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British Journal of Pharmacology logoLink to British Journal of Pharmacology
. 2014 Nov 24;172(1):131–141. doi: 10.1111/bph.12905

The role of adenylyl cyclase isoform 6 in β-adrenoceptor signalling in murine airways

Mark A Birrell 1,2, Sara J Bonvini 1, Michael A Wortley 1, James Buckley 1, Liang Yew-Booth 1, Sarah A Maher 1, Nicole Dale 1, Eric D Dubuis 1, Maria G Belvisi 1,2
PMCID: PMC4280973  PMID: 25205328

Abstract

BACKGROUND AND PURPOSE

Adenylyl cyclase (AC) is a key signalling enzyme for many GPCRs and catalyses the conversion of ATP to cAMP which, in turn, is a crucial determinant of many biological responses. β-Adrenoceptor agonists are prescribed as bronchodilators for asthma and chronic obstructive pulmonary disease, and it is commonly assumed that they elicit their actions via AC-dependent production of cAMP. However, empirical evidence in support of this is lacking and the exact mechanism by which these drugs acts remains elusive. This is partly due to the existence of at least 10 different isoforms of AC and the absence of any truly selective pharmacological inhibitors. Here, we have used genetically modified mice and model systems to establish the role of AC isoforms in the airway responses to β-adrenoceptor agonists.

EXPERIMENTAL APPROACH

Receptors mediating responses to β-adrenoceptor agonists in airway smooth muscle (ASM) and sensory nerve were identified in isolated tissue systems. Expression of mRNA for the AC isoforms in ASM and neurones was determined by qPCR. Functional responses were assessed in AC isoform KO mice and wild-type controls.

KEY RESULTS

Airway and vagal tissue expressed mRNA for various isoforms of AC. AC6 was the most prominent isoform. Responses to β-adrenoceptor agonists in tissues from AC6 KO mice were virtually abolished.

CONCLUSIONS AND IMPLICATIONS

AC6 played a critical role in relaxation of ASM to β1-adrenoceptor agonists and in modulation of sensory nerves by β1-3-adrenoceptor agonists. These results further unravel the signalling pathway of this extensively prescribed class of medicine.

Tables of Links

TARGETS
Enzymesa
AC, adenylyl cyclase
GPCRsb
β1-Adrenoceptors
β2-Adrenoceptors
β3-Adrenoceptors
Ion channelsc
TRPV1
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LIGANDS
ACh, acetylcholine Isoprenaline
BRL 37344 ICI 118551
Capsaicin ONO-AE1-259
CCh, carbachol PGE2
CGP 20712
Denopamine
Fenoterol
Formoterol
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These Tables list key protein targets and ligands in this article which are hyperlinked to corresponding entries in http://www.guidetopharmacology.org, the common portal for data from the IUPHAR/BPS Guide to PHARMACOLOGY (Pawson et al., 2014) and are permanently archived in the Concise Guide to PHARMACOLOGY 2013/14 (a,b,cAlexander et al., 2013a,b,c).

Introduction

Adenylyl cyclase (AC) is believed to be a central component of signalling pathways downstream from many GPCRs. It catalyses the conversion of ATP to cAMP, an intracellular messenger which is involved in many biological processes including cell growth and differentiation, transcriptional regulation, apoptosis and various cellular functions (Patel et al., 2001). The ability of this enzyme to be involved in such a diverse range of effects is due to its tissue distribution, the spatiotemporal compartmentalization of intracellular cAMP gradients, numerous regulatory mechanisms and, crucially, the existence of multiple AC isoforms, each with varying enzymic and pharmacological properties. Ten AC isoforms have been identified of which nine are transmembrane proteins activated by Gαs, whereas AC10 is a soluble isoform that lacks the transmembrane domains and is insensitive to Gαs (see Sunahara et al., 1996; Hanoune and Defer, 2001; Onda et al., 2001; Patel et al., 2001; Pavan et al., 2009; Duan et al., 2010; Brand et al., 2013; Conley et al., 2013).

Activation of β-adrenoceptors (β2-adrenoceptors in humans, β1-adrenoceptors in mice; Henry and Goldie, 1990; Henry et al., 1990) on airway smooth muscle (ASM) increases cAMP (Kume et al., 1994; Kotlikoff and Kamm, 1996; Billington et al., 2013), and inhaled β2-adrenoceptor agonists are used therapeutically as bronchodilators in patients with respiratory diseases such as asthma and COPD (Goldie et al., 1986; Barnes, 1995; Johnson, 1998; Dennis et al., 2000). β2-adrenoceptor agonists also inhibit airway sensory nerve activation and cough in preclinical models (Freund-Michel et al., 2010). Despite β-adrenoceptor agonists being commonly prescribed for numerous respiratory diseases, the exact mechanism by which they act is uncertain (Giembycz and Newton, 2006).

It is generally accepted, based on traditional second messenger signalling paradigms, that AC-linked cAMP production is central to the effects of β-adrenoceptor agonists, although direct evidence in support of this is minimal. Indeed some studies have found that AC inhibitors, while inhibiting cAMP production, do not inhibit β-adrenoceptor agonist-induced relaxation of ASM (Turcato and Clapp, 1999; Koike et al., 2004). Furthermore, the AC isoform responsible for β-adrenoceptor agonist-induced increases in cAMP is yet to be determined and elucidating which isoform is responsible represents a vital step in improving our understanding of the mechanism(s) by which β-adrenoceptor agonists signal. To date, investigation into the role of AC isoforms has been hampered by the lack of available pharmacological tools. Although selective AC isoform inhibitors are becoming available, they currently lack the necessary potency and selectivity required (Iwatsubo et al., 2006; Brand et al., 2013; Conley et al., 2013). In the present studies, the use of murine transgenic systems previously reported has allowed us to circumvent these issues by investigating functional responses in tissues from mice lacking individual AC isoforms.

Methods

Animals

All in vivo protocols were approved by Imperial College London ethical review process committee and we strictly adhered to the Animals (Scientific Procedures) Act 1986 UK Home Office guidelines. Experiments were performed under a Home Office project licence (PPL 70/7212). All studies involving animals are reported in accordance with the ARRIVE guidelines for reporting experiments involving animals (Kilkenny et al., 2010; McGrath et al., 2010). A total of 124 animals were used in the experiments described here.

Male wild-type mouse strains were originally obtained from Harlan UK Limited (Bicester, UK) and bred in our animal facility; food and water were supplied ad libitum. Genetically modified mice (knockout, KO) were back-crossed at least eight times and bred alongside the wild-type mouse strains (FVB/N or C57BL/6). The β-adrenoceptor KO mice (β1, Adrb1−/−; β2, Adrb2−/−; β3, Adrb3−/−) all on FVB/N backgrounds were kindly donated to us by Dr Brad Lowell. The AC6 KO mice (Adcy6−/−) on a C57BL/6 background, were kindly donated to us by Drs Kirk Hammond and Tong Tang. As reported by Tang et al. (2008), the Adcy6−/− mice did not appear to have any phenotypical differences from wild-type mice.

Role of β-adrenoceptor subtypes in mediating functional effects in murine tissues

Tracheal tissue was harvested from wild-type (18–24 g, FVB/N) or Adrb1−/−, Adrb2−/−, Adrb3−/− mice and placed into tissue baths as described by Buckley et al. (2011). Once the tissues had stabilized, they were challenged with ACh (1 mM) three times to confirm viability and to standardize responses. Carbachol (CCh; 1 μM) was used to induce increased tension in tissues over a 30 min period before the addition of test substances. Cumulative concentration–response curves were generated to the non-selective β-adrenoceptor agonist, isoprenaline (with ascorbic acid, 1 μM), and at the end of the experiment the tissues were challenged with papaverine to establish maximal relaxation. In parallel, other samples of trachea were exposed to PGE2, which is known to relax mouse tracheal tissue via activation of the EP2 receptor (Buckley et al., 2011).

To confirm the data generated in tissue from genetically modified mice, selective β-adrenoceptor agonists/antagonists were used. Using the system described above, mouse trachea was exposed to cumulative concentrations of denopamine (pKi of 5.8 at the β1-adrenoceptor, IUPHAR database, http://www.iuphar-db.org/) or fenoterol (pKi of 6.9 at the β2-adrenoceptor, IUPHAR database) in the presence or absence of CGP 20712 (300 nM: pKi 8.5–9.2 at the β1-adrenoceptor, IUPHAR database, ∼10,000 less potent against the β2-adrenoceptor; Dooley et al., 1986) or ICI 118551 (100 nM: pKi 9.2–9.5 at the β2-adrenoceptor, IUPHAR database, ∼100 less potent against the β1-adrenoceptor; Bilski et al., 1983). These concentrations of antagonist were selected from published literature (Hall et al., 1990). To confirm that the pharmacological tools were acting appropriately, fenoterol and ICI 118551 were tested in tracheal tissue collected from male Dunkin-Hartley guinea pigs, using the methods described previously (Buckley et al., 2011).

Vagal tissue was harvested from wild-type (18–24 g, FVB/N), Adrb2−/− or Adrb3−/− mice and mounted in a grease-gap recording system as described previously (Maher et al., 2009). Once a stable baseline was achieved, tissues were exposed to two challenges with a submaximal concentration of the TRPV1 agonist, capsaicin (1 μM), to induce control depolarization, and then incubated with vehicle (0.1% DMSO, v/v) or single concentrations of the selective β1 (denopamine, 100 μM), β2 (formoterol, 10 nM, pKi of 10.1 at the β2-adrenoceptor, IUPHAR database) or β3 (BRL 37344, 10 μM, pKi of 6.4–7.0 at the β3-adrenoceptor, IUPHAR database) adrenoceptor agonists for 10 min. Formoterol (a clinically used long-acting β-adrenoceptor agonist; LABA) was used in these experiments to confirm earlier data with a short-acting β-adrenoceptor agonist (Freund-Michel et al., 2010). Single concentrations were determined from concentration–response experiments and selectivity was confirmed with the respective, receptor-selective antagonists (data not shown). The tissue was then re-challenged with capsaicin in the presence of the vehicle or β-adrenoceptor agonist. Following a washout phase, capsaicin was reapplied to demonstrate tissue viability. Percentage inhibition was calculated by comparing the average depolarization to capsaicin before and after incubation with the vehicle or drug.

Expression of AC isoforms in ASM and neurones

The relative gene expression of individual AC isoforms was measured by real-time PCR, using TaqMan primers and probes, purchased from Applied Biosystems (Life Technology Limited, Paisley, UK). The following TaqMan assays (catalogue #) were used to measure the expression of each respective AC isoform: Adcy1: Mm01187829_m1, Adyc2: Mm00467874_m1, Adcy3: Mm00460371_m1, Adcy4: Mm00475491_m1, Adcy5: Mm00674122_m1, Adcy6: Mm00475772_m1, Adcy7: Mm00545780_m1, Adcy8: Mm00507722_m1, Adcy9: Mm00507743_m1, Adcy10: Mm00557236_m1. Prior to measuring the gene expression of AC isoforms in a multiplex system, each TaqMan assay was first validated to ensure equal amplification efficiency relative to the housekeeping gene, 18S rRNA, using cDNA from tissues (selected from a panel) that expressed the target AC isoform at high levels (data not shown). RNA (subsequently reverse transcribed to cDNA) was extracted by a standard guanidinium thiocyanate-phenol-chloroform method from ASM and vagal sensory ganglia (nodose/jugular ganglia) harvested from wild-type male C57BL/6 mice. Expression levels were determined as described previously (Wong et al., 2009). Briefly, real-time PCR was performed using an ABI PRISM 7000 TaqMan machine (Applied Biosystems). The following amplification protocol was used: one cycle of 50°C for 2 min, one cycle of 95°C for 10 min and 40 cycles of 95°C for 15 s followed by 1 min at 60°C. Data were analysed using Sequence Detection Software (Applied Biosystems).

Role of AC6 in responses to β-adrenoceptor agonists in murine tissues

Tracheal tissue was harvested from wild-type (18–24 g, C57BL/6) or Adcy6−/− mice and placed into tissue baths as described above with a modification to circumvent any possible differences in responses to the muscarinic agonist (we found that reduced contraction to CCh was observed in the trachea from Adcy6−/− mice). Briefly, once the tissues had stabilized and had been challenged with ACh (1 mM) three times, they were incubated with vehicle (0.1% DMSO v/v) or the selective β1-adrenoceptor agonist, denopamine (100 μM), for 10 min. The tissues were then exposed to ACh in increasing concentrations to generate cumulative concentration–response curves and the effects of the β1-adrenoceptor agonist on responses to ACh in tissue from wild-type mice and Adcy6−/− mice were compared.

In parallel experiments, the effect of an EP2 receptor agonist, ONO AE1 259 (10 μM) (Birrell et al., 2013), was determined in tracheal tissue harvested from wild-type and Adcy6−/− mice.

Vagal tissue was collected from wild-type (18–24 g) or Adcy6−/− mice and mounted in a grease-gap recording system as described above. Once a stable baseline was achieved, tissues were exposed to two challenges with capsaicin (1 μM) to induce control depolarizations, and then incubated with vehicle (0.1% DMSO) or selective β1 (denopamine, 100 μM), β2 (formoterol, 10 nM) or β3 (BRL 37344, 10 μM) adrenoceptor agonists for 10 min. The concentrations of agonists used in these experiments were selected from previous concentration ranging studies. At these concentrations the ligands achieved around 80% of their maximal effect. The tissue was then re-challenged with capsaicin in the presence of the vehicle or β-adrenoceptor agonist. Following a wash phase, capsaicin was reapplied to show tissue viability. To determine if the downstream signalling was still intact in the Adcy6−/− mice, we first tried to mimic the effect of the β-adrenoceptor agonist with cAMP analogues that can activate PKA and PKG (8 Br-cAMPS and 8-pCPT-cGMPS, respectively (30 μM; selected from Butt et al., 1992; Freund-Michel et al., 2010). Having established that PKA activation mimicked the effect of a β-adrenoceptor agonist on capsaicin responses in wild-type tissues, the experiment was repeated in tissues from Adcy6−/− mice.

Data analysis

ASM tone experiments

Monophasic agonist concentration–effect curves were fitted by least-squares, non-linear iterative regression following the form of the Hill equation (Prism 5®, GraphPad Software Inc., San Diego, CA, USA).

Vagal depolarization experiments

Data are presented as mean ± SEM of n independent observations. Inhibition of agonist responses in the isolated vagus nerve preparation were analysed by two-tailed paired t-test, comparing responses to agonist in the absence and presence of antagonist in the same piece of nerve. P < 0.05 was taken to indicate significant differences between group means.

Materials

The EP2 receptor agonist (ONO-AE1-259) was a gift from Ono Pharmaceuticals (Osaka, Japan). It was prepared in DMSO (10 mM stock) and stored at −20°C until required. PGE2 was purchased from Cayman Europe (Tallinn, Estonia) and stock solutions of 10 mM were made in ethanol. Papaverine was purchased from Sigma Aldrich (Poole, UK) and dissolved in distilled water at 100 mM. Krebs salts were obtained from BDH (Dorset, UK) and all other chemicals and reagents were purchased from Sigma Aldrich. ACh and CCh were purchased from Sigma Aldrich and dissolved in Krebs solution at 100 and 1 mM respectively. Capsaicin was purchased from Sigma Aldrich and dissolved in DMSO at 1 mM. Denopamine, fenoterol, formoterol and BRL 37344 were purchased from Sigma Aldrich and dissolved in DMSO at 100, 1 and 100 mM respectively. ICI 118551 and CGP 20712 were from Sigma Aldrich. 8 Br-cAMPS and 8-pCPT-cGMPS were bought from Sigma Aldrich and dissolved in DMSO at 30 mM.

Results

Identifying the β-adrenoceptor associated with functional responses in murine airway tissue

The non-selective β-adrenoceptor agonist, isoprenaline, caused a concentration-dependent relaxation of wild-type tracheal smooth muscle tissue that was not different in the trachea harvested from Adrb2−/− and Adrb3−/− mice (Figure 1A and B). The tracheal tissue from the Adrb1−/− mice failed to relax to isoprenaline. In parallel tissues, the PGE2 response appeared not to be altered (Figure 1C and D). These data confirm observations by Henry and Goldie (1990), using pharmacological tools that activation of the β1-, and not the β2-adrenoceptor causes relaxation of murine ASM.

Figure 1.

Figure 1

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Effect of β-adrenoceptor activation on isolated tracheal tissue from wild–type and KO mice. Tracheal tissue from wild-type, age-matched control mice and KO mice (Adrb1−/−, Adrb2−/− or Adrb3−/−), placed in tissue baths and the effect of cumulative concentrations of isoprenaline or PGE2 assessed on tissues pre-contracted with CCh (1 μM). Panel A compares agonist responses in tissues from wild-type (FVB/N) mice with those in tissues from Adrb1−/− (‘β1 AR−/−’) and from Adrb2−/− (‘β2 AR−/−’) mice. Panel B compares responses to the β-adrenoceptor agonist in tissues from wild-type (FVB/N) mice with those in tissues from Adrb3−/− (‘β3 AR−/−’) mice. Panel C compares PGE2 responses in tissues from wild-type (FVB/N) mice with those in tissues from Adrb1−/− (‘β1 AR−/−’) and Adrb2−/− (‘β2 AR−/−’) mice. Panel D compares PGE2 responses in tissues from wild-type (FVB/N) mice with those in tissues from Adrb3−/− (‘β3 AR−/−’) mice. The data shown are the percentage of papaverine relaxation and are presented as mean ± SEM; n = 4–6 animals in each group. The concentration-response curve was fitted using GraphPad Prism.

The β1-adrenoceptor selective agonist, denopamine, caused concentration-dependent relaxation of the mouse trachea with the potency similar to reported values (Figure 2A). The β1–adrenoceptor selective antagonist, CGP 20712, caused a rightward shift in the denopamine response (Figure 2). The β2-adrenoceptor agonist, fenoterol, also caused concentration-dependent relaxation of the mouse trachea but with potency far below that expected in a β2-adrenoceptor-driven system (Figure 2B). Indeed, the data shown in Figure 2C, using the guinea pig trachea, a tissue known to be driven by β2-adrenoceptors (Spicuzza et al., 2001), demonstrate the expected potency of fenoterol.

Figure 2.

Figure 2

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Effect of β-adrenoceptor activation on isolated murine tracheal tissue, using pharmacological tools. Tracheal tissues from male mice, or guinea pigs were placed in tissue baths and the effect of cumulative concentrations of the β1-or β2-adrenoceptor agonists, denopamine or fenoterol, assessed on tissues pre-contracted with CCh (1 μM). Panel A shows responses to denopamine ± the β1-adrenoceptor antagonist, CGP 20712, in mouse tissue. Panel B shows responses to fenoterol ± the β2-adrenoceptor antagonist, ICI 118551, in mouse tissue. Panel C shows responses to fenoterol ± the β2-adrenoceptor antagonist, ICI 118551, in guinea pig tissue. Panel D shows responses to fenoterol ± the β1-adrenoceptor antagonist, CGP 20712, in mouse tissue. The data shown are the percentage of papaverine relaxation and are presented as mean ± SEM; n = 4–6 animals in each group. The concentration-response curve was fitted using GraphPad Prism.

Further, in the mouse trachea, the fenoterol-induced relaxation was suppressed by the β1-adrenoceptor selective antagonist and much less so with the β2-adrenoceptor selective antagonist (Figure 2B and D). However in the guinea pig trachea, the β2-selective antagonist caused a marked rightward shift in the concentration-response curve with a pA2 value of around the reported value, 9.25 (Figure 2C).

All of the selective β–adrenoceptor agonists modulated the responses to capsaicin in vagal tissue from wild-type mice (Figure 3). The β2- and β3-adrenoceptor selective agonists failed to modulate capsaicin responses in vagal tissue from their respective KO mice (Figure 2). The β1-adrenoceptor selective agonist was still effective in the vagus from Adrb2−/− and Adrb3−/− mice, a finding consistent for a role of the β1–adrenoceptor receptor. Recovery capsaicin responses were not altered in these experiments (data not shown).

Figure 3.

Figure 3

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Effect of β-adrenoceptor activation on isolated murine vagal tissue. Vagal tissue from wild-type, age-matched control mice and KO mice (Adrb2−/−;‘β2−/−' or Adrb3−/−;‘β3−/−’) was placed in a grease-gap recording system. The effect of selective β1, β2 and β3-adrenoceptor agonists (denopamine: 100 μM, formoterol: 10 nM, BRL 37344: 10 μM) on capsaicin (1 μM) induced nerve depolarization in each strain was determined. The data (% inhibition of capsaicin responses) are presented as mean ± SEM; n = 4 animals in each group. *P < 0.05, significant inhibition of the capsaicin responses; paired Student's t-test.

AC isoform expression in ASM and vagal sensory ganglia

The data generated using the specific and validated RT-PCR assays demonstrated that AC6 was the dominant isoform expressed both in the mouse ASM and in the vagal sensory ganglia (Figure 4). Although mRNA expression is not necessarily indicative of protein expression or enzyme activity, in the absence of any available selective AC6 antibodies for Western blotting/immunohistochemistry, we focussed on the functional role of AC6 in our further investigations. To this end, Adcy6−/− mice were acquired, a colony established and male mice used for further studies.

Figure 4.

Figure 4

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AC isoform mRNA expression in murine ASM and nodose/jugular ganglia. Tracheal ASM and nodose/jugular ganglia were harvested from male C57BL/6 mice. mRNA was extracted and cDNA prepared for real-time RT-PCR. Validated assays were used to measure the expression profiles of the AC isoforms in ASM (panel A) and ganglia (panel B). The data represented as mean ± SEM; n = 4 animals in each group.

Role of AC6 in β–adrenoceptor agonist responses in murine tissues

Maximal contractile responses to ACh were not reduced in trachea from Adcy6−/− mice compared with wild type (140.3 ± 45.8 mg, logEC50 = −4.9 compared with 205.5 ± 50.9 mg and a logEC50 = −4.8, respectively, n = 6, P > 0.05). Pre-incubation with the β1–adrenoceptor agonist, denopamine caused a rightward shift in the concentration–response curve to ACh in tissue from wild-type mice typical of a bronchodilator drug of this class (Figure 5). In the tissues from the Adcy6−/− mice, denopamine failed to alter the ACh concentration-dependent contractile responses (Figure 5B). This suggests that the AC6 isoform is crucial for to β-adrenoceptor agonist-induced relaxation of murine ASM. The EP2 receptor agonist, ONO AE1 259, also evoked a rightward shift in the concentration–response curve to ACh, which was not changed in tissue from the Adcy6−/− mice (Figure 5C and D). This suggests that, unlike the β–adrenoceptor agonist, in this system, PGE2 acts independently of the AC6 isoform.

Figure 5.

Figure 5

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A role for AC6 isoform in β-adrenoceptor-mediated responses in mouse trachea. Tracheal tissue from wild-type, age-matched control mice and KO mice (Adcy6−/−;‘AC6−/−’) was placed in tissue baths. Vehicle (0.1% DMSO), a β1-adrenoceptor agonist, denopamine (100 μM) or an EP2 receptor agonist, ONO AE1 259 (10 μM), were incubated with the tissue for 10 min prior to the addition of cumulative concentrations of ACh. Panel A compares the effect of vehicle and β1-adrenoceptor agonist on ACh responses in tissues from wild-type mice whereas panel B compares the same responses in tissues from Adcy6−/− (‘AC6−/−’) mice. Panel C compares the effect of vehicle and ONO AE1 259 ACh responses in tissues from wild-type mice whereas panel D compares the same responses in tissues from Adcy6−/− mice. The data shown are the percentage of initial supra-maximal ACh, and were curve fitted using GraphPad Prism and are presented as mean ± SEM; n = 6–7 animals in each group.

Vagal depolarization to capsaicin was not reduced in tissues from the Adcy6−/− mice compared with wild type (0.256 ± 0.04 and 0.311 ± 0.05 mV, respectively, n = 6, P < 0.05). As previously demonstrated β1-, β2- and β3-adrenoceptor selective agonists attenuated capsaicin-induced vagal depolarization in tissues from wild-type mice (Figure 6). These effects were lost in vagal tissue from Adcy6−/− mice, suggesting that the AC6 isoform was also crucial to the responses of mouse sensory nerves to β-adrenoceptor agonists (Figure 6). To determine if signalling downstream of the β-adrenoceptor was still functional in the Adcy6−/− mice, we tested a cAMP analogue (8-Br-cAMPS) that is reported to activate PKA at a specific range of concentrations, while not activating PKG. This analogue was able to mimic the effect of β-adrenoceptor agonists (Figure 6A). Then we used the same tool in vagal tissue from mice missing functional Adcy6−/− and demonstrated that PKA activation modulates capsaicin responses in Adcy6−/− mice to the same extent as tissue from wild-type mice (Figure 7B).

Figure 6.

Figure 6

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A role for AC6 in β-adrenoceptor responses in mouse vagus. Vagal tissue from wild-type, age-matched control mice and KO mice (Adcy6−/−) was placed in a grease-gap recording system. The effect of specific β1, β2 and β3-adrenoceptor agonists (denopamine: 100 μM, formoterol: 10 nM, BRL 37344: 10 μM) on capsaicin-induced nerve depolarization were compared in tissue from wild-type and Adcy6−/− (‘AC6−/−’) mice. The data (% inhibition of capsaicin responses) are presented as mean ± SEM; n = 4 animals in each group. *P < 0.05, significant inhibition of the capsaicin responses; paired Student's t-test.

Figure 7.

Figure 7

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Role for PKA/PKG in Adcy6−/− mouse vagus. Vagal tissue from wild-type, age-matched control mice and KO mice (Adcy6−/−) was placed in a grease-gap recording system. The effect of cAMP analogues that can activate either PKA or PKG (both 30 μM) on capsaicin-induced nerve depolarization in nerve tissue from wild-type mice was determined (panel A). The effect of the cAMP analogue that can activate PKA on capsaicin-induced depolarization was then compared in vagal tissue from wild-type and Adcy6−/− (‘AC6−/−’) mice (panel B). The data (% inhibition of capsaicin responses) are presented as mean ± SEM; n = 4 animals in each group. *P < 0.05, significant inhibition of the capsaicin responses; paired Student's t-test.

Discussion and conclusions

The β-adrenoceptor agonists are one of the most commonly prescribed medicines for patients with respiratory diseases such as asthma and COPD. They are thought to achieve their clinical benefit via AC-dependent production of cAMP but the exact mechanism by which this class of drug acts remains elusive. This is partly due to the fact that there are several AC isoforms and there are currently no truly selective pharmacological inhibitors. The aim of this study was to use tissue from genetically modified mice to establish the role of AC isoforms in β-adrenoceptor agonist responses in the airway.

Before investigating the signalling pathways, the aim was to establish or confirm the functional role of the three β-adrenoceptors (β1, β2 and β3) in the murine tissue based systems. Using tissue from β-adrenoceptor KO mice and pharmacological ligands, we confirmed previous findings that, in the mouse airway, it was activation of β1-adrenoceptors, and not the β2-adrenoceptor (as it is in humans), that led to relaxation of ASM. Although certain experimental conditions might reveal a role for the β2-adrenoceptor (Lin et al., 2012), our data suggest the β1-adrenoceptor plays a dominant role in mediating relaxation of murine tracheal ASM by β-adrenoceptor agonists, consistent with the findings of Henry and Goldie (1990). Interestingly, in a separate publication the same authors demonstrated that the lack of β2-adrenoceptor agonist mediated relaxation was not due to a lack of receptor expression in murine ASM (Henry and Goldie, 1990; Henry et al., 1990). Finally, in these experiments PGE2 still induced relaxation (via activation of the EP2 receptor) in tracheal tissue from mice lacking the β1-adrenoceptor, suggesting that the ability to relax ASM was not compromised by genetic modification in this strain. Having established that the relaxation of ASM in murine trachea was dependent on β1-adrenoceptor activation, further experiments investigated which β-adrenoceptor was involved in another functional response related to airway function. Earlier, we reported that β2-adrenoceptor agonists inhibited sensory nerve activation in guinea pig and human vagal tissue and the cough reflex in a conscious guinea pig model (Freund-Michel et al., 2010). Using tissue from genetically modified mice and pharmacological tools, these results were confirmed but it was also apparent that this property was also shared by the other β-adrenoceptors, namely, the β1 and β3 receptors. These data corroborate previously published data in which agonists of each β-adrenoceptor caused a concentration-related inhibition of capsaicin-induced depolarization in guinea pig, mouse and human vagus. This inhibition was reversed only by respective β1, β2 or β3-adrenoceptor antagonists in guinea pig and human vagus (Wortley et al., 2013).

To investigate which isoform of AC could be central to these β-adrenoceptor agonist-driven responses, we first assessed AC mRNA expression profiles in ASM and sensory nerve cell bodies (contained in the nodose and jugular ganglia). Using validated assays, we found that AC6 was the predominant isoform expressed in both types of tissue. Although mRNA expression is not necessarily indicative of actual protein expression, or the activity of the isoform, it is strongly suggested that AC6 is present in the cell types we were investigating. Although one study describes the presence of AC isoforms (including AC6) in rat dorsal root ganglia (Zhu and Oxford, 2011) and others have presented data from whole murine lung tissue (Sunahara et al., 1996; Hanoune et al., 1997), we are not aware of other reports of AC isoform expression in these specific murine cell types. However, in human ASM, several publications have indicated a similar mRNA expression profile to that observed in the murine tissues presented here, with dominant expression of AC6 (Billington et al., 1999; Xu et al., 2001; Bogard et al., 2011). This would suggest that our data on AC isoform expression in mouse tissue are indicative of that seen in human cells (Billington et al., 1999; Bogard et al., 2011). At this time, the lack of truly selective antibodies prevents us from confirming that the mRNA expression profile is reproduced at the protein level.

In these studies, it was established that AC6 was a prime candidate for mediating the post β-adrenoceptor signalling cascade and functional responses seen in murine tissues. In the absence of selective inhibitors, we performed comparative functional experiments with wild-type tissue and tissues harvested from AC6 KO mice (Tang et al., 2008). The data clearly impy a central role for the AC6 isoform in β1 receptor-relaxation of murine ASM mediated by β1-adrenoceptors and attenuation of sensory nerve activity mediated by β1, β2 and β3-adrenoceptors. To our knowledge this is the first study to provide clear evidence that AC activity is actually involved in the relaxation of ASM by β-adrenoceptor agonists and that it is the AC6 isoform which is key. Furthermore, it would seem that in murine sensory nerves the three β receptors share a common step in their signalling cascade, that is, AC6. Finally, it would seem that activating PKA (with an cAMP analogue), and not PKG, can mimic the effect of the β-adrenoceptor agonists and that AC6 is upstream of PKA as the PKA activator was still functional in the tissue from the Adcy6−/− mice. A previous publication utilizing the Adcy6−/− mice reported that these mice also had lower levels of AC5, but that the apparent reduction was not sufficient to generate the phenotype usually observed in Adcy5−/− mice, suggesting that any reduction in expression was not functionally relevant (Tang et al., 2008). Interestingly, in the same system, relaxation of murine ASM triggered by EP2 receptor agonists was not dependent on the AC6 isoform. This may suggest that other Gs-coupled receptors, such as the EP2 receptor, may preferentially couple to other AC isoforms in the same cell, which has interesting implications for compartmentalized signalling, its regulation and targeting. These data build on a recent publication which suggested that in cultured human ASM cells overexpressing AC6, production of cAMP was enhanced by β-adrenoceptor agonists, but not by EP receptor agonists (Bogard et al., 2011).

In summary, our data demonstrate that AC6 plays a key role in the relaxation of ASM and modulation of sensory nerves induced by β-adrenoceptor agonists. Mice are often the species of choice for preclinical modelling of respiratory diseases and thus our data may help to design and interpret relevant future studies in this field. Furthermore, these data represent a vital step in understanding the mechanism of action of this extensively prescribed class of medicine and leads the way for the next phase of research and determining the role of AC isoforms in human systems.

Acknowledgments

This work would not have been possible without the generous donation by Dr Kirk Hammond and Dr Tong Tang of the Adcy6−/− mice.

Glossary

AC

adenylyl cyclase

Adcy6−/−

AC6 KO mouse

Adrb1−/−, Adrb2−/−, Adrb3−/−

β1-, β2-, β3-adrenoceptor KO mouse

ASM

airway smooth muscle

CCh

carbachol

Author contributions

M. A. B. and M. G. B. devised the experiment and wrote the manuscript; S. J. B., J. B. and N. D. performed and analysed the tissue bath experiments; M. W. performed and analysed the vagal experiments, L. Y.-B. and S. A. M. performed the genotyping and RT-PCR.

Conflict of interest

None.

References

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