Interdependence of hypoxia and β-adrenergic receptor signaling in pulmonary arterial hypertension

Abstract

The β-adrenergic receptor (βAR) exists in an equilibrium of inactive and active conformational states, which shifts in response to different ligands and results in downstream signaling. In addition to cAMP, βAR signals to hypoxia-inducible factor 1 (HIF-1). We hypothesized that a βAR-active conformation (R**) that leads to HIF-1 is separable from the cAMP-activating conformation (R*) and that pulmonary arterial hypertension (PAH) patients with HIF-biased conformations would not respond to a cAMP agonist. We compared two cAMP agonists, isoproterenol and salbutamol, in vitro. Isoproterenol increased cAMP and HIF-1 activity, while salbutamol increased cAMP and reduced HIF-1. Hypoxia blunted agonist-stimulated cAMP, consistent with receptor equilibrium shifting toward HIF-activating conformations. Similarly, isoproterenol increased HIF-1 and erythropoiesis in mice, while salbutamol decreased erythropoiesis. βAR overexpression in cells increased glycolysis, which was blunted by HIF-1 inhibitors, suggesting increased βAR leads to increased hypoxia-metabolic effects. Because PAH is also characterized by HIF-related glycolytic shift, we dichotomized PAH patients in the Pulmonary Arterial Hypertension Treatment with Carvedilol for Heart Failure trial (NCT01586156) based on right ventricular (RV) glucose uptake to evaluate βAR ligands. Patients with high glucose uptake had more severe disease than those with low uptake. cAMP increased in response to isoproterenol in mononuclear cells from low-uptake patients but not in high-uptake patients' cells. When patients were treated with carvedilol for 1 wk, the low-uptake group decreased RV systolic pressures and pulmonary vascular resistance, but high-uptake patients had no physiologic responses. The findings expand the paradigm of βAR activation and uncover a novel PAH subtype that might benefit from β-blockers.

Keywords: hypoxia; metabolism; pulmonary hypertension; β-adrenergic receptor.

Fig. 1.

β-Adrenergic receptor (βAR) ligands differentially regulate hypoxia-inducible factor 1 (HIF-1) and cAMP in vitro. A–C: human embryonic kidney cells overexpressing the β₂AR (HEK293-β₂AR) were treated in normoxic conditions for 20 h with isoproterenol (A), salbutamol (B), or carvedilol (C). HIF-1 activity was measured using a hypoxia-response element (HRE)-luciferase reporter. Error bars represent SE, n = 3–4, in triplicate. **P < 0.01 and ***P < 0.001, relative to vehicle, Bonferroni posttest. D: summary of A–C effects on HRE-luciferase compared with hypoxia-positive control. Isoproterenol (30 µM), 100 µM salbutamol, and 1 µM carvedilol are shown. E: regulation of cAMP in HEK293-β₂AR cells treated with isoproterenol (10 µM), salbutamol (10 µM), or carvedilol (10 µM) + IBMX (500 mM) for 5 min. cAMP was measured via fluorescent assay and normalized to total protein. Error bars represent SE; n = 3–4 in duplicate. **P < 0.01 and ***P < 0.001 relative to vehicle (Bonferroni posttest). F: theoretical model of βAR regulation. The receptor status is an equilibrium of multiple conformations that signal through different pathways. Ligands affect signaling by shifting the equilibrium of receptors toward or away from specific conformations. Results indicate that isoproterenol is an agonist for cAMP and a partial agonist for HIF-1, shifting the equilibrium toward both pathways. Salbutamol is an agonist for cAMP and an inverse agonist for HIF-1, shifting the equilibrium away from HIF-1. Carvedilol is an inverse agonist for cAMP and partial agonist for HIF-1, shifting the equilibrium away from cAMP and toward HIF-1. CAR, carvedilol; ISO, isoproterenol; R, inactive conformation; R*, conformation that activates cAMP; R**, conformation that activates HIF-1; SAL, salbutamol.

Fig. 2.

β-Adrenergic receptor (βAR) ligands differentially affect hypoxia-inducible factor 1 activity in vitro. A–C: human embryonic kidney cells overexpressing the β₂AR (HEK293-β₂AR) were treated for 45 min with isoproterenol (A), salbutamol (B), or carvedilol (C) then exposed to hypoxia (2% O₂) for 20 h. Data are represented as % change in hypoxia-response element (HRE)-luciferase activity compared with the vehicle control. D: summary of A–C HRE-luciferase effects with 30 µM isoproterenol, 100 µM salbutamol, and 1 µM carvedilol. Error bars represent SE; n = 3–4 in triplicate. *P < 0.05, **P < 0.01, and ***P < 0.001 compared with vehicle control (Bonferroni posttest).

Fig. 3.

Isoproterenol (ISO) and salbutamol (SAL) have opposing effects on erythropoietic response in vivo. Mice received 1, 5, or 10 mg/kg isoproterenol, salbutamol, or vehicle via intraperitoneal injection 2 h before euthanization. n = 5 per group for 1 and 10 mg/kg, and n = 10 for 5 mg/kg doses. A: representative Western blot of kidney hypoxia-inducible factor 1 α (HIF-1α) levels. HIF-1α was normalized to Lamin B1. Samples were run on multiple gels in parallel. Data were normalized to vehicle-treated group. Error bars represent SE. **P < 0.01 (Bonferroni posttest). C and D: gating for erythroid progenitor stages measured via flow cytometry. Proerythroblast (stage I), basophilic erythroblasts (stage II), polychromatic erythroblasts (stage III), and orthochromatic erythroblasts (stage IV) were determined based on their expression of CD44, TER119, and size. E–G: quantification of stages I–IV by dose. Normal erythroid development shows a 1:2:4:8 ratio from stage I to stage IV. Each treatment group was normalized to stage I. Error bars represent SE. *P < 0.05, **P < 0.01, and ***P < 0.001 relative to vehicle (Bonferroni posttest).

Fig. 4.

Overexpression of β₂-adrenergic receptor (β₂AR) in human embryonic kidney 293 cells (HEK293) increases basal hypoxia-inducible factor 1 (HIF-1) activity and downstream effects under normoxia. A: β₂AR density on the plasma membrane of HEK293-β₂AR and HEK293-wild-type (WT) cells was determined via radio-ligand binding. Error bars represent SE; n = 3. ***P < 0.001 (Student’s t-test). B: basal HIF-1 activity was measured via hypoxia-response element (HRE)-luciferase activity normalized to Renilla control plasmid in normoxia. Error bars represent SE; n = 18. ***P < 0.001 (Student’s t-test). C: estimated glycolysis was determined using the Seahorse glycolytic stress test. Glycolysis is calculated as maximal glucose-stimulated extracellular acidification rate (ECAR) minus baseline ECAR. Cells were pretreated with HIF-1 inhibitors digoxin (100 nM) or chrysin (30 µM) or vehicle control for 4.5 h. Error bars represent SE; n = 3 in duplicate. P values determined via ANOVA. **P < 0.01 and *** P < 0.001 compared with WT cells with same treatment (Student’s t-test). D–F: vehicle, chrysin, or digoxin-treated cells were measured at baseline, after 10 mM glucose addition to stimulate glycolysis, after 1 µM oligomycin addition to inhibit oxidative phosphorylation, and after 50 mM 2-deoxy-glucose (2-DG) to inhibit glycolysis. Error bars represent SE; n = 3 in duplicate. *P < 0.05, **P < 0.01, and ***P < 0.001, HEK293-β₂AR vs. HEK293-WT (Student’s t-test).

Fig. 5.

Hypoxia blunts cAMP response to isoproterenol and salbutamol in vitro. A and B: human embryonic kidney 293-β₂-adrenergic receptor (HEK293-β₂AR) cells were exposed to hypoxia (2% O₂) or normoxia for 2 h then stimulated with isoproterenol or salbutamol for 5 min. cAMP was measured via fluorescent assay. Data were normalized to vehicle normoxic condition. Error bars represent SE; n = 3–5. *P < 0.05 and **P < 0.01, hypoxia vs. normoxia (paired t-test). C: HEK293-β₂AR cells were exposed to hypoxia (2% O₂) or normoxia for 2–24 h then stimulated with isoproterenol (10 µM) or salbutamol (10 µM) for 5 min. Data were normalized to vehicle normoxic condition. Error bars represent SE; n = 3–5. *P < 0.05, **P < 0.01 and ***P < 0.001 compared with vehicle (Bonferroni posttest). D: HEK293-β₂AR cells were exposed to hypoxia or normoxia for 2 h and then βAR density on plasma membranes and endosomes was measured via [¹²⁵I]-cyanopindolol binding. Error bars represent SE; n = 7. There were no significant differences between any condition (Student’s t-test). E: theoretical model of βAR regulation based on the data, suggesting hypoxia shifts the equilibrium toward hypoxia-inducible factor 1 (HIF-1) activation, reducing availability of R* and thus blunting cAMP signaling. R, inactive conformational state; R*, cAMP-active conformational state; R**, HIF-1 active conformational state.

Fig. 6.

Pulmonary arterial hypertension (PAH) patients with the phenotype of high RV glucose uptake have more severe disease. A: patients with PAH (n = 30) underwent 2-[¹⁸F]fluoro-2-deoxy-d-glucose (FDG)-PET scanning to obtain standardized uptake values (SUVs) of glucose in the right ventricle (RV) determined relative to the left ventricle (LV), echocardiogram to estimate cardiac functions and pulmonary vascular resistance (PVR), and blood draw for measure of serum endothelin-1 and red blood cell counts (RBC). B and C: RV/LV SUV correlates with endothelin-1 and RBC. D: patients were dichotomized into high or low RV glucose uptake based on their RV/LV SUV using the log-transformed median as a cutoff for high and low. E–G: PAH patients with high RV glucose uptake had higher right ventricular systolic pressure (RVSP), PVR, and endothelin-1 levels. Error bars represent SE. P values determined from Student’s t-test.

Fig. 7.

Mononuclear cells from pulmonary arterial hypertension (PAH) patients with the phenotype of high right ventricular (RV) glucose uptake do not produce cAMP in response to isoproterenol (ISO). A: mononuclear cells isolated from PAH patients were treated with 300 µM isoproterenol for 10 min. B: cAMP was measured via fluorescent assay. Error bars represent SE; n = 5–6 per group. P values determined from Student’s t-test. C: β-adrenergic receptor (βAR) density on mononuclear cells from high or low glucose uptake phenotypes of patients is similar. Mononuclear cells were incubated with biotinylated alprenolol, which was detected with phycoerythrin-conjugated streptavidin. Cells were analyzed via flow cytometry to determine relative βAR density based on the median fluorescence intensity (MFI). Error bars represent SE. P values determined from Student’s t-test. D: model of βAR equilibrium in the high glucose uptake PAH phentoype. Patients who have high RV glucose uptake have their βAR shifted toward R** (glycolytic metabolism), and thus less R* availability and inability to activate cAMP signaling. HIF-1, hypoxia-inducible factor 1; R, inactive conformation; R*, conformation that activates cAMP; R**, conformation that activates HIF-1.

Fig. 8.

Pulmonary arterial hypertension (PAH) patients with the high right ventricular (RV) glucose uptake phenotype do not respond to carvedilol. PAH patients were all treated with low-dose carvedilol (3.125 mg 2× day) for 1 wk. A–D: right ventricular systolic pressure (RVSP) and pulmonary vascular resistance (PVR) were measured at baseline and after 1-wk treatment in the low (A and B) and high (C and D) RV glucose groups. Bars show means of populations; n = 15 for RVSP and 10 for PVR. P values determined from paired t-test. E: theoretical model of β-adrenergic receptor equilibrium in PAH. Individuals with high RV glucose uptake are shifted toward R** and have less R* available for carvedilol. HIF-1, hypoxia-inducible factor 1; R, inactive conformation; R*, conformation that activates cAMP; R**, conformation that activates HIF-1.