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Inhibition of ABCC1 Decreases cAMP Egress and Promotes Human Airway Smooth Muscle Cell Relaxation.

ABSTRACT: In most living cells, the second-messenger roles for adenosine 3',5'-cyclic monophosphate (cAMP) are short-lived, confined to the intracellular space, and tightly controlled by the binary switch-like actions of Gα_s (stimulatory G protein)-activated adenylyl cyclase (cAMP production) and cAMP-specific PDE (cAMP breakdown). Here, by using human airway smooth muscle (HASM) cells in culture as a model, we report that activation of the cell-surface β₂AR (β₂-adrenoceptor), a G_s-coupled GPCR (G protein-coupled receptor), evokes cAMP egress to the extracellular space. Increased extracellular cAMP levels ([cAMP]_e) are long-lived in culture and are induced by receptor-dependent and receptor-independent mechanisms in such a way as to define a universal response class of increased intracellular cAMP levels ([cAMP]_i). We find that HASM cells express multiple ATP-binding cassette (ABC) membrane transporters, with ABCC1 (ABC subfamily member C 1) being the most highly enriched transcript mapped to MRPs (multidrug resistance-associated proteins). We show that pharmacological inhibition or downregulation of ABCC1 with siRNA markedly reduces β₂AR-evoked cAMP release from HASM cells. Furthermore, inhibition of ABCC1 activity or expression decreases basal tone and increases β-agonist-induced HASM cellular relaxation. These findings identify a previously unrecognized role for ABCC1 in the homeostatic regulation of [cAMP]_i in HASM that may be conserved traits of the G_s-GPCRs (G_s-coupled family of GPCRs). Hence, the general features of this activation mechanism may uncover new disease-modifying targets in the treatment of airflow obstruction in asthma. Surprisingly, we find that serum cAMP levels are elevated in a small cohort of patients with asthma as compared with control subjects, which warrants further investigation.

SUBMITTER: Cao G

PROVIDER: S-EPMC8803359 | biostudies-literature | 2022 Jan

REPOSITORIES: biostudies-literature

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Inhibition of ABCC1 Decreases cAMP Egress and Promotes Human Airway Smooth Muscle Cell Relaxation.

Cao Gaoyuan G Lam Hong H Jude Joseph A JA Karmacharya Nikhil N Kan Mengyuan M Jester William W Koziol-White Cynthia C Himes Blanca E BE Chupp Geoffrey L GL An Steven S SS Panettieri Reynold A RA

American journal of respiratory cell and molecular biology 20220101 1

In most living cells, the second-messenger roles for adenosine 3',5'-cyclic monophosphate (cAMP) are short-lived, confined to the intracellular space, and tightly controlled by the binary switch-like actions of Gαs (stimulatory G protein)-activated adenylyl cyclase (cAMP production) and cAMP-specific PDE (cAMP breakdown). Here, by using human airway smooth muscle (HASM) cells in culture as a model, we report that activation of the cell-surface β2AR (β2-adrenocept ...[more]

PMID: 34648729

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Project description:BackgroundDopamine signaling is mediated by Gs protein-coupled "D1-like" receptors (D1 and D5) and Gi-coupled "D2-like" receptors (D2-4). In asthmatic patients, inhaled dopamine induces bronchodilation. Although the Gi-coupled dopamine D2 receptor is expressed and sensitizes adenylyl cyclase activity in airway smooth muscle (ASM) cells, the Gs-coupled dopamine D1-like receptor subtypes have never been identified on these cells. Activation of Gs-coupled receptors stimulates cyclic AMP (cAMP) production through the stimulation of adenylyl cyclase, which promotes ASM relaxation. We questioned whether the dopamine D1-like receptor is expressed on ASM, and modulates its function through Gs-coupling.MethodsThe mRNA and protein expression of dopamine D1-like receptor subtypes in both native human and guinea pig ASM tissue and cultured human ASM (HASM) cells was measured. To characterize the stimulation of cAMP through the dopamine D1 receptor, HASM cells were treated with dopamine or the dopamine D1-like receptor agonists (A68930 or SKF38393) before cAMP measurements. To evaluate whether the activation of dopamine D1 receptor induces ASM relaxation, guinea pig tracheal rings suspended under isometric tension in organ baths were treated with cumulatively increasing concentrations of dopamine or A68930, following an acetylcholine-induced contraction with or without the cAMP-dependent protein kinase (PKA) inhibitor Rp-cAMPS, the large-conductance calcium-activated potassium (BKCa) channel blocker iberiotoxin, or the exchange proteins directly activated by cAMP (Epac) antagonist NSC45576.ResultsMessenger RNA encoding the dopamine D1 and D5 receptors were detected in native human ASM tissue and cultured HASM cells. Immunoblots confirmed the protein expression of the dopamine D1 receptor in both native human and guinea pig ASM tissue and cultured HASM cells. The dopamine D1 receptor was also immunohistochemically localized to both human and guinea pig ASM. The dopamine D1-like receptor agonists stimulated cAMP production in HASM cells, which was reversed by the selective dopamine D1-like receptor antagonists SCH23390 or SCH39166. A68930 relaxed acetylcholine-contracted guinea pig tracheal rings, which was attenuated by Rp-cAMPS but not by iberiotoxin or NSC45576.ConclusionsThese results demonstrate that the dopamine D1 receptors are expressed on ASM and regulate smooth muscle force via cAMP activation of PKA, and offer a novel target for therapeutic relaxation of ASM.

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Inhibition of ABCC1 Decreases cAMP Egress and Promotes Human Airway Smooth Muscle Cell Relaxation.

Publications

Inhibition of ABCC1 Decreases cAMP Egress and Promotes Human Airway Smooth Muscle Cell Relaxation.

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