Paradoxical attenuation of β₂-AR function in airway smooth muscle by G_i-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 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. 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 β(2)-AR signaling when AC expression is increased. In contrast, acetylcholine-mediated contraction was preserved. G(αi) expression and ERK1/2 activation were markedly increased in AC5-TG (5- and 8-fold, respectively), and β-AR expression was decreased by ∼40%. Other G proteins, G protein-coupled receptor kinases, and β-arrestins were unaffected. β-agonist-mediated airway relaxation of AC5-TG was normalized to that of nontransgenic mice by pertussis toxin, implicating β(2)-AR coupling to the increased G(i) as a mechanism of depressed agonist-promoted relaxation in these mice. The decrease in β(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 β(2)-AR to AC in ASM by increasing AC fails to improve (and actually decreases) β-agonist efficacy due to counterregulatory events.

Contribution of the Extracellular cAMP-Adenosine Pathway to Dual Coupling of β2-Adrenoceptors to Gsand GiProteins in Mouse Skeletal Muscle

“…jpet.aspetjournals.org rotid artery (Ciccarelli et al, 2007). However, the precise mechanism underlying the switch of ␤ 2 -AR from G s to G i signaling remained obscure because it was primarily based on disruption of G i signaling by PTX or immunoprecipitation of GTP-G i protein complexes induced by ␤ 2 -AR agonists (Daaka et al, 1997;Ciccarelli et al, 2007;Liu et al, 2009;Wenzel et al, 2009;Wang et al, 2011). A plausible explanation for reduced coupling of ␤ 2 -AR to G s is receptor desensitization and reduced receptor efficacy in stimulating AC (Bü nemann et al, 1999).…”

Airway smooth muscle in airway reactivity and remodeling: what have we learned?

“…However, different agonists, or stereoisomers of a single agonist, can work through the same receptor to activate different downstream pathways to different extents, i.e., it is the ligand that determines the direction of signaling, rather than the GPCR, presumably by differential stabilization of the activation state of the receptor. Such biased agonism has been demonstrated in the context of asthma and ASM for ␤ 2 AR, which can activate G s to enhance cAMP (and thus be of benefit by enhancing bronchodilation for example) vs. their ability to work via the ␤-arrestin-mediated MAPK pathway, which may be detrimental in terms of contractility, promoting cell proliferation, and remodeling (60,326). Thus the potential to direct ␤ 2 AR signaling toward a beneficial pathway while avoiding activation of detrimental mechanisms is a highly appealing goal for drug development.…”

Novel cAMP signalling paradigms: therapeutic implications for airway disease

“…In support of this theory, in separate studies, we and Bogard et al, overexpressed AC6 in hASM cells and each found cAMP to be elevated versus empty vector controls (Billington et al, 1999;Bogard et al, 2011) and even versus hASM cells individually overexpressing the other key components of the pathway; b2-adrenoceptor and Gsa (Billington et al, 1999). Wang et al (2011) recently explored this in vivo, hypothesizing that transgenic mice overexpressing AC5 would exhibit augmented b2-adrenoceptor-mediated airway relaxation. Intriguingly, the opposite was found with isoprenalineinduced airway relaxation and cAMP production being significantly reduced.…”

“…This occurred in parallel with increased Gai expression and ERK1/2 activation, suggesting that increased AC5 tips the Gs/Gi balance of b2-adrenoceptor signalling firmly towards Gi. Wang et al (2011) suggest that b2-adrenoceptor signalling may operate within a set range and any alteration of signalling components leading to a deviation from this range would result in the induction of compensatory mechanisms to return to the 'homeostatic set point'. Interestingly, similar observations have been made in transgenic mice overexpressing the b2-adrenoceptor, where airway responsiveness is actually increased with up-regulation of PLC signalling (McGraw et al, 2003).…”