Receptors and signaling pathway underlying relaxations to isoprostanes in canine and porcine airway smooth muscle

Adriana Catalli, Dawei Zhang, and Luke J. Janssen

Asthma Research Group, Father Sean O'Sullivan Research Center, Firestone Institute for Respiratory Health, St. Joseph's Hospital; and Department of Medicine, McMaster University, Hamilton, Ontario, Canada L8N 4A6


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Using muscle bath techniques, we examined the inhibitory activities of several E- and F-ring isoprostanes in canine and porcine airway smooth muscle. 8-Isoprostaglandin E1 and 8-isoprostaglandin E2 (8-iso PGE2) reversed cholinergic tone in a concentration-dependent manner, whereas the F-ring isoprostanes were ineffective. Desensitization with 8-iso-PGE2 and PGE2 implicated isoprostane activity at the PGE2 receptor (EP). We found that the inhibitory E-ring isoprostane responses were significantly augmented by rolipram (a type IV phosphodiesterase inhibitor), while 1H-[1,2,4]-oxadiazolo[4,3-a]quinoxalin-1-one (a guanylate cyclase inhibitor) had no effect, suggesting a role for cAMP in isoprostane-mediated relaxations. 8-Iso-PGE2 did not reverse KCl tone, suggesting that voltage-dependent Ca2+ influx and myosin light chain kinase are not suppressed by isoprostanes. Patch-clamp studies showed marked suppression of K+ currents by 8-iso-PGE2. We conclude that E-ring isoprostanes exert PGE2 receptor-directed, cAMP-dependent relaxations in canine and porcine airway smooth muscle. This activity is not dependent on K+ channel activation or the direct inhibition of voltage-operated Ca2+ influx or myosin light chain kinase.

cyclic nucleotides; potassium channels; calcium channels; prostanoid receptors


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

IT HAS BEEN SUGGESTED THAT the bronchodilatory and contractile effects of free radicals in airways are mediated by isoprostanes (9), a large family of prostaglandin (PG)-like molecules, produced in a cyclooxygenase-independent manner via free radical-mediated peroxidation of the membrane phospholipid arachidonic acid (22-25). These molecules, which differ from PGs in the cis orientation of their side chains at the cyclopentane ring junction, are present at levels in the nanomolar range in normal human plasma and urine (23). Isoprostanes are present in even higher concentrations during periods of oxidative stress (20, 21, 23, 27), in such diseases as asthma (18), cystic fibrosis (19), chronic obstructive pulmonary disorder (17), and atherosclerosis (33), and may contribute to the pathology of these diseases (13, 23, 28).

Researchers have only recently begun to demonstrate the biological responses elicited by isoprostanes in many tissue types. Most research has focused solely on the excitatory activity of 8-iso-PGF2alpha , a vasoconstrictor (16, 28, 40), bronchoconstrictor (9, 13), and inhibitor of platelet aggregation (26).

Very little is known about the signaling pathways or receptors through which inhibitory isoprostanes act. It has been suggested that isoprostanes may act via prostanoid receptors (4, 37); isoprostanes and prostanoids differ only in the orientation of their side chain, thus allowing for many similar binding interactions with receptor active site residues (1, 38).

The prostanoid receptors are categorized as follows: DP, EP, FP, IP, and TP receptors, one each for the five principal metabolites PGD2, PGE2, PGF2alpha , PGI2, and thromboxane A2, respectively (with each receptor having an affinity of >= 1 order of magnitude greater for one prostanoid over the other four) (2, 29). The EP receptor can be further subdivided into the EP1, EP2, EP3, and EP4 subtypes, all of which bind PGE2 with high affinity but differ with regard to physiological activity and/or the signaling pathway induced (29). Evidence supporting subtypes of the EP1, EP3, FP, and TP receptors exists as well (7, 15, 32).

There is evidence to support 8-iso-PGF2alpha acting as an agonist at the vascular TP receptors (16, 28, 40) while conversely acting antagonistically at platelet TP receptors to inhibit aggregation (26). 8-Iso-PGE2 may act at the TP receptor as well in renal vasculature (25). There are also studies that support a role for the FP (39) and the EP prostanoid receptors (34, 38, 39) in excitatory 8-iso-PGE2 activity. It has also been argued that isoprostanes may exert their effects through their own unique class of receptor (28).

Previously, we communicated the effects of seven different isoprostanes on airway smooth muscle, including the first description of relaxations evoked by E-ring isoprostanes (9). The aim of this study was to further enhance the present knowledge of isoprostanes and their inhibitory physiological responses. For this purpose, canine and porcine bronchial smooth muscle was used to examine the relaxant effects of several E- and F-ring isoprostanes as well as the signaling pathway(s) underlying these relaxations. Initial studies were also performed to assess the possibility that the relaxant isoprostanes act through the classic relaxant prostanoid receptors IP, DP, EP2, and EP4.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Tissue preparation. Adult mongrel dogs were euthanized with pentobarbital sodium (100 mg/kg), and lobes of lungs were excised; pig lungs were obtained from an abattoir. Trachea and lungs were placed in ice-cold Krebs solution (see Solutions and chemicals) and secured with dissection pins. The bronchus was carefully dissected by removal of the overlying connective tissue and pulmonary vasculature. Tracheal smooth muscle strips (~1 mm wide) and bronchial ring segments (3-5 mm OD, ~2-5 mm long) were excised and used immediately or stored at 4°C for use up to 48 h later.

Muscle bath studies. Tracheal strips and bronchial smooth muscle ring segments were hung in 4-ml baths. The tracheal strips were tied with silk thread (Ethicon 4-0), such that one end of the strip was anchored, while the other was fastened to a Grass FT.03 force transducer. Hooks inserted through the lumen, one of which was anchored and the other fastened to the transducer, were used to hang the bronchial rings. Changes in isometric force were digitized (at 2 Hz) and recorded on-line using the DigiMed System Integrator program (MicroMed, Louisville, KY).

Protocol. Tissues were maintained in Krebs buffer containing 10-5 M indomethacin (to prevent formation of endogenous PGs, which could cause relaxation), gassed with 95% O2-5% CO2 to give a final pH of 7.4, and maintained at 37°C. Preload tension was applied and kept between 1.0 and 1.5 g (optimal resting tension) to ensure maximal physiological responses. Tissues were equilibrated for 1 h, during which time they were repeatedly washed with Krebs buffer. To establish a reference contraction for standardization of contractile responses and to ensure tissue responsiveness, tissues were challenged with 60 mM KCl. The KCl was then washed out, and tissues were allowed to recover before the experiments were begun.

Tissues were exposed to 10-6 M ICI-192605, a TP prostanoid receptor blocker, >= 15 min before isoprostane additions to inhibit excitatory activities of the isoprostanes at this receptor (9). Relaxant responses were studied in tissues precontracted with the cholinergic agonist carbachol (CCh) or KCl. Once tone stabilized, cumulative concentration-relaxation response relationships were evaluated for the various isoprostanes. All inhibitors {rolipram, atropine, propranolol, and 1H-[1,2,4]-oxadiazolo[4,3-a]quinoxalin-1-one (ODQ)} were added >= 20 min before addition of precontractile agonist to allow establishment of inhibitory activity.

Patch-clamp studies. Tracheal tissues were minced, transferred to dissociation buffer containing collagenase (Sigma blend F; 0.9 U/ml) and elastase (type IV, 12.5 U/ml), incubated at 37°C for 1 h, and then gently triturated to liberate individual myocytes. These were allowed to settle and adhere to the bottom of a recording chamber (1 ml volume) and superfused with standard Ringer solution at room temperature. Electrophysiological responses were tested in cells that were phase dense and appeared relaxed. Recordings were made using the nystatin perforated-patch configuration of the conventional patch-clamp recording technique (8) and pipettes with tip resistances of 3-5 MOmega when filled with electrode solution. Membrane currents were filtered at 5 kHz and sampled at 2 Hz. Acquisition and analysis of data were accomplished using Axopatch 200B and pCLAMP8 software (Axon Instruments, Foster City, CA).

Solutions and chemicals. The Krebs buffer consisted of 116 mM NaCl, 4.6 mM KCl, 2.5 mM CaCl2, 1.3 mM NaH2PO4, 1.2 mM MgSO4, 23 mM NaHCO3, 11 mM D-glucose, and 10-5 M indomethacin bubbled to maintain pH 7.4. The composition of the dissociation buffer was as follows (mM): 125 NaCl, 5 KCl, 1 CaCl2, 1 MgCl2, 10 HEPES, 0.25 EDTA, 10 D-glucose, and 10 L-taurine (pH 7.0). The composition of the electrode solution was as follows: 140 mM KCl, 1 mM MgCl2, 0.4 mM CaCl2, 20 mM HEPES, 1 mM EGTA, and 150 U/ml nystatin (pH 7.2). The composition of Ringer buffer was as follows (mM): 130 NaCl, 5 KCl, 1 CaCl2, 1 MgCl2, 20 HEPES, and 10 D-glucose (pH 7.4). The isoprostanes were dissolved in ethanol, with the exception of 8-iso-PGF3alpha , which was dissolved in methyl acetate; these stock solutions were then diluted with Krebs buffer. Aqueous dilutions were discarded after 24 h. ODQ, S-nitroso-N-acetylpenicillamine (SNAP), rolipram, and ICI-192605 were dissolved in DMSO. All other compounds were dissolved in Krebs buffer. The isoprostanes and PGE2 were obtained from Cayman Chemical (Ann Arbor, MI) and rolipram from Research Biochemicals. Cicaprost, iloprost, and BW-245C were gifts from Dr. Denis Crankshaw (McMaster University). All other chemicals were obtained from Sigma Chemical.

Data analysis. Relaxations were expressed as percent reversal of precontractile tone (minus preload tone). The concentration at which 50% reversal of tone was achieved ([R50] values) was interpolated from the concentration-response relationships. The -log [R50] value (in M) is used to express the potency of the drug; %R is used to describe the percentage of relaxation evoked for the highest drug concentration used (i.e., the maximal response); n is the number of animals used.

Values are means ± SE. One-way ANOVA and Student's t-test were used to ascertain the statistical significance of differences between mean values.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Relaxant responses to isoprostanes in canine and porcine bronchial smooth muscle. Cumulative concentration-response relationships were investigated for various E- and F-ring isoprostanes (Fig. 1). In canine and porcine tissues, 8-iso-PGF1alpha , 8-iso-PGF2alpha , and 8-iso-PGF2beta had little or no effect on cholinergic tone. In canine tissue, 8-iso-PGE1, 8-iso-PGE2, and 8-iso-PGF3alpha were quite effective as bronchodilators, achieving 78 ± 5, 83 ± 4, and 88 ± 4% reversal of tone, respectively, when applied at 10-5 M (Fig. 2A): -log [R50] values were 5.4 ± 0.05, 5.6 ± 0.1, and 5.5 ± 0.04 M, respectively. In porcine tissue, 8-iso-PGE1 and 8-iso-PGE2 at 10-5 M achieved 59 ± 13 and 59 ± 7% reversals at a concentration of 10-5 M (Fig. 2B). The cost of these compounds prevented us from testing at >10-5 M. 


View larger version (8K):
[in this window]
[in a new window]
 
Fig. 1.   Relaxation evoked by 8-isoprostaglandin E2 (8-iso PGE2) in canine bronchial smooth muscle precontracted with 10-6 M carbachol (CCh). Tissue was pretreated with 10-6 M ICI-192605.



View larger version (14K):
[in this window]
[in a new window]
 
Fig. 2.   Isoprostane concentration-relaxation relationships. Inhibitory responses of a variety of isoprostanes were examined using the protocol shown in Fig. 1. A: canine bronchial tissues (n = 4, 10-6 M CCh). B: porcine bronchial tissues (n = 4-5, 3 × 10-7 M CCh).

Receptor identity. It has been suggested that isoprostanes may act via the classic prostanoid receptors (4, 37), three of which, DP, IP, and EP (EP2 and EP4 subtypes), are generally inhibitory in smooth muscle. We first sought to ascertain which inhibitory prostanoid receptors are present in canine bronchial tissue. Cumulative concentration-response relationships were investigated using the receptor-selective agonists cicaprost and iloprost (IP selective), BW-245C (DP selective), and PGE2 (EP selective, but does not distinguish between EP2 and EP4). The DP-selective agonist exerted only minor reversal (25 ± 6%) of cholinergic tone at 10-5 M (Fig. 3), whereas the IP-selective agonists were somewhat more efficacious, their effects beginning in the submicromolar range, but still reversed cholinergic tone by <50% at the highest concentration tested (%R = 51 ± 8% for cicaprost and 37 ± 4% for iloprost). The EP-selective agonist, however, markedly reversed tone (%R = 76 ± 13% at 10-5 M), with -log [R50] estimated to be 6.4 ± 0.1 M. These results indicate that the inhibitory prostanoid receptors in canine bronchi are predominantly of the EP type.


View larger version (15K):
[in this window]
[in a new window]
 
Fig. 3.   Inhibitory EP receptors are present in canine bronchial tissue. Concentration-relaxation relationships for various prostanoid receptor agonists were examined. IP agonists cicaprost and iloprost (n = 3) and DP agonist BW-245C (n = 4) exhibited minor reversal of CCh (10-6 M) tone; EP receptor agonist PGE2 (n = 4) demonstrated marked relaxation.

Before assessing the role of the EP receptors in isoprostane-mediated inhibitory responses, we ascertained the antagonistic effects of the precontractile agent in these studies. CCh, a cholinergic agonist, acts at the muscarinic receptors present on airway smooth muscle to induce phosphoinositide turnover (M3 receptors) and inhibition of adenylate cyclase (M2 receptors). Because the inhibitory EP receptors generally induce adenylate cyclase activity (29), the use of CCh as the precontractile agent may confound the results, leading to attenuation of the EP agonist relaxant response. This functional antagonism has been seen with other bronchodilators such as beta -adrenergic agonists (5). We evaluated the antagonistic effects of CCh using the M2 receptor-selective antagonist AFDX-116 (10-6 M). AFDX markedly enhanced the responses to 8-iso-PGE2 by as much as 50% at 10-7 M, shifting the -log [R50] from 6.0 ± 0.2 to 7.3 ± 0.1 M (Fig. 4). We conclude that activation of the M2 receptor by CCh antagonizes the activity of 8-iso-PGE2 (likely via inhibition of adenylate cyclase).


View larger version (11K):
[in this window]
[in a new window]
 
Fig. 4.   Muscarinic M2 receptor inhibition enhanced 8-iso-PGE2 response. In porcine tracheal smooth muscle precontracted with 3 × 10-7 M CCh, muscarinic M2 receptor-selective inhibitor AFDX-116 (10-6 M) heightened 8-iso-PGE2-evoked responses (n = 6). * Significantly different from control.

Next, we addressed whether the responses to 8-iso-PGE2 are mediated through the EP receptors. Unfortunately, highly selective antagonists for the inhibitory EP receptor subtypes (EP2 and EP4) are not readily available. We therefore investigated the effects of desensitizing the tissues to PGE2 on 8-iso-PGE2-induced relaxations and vice versa. Tissues were exposed to a high concentration of PGE2 or 8-iso-PGE2 (10-5 M) for 3 h before assessment of the concentration-response profiles for these two agonists, as well as isoproterenol. AFDX-116 (10-6 M) was added near the end of the desensitization period for 20 min, and the tissues were washed with CCh (7 × 10-7 M)-supplemented Krebs solution (to remove the desensitizing agent). After 15 min, during which time cholinergic tone became reasonably stable, the responses to PGE2, 8-iso-PGE2, and isoproterenol were compared.

The data are summarized in Fig. 5. After the tissues were desensitized to 8-iso-PGE2, responses to the isoprostane and the PG were reduced by as much as 45 and 64% for 8-iso-PGE2 (10-5 M) and PGE2 (10-7 M), respectively, whereas the isoproterenol response was essentially unaltered. Similarly, desensitization with PGE2 resulted in marked suppression of responses to PGE2 (79% at 10-7 M) and 8-iso-PGE2 (37% at 10-5 M), but not to isoproterenol. The lack of desensitization in the case of isoproterenol, which does not induce EP receptor activity, indicates that the attenuation of PGE2- and 8-iso-PGE2-induced relaxations was due to homologous desensitization.


View larger version (11K):
[in this window]
[in a new window]
 
Fig. 5.   8-Iso-PGE2 appears to act via EP receptors. In porcine tracheal tissue, relaxations were evoked by 8-iso-PGE2 (A), PGE2 (B), or isoproterenol (C) in control tissues and in tissues that had been desensitized to 8-iso-PGE2 or PGE2. Relaxations are expressed as percent reversal of tone evoked by 7 × 10-7 M CCh. All tissues were exposed to 10-6 M AFDX-116 (n = 4-6). * Significantly different from control.

Equieffective concentrations of the isoprostane and prostanoid were used in similar but limited studies on canine bronchial smooth muscle. After the tissues were desensitized to 8-iso-PGE2, responses to 8-iso-PGE2 (10-5 M) and PGE2 (10-6 M) decreased equivalently (40 and 39%, respectively; Table 1). Similarly, after the tissues were desensitized with PGE2, both responses were reduced to a similar degree (30 and 37%, respectively; Table 1). These results are consistent with 8-iso-PGE2 acting at an EP receptor.

                              
View this table:
[in this window]
[in a new window]
 
Table 1.   Reversal of cholinergic tone by 8-iso-PGE2 or PGE2 in 8-iso-PGE2- or PGE2-desensitized canine bronchial tissues

Role of cyclic nucleotides. The EP2 and EP4 receptors generally couple to the Gs protein to induce adenylate cyclase activity, resulting in increased cAMP levels and subsequent activation of protein kinase A, which in turn phosphorylates various targets, leading to smooth muscle relaxation (14, 29). cAMP can also cause elevation of cGMP levels by various means, leading to powerful relaxations (31). We therefore investigated the role of cAMP vs. cGMP in isoprostane-induced relaxations.

The effects of ODQ, an inhibitor of soluble guanylate cyclase, on 8-iso-PGE2- and SNAP-evoked responses were compared. ODQ had essentially no effect on 8-iso-PGE2-induced reversal of tone evoked by 10-7 M CCh, whereas SNAP-induced relaxations were abolished (Fig. 6). We conclude that guanylate cyclase plays little or no role in 8-iso-PGE2-induced relaxations. To ascertain the involvement of cAMP in these relaxations, we examined relaxations evoked by 8-iso-PGE2 (in canine tissues preconstricted with 10-7 M CCh) and 8-iso-PGE1 (in porcine tissues preconstricted with 3 × 10-7 M CCh) in the presence or absence of the cAMP-dependent phosphodiesterase inhibitor rolipram (type IV phosphodiesterase blocker, 10-5 M). Rolipram significantly augmented the relaxations at 10-5 and 10-6 M for 8-iso-PGE1 and at 10-6 M for 8-iso-PGE2 (Fig. 7). These data suggest that cAMP may play a role in the signaling pathway activated by the isoprostanes.


View larger version (9K):
[in this window]
[in a new window]
 
Fig. 6.   8-Iso-PGE2-induced relaxations are not mediated by cGMP. In canine bronchial smooth muscle precontracted with 10-7 M CCh, 10-5 M 1H-[1,2,4]-oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) had no effect on concentration-relaxation relationship for 8-iso-PGE2 but essentially abolished that for S-nitroso-N-acetylpenicillamine (SNAP, A and B, respectively; n = 4 for both). * Significantly different from control.



View larger version (19K):
[in this window]
[in a new window]
 
Fig. 7.   E-ring isoprostanes evoke relaxations via second messenger cAMP. In porcine bronchial smooth muscle precontracted with 3 × 10-7 M CCh (right), phosphodiesterase inhibitor rolipram (10-5 M) heightened 8-iso-PGE1-evoked responses (n = 5). Rolipram also enhanced 8-iso-PGE2 responses in canine bronchial smooth muscle precontracted with 10-7 M CCh (left; n = 5).

Inhibition of Ca2+ channels or myosin light chain kinase. Relaxant agonists may act by inhibiting Ca2+ influx and/or suppressing myosin light chain kinase (MLCK) activity (36). To test whether isoprostanes exert either of these effects, we used tissues preconstricted with KCl (60 mM), which evokes contractions that are entirely dependent on both of these processes. Tissues were pretreated with atropine and propranolol to eliminate the contributions of cholinergic and adrenergic nerves stimulated by KCl. 8-Iso-PGE2 did not substantially reverse KCl tone, inducing <15% relaxation, compared with the complete reversal of tone in matching tissues preconstricted with 10-7 M CCh (Fig. 8). The relative lack of effect of the isoprostane on KCl-evoked tone indicates that 8-iso-PGE2 does not have a major inhibitory effect on voltage-gated Ca2+ channels or MLCK.


View larger version (12K):
[in this window]
[in a new window]
 
Fig. 8.   8-Iso-PGE2 does not inhibit voltage-dependent Ca2+ influx or myosin light chain kinase. In canine bronchial tissues precontracted with 60 mM KCl, 8-iso-PGE2 failed to evoke a relaxant response. In tissues precontracted with 10-7 M CCh, 8-iso-PGE2 completely reversed tone (n = 4).

Activation of K+ channels. It is often suggested that bronchodilators mediate their effects in part by opening K+ channels, leading to membrane hyperpolarization and cessation of voltage-dependent Ca2+ influx. We used the patch-clamp electrophysiological technique to test directly whether isoprostanes activate K+ currents. Figure 9A shows representative traces of currents evoked using depolarizing step commands from a holding potential of -60 mV; the current-voltage relationship is given in Fig. 9B. 8-Iso-PGE2 (10-5 M) in the application pipette caused a marked suppression of these K+ currents: on average, currents evoked by pulses to +10 mV were reduced 72 ± 13% (n = 8). Clearly, then, isoprostanes do not mediate their relaxant effects via activation of K+ channels.


View larger version (26K):
[in this window]
[in a new window]
 
Fig. 9.   K+ currents are suppressed by 8-iso-PGE2. A: representative trace showing outward currents evoked in a canine tracheal myocyte by depolarizing pulses (from -60 to +40 mV, in 10-mV increments) from a holding potential of -70 mV, in the presence and absence of 8-iso-PGE2 (10-5 M in application pipette). B: mean amplitude of outward current evoked by individual depolarizing pulses to +40 mV at 15-s intervals (open circle ) or as part of protocol in A ().


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Researchers have only begun to characterize the biological effects of isoprostanes. Most studies of isoprostane-elicited responses have focused solely on the excitatory activity of 8-iso-PGF2alpha on various types of tissues. Only recently has attention been focused on their potential inhibitory effects.

Previously, we reported the effects of seven different isoprostanes on airway smooth muscle, including the first description of relaxations evoked by E-ring isoprostanes (9). The results of this study demonstrate that 8-iso-PGE1 and 8-iso-PGE2 induce relaxations in canine and porcine bronchial smooth muscle with similar potencies, whereas F-ring isoprostanes are relatively ineffective in this respect. E-ring isoprostane-induced relaxations were conserved in epithelium-denuded porcine tracheal tissues as well as in N-nitro-L-arginine- and indomethacin-treated, epithelium-intact bronchial tissues (data not shown). This finding is consistent with isoprostanes acting directly on smooth muscle tissues, although a role for epithelium-derived relaxant mediators cannot be entirely discounted.

Isoprostanes and prostanoids share a similar structure, differing only in the orientation of their side chains; thus it is reasonable to assume that they are capable of forming similar binding interactions with receptor active site residues (23). Indeed, Breyer (1) and Ungrin et al. (38) shed some light on the structural features of PGE2 required for binding to the EP1 prostanoid receptor, including the hydroxyl group on the cyclopentane ring and the omega  side chain, both of which are present on the isoprostanes tested in this study. There is evidence to support 8-iso-PGF2alpha (16, 26, 28, 40) and 8-iso-PGE2 (25) activity at the TP prostanoid receptor. There are also studies supporting the role of FP (39) and EP excitatory prostanoid receptors (34, 38, 39) in 8-iso-PGE2 activity. On the other hand, others argue for the existence of a unique class of isoprostane receptors (28).

The EP2 prostanoid receptor, along with the IP and, to a lesser extent, the DP receptor, is known to be involved in human bronchial smooth muscle relaxations, whereas EP4 is not (30). EP2 was also shown to mediate airway smooth muscle relaxations in mice (6, 35). Our data suggest that IP and DP receptors are largely unimportant with respect to prostanoid relaxations in canine bronchial tissue, since only partial responses to the IP- and DP-selective agonists are seen and only at relatively high concentrations. Instead, these IP- and DP-selective agonists may be exerting nonspecific actions at inhibitory EP receptors, which are present and are likely to be involved in isoprostane-induced relaxations, as was demonstrated using receptor desensitization techniques. Using this approach, we determined that PGE2 desensitization of the 8-iso-PGE2 response and vice versa were due to homologous desensitization, inasmuch as neither 8-iso-PGE2 nor PGE2 induced heterologous desensitization of the isoproterenol response.

The EP2 and EP4 receptors couple to the Gs protein to induce adenylate cyclase activity, resulting in increased cAMP levels (14, 29). Inhibition of type IV phosphodiesterase activity with rolipram enhanced the relaxations to 8-iso-PGE1 and 8-iso-PGE2 in porcine and canine tissues. These results suggest that the two E-ring isoprostanes may induce relaxations through the accumulation of cAMP and that type IV phosphodiesterase opposes this. Further studies involving the inhibition of adenylate cyclase and protein kinase A would lend additional support to this conclusion. In contrast, the guanylate cyclase inhibitor ODQ had no effect on 8-iso-PGE2-induced relaxations, thus ruling out the involvement of cGMP in these responses.

Isoprostanes may induce relaxations by downregulating MLCK activity directly or indirectly via inhibition of Ca2+ influx from voltage-gated Ca2+ channels. KCl was employed to elicit contractions via membrane depolarization, promoting the opening of voltage-gated Ca2+ channels. The resulting influx of cytosolic Ca2+ activates MLCK, which phosphorylates myosin, thus allowing for interaction with actin filaments (36). Essentially no reversal of KCl tone was observed for 8-iso-PGE2, thus implying no direct inhibition of the voltage-gated Ca2+ channels or MLCK by 8-iso-PGE2 occured. However, no direct measurements of MLCK activity were performed.

Alternatively, 8-iso-PGE2 may activate K+ channels in the membrane. Bronchodilators such as beta -adrenergic agonists and nitric oxide are proposed to induce relaxations in airways via activation of large-conductance Ca2+-dependent K+ channels (3, 10-12). We tested this directly using patch-clamp techniques and found that K+ currents were rapidly and markedly suppressed by isoprostanes.

Other signaling pathways by which isoprostanes might exert a relaxant effect include 1) inhibition of the Rho A-Rho kinase pathway, which increases the Ca2+ sensitivity of the contractile apparatus by inhibiting myosin light chain phosphatase, 2) inhibition of the phosphoinositide pathway, which increases cytosolic Ca2+ by activating sarcoplasmic reticulum inositol trisphosphate Ca2+ channels, 3) activation of sarcoplasmic/endoplasmic reticulum Ca2+-ATPase, which decreases cytosolic Ca2+, or 4) activation of myosin light chain phosphatase, which dephosphorylates and deactivates myosin.

Most of the experiments in this study involved the use of CCh, a cholinergic agonist that acts at the M2 and M3 muscarinic receptors present on airway smooth muscle to induce contraction. Activation of M3 receptors leads to the induction of phosphoinositide turnover, whereas M2 receptor activation results in the inhibition of adenylate cyclase and thus a decreased level of cAMP. Because inhibitory isoprostanes appear to act through induction of adenylate cyclase activity, the use of CCh as the precontractile agent confounds the results, leading to an attenuation of isoprostane-induced relaxation. We have demonstrated that this functional antagonism could be overcome using the M2 receptor-selective inhibitor AFDX-116.

Isoprostanes are present in the nanomolar range in normal human plasma (23) and increase up to 100- to 200-fold during periods of oxidative stress, which characterize a variety of disease states (20, 21, 23, 27). The physiological responses of isoprostanes commence in the submicromolar range and are thus clinically relevant. Therefore, a better understanding of the mechanisms of action of isoprostanes may provide further insight into the pathology of oxidative stress.

In conclusion, it was found that 8-iso-PGE2 and 8-iso-PGE1 induce relaxations in canine and porcine bronchial smooth muscle, likely because of activity at the EP prostanoid receptor. The relaxant responses to the E-ring compounds appear to be mediated by the second messenger cAMP. Although the downstream effector(s) is unclear, we have ruled out activation of K+ channels and have evidence to suggest that there is no direct inhibition of voltage-gated Ca2+ channels or of MLCK.


    ACKNOWLEDGEMENTS

We thank Dr. D. Crankshaw (McMaster University) for the kind gift of cicaprost, iloprost, and BW-245C.


    FOOTNOTES

These studies were supported by operating grants from the Canadian Institutes of Health Research and the Ontario Thoracic Society, a studentship from the National Science and Engineering Research Council (A. Catalli), and a Medical Research Council of Canada Scientist Award (L. J. Janssen).

Address for reprint requests and other correspondence: A. Catalli, Dept. of Medicine, McMaster University, St. Joseph's Hospital, 50 Charlton Ave. East, Hamilton, ON, Canada L8N 4A6 (E-mail: janssenl{at}mcmaster.ca).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

June 28, 2002;10.1152/ajplung.00038.2002

Received 24 January 2002; accepted in final form 13 June 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Breyer, RM. Prostaglandin EP1 receptor subtype selectivity takes shape. Mol Pharmacol 59: 1357-1359, 2001[Free Full Text].

2.   Coleman, RA, Smith WL, and Narumiya S. International Union of Pharmacology classification of prostanoid receptors: properties, distribution, and structure of the receptors and their subtypes. Pharmacol Rev 46: 205-229, 1994[ISI][Medline].

3.   Corompt, E, Bessard G, Lantuejoul S, Naline E, Advenier C, and Devillier P. Inhibitory effects of large Ca2+-activated K+ channel blockers on beta -adrenergic- and NO-donor-mediated relaxations of human and guinea-pig airway smooth muscles. Naunyn Schmiedebergs Arch Pharmacol 357: 77-86, 1998[ISI][Medline].

4.   Elmhurst, JL, Betti PA, and Rangachari PK. Intestinal effects of isoprostanes: evidence for the involvement of prostanoid EP and TP receptors. J Pharmacol Exp Ther 282: 1198-1205, 1997[Abstract/Free Full Text].

5.   Fernandes, LB, Fryer AD, and Hirshman CA. M2 muscarinic receptors inhibit isoproterenol-induced relaxation of canine airway smooth muscle. J Pharmacol Exp Ther 262: 119-126, 1992[Abstract].

6.   Fortner, CN, Breyer RM, and Paul RJ. EP2 receptors mediate airway relaxation to substance P, ATP, and PGE2. Am J Physiol Lung Cell Mol Physiol 281: L469-L474, 2001[Abstract/Free Full Text].

7.   Hirata, T, Ushikubi F, Kakizuka A, Okuma M, and Narumiya S. Two thromboxane A2 receptor isoforms in human platelets. Opposite coupling to adenylyl cyclase with different sensitivity to Arg60-to-Leu mutation. J Clin Invest 97: 949-956, 1996[Abstract/Free Full Text].

8.   Horn, R, and Marty A. Muscarinic activation of ionic currents measured by a new whole-cell recording method. J Gen Physiol 92: 145-159, 1988[Abstract].

9.   Janssen, LJ, Premji M, Netherton S, Catalli A, Cox G, Keshavjee S, and Crankshaw DJ. Excitatory and inhibitory actions of isoprostanes in human and canine airway smooth muscle. J Pharmacol Exp Ther 295: 506-511, 2000[Abstract/Free Full Text].

10.   Jones, TR, Charette L, Garcia ML, and Kaczorowski GJ. Selective inhibition of relaxation of guinea-pig trachea by charybdotoxin, a potent Ca++-activated K+ channel inhibitor. J Pharmacol Exp Ther 255: 697-706, 1990[Abstract].

11.   Jones, TR, Charette L, Garcia ML, and Kaczorowski GJ. Interaction of iberiotoxin with beta -adrenoceptor agonists and sodium nitroprusside on guinea pig trachea. J Appl Physiol 74: 1879-1884, 1993[Abstract].

12.   Kannan, MS, and Johnson DE. Modulation of nitric oxide-dependent relaxation of pig tracheal smooth muscle by inhibitors of guanylyl cyclase and calcium-activated potassium channels. Life Sci 56: 2229-2238, 1995[ISI][Medline].

13.   Kawikova, I, Barnes PJ, Takahashi T, Tadjkarimi S, Yacoub MH, and Belvisi MG. 8-Epi-PGF2alpha , a novel noncyclooxygenase-derived prostaglandin, constricts airways in vitro. Am J Respir Crit Care Med 153: 590-596, 1996[Abstract].

14.   Knox, AJ, and Tattersfield AE. Airway smooth muscle relaxation. Thorax 50: 894-901, 1995[ISI][Medline].

15.   Kotani, M, Tanaka I, Ogawa Y, Usui T, Mori K, Ichikawa A, Narumiya S, Yoshimi T, and Nakao K. Molecular cloning and expression of multiple isoforms of human prostaglandin E receptor EP3 subtype generated by alternative messenger RNA splicing: multiple second messenger systems and tissue-specific distributions. Mol Pharmacol 48: 869-879, 1995[Abstract].

16.   Lahaie, I, Hardy P, Hou X, Hassessian H, Asselin P, Lachapelle P, Almazan G, Varma DR, Morrow JD, Roberts LJ, and Chemtob S. A novel mechanism for vasoconstrictor action of 8-isoprostaglandin F2alpha on retinal vessels. Am J Physiol Regul Integr Comp Physiol 274: R1406-R1416, 1998[Abstract/Free Full Text].

17.   Montuschi, P, Collins JV, Ciabattoni G, Lazzeri N, Corradi M, Kharitonov SA, and Barnes PJ. Exhaled 8-isoprostane as an in vivo biomarker of lung oxidative stress in patients with COPD and healthy smokers. Am J Respir Crit Care Med 162: 1175-1177, 2000[Abstract/Free Full Text].

18.   Montuschi, P, Corradi M, Ciabattoni G, Nightingale J, Kharitonov SA, and Barnes PJ. Increased 8-isoprostane, a marker of oxidative stress, in exhaled condensate of asthma patients. Am J Respir Crit Care Med 160: 216-220, 1999[Abstract/Free Full Text].

19.   Montuschi, P, Kharitonov SA, Ciabattoni G, Corradi M, van Rensen L, Geddes DM, Hodson ME, and Barnes PJ. Exhaled 8-isoprostane as a new non-invasive biomarker of oxidative stress in cystic fibrosis. Thorax 55: 205-209, 2000[Abstract/Free Full Text].

20.   Morrow, JD, Awad JA, Boss HJ, Blair IA, and Roberts LJ. Non-cyclooxygenase-derived prostanoids (F2-isoprostanes) are formed in situ on phospholipids. Proc Natl Acad Sci USA 89: 10721-10725, 1992[Abstract].

21.   Morrow, JD, Awad JA, Kato T, Takahashi K, Badr KF, Roberts LJ, and Burk RF. Formation of novel non-cyclooxygenase-derived prostanoids (F2-isoprostanes) in carbon tetrachloride hepatotoxicity. An animal model of lipid peroxidation. J Clin Invest 90: 2502-2507, 1992[ISI][Medline].

22.   Morrow, JD, Harris TM, and Roberts LJ. Noncyclooxygenase oxidative formation of a series of novel prostaglandins: analytical ramifications for measurement of eicosanoids. Anal Biochem 184: 1-10, 1990[ISI][Medline].

23.   Morrow, JD, Hill KE, Burk RF, Nammour TM, Badr KF, and Roberts LJ. A series of prostaglandin F2-like compounds are produced in vivo in humans by a non-cyclooxygenase, free radical-catalyzed mechanism. Proc Natl Acad Sci USA 87: 9383-9387, 1990[Abstract].

24.   Morrow, JD, Minton TA, Badr KF, and Roberts LJ. Evidence that the F2-isoprostane, 8-epi-prostaglandin F2alpha , is formed in vivo. Biochim Biophys Acta 1210: 244-248, 1994[ISI][Medline].

25.   Morrow, JD, Minton TA, Mukundan CR, Campbell MD, Zackert WE, Daniel VC, Badr KF, Blair IA, and Roberts LJ. Free radical-induced generation of isoprostanes in vivo. Evidence for the formation of D-ring and E-ring isoprostanes. J Biol Chem 269: 4317-4326, 1994[Abstract/Free Full Text].

26.   Morrow, JD, Minton TA, and Roberts LJ. The F2-isoprostane, 8-epi-prostaglandin F2alpha , a potent agonist of the vascular thromboxane/endoperoxide receptor, is a platelet thromboxane/endoperoxide receptor antagonist. Prostaglandins 44: 155-163, 1992[Medline].

27.   Morrow, JD, and Roberts LJ. Mass spectrometry of prostanoids: F2-isoprostanes produced by non-cyclooxygenase free radical-catalyzed mechanism. Methods Enzymol 233: 163-174, 1994[ISI][Medline].

28.   Morrow, JD, and Roberts LJ. The isoprostanes. Current knowledge and directions for future research. Biochem Pharmacol 51: 1-9, 1996[ISI][Medline].

29.   Narumiya, S, Sugimoto Y, and Ushikubi F. Prostanoid receptors: structures, properties, and functions. Physiol Rev 79: 1193-1226, 1999[Abstract/Free Full Text].

30.   Norel, X, Walch L, Labat C, Gascard JP, Dulmet E, and Brink C. Prostanoid receptors involved in the relaxation of human bronchial preparations. Br J Pharmacol 126: 867-872, 1999[Abstract/Free Full Text].

31.   Pelligrino, DA, and Wang Q. Cyclic nucleotide crosstalk and the regulation of cerebral vasodilation. Prog Neurobiol 56: 1-18, 1998[ISI][Medline].

32.   Pierce, KL, and Regan JW. Prostanoid receptor heterogeneity through alternative mRNA splicing. Life Sci 62: 1479-1483, 1998[ISI][Medline].

33.   Pratico, D, Iuliano L, Mauriello A, Spagnoli L, Lawson JA, Rokach J, Maclouf J, Violi F, and FitzGerald GA. Localization of distinct F2-isoprostanes in human atherosclerotic lesions. J Clin Invest 100: 2028-2034, 1997[Abstract/Free Full Text].

34.   Sametz, W, Hennerbichler S, Glaser S, Wintersteiger R, and Juan H. Characterization of prostanoid receptors mediating actions of the isoprostanes, 8-iso-PGE2 and 8-iso-PGF2alpha , in some isolated smooth muscle preparations. Br J Pharmacol 130: 1903-1910, 2000[Abstract/Free Full Text].

35.   Sheller, JR, Mitchell D, Meyrick B, Oates J, and Breyer R. EP2 receptor mediates bronchodilation by PGE2 in mice. J Appl Physiol 88: 2214-2218, 2000[Abstract/Free Full Text].

36.   Somlyo, AP, and Somlyo AV. Signal transduction and regulation in smooth muscle. Nature 372: 231-236, 1994[ISI][Medline].

37.   Takahashi, K, Nammour TM, Fukunaga M, Ebert J, Morrow JD, Roberts LJ, Hoover RL, and Badr KF. Glomerular actions of a free radical-generated novel prostaglandin, 8-epi-prostaglandin F2alpha , in the rat: evidence for interaction with thromboxane A2 receptors. J Clin Invest 90: 136-141, 1992[ISI][Medline].

38.   Ungrin, MD, Carriere MC, Denis D, Lamontagne S, Sawyer N, Stocco R, Tremblay N, Metters KM, and Abramovitz M. Key structural features of prostaglandin E2 and prostanoid analogs involved in binding and activation of the human EP1 prostanoid receptor. Mol Pharmacol 59: 1446-1456, 2001[Abstract/Free Full Text].

39.   Unmack, MA, Rangachari PK, and Skadhauge E. Effects of isoprostanes and prostanoids on porcine small intestine. J Pharmacol Exp Ther 296: 434-441, 2001[Abstract/Free Full Text].

40.   Wagner, RS, Weare C, Jin N, Mohler ER, and Rhoades RA. Characterization of signal transduction events stimulated by 8-epi-prostaglandin (PG) F2alpha in rat aortic rings. Prostaglandins 54: 581-599, 1997[Medline].


Am J Physiol Lung Cell Mol Physiol 283(5):L1151-L1159
1040-0605/02 $5.00 Copyright © 2002 the American Physiological Society