Tetrodotoxin Does Not Block the Epithelium-dependent Release of Prostaglandin E, Induced by Electrical Field Stimulation in Isolated Ferret Trachea A. Ullman, G. Ciabattoni, N. Svedmyr, B.-E. Skoogh, and c.-G. Lofdahl Departments of Clinical Pharmacology and Pulmonary Medicine, Gothenburg University, Sweden, and Department of Pharmacology, Catholic University, Rome, Italy

Electrical field stimulation (EFS) has previously been shown to induce the release of prostaglandin (PG) B.! from ferret tracheal epithelium. We have now conducted a study to see whether this effect of EFS is due to the activation of nerves or whether it is a non-neural effect. The release of PGE2 and 6-keto-PGF 1a into the bath fluid was assayed in isolated ferret tracheas with (E") or without (E-) epithelium, stimulated by either EFS or direct vagal nerve stimulation (DNS) repeatedly for 120 min. EFS-stimulated E" preparations showed a gradual decline in the contractile responses (30 ± 1% of baseline) and art increase in PGE2 to 296 ± 38 pg/ml. In EFS-stimulated, epithelium-denuded (E-) preparations, the decline was significantly lower (11 ± 5%), as well as the final concentration of PGE 2 (107 ± 21 pg/ml). In DNSstimulated E+ preparations, the contraction decline was 8 ± 1% and the final concentration of PGE 2 was < 6 pg/ml. Although tetrodotoxin (TTX) abolished the contractile response in EFS-stimulated E" preparations, it did not significantly reduce the release of PGE 2 (260 ± 6 pg/ml), whereas atropine partly counteracted the release. The bath concentration of 6-keto-PGF lc increased, independently of the electrical stimulation, contractile response, or presence of the epithelium. We conclude that EFS activates the epithelium-dependent release of PGE 2 by a TTX-resistant mechanism. This may be due to an activation of TTX-resistant nerves, or possibly to a non-neural effect, such as a direct effect on the epithelial cells. The results indicate that the airway epithelium has the ability to respond to certain stimuli with a pronounced release of PGE 2 , thereby counteracting bronchoconstriction.

Over the past five years, possible interactions between airway epithelium and smooth muscle have been studied in different in vitro models. In a number of studies, it has been demonstrated that removal of the mucosa or epithelium results in an increased sensitivity to different contractile agonists and decreased sensitivity to relaxing agonists (1-3). One possible mechanism for such an interaction is the postulated epithelium-derived release of relaxing or inhibitory factors in analogy with the endothelium-derived relaxing factor (EDRF) in the blood vessels (4). It has been debated whether or not prostanoids are involved in these interactions and conflicting data have been

(Received in original form February 21, 1990 and in revised form July 27, 199{}) Address correspondence to: Dr. A. Ullman, Department of Clinical Pharmacology, Sahlgren's University Hospital, S-413 45 Gothenburg, Sweden. Abbreviations: direct vagal nerve stimulation, DNS; ferret tracheas with epithelium, E+; ferret tracheas without epithelium, E-; endothelium-derived relaxing factor, EDRF; electrical field stimulation, EFS; KrebsRinger's solution, KR; prostaglandin, PG; radioimmunoassay, RIA; tetrodotoxin, TTX. Am. J. Respir. Cell Mol. BioI. Vol. 4. pp. 243-247, 1991

presented (1, 2, 5, 6). However, it has been clearly demonstrated in several species that prostaglandin (PG) E2 can be released from the airway epithelium (7-13). Thus, PGE2 , which has bronchodilatoryproperties and inhibits cholinergic neurotransmission (14), may be released from the intact airway epithelium to counteract bronchoconstrictor stimuli. We have previously demonstrated the epithelium-dependent release of PGE 2 when an isolated nerve-muscle preparation of ferret trachea was stimulated repeatedly using transmural electrical field stimulation (EFS) (15). In this study, we also found that the release of prostacycline measured as the bath concentration of its stable metabolite, 6-ketoPGF 1a , did not appear to originate from the epithelium. In the present investigation, we studied the activating mechanism for the epithelium-dependent release of PGs. This was done by comparing the effect of transmural EFS and direct vagal nerve stimulation (DNS). The involvement of neural mechanisms was evaluated by use of tetrodotoxin and atropine.

Materials and Methods The study was approved by the Ethics Committee for animal experiments at Gothenburg University. A previously de-

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scribed in vitro nerve-muscle preparation of ferret trachea was used (16, 17). Ferrets were rendered unconscious by electric shocks and were killed by exsanguination. The trachea, with the right recurrent and vagus nerves intact, was rapidly removed and immersed in an organ bath filled with 200 ml Krebs-Ringer's (KR) solution with the following composition (in mM): NaCI (118), KCI (5.9), CaClz (2.5), MgS04 (1.2), NaH zP04 (1.2), NaHC03 (25.5), and glucose (5.6), maintained at 38 0 C and aerated with 94% O2/6% CO 2 • Ascorbic acid was added to the KR solution in a concentration of 0.3 mM to prevent the formation of oxygen free radicals. In the organ bath, the trachea was split along the anterior long axis and opened. One side was attached by pins and the opposite side was connected to three strain gauges (Grass FT 03) in order to record isometric muscle tension at optimum resting tension (7 to 10 g). The epithelium was either left intact (E+) or removed (E-). In E- preparations, only the mucosa covering the tracheal muscle was removed. This was done by cutting along both of the borders between the membranous part and the cartilaginous part and then peeling off the intervening mucosa. Care was taken not to damage the epithelium in other parts of the trachea. The preparations were mounted with the tracheal muscle between two platinum electrodes for transmural EFS. The right vagal and recurrent nerves were dissected and freed and were connected to suction electrodes for DNS. EFS was given with biphasic square waves with a duration of 0.50 ms of each wave and a current of 1,000 mAo DNS was given with biphasic square waves with a duration of 0.25 ms and a current of 22 rnA. The frequency used was 12 Hz for both EFS and DNS. After pre-amplification, the signal from the FT-03-transducer was connected to a computerized system based on a Macintosh II computer with a NB-MIO-16 analog/digital converting board and the signal-processing software LabVIEW (National Instruments, Austin, TX). This system permits the real-time monitoring of the experiment and the simultaneous and continuous saving of data for subsequent evaluation. Five experimental series were performed. In all of them, contractions were induced by electrical stimulation with either EFS or DNA for 20 severy 2 min for 120 min. In one series with intact epithelium and one with removed epithelium, contractions were induced by EFS to verify the expected difference (15) in released PGE 2 and corresponding differences in contractile responses. In two further series with intact epithelium, stimulations were given with EFS in the presence of tetrodotoxin (TTX) 3 X 10-7 M or 10-6 M atropine. In one series with intact epithelium, contractions were induced by DNS. The preparations were equilibrated for 60 min at 10-g resting tension. During the equilibration period, the bath was continuously flushed with prewarmed KR solution, 10 ml/min. At the start of the experiment, the flushing was stopped and stimulations were given for 120 min. During this period, new KR solution was added only to balance sample volume. Samples (2 ml) of the bath fluid for radioimmunoassay (RIA) for PGEz and 6-keto-PGF la were collected immediately before the start of the stimulations and then af-

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Figure 1. The time course of the contractile response to EFS (12 Hz) in preparations with intact (closed circles) and removed (open circles) epithelium and to DNS (12 Hz) in preparations with intact epithelium (closed squares). The results are expressed as a percentage of baseline, i.e., the contractile response after 30 min of stimulation, and are the mean ± SEM; n = 6.

ter 60 and 120 min. The samples were aspirated by plastic pipettes and were frozen immediately in plastic tubes and stored at -70 C. The RIAs for PGE z and 6-keto-PGF l a were performed using a previously described method, with a documented high specificity and low cross-reactivity (18, 19). 0

Drugs TTX (Sigma Chemical Co., St. Louis, MO) and atropine (Sigma) were dissolved in distilled water before being added to the bath fluid. Statistics Values are expressed as the mean ± SEM. Statistical comparisons were made using a one-way analysis of variance with significance testing at 95 % and 99 % levels using Fisher's protected least significant difference. All the significance tests of bath concentrations of prostanoids were performed after logarithmic transformation of the data.

Results Contractile Responses The baseline contractile response 30 min after the start of the stimulation was 37.9 ± 1.2 g (mean ± SEM, n = 6) in the EFS series with intact epithelium and 40.1 ± 2.3 g in the EFS series with removed epithelium. During the additional90 min of phasic stimulations, there was a significantly more pronounced decrease in the contractile response in epithelium-intact compared with epithelium-denuded preparations (Figure 1). During the 120 min, the contractile response diminished to 89.0 ± 1.2% of baseline in epitheliumdenuded preparations compared with 70.5 ± 0.8% (P < 0.001) in preparations with intact epithelium. In preparations with intact epithelium stimulated with DNS, the baseline contractile response was 31.0 ± 2.6 g. At the end of the experiment, the contractile response was 91.5 ± 2.7% of baseline. This decline was significantly (P < 0.001) lower compared with the decline in EFS-stimulated preparations with intact epithelium but not significantly different compared with epithelium-denuded preparations.

Ullman, Ciabattoni, Svedmyr et al.: PGEz Release from Tracheal Epithelium

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Tetrodotoxin does not block the epithelium-dependent release of prostaglandin E2 induced by electrical field stimulation in isolated ferret trachea.

Electrical field stimulation (EFS) has previously been shown to induce the release of prostaglandin (PG) E2 from ferret tracheal epithelium. We have n...
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