Serotonin decreases alveolar epithelial fluid transport via a direct inhibition of the epithelial sodium channel

Abstract

Hypoxia and epithelial stretch that are commonly observed in patients with acute lung injury have been shown to promote the release of serotonin (5-hydroxytryptamine, 5-HT) in vitro. However, whether 5-HT contributes to the decrease of alveolar epithelial fluid transport, which is a hallmark of lung injury, is unknown. Thus, we investigated the effect of 5-HT on ion and fluid transport across the alveolar epithelium. 5-HT caused a dose-dependent inhibition of the amiloride-sensitive current across primary rat and human alveolar epithelial type II cell monolayers, but did not affect Na(+)/K(+) ATPase function. Furthermore, we found that the 5-HT induced inhibition of ion transport across the lung epithelium was receptor independent, as it was not prevented by the blockade of 5-HT2R (5-HT receptor 2), 5-HT3R (5-HT receptor 3), or by pretreatment with an intracellular calcium-chelating agent, BAPTA-AM (1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetra(acetoxymethyl) ester). In addition, the stimulation of 5-HT1R (5-HT receptor 1), 5-HT2R (5-HT receptor 2), 5-HT4R (5-HT receptor 4), and 5-HT7R (5-HT receptor 7) failed to reproduce the 5-HT effect on amiloride-sensitive sodium transport. We ascertained that 5-HT directly inhibited the function of rat alphabetagamma epithelial sodium channel (ENaC), as determined by heterologous expression of rat ENaC in Xenopus oocytes that do not express endogenous ENaC nor 5-HT receptors (5-HTR). Exposure of mice to hypoxia for 1 hour induced a 30% increase of 5-HT secretion into the distal airways of mice. Finally, the intratracheal instillation of 5-HT inhibited the amiloride-sensitive fraction of alveolar fluid clearance in mice. Together, these results indicate that 5-HT inhibits the amiloride-sensitive fraction of the alveolar epithelial fluid transport via a direct interaction with ENaC, and thus can be an endogenous inhibitor of this ion channel.

Figure 1.

Serotonin (5-HT) decreases the amiloride-sensitive fraction of the short-circuit current (Isc) across rat distal lung epithelial cell monolayers. (A) 5-HT induces a rapid decrease in the Isc across primary cultures of polarized rat alveolar epithelial type II (ATII) cell monolayers. Rat ATII cell monolayers cultured at an air–liquid interface for 4 days were placed in Ussing chambers and exposed to 5-HT (4 mM), then to amiloride (10 μM). Exposure to amiloride further decreased the Isc across rat ATII cell monolayers. One representative of four experiments is shown. Open squares represent averaged tranepithelial resistances (±SD) before and after 5-HT treatment. The transepithelial resistance scale is displayed on the left. (B) Pretreatment with amiloride abolishes the 5-HT–induced decrease in Isc across primary cultures of polarized rat ATII cell monolayers. Rat ATII cell monolayers cultured at an air–liquid interface for 4 days were placed in Ussing chambers and exposed to amiloride (10 μM), then 5-HT (4 mM). Exposure to 5-HT (4 mM) did not further inhibit the Isc. In all experiments, the effect of 5-HT was reversible. All the experiments were performed at 37°C under symmetrical conditions with saline solution (140 mM NaCl, 4 mM KCl, 1 mM MgCl₂, 1 mM CaCl₂, 10 mM Hepes, and 15 mM glucose [pH = 7.3, adjusted with NaOH]). The EasyMount system, from Physiological Instruments, Inc. (San Diego, CA) was used for all experiments. The voltage was clamped at 0 mV during the whole experiments. One representative experiment is shown. For all experiments, histogram data are the means (±SD) of four experiments done in triplicate; *P < 0.05. NS, not significant; TER, transepithelial electrical resistance.

Figure 2.

5-HT induces a dose-dependent decrease in TEC across primary cultures of polarized rat ATII cell monolayers. Rat ATII cell monolayers cultured at an air–liquid interface for 4 days were exposed to 5-HT (250 μM−38 mM). All experiments were performed under symmetric conditions with saline solution (140 mM NaCl, 4 mM KCl, 1 mM MgCl₂, 1 mM CaCl₂, 10 mM Hepes, and 15 mM glucose [pH = 7.3, adjusted with NaOH]). The EasyMount system from was used for all experiments. The voltage was clamped at 0 mV during the whole experiments. The polynomial regression provided an IC₅₀ (half maximal inhibitory concentration) of 7.6 mM and a Hill coefficient of 0.96 (Microcal Origin Software 5.0; Origin Laboratories, Northampton, MA). Curve fitting was performed with a logistic Hill regression. Each point represents the mean of at least three experiments (±SD).

Figure 3.

5-HT reversibly inhibits the amiloride-sensitive current in Xenopus oocytes expressing rat αβγ epithelial sodium channel (ENaC). (A) 5-HT induces a rapid decrease of inward current in Xenopus oocytes expressing rat αβγENaC. The sodium current sensitive to 10 μM amiloride was measured at −60 mV holding potential before and during exposure to 4 mM 5-HT. This representative experiment also shows the reversibility of 5-HT–induced current inhibition. No effect of 4 mM 5-HT could be detected in noninjected oocytes (n = 5). (B) 5-HT induces a dose-dependent decrease of inward current in Xenopus oocytes expressing rat αβγENaC. This representative experiment was performed with increasing 5-HT doses (0.25, 0.5, 1, 2, and 4 mM). (C) 5-HT induces a dose-dependent decrease of inward current in Xenopus oocytes expressing rat αβγENaC. Mean values of amiloride-sensitive sodium current measured before and after perfusion with 0.25, 0.5, 1, 2, or 4 mM 5-HT are shown (n = 26, 15, 25, 20, 14, and 26, respectively). For all experiments, histogram data are the means (±SD); *P < 0.05 from control animals.

Figure 4.

5-HT–induced inhibition of the amiloride senitive Na⁺ transport across primary cultures of polarized rat ATII cell monolayers is not mediated through the Na⁺/K⁺ ATPase. (A) Neither the kinetic of the 5-HT–mediated inhibition nor the magnitude of 5-HT effect was affected by the Na⁺/K⁺ ATPase blockade and basolateral membrane permeabilization. Permeabilization was achieved with nystatin (400 μg/ml) in the basolateral chamber. All experiments were performed under asymmetric conditions with different saline solutions for apical and basolateral chambers (apical: 140 mM NaCl, 4 mM KCl, 1 mM MgCl₂, 1 mM CaCl₂, 10 mM Hepes, and 15 mM glucose [pH = 7.3, adjusted with NaOH]; basolateral: 10 mM NaCl, 130 mM N-methyl-D-glucamine, 4 mM KCl, 1 mM MgCl₂, 1 mM CaCl₂, 10 mM Hepes, and 15 mM glucose [pH = 7.3, adjusted with HCl). The EasyMount system was used for all experiments. The voltage was clamped at 0 mV during the whole experiments. (B) This histogram summarizes the effect of 5-HT after Na⁺/K⁺ ATPase inhibition and permeabilization of the basolateral membrane of ATII cell monolayers. The results are means (±SD) of four independent experiments; *P < 0.05 from controls.

Figure 5.

5-HT–induced inhibition of ENaC is receptor independent in rat ATII cell monolayers. (A) Pretreatment with ketanserin, a 5-HTR(2) receptor antagonist, did not affect the magnitude of 5-HT–induced decrease in Isc across primary cultures of polarized rat ATII cell monolayers. Rat ATII cell monolayers cultured at an air–liquid interface for 4 days were placed in Ussing chambers and exposed to 5-HT (4 mM). In some experiments, ketanserin (100 μM) was added 10 minutes before 5-HT, and amiloride (10 μM) was added 3 minutes after 5-HT treatment. (B) Pretreatment with Y-25130, an 5-HTR(3) (5-HT receptor 3) receptor antagonist, did not affect the magnitude of 5-HT–induced decrease of Isc across primary cultures of polarized rat ATII cell monolayers. Rat ATII cell monolayers cultured at an air–liquid interface for 4 days were placed in Ussing chambers and exposed to 5-HT (4 mM). In some experiments, Y-25130 (100 μM) was added 10 minutes before 5-HT, and amiloride (10 μM) was added 3 minutes after 5-HT treatment. (C) Pretreatment with BAPTA-AM (1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetra(acetoxymethyl) ester), a calcium chelator, did not affect the magnitude of 5-HT–induced decrease of Isc across primary cultures of polarized rat ATII cell monolayers. Rat ATII cell monolayers, cultured at an air–liquid interface for 4 days, were placed in Ussing chambers and exposed to 5-HT (4 mM). BAPTA-AM (100 μM) was added 35 minutes before 5-HT, and amiloride (10 μM) was added 3 minutes after 5-HT treatment. (D) 5-HT2R (5-HT receptor 2) stimulation by (±)-2,5-dimethoxy-4-iodoamphetamine (DOI) hydrochloride did not reproduce the effect of 5-HT on Isc. Rat ATII cell monolayers, cultured at an air–liquid interface for 4 days, were placed in Ussing chambers and successively exposed to DOI (1 mM), 5-HT (4 mM), and amiloride (10 μM). (E) Nonspecific stimulation of 5-HT1R (5-HT receptor 1), 5-HT4R (5-HT receptor 4), and 5-HT7R (5-HT receptor 7) by 5-carboxamidotryptamine (5-CT) did not reproduce the effect of 5-HT on Isc. Rat ATII cell monolayers, cultured at an air–liquid interface for 4 days, were placed in Ussing chambers and successively exposed to 5-CT (1 mM), 5-HT (4 mM), and amiloride (10 μM). All these experiments were performed at 37°C under symmetrical conditions with saline solution (140 mM NaCl, 4 mM KCl, 1 mM MgCl₂, 1 mM CaCl₂, 10 mM Hepes, and 15 mM glucose [pH = 7.3, adjusted with NaOH]). The EasyMount system was used for all experiments; the voltage was clamped at 0 mV during the whole experiments. (F) Both DOI and 5-CT cause an increase in intracellular calcium in rat ATII cells. Rat ATII cells were cultured on glass coverslips for 15 hours before experiments. The cells were loaded with 5 μM FURA2-AM. DOI and 5-CT (curves 1 and 2, respectively) elicited a significant increase in intracellular calcium. One representative of four experiments is shown. For all experiments, the histogram results are expressed as means (±SD) of four experiments repeated in triplicate; *P < 0.05. NS, not significant.

Figure 6.

Amiloride- and 5-HT–mediated inhibition of ENaC are charge dependent. (A) Amiloride-induced inhibition of Isc across primary cultures of polarized rat ATII cell monolayers is pH dependent. Rat ATII cell monolayers, cultured at an air–liquid interface for 4 days, were placed in Ussing chambers and exposed to amiloride (10 μM). The two sets of experiments were performed in symmetrical conditions at two different pH levels. The average amiloride-induced inhibition of TEC (transepithelial current) was 48% lower at pH 8.3 than at physiological pH (7.36). (B) 5-HT–induced inhibition of Isc across primary cultures of polarized rat ATII cell monolayers is pH dependent. Rat ATII cell monolayers, cultured at an air–liquid interface for 4 days, were placed in Ussing chambers and exposed to 5-HT (2 mM). The two sets of experiments were performed in symmetrical conditions at two different pH levels. The average 5-HT–induced inhibition of TEC was 24% greater at pH 6.0 than at physiological pH (7.36). All the experiments were performed at 37°C under symmetrical conditions with saline solution (140 mM NaCl, 4 mM KCl, 1 mM MgCl₂, 1 mM CaCl₂, 10 mM Hepes, and 15 mM glucose). The EasyMount system was used for all experiments. The voltage was clamped at 0 mV during the whole experiments. Results are the means (±SD) of at least three experiments done in triplicate; *P < 0.05 from cell monolayers exposed to a pH of 7.36.

Figure 7.

5-HT and amiloride do not compete to mediate ENaC inhibition in A549 cells. A549 cells were used 24 hours after plating on glass coverslips. A549 cells were cultured in the presence of 10 nM dexamethasone. All these experiments were performed in whole-cell configuration. Closed squares represent the dose–response curve for amiloride alone. Open squares represent the dose–response curve for amiloride in the presence of 2 mM 5-HT. The two curves were obtained after fitting experimental data with the Boltzman equation. Exposure to serotonin did not modify amiloride's K_d in A549 cells. We observed that the inhibition of the amiloride-sensitive current by 5-HT was completely reversible (data not shown). Results are the means (±SD) of at least four experiments done in triplicate.

Figure 8.

5-HT inhibits the amiloride-sensitive fraction of the alveolar epithelial fluid transport in mice. (A) 5-HT is present in distal airways of mice, and its concentration is increased after exposure to hypoxia. C57BL6 mice were either exposed to 11–13% fraction of inspired oxygen for 1 hour or to air. The results are the means (±SD) for 10 mice in each group; *P < 0.03 from mice exposed to air. (B) Intratracheal instillation of 5-HT decreases the amiloride-sensitive fraction of the alveolar epithelial fluid transport in mice. Alveolar fluid clearance (AFC) was measured as described in Materials and Methods. Results are the means (±SD) of 5–7 mice per experimental group. The addition of 5-HT (4 mM) to the alveolar instillate did not cause an additional significant decrease in AFC of rats instilled with amiloride (100 μM). *P < 0.05 from control animals instilled with albumin solution alone. NS, not significant.