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. 2023 Jul 31;69(5):584–591. doi: 10.1165/rcmb.2022-0445OC

Prorelaxant E-type Prostanoid Receptors Functionally Partition to Different Procontractile Receptors in Airway Smooth Muscle

Ajay P Nayak 1,*, Elham Javed 1,*, Dominic R Villalba 1, Yinna Wang 2, Henry P Morelli 1, Sushrut D Shah 1, Nicholas Kim 4, Rennolds S Ostrom 3, Reynold A Panettieri Jr 4, Steven S An 4, Dale D Tang 2, Raymond B Penn 1,✉
PMCID: PMC10633839  PMID: 37523713

Abstract

Prostaglandin E2 imparts diverse physiological effects on multiple airway cells through its actions on four distinct E-type prostanoid (EP) receptor subtypes (EP1–EP4). Gs-coupled EP2 and EP4 receptors are expressed on airway smooth muscle (ASM), yet their capacity to regulate the ASM contractile state remains subject to debate. We used EP2 and EP4 subtype-specific agonists (ONO-259 and ONO-329, respectively) in cell- and tissue-based models of human ASM contraction—magnetic twisting cytometry (MTC), and precision-cut lung slices (PCLSs), respectively—to study the EP2 and EP4 regulation of ASM contraction and signaling under conditions of histamine or methacholine (MCh) stimulation. ONO-329 was superior (<0.05) to ONO-259 in relaxing MCh-contracted PCLSs (log half maximal effective concentration [logEC50]: 4.9 × 10−7 vs. 2.2 × 10−6; maximal bronchodilation ± SE, 35 ± 2% vs. 15 ± 2%). However, ONO-259 and ONO-329 were similarly efficacious in relaxing histamine-contracted PCLSs. Similar differential effects were observed in MTC studies. Signaling analyses revealed only modest differences in ONO-329– and ONO-259–induced phosphorylation of the protein kinase A substrates VASP and HSP20, with concomitant stimulation with MCh or histamine. Conversely, ONO-259 failed to inhibit MCh-induced phosphorylation of the regulatory myosin light chain (pMLC20) and the F-actin/G-actin ratio (F/G-actin ratio) while effectively inhibiting their induction by histamine. ONO-329 was effective in reversing induced pMLC20 and the F/G-actin ratio with both MCh and histamine. Thus, the contractile-agonist–dependent differential effects are not explained by changes in the global levels of phosphorylated protein kinase A substrates but are reflected in the regulation of pMLC20 (cross-bridge cycling) and F/G-actin ratio (actin cytoskeleton integrity, force transmission), implicating a role for compartmentalized signaling involving muscarinic, histamine, and EP receptor subtypes.

Keywords: ASM, prostaglandin, asthma, compartmentalization, G protein-coupled receptor

Clinical Relevance

Findings from the present study demonstrate different abilities of EP2 and EP4 receptor agonists to regulate airway smooth muscle contraction, depending on the type of contractile agonist. The findings suggest that: 1) compartmentalization of signaling causes these differences; and 2) future asthma therapies using EP receptor targeting may be influenced by the nature of the pro-mitogenic agents in the airway.

Prostaglandin E2 (PGE2) is a major mediator of various physiological and pathophysiological responses. PGE2 exerts diverse physiological effects through four distinct E-type prostanoid (EP) receptor subtypes (EP1–EP4) that couple to different heterotrimeric G proteins (1, 2). The potential of PGE2 as an asthma therapeutic with respect to its bronchorelaxant and antiproliferative properties has been well established in cell-based, in vivo (animal) and ex vivo (human and animal) studies (3–8). We and others have demonstrated a strong ability of PGE2 to relax contracted human airway smooth muscle (ASM) and inhibit mitogen-stimulated ASM proliferation, both effects that are dependent on cAMP/protein kinase A (PKA) signaling (9–11) mediated through Gs-coupled EP2 or EP4 receptors.

Clinically, the bronchodilatory effects of PGE2 have also been established in patients with asthma and chronic bronchitis, and they protect against exercise-induced (12) and aspirin-induced (13, 14) bronchoconstriction. However, PGE2 inhalation stimulates both cough (through EP3/Gαi signaling) and substernal burning (15–17). Other studies have suggested that the mixed effects of PGE2 on multiple EP receptor subtypes activating multiple heterotrimeric G protein subtypes limit the use of PGE2 as a therapeutic (18). Consequently, the therapeutic utility of EP receptor agonism for asthma likely relies on the development of effective and safe EP2 or EP4 subtype-specific agonists. Efforts to develop EP subtype-specific ligands (such as EP2 agonist AH13205) have been fraught with difficulties, including weak intrinsic EP2 receptor activity and selectivity, as well as efficacy challenges (19, 20); as a result, they have not progressed in clinical development (21). More recently, ONO-AE1–259 (EP2-specific agonist; hereinafter ONO-259) and ONO-AE1–329 (EP4-specific agonist; hereinafter ONO-329) were developed as subtype-specific EP2 and EP4 receptor agonists, respectively (11). However, Buckley and colleagues (22) reported that EP4-selective ONO-329, but not EP2-specific ONO-259, potently relaxed contracted human airways ex vivo, suggesting that EP4 is a more suitable, druggable target relative to EP2.

However, we recently published a study where we tested subtype-specific agonists of EP receptors in recombinant receptor expression systems as well as human ASM and demonstrated strong capabilities of EP2-specific agonism to both signal and regulate mitogen-stimulated ASM proliferation (23).

To clarify the role of EP receptor subtypes in human ASM with respect to contractile regulation, the present study characterized signaling and functional effects of subtype-selective EP agonists in multiple models of human ASM contraction, induced by different contractile agonists. The results suggest a nuanced role for EP2 and EP4 as prorelaxant G protein–coupled receptors (GPCRs) in ASM, likely dictated by compartmentalization of EP2/EP4 signaling.

Methods

Reagents and Antibodies

The antibody against phosphorylated (at Ser16) small heat-shock protein 20 (HSP20) was purchased from Abcam. Antibodies against phosphorylated (at Thr18 and Ser19) myosin light chain 20 (MLC20) and phosphorylated (at Thr696) myosin light chain phosphatase were purchased from Cell Signaling Technology. Anti–vasodilator-stimulated phosphoprotein (anti-VASP) antibody was purchased from BD Biosciences. Human β-actin (A5441) antibody and PGE2 were obtained from Sigma. ONO-259 and ONO-329 were provided by ONO Pharmaceuticals. Protease and phosphatase inhibitors were from Bimake. Secondary antibodies conjugated to IRDye 680 and 800 were purchased from LI-COR.

Human Cells and Human Precision-Cut Lung Slices

Primary ASM cultures were established from human tracheae, as described previously (24). In addition, senescence-resistant human ASM cultures stably expressing human telomerase reverse transcriptase (25) and retaining expression of the m3 muscarinic acetylcholine receptor were also used. As described previously (26), cells were maintained in medium containing 10% FBS and then switched to serum-free medium 24 hours before stimulation to enhance contractile signaling and response in cells.

Human lung tissue cores were obtained from Rutgers University and were previously procured by the National Disease Resource Interchange. The use of these tissues, as well as of ASM cultures derived from donor lungs, for research is approved by the respective institutional review boards of Thomas Jefferson University and Rutgers University and has been judged as “not human subjects research” because of the deidentified nature of the cells and tissue.

Human lung cores are generated from (well-preserved) lungs within 24 hours of resection. Cores were cut into 300-μm slices and stored in serum-free Ham’s F12 medium with Primocin (InvivoGen), a broad-spectrum antibiotic against Gram-positive and -negative bacteria, mycoplasma, and fungi. Representative slices from individual cores were tested for responsiveness and efficacy with methacholine (MCh; 1 μM). Treatments were conducted in slices generated from the same lung core to ensure very similar tissues, optimal matching, and consistency for treatment effects. No cryopreservation was performed on the cores or the slices.

The EVOS FL Auto Imaging System (Thermo Fisher Scientific) was used to capture images of the slices. Images of slices were taken before treatment, after a 15-minute stimulation with MCh (1 μM) or histamine (10 μM), and 5 minutes after each dose of ONO-259 or ONO-329. Slices were analyzed post hoc using ImageJ software, and luminal airway was measured using an airway diameter measurement as described elsewhere (27). Data are represented as (relaxant agent–mediated) change in airway lumen area relative to the area measured after contractile agonist stimulation and before (relaxant) drug administration.

Immunoblotting

Human ASM cells were grown to near confluence in 12-well plates and switched to serum-free media 24 hours before stimulation. Cells were stimulated as indicated, lysed, sonicated briefly, electrophoresed on an 8% or 10% SDS-polyacrylamide gel, transferred to nitrocellulose membranes, and subsequently probed with the indicated primary and secondary antibodies conjugated with infrared fluorophores (28, 29). Typically, to limit experiment complexity in the signaling analyses, we used 10-nM concentrations of ONO-259 and ONO-329; given at these concentrations, large differences in the abilities of drugs to regulate MCh-induced, but not histamine-induced, ASM contraction in precision-cut lung slices (PCLSs) were observed. For PCLS experiments, concentrations of 1 μM MCh and 10 μM histamine were used to contract airways, on the basis of others’ findings and our findings that these concentrations produce suitable contraction and also enable a sufficient dynamic range of regulation by relaxant agents (30, 31). For all cell-based assays, 10 μM MCh and 1 μM histamine were used, given our prior studies, suggesting these concentrations generate sufficient contractile and signaling effects that are regulated by Gs-coupled receptors (29, 31). Bands were visualized, and signals (infrared emission) were quantified directly using the Odyssey Infrared Imaging System (LI-COR), as described previously (28).

Magnetic Twisting Cytometry

We performed a single-cell contraction assay by examining dynamic changes in cell stiffness in isolated primary human ASM. Magnetic beads, functionalized to the cytoskeleton through cell-surface integrin receptors, were used for attachment to the ASM cytoskeleton as described previously (32, 33). Baseline ASM cell stiffness measurements were obtained for the first 60 seconds, after which the cell stiffness was increased by treating cells with either MCh (10 μM) or histamine (1 μM) and measured for an additional 240 seconds. Contracted ASM cells were subsequently treated with either ONO-259 or ONO-329 (10 nM), and stiffness was measured continuously for 300 seconds. For each cell, EP agonist–induced changes in cell stiffness were normalized to stiffness induced by the specific contractile stimulus.

Actin Cytoskeleton Regulation by EP Agonists

Differential regulation of actin cytoskeleton by EP agonists was initially examined by western blot analysis of F-actin and G-actin pools as described previously (34–37). Briefly, cells were fixed with 4% paraformaldehyde and subsequently stained with rhodamine-phalloidin (F-actin) and Alexa Fluor 488-DNase I (G-actin) (Life Technologies). For the assessment of dose-dependent effects of EP agonists, we performed fluorescence imaging using a Leica DMI 6000 microscope as described previously (29). Fluorescence signal intensities for rhodamine and Alexa Fluor 488 were compared to determine the F/G-actin ratio (29).

Statistical Analysis

Data are presented as mean ± SE. Experiments using human ASM cultures were repeated with cultures from n different donors to create the indicated n values. Statistically significant differences among vehicle/treatment groups were assessed by ANOVA, and Bonferroni’s post hoc analysis or Student’s t test were applied in studies where appropriate, with values of P < 0.05 sufficient to reject the null hypothesis, using Prism 7.0 software (GraphPad).

Results

The Prorelaxant Effect of Targeting EP2 and EP4 Receptor Subtypes Depends on the Contractile Agonist

Consistent with the findings of Buckley and colleagues (22), our studies using human PCLCs (hPCLSs) show that, in the presence of MCh, the EP2 receptor agonist ONO-259 demonstrates relatively poor efficacy in relaxing airways (Figure 1). The EP4 agonist ONO-329 was superior to ONO-259 in relaxing MCh-contracted hPCLSs: logEC50, 4.9 × 10−7 (ONO-329) versus 2.2 × 10−6 (ONO-259); maximal bronchodilation ± SE, 35 ± 2% (ONO-329) versus 15 ± 2% (ONO-259), respectively; both Ps < 0.05 (Figure 1A). However, when histamine was used as the contractile stimulus, both EP4 and EP2 agonists significantly (P < 0.05) inhibited histamine-induced contraction in hPCLSs: logEC50, 2.4 × 10−7 (ONO-329) versus 1.0 × 10−7 (ONO-259); maximal bronchodilation ± SE, 17 ± 6% (ONO-329) versus 22 ± 2% (ONO-259) (Figure 1B).

Figure 1.

Figure 1.

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Differential regulation of airway contraction by EP2- and EP4-specific agonists in human precision cut lung slices. Human precision-cut lung slices were contracted with (A) MCh (1 μM) or (B) His (10 μM). After achieving a steady-state level of contraction, increasing concentrations (0.1–1,000 nM) of ONO-259 or ONO-329 were added and changes on airway luminal area were recorded. N = 4–7 distinct donors. EP = E-type prostanoid receptor subtype; His = histamine; log M = log molar; MCh = methacholine.

Although hPCLSs are a useful translational tool to study the regulation of airway contraction, they are a complex integrated system involving multiple cell types (including epithelial cells), other than ASM, that also express EP2 and EP4 receptors. Therefore, we used a single-cell contraction assay to assess EP2 and EP4 relaxant effects, specifically in ASM cells, using magnetic twisting cytometry. Consistent with our findings from hPCLSs, EP4 agonist ONO-329 (but not EP2 agonist ONO-259) was able to reverse MCh-induced change in cell stiffness (Figure 2A). In ASM cells contracted with histamine, ONO-329 and ONO-259 were comparable in reversing cell stiffness (Figure 2B). Thus, in both tissue- and cell-based models of ASM contraction, inhibition by EP2 agonism occurs under conditions of histamine, but not MCh, stimulation.

Figure 2.

Figure 2.

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A differential regulation of airway cell contraction by EP2- and EP4-specific agonists in airway smooth muscle (ASM) by magnetic twisting cytometry. (A) Effects of EP2 agonist ONO-259 and EP4 agonist ONO-329 (50 nM each) on MCh (10 μM)-induced contraction of individual ASM cells (ns = 112 and 95, respectively). (B) Effects of ONO-259 and ONO-329 on His (1 μM)-induced contraction of individual ASM cells (ns = 169 and 80, respectively). Data represent mean ± SE normalized to MCh- or His-induced contraction.

Regulation of Contractile Signaling Pathways by EP Receptor Subtypes

To assess the mechanistic basis of the differential regulation of ASM function by EP receptor subtypes, we characterized the regulation of downstream signaling events by the EP agonists. In our recent report, we established the responsiveness of ASM cells to EP2 agonist ONO-259 and EP4 agonist ONO-329 (23). We demonstrated that stimulation of ASM cells with each of these agonists resulted in comparable accumulation of cAMP and activation of downstream PKA-mediated signaling marked by phosphorylation of its substrate VASP. Phosphorylation of VASP by PKA at Ser157 causes a mobility shift on electrophoresis, with the phosphorylated species of VASP (pVASP) migrating at ∼50 kDa and the unphosphorylated species migrating at ∼46 kDa (23, 28, 38). In the present study, stimulation of primary ASM cells with ONO-259, ONO-329, or PGE2 induced comparable levels of pVASP (mean values of percent VASP shift ± SE: ONO-259, 64 ± 6%; ONO-329, 68 ± 4%; and PGE2, 68 ± 3%) (Figures 3A and 3B). Costimulation of ASM cells with contractile agonists (histamine or MCh) did not significantly alter pVASP levels (mean values ± SE for histamine vs. MCh: EP2, 57 ± 9% vs. 46 ± 10%; EP4, 51.18 ± 8% vs. 55 ± 6%; and PGE2, 59 ± 3% vs. 51 ± 6%).

Figure 3.

Figure 3.

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Differential regulation of vasilodilator-stimulated phosphoprotein (VASP), phosphorylation of heat shock protein 20 (pHSP20), and phosphorylation of the regulatory myosin light chain (pMLC20) by EP receptor subtypes (EP2 and EP4) and contractile receptor agonists. Primary human ASM cultures were treated for 10 minutes with vehicle (Veh), ONO-259 (10 nM), ONO-329 (10 nM), or prostaglandin E2 (PGE2) (10 nM), with or without Veh, MCh (10 μM), or His (1 μM). (A) Representative western blot of total VASP. (B) Graphical presentation of mean ± SE for data of VASP shift (calculated as 50-kDa band intensity divided by the sum of 46 kDa + 50 kDa band intensities). n = 12 distinct primary human ASM cell cultures. (C) Representative western blot for pHSP20. (D) Graphical presentation of mean ± SE for data of percent maximal response, with band intensity values normalized to corresponding β-actin band intensities. n = 10 distinct primary human ASM cell cultures. (E) Representative western blot for pMLC20. (F) Graphical presentation of mean ± SE for data of fold change of pMLC20, with band intensity values normalized to corresponding β-actin band intensities. n = 9 distinct primary human ASM cell cultures. *P < 0.05. pVASP = phosphorylated species of vasilodilator-stimulated phosphoprotein.

Although pSer157 VASP induction tends to faithfully track with cAMP/PKA-mediated signaling induced by the activation of GPCRs that couple to heterotrimeric G protein (Gs), its ability to regulate contraction of ASM is not established. However, phosphorylation of another PKA substrate, heat shock protein 20 (pHSP20), has been shown to have a prorelaxant effect on ASM (39, 40). Stimulation of primary ASM cells with ONO-259 or ONO-329 resulted in similar levels of pHSP20 (Figures 3C and 3D). When ASM cells stimulated with EP2 agonist ONO-259 were costimulated with histamine, we observed no reduction in pHSP20 levels; however, when ASM cells were costimulated with EP4 agonist ONO-329 and histamine, we observed a significant reduction in pHSP20 level. Finally, when the contractile agonist in these experiments was MCh, we observed significant reduction in pHSP20 levels induced by EP2 and EP4 agonists; however, no difference was observed between the magnitudes of effect of the two EP agonists.

EP2 and EP4 Regulation of MCh- and Histamine-induced Pharmacomechanical Coupling

To further detail the mechanisms mediating differential regulation of ASM contractility by EP receptor subtypes, we pretreated normal donor human ASM cells with EP subtype-selective agonists before stimulation of cells with MCh or histamine. Stimulation of ASM cells with EP2 or EP4 agonist alone did not result in any changes in phosphorylation of MLC20 (pMLC20) (EP2, 0.9-fold and EP4, 1.1-fold, relative to vehicle-treated cells), whereas treatment of primary ASM cells with histamine (∼13.5-fold over baseline) or MCh (∼4.6-fold over basal) significantly increased pMLC20 levels (Figures 3E and 3F). Costimulation with EP2 agonist ONO-259 significantly inhibited histamine-induced pMLC20 but not MCh-induced pMLC20, whereas EP4 agonist ONO-329 significantly inhibited pMLC20 induced by either histamine or MCh. These studies suggest that, whereas EP4 activation results in efficient regulation of pharmacomechanical coupling induced by either contractile agonist, EP2 activation results in preferential regulation of histamine-induced signal (pMLC20) but not that induced by MCh.

EP2 and EP4 Regulation of Actin Cytoskeletal Dynamics

Previous studies have established that changes in the actin cytoskeleton, reflected in an increase the ratio of F-actin to G-actin (F/G-actin ratio), contribute to the development of contractile force in ASM and that various approaches that reduce the F/G-actin ratio cause ASM relaxation (29, 41, 42). Consistent with these findings, we observed an increase in F/G-actin ratio in ASM cells stimulated with either MCh or histamine (Figure 4). Costimulation with the EP4 agonist ONO-329 reversed this increase in both MCh- and histamine-stimulated cells (Figures 4B and 4D). However, the EP2 agonist ONO-259 reversed the histamine-stimulated increase in the F/G-actin ratio (Figure 4C) but not in that stimulated by MCh (Figure 4A).

Figure 4.

Figure 4.

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Regulation of actin cytoskeleton elements by EP ligands. Western blot analysis of F- and G-actin pools in primary ASM cells treated for 10 minutes with different combinations of contractile and (EP receptor) agonists: (A) MCh + EP2 agonist ONO-259 (50 nM), (B) MCh + EP4 agonist ONO-329 (50 nM), (C) His + EP2 agonist ONO-259 (50 nM), or (D) His + EP4 agonist ONO-329 (50 nM).

Dose-Dependent Effects of EP Agonists on Mechanism Underlying Contractile Regulation

To further establish the robustness of our mechanistic findings, we examined the dose-dependent effect of ONO-259 and ONO-329 in regulating pMLC20 induction by histamine (Figures 5A–5D). ONO-259 and ONO-329 demonstrated comparable efficacy in inhibiting pMLC20 induced by histamine (−log half maximal inhibitory concentration [logIC50]: −3.254 × 10−7 vs. −logIC50: −4.476 × 10−7). Similarly, a dose-dependent effect of both ONO-259 and ONO-329 was observed in the regulation of histamine-induced F/G-actin ratios (Figures 5E–5G).

Figure 5.

Figure 5.

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Dose-dependent regulation of His-induced pMLC20 and F-actin/G-actin ratios by EP receptor ligands. (A and B) Primary human ASM cultures were treated for 10 minutes with Veh, (A) ONO-259 (0.1 nM–50 nM), or (B) ONO-329 (0.1–50 nM) with or without His (1 μM). Lysates were harvested and subjected to immunoblot analysis for pMLC20. (C and D) Graphical representation of dose-dependent effects of (C) ONO-259 and (D) ONO-329 of His-induced pMLC20. n = 3–4 distinct primary human ASM cell cultures. (E–G) Dose-dependent regulation of actin cytoskeleton elements by EP ligands. Fluorescence analysis of F-actin (phalloidin) and G-actin (DNase I) in ASM cells treated with Veh, His (1 μM), or His plus different concentrations of EP agonists. Data are presented as mean ± SE from five independent experiments. (E) Representative fluorescence images showing the effects of His on F- and G-actin. Scale bars, 20 μm. (F and G) Treatment with (F) ONO-259 or (G) ONO-329 induces dose-dependent inhibition of F/G-actin ratios stimulated by His. Data are presented as mean ± SE from three to four independent experiments. Ctrl = control.

Discussion

The potential for PGE2 as an asthma therapeutic has been recognized for many years. PGE2 exerts its actions primarily through the EP receptor subtypes (EP1–EP4). EP2 and EP4 have gained particular interest in the field, given that each activates canonical Gs/cAMP/PKA signaling known to inhibit procontractile signaling in ASM (43).

On the basis of mRNA transcript levels and signaling analyses, we previously determined human ASM to express EP2, EP3, and EP4 receptors, with little to no EP1 receptor expression (23, 44–46). EP3 in ASM promotes contraction (46, 47) and also promotes cough in the lung (48); thus, selective EP2/EP4 targeting represents the most logical approach for an effective antiasthma therapy. We have recently demonstrated that activation of EP2 and EP4 in ASM cells—using ONO-259 and ONO-329, respectively—results in comparable increases in cAMP accumulation as well as comparable increases in whole-cell PKA-phosphorylated proteins (23). However, studies have consistently demonstrated that targeting EP4, and not EP2, inhibits ASM contraction (22). EP2 and EP4 exhibit weak sequence homology (sharing ∼30% amino acid sequence) and differ in structural and regulatory features (49, 50). Studies to date have demonstrated that, although the EP2 receptor promotes canonical Gs-adenylyl cyclase signaling that should relax ASM, targeting ASM with EP2-selective agonists has little or no prorelaxant effect.

The endogenous ligand of EP receptor subtypes, PGE2, can inhibit both histamine- and MCh-induced bronchoconstriction (15, 51, 52). The extent to which activation of each of the EP subtypes influences this prorelaxant effect is not established, although the findings of Buckley and colleagues would suggest a dominant role of EP4. For quite some time now, establishing a clear mechanistic link between EP2 and EP4 signaling and physiological function has been hindered because of a lack of highly selective agonists for EP receptor subtypes. The recent development of superior subtype-selective agonists has enabled greater clarity into the signaling and functional effects of EP receptor subtypes (23).

Whereas previous attempts to target the EP2 receptor for asthma management likely failed because of the poor pharmacological profile (selectivity, efficacy) of the ligands used (20), our studies suggest that the nature of contractile stimulus is important in determining the functional/therapeutic effect of EP subtype agonism. Given that EP2 and EP4 receptor agonists stimulate comparable levels of cAMP (23), we contend that spatiotemporal compartmentalization (and not total cAMP induction) of cAMP/PKA-mediated signaling is a key determinant of how these receptors regulate pMLC20/cross-bridge and actin cytoskeleton dynamics that promote contraction. How such compartmentalized signaling occurs is currently unclear. Partition of the GPCRs as well as proteins and their downstream membrane effectors in or out of lipid rafts/caveolin-rich domains is one potential means of compartmentalizing signaling in ASM cells, as suggested by elegant studies by the Halayko (53–55) and Ostrom and colleagues (56–58) labs. Indeed, Agarwal and colleagues used targeted fluorescence resonance energy transfer–based cAMP biosensors to show that EP2 receptors couple selectively to AC2 in lipid raft microdomains and that PDE4 activity restricted diffusion of these cAMP signals (56). In addition, the Rich lab has proposed models suggesting the ability of more downstream regulatory elements (e.g., A kinase–anchoring proteins, phosphodiesterases, and physical barriers) to shape subcellular cAMP flow and abundance (59, 60).

In summary, the present study demonstrates that the relaxant effect of EP2 and EP4 agonists on human ASM is dependent on the type of procontractile stimuli. These differential effects are not explained by changes in global cell signals, which implies a role for compartmentalized signaling involving muscarinic, histamine, and EP receptor subtypes. Collectively, our studies demonstrate that EP2 receptor agonism may be equally efficacious to that of the EP4 receptor, dependent on context, particularly when histamine is the mediator of bronchoconstriction. Our studies suggest that, in those scenarios of allergic airway inflammation in which histamine might contribute significantly to bronchoconstriction, targeting the EP2 receptor may have therapeutic utility.

Footnotes

Supported by National Heart, Lung, and Blood Institute (NHLBI) grants HL136209 and HL58506 (to R.B.P.), HL169522 (to R.B.P. and R.S.O.), and HL145392 (to R.B.P and D.D.T.). The content is solely the responsibility of the authors and does not necessarily represent the official views of the NHLBI.

Author Contributions: A.P.N., S.S.A., D.D.T., R.S.O., and R.B.P. designed the studies, analyzed and interpreted the data, and wrote the manuscript. A.P.N., E.J., D.R.V., H.P.M., S.D.S., S.S.A., N.K., and Y.W. performed experiments. R.A.P. provided lung cores for generating human precision-cut lung slices. All authors provided comments contributing to the final content of the manuscript.

Originally Published in Press as DOI: 10.1165/rcmb.2022-0445OC on July 31, 2023

Author disclosures are available with the text of this article at www.atsjournals.org.

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