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E-Ring Isoprostanes Stimulate a Cl^– Conductance in Airway Epithelium via Prostaglandin E₂-Selective Prostanoid Receptors

¹ Firestone Institute for Respiratory Health, St. Joseph's Healthcare, and Department of Medicine, McMaster University, Hamilton, Ontario, Canada

Correspondence and requests for reprints should be addressed to Dr. L. J. Janssen, L-314, St. Joseph's Hospital, 50 Charlton Ave. East, Hamilton, ON, L8N 4A6 Canada. E-mail: janssenl{at}mcmaster.ca

	Abstract

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Isoprostanes comprise a class of membrane lipid metabolites produced during oxidative stress, including asthma, chronic obstructive pulmonary disease, and cystic fibrosis. They are widely recognized to evoke a variety of biological responses in airway and pulmonary vascular smooth muscle, lymphatics, and innervation. However, their effects on airway epithelium are largely unstudied. We examined the electrophysiological responses evoked by several different isoprostane species in bovine airway epithelium using the Ussing chamber technique. The E-ring isoprostanes 15-E_1t-IsoP and 15-E_2t-IsoP evoked a substantial increase in short-circuit current (I_SC), whereas four different F-ring isomers were ineffective. 15-E_2t-IsoP–evoked I_SC was mimicked by the prostaglandin E₂-selective prostanoid receptor (EP)-agonist prostaglandin E₂ but not by agonists of EP₁/EP₃-, FP-, or TP receptors (sulprostone, fluprostenol, and U46619, respectively). This response was significantly reduced by the EP₄-receptor blocker GW627386 but not by blockers of other prostanoid receptors (ICI 192,605 [TP-selective], SC19220 [EP₁-selective], AH6809 [DP/EP₁/EP₂-selective], and AL8810 [FP-selective]). 15-E_2t-IsoP–evoked I_SC was reduced by blockers of Cl^– channels (niflumic acid and 5-nitro-2-(3-phenylpropylamino)-benzoic acid), of Na⁺/K⁺/2Cl^– co-transport (furosemide and bumetanide), of adenylate cyclase (MDL 12,330A), or of guanylate cyclase (1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one) but not by blockers of Na⁺ conductances (amiloride). We conclude that 15-E_2t-IsoP activates a transepithelial Cl^– conductance in bovine airway epithelium through an EP₄ receptor coupled to adenylate cyclase and soluble guanylate cyclase.

Key Words: chloride conductance • isoprostane • oxidative stress • prostanoid receptor • secretion

CLINICAL RELEVANCE Top Abstract CLINICAL RELEVANCE MATERIALS AND METHODS RESULTS DISCUSSION References   This article provides the first description of the physiological response of native airway epithelium to isoprostanes, a novel class of inflammatory mediators. There is only one other such report in which cultured epithelial cells were used.

 
Isoprostanes are a class of molecules produced by peroxidative attack of membrane lipids. Peroxides and superoxides oxidize the double bonds within lipids such as arachidonic acid, producing a large family of molecules, some of which are isomers of prostaglandins (1). Their relative stability and the fact that their production closely parallels the degree of free radical production make them excellent tools as markers of oxidative stress. Concentrations of these molecules are elevated in the blood and bronchoalveolar lavage fluid of those who inhale allergen, ozone, or cigarette smoke (2–5) or suffer from asthma (6–9), cystic fibrosis (10, 11), chronic obstructive pulmonary disease (5, 12–14), or sleep apnea (15).

More recently, many groups have shown these molecules to have important biological activity (16–19). They were first shown to be powerful vasoconstrictors in the renal vasculature (20) and have since been found to evoke the same response in almost every vascular preparation in which they have been tested (in some preparations or under certain conditions, they can also evoke vasodilation [21]). In fact, they elicit a biological response in virtually every cell type found in the lung, including airway smooth muscle (17, 22–24), pulmonary (18, 25, 26) and bronchial (19) vasculature, lymphatics (27), innervation (28, 29), and inflammatory cells (30, 31). However, there has been only one study describing their effects in airway epithelium (16), although that study used cultured transfected epithelial cells, which can undergo substantial changes in phenotype during the course of cell culturing and passage.

A primary function of the airway epithelium is to generate mucus (via active secretion) and move it (via ciliary beating) up the airway tree. Secretion of water (the major determinant of mucous volume and consistency) across the epithelial layer is driven by osmotic gradients resulting from carefully orchestrated ion transport mechanisms. Under basal conditions, a Na⁺/K⁺-ATPase located on the basolateral membrane is responsible for setting up concentration gradients for those two ions, which are used to drive the movement of other ions (32). Luminal reabsorption of Na⁺ occurs via an amiloride-sensitive channel (32). A bumetanide- and furosemide-sensitive Na⁺/K⁺/2Cl^– cotransporter on the basolateral membrane moves all three ions into the cell using the energy stored in the Na⁺-gradient (32). The subsequent accumulation of Cl^– and K⁺ resulting from Na⁺/K⁺/2Cl^– cotransport and Na⁺/K⁺-ATPase activities is relieved by passive efflux through Cl^– and K⁺ channels on the basolateral membrane (32). As such, there is a net transepithelial absorption of Na⁺ and secretion of Cl^–.

Cl^– secretion predominates over the Na⁺ absorption in canine tracheal epithelia (33), whereas the opposite is true in the ferret and cat (34), and the two ion movements are relatively equal in bovine epithelia (35, 36) and in humans (37). Cl^– secretion is likely mediated by a number of different channel types, including the cystic fibrosis transmembrane conductance regulator, outwardly rectifying Cl^– channels and Ca²⁺-activated Cl^– channels. The cAMP-regulated cystic fibrosis transmembrane conductance regulator is the most abundant on the luminal membrane and thus has been the most extensively studied in airway epithelium (38).

In this study, we examined the electrophysiological responses evoked in fresh bovine airway epithelium by six structurally related isoprostanes (two E-ring and four F-ring). We found these to stimulate a substantial transepithelial Cl^– conductance via EP receptors coupled to cyclic nucleotide signaling pathways.

	MATERIALS AND METHODS

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Preparation of Bovine Bronchial Epithelial Tissues
All experimental procedures were approved by the McMaster University Animal Care Committee and conform to the guidelines set out by the Canadian Council on Animal Care. Tracheas were obtained from adult cows (135–455 kg) killed at a local abattoir and transported to the laboratory in 37°C Krebs buffer solution (composition below), which had been bubbled with 5% CO₂/95% O₂ mixture to pH 7.4. In the laboratory, loosely adherent connective tissue and vasculature were excised from the dorsal aspect of the whole trachea, and then the epithelium was dissected away from the underlying connective tissue on the smooth muscle surface of the tracheal lumen. Strips (15 mm x 5 mm) were cut with caution so as to not impale the uniform sheet before mounting for experiments. Tissues were used the same day or were stored overnight in sterile Dulbecco's modified Eagle medium supplemented with Penn/Strep (10,000 U/ml), amphotericin B (250 ng/ml), L-ascorbic acid (35 µg/m), transferrin (5 µg/ml), selenium (3.25 ng/ml), and insulin (2.85 µg/ml) at 4°C. It was our experience that the tissues survived overnight storage better in this medium, and we did not find the latter to alter electrophysiological responses; the majority of the data presented here were collected on the same day, with exploratory experiments being performed on the second day.

Ussing Chamber Technique
Tracheal epithelial strips were mounted on Lucite chambers with 0.6-cm² openings and inserted into an Ussing apparatus (World Precision Instruments, Sarasota, FL) in which luminal and basolateral faces were perfused with Krebs buffer, maintained at 37°C by heated water jackets, and bubbled continuously with a 95% O₂/5% CO₂ mixture. The transepithelial potential difference was recorded using Ag-AgCl electrodes connected to the Lucite chambers via agar bridges (3%, 1 M KCl). Each epithelial preparation was voltage-clamped to 0 mV using a DVC1000 voltage clamp (World Precision Instruments) and a pair of calomel electrodes connected via similar agar bridges. The short-circuit current (I_SC) required to maintain voltage-clamp was digitized and sampled at 50 Hz, and tissue conductance was calculated from the changes in I_SC during voltage pulses (5 mV; 0.5-s duration; 50-s intervals) across the epithelium using Ohm's law. Before experimentation, the voltage clamp and measurement electrodes were equilibrated in open circuit mode, after which potential differences between electrodes were nulled. Measurements of transepithelial potential and I_SC were stored to the hard drive for subsequent analysis using AcqKnowledge 3.8.5 (Goleta, CA) and SigmaPlot 2000 software (SPSS Inc., Chicago, IL).

Solutions and Chemicals
Tissues were maintained in standard Krebs-Ringer's buffer containing (in mM) NaCl, 116; KCl, 4.2; CaCl₂, 2.5; NaH₂PO₄, 1.6; MgSO₄, 1.2; NaHCO₃, 22; D-glucose, 11 bubbled to maintain pH at 7.4. Indomethacin (10 µM) was added to prevent endogenous generation of cyclooxygenase metabolites of arachidonic acid.

Isoprostanes, prostaglandins, and agonists/antagonists of prostanoid receptors were obtained from Cayman Chemicals (Ann Arbor, MI). All other chemicals were obtained from Sigma Chemical Co. or Gibco (Grand Island, NY). Pharmacological tools were prepared in 70% ethanol (all isoprostanes and prostanoids, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one [ODQ], niflumic acid, bumetanide, furosemide, and 3-isobutyl-1-methylxanthine [IBMX]) or dimethyl sulfoxide (amiloride, SC 19220, MDL 12330A). Agents were applied to the lumenal and/or adventitial chambers of the Ussing apparatus, as indicated.

Data Analysis
Unless indicated otherwise, I_SC was expressed as a percent change from baseline. All responses are reported as mean ± SEM; n refers to the number of animals. Statistical comparisons were made using Student's t test (for single pairwise comparisons); P < 0.05 was considered statistically significant. pEC₅₀ values were obtained by nonlinear fit analysis of concentration-response curves using SigmaPlot 2000 (SPSS Inc.).

	RESULTS

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Transepithelial Currents at Rest
Before investigating the effects of various ion channel blockers on isoprostane-evoked responses, we first characterized their effects on baseline I_SC (Figure 1).

View larger version (13K): [in this window] [in a new window]   Figure 1. Pharmacology of baseline transepithelial current in bovine airway epithelium. Mean changes (± SEM) in baseline short-circuit current (ISC) exerted by dimethyl sulfoxide, ethanol, or amiloride (50 µM). Also shown are the mean changes (± SEM) in ISC exerted by the Cl– channel blockers niflumic acid (100 µM) or 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB) (50 µM); the Na+/K+/Cl– cotransporter inhibitors furosemide (100 µM) or bumetanide (10 µM); or the nonselective phosphodiesterase inhibitor, isobutyl-1-methylxanthine (IBMX) (10 µM), in the presence or absence of amiloride (50 µM) (n > 6 for all). # Significantly different from vehicle control. * Statistically significant effect of amiloride.

Luminal application of the Na⁺ channel blocker amiloride (50 µM) caused a substantial decrease in I_SC, which peaked within 20 seconds. The mean reversal of I_SC in tissues was 10.57 ± 0.43 µA/cm² (n > 50). Adventitial application of amiloride also caused a comparable decrease in the magnitude of I_SC, but this required up to 20 minutes to stabilize.

The Cl^– channel blockers niflumic acid (100 µM) and 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB) (50 µM) also decreased I_SC: the mean reversals were 6.13 ± 0.74 (n = 9) and 3.85 ± 0.74 (n = 9) µA/cm², respectively, in the absence of amiloride or 1.0 ± 0.17 (n = 9) and 3.18 ± 0.58 (n = 15) µA/cm², respectively, in the presence of 50 µM amiloride.

I_SC was also modestly sensitive to inhibition of Na⁺/K⁺/2Cl^– cotransport. In the absence of amiloride, furosemide (100 µM) reversed this by 1.65 ± 0.31 µA/cm² (n = 6), whereas bumetanide (10 µM) reversed this by 2.64 ± 1.06 µA/cm² (n = 6). In the presence of amiloride (50 µM), these values were 0.14 ± 0.17 (n = 11) and 0.91 ± 0.55 µA/cm² (n = 6), respectively.

Collectively, these observations indicate a substantial Na⁺-permeable conductance on the lumenal face of the bovine airway epithelium, with a Cl^– channel and Na⁺/K⁺/2Cl^– cotransport located primarily at the adventitial face of the epithelium.

Finally, it seems that I_SC is increased by accumulation of cyclic nucleotides because IBMX increased I_SC by 3.82 ± 0.82 (n = 10) and 5.31 ± 0.69 (n = 14) µA/cm² in the absence or presence of amiloride, respectively.

Responsiveness of Bovine Airway Epithelium to Isoprostanes
Two different E-ring isoprostanes and four different F-ring isoprostanes were applied at a concentration of 10 µM in the presence of amiloride to both faces of the epithelium to identify those that were pharmacologically active. 15-E_1t-IsoP and 15-E_2t-IsoP increased I_SC by 26.66 ± 6.02 (n = 5) and 15.22 ± 2.01 µA/cm² (n = 24), respectively, whereas none of the F-ring isoprostanes statistically significantly altered I_SC when applied at this supramicromolar concentration (n = 3–4) (Figure 2). Given its efficacy relative to the other isoprostanes and structural similarity to prostaglandin (PG)E₂ (a prostanoid that was also found to evoke a substantial change in I_SC; see below), subsequent characterizations of the ion conductances and signaling pathways activated by isoprostanes were performed using 15-E_2t-IsoP.

View larger version (11K): [in this window] [in a new window]   Figure 2. Relative efficacies of six different isoprostanes. Mean increase in ISC (± SEM) upon luminal addition of two different E-ring and four F-ring isoprostanes, all at 10 µM, as indicated (n = 3).

View larger version (24K): [in this window] [in a new window]   Figure 3. Ion conductances underlying the isoprostane response. (A) Mean change (± SEM) in baseline ISC evoked by luminal application of 10 µM 15-E2t-IsoP after pretreatment for 10 minutes with vehicle (n = 24), the Cl– channel blockers niflumic acid (100 µM; n = 10) or NPPB (50 µM; n = 12), the Na+/K+/2Cl– cotransport inhibitors furosemide (100 µM; n = 13) or bumetanide (10 µM; n = 6), or the phosphodiesterase inhibitor IBMX (10 µM; n = 12). * Significantly different from vehicle. (B) Mean reversal (± SEM) of the 15-E2t-IsoP- (10 µM) enhanced ISC upon addition of niflumic acid (100 µM; n = 8), NPPB (50 µM; n = 8), bumetanide (10 µM; n = 6), or IBMX (10 µM; n = 8).

Potential Involvement of Prostanoid Receptor(s) in the 15-E_2t-IsoP Response
Because many previous studies of isoprostane pharmacology have found these to act through prostanoid receptors (usually TP or EP subtypes), we examined the effects of various blockers of prostanoid receptors on the potency of 15-E_2t-IsoP. Epithelial tissues were pretreated with amiloride (50 µM) alone or in conjunction with one of the prostanoid receptor blockers for 10 to 15 minutes before examining the full 15-E_2t-IsoP concentration-response relationship. None of these prostanoid receptor blockers had any significant effect on baseline I_SC compared with vehicle alone. The TP-receptor blocker ICI 192,605 (39) and the DP/EP₁/EP₂-receptor blocker AH 6809 (40, 41) each caused a small but statistically insignificant rightward shift in the concentration response relationship (Figure 4A; Table 1). Neither the EP₁-selective blocker SC19220 (41–43) nor the FP-receptor blocker AL 8810 (44) had affected the isoprostane concentration-response relationship (Figure 4A; Table 1). In a separate set of tissues, we examined the effects of the EP₄-selective blocker GW627386 (10^–7 M). The mean response to a half-maximally effective concentration of 15-E_2t-IsoP (10^–7 M) was significantly reduced in the presence of this agent compared with tissues treated with vehicle (2.5 ± 1.5 µA/cm² and 10.8 ± 3.0 µA/cm², respectively; n = 4), whereas the maximal response to isoprostane was not affected (41.1 ± 5.0 µA/cm² and 40.0 ± 4.5 µA/cm², respectively).

View larger version (14K): [in this window] [in a new window]   Figure 4. Involvement of prostanoid receptors in the isoprostane response. (A) Mean (± SEM) concentration–response relationships for 15-E2t-IsoP in tissues pretreated with amiloride alone (50 µM; n = 38) or in conjunction with the EP1 antagonist SC 19220 (10 µM; n = 8), the TP antagonist ICI 192605 (10 µM; n = 5), the DP/EP1/EP2 antagonist AH 6809 (10 µM; n = 5), or the FP antagonist AL 8810 (10 µM; n = 7); all blockers were added bilaterally. (B) Mean (± SEM) concentration–response relationships for 15-E1t-IsoP, 15-E2t-IsoP or prostaglandin (PG)E2 added luminally only (n = 3, n = 14, and n = 5, respectively) or basolaterally only (n = 12, n = 8, and n = 6, respectively). (C) Mean (± SEM) concentration–response relationships for 15-E2t-IsoP (n = 38), the TP-agonist U46619 (n = 11), the EP3-agonist sulprostone (n = 4), or the FP-agonist fluprostenol (n = 4), all added luminally.

Collectively, these data indicate that I_SC in the bovine epithelium is not regulated by TP, DP, or FP receptors and that 15-E_2t-IsoP may regulate I_SC through EP receptors that are not of the EP₁, EP₂, or EP₃ subtypes.

Elucidating the Second Messenger Involved in 15-E_2t-IsoP Response
Given that I_SC was augmented by IBMX (see Figure 1), and that EP₂/EP₄ receptors are known to couple through adenylate cyclase, we examined the effects of the adenylate cyclase inhibitor MDL 12,330A (46) on the responses to 15-E_2t-IsoP, making comparisons with the guanylate cyclase inhibitor ODQ (47). When tissues were pretreated bilaterally with 10 µM MDL 12,330A for 10 to 15 minutes, the maximal change in I_SC evoked by 15-E_2t-IsoP was decreased significantly from 21.75 ± 1.23 µA/cm² to 10.98 ± 3.22 µA/cm² (n = 6) (Figure 5A). Bilateral pretreatment of the tissues with 10 µM ODQ, on the other hand, effectively abolished responsiveness to 15-E_2t-IsoP (Figure 5A). In another set of tissues that were first stimulated with 15-E_2t-IsoP, subsequent bilateral addition of MDL 12,330A (10 µM; n = 6) only partially reversed I_SC, whereas bilateral addition of ODQ (10 µM; n = 6) completely reversed the 15-E_2t-IsoP response (Figure 5B).

View larger version (20K): [in this window] [in a new window]   Figure 5. Involvement of cyclic nucleotides in the isoprostane response. (A) Mean (± SEM) concentration–response relationships for 15-E2t-IsoP in tissues pretreated with amiloride alone (50 µM; n = 38) or in conjuction with bilateral application of MDL 12330A (10 µM; n = 6) or 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) (10 µM; n = 6). * Significantly different from amiloride control. (B) Mean (± SEM) reversal of 10 µM 15-E2t-IsoP–evoked ISC upon bilateral addition of MDL 12,330A or ODQ (both 10 µM; both n = 6).

	DISCUSSION

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This electrophysiological response is clearly mediated by an action on some relatively selective receptor because it is able to distinguish completely between the E-ring and F-ring isoprostanes, which differ only with respect to the nature of an oxygen group on the central cyclopentane ring: a ketone group in the E-ring molecule and a hydroxyl group in the F-ring molecule. Whichever receptor is involved, it seems to be located on the luminal face of the epithelium. Many previous studies of the pharmacology of isoprostane-evoked responses have concluded that they act through prostanoid receptors, usually of the TP or EP types (1). Our observation that U46619 did not mimic the electrophysiological effect of 15-E_2t-IsoP indicates that TP receptors, if present in these tissues, clearly do not couple to the signaling pathway, which is triggered by that isoprostane. Consistent with that conclusion, we did not find the TP-receptor blocker ICI 192,605 to have any statistically significant effect on the isoprostane-evoked response, even when applied at 10 µM (orders of magnitude above its published pA₂ value of 7.5) (39, 43). Likewise, the fact that 15-E_2t-IsoP responses were not blocked by AL 8810 nor mimicked by fluprostenol indicates that FP receptors are not involved. Our use of 10 µM AH6809 and the fact that all experiments were performed in the presence of indomethacin rule out the involvement of DP receptors. Instead, the responses were mimicked by PGE₂, suggesting an involvement of EP receptors. Given that they were not mimicked by sulprostone or blocked by AH 6809 or SC 19220, we conclude that EP₁, EP₂, and EP₃ receptors are not involved. On the other hand, the isoprostane-evoked response was markedly and statistically significantly suppressed by the EP₄-receptor blocker GW627386. Altogether, the data clearly advocate for an action of 15-E_2t-IsoP through EP₄-receptors.

EP₄ receptors typically mediate their responses through stimulation of adenylate cyclase activity. We found that I_SC was augmented by IBMX, which, as a phosphodiesterase inhibitor, causes accumulation of cyclic nucleotides. Pretreatment with the adenylate cyclase inhibitor MDL 12330A substantially suppressed the isoprostane concentration-response relationship, decreasing its efficacy (a measure of receptor–effector coupling) without altering its potency (an indicator of receptor–ligand interactions). However, ODQ, an inhibitor of soluble guanylate cyclase activity, effectively abolished responsiveness to the isoprostane. It seems, then, that cGMP plays a major role in mediating the isoprostane-evoked response. Interactions ("cross-talk") between the cAMP/PKA and cGMP/PKG signaling pathways are common. For example, Namkoong and colleagues (49) have recently described an effect of PGE₂ on human umbilical vein endothelial cells that involves stimulation of soluble guanylate cyclase via cAMP-dependent, PKA-mediated phosphorylation of endothelial nitric oxide synthase. Future studies are needed to unravel potential interactions between these two signaling pathways in the response of airway epithelium to isoprostanes.

Airway epithelial cells are subject to oxidative stress. Free radicals arising during inadequate ventilation/perfusion matching or released by inflammatory cells are known to lead to the generation of isoprostanes. In fact, isoprostanes have been shown to accumulate to substantial levels in a variety of airway-related disease states including asthma (6–8), chronic obstructive pulmonary disease (5, 12, 13), and cystic fibrosis (10, 11), as well as after inhalation of allergen, ozone, or cigarette smoke (2–4). Therefore, the finding that isoprostanes alter airway epithelial function is of major clinical relevance. For example, stimulation of a chloride conductance could account for the increased mucus production typically seen in these pathologies because this leads to the secretion of salt and water into the lumen of the airways and consequent hydration of mucus. It remains to be seen whether isoprostanes alter expression and release of cytokines and other signaling molecules because they have been shown to do so in airway smooth muscle (50).

In conclusion, we have found that the E-ring isoprostane 15-E_2t-IsoP (but not the F-ring) isoprostanes stimulate a transepithelium chloride conductance in bovine airway through activation of an EP₄ receptor on the basolateral membrane coupled through a cyclic nucleotide signaling pathway. Further studies are needed to further elucidate the underlying signaling mechanisms.

	Footnotes

 
These studies were supported by operating grants from the Canadian Institutes of Health Research and the Ontario Thoracic Society.

Originally Published in Press as DOI: 10.1165/rcmb.2007-0117OC on August 2, 2007

Conflict of Interest Statement: L.J.J. has received $70,000 from Astra-Zeneca to study the mechanisms underlying B-adrenoceptor desensitization in airway smooth muscle and $75,000 from Glaxo-SmithKline to study isoprostane-induced airway hyperresponsiveness. None of the other authors has a financial relationship with a commercial entity that has an interest in the subject of this manuscript.

Received in original form April 4, 2007

Accepted in final form July 5, 2007

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