Triiodo-L-thyronine rapidly stimulates alveolar fluid clearance in normal and hyperoxia-injured lungs

Abstract

Rationale: Edema fluid resorption is critical for gas exchange and requires active epithelial ion transport by Na, K-ATPase and other ion transport proteins.

Objectives: In this study, we sought to determine if alveolar fluid clearance (AFC) is stimulated by 3,3',5 triiodo-L-thyronine (T(3)).

Methods: AFC was measured in in situ ventilated lungs and ex vivo isolated lungs by instilling isosmolar 5% bovine serum albumin solution with fluorescein-labeled albumin tracer and measuring the change in fluorescein isothiocyanate-albumin concentration over time.

Measurements and main results: Systemic treatment with intraperitoneal injections of T(3) for 3 consecutive days increased AFC by 52.7% compared with phosphate-buffered saline-injected control rats. Membranes prepared from alveolar epithelial cells from T(3)-treated rats had higher Na, K-ATPase hydrolytic activity. T(3) (10(-6) M), but not reverse T(3) (3,3',5' triiodo-L-thyronine), applied to the alveolar space increased AFC by 31.8% within 1.5 hours. A 61.5% increase in AFC also occurred by airspace instillation of T(3) in ex vivo isolated lungs, suggesting a direct effect of T(3) on the alveolar epithelium. Exposure of rats to an oxygen concentration of greater than 95% for 60 hours increased wet-to-dry lung weights and decreased AFC, whereas the expression of thyroid receptor was not markedly changed. Airspace T(3) rapidly restored the AFC in rat lungs with hyperoxia-induced lung injury.

Conclusions: Airspace T(3) rapidly stimulates AFC by direct effects on the alveolar epithelium in rat lungs with and without lung injury.

Figure 1.

Effect of intraperitoneal injections of 30 μg triiodo-l-thyronine (T₃) for 3 consecutive days on alveolar fluid clearance from rat lungs. Alveolar fluid clearance was measured over 90 minutes in ventilated animals. Data are presented as a box and whiskers plot showing median, 25th percentile, and 75th percentile, with the whiskers demonstrating the range. *P < 0.05 versus control. PBS = phosphate-buffered saline.

Figure 2.

Effect of intraperitoneal (IP) injections of 30 μg of triiodo-l-thyronine (T₃) for 3 consecutive days on peak Na,K-ATPase hydrolytic activity. Type 2 alveolar epithelial cells were isolated from T₃-treated rats. Na,K-ATPase activity was measured on cell membranes preparations on the same day by measuring the generation of inorganic phosphate under V̇max conditions. Cell isolation and Na,K-ATPase activity were measured in two animals in each group on the same day. Each data point represents the ratio of individual value to mean of controls for that day. The solid lines represent the median value. *P < 0.05 versus controls (IP phosphate-buffered saline [PBS]).

Figure 3.

Effect of intraperitoneal triiodo-l-thyronine (T₃) on steady-state Na,K-ATPase α₁ subunit protein levels. Alveolar epithelial cell isolation and cell membrane preparation as in Figure 2. Protein separation was done on 7.5% sodium dodecyl sulfate gel under denaturing condition after equal loading. Data are presented as medians, with individual time point presented as ratio to tubulin, P = 0.11 versus control. IP = intraperitoneal; PBS = phosphate-buffered saline.

Figure 4.

Effect of intraperitoneal (IP) triiodo-l-thyronine (T₃) on steady-state Na,K-ATPase β₁ subunit protein levels. Western blot of Na,K-ATPase β₁ subunit protein was performed after cell isolation from rats with or without in vivo T₃ treatment as in Figure 2. Enzymatic deglycosylation was performed using peptide-N-glycosidase (PNGAse) F and separated on 10% sodium dodecyl sulfate gel under denaturing conditions. Protein isolates from kidney were used as positive controls. As expected, with deglycosylation (Dgly), the size of the α₁ subunit protein shifts from 45–50 kD (arrows) to 30 kD (arrows). For alveolar epithelial cell (but not kidney) lysates, deglycosylation was not complete and there was a decrement in the total immunoreactivity, as previously reported (25). A modest increase in the levels of total and deglycosylated Na,K-ATPase β₁ subunit protein was seen in two independent experiments.

Figure 5.

Effect of intrapritoneal triiodo-l-thyronine (T₃) treatment on Na,K-ATPse mRNA levels. T₃ treatment and cell isolation were performed as in Figure 2. Steady-state mRNA levels measured using reverse transcriptase–polymerase chain reaction with α₁ and β₁ Na,K-ATPase-specific primers. Each time point represents ratio to tubulin. *P < 0.05 versus controls. PBS = phosphate-buffered saline.

Figure 6.

Effect of airspace instillation of triiodo-l-thyronine (T₃) on alveolar fluid clearance in in situ ventilated rat lungs. Alveolar fluid clearance was measured in ventilated rats and data are presented as median and range as in Figure 1. A concentration of 10⁻⁶ M T₃ or reverse T₃ (rT₃) was included in the isosomolar bovine serum albumin solution to determine the effect on alveolar fluid clearance by direct instillation into the airspace. The data were log-transformed and analysis of variance was performed on the transformed data. Post hoc testing used the least significant difference method; *P < 0.05, airspace T₃ versus control or rT₃.

Figure 7.

Airspace instillation of triiodo-l-thyronine (T₃) increases alveolar fluid clearance in normal and injured ex vivo rat lungs. Alveolar fluid clearance was measured in isolated ex vivo lungs; data are presented as median and range as in Figure 1. Statistical analysis performed was similar to Figure 6. Intact rat lungs had a higher rate of removal of alveolar fluid when 10⁻⁶ M T₃ was included in the instillate (*P < 0.05 vs. control intact lungs). With exposure to an oxygen concentration of more than 95%, there is a numerical trend toward a decrease in alveolar fluid clearance (P = 0.09), but inclusion of 10⁻⁶ M T₃ in the instillate increased alveolar fluid clearance; solid diamond, P < 0.05 versus hyperoxia.

Figure 8.

Increase in wet:dry lung weight with 60 hours of exposure to hyperoxia. Rats were exposed to an oxygen concentration of more than 95% for 60 hours and wet-to-dry lung weights were measured. Each data point represents individual wet:dry weight. Solid lines represent the median value. *P < 0.05 versus no hyperoxia exposure.

Figure 9.

Lung injury caused by exposure to an oxygen concentration of more than 95% for 60 hours does not alter the expression of thyroid receptors. Type 2 alveolar epithelial cells were isolated from rats exposed to an oxygen concentration of more than 95%. Type 2 alveolar epithelial cells from rats maintained on room air were used as controls. (A) Freshly isolated cells were cytospun on glass slides for immunofluorescence (higher dilutions were used for injured cells). No primary controls had any immunofluorescence (data not shown). (B) Cell lysates from these cells were separated on 12.5% sodium dodecyl sulfate gel under denaturing conditions. Protein isolates from the liver and heart were used as positive controls and HeLa cells were used as negative controls because they have minimal protein expression for the thyroid hormone receptors. (C) More than one isoform of thyroid receptor (TR) α and β were detected by Western blotting, but no significant difference in the total amount of thyroid receptor was detected by densitometry. H = hyperoxia; N = normoxia.

Figure 9.

Lung injury caused by exposure to an oxygen concentration of more than 95% for 60 hours does not alter the expression of thyroid receptors. Type 2 alveolar epithelial cells were isolated from rats exposed to an oxygen concentration of more than 95%. Type 2 alveolar epithelial cells from rats maintained on room air were used as controls. (A) Freshly isolated cells were cytospun on glass slides for immunofluorescence (higher dilutions were used for injured cells). No primary controls had any immunofluorescence (data not shown). (B) Cell lysates from these cells were separated on 12.5% sodium dodecyl sulfate gel under denaturing conditions. Protein isolates from the liver and heart were used as positive controls and HeLa cells were used as negative controls because they have minimal protein expression for the thyroid hormone receptors. (C) More than one isoform of thyroid receptor (TR) α and β were detected by Western blotting, but no significant difference in the total amount of thyroid receptor was detected by densitometry. H = hyperoxia; N = normoxia.

Figure 9.

Lung injury caused by exposure to an oxygen concentration of more than 95% for 60 hours does not alter the expression of thyroid receptors. Type 2 alveolar epithelial cells were isolated from rats exposed to an oxygen concentration of more than 95%. Type 2 alveolar epithelial cells from rats maintained on room air were used as controls. (A) Freshly isolated cells were cytospun on glass slides for immunofluorescence (higher dilutions were used for injured cells). No primary controls had any immunofluorescence (data not shown). (B) Cell lysates from these cells were separated on 12.5% sodium dodecyl sulfate gel under denaturing conditions. Protein isolates from the liver and heart were used as positive controls and HeLa cells were used as negative controls because they have minimal protein expression for the thyroid hormone receptors. (C) More than one isoform of thyroid receptor (TR) α and β were detected by Western blotting, but no significant difference in the total amount of thyroid receptor was detected by densitometry. H = hyperoxia; N = normoxia.