Intracellular interactions of umeclidinium and vilanterol in human airway smooth muscle

Figures & data

Figure 1 Proposed mechanisms of action of the long-acting MR agonist UMEC and the long-acting β₂R agonist VI.

Abbreviations: AC, adenylyl cyclase; ACh, acetylcholine; ADP, adenosine diphosphate; ATP, adenosine triphosphate; β₂R, β₂-adrenoreceptor; cAMP, cyclic adenosine monophosphate; CRE, cAMP-responsive element; CREB, CRE binding protein; G_i, inhibitory G proteins; G_s, stimulatory G protein; IP₃, inositol 1,4,5-trisphosphate; M₂, muscarinic receptor 2; M₃, muscarinic receptor 3; MR, muscarinic receptor; PLC, phospholipase C; RGS2, regulator of G-protein signaling 2; UMEC, umeclidinium; VI, vilanterol.

Figure 2 cAMP production following β₂R agonism.

Notes: (A) Time course of cAMP production following treatment with VI (10⁻⁶ M) and salmeterol (10⁻⁶ M) in human ASMCs (n=3). (B) Concentration–response curves for cAMP production in response to VI and salmeterol at 60 minutes poststimulation (n=7).
Abbreviations: ASMCs, airway smooth-muscle cells; cAMP, cyclic adenosine monophosphate; VI, vilanterol.

Figure 3 VI signaling through β₂R in human ASMCs.

Notes: (A) Concentration–response curves for the production of cAMP in response to VI in the presence of β₂R antagonists propranolol and ICI 118.551 (n=3–4). (B) Antagonist concentration–response curves for the production of cAMP in response to VI (10⁻⁸ M) in the presence of propranolol and ICI 118.551 (n=5–7).
Abbreviations: ASMCs, airway smooth-muscle cells; β₂R, β₂-adrenoreceptor; cAMP, cyclic adenosine monophosphate; VI, vilanterol; EC₅₀, half-maximal effective concentration.

Figure 4 UMEC antagonism of muscarinic receptors reverses MCh-induced attenuation of VI-mediated cAMP production.

Notes: (A) cAMP production in human ASMCs with VI for 1 hour following 30-minute pretreatment with MCh (5×10⁻⁶ M; n=8; *P<0.05, **P<0.01, ***P<0.001 VI versus VI+MCh). (B) VI concentration–response curves for cAMP production at 1 hour in human ASMCs (n=8; ^#P<0.05, ^##P<0.01 VI+MCh versus VI+MCh+UMEC; ^§P<0.05 VI+MCh versus VI+UMEC).
Abbreviations: ASMCs, airway smooth-muscle cells; cAMP, cyclic adenosine monophosphate; MCh, methacholine; UMEC, umeclidinium; VI, vilanterol.

Figure 5 MR antagonism with UMEC suppresses MCh-induced Ca²⁺ release.

Notes: (A) Concentration–response curve for the release of Ca²⁺ in response to MCh. (B) Concentration–response curve for the release of Ca²⁺ in human ASMCs pretreated with UMEC for 2 minutes, then stimulated with MCh (5×10⁻⁶ M; examined using one-way ANOVA [Kruskal–Wallis test] followed by a Dunn’s multiple comparison test). (C) Concentration–response curve for the release of Ca²⁺ in human ASMCs in response to MCh after pretreatment with UMEC for 2 minutes. *P<0.05, **P<0.01 MCh versus UMEC (10⁻⁶ M).
Abbreviations: ANOVA, analysis of variance; ASMCs, airway smooth muscle cells; MCh, methacholine; MR, muscarinic receptor; UMEC, umeclidinium.

Figure 6 β R agonist VI augments UMEC-mediated attenuation of MCh-induced release in [Ca²⁺]_i.

Notes: (A) Concentration–response curves for Ca²⁺ release in ASMCs pretreated with UMEC alone or in combination with VI for 2 minutes, followed by stimulation with MCh (n=6–8). (B) Ca²⁺ release following treatment with MCh (5×10⁻⁶ M), with VI alone for 2 minutes, UMEC alone, and a combination of UMEC with a selected range of VI concentrations. **P<0.01 UMEC 10⁻¹² M versus UMEC + VI 10⁻⁶ M (n=6–8).
Abbreviations: ASMCs, airway smooth-muscle cells; β₂R, β₂-adrenoreceptor; MCh, methacholine; UMEC, umeclidinium; VI, vilanterol.

Figure 7 β₂R-agonist-induced RGS2 expression is enhanced by MR antagonism.

Notes: Time course for the expression of RGS2 messenger RNA in response to VI (10⁻⁸ M), UMEC (10⁻⁸ M), or UMEC/VI (both at 10⁻⁹ M) (n=6–8). *P<0.05 VI versus VI + UMEC, paired t-test.
Abbreviations: β₂R, β₂ adrenoreceptor; RGS2, regulator of G-protein signaling; MR, muscarinic receptor; UMEC, umeclidinium; VI, vilanterol.