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Paradoxical attenuation of ?2-AR function in airway smooth muscle by Gi-mediated counterregulation in transgenic mice overexpressing type 5 adenylyl cyclase.

ABSTRACT: The limiting component within the receptor-G protein-effector complex in airway smooth muscle (ASM) for ?(2)-adrenergic receptor (?(2)-AR)-mediated relaxation is unknown. In cardiomyocytes, adenylyl cyclase (AC) is considered the "bottleneck" for ?-AR signaling, and gene therapy trials are underway to increase inotropy by increasing cardiac AC expression. We hypothesized that increasing AC in ASM would increase relaxation from ?-agonists, thereby providing a strategy for asthma therapy. Transgenic (TG) mice were generated with approximately two- to threefold overexpression of type 5 AC (AC5) in ASM. cAMP and airway relaxation in response to direct activation of AC by forskolin were increased in AC5-TG. Counter to our hypothesis, isoproterenol-mediated airway relaxation was significantly attenuated (?50%) in AC5-TG, as was cAMP production, suggesting compensatory regulatory events limiting ?(2)-AR signaling when AC expression is increased. In contrast, acetylcholine-mediated contraction was preserved. G(?i) expression and ERK1/2 activation were markedly increased in AC5-TG (5- and 8-fold, respectively), and ?-AR expression was decreased by ?40%. Other G proteins, G protein-coupled receptor kinases, and ?-arrestins were unaffected. ?-agonist-mediated airway relaxation of AC5-TG was normalized to that of nontransgenic mice by pertussis toxin, implicating ?(2)-AR coupling to the increased G(i) as a mechanism of depressed agonist-promoted relaxation in these mice. The decrease in ?(2)-AR may account for additional relaxation impairment, given that there is no enhancement over nontransgenic after pertussis toxin, despite AC5 overexpression. ERK1/2 inhibition had no effect on the phenotype. Thus perturbing the ratio of ?(2)-AR to AC in ASM by increasing AC fails to improve (and actually decreases) ?-agonist efficacy due to counterregulatory events.

SUBMITTER: Wang WC

PROVIDER: S-EPMC3064290 | biostudies-literature | 2011 Mar

REPOSITORIES: biostudies-literature

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Paradoxical attenuation of β2-AR function in airway smooth muscle by Gi-mediated counterregulation in transgenic mice overexpressing type 5 adenylyl cyclase.

Wang Wayne C H WC Schillinger Rachel M RM Malone Molly M MM Liggett Stephen B SB

American journal of physiology. Lung cellular and molecular physiology 20101203 3

The limiting component within the receptor-G protein-effector complex in airway smooth muscle (ASM) for β(2)-adrenergic receptor (β(2)-AR)-mediated relaxation is unknown. In cardiomyocytes, adenylyl cyclase (AC) is considered the "bottleneck" for β-AR signaling, and gene therapy trials are underway to increase inotropy by increasing cardiac AC expression. We hypothesized that increasing AC in ASM would increase relaxation from β-agonists, thereby providing a strategy for asthma therapy. Transgen ...[more]

PMID: 21131397

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Project description:Background and purposeDespite the view that only ?2- as opposed to ?1-adrenoceptors (?ARs) couple to G(i), some data indicate that the ?1AR-evoked inotropic response is also influenced by the inhibition of Gi. Therefore, we wanted to determine if Gi exerts tonic receptor-independent inhibition upon basal adenylyl cyclase (AC) activity in cardiomyocytes.Experimental approachWe used the Gs-selective (R,R)- and the Gs- and G(i)-activating (R,S)-fenoterol to selectively activate ?2ARs (?1AR blockade present) in combination with Gi inactivation with pertussis toxin (PTX). We also determined the effect of PTX upon basal and forskolin-mediated responses. Contractility was measured ex vivo in left ventricular strips and cAMP accumulation was measured in isolated ventricular cardiomyocytes from adult Wistar rats.Key resultsPTX amplified both the (R,R)- and (R,S)-fenoterol-evoked maximal inotropic response and concentration-dependent increases in cAMP accumulation. The EC50 values of fenoterol matched published binding affinities. The PTX enhancement of the Gs-selective (R,R)-fenoterol-mediated responses suggests that Gi regulates AC activity independent of receptor coupling to Gi protein. Consistent with this hypothesis, forskolin-evoked cAMP accumulation was increased and inotropic responses to forskolin were potentiated by PTX treatment. In non-PTX-treated tissue, phosphodiesterase (PDE) 3 and 4 inhibition or removal of either constitutive muscarinic receptor activation of Gi with atropine or removal of constitutive adenosine receptor activation with CGS 15943 had no effect upon contractility. However, in PTX-treated tissue, PDE3 and 4 inhibition alone increased basal levels of cAMP and accordingly evoked a large inotropic response.Conclusions and implicationsTogether, these data indicate that Gi exerts intrinsic receptor-independent inhibitory activity upon AC. We propose that PTX treatment shifts the balance of intrinsic G(i) and Gs activity upon AC towards Gs, enhancing the effect of all cAMP-mediated inotropic agents.

Project description:Dopamine receptors are G protein-coupled receptors that are divided into two subgroups, "D(1)-like" receptors (D(1) and D(5)) that couple to the G(s) protein and "D(2)-like" receptors (D(2), D(3), and D(4)) that couple to G(i). Although inhaled dopamine has been reported to induce bronchodilation in patients with asthma, functional expression of dopamine receptor subtypes has never been described on airway smooth muscle (ASM) cells. Acute activation of G(i)-coupled receptors inhibits adenylyl cyclase activity and cAMP synthesis, which classically impairs ASM relaxation. In contrast, chronic activation of G(i)-coupled receptors produces a paradoxical enhancement of adenylyl cyclase activity referred to as heterologous sensitization. We questioned whether the dopamine D(2)-like receptor is expressed on ASM, whether it exhibits classical G(i)-coupling, and whether it modulates ASM function. We detected the mRNA encoding the dopamine D(2) receptor in total RNA isolated from native human ASM and from cultured human airway smooth muscle (HASM) cells. Immunoblots identified the dopamine D(2) receptor protein in both native human and guinea pig ASM and cultured HASM cells. The dopamine D(2) receptor protein was immunohistochemically localized to both human and guinea pig ASM. Acute activation of the dopamine D(2) receptor by quinpirole inhibited forskolin-stimulated adenylyl cyclase activity in HASM cells, which was blocked by the dopamine D(2) receptor antagonist L-741626. In contrast, the chronic pretreatment (1 h) with quinpirole potentiated forskolin-stimulated adenylyl cyclase activity, which was inhibited by L-741626, the phospholipase C inhibitor U73122, or the protein kinase C inhibitor GF109203X. Quinpirole also stimulated inositol phosphate synthesis, which was inhibited by L-741626 or U73122. Chronic pretreatment (1 h) of the guinea pig tracheal rings with quinpirole significantly potentiated forskolin-induced airway relaxation, which was inhibited by L-741626. These results demonstrate that functional dopamine D(2) receptors are expressed on ASM and could be a novel therapeutic target for the relaxation of ASM.

Project description:?-aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the mammalian central nervous system, and exerts its actions via both ionotropic (GABA(A)) and metabotropic (GABA(B)) receptors. Although the functional expression of GABA(B) receptors coupled to the G(i) protein was reported for airway smooth muscle, the role of GABA(B) receptors in airway responsiveness remains unclear. We investigated whether G(i)-coupled GABA(B) receptors cross-regulate phospholipase C (PLC), an enzyme classically regulated by G(q)-coupled receptors in human airway smooth muscle cells. Both the GABA(B)-selective agonist baclofen and the endogenous ligand GABA significantly increased the synthesis of inositol phosphate, whereas GABA(A) receptor agonists, muscimol, and 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol exerted no effect. The baclofen-induced synthesis of inositol phosphate and transient increases in [Ca(2+)](i) were blocked by CGP35348 and CGP55845 (selective GABA(B) antagonists), pertussis toxin (PTX, which inactivates the G(i) protein), gallein (a G(??) signaling inhibitor), U73122 (an inhibitor of PLC-?), and xestospongin C, an inositol 1,4,5-triphosphate receptor blocker. Baclofen also potentiated the bradykinin-induced synthesis of inositol phosphate and transient increases in [Ca(2+)](i), which were blocked by CGP35348 or PTX. Moreover, baclofen potentiated the substance P-induced contraction of airway smooth muscle in isolated guinea pig tracheal rings. In conclusion, the stimulation of GABA(B) receptors in human airway smooth muscle cells rapidly mobilizes intracellular Ca(2+) stores by the synthesis of inositol phosphate via the activation of PLC-?, which is stimulated by G(??) protein liberated from G(i) proteins coupled to GABA(B) receptors. Furthermore, crosstalk between GABA(B) receptors and G(q)-coupled receptors potentiates the synthesis of inositol phosphate, transient increases in [Ca(2+)](i), and smooth muscle contraction through G(i) proteins.

Dataset Information

Paradoxical attenuation of ?2-AR function in airway smooth muscle by Gi-mediated counterregulation in transgenic mice overexpressing type 5 adenylyl cyclase.

Publications

Paradoxical attenuation of β2-AR function in airway smooth muscle by Gi-mediated counterregulation in transgenic mice overexpressing type 5 adenylyl cyclase.

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