Abstract
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.
Highlights
Dopamine signaling is mediated by Gs protein-coupled “D1-like” receptors (D1 and D5) and Gicoupled “D2-like” receptors (D2-4)
A recent study suggested that the dopamine D1-like receptor antagonist SCH23390 inhibited the bronchodilator effect of dopamine in acetylcholine-contracted rat tracheal rings suspended in organ baths [13], suggesting that the dopamine D1-like receptor is expressed on airway smooth muscle and induces bronchodilation
Immunoblot analysis of dopamine D1-like receptors in human and guinea pig airway smooth muscle A single immunoreactive band of expected molecular mass of approximately 75 kDa for dopamine D1 receptor was identified in freshly dissected native human airway smooth muscle, cultured human ASM (HASM) cells from all three different sources [#1: cultures of immortalized HASM cells gifted from Dr William Gerthoffer, #2: primary cultured cells gifted from Dr Reynold A Panettieri, Jr., and #3: primary cultured cells obtained from Lonza (Walkersville, MD)], and in human whole kidney (Figure 2A)
Summary
Dopamine signaling is mediated by Gs protein-coupled “D1-like” receptors (D1 and D5) and Gicoupled “D2-like” receptors (D2-4). 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. Dopamine is a ubiquitous endogenous catecholamine neurotransmitter in the mammalian central nervous system, where it controls a variety of physiological responses including emotion, cognition, and endocrine functions [1,2,3] This catecholamine modulates cardiovascular function, hormone secretion, vascular tone, renal function, and gastrointestinal motility through the specific dopamine receptor subtypes expressed in peripheral organs and tissues [3,4,5,6,7]. Chronic activation of the dopamine D2 receptor has been shown to sensitize adenylyl cyclase and facilitate forskolin-induced airway smooth muscle relaxation [14], the functional expression of the dopamine D1-like receptor subtype on airway smooth muscle has not been described
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