Abstract
Intracellular Ca2+ and cAMP typically cause opposing effects on airway smooth muscle contraction. Receptors that stimulate these pathways are therapeutic targets in asthma and chronic obstructive pulmonary disease. However, the interactions between different G protein-coupled receptors (GPCRs) that evoke cAMP and Ca2+ signals in human bronchial airway smooth muscle cells (hBASMCs) are poorly understood. We measured Ca2+ signals in cultures of fluo-4-loaded hBASMCs alongside measurements of intracellular cAMP using mass spectrometry or [3H]-adenine labeling. Interactions between the signaling pathways were examined using selective ligands of GPCRs, and inhibitors of Ca2+ and cAMP signaling pathways. Histamine stimulated Ca2+ release through inositol 1,4,5-trisphosphate (IP3) receptors in hBASMCs. β2-adrenoceptors, through cAMP and protein kinase A (PKA), substantially inhibited histamine-evoked Ca2+ signals. Responses to other Ca2+-mobilizing stimuli were unaffected by cAMP (carbachol and bradykinin) or minimally affected (lysophosphatidic acid). Prostaglandin E2 (PGE2), through EP2 and EP4 receptors, stimulated formation of cAMP and inhibited histamine-evoked Ca2+ signals. There was no consistent relationship between the inhibition of Ca2+ signals and the amounts of intracellular cAMP produced by different stimuli. We conclude that β-adrenoceptors, EP2 and EP4 receptors, through cAMP and PKA, selectively inhibit Ca2+ signals evoked by histamine in hBASMCs, suggesting that PKA inhibits an early step in H1 receptor signaling. Local delivery of cAMP within hyperactive signaling junctions mediates the inhibition.
Highlights
Bronchial asthma and chronic obstructive pulmonary disease (COPD) are associated with inflammation, hyper-responsiveness and airway obstruction leading to restricted airflow
Ca2+ signals are usually initiated by receptors that stimulate phospholipase Cβ (PLCβ) and Abbreviations: AC, adenylyl cyclase; 2-APB, 2-aminoethoxyphenylborane; ASM, airway smooth muscle; cAMP, 3',5'-cyclic AMP; COPD, chronic obstructive pulmonary disease; [Ca2+]i, intracellular free Ca2+ concentration; DMSO, dimethyl sulfoxide; EC50 (IC50), half-maximally effective concentration; Epac, exchange protein activated by cAMP; GPCR, G protein-coupled receptor; hBASMC, human bronchial airway smooth muscle cell; HEPES-buffered saline (HBS), Hepes-buffered saline; Hank's balanced salt solution (HBSS), Hank’s balanced salt solution; IBMX, 3-isobutyl-1-methylxanthine; IP3, inositol 1,4,5-trisphosphate; IP3 receptors (IP3R), IP3 receptor; LPA, 18:1 lysophosphatidic acid; pEC50, -logEC50; protein kinase A (PKA), cyclic AMP-dependent protein kinase; Prostaglandin E2 (PGE2), prostaglandin E2; PKI-myr, myristoylated PKA inhibitor; PLCβ, phospholipase C β; Pertussis toxin (PTX), pertussis toxin
Stimuli of several GPCRs reported to be expressed in ASM evoked increases in [Ca2+]i in hBASMCs (Fig. 1A)
Summary
Bronchial asthma and chronic obstructive pulmonary disease (COPD) are associated with inflammation, hyper-responsiveness and airway obstruction leading to restricted airflow. Ca2+ signals are usually initiated by receptors that stimulate phospholipase Cβ (PLCβ) and Abbreviations: AC, adenylyl cyclase; 2-APB, 2-aminoethoxyphenylborane; ASM, airway smooth muscle; cAMP, 3',5'-cyclic AMP; COPD, chronic obstructive pulmonary disease; [Ca2+]i, intracellular free Ca2+ concentration; DMSO, dimethyl sulfoxide; EC50 (IC50), half-maximally effective (inhibitory) concentration; Epac, exchange protein activated by cAMP; GPCR, G protein-coupled receptor; hBASMC, human bronchial airway smooth muscle cell; HBS, Hepes-buffered saline; HBSS, Hank’s balanced salt solution; IBMX, 3-isobutyl-1-methylxanthine; IP3, inositol 1,4,5-trisphosphate; IP3R, IP3 receptor; LPA, 18:1 lysophosphatidic acid; pEC50, -logEC50; PKA, cyclic AMP-dependent protein kinase; PGE2, prostaglandin E2; PKI-myr, myristoylated PKA inhibitor; PLCβ, phospholipase C β; PTX, pertussis toxin. A major limitation of cultured human ASM has been loss of the muscarinic receptors [20,21] that both contribute to the contractile responses in COPD and asthma, and provide important targets for therapy. These include prostaglandin E2 (PGE2), which can stimulate adenylyl cyclase (AC), primarily through EP2 and EP4 receptors [22]. There is a need to determine in human ASM the interplay between the different G protein-coupled receptors (GPCRs) that stimulate Ca2+ and cAMP signals
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