a dominant role of AC VI in HASM, and points to important differences in ASM AC isoform expression among species. Additional studies identify AC as the limiting component in beta(2)AR-G(s)-AC signaling in HASM, and thus a potentially important target of therapeutic strategies designed to influence airway contractile state.

Modulation of human airway smooth muscle proliferation by type 3 phosphodiesterase inhibition.

Elevation in cell cAMP content can inhibit mitogenic signaling in cultured human airway smooth muscle (HASM) cells. We studied the effects of the type 3-selective phosphodiesterase inhibitor siguazodan, the type 4-selective phosphodiesterase inhibitor rolipram, and the nonselective inhibitor 3-isobutyl-1-methylxanthine (IBMX) on proliferation of cultured HASM cells. At concentrations selective for the type 3 phosphodiesterase isoform, siguazodan inhibited both [3H]thymidine incorporation (IC50 2 microM) and the increase in cell number (10 microM; 64% reduction) induced by platelet-derived growth factor-BB (20 ng/ml). These effects were mimicked by IBMX. At concentrations selective for type 4 phosphodiesterase inhibition, rolipram was without effect. A 20-min exposure to siguazodan and rolipram did not increase whole cell cAMP levels. However, in HASM cells transfected with a cAMP-responsive luciferase reporter (p6CRE/Luc), increases in cAMP-driven luciferase expression were seen with siguazodan (3.9-fold) and IBMX (16.5-fold). These data suggest that inhibition of the type 3 phosphodiesterase isoform present in airway smooth muscle results in inhibition of mitogenic signaling, possibly through an increase in cAMP-driven gene expression.

Regulation of proliferation of human colonic subepithelial myofibroblasts by mediators important in intestinal inflammation.

An increase in myofibroblast number may be necessary for wound healing but may also lead to postinflammatory scarring. We have, therefore, studied the role of mediators important in inflammatory bowel disease in regulating proliferation of human colonic myofibroblasts. Using primary cultures of these cells, we have shown increases in [3H]thymidine incorporation in response to platelet-derived growth factor (EC50 = 14 ng/ml), basic fibroblast growth factor (EC50 = 2.2 ng/ml), and epidermal growth factor (EC50 = 1.1 ng/ml). Coulter counting of cell suspensions demonstrated increases in cell number with these growth factors along with insulin-like growth factor-I and -II. In addition the proinflammatory cytokines IL-1beta and TNF-alpha produced increases in [3H]thymidine incorporation. IL-1beta and platelet-derived growth factor together produced an increase in [3H]thymidine greater than either agonist alone; this effect was not, however, seen when we examined changes in cell numbers. Finally, we demonstrate a mechanism whereby these responses may be downregulated: vasoactive intestinal peptide (1 microM) elevates cyclic AwMP in these cells 4. 2-fold over control and produces a dose-related inhibition of platelet-derived growth factor-driven proliferation with a maximum inhibition of 33% at 1 microM.