( M) did not affect the resting membrane potential. At a concentration of

Transcription

1 Journal of Physiology (1992), 449, pp With 9 figures Printed in Great Britain AIRWAY EPITHELIAL CELLS REGULATE MEMBRANE POTENTIAL, NEUROTRANSMISSION AND MUSCLE TONE OF THE DOG AIRWAY SMOOTH MUSCLE BY ZHUOQIU XIE, HIROYUKI HAKODA AND YUSHI ITO From the Department of Pharmacology, Faculty of Medicine, Kyushu University, Fukuoka 812, Japan (Received 3 May 1991) SUMMARY 1. The effects of epithelial cells were investigated on resting membrane potential and neuro-effector transmission in smooth muscle cells of the dog tracheal and bronchiolar tissues. 2. The mean value of the resting membrane potential of the epithelium-intact bronchiolar smooth muscle cells of the dog was mv (± S.D., n = 4) and mechanical denudation of the epithelial layer depolarized the membrane to '5 mv (± S.D., n = 4). Application of isolated and dispersed epithelial cells (> 2 x 15 cells/ml) to the perfusing solution repolarized the membrane of epithelium-denuded bronchiolar smooth muscle cells to mv (± S.D., n = 2). The mean resting membrane potential of the mucosa-free tracheal smooth muscle cells was mv (± S.D., n = 5), and application of isolated and dispersed cells (> 2 x 15 cells/ml) hyperpolarized the membrane to '8 mv (± S.D., n = 5). These repolarizing actions were not modified by indomethacin (1-5 M). 3. In the epithelium-denuded bronchioles, ACh (> 1-9 M) dose-dependently depolarized the smooth muscle cells, while in the epithelium-intact bronchioles, ACh ( M) did not affect the resting membrane potential. At a concentration of 1-7 M, ACh significantly depolarized the membrane. 4. Electrical field stimulation (EFS; 5,ts in duration and about 1-2 V in strength) applied to ring preparations of the bronchioles evoked twitch-like contractions (hereafter referred as twitch contraction), and size of the twitch contractions gradually and continuously decreased in the presence or absence of indomethacin (1-5 M) and guanethidine (1-6 M). When similar experiments were performed using epithelium-denuded bronchiolar ring preparations, in no case was there a prominent reduction in the amplitude of the twitch contractions in the presence of indomethacin and guanethidine. 5. The decremental response of the twitch contraction observed in the epitheliumintact bronchioles was overcome by application of the leukotriene synthesis inhibitor AA861 (1-6 M) and the leukotriene antagonist ON178 (1-5 M). 6. Leukotrienes C4 and D4 (LTC4 and LTD4, > 1-8 M) evoked muscle contraction with a steady increase in muscle tone, up to a certain level. However, at 1-9 M, LTC4 increased and LTD4 decreased the amplitude of the twitch contractions evoked by MS 9432

2 62 Z. XIE, H. HAKODA AND Y. ITO EFS in the epithelium-intact bronchioles. On the other hand LTE4 (1-8 M) consistently suppressed the relationship between the number of stimuli and the relative amplitude of the twitch contractions. 7. In the epithelium-denuded bronchioles, indomethacin (1- M) markedly enhanced the amplitude of the excitatory junction potentials (EJPs, to of the control value, + S.D., n = 4). However, this compound only slightly increased the EJP amplitude in the epithelium-intact bronchioles. The leukotriene synthesis inhibitor AA861 (1-6M) enhanced the EJP amplitude to times the control value (± S.D., n = 4) in the epithelium-intact bronchioles, but had little effect on the epithelium-denuded bronchioles. In the intact bronchioles LTC4 (1-8 M) markedly and LTB4, D4 and E4 (1-8 M) slightly enhanced the EJP amplitude. 8. Application of isolated and dispersed epithelial cells (3-5 x 15 cells/ml) to the bath significantly suppressed the EJP amplitude of mucosa-free trachealis to (± S.D., n = 5) times the control value, and these effects were only partially suppressed by indomethacin (1- M). In the presence of indomethacin (1-5 M), the dispersed epithelial cells suppressed the EJP amplitude to times the control, and the additional application of AA861 (1-6 M) further enhanced the EJP amplitude to (±S.D., n = 4). 9. Hydroxytryptamine (1' M) produced a tonic contraction of the intact bronchiole, and electrical field stimulation (EFS) applied during tonic contraction produced an initial phasic contraction and a subsequent relaxation in the presence of indomethacin (1-5 M) and guanethidine (1-6 M). Atropine (16 M) and mechanical denudation or hypo-osmolarity shock selectively abolished the phasic contraction and relaxation, respectively. 1. These observations suggest that a factor(s) released from epithelial cells in the airway plays multiple roles in controlling the resting membrane potential, neuroeffector transmission and muscle tone of the smooth muscle cells. INTRODtUCTION Removal of the epithelium modulates responsiveness of the airway smooth muscle to various spasmogenic and relaxant agonists, in variety of animal species, including humans (see, for example, Goldie, Fernandes, Farmer & Hay, 199). Evidence for the release of an inhibitory factor by airway epithelium comes from cascade experiments (Flavahan & Vanhoutte, 1985), sandwich experiments (Tschirhart & Landry, 1986) and co-axial bioassay system consisting of an epitheliumintact guinea-pig tracheal tube surrounding an epithelium-denuded rabbit aortic strip (Ilhan & Sahin, 1986), rat anococcygeus (Guie, Ilhan & Kayaalp, 1988) and rat aorta (Fernandes, Paterson & Goldie, 1989; Fernandes & Goldie, 199). Thus, contractions of airway and vascular smooth muscle evoked by various agonists can be modulated by factor(s) released from accessory cells, such as the epithelium, upon stimulation with spasmogenic agents, and the main technique previously used to study the regulatory role of the epithelium on smooth muscle has been mechanical recording of isometric tension of the airway and vascular smooth muscle in the presence or absence of epithelial cells. To elucidate the role of epithelial cells on membrane activities of airway smooth muscles, we observed the effects of dispersed epithelial cells as well as epithelial

3 EPITHELIAL REGULATION OF AIR WA Y MUSCLES denudation on the resting membrane potential of bronchial and tracheal smooth muscle cells of the dog, using a microelectrode method. We also studied the possible role of epithelial cells on excitatory neuro-effector transmission in the airway smooth muscle tissues. We report here our observations of dispersed epithelial cells and epithelial denudation on excitatory junction potentials (EJPs) and contractions recorded from tracheal and bronchiolar smooth muscle tissues in response to electrical field stimulation (EFS), using tension recording, double sucrose-gap and microelectrode methods. 621 METHODS Adult mongrel dogs of either sex, weighing 1-13 kg were anaesthetized with i.v. administration of pentobarbitone (3 mg/kg). Segments of the cervical trachea were excised and whole pulmonary lobes were quickly resected from the main bronchus. A dorsal strip of transversely running tracheal smooth muscles was separated from the cartilage, and the mucosa and adventitial areolar tissue were carefully removed. The mucosa from the tracheal muscle was removed to leave only smooth muscle. The tracheal smooth muscle was cut to a width of mm and a length of about 15 mm for use in the double sucrose-gap method. Small airways (about 1 mm in diameter) were carefully excised from the lung tissue (under the microscopic observation), and lung parenchyma and pulmonary vessels running along a bronchiolar branch were removed. Histological investigations revealed that the tissue used for the present experiments had a diameter of 8-1P1 mm and was composed of smooth muscle layers and mucous membrane but lacked cartilage, thereby indicating that the tissue comprised bronchioles (Cumming, 1972; Inoue & Ito, 1986; Ito & Inoue, 1989). A piece of bronchiole 1 mm in diameter, 4-5 mm in length),and ring preparations ( 8-1 mm in diameter, 1-2 mm in length) were used for the microelectrode and tension-recording experiments, respectively. The preparation was bathed in a modified Krebs's solution of the following ionic concentration (mm): Na+, 137-4; K+, 5 9; Mg2+, 12; Ca2+, 2F5; C1, 134F; 2P4, 1F2; HCO23, 155 and glucose, The solution was aerated with 97% 2 and 3% CO2 and the ph was The double sucrose-gap method was used to record the membrane potential and tension development from the tracheal smooth muscles. The chamber used has been described elsewhere (Ito & Tajima, 1981). To produce neurogenic responses, EFS was applied by a ring electrode placed in the centre pool of the apparatus, using an electrode stimulator (Nihon Kohden SEN-713). Single and repetitive stimulation was applied with a 1-2 V current pulse of 5,ss in duration. The voltage of the current pulse was adjusted so that an EJP of a defined amplitude was evoked by a single pulse. Drugs were dissolved in Krebs solution and applied to the tissue through the centre pool of the double sucrose-gap apparatus, using a multi-way tap (dead time approximately 3 s). For intracellular recording of the membrane potential from a single cell, thin strips of tracheal tissue 1-15 mm in length, 4-5 mm in width and 3-{4 mm thick or a piece of intact bronchiole (1 mm in diameter, 4-5 mm in length) were used. The dog tracheal preparations used in these experiments were entirely dissected free from the overlying mucosal layer and its cartilaginous attachments. In the case of bronchioles, the airway tissues were dissected out from the lung tissue (under microscopic observation), and smooth muscle cells were impaled with a microelectrode from the outer surface of the tissue leaving the inner lumen intact. Thus, measurements of the resting membrane potential of bronchiolar smooth muscle cells were made with an intact epithelial layer inside the lumen. To study the effects of epithelium removal on the resting membrane potential of the bronchiolar smooth muscle cells, a fine silver wire (2,um in diameter) was inserted into the lumen and the epithelial layer was removed mechanically by gentle rolling of the tissue back and forth in the chamber. We measured the resting membrane potential of the bronchiolar muscle immediately after (5-1 min) the epithelium removal, and after equilibrating the tissue in normal Krebs solution for 1-2 h after the removal. No differences in the resting membrane potential were observed in two cases; hence mechanical agitation probably did not damage the muscle cells. A conventional microelectrode filled with 3 M-KCl (3-5 MQ) was used for all experiments. Field stimulation was applied to the nerves through a pair of Ag-AgCl wires 3-5 mm apart and placed so that a current pulse would pass transversely across the tissue. Single and repetitive stimuli at 2 Hz were applied, with a pulse of 3-1,us duration and 3-5 V, using an electronic stimulator (Nihon Kohden SEN-713). The chamber in which the strips were mounted had a volume of 2 ml,

4 622 Z. XIE, H. HAKODA AND Y. ITO and was superfused at a rate of 3 ml/min at a temperature of 'C. To avoid recording artifacts due to twitch contractions of the muscle tissue, the preparation was immobilized on the rubber plate in the chamber using insect pins with a diameter of 1,um. Histological methods were used to determine the extent and effects of mechanical denudation of epithelial cells. Two control and five mechanically agitated bronchioles were fixed after the experiments by immersion in 1 % of phosphate-buffered formalin and were then embedded in paraffin. For each bronchiole, serial tissue sections (3,am thick and 3 mm long) were cut and stained with Haematoxylin and Eosin. Twenty serial sections from each preparation were examined by two observers with no knowledge as to their source. A light microscope at a magnification x 3 was used. In each section from the control bronchioles, the complete epithelial luminal surface was identified. However, in the mechanically agitated bronchioles, almost all the examined sections (95%) had lost 6-9% of the epithelial luminal surface. We also used hypo-osmolarity shock to destroy the epithelial layer, as is done for endothelial cells in blood vessels (Bolton, Lang & Takewaki, 1984; Nagao & Suzuki, 1987). Histological examination of the bronchioles revealed that the epithelial luminal surface was completely destroyed after the hypo-osmolarity shock, as has been noted in blood vessels (Nagao & Suzuki, 1987). To measure the mechanical responses of the bronchiole, ring preparations (-8-1- mm in diameter and 1 mm in length) were hooked by a pair of right-angled fine needles which were reduced in diameter by electrolysis to about 5,um as observed under a microscope. One of the needles was fixed at the bottom of the chamber. The other needle was connected to a manipulator at one end and at the other end to an isometric tension transducer. The fixed needle was also used for electrical stimulation of ring preparations of the bronchiole. As required for the study, the epithelial cells were removed mechanically by rubbing the internal surface of the bronchioles with a fine silver wire (2,Im diameter) or by hypo-osmolarity shock by exposing the inner lumen of the bronchioles to distilled water for about 3-6 s as has previously been done in vascular tissues for removal of endothelial cells (Bolton et al. 1984; Nagao & Suzuki, 1987). To prepare the isolated and dispersed epithelial cells, we used tracheal mucosa, i.e. tracheal mucosa freed from cartilage and smooth muscle layers was cut into small pieces (about 1 x 1-5 x 2-5 mm) and bathed in the phosphate buffer solution (PBS) containing NaCl, (mm); KCl, 2 7; Na2HPO4, 8 1; KH2PO4, 1 5. After rinsing the chopped mucosa three times with PBS, we exposed the tissue to protease (dispase, 1 unit/ml) for 2-5 h at 37 'C, and then centrifuged the cells (8-1 r.p.m. for 1 min). We repeated this procedure three times and the precipitate was diluted with Krebs solution to count the number of dispersed cells. Most of the cells counted (> 95 %) showed ciliary beating, and hence were mostly epithelial cells. The following drugs were used: indomethacin, acetylcholine hydrochloride, 5-hydroxytryptamine, prazosin, yohimbine, 1-nitro-arginine, oxyhaemoglobin, leukotrienes B4, C4, D4, E4, (Sigma), guanethidine (Tokyo Kasei), dispase (Sanko Junyaku), tetrodotoxin (Sankyo), the leukotriene synthesis inhibitor AA861 (Takeda), the leukotriene antagonist ON178 (Ono) and atropine sulphate (Daiichi). Results (membrane potential, amplitude of contractions, relaxations and EJPs) are expressed as means + S.D. and were analysed using Student's t test for paired (amplitude of contractions, relaxations or EJPs) or unpaired data (membrane potential) to estimate the significance of differences between means, and P < 5 was judged to have statistical significance. For measurements of the resting membrane potential, twenty to thirty cells were impaled before and after drug application of the same preparation, and the experiments were repeated using two to five preparations. The mean values were calculated using the collected data (unpaired data). Microelectrode or double sucrose-gap recordings of EJPs were obtained from the same cell or the same tissue throughout the experiments, before and after the drug application, and data obtained from five to eight cells or three to eight preparations were analysed statistically (paired data). RESULTS Effects of epithelium denudation, isolated and dispersed epithelial cells on the resting membrane potential of bronchiolar and tracheal smooth muscle cells Mechanical denudation of the bronchiole epithelium (see Methods) depolarized the resting membrane potential of bronchiole smooth muscle cells (Fig. 1A). When dispersed epithelial cells (>5 x 1O4 cells/ml) were added to the perfusing Krebs

5 EPITHELIAL REGULATION OF AIRWAY MUSCLES 623 solution the membrane potential of the bronchiole smooth muscle cells reverted to values not significantly different from those seen in the epithelium-intact tissue (Fig. 1A). Similarly, the addition of dispersed tracheal cells (> 2 x 15 cells/ml) to the bath significantly hyperpolarized the mucosa-free tracheal smooth muscle cells (Fig. A Bronchiole B Trachea > E ) E(-) E(+) T 2x14 cells/ml E() E(+) c -6 6Cl14 Control 2x<1' 5x14 itt5 cells/ml CL E _7; -7 Eict+ i* I-7 2 :(1 5 1X 5 Fig. 1. A, effects of the presence or absence of epithelial cells, and dispersed epithelial cells (2 x 14-5 x 15 cells/ml) on the resting membrane potential of bronchial smooth muscle cells. E intact or E(-) indicate bronchioles with or without intact epithelium, respectively. Mechanical denudation of the bronchiolar epithelium depolarized the membrane from mv to mv (n = 4, P < -1). Addition of dispersed cells (> 5 x 14 cells/ml) repolarized the epithelium-denuded bronchioles. B, effects of dispersed epithelial cells on the resting membrane potential of mucosa-free tracheal smooth muscle cells. E( +) or E(-) indicate the presence or absence of isolated and dispersed epithelial cells. Each point is the mean value derived from fifteen to thirty penetrations (n = 15-3) using three to five preparations and vertical bars indicate 2 X S.D. ** Significantly different from the control (P < 1). 1 B). However, in the case of trachealis, a higher concentration of epithelial cells (> 2 x 15 cells/ml) was needed to evoke membrane hyperpolarization. It has been reported that bradykinin causes an increase in the rate of the release of prostaglandin (PG) E2 from primary cultures of dog or human tracheal epithelium (Widdicombe, Ueki, Emery, Margolskee, Yergey & Nadel, 1989) and it was recently shown that ACh induces concentration-dependent relaxation of pre-contracted rat aorta via the release of an epithelium-derived inhibitory factor from guinea-pig tracheal epithelium, determined using a co-axial bioassay system (Fernandes & Goldie, 199). Therefore, we observed the effects of bradykinin and ACh on the hyperpolarizing effect of epithelial cells on tracheal smooth muscle cells. Figure 2A shows the effects of bradykinin (1-v M) and ACh (1-9 and 1-8 M) in the presence or absence of dispersed epithelial cells on the resting membrane potential of mucosafree tracheal smooth muscle cells. Bradykinin (1-7 M) and ACh (1-9 and 1-s M) did not alter the resting membrane potential of tracheal smooth muscle cells in the absence of epithelial cells. After exposing the tissue to the dispersed epithelial cells (> 2 x 15 cells/ml) in the presence of bradykinin and ACh, the mean values of the resting membrane potential were mv (n = 25) or mv (n = 3), respectively. This means that bradykinin (1-7 M) or ACh (1-8 and 1-9 M) does not modify the membrane potential of the tracheal smooth muscle cells in the

6 624 Z. XIE, H. HAKODA AND Y. ITO A Trachea EH- E(+) E Control o-7 1o6 io i -5 ACh ACh ACh Brad Hb 1-nArg 2x14 2x15 5 2x15 5 5x15 5 2x15 2x15 ) 1-5 i- I ACh ACh ACh Brad Hb 1-nArg Indo io : ; + 3 ndo -- (X*-6 - E 8) CD -7 - a -41-T;E() f B Bronchiole -3 E-4EH E intact -8 Control ACh (M) Fig. 2. A, effects of ACh (1-' and 1-8 M), bradykinin (Brad, 1-7 M-, oxyhaemoglobin (Hb, 1-5 M), indomethacin (Indo, 1-5 M) or 1-nitro-arginine (1-nArg, 1- M) on the resting membrane potential of tracheal smooth muscle cells in the presence or absence of dispersed epithelial cells. ACh (1-' M) or bradykinin (1-7 m) did not change the resting membrane potential of the tracheal smooth muscle cells, however, additional application of epithelial cells hyperpolarized the membrane to the same extent as was induced by application of epithelial cells alone. Membrane hyperpolarization induced by epithelial cells in the presence of ACh was not affected at all by indomethacin (1-5 M). Oxyhaemoglobin or 1-nitro-arginine did not affect the resting membrane potential in the presence or absence of epithelial cells. B, effects of ACh ( M) on the resting membrane potential of epithelium-intact (@, E intact) or epithelium-denuded (, E(-)) bronchiolar smooth muscle cells. Each point is the mean value derived from fifteen to twenty-five experiments (n = 15-25) using two to four preparations and vertical bars indicate 2 X S.D. ** Significantly different from the control (P < -1) (see Methods).

7 EPITHELIAL REGULATION OF AIRWAY MUSCLES 625 presence or absence of dispersed epithelial cells. Figure 2A also shows the effects of indomethacin (1-5 M), oxyhaemoglobin (1-5 M) and 1-nitro-arginine (1-5 M) on the resting membrane potential of the tracheal smooth muscle cells, in the presence or absence of dispersed epithelial cells. None of these agents had any effect. The effects of various concentrations of ACh (1-11-1O-7 M) were also observed on the resting membrane potential of intact or epithelium-denuded bronchiolar smooth muscles. In the epithelium-denuded tissues, ACh (> 1-9 M) dose-dependently depolarized the membrane of smooth muscle cells, while in the intact bronchiole, 1-9 M-ACh slightly hyperpolarized the membrane (although the value was not statistically significant), and at a concentration of 1-7 M, ACh significantly depolarized the smooth muscle membrane, thereby indicating that ACh may stimulate the release of epithelium-derived hyperpolarizing factor from the epithelial layer in addition to the continuous release (Fig. 2B). Effects of EFS on the contractile response of bronchiolar rings with or without epithelium Effects of EFS on the contractile response of intact and epithelium-denuded bronchiolar rings were observed in the presence of guanethidine (1-6 M). The dog airway smooth muscle cells are innervated by both cholinergic and adrenergic nerves (Russel, 198) and noradrenaline released from sympathetic nerves can activate prejunctional fl-adrenoceptors to inhibit cholinergic transmission (Danser, Van Den Ende, Lorenz, Flavahan & Vanhoutte, 1987; Ito, 1988). Size of the twitch contractions elicited by EFS and recorded from the ring preparation of bronchioles with the epithelial layer, gradually and continuously decreased and practically disappeared about 3-6 min after the start of stimulation (Fig. 3A). We repeated the experiments in the presence of indomethacin (1-5 M) and guanethidine (16 M), since it was reported that 1-5 M-indomethacin suppressed the decremental responses in the helically cut bronchiolar tissues (Inoue & Ito, 1986). However, the twitch contraction of the intact bronchiolar ring evoked by EFS gradually decreased as shown in Fig. 3B, and indomethacin slowed the rate of this decremental response. When the experiments were performed using epithelium-denuded bronchiolar ring preparations in the presence of indomethacin (1-5 M) and guanethidine (1-6 M), in no case was there a prominent reduction in amplitude of twitch contractions, as is shown in Fig. 3C. The gradual and continuous reduction in size of the twitch contraction evoked by EFS is probably due to factor(s) released from the epithelium. The decline in the twitch contractions elicited every 3 min by EFS was overcome by adding to the bath AA861 (1-6 M) and ON178 (1-s M) in the presence of indomethacin (1-5 M). In the experiments shown in Fig. 4, the tissues were treated with A861 or ONO min after the start of stimulation in the presence of indomethacin (1-5 M) with guanethidine (1-6 M). Application of AA861 (16 M) or ONO178 (1-5 M) during the gradual reduction in the amplitude of twitch contractions prevented this reduction, and restored the amplitude of the contraction to the initial level (ON178) or beyond the initial level (AA861). However, at concentrations of 1-7, or 1-6 M respectively, AA861 or ON178 only slightly enhanced the amplitude of contractions. As the gradual and continuous reduction in the size of twitch contraction of the epithelium intact bronchiolar ring preparations

8 626 Z. XIE, H. HAKODA AND Y. ITO evoked by EFS was overcome by a leukotriene synthesis inhibitor and a leukotriene antagonist, lipoxygenase metabolites of arachidonic acid are involved in the decremental response seen in these ring preparations. We then observed the effects of leukotriene B4, C4, D4 and E4 on the amplitude of the twitch contractions evoked by EFS in the presence of AA861. Figure 5 shows the A *45 9 B 1. 3 min C b+ii+ g) ii + i; 4 < i < i<_ -2 3 min.5 1. Time (h) Fig. 3. Effects of repetitive stimulation on twitch contractions evoked by field stimulations. Stimulations (5 stimuli at 2 Hz) were applied to the whole tissue every 3 min in the presence of guanethidine (1-6 M). The amplitude of twitch contractions evoked by the first stimulations was registered as a relative tension of 1l. Each point is the mean value of five to eight experiments (n = 5-8). A and B, records from intact bronchiolar rings. B, the effects of indomethacin (1-5 M). C, records from epitheliumdenuded bronchioles, and in the presence of indomethacin. Insets are the actual traces of twitch contractions. The absolute value of tension development ranged between -2 and -7 g. effects of leukotrienes on the relationship between the number of stimuli at 2 Hz and the relative amplitude of twitch contractions, where the amplitude of contraction evoked by 3 stimuli was defined as a relative amplitude of 1. LTB4 (1-' M) had

9 EPITHELIAL REGULATION OF AIRWAY MUSCLES 627 no apparent effect on the relationship, while LTC4 and LTD4 showed both direct and indirect actions on the bronchioles, at relatively low and high concentrations, respectively, i.e. LTC4 or LTD4 (> 1-8 M) evoked a muscle contraction with a steady increase in muscle tone, up to a certain level (Fig. 5G and I). To observe the A 1. - ON io-5 M ON178 io-5m c C 6)._a a) B l-" I 5, jllliiio. 1 i2 3 min a) AA861 {1-6 [-L-~ AA861 1~-M l.5 Time (h) A 1 ~ ~ ~~.59 L i L IJ-2 3 min 1. Fig. 4. Effects of ONO 178 or AA861 on twitch contractions evoked by field stimulation (5 stimuli at 2 Hz) applied to the whole tissue every 3 min in the presence of guanethidine (1-6 M) and indomethacin (1-5 M). The amplitude of twitch contractions evoked by the first field stimulation was registered as a relative tension of 1.. A, effects of ON178 (1-5 M). B, effects of AA861 (1-6 M). Arrows indicate the application of ONO178 or AA861. Each point is the mean value of five to nine experiments (n = 5-9), and bars are 2 x S.D. Absolute value of tension development ranged between -2 and -8 g. Insets are the examples of actual traces of twitch contractions before and after application of chemicals. effects of LTC4 and LTD4 on the twitch contraction, we performed experiments using a concentration of less than 1-9 M. At 1-1 M LTC4 did not affect the twitch contraction, but at a concentration of 1-9 M it did significantly enhance the amplitude of the twitch contractions. On the other hand, 1-9 M-LTD4 and 1-8 M LTE4 suppressed the relationship between the number of stimuli and the relative amplitude of twitch contractions (Fig. 5 C and D). Effects of isolated and dispersed epithelial cells on the amplitude of excitatory junction potentials (EJPs) recorded from bronchioles and trachealis As the amplitude of twitch contractions evoked by nerve stimulation strictly correlates to that of EJPs (Inoue, Ito & Takeda, 1984), we examined the role of

10 628 Z. XIE, H. HAKODA AND Y. ITO A o Control * LTB4, 14 M B o Control * LTC4, 1-9 M c i*i* i ** a {** p 8) Co C C ) a) cr Co o Control * LTD4, 1-9 M Ir-** D - _ ZY t ** f** o Control * LTE4, 1-9 M A LTE4, 1-8 M i** ** c Number of stimuli at 2 Hz 2 3 E Control LTB4, 1-8 M H Control LTD4, 1-9 M SO m 3 m I,1,_ Q CControl LTD4, 1-8 M F Control LTC4, 1-9 M -< * LTC4, 1-8 M G Control J Control LTE4, -J8 M / Fig. 5. Effects of various leukotrienes (LTB4, C4, D4 and E4) on contractions evoked by field stimulation (5, 1, 2 and 3 stimuli at 2 Hz) of bronchioles. The amplitude of twitch contractions evoked by 3 stimuli at 2 Hz in normal Krebs solution was defined

11 EPITHELIAL REGULATION OF AIR WA Y MUSCLES epithelial cells involved in the decremental response of the twitch contractions recorded from the ring preparations of the bronchiole. Here we observed the effects of indomethacin, AA861 and leukotrienes on the amplitude of EJPs recorded from the bronchioles, using the microelectrode procedure. As shown in Fig. 6, indomethacin (1-5 M) markedly enhanced the amplitude of the EJPs in the epithelium-denuded bronchioles, but there was only a slight increase in the EJP amplitude in the intact bronchioles. On the other hand, AA861 markedly enhanced the EJP amplitude in the epithelium-intact bronchioles, but had little effect in the case of epithelium-denuded bronchioles. Thus, lipoxygenase products released from the epithelial cells may modify the excitatory neuro-effector transmission. We then observed the effects of leukotrienes on the amplitude of EJPs in the presence of AA861, using epithelium-intact bronchiolar preparations. LTC4 (1-8 M) markedly enhanced the EJP amplitude, although LTB4 (1'8 M) had little effect. LTD4 and LTE4 (1-8M) slightly enhanced the EJP amplitude. We also observed the effects on the amplitude of EJP of exposure of the mucosafree trachealis to dispersed epithelial cells-. Figure 7 shows an example of the effects of epithelial cells (3'5 x 15 cells/ml) on the amplitude of EJP and contraction of the trachealis evoked by EFS using the double sucrose-gap method. With application of dispersed epithelial cells (3 5 x 15 cells/ml) the amplitudes of EJPs and contraction were markedly reduced and these effects were partially suppressed by indomethacin (1-5 M). After exposure to epithelial cells ( x 15 cells/ml), the EJP amplitude was reduced to -48 times the control, and indomethacin (1-' M) restored the amplitude to -66 times the control of the initial value. To examine the action of agonist on the effects of epithelial cells, we used ACh in the presence of indomethacin (1-' M). As noted in other experiments (Ito & Yoshitomi, 1988), exogenous ACh (1-8M) reduced the amplitude of EJPs to -52 times the control due to the inhibitory effects on ACh release from vagal nerve terminals through activation of pre-junctional muscarinic autoreceptors in the presence of indomethacin. An additional application of epithelial cells (2- x 15 cells/ml) further reduced the amplitude to -31 times the control. It would thus appear that epithelial cells release a factor which inhibits neuro-effector transmission and that ACh further stimulates the release of this factor from epithelial cells. The effects of AA861 on the inhibitory effects of epithelial cells on the EJP amplitude of the trachea were also observed. With application of dispersed epithelial cells, the EJP amplitude were suppressed to -64 times the control in the presence of indomethacin (1-5 M), and additional application of AA861 (1-6 M) restored the amplitude of EJP to 7 times the control value. All these observations suggest the possible role of leukotrienes in the inhibitory effects of epithelial cells on excitatory neuro-effector transmission. 629 as a relative tension of 1. Each point is the mean value of five to eight experiments (n = 5-8) and vertical lines represent 2 x S.D. The absolute value of tension development ranged between -2 and 5 g. ** Significantly different from the control (P < -1). E-J, actual traces of twitch contractions in the presence or absence of LTB4 (1-8 M), LTC4 (1-1 M and 1-8 M), LTD4 (1-9 and 1-8 M) and LTE4 (1-8 M). Calibration values in E apply to all traces.

12 63 Z. XIE, H. HAKODA AND Y. ITO Effects of EFS on ring bronchiolar preparations during 5-hydroxytryptamine (5-HT)-induced contraction To observe the effects of EFS on the muscle tone of dog bronchiolar rings with an intact epithelium, we used 5-hydroxytryptamine (5-HT; 1-5 M), since it was found A Relative amplitude of bronchiolar EJPs ; E Control (+) Indo, 1-5 ( Indo, 1-5 H- AA861, 16 (+) AA86 1, 1 - (-) I l~~~~~~ -i I AA861, 16 (+) + LTB4, 1-8(+) + LTC4, 1-8 ( + LTD4, 1-8 (+) + LTE 4, 1-8 (+) I ihi** Control AA861, 1-6 M Control Indo, 1 M B -59 mv D I7 mv 1 s 1 s X [I:I [-6 mv E(-) 6 \, A, 1 1 s 1 S Fig. 6. Effects of indomethacin (Indo), AA861 and various leukotrienes (LTB4, C4, D4 and E4) on the amplitude of EJPs recorded using a microelectrode in intact (E(+)) and epithelium-denuded (E(-)) bronchioles. EJPs were recorded from the same cell throughout the experiments, using intact and epithelium-denuded bronchioles. The amplitude of EJPs before the application of chemicals was defined as a relative amplitude of 1. Each value indicates mean of five or six experiments (n = 5 or 6), and bars are 2 x S.D. * P < -5; ** P < -1. Absolute values of EJPs ranged between 5 and 17 mv. that this compound at this concentration induces sustained contraction in bronchioles (Inoue & Ito, 1986). EFS applied during contraction evoked by 5-HT (1-5 M) produced an initial phasic contraction with a subsequent phasic relaxation.

13 EPITHELIAL REGULATION OF AIRWAY MUSCLES 631 A Relative amplitude of tracheal EJPs.5 1. Control E(+) E(+) + Indo, 1-5 M I~~~~~~~~~~~~~~~~~ I ACh, 1- M + Indo, 1-5 M E(+) + ACh, 14 M + Indo, 1-5 M E(+) + Indo, 1-5 M E(+)+ Indo, 1-5 M + AA861, 16 M i B Control C Epithelial cells 3*5x 15 Wash D [ī5 8 ~~mv 8 - ~~-6 E Control Epithelial cells F Indo 15 Wash G, mg 1 [ mg 5 5 s Fig. 7. Effects of epithelial cells on the amplitude of EJPs and twitch contraction of tracheal muscles recorded using the double sucrose-gap method and in the presence of various chemicals. EJPs were recorded from six to eight preparations before and after the application of chemicals in the presence or absence of dispersed epithelial cells. Each value indicates mean of six to eight experiments (n = 6-8), and bars are 2 x S.D. * P < -5; ** P < 1. Absolute values of EJPs and twitch contractions ranged between 1 and 17 mv and 2 and 5 mg. 21 PHY 449

14 632 Z. XIE, H. HAKODA AND Y. ITO Atropine Atropine + propranolol + prazosin + yohimbine A HT Indo AA861 wl : : ^~Ir -2 9 B 5-HT + TT: 'X C min.7 L-2 9 c x X 1- a)._ ) 'a Number of stimuli at 2 Hz Fig. 8. Effects of electrical field stimulation of ring preparations of bronchioles during elevated muscle tone induced by 5-HT (1-5 M) in the presence of guanethidine. A and B, effects of field stimulation (5 stimulation at 2 Hz, 1 /ts stimulus duration and 2 V in intensity) in the presence or absence of atropine (1-6 M), prazosin (1-6 M), yohimbine (1-6 M), tetrodotoxin (1-' M), indomethacin (1-5 M) and AA861 (1-5 M). C, shows the relationship between the stimulus number at 2 Hz and relative amplitude of phasic relaxation, where the amplitude of muscle relaxation evoked by 3 stimuli at 2 Hz was taken as a relative amplitude of 1. Each point indicates mean of five to seven experiments (n = 5-7), and bars are 2 X S.D. Atropine (1-6 M) or tetrodotoxin (1-v M) abolished the initial phasic contraction but did not affect the subsequent relaxation (Fig. 8), thereby indicating that the phasic relaxation of the pre-contracted tissue is not due to activation of neural

15 EPITHELIAL REG ULA TION OF AIR WA Y MUSCLES 633 elements in the tissue. Furthermore, a- and,-adrenoceptor blocking agents (propranolol, prazosin and yohimbine (1-' M) each) did not alter the amplitude of the phasic relaxation. To investigate the property of the phasic relaxation, we applied EFS at various intensities and duration. A single stimulus of short duration (1 its) and 2 V in B C H2 5-HT + TTX D / 7 A * 5-HT + TTX.2 E* 3 min I G Fig. 9. Effects of hypo-osmolarity shock on the phasic relaxation induced by electrical field stimulation of bronchiole ring preparations during contraction evoked by 5-HT (1-5 M) and in the presence of tetrodotoxin (TTX, 1-7 M). A, effects of 5-HT (1-5 M) on muscle tone in the presence of tetrodotoxin (1-7 M). B, during the 5-HT contraction, electrical field stimulations (5, 1, 2 at 2 Hz) evoked phasic muscle relaxations. C, application of distilled water for 3 s elevated the muscle tone and the tissue was re-perfused with Krebs solution containing 5-HT and tetrodotoxin. D, EFS (5, 1 and 2 at 2 Hz) evoked no muscle relaxation after exposure to distilled water. E, the tissue was rinsed with normal Krebs solution for 1-15 h. F and G, twitch contractions evoked by EFS and which were sensitive to tetrodotoxin (1-7 M). intensity evoked a phasic relaxation and repetitive stimulation applied at 3 min intervals evoked a relaxation with a constant amplitude. Increase in the stimulus number at high frequency (2 Hz) with the fixed stimulus duration (1,ts) and intensity increased the amplitude of phasic relaxation. Figure 8 C shows the relationship between the number of stimuli and the relative amplitude of phasic relaxation. The amplitude of phasic relaxation evoked by 3 stimuli at 2 Hz was defined as a relative amplitude of 1P. The repetitive stimulation (1-3 stimuli) increased the amplitude of phasic relaxation to times the value evoked by a single stimulus. To examine the property of the phasic relaxation evoked by EFS, the effects of epithelial cells were observed. Following mechanical denudation of the epithelial layer, EFS evoked no muscle relaxation during the 5-HT-induced 21-2

16 634 Z. XIE, H. HAKODA AND Y. ITO contraction (data not shown). With application of 1-nitro-arginine, oxyhaemoglobin, indomethacin, or AA861 the amplitude and duration of phasic relaxation observed in intact bronchiolar muscle tissues remained unchanged. As shown in Fig. 9, EFS (5, 1 and 2 stimuli at 2 Hz) evoked phasic relaxation during the 5-HT-induced contraction in the presence of TTX (1-7 M) (Fig. 9B). When the tissue was exposed to distilled water for 3-6 s there was a gradual increase in muscle tone (Fig. 9 C), we then re-perfused the tissues with Krebs solution containing 5-HT and TTX (1-7 M). After this hypo-osmolarity shock, EFS failed to evoke phasic relaxation, as shown in Fig. 9D. However, after repeated rinses of the tissue with normal Krebs solution, EFS evoked a twitch contraction which was sensitive to TTX or atropine (data not shown), thereby indicating that the hypoosmolarity shock did not cause degeneration of the nervous tissue and smooth muscle. However, histological examination revealed that hypo-osmolarity shock did destroy the epithelial layer. All these data taken together indicate that the EFS stimulates the epithelial cells to release a factor(s) that induces phasic relaxation during the elevated tone of the ring preparations. DISCUSSION The resting membrane potential of tracheal smooth muscle cells of the dog was reported to be about -6 mv (Suzuki, Morita & Kuriyama, 1976), and this value has been confirmed by other investigators (Farley & Miles, 1977; Ito & Tajima, 1981; Souhrada, Klein, Berend & Souhrada, 1983). Although no differences were found in membrane potential among upper, middle, and lower segments of the trachea, it was reported that the resting membrane potential of the smooth muscle cells of the bronchial airway (second generation) is -63 mv, that is, slightly larger than that of the trachealis (Souhrada et al. 1983). We found that the resting membrane potential of the bronchiolar smooth muscle cells (about 1 mm in diameter) is about -7 mv (Inoue & Ito, 1986; Ito & Inoue, 1989; present study), a value significantly larger than those observed in the muscle cells in trachealis or bronchial wall. Thus, the smaller the airway, the larger the resting membrane potential of the airway smooth muscle cell, and it was assumed that the lower membrane potential in the trachealis, compared to that of bronchioles, is probably due to a large sodium conductance rather than to a smaller potassium conductance in the membrane (Ito & Inoue, 1989). All these experiments were performed using the epithelium-denuded trachealis and epithelium-intact bronchioles. It has become evident that accessory cells such as endothelium or epithelium modulate the smooth muscle tone in vascular and airway tissues (see for example Goldie et al, 199; Suzuki & Chen, 199). The present study clearly showed that mechanical denudation of the epithelium depolarizes the membrane of smooth muscle cells by about 1 mv. Conversely, exposure to dispersed epithelial cells (> 5 x 14-2 x 15 cells/ml) hyperpolarized the membrane of mucosa-free tracheal and epithelium-denuded bronchiolar smooth muscle cells. These observations suggest that a factor released from airway epithelial cells regulates the resting membrane potential of airway smooth muscle cell (epithelium-derived hyperpolarizing factor, EpDHF). In endothelial cells in vascular tissue, endothelium-derived hyperpolarizing factor (EDHF) is released in response to Ml-muscarinic receptor

17 EPITHELIAL REGULATION OF AIR WAY MUSCLES activation followed by a transient membrane hyperpolarization of vascular smooth muscle cells (Komori & Suzuki, 1987; Chen, Suzuki & Weston, 1988; Komori, Lorenz & Vanhoutte, 1988; Chen & Suzuki, 199). In bronchioles, this transient membrane hyperpolarization was not observed after the application of ACh, yet isolated and dispersed epithelial cells did hyperpolarize the membrane of tracheal or bronchiolar smooth muscle cells, indicating that there is a continuous release of EpDHF from epithelial cells, in the absence of any specific stimulation. This continuous release of the epithelium-dependent factor is worthy of consideration, since there is evidence for a continuous transepithelial transport of solutes and water in airway epithelium (Frizzell, Welsh & Smith, 1981). Removal of the epithelium modulates airway smooth muscle responsiveness to a variety of spasmogenic and relaxant agonists (see for example Goldie et al. 199). It has also been shown that epithelial cells release known and unknown substances in response to stimulations by various agents. Cyclo-oxygenase is present in the rabbit pulmonary epithelium (Szarek, Butler, Adler & Evans, 1986) and tracheal epithelial cells generate arachidonic acid metabolites (Widdicombe et al. 1989). Furthermore, it was demonstrated that arachidonic acid contracted epithelium-free guinea-pig tracheal strips, but relaxed intact tracheal strips (Nijkamp & Folkerts, 1986). In contrast, when tracheal strips were pre-contracted with histamine or carbachol, exogenous arachidonic acid had no apparent effect on epithelium-free preparations yet did induce a concentration-dependent relaxation of intact tracheal strips. These effects of arachidonic acid were blocked by indomethacin or aspirin (Tschirhart, Frossard, Bertrand & Landry, 1987). Recent studies have shown that bradykinin, platelet-activating factor (PAF) and the calcium ionophore A23187, increase the release of PGE2 with smaller increases in PGF2a, 6-keto PGF1. and thromboxane A2 from cultured dog and human tracheal epithelial cells. Gas chromatography-mass spectrometry showed that the ratio of PGE2 to PGD2 released from the dog epithelial cells by A23187 was 3: 1, and responses to bradykinin and A23187 were reduced by pre-treatment with indomethacin (Widdicombe et al. 1989). These and other results (Ulman, Lofdahl, Svedmyr & Skoogh, 199) suggested that airway epithelial cells release cyclo-oxygenase metabolites of arachidonic acid, and mainly PGE2 (Widdicombe et al. 1989). However, it was also reported that prostanoids such as PGE1, PGE2, PGF2.or PGI2 do not affect the resting membrane potential of airway smooth muscle cells in a concentration range between 1-1 to 1' M (Ito & Tajima, 1981; Inoue & Ito, 1985; Inoue et al. 1986). As the present experiments were performed in the presence of indomethacin the EpDHF may not be a prostanoid. Similarly, Barnes, Cuss & Palmer (1985) noted the lack of effects of indomethacin on both EC5 and maximal effect in ACh concentration-response curves for intact and epithelium-free tracheal strips and concluded that the epithelium-dependent relaxing factor (EpDRF) of bovine trachea was not a cyclo-oxygenase product. The failure of Methylene Blue, oxyhaemoglobin and 1-nitro-arginine to inhibit membrane hyperpolarization induced by epithelial cells in tracheal smooth muscle cells and phasic relaxation induced by EFS indicates that EpDHF and EpDRF do not act through stimulation of soluble guanylate cyclase, and therefore the factor was not NO or related substances. Komori et al. (1988) reported that NO caused a concentration-dependent relaxation in rings of canine artery, with or without endothelium, but did not alter the membrane potential in either the presence or 635

18 636 Z. XIE, H. HAKODA AND Y. ITO absence of the endothelium, the relaxation was inhibited by Methylene Blue or oxyhaemoglobin. In addition, ACh induces concentration-dependent relaxations and transient hyperpolarization of the cell membrane of the vascular muscle in arteries with an intact endothelium. Thus it seems reasonable to conclude that EpDRF differs from the EDRF released in the vascular tissues. One of the features of twitch contractions elicited by EFS in bronchial ring preparations is a gradual and continuous reduction in the amplitude of contraction during superfusion, with or without indomethacin. This observation is in sharp contrast to the previous one in which size of the twitch contractions of the helically cut preparations of bronchiole elicited by nerve stimulation in the presence of propranolol gradually decreased, but the reduction in the amplitude was relatively small (Inoue & Ito, 1986), compared to that observed in the dog trachea (Inoue et al. 1984). Namely, the decline of the twitch contraction of the helically cut preparation of bronchioles evoked by nerve stimulation never decreased to less than 5 % of the initial value during prolonged perfusion, and was overcome in the presence of indomethacin. The decline of twitch contractions observed in the dog trachea and helical preparations of bronchiole (epithelium-denuded preparations) was prevented by the cyclo-oxygenase inhibitor, indomethacin and a prostaglandin antagonist SC1922 (Ito & Tajima, 1981; Inoue et al. 1984; Inoue & Ito, 1986). Thus, the decremental responses observed in the mucosa-free trachealis and epitheliumdenuded bronchioles are due to endogenous prostaglandins which inhibit ACh release from nerve terminals of the vagus following stimulation of nervous elements in muscle tissues (Inoue et at. 1984; Inoue & Ito, 1986; Ito, 1991). Using cultured tracheal epithelial cells and intact tissues, it was found that a cyclo-oxygenasedependent inhibitory factor is produced by the epithelium, and that the factor is PGE2 (Barnett, Jacoby, Nadel & Lazarus, 1988). The present experiments were performed in the presence of indomethacin in order to inhibit cyclo-oxygenase activity, yet there was- evidence of decremental responses. The inhibitory factor may not be a cyclo-oxygenase product of arachidonic acid. After epithelium denudation of the ring preparation indomethacin suppressed the decremental response, as was noted using helical preparations of the dog bronchioles (Inoue & Ito, 1986). The lipoxygenase inhibitory AA861 or the leukotriene antagonist ONO 178 suppressed the decremental response of the twitch contraction and enhanced the amplitude of EJPs recorded from the intact bronchioles. These observations suggest a role of lipoxygenase metabolites released from the epithelium in the decremental response of the twitch contractions in the ring preparations. To examine this possibility, we observed the effects of exogenous leukotrienes on the amplitude of twitch contractions and EJPs recorded from bronchioles. We found the leukotriene D4 and E4 suppressed the amplitude of twitch contractions, but the concentration of exogenous LTD4 and LTE4 required for this suppression ranged between 1-8 and 1- M. Exogenous PGEE1 or PGE2 in very low concentrations ( M) were found to prejunctionally inhibit the excitatory neuro-effector transmission (Ito & Tajima, 1981). On the other hand, LTD4 and LTE4 did not suppress the EJP amplitude but did slightly enhance the EJP amplitude. These conflicting observations indicate that leukotrienes have multiple actions on epithelial cells and on excitatory neuro-effector transmission.

19 EPITHELIAL REGULATION OF AIRWA Y MUSCLES The actions of leukotrienes on intact and epithelium-denuded tissues were not simple. It has been suggested that leukotrienes may stimulate epithelial cells and produce an airway smooth muscle-sensitive inhibitory factor (Hay, Farmer, Raeburn, Muccitelli, Wilson & Fedan, 1987; Hisayama, Takayanagi, Nakazato & Hirano, 1988) in addition to direct actions on the smooth muscle cells (Smedegard, Hedqvist, Dahlen, Revenas, Hammarstrom & Samuelsson, 1982). On the other hand, judging from the actions of antagonists for leukotrienes (BW755C and FPL55712), leukotrienes were thought to be involved in relaxing and contracting events on contractions evoked by histamine or carbachol (Tschirhart et al. 1987). FPL55712 and BW755C enhanced the maximum contractile response to carbachol in epithelium-free tissues but not in intact tissues. Thus, the actions of leukotriene D4 and E4 observed in intact ring preparations probably include the direct effects on the excitatory neuro-effector transmission, and indirect effects through inhibitory factors released from the epithelium. Alternatively, EpDRF, which is released in response to electrical field stimulation of a short duration (1,us) may also inhibit excitatory neuro-effector transmission. In the ferret trachea, a similar mucosadependent decremental response of twitch contractions was reported to be mediated by both a prostanoid and a non-prostanoid factor (Ulman et al. 199). An epithelium-derived inhibitory factor (EpDIF) released from the guinea-pig tracheal epithelium was evaluated in a co-axial bioassay system consisting of an intact guinea-pig tracheal tube surrounding by an endothelium-denuded rat aortic strip (Fernandes & Goldie, 199). They concluded that histamine as well as muscarinic agonists cause a concentration-dependent relaxation through the release of EpDIF, a compound which is neither a cyclo-oxygenase nor a lipoxygenase product of arachidonic acid, and that the target of this factor is the microcirculation in the airway wall. However, our present experiments show that EFS releases EpDRF which in turn induces a phasic relaxation of pre-contracted bronchioles, with no change in the membrane potential. Since microelectrode experiments showed that EFS evokes no change in the membrane potential in the presence of atropine (data not shown), EpDRF probably differs from the EpDHF observed in the present experiments. A similar epithelial-dependent relaxation was noted in the guinea-pig trachea using osmotic stimuli (Munakata, Mitzner & Menkes, 1988). However, EFS is simpler compared to osmotic stimuli and the response is reproducible. We propose that in airway smooth muscle tissues epithelial cells release more than two factors, one of which selectively modulates the resting membrane potential and is released continuously even without electrical or chemical stimulation. The other factor is released in response to electrical field stimulation and modulates the smooth muscle tone or excitatory neuro-effector transmission. Thus, multiple actions induced by epithelium-derived factors may regulate the resting membrane potential, neuro-effector transmission, and muscle tone of the airway smooth muscles. 637 REFERENCES BARNES, P. J., Cuss, F. M. & PALMER, J. B. (1985). The effect of airway epithelium on smooth muscle contractility in bovine trachea. British Journal of Pharmacology 86,

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