Abstract
Catecholamines, including β-adrenergic and dopaminergic neurotransmitters, have an essential role in regulating the “fight or flight” reflex and also affects immune cell proinflammatory action. However, little is known about whether catecholamines prevent dysfunction of metabolic pathways associated with inflammatory organ injury, including development of acute lung injury (ALI). We hypothesize that selected catecholamines may reduce metabolic alterations in LPS-stimulated macrophages and in the lungs of mice subjected to endotoxin-induced ALI, a situation characterized by diminished activity of AMP-activated protein kinase (AMPK). We found that activation of the dopamine 1 receptor (D1R) with fenoldopam, but not stimulation of adrenergic receptors with norepinephrine, resulted in a robust activation of AMPK in peritoneal macrophages, human monocytes, or alveolar epithelial cells (AECs). Such AMPK activation was mediated by a phospholipase C (PLC)–dependent mechanism. Unlike norepinephrine, D1R activation also prevented Thr172–AMPK dephosphorylation and kinase inactivation in LPS-treated macrophages. Furthermore, we show that a culture of AECs with either fenoldopam or the AMPK activator metformin effectively diminished IL-1β–induced release of adverse paracrine signaling, which promotes the macrophage proinflammatory response. In vivo, fenoldopam reduced the severity of LPS-induced ALI, including development of pulmonary edema, lung permeability, and production of inflammatory cytokines TNF-α, MIP-2, or KC and HMGB1. Fenoldopam also prevented AMPK dephosphorylation in the lungs of LPS-treated mice and prevented loss of mitochondrial complexes NDUFB8 (complex I) and ATP synthase (complex V). Collectively, these results suggest that dopamine is coupled to AMPK activation, which provides a substantial anti-inflammatory and bioenergetic advantage and reduces the severity of endotoxin-induced ALI.
Keywords: ARDS, catecholamines, dopamine, immune homeostasis, LPS, pulmonary
Introduction
Dopamine, norepinephrine, and epinephrine are the most abundant catecholamines. They are produced by enzymatic conversion of tyrosine, predominantly in the adrenal medulla and central nervous system [1, 2]. Besides a regulatory role in the “fight or flight” reflex, catecholamines also affect pro- and anti-inflammatory responses in immune cells and peripheral tissues, mediated by the variety and bioavailability of adrenergic and dopaminergic receptors [2–4]. For example, adrenergic pro- and anti-inflammatory signaling is dependent on the abundance of α and β receptors [5, 6]. Besides adrenergic signaling, recent studies have underscored the importance of dopaminergic pathways in moderating inflammatory conditions in experimental models of organ injury [7, 8]. The physiologic effects of dopamine are mediated by dopamine receptors that consist of the D1-like (D1, D5) and the D2-like (D2, D3, D4) subtypes. Recently, D1 dopaminergic signaling has been shown to diminish mortality in experimental sepsis, an important predisposition for development of ARDS [8–10]. Although anti-inflammatory action can be considered a major effect of catecholamines, preservation of immune and peripheral tissue metabolic and bioenergetic homeostasis may have an equally important effect in organ injury. In particular, metabolic reprogramming and loss of bioenergetic plasticity of immune cells are related to mitochondrial dysfunction and may contribute to development and likely insufficient resolution of inflammatory conditions [11]. Recent studies suggest that the bioenergetic profile is differentially regulated by α- or β-adrenergic receptor signaling pathways in human PBMCs [12]. However, it is not known whether selective catecholamines affect the function of major bioenergetic sensors and metabolic regulators, such as AMPK.
The ability of AMPK to sense energy demand and preserve bioenergetic and redox homeostasis suggests that AMPK is a plausible target in sepsis, hemorrhage, or other inflammatory conditions associated with development of ARDS [13–15]. AMPK is a serine/threonine protein kinase that consists of the α-catalytic subunit and β- and γ-regulatory subunits. The AMPK α/β/γ heterotrimer has a unique mechanism of activation during bioenergetic imbalance, which may be triggered by limited access to oxygen and nutrients [16]. The activation process is initiated by binding of AMP and ADP to the γ-subunit [17–19] that allows for phosphorylation of the AMPK α subunit by upstream kinases [16, 20, 21]. Once activated, AMPK effectively preserves energy expenditure by switching from anabolic to catabolic metabolism [16]. Pharmacologic AMPK activators, for example, AICAR and metformin, have substantial protective effects on the liver, kidney, or heart in murine models of inflammatory organ injury [15, 22–25]. Besides anti-inflammatory action, AMPK activation is linked to stimulation of autophagy/mitophagy, mitochondrial biogenesis, and normalization of mitochondrial redox status [26–29]; pathways known to be dysregulated in sepsis and ALI.
Previous studies indicate that α- or β-adrenergic pathways are implicated in regulating AMPK activity and glucose homeostasis in skeletal muscle cells and adipocytes, respectively [30, 31]. However, the potential effect of catecholamines on AMPK activity in immune cells and lung epithelial cells during inflammatory conditions remains to be determined. We hypothesized that activation of AMPK by specific adrenergic and/or dopaminergic systems would affect the severity of endotoxin-induced ALI.
MATERIALS AND METHODS
Mice
Male C57BL/6 mice were purchased from the National Cancer Institute (Frederick, MD, USA). Mice 10–12 wk old were used for experiments. Mice were given food and water ad libitum and kept on a 12-h light–dark cycle. All experiments were conducted in accordance with approved protocols by the University of Alabama at Birmingham Animal Care and Use Committee.
Reagents and Abs
The dopamine D1 receptor agonist fenoldopam, m-3M3FBS, IL-1β, and anti-HMGB1 Ab were purchased from R&D Systems (Minneapolis, MN, USA). Histopaque, LPS, dopamine, norepinephrine, salbutamol, and phenylephrine were obtained from Sigma-Aldrich (St. Louis, MO, USA). Antibodies for phospho-Thr172–AMPK, total AMPK, and phospho-Ser79–ACC were obtained from Cell Signaling Technologies (Danvers, MA, USA). HRP-conjugated β-actin Ab was obtained from Santa Cruz Biotechnologies (Santa Cruz, CA, USA). Custom Ab mixtures and negative selection columns for mouse neutrophil isolation were obtained from Stem Cell Technologies (Vancouver, BC, Canada). Dispase, Anti-Mouse CD 16/32 and Anti-Mouse CD 45 were obtained from BD Bioscience (San Jose, CA, USA). MACS human CD16 Microbeads was obtained from Miltenyi Biotec Inc. (Auburn, CA, USA). PLC inhibitor U-73122 was purchased from Cayman Chemical (Ann Arbor, MI, USA). DMEM was obtained from Corning (Cellgro; (Manassas, VA, USA). RPMI-1640 was purchased from GE Healthcare Life Sciences (Township, NJ, USA). FBS was purchased from Atlanta Biologicals (Flowery Branch, GA, USA). F12K medium was purchased from Gibco (Grand Island, NY, USA).
Peritoneal macrophages, neutrophils, and monocytes isolation and culture
Peritoneal macrophages were isolated, as previously described [32]. Macrophages were elicited in 10–12-wk-old mice by i.p. application of Brewer thioglycollate. Cells were collected 4 d after thioglycollate injection. Peritoneal macrophages were cultured in RPMI-1640 media supplemented with 8% FBS at 37°C. In select experiments macrophages were incubated with RPMI-1640 media supplemented with 0.5% FBS for 2 h before LPS exposure, as indicated in the figure legends. Bone marrow neutrophils were isolated using a negative selection method, as previously described [33]. Neutrophil purity was consistently >97%, as determined by Wright-Giemsa–stained cytospin preparations. Neutrophils were cultured in RPMI-1640 medium containing 8% FBS and treated as indicated in the figure legends. Neutrophil viability under experimental conditions was determined by trypan blue staining and was consistently >95%.
Monocyte isolation was processed according to University of Alabama, Birmingham, Institutional Review Board–approved protocols. Monocytes were isolated from blood samples using MACS Microbeads, selective for CD14+CD16+ cells, and in accord with the manufacturer’s recommended protocol (Miltenyi Biotec). Monocytes were cultured in RPMI-1640 media supplemented with 8% FBS at 37°C.
Mouse alveolar lung epithelial cell isolation and culture
Epithelial cells were isolated, as previously described [34]. Lungs were perfused through the heart using PBS. Dispase was instilled into the lungs through the trachea, followed by digestion for 60 min at room temperature. Lungs were minced, incubated in DMEM containing DNase I, and then pipetted and passed through sequentially smaller pore size filters. Epithelial cells were collected from the cell suspension using the negative selection process, and cell suspension was plated overnight. Next, nonadherent epithelial cells were transferred to collagen-coated culture plates and cultured in DMEM medium with 8% FBS. In selected experiments, human lung epithelial cell line A549 cells (ATCC, Manassas, VA, USA) were cultured and treated in F12K supplemented with 8% FBS.
Cytokine ELISA
ELISA was used to measure cytokine levels in culture media and BAL fluids, as previously described [33]. Levels of TNF-α, MIP-2, and KC were determined using commercially available ELISA kits (R&D Systems) according to manufacturer’s instructions.
Western blot analysis
Western blot analysis was performed as described previously [35]. Each experiment was carried out 3 or more times with cell populations obtained from separate groups of mice. In selected experiments, BAL fluids (30 μl) were mixed with Laemmli sample buffer (Boston BioProducts, Ashland, MA, USA) and boiled for 5 min followed by Western Blot analysis with anti-HMGB1 Ab.
A mouse model for endotoxin-induced lung injury
Lung injury was induced by i.t. administration of LPS (2 mg/kg), as previously described [22, 33]. Characterization of lung injury was by neutrophil infiltration into the interstitium and airways of the lungs, interstitial edema development, and increased proinflammatory cytokine production. In addition to mononuclear/neutrophilic infiltrates, cellular debris and proteinaceous material were present in the alveolar space. Alveolar walls were also thickened, and the septa were edematous. To induce ALI, mice were anesthetized with isoflurane and then suspended by their upper incisors on a 60° incline board. The tongue was gently extended and LPS or PBS solution was deposited into the pharynx followed by aspiration to the lungs [36]. Fenoldopam (10 mg/kg, previously described [8]) in 0.5 ml of DMSO/saline or the control vehicle (DMSO/saline) was injected i.p. for 18 and 0.5 h before LPS i.t. instillation. Mice were euthanized 24 h after LPS administration. BALs were obtained by lavaging the lungs 3 times with 1 ml PBS.
Statistical analysis
Statistical analysis was performed using 3 or more independent experiments. Multigroup comparisons were performed using 1-way ANOVA with Tukey’s post hoc test. Values were normally distributed. Statistical significance was determined by the Student’s t test for comparisons between 2 groups. A value of P < 0.05 was considered significant. Analyses were performed on SPSS version 16.0 (IBM, Armonk, NY, USA) for Windows (Microsoft Corp., Redmond, WA, USA).
RESULTS
Dopaminergic and adrenergic signaling have distinct effects on AMPK activity in macrophages and neutrophils
In the first set of experiments, Thr172-AMPK phosphorylation (activation) was determined in murine peritoneal macrophages before and after stimulation of dopaminergic or adrenergic receptors. As shown in Fig. 1A and B, exposure to dopamine or fenoldopam, a potent and highly selective D1R dopaminergic agonist, resulted in a robust increase in T172-AMPK phosphorylation. Although the dopaminergic pathway increased AMPK activation, inclusion of the adrenergic agonist norepinephrine or salbutamol (a β2 adrenergic agonist) had little or no effect (Fig. 1D and E and Supplemental Fig. 1). Consistent with results obtained from murine macrophages, culture of human monocytes with fenoldopam also increased T172-AMPK phosphorylation, whereas norepinephrine had no effect (Fig. 1F). Next, we examined whether similar mechanisms are operational in neutrophils. However, we found that AMPK phosphorylation was unaltered in fenoldopam-treated neutrophils, whereas norepinephrine caused a substantial T172-AMPK dephosphorylation (Supplemental Fig. 2). These results suggest that the dopamine/D1R, but not norepinephrine/adrenergic, signaling pathway is coupled with AMPK activation in murine macrophages and human monocytes.
Figure 1. Dopaminergic and adrenergic signaling have distinct effects on AMPK activity in murine macrophages and human monocytes.
Western blot and quantitative analysis show the amounts of pT172–AMPK, total AMPK, and β-actin in mouse peritoneal macrophages treated dose dependently with dopamine (A), D1R agonist fenoldopam (B), or norepinephrine (D) for 2 h. Cells were also incubated with fenoldopam (C) (1 μM) or norepinephrine (E) (1 μM) for the indicated time. (F) Human blood monocytes were treated with fenoldopam (0 or 1 μM) or norepinephrine (0 or 1 μM) for 2 h followed by Western blot analysis of pT172–AMPK, total AMPK, and β-actin. Means ± sd, n = 3, *P < 0.05.
D1R and phospholipase C signaling increases AMPK activation in fenoldopam-treated macrophages
To determine the specific components of the D1R signaling implicated in AMPK activation, we examined the effects of adenylyl cyclase/cAMP, PKA, and PLC in mouse peritoneal macrophages. As shown in Fig. 2A, activation of adenylyl cyclase by forskolin or pretreatment with the phosphodiesterase inhibitor IBMX to prevent cAMP degradation did not activate AMPK. Of note, a modest activation of AMPK was observed after inclusion of PKA inhibitor H-9 (Fig. 2A). Next, we examined the effects of PLC. As shown in Fig. 2B, inclusion of PLC inhibitor U-73122 prevented the ability of fenoldopam to increase AMPK phosphorylation. In contrast to U-73122, culture of cells with PLC activator m-3M3FBS resulted in activation of AMPK (Fig. 2C). These results indicate that stimulation of D1 dopaminergic signaling and activation of PLC, but not adenylyl cyclase/cAMP or PKA, was implicated in activation of AMPK in peritoneal macrophages. It is important to note that such a mechanism of AMPK activation by PLC is likely cell population specific, as a recent study proposed that both cAMP/PKA and PLC may affect AMPK activity in skeletal muscle cells [37].
Figure 2. The D1R–PLC signaling axis, but not cAMP–PKA, induces AMPK activation in peritoneal macrophages.
(A) Cells were preincubated with forskolin (0 or 10 μM), H-9 (0 or 10 μM), or a combination of IBMX (100 μM) and forskolin (10 μM) for 60 min. Next, cells were treated with fenoldopam (0 or 1 μM) for an additional 2 h. pT172–AMPK, total AMPK, and β-actin are shown. (B) Macrophages were pretreated with PLC inhibitor U-73122 (0 or 5 μM) for 60 min and then incubated with fenoldopam (1 µM) for 2 h. (C) Macrophages were treated with PLC activator m-3M3FBS (0 or 5 μM) for 2 h followed by Western blot analysis of AMPK. Means ± sd, n = 3, *P < 0.05.
Stimulation of D1 dopaminergic pathway prevents T172-AMPK dephosphorylation in LPS-treated macrophages
Previous studies have shown that inflammatory conditions, including engagement of LPS/TLR4, are associated with T172-AMPK dephosphorylation and decreased kinase activity in macrophages and other cell populations [22, 25, 38, 39]. As shown in Fig. 3, exposure to LPS resulted in a time-dependent decrease in T172-AMPK phosphorylation. Importantly, pretreatment with fenoldopam diminished the effect of LPS (Fig. 3A and B). Of note, AMPK dephosphorylation was not affected by norepinephrine (Fig. 3C and D). These results suggest that selective activation of the D1 dopaminergic pathway preserved AMPK activity in LPS-treated macrophages.
Figure 3. Engagement of fenoldopam/D1R signaling prevents AMPK dephosphorylation in LPS-treated macrophages.
Peritoneal macrophages were cultured with fenoldopam (A and B) (0 or 1 μM) or norepinephrine (C and D) (0 or 1 μM) for 60 min followed by exposure to LPS (300 ng/ml) for the indicated time. Representative Western blots show the amounts of pT172-AMPK, total AMPK, and β-actin. Means ± sd, n = 3, *P < 0.05 compared with the control (untreated); #P < 0.05 compared with fenoldopam + LPS treatment for 30 or 60 min with LPS alone (30 or 60 min).
In additional experiments, we confirmed the anti-inflammatory effect of the D1R signaling pathway. In particular, pretreatment with the D1R agonist fenoldopam reduced TNF-α and MIP-2 production by LPS-stimulated macrophages (Fig. 4). Because anti-inflammatory effects have been previously linked to the cAMP-PKA signaling [40, 41], we tested whether H-9 (PKA inhibitor) moderates fenoldopam action in LPS-treated macrophages. However, PKA inhibitor did not diminish the ability of fenoldopam to reduce TNF-α production by LPS-stimulated macrophages (Supplemental Fig. 3). These findings suggest that PLC, but not PKA, mediates anti-inflammatory action of fenoldopam.
Figure 4. Activation of the D1 dopaminergic pathway reduces LPS-mediated macrophage proinflammatory cytokine production.
Macrophages obtained from 3 mice were pretreated with dopaminergic agonist fenoldopam (0 or 1 μM) for 60 min and then treated with LPS (0, 0.1, 1, or 10 ng/ml) for an additional 4.5 h. The amounts of TNF-α (A) and MIP-2 (B) cytokines in culture media were determined using ELISA. Means ± sd, n = 3 independent experiments, each with 4 biologic replicates. *P < 0.05.
Stimulation of D1R or direct AMPK activation diminished the adverse epithelial paracrine signaling on macrophage proinflammatory response
In addition to the effects of dopaminergic signaling in macrophages and neutrophils, we examined whether D1R stimulation affects AMPK activity in type II AECs. Primary mouse AECs were treated with D1R-agonist fenoldopam or AMPK-activator metformin. As shown in Fig. 5A and B, nearly a 3-fold increase of phosphoT172-AMPK was observed after treatment with fenoldopam for 4 h compared with the untreated (control) cells. Furthermore, we found that activation of AMPK also diminished the epithelial paracrine signaling, which promotes the macrophage proinflammatory response (Fig. 5C and D). In these experiments, AECs were incubated with metformin (0 or 300 μM) or fenoldopam (0 or 1 μM) for 2 h followed by inclusion of IL-1β (0 or 10 ng/ml) for an additional 4 h. Next, cells were washed to remove AMPK activators and IL-1β and then incubated for 24 h. Conditioned medium was further used to incubate peritoneal macrophages for 24 h, and the amounts of TNF-α were determined by ELISA. These results suggest that AMPK activation in epithelial cells reduced the effects of the paracrine signaling that caused macrophage proinflammatory activation.
Figure 5. D1R–AMPK axis diminished the adverse epithelial paracrine signaling that promotes macrophage proinflammatory response.
(A and B) Primary type II AECs were incubated with metformin (0 or 300 μM) or fenoldopam (0 or 1 μM) for 4 h followed by Western blot analysis of phospho-T172-AMPK and total AMPK. Means ± sd, n = 3, *P < 0.05 compared with the control (untreated). (C and D) AECs were pretreated with metformin or fenoldopam for 4 h followed by inclusion of IL-1β (0 or 10 ng/ml) for an additional 4 h. Cells were then washed, and the medium was collected after incubation for 24 h. Conditioned medium (1:1 normal:conditional medium) was used to treat peritoneal macrophages for 24 h followed by TNF-α ELISA. Means ± sd, n = 3, *P < 0.05 compared with the control (untreated cells).
Stimulation of D1R dopaminergic signaling diminished the severity of LPS-induced acute lung injury
Fenoldopam (10 mg/kg, i.p.) or vehicle (saline, i.p) was injected for 18 h, and then a second dose of fenoldopam or saline was applied 1 h before LPS i.t. instillation (2 mg/kg; i.t.). The amount of inflammatory cytokines in BAL fluids, the extent of pulmonary edema and lung permeability, as well as T172-AMPK phosphorylation status in lung homogenates were determined 24 h after exposure to LPS. Representative images of lung sections (Fig. 6A) demonstrate that fenoldopam effectively prevented LPS-mediated neutrophil accumulation (fenoldopam + LPS group) and, overall, preserved lung architecture compared with mice treated with LPS alone. Reduced pulmonary edema and vascular permeability were evidenced by a decrease in the wet-to-dry ratios and BAL protein content in fenoldopam and LPS mice compared with the LPS group (Fig. 6B). Consistent with results obtained from peritoneal macrophages (Fig. 4), fenoldopam also diminished BAL cytokines, including TNF-α, MIP-2, and KC (Fig. 7A). HMGB1, an important marker and mediator of lung injury, was significantly increased in BAL fluids of LPS-treated mice. Importantly, fenoldopam effectively prevented such accumulation (Fig. 7B and C). These results indicate that D1 dopaminergic agonist fenoldopam reduced the severity of endotoxin-induced ALI.
Figure 6. Activation of the D1R dopaminergic signaling pathway diminished severity of LPS-mediated ALI.
Mice were first treated with vehicle (control; saline 500 μl, i.p.) or fenoldopam (10 mg/kg; 500 μl, i.p.) for 18 h and a second dose of saline or fenoldopam was applied 15 min before i.t. instillation of LPS (saline or 2 mg/kg; 50 μl, i.t.). (A) Representative images show H&E-stained lung sections obtained from control mice or mice treated with fenoldopam, LPS, or a combination of fenoldopam and LPS. Scale bars, 100 µm (10×) or 1000 µm (40×). (B) Increase in lung wet-to-dry ratios, number of lung neutrophils in BAL fluids, and BAL proteins were obtained 24 h after exposure to LPS alone or a combination of LPS and fenoldopam. Means ± sd, n = 4, *P < 0.05 comparing fenoldopam + LPS to mice treated with LPS alone.
Figure 7. The D1R dopaminergic signaling agonist fenoldopam diminished production of proinflammatory cytokines in lungs of mice subjected to LPS-induced ALI.
(A) TNF-α, MIP-2, and KC were measured in BAL fluids obtained from control (saline), LPS, or fenoldopam and LPS–treated mice (as described in Fig. 6). Means ± sd, n = 5, *P < 0.05. Representative Western blot (B) and quantitative analysis (C) show the amounts of HMGB1 in BALs obtained from 3 sets of mice: control, LPS alone, or treated with a combination of fenoldopam and LPS. Means ± sd; n = 3; ***P < 0.001.
The D1R signaling pathway preserved AMPK activity along with major components of ETC complexes in lung tissue of mice subjected to LPS-induced ALI
The amounts of phospho-T172-AMPK and phospho-S79–ACC, an AMPK downstream target, were measured in whole lung homogenates obtained from vehicle- (saline), LPS-, or fenoldopam and LPS-treated mice. Western blot analysis of lung homogenates showed a marked decrease in T172-AMPK and S79-ACC phosphorylation after i.t. instillation of LPS, as compared with the control (vehicle) group (Fig. 8A and B). Importantly, application of fenoldopam was sufficient to prevent both AMPK and ACC dephosphorylation in mice treated with LPS. These results suggest that stimulation of the D1R dopaminergic pathway preserved AMPK activity in lungs of mice subjected to LPS (Fig. 9A).
Figure 8. The D1R dopaminergic agonist fenoldopam prevented T172-AMPK dephosphorylation in lungs of mice subjected to LPS-induced ALI.
Mice were treated with LPS or fenoldopam and LPS for 24 h (as described in Fig. 6). Representative Western blots (A) and quantitative analysis (B) show the extent of T172-AMPK and S79-ACC phosphorylation in 3 sets of lung homogenates. (C and D) The extent of major components of ETC complexes was determined in lung homogenates. Means ± sd, n = 3–4 mice/group, *P < 0.05.
Figure 9. (A) LPS/TLR4 engagement promotes AMPK dephosphorylation and decreased kinase activity in peritoneal macrophages.
(A) In turn, stimulation of dopaminergic D1R signaling with dopamine or fenoldopam followed by PLC activation prevented LPS-mediated dephosphorylation of AMPK. (B) Stimulation of D1R with fenoldopam and subsequent activation of AMPK diminished the adverse paracrine signaling from IL-1β–treated AECs and, therefore, prevented macrophage proinflammatory activation and reduced the severity of LPS-induced ALI.
As shown in Fig. 8C and D, a substantial decrease in major components of the mitochondrial ETC complexes occurred in lungs of LPS-treated mice, including NDUFB8 (complex I) and ATP synthase α subunit (complex V). Importantly, fenoldopam effectively prevented such loss of NDUFB8 and the ATP synthase α subunit. These findings are consistent with previous studies that showed that preservation of mitochondrial structure and function reduced the severity of LPS-induced lung injury [42, 43].
DISCUSSION
In this study, we found that dopamine-mediated stimulation of the D1R pathway was associated with AMPK activation in peritoneal macrophages, monocytes, and AECs. Activation of D1R signaling prevented T172-AMPK dephosphorylation (inactivation) when fenoldopam was applied before LPS exposure. We also found that fenoldopam-dependent activation of AMPK in AECs diminished the IL-1β–induced adverse paracrine signaling that promoted macrophage proinflammatory response. Furthermore, our results indicate that administration of fenoldopam effectively preserved AMPK activity along with major mitochondrial ETC components in lungs of mice subjected to i.t. instillation of LPS. Importantly, activation of the D1R-AMPK signaling axis before LPS administration reduced the severity of ALI.
The ability of D1R to preserve AMPK activity is likely an important event to reduce exaggerated macrophage proinflammatory activation and, therefore, diminish the extent of ALI in LPS-treated mice. This possibility is supported by recent studies that demonstrated the effects of AMPK activators metformin or AICAR to diminish LPS-mediated organ injury, including lung, heart, kidney, or liver injury [33, 44–48]. Besides anti-inflammatory effects associated with inhibition of the NF-κB signaling cascade, AMPK activation may also accelerate endothelial barrier recovery and decrease lung permeability, processes known to be dysregulated in sepsis and ARDS [22, 48, 49]. In addition to dopamine and AMPK anti-inflammatory effects, dopamine signaling has also been implicated in increasing a liquid clearance in lung epithelium [7, 50]. Of note, dopamine/D1R signaling may also improve lung mechanics because of airway smooth muscle relaxation [51]. Furthermore, the dopaminergic–vagal axis has been recently shown to increase survival in a murine model of polymicrobial sepsis [8].
Although dopamine-D1R signaling are linked to the cAMP/PKA anti-inflammatory signaling cascade [40, 41], we also observed cAMP/PKA-independent, but PLC-dependent, AMPK activation in macrophages (Fig. 9A). Given the importance of AMPK in diminishing macrophage proinflammatory activation, our results suggest that both cAMP/PKA and PLC/AMPK pathways may lessen detrimental inflammation synergistically in LPS-treated mice. Furthermore, the PLC/AMPK pathway may also prevent bioenergetic dysfunction in immune cells and in the lung tissue of mice subjected to ALI. Although AMPK activation in peritoneal macrophages occurs after stimulation of D1R-PLC axis, but not adenylyl cyclase/cAMP or PKA, such a mechanism of AMPK activation is likely specific to cell type. For example, both cAMP/PKA and PLC were implicated in AMPK activation in skeletal muscle cells [37]. Despite mechanistic differences and synergy between dopamine receptors, our findings suggest that AMPK activation is a plausible target for preventing development of ARDS and possibly other inflammatory conditions associated with organ dysfunction.
Our results show a benefit of fenoldopam/D1-AMPK axis, although previous studies have also demonstrated the importance of D2 receptors in LPS-induced ALI [7]. The exact crosstalk between D1 and D2 receptors, including implication of D2 deficiency on D1 signaling is not known. Although D1R and D2R have distinct signaling cascades, dopamine likely has a beneficial synergistic effect on ALI through use of both receptors. For example, D1R-mediated activation promotes the AMPK-bioenergetic pathway, whereas engagement of dopamine-D2R diminishes vascular lung permeability [7]. Of note, recent studies indicate a possible formation of D1R–D2R heteromer receptors and downstream effects mediated by phospholipase C‑mediated calcium signaling [52, 53].
Understanding the relationship between inflammation and metabolism creates an opportunity to develop effective pharmacological interventions, including the possibility of repurposing AMPK activators to reduce adverse inflammation in conjunction with preservation of metabolic homeostasis. Although both D1R and β-adrenergic pathways have a potent anti-inflammatory effect, D1R signaling may provide an advantage because of the activation of AMPK in immune cells and peripheral lung tissue. In addition to reducing LPS/TLR4-mediated neutrophil and macrophage proinflammatory activation [13, 22, 39], AMPK has much broader implications in regulating cellular bioenergetics and redox homeostasis in immune cells and lung tissue [16, 54]. Given that mitochondrial dysfunction is associated with unfavorable outcome of sepsis and sepsis-related ARDS [55, 56] and that AMPK affects mitochondrial quality control (autophagy/mitophagy) and biogenesis [26], AMPK activation is likely an important target for diminishing the severity of organ injury [26, 57]. Indeed, we found that fenoldopam effectively prevented the decrease of mitochondrial complex I (NDUFB8) and complex V (ATP synthase α-subunit) in mice subjected to i.t. instillation of LPS (Fig. 8C and D). This validates recently published findings that preservation of AMPK activity reduces the extent of mitochondrial dysfunction in lungs of septic mice [43] and renal injury in a murine model of diabetes [29].
Mortality rates from ARDS remain high (20–30%), as described in recent studies [58–61]. Despite progress in understanding mechanisms associated with development and perpetuation of acute lung injury, no pharmacological approach to diminish the severity or to improve survival from this condition is available for critically ill patients. Although selective catecholamines have been shown to prevent lung injury in experimental models, clinical trials with β-adrenergic agonists provided no benefit to patients with ARDS [62, 63]. The exact mechanism related to such limited efficacy of β-adrenergic agonists is not well understood. It is possible that inflammatory conditions, including hemorrhagic shock or sepsis, are associated with desensitization of adrenergic pathways [64–67]. Another possibility is that β-adrenergic signaling alone is not preserving AMPK function in inflammatory settings and, therefore, is not preventing metabolic and bioenergetic dysfunction of immune and stromal cells. In contrast, dopamine appears to have both anti-inflammatory effects and the capacity to stimulate AMPK activity in macrophages and in alveolar epithelial cells. Further studies should also delineate whether the bioavailability of D1R is affected in critically ill patients.
Taken together, our results suggest that engagement of dopamine signaling followed by AMPK activation may provide a substantial advantage linked to both AMPK anti-inflammatory and bioenergetic functions. These findings also suggest that activation of the dopamine–AMPK signaling pathway effectively protects the lungs in a murine model of endotoxin-induced ALI.
AUTHORSHIP
N.B.B. and Z.L. performed the experiments. N.B.B., J.-F.P., and J.W.Z. interpreted the results and drafted the manuscript. N.B.B. and J.W.Z revised and edited the final version of manuscript.
Supplementary Material
Acknowledgments
This study was supported by U.S. National Institutes of Health Grant HL107585 to J.W.Z.
Glossary
- ACC
acetyl-CoA carboxylase
- AEC
alveolar epithelial cell
- ALI
acute lung injury
- AMPK
AMP-activated protein kinase
- ARDS
acute respiratory distress syndrome
- BAL
bronchoalveolar lavage
- D1R
dopamine 1 receptor
- D2R
dopamine 2 receptor
- ETC
electron transport chain
- HMGB1
high mobility group box 1
- IBMX
Isobutylmethylxanthine
- i.t.
intratracheal
- KC
keratinocyte-derived chemokine
- PKA
protein kinase A
- PLC
phospholipase C
Footnotes
The online version of this paper, found at www.jleukbio.org, includes supplemental information.
SEE CORRESPONDING EDITORIAL ON PAGE 351
DISCLOSURES
The authors declare no conflicts of interest.
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