Adenosine regulation of alveolar fluid clearance

Abstract

Adenosine is a purine nucleoside that regulates cell function through G protein-coupled receptors that activate or inhibit adenylyl cyclase. Based on the understanding that cAMP regulates alveolar epithelial active Na(+) transport, we hypothesized that adenosine and its receptors have the potential to regulate alveolar ion transport and airspace fluid content. Herein, we report that type 1 (A(1)R), 2a (A(2a)R), 2b (A(2b)R), and 3 (A(3)R) adenosine receptors are present in rat and mouse lungs and alveolar type 1 and 2 epithelial cells (AT1 and AT2). Rat AT2 cells generated and produced cAMP in response to adenosine, and micromolar concentrations of adenosine were measured in bronchoalveolar lavage fluid from mice. Ussing chamber studies of rat AT2 cells indicated that adenosine affects ion transport through engagement of A(1)R, A(2a)R, and/or A(3)R through a mechanism that increases CFTR and amiloride-sensitive channel function. Intratracheal instillation of low concentrations of adenosine (< or =10(-8)M) or either A(2a)R- or A(3)R-specific agonists increased alveolar fluid clearance (AFC), whereas physiologic concentrations of adenosine (> or =10(-6)M) reduced AFC in mice and rats via an A(1)R-dependent pathway. Instillation of a CFTR inhibitor (CFTR(inh-172)) attenuated adenosine-mediated down-regulation of AFC, suggesting that adenosine causes Cl(-) efflux by means of CFTR. These studies report a role for adenosine in regulation of alveolar ion transport and fluid clearance. These findings suggest that physiologic concentrations of adenosine allow the alveolar epithelium to counterbalance active Na(+) absorption with Cl(-) efflux through engagement of the A(1)R and raise the possibility that adenosine receptor ligands can be used to treat pulmonary edema.

Fig. 1.

Adenosine receptor expression in lung and alveolar epithelial cells. (A) RT-PCR for A₁R, A_2aR, A_2bR, and A₃R in total RNA harvested from (left to right) mouse AT2 cells, mouse distal lung, rat AT1 cells, rat AT2 cells, rat distal lung. (B) LCM. Photomicrographs show alveoli before and after microdissection. A photo of representative cells collected is shown (Center). Graph is A₁R, A_2aR, A_2bR, and A₃R mRNA expression (n = 6 rats) measured by using real-time, quantitative RT-PCR, and normalized to GAPDH mRNA. (C) Western blot analysis of AR expression (left to right) mouse AT2 cell homogenates, peripheral lung homogenates, whole-cell membranes from peripheral lung (T), peripheral lung membrane enriched for apical (A), and basolateral (B) membrane domains. Blots for A₁R and A_2aR are 10 μg of protein per lane, and A_2bR and A₃R are 20 μg of protein per lane. (D) Western blot analysis of AR expression (left to right) rat AT1 and AT2 cell homogenates, peripheral lung homogenates, whole cell membranes from peripheral lung (T), peripheral lung membrane enriched for apical (A) and basolateral (B) membrane domains. Blots for A₁R and A_2aR used 10 μg of protein per lane, and A_2bR and A₃R used 20 μg of protein per lane.

Fig. 2.

A₁R and A_2aR function in rat AT2 cells. (A) Change in whole-cell cAMP concentration in response to the A₁R agonist CCPA (10⁻⁵ M for 10 min). Cells were pretreated with the adenylyl cyclase activator forskolin (2 μM for 10 min) with and without pertussis toxin (100 ng/ml for 4 h) before addition of CCPA for 10 min. ∗, P = 0.03 vs. cells treated with vehicle only (Ctl), ∗∗, P < 0.01 vs. cells treated with forskolin only. (B) Whole-cell cAMP concentration in rat AT2 cells treated with incremental concentrations of the A_2aR agonist CGS 21680 (10 min). ∗, P < 0.02 vs. vehicle-treated controls (Ctl).

Fig. 3.

Electrophysiologic studies in rat AT2 cell monolayers. (A) Typical tracing of short circuit current (I_sc) before and after addition of adenosine and amiloride. (B) Change in short circuit current (ΔI_sc) across high-resistance monolayers of rat AT2 cells after 4 d in culture in the presence or absence of adenosine. Change is calculated as adenosine-induced current − baseline current measured in the presence of vehicle (water). ∗, P < 0.009 vs. baseline current. (C) Monolayer resistance of adenosine- or vehicle (control)-treated AT2 cells. Baseline values for all I_sc measurements were obtained after stabilization of I_sc and before addition of adenosine or vehicle. (D) Change in short circuit current (ΔI_sc) across monolayers of rat AT2 cells treated with the doses shown of the A₁R agonist CPA. ∗, P < 0.01 vs. control (n = 3). (E) ΔI_sc produced by AT2 cells treated with the doses shown of the A_2aR agonist CGS 21680. ∗, P < 0.04 vs. baseline (n = 3). (F)(ΔI_sc produced by rat AT2 cells concomitantly treated with (adenosine, 10⁻⁶ M) and the A_2aR antagonist ZM 241385 (10⁻⁶ M). ∗, P < 0.01 vs. vehicle (water)-treated control (n = 3). (G) ΔI_sc in rat AT2 cells treated with the nonspecific type 2 receptor agonist NECA (3 × 10⁻⁶ M) with and without the A_2bR-specific antagonist MRS 1706 (10⁻⁵ M). n = 3 filters. (H) ΔI_sc from baseline in rat AT2 cells treated with the A₃R-specific agonist MECA at the doses shown. n = 3 filters. ∗, P < 0.02 vs. baseline I_sc.

Fig. 4.

Effect of adenosine on Na⁺ and Cl⁻ transporter. (A) Tracing from a representative study of cells with basal membranes permeabilized with amphotericin B (10 μM) before application of adenosine (10⁻⁶ M). (B) Net effect of amiloride (10 μM) on I_sc in permeabilized, adenosine (10⁻⁶ M)- treated cells. Data are peak I_sc of basally permeabilized AT2 cells in the presence of an apical-to-basal Na⁺ gradient (145:25 mM). n = 3 (adenosine) or 4 (control and amiloride) filters per condition. ∗, P = 0.04 vs. baseline I_sc. (C) I_sc across monolayers of AT2 cells treated with a fixed dose of adenosine (10⁻⁴ M) before and during application of incremental concentrations of amiloride. Data are normalized to maximal I_sc for each condition. n = 4 (control) and 5 (adenosine). (D) I_scs in AT2 cells treated with amiloride (10 μM) before application of the indicated concentrations of adenosine. n = 5 filters. Base, baseline current measured after stabilization and before addition of amiloride. ∗, P = 0.001 vs. baseline, ∗∗, P = 0.0.04 vs. amiloride-treated cells; ∗∗∗, P < 0.005 vs. amiloride-treated cells. (E) Effect of CFTR inhibition on I_sc in rat AT2 cells. Baseline I_sc was measured before addition of amiloride (10⁻⁵ M), followed by adenosine (10⁻⁵ M) and then CFTR_inh-172 (CFTR_i, 50 μM) or just CFTR_inh-172. I_scs shown were measured after stabilization of I_sc. n = 5 filters. ∗, P = 0.05 vs. baseline; ∗∗, P < 0.05 vs. amiloride and CFTR_inh-172; ∗∗∗, P < 0.05 vs. all other groups.

Fig. 5.

AFC in C57bl6 mice. (A) Adenosine effects on AFC. n = 8 mice per group. ∗, P = 0.03 vs. vehicle-treated controls (Ctl). (B) AFC in mice treated with the A₁R agonist CPA (10⁻⁶ M, n = 5). ∗, P = 0.001 vs. vehicle-treated controls (Ctl). (C) AFC in the presence of the nonspecific A₂ agonist NECA (10⁻⁴ M) with and without the A_2aR antagonist CSC-caffeine (10⁻⁶ M). A_2bR function was assessed by using a dose of adenosine that increases AFC (10⁻⁸ M) with and without the A_2bR antagonist MRS 1706 (10⁻⁶ M). n = 8 mice per group. ∗, P < 0.02 vs. vehicle-treated control (Ctl). (D) A₃R function was assessed by using the A₃R agonist IB-MECA at the doses shown. n = 8 mice per group. ∗, p = 0.001 vs. vehicle-treated controls (Ctl). (E) Alveolar fluid clearance in mice with targeted deletions of the A₁R (A₁R^−/−) in the presence and absence of a dose of adenosine that decreases AFC (10⁻⁴ M). n = 8 mice per group. P = 0.04 vs. untreated A₁R^−/− mice. (F) Effect of Cl⁻ transport inhibitors glibenclamide (10⁻⁶ M) with bumetinide (10⁻⁶ M) (G+B) or CFTR_inh-172 (CFTR_i,10⁻⁶ M) on adenosine (10⁻⁴ M) induced reduction of AFC. n = 6 mice. ∗, P = 0.03 vs. controls treated with vehicle only (Ctl). (G). Wet–dry weight ratios of mice treated with the doses of adenosine shown (in 50 μl of 0.9% NaCl, intratracheal) or an equal volume of vehicle (0.9% NaCl) for 30 min (Ctl). The role of CFTR in this model was assessed by instillation of the CFTR inhibitor CFTR_inh-172 (CFTR_i, 10⁻⁶ M) with and without adenosine (10⁻⁴ M). n = 4 mice. ∗, P = 0.002 vs. vehicle-treated control (Ctl).