Pivotal role of the alpha(2A)-adrenoceptor in producing inflammation and organ injury in a rat model of sepsis

Abstract

Background: Norepinephrine (NE) modulates the responsiveness of macrophages to proinflammatory stimuli through the activation of adrenergic receptors (ARs). Being part of the stress response, early increases of NE in sepsis sustain adverse systemic inflammatory responses. The intestine is an important source of NE release in the early stage of cecal ligation and puncture (CLP)-induced sepsis in rats, which then stimulates TNF-alpha production in Kupffer cells (KCs) through the activation of the alpha(2)-AR. It is important to know which of the three alpha(2)-AR subtypes (i.e., alpha(2A), alpha(2B) or alpha(2C)) is responsible for the upregulation of TNF-alpha production. The aim of this study was to determine the contribution of alpha(2A)-AR in this process.

Methodology/principal findings: Adult male rats underwent CLP and KCs were isolated 2 h later. Gene expression of alpha(2A)-AR was determined. In additional experiments, cultured KCs were incubated with NE with or without BRL-44408 maleate, a specific alpha(2A)-AR antagonist, and intraportal infusion of NE for 2 h with or without BRL-44408 maleate was carried out in normal animals. Finally, the impact of alpha(2A)-AR activation by NE was investigated under inflammatory conditions (i.e., endotoxemia and CLP). Gene expression of the alpha(2A)-AR subtype was significantly upregulated after CLP. NE increased the release of TNF-alpha in cultured KCs, which was specifically inhibited by the alpha(2A)-AR antagonist BRL-44408. Equally, intraportal NE infusion increased TNF-alpha gene expression in KCs and plasma TNF-alpha which was also abrogated by co-administration of BRL-44408. NE also potentiated LPS-induced TNF-alpha release via the alpha(2A)-AR in vitro and in vivo. This potentiation of TNF-alpha release by NE was mediated through the alpha(2A)-AR coupled Galphai protein and the activation of the p38 MAP kinase. Treatment of septic animals with BRL-44408 suppressed TNF-alpha, prevented multiple organ injury and significantly improved survival from 45% to 75%.

Conclusions/significance: Our novel finding is that hyperresponsiveness to alpha(2)-AR stimulation observed in sepsis is primarily due to an increase in alpha(2A)-AR expression in KCs. This appears to be in part responsible for the increased proinflammatory response and ensuing organ injury in sepsis. These findings provide important feasibility information for further developing the alpha(2A)-AR antagonist as a new therapy for sepsis.

Figure 1. Upregulation of KC α_2A-AR expression in CLP-induced sepsis.

Gene expression of α_2A-adrenergic receptor (AR) in Kupffer cells isolated from animals at 2 h after cecal ligation and puncture (CLP) or sham-operation. Relative expression of mRNA was calculated by the ΔΔCt-method, and results expressed as fold change with respect to the housekeeping gene GAPDH. Values (n = 6/group) are presented as mean±SE and compared by Student's t-test. *P<0.05 vs. Sham.

Figure 2. Increased KC α₂-AR binding capacity and affinity in sepsis.

Changes in α₂-receptor binding of the radiolabeled specific ligand [³H]-yohimbine in rat Kupffer cells obtained from sham-operated and cecal ligation and puncture (CLP) animals (A). B_max and K_d were estimated using the the Scatchard analysis (B). The data points represent the average of three different experiments. Best fit analysis and two-way ANOVA showed that curves are different for Sham and CLP (P = 0.0036).

Figure 3. Stimulation with NE increases TNF-α release from KCs via α_2A-ARs.

Alterations of TNF-α after stimulation of isolated KCs with NE (20 nM) with or without BRL-44408 maleate (1 µM), a specific α_2A-AR antagonist, for 4 h. Percentage values (n = 6/group) are presented as mean±95% confidence and compared by ANOVA on Ranks and Student-Newman-Keul's method. *P<0.05 vs. Medium; #P<0.05 vs. NE.

Figure 4. Portal infusion of NE induces α_2A-AR-dependent TNF-α production in KCs.

In vivo Alterations in Kupffer cell gene expression (A) and protein levels of TNF-α (B) as well as serum TNF-α levels (C) after administration of NE, NE combined with BRL-44408 maleate for 2 h through the portal vein. A representative gene and ratio of TNF-α and housekeeping gene G3PDH are presented in panel (A). Values (n = 5/group) are presented as mean±SE and compared by one-way ANOVA and Student-Newman-Keul's method. *P<0.05 vs. Control; #P<0.05 vs. NE.

Figure 5. NE-mediated potentiation of LPS-mediated TNF-α release through the α_2A-AR.

BRL-44408 maleate blocks TNF-α production in LPS-stimulated KCs after co-incubation with NE in vitro and in vivo. (A) Alterations in TNF-α release from cultured KCs 24 h after stimulation with NE (20 nM) and LPS (100 ng/ml), with or without BRL-44408 (BRL, 1 µM). Data are presented as mean±SE (n = 8) and compared by one-way ANOVA and Student-Newman-Keuls test. *P<0.05 vs. Control; #P<0.05 vs. LPS alone; †P<0.05 vs. NE+LPS. (B) Alterations in plasma levels of TNF-α after administration of LPS (7.5 mg/kg, intra-peritoneal) and NE (20 µM NE for 2 h at 13 µl/min), with or without BRL-44408 (2.5 mg/kg BW, intra-portal). Data are presented as mean±SE (n = 4–6) and compared by one-way ANOVA and Student-Newman-Keul's method. *P<0.05 vs. Control; #P<0.05 vs. LPS alone; †P<0.05 vs. NE+LPS.

Figure 6. NE-mediated potentiation of p38 MAP kinase phosphorylation via α_2A-ARs.

(A) Alterations in p38 MAP kinase after 1 h culture with NE alone (20 nM), NE+BRL-44408 (BRL, 1 µM), or NE+pertussis toxin (PTX, 100 ng/ml). The relative percentage of phosphorylated p38/total p38 MAP kinase is presented as mean±95% confidence (n = 4–6) and compared by ANOVA on Ranks and Student-Newman-Keul's method. The Medium group is considered as 100%. *P<0.05 vs. Medium; #P<0.05 vs. NE alone. Representative gels are presented. (B) Enhanced activation of p38 MAP kinase after 1 h culture with a combination of NE (20 nM) and LPS (100 ng/ml). The relative percentage of phosphorylated p38/total p38 MAP kinase ratio is presented as mean±95% confidence (n = 4–6) and compared by ANOVA on Ranks and Student-Newman-Keul's method. Medium group is considered as 100%. *P<0.05 vs. Medium; #P<0.05 vs. LPS alone. Representative gels are also presented. (C) Suppression of TNF-α release from cultured KCs after stimulation with NE (20 nM)+LPS (100 ng/ml) with or without pertussistoxin (PTX, 100 ng/ml), a G_αi-protein inhibitor, or SB203580 (10 µM), a p38 MAP kinase inhibitor. Data are presented as mean±SE (n = 4–6) and compared by one-way ANOVA and Student-Newman-Keul's method. *P<0.05 vs. Medium; #P<0.05 vs. LPS+NE.

Figure 7. Beneficial effects of α_2A-AR inhibition in sepsis.

(A–B) BRL-44408 mediated suppression of TNF-α gene expression in Kupffer cells (A) and plasma concentrations (B) 20 h after CLP. (C–F) BRL-44408 mediated improvement of organ damage parameters. Rats underwent CLP and 20 h later ALT (C), AST (D), creatinine (E) and lactate (F) levels were measured as described in the methods. Values (n = 5/group) are presented as means±SE and compared by one-way ANOVA and Student-Newman-Keul's method. *P<0.05 vs. Sham; #P<0.05 vs. CLP+Vehicle.

Figure 8. The α_2A-AR inhibitor BRL-44408 improves survival in septic rats.

Rats underwent CLP (n = 20/group) and received either Vehicle treatment or BRL-44408 maleate iv 2.5 mg/kg BW. Cecums were removed 20 h later and animals observed for up to 10 days. *P<0.05 vs. Vehicle, Kaplan-Meyer logrank test.

Figure 9. BRL-44408 has no effects on MAP and heart rates in normal rats.

Effects of α_2A-AR inhibitor BRL-44408 on mean arterial pressure (MAP, A) and heart rates (B) in normal rats. Normal rats received either Vehicle treatment or BRL-44408 maleate iv 2.5 mg/kg BW over a period of 30 min. Data are presented as mean±SE (n = 5/group), and compared by Student's t-test. No statistical difference was found.