Connexin-hemichannels-mediated ATP release causes lung injury following chlorine inhalation

Abstract

Chlorine (Cl₂) is a highly reactive halogen gas that undergoes rapid hydrolysis in lung epithelial lining fluid (ELF) upon inhalation, forming hypochlorous acid (HOCl) and hydrochloric acid (HCl). These products subsequently, through chemical reactions, modify the structure and the function of membrane proteins. Herein, we investigated the effects of Cl₂ on connexin-hemichannels and the release of ATP in the ELF. Adult C57BL/6 mice were subjected to 400 ppm Cl₂ for 30 minutes. Subsequent analysis revealed a marked increase in ATP levels within the BAL, with concentrations reaching 43.952 ± 9.553 nM at 2 hours and 30.554 ± 7.383 nM at 24 hours post-exposure, relative to control. Additionally, at 24 hours post-exposure, the lung wet/dry (W/D) ratio significantly increased from 4.48 ± 0.142 to 5.067± 0.359, while alveolar fluid clearance (AFC) decreased from 0.249 ± 0.019 to 0.145 ± 0.018. Electrophysiological recordings in alveolar type 2 (AT2) cells revealed reduced open probabilities (P_o) of both ENaC (4 pS) and a cation channel (18 pS), declining from 0.323 ± 0.021 and 0.202 ± 0.022 to 0.151 ± 0.042 and 0.091 ± 0.019, respectively. Instillation of 50 μl of 100 μg/ml Gap27—a connexin mimetic peptide selectively inhibiting connexin-hemichannels—administered 30 minutes post-exposure, restored ATP to control, normalized the W/D ratio, improved AFC, and reestablished ENaC function. Moreover, Gap27 normalized airway resistance following methacholine challenge. In human airway smooth muscle cells (hASMCs), 100 μM ATP induced Ca_i²⁺ elevation and depolarized V_m to -40 mV, with both effects partially reversed by P2X₇R inhibitor, A804598.

Graphical Abstract

The oxidative stress generated by the inhalation of chlorine and its subsequent reaction with ELF causes connexin-hemichannels to open and facilitate the release of ATP that signals danger, cell death, and causing lung injury. Created in BioRender. Lazrak, A. (2025) https://BioRender.com/deom03g.

Introduction

Chlorine (Cl₂) poses a significant chemical threat primarily due to the potential for its release during transportation, industrial accidents, and wartime activities. Inhalation of Cl₂ is associated with considerable morbidity and mortality for both humans and animals (1–3). The most extensive human exposure to Cl₂ historically occurred during World War I when it was utilized as a chemical weapon. Cl₂ inhalation leads to lung injuries through the interaction of its hydration products, hypochlorous acid (HOCl) and hydrochloric acid (HCl), with components of the cell membrane. This interaction triggers the release of substances such as polyamines, polycations, and chlorinated lipids and proteins (4–6). Acute Cl₂ exposure, common in both industrial and environmental settings, induces various lung injuries. These injuries range in severity depending on the dose, from respiratory airway hyperreactivity to alveolar injury and pulmonary edema (7).

The detrimental effects of Cl₂ inhalation extend to active ion transport across various lung cell types, including alveolar epithelial cells, bronchiolar epithelial cells, endothelial cells, and smooth muscle cells (4–6). It increases the permeability of the blood-gas barrier to plasma proteins (8) and induces airway hyperreactivity (AHR) (6). In the lung, gap junction channels and connexons (connexin-hemichannels) are crucial for direct, autocrine, and paracrine cell-to-cell communication and signaling. While newly formed connexin-hemichannels can move freely within the plasma membrane, gap junction channels remain confined to the junctional membrane between two adjacent cells. These channels form plaques at the cell junctions and consist of intercellular channels facilitated by the interaction of connexons from each adjacent cell. Each connexin-hemichannel comprises six connexin proteins. Connexins, membrane proteins with four transmembrane domains connected by a cytoplasmic loop and two extracellular loops with cytoplasmic C and N termini, have 23 recognized mammalian types classified by their molecular weight.

Gap Junctional Intercellular Communication (GJIC), which allows the intercellular transfer of small hydrophilic molecules under 1.2 kD, is regulated by voltage, pH, and Ca²⁺, along with posttranslational modifications of connexins such as S-nitrosylation, sumoylation, and phosphorylation(9–13). Although many connexins are expressed in lung tissue, Cx40 and Cx43 are uniquely ubiquitous across all lung tissues, from the epithelia of the upper airways to the distal lung, as well as in the endothelium and airway smooth muscle (14, 15). Gap junctions involving Cx40 and Cx43 coordinate the ciliary movements of airway cells for mucus clearance (16), facilitate surfactant release by alveolar epithelial type 2 cells (17, 18), and regulate airway smooth muscle contractions and relaxation (19, 20). Recent evidence suggests that Cx43 may also synchronize inflammatory signaling indices, such as adhesion molecule expression and leukocyte infiltration(21, 22).

Connexin-hemichannels, usually, remain closed. However, they open under oxidative stress and allow the exchange of ions and small metabolites with the extracellular milieu such as ATP, ADP, and AMP. ATP when released in small quantities contributes to autocrine and paracrine signaling; however, at higher concentrations, it functions as a damage-associated molecular pattern (DAMP), signaling cellular danger and injury (23–27). Through its interaction with P2Y and P2X purinoreceptors, ATP induces intracellular signaling cascades and opens cation channels, permitting the entry of sodium and calcium ions into the cell, consequently causing membrane depolarization.

Both ATP paracrine signaling and direct cell-to-cell communication through gap junctions contribute to the spread of depolarization and calcium waves across lung tissues. This results in damage to ENaC, lung edema, and triggers airway smooth muscle contraction, leading to AHR.

In our study, we exposed mice to chlorine gas at concentrations approximating those found near industrial accidents (400 ppm for 30 minutes). At 24 hours post-exposure, we measured ATP levels in bronchoalveolar lavage fluid, assessed airway hyperresponsiveness, evaluated alveolar fluid clearance, and determined ENaC open probabilities in alveolar epithelial type 2 cells using cell-attached patch clamp techniques and precision cut lung slices. These measurements were repeated following administration of Gap27, a connexin mimetic peptide corresponding to the Ser-Arg-Pro-Thr-Glu-Lys-Thr-Ile-Phe-Ile-Ile amino acid sequence (amino acids 204–214) located on the second extracellular loop of Cx43. Our results demonstrate that intranasal Gap27 administration significantly reduced ATP levels in bronchoalveolar lavage fluid, resolved pulmonary edema, restored alveolar fluid clearance, normalized ENaC activity in type 2 alveolar epithelial cells, and alleviated airway hyperresponsiveness. Complementary experiments involved exposing human airway smooth muscle cells to ATP, during which we recorded intracellular Ca2+ changes and evaluated junctional coupling using double whole-cell patch clamp techniques. The exposure to ATP resulted in increased intracellular Ca2+ levels accompanied by cell-to-cell uncoupling. Taken collectively, these data establish that ATP is a key mediator of chlorine-induced lung injury, while Gap27 prevents injury by inhibiting ATP release through connexin hemichannels and blocking the propagation of calcium signals effectively.

Methods

Mice.

C57BL/6 mice, male and female, were purchased from Jackson Laboratory (Bar Harbor, ME). Mice were allowed to acclimate in our facilities at UAB for one week before using them for experiments.

Mice exposure to Chlorine (Cl₂).

Adult male and female C57BL/6 mice (8–10 wks. old; 20–25 g body weight) were exposed to 400ppm Cl₂ for 30 min as previously reported (28). Control mice were exposed to air for 30 min under the same experimental conditions. After exposure, the mice were returned to their cages and had access to food and water ad libitum. To study the role of connexin hemichannels in the lung injury caused by Cl₂ inhalation, mice were instilled with 50 μl PBS (control) or 50 μl of 100 μg/ml Gap27, a gap junction channels and hemichannels specific inhibitor, 30 min post exposure to air or 400 ppm Cl₂ for 30 min.

Measurement of ATP in mouse BAL.

One milliliter saline was instilled to mice and 1 min later the BAL was withdrawn using a 1ml syringe. ATP concentration in the BAL was measured using ATPlite luciferase kit (PerkinElmer). Luminescence from ATP reaction with firefly luciferase was detected using a microplate reader (SpectraMax^® M3).

Lung edema measurement: Wet to Dry ratio weight (W/D).

To measure total lung water, lung weight is measured immediately after excision (wet weight), and after lung tissue is dried at 60°C for 48 h and reweighted again. The wet/dry ratio, W/D, is calculated by dividing the wet weight of the lung by its dry weight (29). For the experiment, control mice were instilled with 50μl PBS, to test the effect of Gap27 (Tocris Biosciences, cat# 1476)) on lung W/D we instilled control and experimental mice with 50μl of 100 μg/ml Gap27. Twenty-four hours later mice were sacrificed, and their lungs were surgically removed and weighted. To measure W/D of each group, lungs were placed in an oven at 60ºC degrees for 48 hours, then weighed again and the ratio calculated.

Alveolar Fluid Clearance (AFC).

Twenty-four hours post mice exposure to air or 400 ppm Cl₂ for 30 min, AFC was measured as follow: 1. Mice were anesthetized with an intraperitoneal injection of Ketamine 80–100mg/kg and xylazine 5–10mg/kg, depending on mouse weight. For reproducibility mice were paralyzed with an intraperitoneal injection of pancuronium bromide (0.04mg). 2. The trachea was exposed and cannulated with an 18-gauge intravenous catheter trimmed to 0.5 inch long. 3. Mice were positioned in the left decubitus position, and 0.5ml of 5% BSA was instilled over 30 sec and infused with 0.1ml air in the catheter to clear the dead space and position the fluid in the alveolar spaces. 4. Mice were ventilated for 30min to allow fluid clearance from the alveolar spaces. At the end of this period, the remaining alveolar fluid was collected using a 1ml syringe. 5. AFC expressed as a percentage of total instilled volume, was calculated using the following formula: AFC = (1- Ci/C30)/0.95, where Ci and C30 are BSA concentrations at time zero and 30 min, respectively, measured using the bicinchoninic acid (BCA) protein assay(30).

ENaC activity in mouse AT2 cells in-situ.

Precision cut lung slices were prepared from mice instilled with 50μl saline (control) or 50μl of 100μg/ml Gap27 at 30 min following their exposure to air or 400 ppm Cl₂ for 30min. Twenty-four hours post instillations, mice were euthanized using a lethal dose of Ketamine and Xylazine, then their chests were cut open, the lungs were washed with Krebs-Ringer solution containing (in mM): 140 NaCl, 3 KCl, 2.5 CaCl₂, 1 MgCl₂, 10 glucose, and 10 HEPES (pH ≈7.35–7.4), and subsequently filled with low temperature melting agar through an incision in the trachea to stiffen the tissue for slicing with a microtome (Precisionary Instruments. Greenville, NC). Lower lobe of the right lung was dissected and mounted on the microtome for slicing. The slices, 250 μm thick, were incubated in DMEM at 37°C in a humidified atmosphere of 5% CO₂. For an experiment, a slice was transferred to the recording chamber filled with a solution of the following composition (in mM): 130 K-gluconate, 2 MgCl₂, 10 KCl, 5 glucose, and 10 HEPES (pH 7.4, KOH) and held in place with an anchor (Warner Instruments, Boston, MA). The recording pipette resistance ranged from 5–6 MΩ when filled with a solution of the following ionic composition (in mM): 140 NaCl, 3 KCl, 2.5 CaCl₂, 1 MgCl₂, 5 glucose, and 10 HEPES (pH = 7.4). For ENaC activity measurement, the recording chamber was mounted onto the stage of an upright Olympus microscope EX51WI (Olympus, Center Valley, PA) in order to assess ENaC activity in AT2 cells by patch clamp (31). AT2 cells within the slices were visualized by the accumulation of LysoTracker green (1 μM, ThermoFisher) under UV light. Data were recorded and stored onto the hard drive of a computer equipped with pClamp software (Molecular Devices, San Jose, CA) and interfaced to an Axon amplifier Axopatch 200B (molecular devices) with Digitata 1440A (Molecular devices). Data were analyzed using Clampfit (molecular devices). The experimental groups were compared using ordinary ANOVA, p<0.05 was considered significant.

Airway resistance measurement (AHR).

At 30 min post mice exposure to air or 400 ppm Cl₂, mice were instilled with PBS, or 50 μl of 100 μg/ml Gap27 or 125mg/Kg Carbenoxolone (CBX). At 24 h post exposure, mice were anesthetized with pentobarbital sodium (50 mg/kg ip.; Vortech Pharmaceuticals, Dearborn, MI) and paralyzed with pancuronium (4 mg/kg ip; Gensia Sicor Pharmaceuticals, Irvine, CA), intubated, then connected, and mechanically ventilated by a FlexiVent FX system (SCIREQ, Montreal, QC, Canada). The animals were ventilated at a rate of 160 breaths per minute at a tidal volume of 0.2 ml with a positive end-expiratory pressure of 3 cm H₂O, as previously reported (28). The baseline value of lung resistance (R) was set via deep inhalation controlled by the Flexivent system. Lungs were challenged by increased concentrations of methacholine (0 to 40 mg/mL) aerosolized within 10 s from the start of each measurement and airway responses were recorded every 15 s for a period of 3 min.

Measurement of electrical coupling between human smooth muscle cell pairs in culture.

Human smooth muscle cells (hSMCs) cultured overnight on glass coverslips were transferred the recording chamber mounted onto the stage of an Olympus microscope (Olympus, city, state) for electrophysiology measurements. The cells were continuously perfused at 0.5 ml/min (T = 22 + 1.5°C). All the experiments were performed using the double whole-cell patch-clamp procedure (Lazrak and Peracchia, 1993; Weingart, 1986; White et al., 1985; Neyton and Trautmann, 1985). Briefly, the pipettes were made from capillary tubing (LG16, Company, city, state)) with a vertical puller (Narishige, Japan). The resistance of pipettes ranged from 2–3 MΩ when filled with intrapipette solution of the following ionic composition, in mM, KCl 135, NaCl 12, MgCl₂ 1, CaCl₂ 1.8, EGTA 10, HPES 10, Glucose 5, pH 7.2. Each pipette was connected to a separate patch clamp amplifier (Axopatch 200 B; Molecular devices, foster city, CA). The pipettes were lowered to the cells by two micromanipulators (Sutter Instruments, City, State) and the steps involved in the formation of the giga-seals and whole-cell configuration were followed on a computer monitor. The head stage of each amplifier was connected to the cell interior through a series resistance (R_p1 and R_p2). The pipette resistance (R_p1 and R_p2) increased two to four times following membrane patch rupture and establishment of whole-cell configuration (10–20 MΩ); 70–90% of this resistance was compensated by the amplifiers. The initial resistance was less than 10% of the parallel combination of plasma membrane (R_m) and seal (R_s) resistances, ≤ 5 GΩ. Pulse generation and data acquisition were performed by means of a computer equipped with Pclamp software 10 (Molecular devices, Foster City, CA) and A/D-D/A interface (Digidata 1400, Molecular devices). During acquisition the current traces were filtered at 5 kHz, and current-voltage (I-V) curves were plotted using Clampfit (Molecular Devices). For studying the electrical properties of the junctional membrane, the cells were initially voltage-clamped to the same holding potential (V_h= -60 mV), so that no junctional current would flow at rest (I_j = 0 pA). A V_j gradient was created by imposing a voltage step (V₁) to cell 1, while maintaining V₂ at -60 mV: thus, V_j = V1. The negative feedback current (I₂), injected by the amplifier 2 connected to cell 2 to maintain V₂ constant at -60 mV was used for calculating G_j, as it is identical in magnitude to the junctional current (I_j), but of opposite sign (I_j = -I₂); using Ohm’s law: G_j = I_j/V_j.

Calcium measurement.

Intra-cellular calcium (Ca_i²⁺) measurements in hASMCs were performed following the protocol described previously (5). Briefly, hASMCs, seeded on 25-mm glass coverslips in six-well cell culture plates with normal medium, were exposed to air or 100 ppm Cl₂ for 10 min. To measure intracellular calcium, hASMCs were incubated for 20 min with fura-2 AM (3 μg/ml) in HBSS containing 25 mM MOPS at room temperature and pH 7.4. Cells were rinsed for an additional 20 min using HBSS + MOPS buffer to remove excess fura-2 AM. Fresh HBSS + MOPS was added to the cells upon transfer of a coverslip to the Attofluor chamber. Data were acquired using a Nikon Eclipse Ti inverted microscope fitted with a 40X oil immersion objective and Nikon Elements software. Intracellular calcium concentrations were calculated from the emission ratio R of calcium-bound Fura2 (340 nm) to calcium-unbound Fura2 (380 nm) according to Grynkiewicz et al (1985) (32), using the calibration procedure based on ionophore permeabilization suggested by Williams et al. (1990) (33).

Connexins 40 and 43 expressions.

Cx40 and Cx43 proteins were measured at 2- and 24 h post mice exposure to air (control) or 400 ppm Cl₂ for 30 min using Western Blotting (WB) technique. Commercially available antibodies to Cx40 (Abcam cat# ab183648), Cx43 (Abcam cat# ab11370), and β-actin (ThermoFisher cat#15G5A11/E2), were used to assess the expression of each connexin in the lung tissue in control and 24hrs post exposure to Cl₂. These measurements were repeated in mice instilled with 50 μl 100 μg/ml Gap27.

Statistics.

Data analysis and presentation were perform using Clampfit (Molecular Devices) GraphPad Prism 10.4 (GraphPad Software, San Diego, CA) and Igor9 (WaveMetrics, Portland, OR). Data were summarized as means ± SD. Statistical significance between groups was performed using One-way ANOVA, followed by Tukey’s test for multigroup comparisons. t-test was performed when comparing two groups. P < 0.05 was considered significant.

Study approval.

C57BL/6, 8 to 12 weeks old mice (20–25 g body weight), male or female, were purchased from Jackson Laboratories (Bar Harbor, ME USA). All experimental procedures were approved by the University of Alabama at Birmingham Institutional Animal Care and Use Committee (IACUC).

Results.

Chlorine inhalation results in the onset of pulmonary edema and airway hyperreactivity (AHR) through the induction of ATP release. In a controlled study, bronchoalveolar lavage (BAL) was collected at 2 hours and 24 hours following mice exposure to either ambient air or chlorine gas (Cl₂). The ATP concentration in BAL was measured using the luciferase method. As depicted in Figure 1A, ATP concentration was absent in the BAL from mice exposed to air at both timepoints; conversely, ATP was detected in mice exposed to 400 ppm Cl₂ for 30 minutes at both the 2-hour and 24-hour marks. Notably, ATP became detectable as early as 2 hours post-exposure and remained elevated for a minimum of 24 hours post-Cl₂ exposure. This ATP surge returned to baseline levels in Cl₂-exposed mice that were treated with 50 μl of 100 μg Gap27 instilled 30 minutes post-exposure. Gap27 exhibited no influence on ATP levels in control mice that were exposed to air.

Figure 1.

A. The concentration of adenosine triphosphate (ATP) in bronchoalveolar lavage (BAL) fluid from mice exposed to either air or 400 ppm chlorine (Cl₂) for 30 minutes was measured at 2- and 24-hours post-exposure. No ATP was detected in the BAL fluid of mice exposed to air at either time point. However, a marked increase in ATP concentration was observed as early as 2 hours post-Cl₂ exposure and remained elevated for the subsequent 24 hours. Administration of 50 μl of 100 μg/ml Gap27, a connexin-hemichannels inhibitor, 30 minutes after exposure, significantly reduced the ATP concentration at both time points. Phosphate-buffered saline (PBS) alone exhibited no impact. B. The weight-to-dry weight (W/D) ratio of lungs from mice exposed to air or Cl₂, and subsequently instilled with PBS or Gap27, was assessed. Chlorine exposure resulted in a 13% increase in W/D ratio within 24 hours, whereas Gap27 administration post-exposure restored the W/D ratio close to control levels. C. Alveolar fluid clearance (AFC) was reduced 24 hours post-exposure. Mice exposed to Cl₂ and instilled with PBS exhibited a significant decrease in AFC, a reduction not observed in the group receiving Gap27. D. GlyH-101 impact on AFC in mice following Cl₂ exposure. The data clearly show that CFTR is not implicated in Cl₂-induced AFC inhibition, nor in its restoration via Gap27 instillation administered 30 minutes post exposure. Means ± SD were calculated from sample sizes of n = 5–10, with statistical significance assessed via one-way ANOVA and Tukey’s post hoc test.

The mice subjected to 400 ppm Cl₂ developed noticeable lung edema. This was determined by comparing the wet-to-dry (W/D) lung weight ratio of Cl₂-exposed mice to that of air-exposed controls, as illustrated in Figure 1B. The administration of 50 μl of 100 μg Gap27 effectively prevented fluid accumulation in the lung tissue. To further elucidate the mechanism through which Cl₂ inhalation fostered pulmonary edema, we measured the ability of lungs to clear saline with 5% bovine serum albumin (BSA) and determine alveolar fluid clearance (AFC) rate over a 30-minute timeframe. When compared to controls, mice exposed to Cl₂ exhibited a 50% reduction in AFC, Figure 1C. However, instilling 50 μl of 100 μg/ml Gap27 in these mice post-exposure ameliorated fluid retention, as shown in Fig. 1B, and returned AFC to baseline, Fig. 1C. Gap27 operates as a selective inhibitor of gap junctions and connexin-hemichannels located in the plasma membrane. The data clearly demonstrate that inhibiting connexin-hemichannels within alveolar epithelial cells mitigated fluid accumulation in the alveolar spaces, but it did not reverse proteins concentration in the BAL (data not shown). Inhibition of AFC may result from reduced Na⁺ reabsorption or enhanced fluid secretion. Treatment with Gly-H-101, a water-soluble CFTR inhibitor, did not alter AFC levels following Cl₂ exposure. As depicted in Figure 1D, CFTR does not contribute to the inhibition or restoration of AFC by Gap27 after mice are exposed to Cl₂. Using precision-cut lung slices, we analyzed sodium single channel activities in alveolar type cells in situ to understand the mechanisms behind reduced alveolar fluid clearance. Measurements were taken from control mice, and those 24 hours post chlorine exposure, enabling direct comparison. Figure 2 provides a graphical representation of ENaC activity in AT2 cells in situ: Panel A illustrates control activity of a 4 pS channel from AT2 cells in a lung slice following exposure to air and PBS instillation, while Panel B demonstrates activity in an AT2 cell post-air exposure and Gap27 administration. Conversely, Panel C showcases the reduced activity of the 4 pS channel in an AT2 cells 24 hours after Cl₂ exposure and PBS instillation. Panel D confirms that Gap27 effectively prevented inhibition of the 4 pS channel. Similar observations were made for an 18 pS channel in AT2 cells; Panel F reflects control activity, Panel G exhibits activity in a Gap27-treated AT2 cell harvested from an air-exposed mouse, Panel H indicates decreased activity in an AT2 cell from a Cl₂-exposed mouse treated with PBS, and Panel I verify that Gap27 negated the Cl₂-induced inhibition of the 18 pS channel. Panels E and J depict reduced open probabilities (P_o) for both channels upon Cl₂ inhalation. The channels activities were reverted following Gap27 administration at 30 minutes post-Cl₂ exposure, indicating ATP’s potential role in ENaC inhibition. Notably, PBS instillation bore no measurable effects. Panel F illustrates the inhibition of ENaC activity within an AT₂ cell situated in a lung slice upon exposure to ATP at a concentration of 100 μM. ATP was administered through the intrapipette solution, resulting in suppression of ENaC activity within 46 seconds following Gigaseal formation. Regarding airway hyperreactivity, it was measured 24 hours post-exposures using a FlexiVent system, as lungs were subjected to incremental concentrations of methacholine. Mice exposed solely to air and instilled with either PBS or Gap27 showed minimal reactivity to methacholine (Figure 3A and 3B). In stark contrast, mice exposed to 400 ppm Cl₂ for 30 minutes and instilled with PBS exhibited pronounced reactivity under methacholine challenge. The increased reactivity of airway smooth muscle to methacholine was most likely attributable to elevated epithelial permeability rather than to an increase in muscarinic receptors in response to chlorine exposure (data not shown). Figures 3A and 3B demonstrate that this reactivity was circumvented in mice treated with 50 μl of 100 μg/ml Gap27 or 25mg/kg CBX, identifying the contributory role of connexin-hemichannels in Cl₂-induced AHR. In terms of connexin expression following Cl₂ inhalation, lung tissues harvested at 2- and 24-hours post-exposure showed an increase in Cx40 and Cx43 expression, as evaluated via western blotting with specific antibodies, Figure 4A. The upregulation of connexins was evident at 2 hours and persisted for at least 24 hours. Figures 4B and 4C reveal a two-fold surge in Cx40 and Cx43 expression at 2 hours post-exposure and an even larger 3.5-fold increase in Cx43 expression at 24 hours. Moreover, mice treated with 50 μl of 100 μg/ml Gap27 post-exposure demonstrated a decline in Cx40 expression across both air and Cl₂ conditions (Fig. 4A), though Cx43 levels were unchanged in air-exposed mice.

Figure 2.

Epithelial sodium channel (ENaC) activity in alveolar epithelial Type 2 (AT2) cells was evaluated in situ using freshly cut lung slices 24 hours after exposure to air or Cl₂, with PBS or Gap27 instillation occurring 30 minutes post-exposure. A. Control channel activity (4 pS) in AT2 cells from mice exposed to air and instilled with PBS exhibited an open probability (P_o) of 0.323 ± 0.021. B. Consistent findings were noted in the cohort exposed to air and treated with Gap27 (P_o = 0.325 ± 0.026), indicating no influence of Gap27 on ENaC activity. C. Exposure to 400 ppm Cl₂ and subsequent PBS instillation resulted in a 50% reduction in channel activity (P_o = 0.151 ± 0.042). D. Gap27 administration 30 min post-Cl₂ exposure restored ENaC activity near control levels (P_o = 0.267 ± 0.038). E. The Po summary for the 4 pS channel confirmed Chlorine-mediated activity reduction at 24 hours, mitigated by Gap27 administration. Saline did not alter ENaC functionality in either group. F, G, H, I. These panels illustrate the activity of the 18 pS channel under similar conditions. Like the 4 pS channel, chlorine exposure reduced channel activity, while Gap27 administration prevented this decline. J. The P_o summary for the 18 pS conductance affirmed Cl₂’s inhibitory impact on channel activity at 24 hours post-exposure, successfully counteracted by Gap27. Saline had no effect on channel function for either exposure group. K. addition of ATP (100 μM) to the intrapipette solution induced marked inhibition of ENaC activity. This inhibition was consistently observed approximately 50 seconds after establishing the Gigaseal between the pipette and the membrane of an alveolar type 2 cell in situ within a precision‐cut lung slice from a mouse exposed to air. Means ± SD were determined with a sample size of n = 10. Statistical significance was verified through one-way ANOVA and Tukey’s post hoc test.

Figure 3.

A. Airway hyperreactivity was assessed in mice 24 hours following exposure to air or Cl₂ and subsequent instillation of either PBS or Gap27. Employing a Flexivent system enabled the administration of incremental methacholine concentrations while quantifying lung reactivity. Mice subjected to 400 ppm Cl₂ for 30 minutes and instilled with PBS exhibited pronounced methacholine reactivity at low concentrations. Conversely, Gap27 administration prevented Cl₂-induced hyperreactivity. Air exposure was without effect in both PBS- and Gap27-instilled mice. B. Experimental conditions mirrored (A), substituting Gap27 with 25 mg/kg carbenoxolone (CBX), another gap junction and connexin-hemichannel inhibitor. CBX administration similarly reduced Cl₂-induced lung hyperreactivity to methacholine. Means ± SD were calculated from n = 6, with statistical analysis performed using one-way ANOVA and Tukey’s post hoc test.

Figure 4.

A. Expression levels of connexins Cx40 and Cx43 in mouse lungs at 2- and 24-hours post-exposure to air or 400 ppm Cl₂ for 30 minutes, followed by PBS or Gap27 instillation, were quantified. Both connexins exhibited a twofold increase at the 2-hour and 24-hour marks post-Cl₂ exposure. B & C. Western blot analyses from six mice confirmed these findings. PBS administration exerted no impact on Cx40 and Cx43 expression in air- or Cl₂-exposed mice. Data are represented as Means ± SD, with n = 5–8.

Examining ATP-induced effects in human airway smooth muscle cells (hSMCs), exposure to 100 ppm Cl₂ for 10 minutes catalyzed ATP release into culture media, Figure 5A. Adjustments to Cl₂ concentration and exposure duration were made to avoid cytotoxicity while ensuring observable effects. ATP concentrations in the medium mirrored those found in BALF collected post-mice exposure to 400 ppm Cl₂ for 30 minutes (Fig. 1A). These results infer that local ATP concentrations near cells could be substantially higher than the measured values (Fig. 1A). Given the ELF’s dilution of at least 1000 times while performing the bronchoalveolar lavage, the estimates suggest local ATP concentrations of 40 μM and 20 μM at 2 hours and 24 hours, respectively, post-exposure. Utilizing a 100 μM ATP concentration in the study of hSMC electrophysiology effects induced membrane potential depolarization from -64 ± 3.4 mV to -39.6 ± 2.7 mV, Figure 5B. Partial restoration was achieved through P2X₇ purinoreceptor inhibition, which reversed membrane depolarization to -55 ± 4.2 mV. However, the inhibition by 100 nM A804598 failed to fully restore the membrane potential to its control value.

Figure 5.

A. Human smooth muscle cells (hSMCs) were exposed to air or 100 ppm Cl₂ for 10 minutes in culture, with resultant ATP release into the medium assayed. Air exposure exerted no effect on medium ATP concentration. Conversely, Cl₂ exposure yielded a substantial ATP increase (60 ± 10 nM). B. Perfusion of hSMCs with 100 μM ATP prompted membrane potential depolarization, partially rectified via P2X₇ purinoreceptor inhibition with 100 nM A804598. C, D, E. Intracellular calcium concentrations, assessed via Fur2 technique, indicated PBS exerted no effect, whereas 100 μM ATP significantly elevated Ca²⁺ concentrations, partially mitigated by P2X₇ inhibition. Means ± SD represent data from n = 7–10. Statistical significance was evaluated using one-way ANOVA and Tukey’s post hoc test.

Additionally, ATP heightened intracellular calcium levels measured via the Fura-2 technique (Figures 5C–E). ATP induced significant Ca_i²⁺ increase (Figure 5D), and P2X₇R inhibition only marginally blocked these intracellular increments (Figure 5E), supporting a tentative ATP involvement in ENaC inhibition through intracellular calcium modulation. Finally, Figure 6 denotes cell-to-cell coupling assessments in hSMC pairs using double whole-cell patch clamp recording. By applying voltage steps to one cell, while maintaining the other at the common holding potential of -60 mV, an intercellular current was recorded, reflecting junctional coupling (Figure 6A). Gap27 and ATP treatment resulted in decoupling between hSMC pairs, as shown in Figures 6B and 7B. Inhibiting P2X₇R slightly mitigated the ATP-induced uncoupling (Figure 7C), implying ATP caused impairment through intracellular calcium surges (Figure 7D). Despite partial recovery following P2X₇R inhibition, the results advocate the involvement of other ATP receptors and pathways in modulating hSMC interactions, primarily via elevated intracellular calcium levels (9, 10, 34, 35).

Figure 6.

A. Electrical coupling between hSMC pairs was investigated using double whole-cell patch clamp recording. Cells connected to pipettes in whole-cell mode underwent voltage clamping at –60 mV. Voltage steps (±50 mV) applied to one cell elicited two currents: I₁ in the stimulated cell and I₂, the junctional current (I_j), in the paired cell. B. Gap27 perfusion prompted gap junction inhibition, evidenced by reduced junctional current.

Figure 7.

A. Junctional coupling in hSMC pairs assessed via the double whole-cell patch clamp technique identified ATP-induced coupling inhibition. B. Perfusion with 100 μM ATP induced significant junctional current reduction, observed as decreased coupling from cell2 when a ±50 mV pulse was applied to cell1. C. P2X₇ inhibition with 100 nM A804598 partially reversed ATP’s effect on junctional coupling. D. I/V relationships for cell pairs demonstrated ATP’s modulating impact on junctional coupling, reflecting control and P2X₇ inhibitor conditions. Data are Means ± SD, n = 10, with significance determined through one-way ANOVA and Tukey’s test.

Discussion.

Exposure to significant concentrations of chlorine (Cl₂) can lead to severe pulmonary injuries, such as pulmonary edema, and heightened airway reactivity. Despite extensive studies, the underlying pathophysiology remains incompletely understood. This research focuses on examining the role of connexin-40 and connexin-43 (Cx40 and Cx43) hemichannels in Cl₂-induced pulmonary damage by employing the gap junction inhibitor Gap27. Our findings reveal that administering this peptide to mice exposed to 400 ppm of Cl₂ for 30 minutes effectively mitigated pulmonary edema and airway hyperreactivity (AHR) 24 hours post-exposure.

Upon inhalation, Cl₂ gas quickly reacts within the epithelial lining fluid (ELF), forming hydrochloric acid (HCl) and hypochlorous acid (HOCl). These derivatives irritate airways, interact with cellular membrane lipids, proteins, and antioxidants, thereby altering ion channel functions in epithelial cells. The study also investigates how oxidative stress from extracellular Cl₂ hydration affects connexin-hemichannels in plasma membranes. These channels mediate ATP release into the extracellular environment, contributing to pulmonary edema and AHR. In mice, a significant increase in ATP concentration in the bronchoalveolar lavage (BAL) was detected within two hours post-exposure, persisting for 24 hours. Administration of Gap27 returned ATP levels to baseline, preventing edema and AHR, indicating the critical role of open connexin-hemichannels in ATP release within ELF. This result was corroborated using carbenoxolone a known inhibitor of connexin-hemichannels. However, Panax-10, which selectively inhibits pannexin channels, failed to produce the same effect (data not shown), suggesting pannexin channels non-involvement in Cl₂ effects.

Further exploration demonstrated an increase in Cx40 and Cx43 expression post-inhalation, leading to more connexin-hemichannels formation and sustained ATP release up to 24 hours post-exposure, exacerbating lung damage. Under normal physiological conditions, connexin-hemichannels generally remain closed in the presence of typical extracellular calcium (Ca²⁺) levels of 1–2 mM. However, connexin-hemichannels open to facilitate molecular exchange when extracellular Ca²⁺ is depleted. Research by Li et al. (1986) (11) showed that removing extracellular Ca²⁺ led to cell uptake of 5(6)-carboxyfluorescein, with decreased uptake and lower membrane resistance in Cx43 antisense-transfected cells compared to controls. These findings initially demonstrated the open state of connexin-hemichannels allowing cytoplasm-extracellular milieu communication involving small molecules (<1.2 kD). Connexin-hemichannels are instrumental in managing both the release and uptake of ions and small molecules, under both physiological and pathophysiological contexts.

Though typically closed under resting conditions, connexin-hemichannels may spontaneously open, allowing the release of signaling molecules such as ATP, UDP, and cAMP, among other metabolites and ions of <1.2 kD in molecular weight. The regulation of connexin-hemichannels open probability is complex; physiological extracellular Ca²⁺ is crucial for keeping them closed, while its removal prompts opening (11). Despite the unclear mechanism, Lopez et al. (36) proposed that Ca²⁺ binding to connexin proteins stabilizes a closed channel configuration. Conversely, intracellular Ca²⁺ concentrations also influence connexin-hemichannels behavior. Under 100 nM Ca_i²⁺ is physiological, but an increase inhibits gap junction communication below 500 nM (10), with complete inhibition occurring at micromolar concentrations (9, 10, 34). Bayraktar et al. (37) identified that minor intracellular Ca²⁺ increases (<500 nM) enhance hemichannel open probability, permitting signal molecule and ion exchanges (see (38) for review). Connexin-hemichannels are also susceptible to influences from post-translational modifications of connexin proteins, metabolic stress, and oxidative stress from free radical-antioxidant imbalances (39–41), conditions well documented following Cl₂ inhalation (5, 42, 43). ATP functions as a neurotransmitter (44–46), playing significant roles in autocrine and paracrine signaling (47–49).

Extracellular ATP has emerged as a critical mediator of inflammatory lung injury(50, 51). Its release from apoptotic cells occurring primarily through connexin-based hemichannels and pannexin channels (22, 52), enabling the molecule to function as a potent extracellular signaling agent (53, 54). Upon liberation, ATP engages both the P2X family of ligand-gated ion channels and the P2Y family of G-protein-coupled receptors, thereby initiating a cascade of cellular responses (55, 56). Multiple studies have demonstrated that extracellular ATP exacerbated ventilator-induced lung injury by increasing pulmonary permeability, promoting edema formation, and stimulating the release of pro-inflammatory cytokines (57–60). These actions result in further immune cell infiltration and subsequent tissue damage (54, 61, 62). Nevertheless, the precise role of eATP in the pathogenesis of lung disease remains incompletely understood. For example, in a mouse model of LPS-induced acute lung inflammation, ATP administration was observed to reduce pulmonary inflammation and improve endothelial barrier integrity, suggesting a potential protective effect(63). Similarly, in a hyperoxia model, activation of the P2X7 receptor by ATP was deemed essential for the function of pulmonary invariant natural killer T (iNKT) cells, while the absence of the ectonucleotidase CD39 conveyed protection against hyperoxic injury (64). Additionally, eATP contributes to alveolar surfactant homeostasis and may regulate the lung microbiome, as mechanical stretching of type I alveolar epithelial cells induces ATP release that can desensitize receptors on type II cells, ultimately impairing surfactant production and worsening edema(53) (65) by inhibiting alveolar fluid clearance and the epithelial sodium channels (ENaC).

Epithelial sodium channels (ENaC) are essential for sodium absorption and the regulation of alveolar fluid in the lungs, directly influencing ionic balance and gas exchange. Within lung epithelia, two distinct sodium channels are present: a highly selective 4 pS channel specific for Na⁺ ions (66, 67) and a non-selective cation channel with an 18 pS conductance that permits the passage of both Na⁺ and K⁺ ions (4, 68, 69). ENaC typically consists of at least three subunits—α, β, and γ—which are crucial for maintaining the ionic composition of the epithelial lining fluid (ELF). This maintenance is vital for optimal muco-ciliary clearance, efficient gas exchange, and innate defense against pathogens(70–72). In a recent study by Trac et al (73), the non-selective channel was found formed by an α-ENaC subunit in conjunction with one or more acid-sensing channel 1a (ASIC1a) proteins (73). Importantly, the knockdown of either the β or γ-ENaC subunits significantly impacted the expression and functionality of highly selective channels but not that of the non-selective cation channel. Precisely, the knockdown of ASIC1a eliminated the non-selective channel while leaving the highly selective channel unaffected. Furthermore, the regulation of sodium channels exhibits differential responses to varying oxygen tensions. Under hypoxic conditions, there is a reduction in both the expression and density of ENaC subunits at cells’ apical membranes, which correlates with decreased sodium transport and an increased risk of alveolar flooding(74). In contrast, the expression of the non-selective channel rises in response to hypoxia(73). Although the non-selective cation channel is less efficient at sodium transport compared to the highly selective variant, its activation during moderate hypoxia can help mitigate the risk of alveolar flooding. However, in cases of severe hypoxia, the impaired function of the basolateral Na,K-ATPase hinders sodium extrusion, ultimately leading to alveolar flooding despite the activation of non-selective channels(74). This nuanced regulation of sodium channels under varying oxygen levels underscores their critical role in pulmonary homeostasis and highlights potential therapeutic targets for conditions associated with disrupted alveolar fluid balance.

Thus, the oxidative stress resulting from Cl₂ hydration in ELF leads to both HCl and HOCl contributing to lung injury via membrane lipid and protein modification, impacting their structure and function. Connexin-hemichannels, primary oxidative stress targets, open large pore channels enabling molecules and ions exchange up to 1.2 kD. These channels allow HOCl and HCl intracellular access, damaging cytoplasmic components and organelles such as mitochondria, nuclei, and the endoplasmic reticulum. Cellular metabolites also exit through connexin-hemichannels. If unchecked as demonstrated by Gap27’s effectiveness, these channels would continue propagating lung damage, resulting in edema and increased lung sensitivity to further injurious materials like cell matrix hyaluronan breakdown post-Cl₂ exposure (75–77). Many studies highlighted the importance of early interventions as vital in mitigating Cl₂ exposure effects(1, 3, 5, 42, 78–80). Early treatments significantly lowered or reversed Cl₂-induced damage. Without such timely interventions, damage exacerbates through immune responses, prolonging or intensifying initial injuries (6, 42, 75, 81).

In summary, Cl₂ and its derivatives induce inflammation that opens connexin-hemichannels, allowing small metabolites and K⁺ egress as well as Na⁺ and Ca²⁺ ingress, causing cellular depolarization, Ca_i²⁺ elevation, and inhibiting intercellular communication via gap junctions. This inhibition potentially serves as a protective mechanism, curbing injury propagation beyond initial exposure sites.

New and noteworthy.

Inhaled chlorine gas reacts with lung epithelial lining fluid to form hypochlorous and hydrochloric acids that alter membrane protein structure and function. Under oxidative stress, connexin hemichannels open, releasing ions and metabolites, such as ATP. The released ATP signals danger, cell death, and tissue injury. Early administration of Gap27, a connexin‐hemichannel inhibitor, at 30 minutes post‐exposure preserves ENaC function and prevents the subsequent development of pulmonary edema. These compelling findings underscore a promising therapeutic strategy.