Endothelin regulation of mucus glycoprotein from feline tracheal submucosal glands
secretion
S. SHIMURA, H. ISHIHARA, M. SATOH, T. MASUDA, N. NAGAKI, H. SASAKI, AND T. TAKISHIMA First Department of Internal Medicine, Tohoku University School of Medicine, Sendai 980, Japan Shimura, S., H. Ishihara, M. Satoh, T. Masuda, N. Nagaki, H. Sasaki, and T. Takishima. Endothelin regulation of mucus glycoprotein secretion from feline tracheal submucosal glands. Am. J. Physiol. 262 (Lung Cell. Mol. Physiol. 6): L208-L213, 1992.-We examined the effects of endothelin on both the trichloroacetic acid precipitable 3H-labeled glycoconjugate release and intracellular Ca2+ concentration ( [Ca”+]i]) measured by the usage of fura- in submucosal glands isolated from feline trachea. Endothelin-1 produced a significant increase in glycoconjugate release from the isolated glands in a dose-dependent fashion, reaching a response of 161% of the control at 10m6 M. Atropine, propranolol, phentolamine, or indomethacin did not produce any significant alterations in the ET-l-evoked glycoconjugate secretion from the isolated glands. In contrast, in tracheal explants which contained epithelium, ET-l produced a significant reduction in the glycoconjugate secretion in a dose-dependent fashion, reaching a response of 59% of the control at low6 M. In the presence of cultured epithelial cells, ET-l also produced a significant reduction in the glycoconjugate secretion from isolated glands. In isolated glands, ET- 1 produced a sustained increase in the [ Ca”‘] i which was abolished by the removal of Ca2+ from the medium or by the presence of cultured epithelial cells. Pretreatment with indomethacin failed to alter the epithelial inhibitory action evoked by ET-l in both the glycoconjugate secretion and the [Ca2+]; in isolated glands. ET-2 and ET-3 failed to produce significant alterations in the glycoconjugate secretion or [Ca2+];. These findings indicate 1) that ET-l induces mucus glycoprotein secretion via a Ca2+ influx and 2) that it possibly augments the an epithelial action inhibitory to the mucus glycoprotein secretion from airway submucosal glands. endothelin-I, tration
feline trachea,
intracellular
calcium
ion concen-
is a newly discovered polypeptide secreted from the vessel wall endothelium in response to various stimuli such as hypoxemia (17,34). Further, the mRNAs for endothelin have been shown by in situ hybridation in the airway epithelial cells (10). Endothelin-1 and human endothelin-3 are released from cultured canine, porcine, and human tracheobronchial epithelial cells (1, 12). Since airway mucosa consists of a large number of small vessels, smooth muscles, submucosal glands, connective tissue, and epithelium, it is possible that endothelin is locally produced in the airway mucosa and affects smooth muscle, glandular, and epithelial functions. Recently, endothelin was found to constrict guinea pig, ferret, and rat airway smooth muscle (7, 8, 13, 21, 29, 30) and is implied to act via the release of cyclooxygenase products of arachidonates or by the activation of the calcium channel. Thus endothelin may play some role in the pathophysiological aspects of various respiratory diseasesincluding bronchial asthma (14, 26). Nomura et al. (14) have reported that a patient showed bronchial washing fluid with a raised endothelin-immu-
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noreactive level during status asthmatics, suggesting that endothelin plays a part in bronchoconstriction in bronchial asthma. Further, Ninomiya et al. (13) have observed that endothelin stimulates the mast cells to release histamine via a Ca2+ influx and plays a role in guinea pig tracheal constriction. Although both mucus hypersecretion and bronchoconstriction are characteristic of asthma, as well as of other pulmonary diseases, to our knowledge, the effect of endothelin on airway mucus secretion has not been examined. We have been successful in isolating submucosal glands from the trachea (22, 25), which play a principal role in mucus secretion (16). The purpose of the present study is to examine the effects of endothelin both on the mucus glycoprotein secretion and on the intracellular Ca2+ concentration ([Ca”‘];) of acinar cells in isolated feline tracheal submucosal glands, since a number of experiments have implicated [Ca2+]i as a second messenger in other tissues stimulated by endothelin (6, 8, 27, 28, 30, 33). METHODS Isolation of submucosal glands. The tracheae were removed from 20 adult cats (3.0-4.5 kg of body wt) under an anesthesia of intramuscular ketamine hydrochloride and intravenous thiopental sodium (each 30 mg/kg body wt). Each trachea was cut into rings 3-4 cm long and fixed with pins, posterior (membranous) wall side up, in Krebs-Ringer bicarbonate (KRB) solution at 4°C. Fresh unstained submucosal glands were mechanically isolated under a stereomicroscope using two pairs of sharpened tweezers and microscissors as described previously (22, 25). The entire procedure was performed in a KRB solution which contained (in mM) 125 NaCl, 5 KCl, 1.2 MgC12, 1.0 CaC12, 25 NaHC03, 1.2 NaH2P04, and 11 glucose, gassed with 5% CO,95% 02, pH 7.4, at 20°C. Measurement of 3H-labeled glycoconjugate release. Measurement of 3H-labeled mucus glycoconjugate release from isolated submucosal glands or from tracheal explants was made by a previously reported method (12, 13, 14). Membranous portions of the trachea were fragmented into 3 X 3 mm explants which contained surface epithelium and 5-10 submucosal glands (22). Five to 10 isolated glands or one tracheal explant were incubated for 16 h with 2 ml of medium 199 which contained D-[6“Hlglucosamine hydrochloride (2.5 &i/ml, specific activity 2740 &i/mmol, Amersham Japan, Tokyo), penicillin (100 U/ml) and streptomycin (100 pg/ml) in a controlled atmosphere chamber. The chamber was constantly gassed with 5% C02-55% N240%02 at 37°C. After being washed with the culture medium, samples were allowed to further incubate at 37°C for two successive 2-h periods (periods I and II) in 2 ml of the culture medium without a radiolabeled precursor. At the end of each period, the culture media was harvested and precipitated with 10% trichloroacetic acid (TCA) and 1% phosphotungstic acid (PTA) at 4°C overnight. The precipitated radioactivity was measured by a liquid scintillation counter (LSC-1000, Aloka, Tokyo). The effect of endothelin or other drugs on the release
0 1992 the American
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of 3H-labeled mucus glycoconjugate was determined by adding these to the culture medium at the beginning of period II. Each antagonist was added to the medium 15 min before the addition of the agonist. The ratio of precipitated radioactivity (dpm) from period II to that from period I was determined for each sample and was taken as the secretory index. The effects of the pharmacological agents were determined by comparing the secretory indices of the manipulated samples with those of the matched, unmanipulated control samples. [Ca2+/i measurement. The [Ca2+]; of acinar cells in isolated submucosal glands was measured by a method previously reported (5). The isolated submucosal glands were incubated at room temperature for 2-4 h in a loading solution, which contained 5 PM acetoxymethyl ester of fura(fura-2/AM) and 0.2% of the detergent, Cremophor EL, to increase the solubility of the fura-2/AM. Cremophor (0.2%) itself does not alter the secretory response of isolated glands (5). After the fura- loading, the glands were rinsed with KRB three times at intervals of 5 min (for IO-20 min) to wash out any fura-2/AM attached to the glands. The fura- fluorescence was determined by an apparatus consisting of an inverted microscope (TMD, Nikon, Tokyo) with an objective lens (Flour, x40, Nikon), a xenon lamp, and a photomultiplier for measurement of the furafluorescence of live tissue. The time course of the fura- signal was continuously recorded with a fluorimeter (CAM-220, Japan Spectroscopic, Tokyo) at 37°C in KRB solution. After furaloading, each gland was put in a Rose chamber (0.25 ml in volume, RKI, Ikemoto, Tokyo) on the stage of the inverted microscope and was perfused with warmed (37°C) KRB solution gassed with 5% C02-95% O2 at a constant rate. The microscopic field was adjusted to the acinar portion of the gland at a magnification of x400. The excitation light was automatically alternated between 340 and 380 nm at intervals of 100 Hz, and the fluorescence from the glands was collected by the photomultiplier through a 510 nm filter. The ratio of the fluorescence intensity due to 340 nm (F340) and to that due to 380 nm (F380) was calculated and recorded as a function of time. We used different gland samples for each measurement of the [Ca”+];, since the decrease in the fluorescence intensity was too large to repeat the measurement in the same sample (5). The ratio was then converted to [Ca”+]; using the method of Grynkiewicz et al. (4) and Scanlon et al. (20), i.e., using the equation [Ca2+]; = (R - R,in)/(R,,x - R) & S where R is the experimentally determined minimal and maximal fura- fluorescence ratio (F340/F380), & is the dissociation constant, and S is the ratio of the fluorescence at 380 nm in a Ca2+-free solution to that in a Ca2+-containing solution in the presence of ionomycin. The & was assumed to be 224 nM (4). R,,, and I&in were determined by the addition of 10s5 M ionomycin and 5 mM ethylene glycol-bis(P-aminoethyl ether)-N,N,N’,N’-tetraacetic acid (EGTA), respectively. Adenosine 3’,5’-cyclic monophosphate (CAMP) measurements. After a lo-min preincubation, isolated glands were incubated at 20°C with 10v5 M isoproterenol or low6 M endothelin-1 for 30 min in a KRB solution (pH 7.4). The KRB solution contained 1% bovine serum albumin (BSA) and 0.5 mM 3isobutyl-1-methylxanthine (IBMX) as a CAMP phosphodiesterase inhibitor. The reaction was terminated by the addition of 10% TCA for measurement of CAMP concentration (3, 31). For each measurement, the solution from 20 to 30 glands was extracted with water-saturated ether. The aqueous phase was dried and kept at -20°C until the CAMP assay. Before the assay, sodium acetate buffer (pH 6.2) was added to each sample and acetylated to enhance the sensitivity for the immunoassay. The amount of CAMP was determined in duplicate by the kits for 125I radioimmunoassay (Yamasa Shoyu, Ltd., Tokyo). The precipitates were dissolved in 1 N NaOH and assayed for protein by the method of Lowry et al. (9). Results are expressed
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as picomoles of CAMP per milligram of protein. The effects of isoproterenol and endothelin-1, were compared with the control values obtained by using tissues from the same animals. Isolation of epithelial cells. Immediately after removal, the tracheae were immersed and washed in KRB solution at 4°C with penicillin (50 IU/ml), streptomycin (50 IU/ml), and gentamycin (50 pg/ml). Cartilaginous portions were fixed by pins in KRB solution at 4°C and bubbled with 5% C02-95% 02. Shallow longitudinal incisions on each specimen were made by sharpened scissors at l-mm intervals, and the epithelium was peeled by microforceps. The strips thus obtained were immersed in 4 ml of cold protease (Sigma protease XIV, 0.1% in Eagles minimal essential medium, 4°C) overnight. The digestion was stopped by the addition of fetal bovine serum (FBS, 10% vol/vol) and then filtered through a stainless mesh with holes 100 ,ccrn in diameter. The epithelial cells released by the protease were collected by centrifugation (500 g, 5 min) and again washed with Ham’s F-12 with hormone supplement. Trypan blue exclusion test showed that viability was over 95%. The epithelial cells (1 x IO”) were seeded on collagen-coated plastic Petri dishes (35 mm in diameter) and cultured in Ham’s F-12 nutrient mixture supplemented with penicillin (50 IU/ ml), streptomycin (50 IU/ml), and gentamycin (50 pg/ml), containing 10% FBS, hydrocortisone (0.36 pg/ml), transferrin insulin (IO ,pg/ml), and endothelial (O-5 dml), T3 (0.2 kdml), cell growth supplement (ECGS, 7.5 pg/ml). The epithelial cells were then incubated in a 95% air-5% CO2 humidified incubator at 37°C. Epithelial cells in a dish showed a confluent appearance 3 or 4 days after the seeding and each dish contained 5 x lo6 epithelial cells. Epithelial cells showing a confluent appearance were fixed with glutaraldehyde, embedded in Epon 812 and cut into l-pm sections. A light-microscopic examination showed that the cultured epithelial cells consisted of basal cells and a few ciliated cells with a monolayer appearance and were not contaminated with fibroblasts. We used the dishes that contained cultured epithelial cells with a confluent appearance in period II and examined the effect of epithelial cells on the glycoconjugate secretion from isolated submucosal glands. Reagents. Drugs used in the present study were endothelin1 (human/porcine), endothelin-2 (human), endothelin-3 (human/rat) (Peptide Institute, Osaka, Japan), acetyl-P-methylcholine chloride (methacholine), propranolol, phentolamine, EGTA, (Wako Pure Chemicals, Osaka), fura-2/AM (Dojin, Kumamoto, Japan), Cremophor EL (Nakarai Chemicals, Kyoto, Japan), ionomycin (Calbiochem, La Jolla, CA), FBS, Eagle’s MEM, -Ham’s F-12 (GIBCO, Grand Island, NY), atropine sulfate, protease XIV, Eagle’s MEM, hydrocortisone, transferrin, T3, insulin, ECGS, IBMX, BSA, fraction V (Sigma Chemical, St. Louis, MO), medium 199 (Flow, McLean, VA), and Aquasol 2 (New England Nuclear, Boston, MA). Prostaglandins E1, E2 and 12-analogue OP-41483 were generous gifts from Ono Pharmaceutical, Tokyo, Japan. Statistical analysis. Data are means t SE. For mean comparisons, the two-tailed paired or unpaired Student’s t test was used, and the Cochran Cox t test was used when Bartlett’s test for uniformity of variance showed it to be nonuniform. A P < 0.05 was considered statistically significant. RESULTS
Glycoconjugate secretion. Endothelin-1 produced an increase in the TCA-precipitable glycoconjugate secretion from isolated glands in a dose-dependent fashion as shown in Fig. 1. Endothelin-1 produced the responses of 104, 136, 141, 150, and 161% of control at 10-l’, lo-‘, lo-s, 10s7, and 10Y6 M, respectively. A mixture of atropine (10B6 M), propranolol (10s5 M), and phentolamine
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18Q-
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10’‘M ET-l
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10’“M Et-1
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PHE
10’“M ET-1
IND
Fig. 1. Endothelin-1 (ET-l)-induced glycoconjugate secretion from feline tracheal isolated glands. ET-l produced an increase in glycoconjugate secretion in a dose-dependent fashion, which was not altered by treatment with 3 antagonists or with indomethacin. ATR, lOA M atropine; PRP, lo-” M propranolol; PHE, low5 M phentolamine, IND, lOA” M indomethacin. Means t SE of 5-10 experiments. * P < 0.05, ** P < 0.01, compared with controls.
10""M ET-l
i
10'"M ET-l
10'"M ET-l
10"M ET-l
10'"M ET-l
10’“M ET-l t IND
*
Fig. 2. Effect of endothelin-1 (ET-l) on glycoconjugate secretion from feline tracheal explants. In contrast to isolated glands, ET-1 induced a reduction in glycoconjugate secretion from tracheal explants, which was partially inhibited by indomethacin. Mean k SE of 5-9 experiments. tt P < 0.01, compared with isolated glands simulated by 10m6 M ET-l. §P < 0.05, compared with explants stimulated by 10V6 M ET1. Other symbols as in Fig. 1.
( 10m5M) did not alter the endothelin-1 ( 10s6 M)-evoked glycoconjugate secretion (163 t 8% of control, n = 6) (Fig. 1). Pretreatment with indomethacin (10V5 M) did not significantly alter endothelin-1 (10B6M) evoked glycoconjugate secretion (158 t 4% of control, n = 5) from isolated glands, as in our previous report (18). In contrast, endothelin-1 produced a significant decrease in glycoconjugate secretion from the tracheal explants in a dose-dependent fashion, reaching a response of 59 t 8% of control at 10s6 M (n = 5, P < O.Ol), as shown in Fig. 2. Pretreatment with 10m5M indomethacin partially inhibited the endothelin-1 ( 10B6M)-evoked decreases in glycoconjugate secretion from tracheal explants. This is, indomethacin significantly increased the endothelin-l-evoked response from 59 & 8% of control to 81 t 4% of control by endothelin-1 alone (P < 0.05) (Fig. 2). However, the secretory response to endothelin1 in indomethacin-treated explants was still much smaller than that evoked by endothelin-1 in isolated glands (P < 0.01) (Figs. 1 and 2), indicating that indomethacin failed to abolish the inhibition action. Since indomethacin ( l.0m5M) itself reduced glycoconjugate secretion from tracheal explant (88 t 6% of control, n = 4, P < 0.05), we used explants treated with indomethacin
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as the control for this experiment. The use of the epithelial cell-containing dish in period II produced secretory indices of 0.68 t 0.06 (n = 7), which were similar to 0.70 t 0.01 (n = 56) from corresponding samples, as was the case in our previous report (18). Hence, in the experiment of cultured epithelial cells, we used a dish with no epithelial cells in the period II as the corresponding control sample. Dry weight of isolated glands represents the amount of secretory cells in isolated glands and correlates negatively with the secretory index under the definite number of epithelial cells (11, 23). To avoid any variations due to differing sizes of glands, the secretory index in each sample was divided by the dry weight of the isolated glands (23) and compared with that of a corresponding control in the following experiment of epithelial inhibition. Endothelin-1 produced an inhibition of the glycoconjugate secretion from isolated glands in the presence of cultured epithelial cells (5 x 106) in a dose-dependent fashion, and endothelin-1 (10B6M) produced a response of 70 t 4% of corresponding control containing cultured epithelial cells (n = 9, P c O.Ol), as shown in Fig. 3. Pretreatment with 10B5 M indomethacin did not significantly alter the epithelial inhibitory action evoked by 10B6M endothelin-1 in the glycoconjugate secretion from isolated glands (74 t 6% of control, n = 4) (Fig. 3). Glycoconjugate secretion from indomethacin ( 10B5M) -treated isolated glands with cultured epithelial cells in response to endothelin-1 was still significantly smaller than that from isolated glands in response to endothelin-1 (Fig. 3). Indomethacin (10B5M) itself did not alter the glycoconjugate secretion from isolated glands with cultured epithelial cells (98 t 6% of control, n = 4). To examine the effect of extracellular Ca ions, Ca2’free KRB solution with 0.2 mM EGTA was used instead of medium 199 in periods I and II. In Ca2+-containing KRB solution, endothelin-1 (10m6M) produced a significant increase in the glycoconjugate secretion from isolated glands (143 t 8% of control, n = 5). In Ca2+-free KRB, however, there was no significant stimulation of the glycoconjugate secretion by endothelin-1 in isolated glands by 10s6M endothelin-1 (110 t 15% of control, n
- 5)
Neither endothelin-2 nor endothelin-3 significantly altered glycoconjugate secretion from isolated glands in the absence or in the presence of cultured epithelial cells. For example, endothelin-2 and endothelin-3 (10B6 M 10""M 10'"M 10'"M ET-l ET-l ET-l
10''M ET-l
10’8M ET-l
10'"M ET-l
t
IND
Fig. 3. Effect of endothelin-1 (ET-l) on glycoconjugate secretion from feline isolated glands in presence of cultured epithelial cells. ET-l produced a reduction in glycoconjugate secretion from isolated glands with culture epithelial cells in a dose-dependent fashion, and reduction was not altered even after treatment with indomethacin. Means k SE of 4-8 experiments. tt P < 0.01 compared with isolated glands alone stimulated by ET-l (10m6 M). Other symbols as in Fig. 1.
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each) induced responses of 99 t 6% (n = 5) and 103 t 180 (n = 6) of control in isolated glands, respectively, and responses of 103 t 5% (n = 5) and 98 t 7% of control (n = 5) in tracheal explants. As shown in Fig. 4, prostaglandin Fzcuproduced a significant increase in the glycoconjugate secretion from isolated glands (122 t 7% of control at lOa M, n = 5, P < 0.05), and prostaglandin 1, analogue, OP-41483, produced a significant reduction in the glycoconjugate secretion from isolated glands (86 t 4% of control at 10m5 M, n = 5, P < 0.05). Prostaglandin E1 (10m4M, n = 5) or E, (10B4 M, n = 5) did not produce any significant 80 J h I I I I I 1 alteration in the glycoconjugate secretion from isolated 0123 5 7 10min glands (Fig. 4). Fig. 6. Time courses of the [Ca2+]i in acinar cells of isolated glands [Ca2+/ in acinar cells. Endothelin-1 produced signifi(ET-l) in absence (0) and presence cant increases in [Ca2+]i in the acinar cells of isolated stimulated by 10V6 M endothelin-1 of cultured epithelial cells (0). Mean k SE of 5 experiments. ET-l glands. The time course of the [Ca2+]i rise evoked by produced an increase in [Ca2+]i without any initial transient increase, endothelin-1 showed a prolonged increase without any which was abolished in the presence of cultured epithelial cells. * P < initial transient increase. A representative example is 0.05, ** P < 0.01, compared with baseline values. t P < 0.05, t"f P < shown in Fig. 5A. At low6 M, [Ca2+]; was 120, 131, 146, 0.01, compared with those in absence of cultured epithelial cells. 158, 158, and 160% of the prior baseline value at 1, 2, 3, 180, 5, 7, and 10 min after stimulation, respectively (Fig. 6). ** Because the resting levels of [Ca2+]; varied greatly from 2s z MO68 to 210 nM among samples as in our previous experi> ment (5), the plateau level (5-10 min after stimulation) z as a percentage of the prior baseline level was used in 3 1401 the following analysis of [Ca2+]i data. As shown in Fig. ES + 4%
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Fig. 4. Effects of prostaglandins Fza (PGF2,, 10m4 M)), E, (PGE1, 10m5 M), E2 (PGE,, 10m5 M), and prostaglandin 12 analogue (OP-41483, 10m4 M) on glycoconjugate secretion from feline isolated glands. PGF2, produced a slight increase in the glycoconjugate secretion from isolated glands, whereas PGIz produced a slight but significant decrease. Means t SE of 5 experiments. P < 0.05, compared with controls.
[Ca++li
A
(nM)
-y+--
1::: 50
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200 150 100
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Fig. 5. Representative examples of time course of [Ca2+]; in acinar cells of isolated glands stimulated by 10V6 M endothelin-1. Endothelin1 produced an increase in [Ca2+]; without the initial transient rises as shown in A. In contrast, in presence of cultured epithelial cells, it failed to produce an increase in [Ca2+]; as shown in B. Arrows, beginning of stimulation by endothelin1.
10""M ET-1
10"M ET-1
10'"M ET-1
10"M ET-1
10'"M ET-l
1O'"M 10'"M ET-1 ET-1 t t ATR IND PRP PHE Fig. 7. Endothelin-1 (ET-l) induced [Ca2+]i rise in acinar cells of isolated glands. [Ca2+]i rise is expressed as a % plateau level of before baseline levels in each sample. Mean k SE of 4-8 experiments. ET-l produced an increase in the [Ca”‘] in a dose-dependent fashion, which was not affected bY treatment with indomethacin (lO-5 M). Symbols as in Fig. 1.
7, the endothelin-l-evoked [Ca2+]i rise was dose dependent at concentrations of 10-l’ to 10B6 M. Removal of Ca2+ from the medium abolished the endothelin-1 (lOa M)-evoked increases in [Ca2+]i (98 t 10% of the baseline value, n = 5). To understand the effect of epithelial cells on the [Ca2+]i of acinar cells in isolated glands, we measured the [Ca2+]i in isolated glands 0.3 x 0.5 mm in size placed in the center of the Rose chamber containing cultured epithelial cells on collagen-coated plastic disk (4 x 106/ chamber). Endothelin-1 did not produce any significant alterations in [Ca”‘] of acinar cells in isolated glands in the presence of cultured epithelial cells, and 10B6 M endothelin-1 produced a response of 96 t 5% of the baseline value in isolated glands in the presence of cultured epithelial cells (n = 5) (Figs. 5A and 6). CAMP concentration of isolated glands. Isoproterenol (10Y5 M) produced significant increases in the CAMP concentration of isolated glands (46 t 5 pM/mg protein, n = 5), compared with untreated control glands (8 t 2 pM/mg protein, n = 3, P < 0.01). However, 10B5 M
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endothelin-1 failed to produce any significant increases in the CAMP concentration of isolated glands (10 t 6 pM/mg protein, n = 5), compared with controls. DISCUSSION
Our previous experiment (23) revealed that the TCAprecipitable glycoconjugates from isolated submucosal glands represent mucus glycoprotein. Therefore, the present study indicates that endothelin induces mucus glycoprotein secretion from feline tracheal isolated submucosal glands, inducing a [Ca2+]i rise in the acinar cells, whereas endothelin inhibits mucus glycoprotein secretion from submucosal glands in the presence of epithelial cells. In the present study, we chose 2 h for the collection periods as in our previous experiments (18, 23, 24), and it is possible that transient increases induced by endothelin-1 were averaged out. Endothelin may produce a glandular contraction and a resultant initial and transient mucin secretion as do methacholine and substance P (24, 25). However, since it is known that epithelium does not have the inhibitory action on the glandular contraction (23)) the increase in glycoprotein secretion for the 2-h collection period represents mainly the secretion from secretory cells in the submucosal glands. Muscarinic, a- and ,&adrenergic antagonists, and indomethacin all failed to alter the endothelin-evoked mucus glycoprotein secretion from isolated glands. These findings indicate that endothelin stimulates its own receptors of secretory cells inducing a mucus glycoprotein secretion, although some effects of endothelin on the bronchopulmonary system are known to be due to cyclooxygenase products (7, 21). In ferret airways, endothelin evokes smooth muscle contraction through the activation of its own receptors (8), and endothelin-1 receptors have been found in human and rat bronchial smooth muscle cells (12, 29). The present study suggests that stimulus-secretion coupling in an endothelin-evoked mucus glycoprotein secretion involves Ca ions as shown in other tissues (6, 8, 27, 28, 30, 33), since it produced a rise in the [ Ca2+]; of acinar cells and failed to alter the [cAMP]i levels of isolated glands. The removal of Ca2+ from the medium abolished both the endothelin-evoked mucus glycoprotein secretion and the [Ca”‘] rise in the present study, indicating that the endothelin-evoked rise in [Ca2+]i is mainly due to a Ca2+ influx from the extracellular solution. The Ca2+ influx is though to be more important in the mucus glycoprotein secretion from airway submucosal glands than the Ca2+ release from intracellular storages (5), as in other exocrine cells (15, 19). In contrast, in the tracheal explants or isolated glands with cultured epithelial cells, endothelin produced significant reductions in the glycoconjugate secretion from submucosal glands. Although the cultured epithelial cells consisted mainly of basal cells (different from normal epithelium), they showed fundamentally a similar action in submucosal gland secretion to that in airway explants as well as in the case of epithelial ion transport (32). Since airway epithelium has been implied to have an inhibitory action on mucus secretion evoked by secretagogues from submucosal glands (18), it is possible that the decreases in the mucus glycoprotein secretion are
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due to an epithelial inhibitory action stimulated by endothelin. Indomethacin partially inhibited the endothelin-induced reductions in the glycoconjugate secretion from tracheal explants but did not do so in the isolated glands with cultured epithelial cells, suggesting that cyclooxygenase products of arachidonates from sources other than epithelium have, in part, a role in the inhibitory action in the tracheal explants. Presumably, prostacyclin released from the vascular endothelial cells plays a role in the reduction of the glycoconjugate secretion, since OP-41483, a prostaglandin I2 analogue, induced a slight but significant reduction in the glycoconjugate secretion from isolated glands and also since endothelin is known to stimulate prostacyclin release from vascular endothelium in other tissues (2). On the other hand, since epithelial cells have an inhibitory action on the mucus glycoprotein secretion from isolated submucosal glands even after treatment with indomethacin, epithelium secretes an inhibitory factor other than prostaglandins. Our previous experiment (11, 18) implied the presence of a factor inhibitory to mucus secretion, which is derived from epithelium but differs from the products of arachidonates. The present experiment, which showed the diverse secretory responses to endothelin-1 in the presence or absence of epithelial cells, confirmed the presence of an epithelial factor inhibitory to the mucus glycoprotein secretion from airway submucosal glands. Such an inhibitory factor release evoked by endothelin may represent an autoregulation because epithelium can secrete endothelin (1, 10,12). Since the definite chemical nature of this factor has not yet been determined, the possibility that a metabolite of endothelin produced by the epithelial cells was responsible for the inhibition of mucin secretion from isolated glands remained unresolved. From the present findings, however, one can speculate that endothelin does inhibit the mucus secretion from submucosal glands in normal and intact airways, whereas it does induce mucus hypersecretion in diseased airways with decreased, impaired or absent epithelial cells. In conclusion, the present study showed that endothelin produces a mucus glycoprotein secretion from airway submucosal glands, mediating intracellular Ca ions as a second messenger. Further, it is possible that endothelin stimulates an inhibitory factor release from airway epithelial cells. We acknowledge Dr. Ronald Scott for suggestions concerning English and K. Shibuya for typing the manuscript. This study was supported by Grant-in-Aid (No. 01570422) from the Ministry of Education, Science, and Culture, Japan. Address for reprint requests: T. Takishima, First Dept. of Internal Medicine, Tohoku Univ. School of Medicine, l-1 Seiryo-machi, Aobaku, Sendai 980, Japan. Received
1 May
1991; accepted
in final
form
12 September
1991.
REFERENCES 1. Black, P. N., M. A. Ghatei, K. Takahashi, Watt, T. Krausz, and S. R. Bloom. Formation cultured airway epithelial cells. FEBS Lett. 255: 2. De Nucci, G., R. Thomas, P. D’Orleans-Juste, C. Walder, T. D. Warne, and J. R. Vane. circulating endothelin are limited by its removal
D. Brethertonof endothelin by 129-132, 1989. E. Antunes, Pressor effects of in the pulmonary
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3.
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circulation and by the release of prostacyclin and endotheliumderived relaxing factor. Proc. N&Z. Acad. Sci. USA 85: 9797-9800, 1988. Gespach, C., Y. Cherel, and G. Rosselin. Development of sensitivity to CAMP-inducing hormones in the rat stomach. Am. J. PhysioZ. 247 (Gastrointest. Liver Physiol. 10): G231-G239, 1984. Grynkiewicz, G., M. Poienie, and R. Y. Tsien. A new generation of Ca2+ indicators with greatly improved fluorescence properties. J. Biol. Chem. 260: 3440-3450, 1985. Ishihara, H., S. Shimura, M. Sato, T. Masuda, N. Ishide, H. Sasaki, T. Sasaki, and T. Takishima. Intracellular calcium concentration of acinar cells in feline tracheal submucosal glands. Am. J. Physiol. 259 (Lung CeZl MOL. Physiol. 3): L345-L350, 1990. Kelly, R. A., H. Eid, B. K. Kramer, M. O’Neill, B. T. Liang, M. Reers, and T. W. Smith. Endothelin enhances the contractile responsiveness of adult rat ventricular myocytes to calcium by a pertussis toxin-sensitive pathway. J. CZin. Inuest. 86: 1164-1171, 1990. Lagente, V., P. E. Chabrier, J. -M. Memcia-Huerta, and P. Braquet. Pharmacological modulation of the bronchopulmonary action of the vasoactive peptide, endothelin, administered by aerosol in the guinea pig. Biochem. Biophys. Res. Commun. 158: 625632,1989. Lee, H-K., G. D. Leikauf, and N. Sperelakis. Electromechanical effects of endothelin on ferret bronchial and tracheal smooth muscle. J. Appl. Physiol. 68: 417-420, 1990. Lowry, 0. H., N. J. Rosenbrough, A. L. Farr, and R. J. Randall. Protein measurement with the folin phenol reagent. J. Biol. Chem. 193: 263-275,195l. MacCumber, M. W., C. A. Ross, B. M. Glaser, and S. H. Snyder. Endothelin: visualization of mRNAs by in situ hybridization provides evidence for local action. Proc. N&Z. Acud. Sci. USA 86: 7285-7289, 1989. Masuda, T., S. Shimura, H. Ishihara, M. Sato, T. Sasaki, Y. Andoh, H. Sasaki, and T. Takishima. Cultured epithelial cells inhibit mucus glycoprotein secretion from isolated submucosal glands (Abstract). Am. Reu. Respir. Dis. 141: A911, 1990. Mattoli, S., M. Mezzetti, G. Riva, L. Allergra, and A. Fasoli. Specific binding of endothelin on human bronchial smooth muscle cells in culture and secretion of endothelin-like material from bronchial epithelial cells. Am. J. Respir. CeZZ Mol. BioZ. 3: 145-151, 1990. Ninomiya, H., Y. Uchida, M. Saotome, A. Nomura, and S. Hasegawa. Histamine release from guinea pig mast cells in endothelin induced tracheal contraction (Abstract). Am. Reu. Respir. Dis. 139: A118, 1989. Nomura, A., Y. Uchida, M. Kameyama, M. Saotome, K. Oki, and S. Hasegawa. Endothelin and bronchial asthma. Luncet 2: 747-748,1989. Putney, J. W., Jr. Muscarinic, alpha-adrenergic and peptide receptors regulate the same calcium influx sites in the parotid gland. J. Physiol. Lond. 268: 139-149, 1977. Reid, L. Measurement of the bronchial mucous gland layer: a diagnostic yardstick in chronic bronchitis. Thorax 15: 132-141, 1960. Rubanyi, G. M., and P. M. Vanhoutte. Hypoxemia releases a vasoconstrictor substance from the canine vascular endothelium. J. Physiol. Lond. 364: 45-46, 1985. Sasaki, T., S. Shimura, H. Sasaki, and T. Takishima. Effect of epithelium on mucus secretion from feline tracheal submucosal glands. J. Appl. Physiol. 66: 764-770, 1989.
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19. Sato, K., and F. Sato. Role of calcium in cholinergic and adrenergic mechanisms of eccrine sweat secretion. Am. J. Physiol. 241: (Cell Physiol. 10): Cll3-C120, 1981. 20. Scanlon, M., D. A. Williams, and F. S. Fay. A Ca2+-insensitive form of Furaassociated with polymorphonuclear leukocytes. J. BioZ. Chem. 262: 6308-6312, 1987. 21. Schumacher, W. A., T. E. Steinbacher, G. T. Allen, and M. L. Ogletree. Role of thromboxane receptor activation in the bronchospastic response to endothelin. Prostuglundins 40: 71-79, 1990. 22. Shimura, S. Methods for the morphological study of tracheal and bronchial glands. In: Models of Lung Disease. Microscopy and Structural Methods, edited by J. Gil. New York: Dekker, 1990, p. 309-358. (Lung Biol. Health Dis. Ser.) 23. Shimura, S., T. Sasaki, K. Ikeda, K. Yamauchi, H. Sasaki, and T. Takishima. Direct inhibitory action of glucocorticoid on glycoconjugate secretion from airway submucosal glands. Am. Reu. Respir. Dis. 141: 1044-1049, 1990. 24. Shimura, S., T. Sasaki, H. Okayama, H. Sasaki, and T. Takishima. Effect of substance P on mucus secretion of isolated submucosal gland from feline trachea. J. Appl. Physiol. 63: 646653, 1987. 25. Shimura, S., T. Sasaki, H. Sasaki, and T. Takishima. Contractility of isolated single submucosal gland from trachea. J. Appl. PhrysioZ. 60: 1237-1247, 1986. 26. Springall, D. R., P. H. Howarth, H. Counihan, R. Djukanovic, S. T. Holgate, and J. M. Polak. Endothelin immunoreactivity of airway epithelium in asthmatic patients. Luncet 337: 697-701, 1991. 27. Stojilkovic, S. S., F. Merelli, T. Iida, L. Z. Krsmanovic, and K. J. Catt. Endothelin stimulation of cytosolic calcium and gonadotropin secretion in anterior pituitary cells. Science Wash. DC 248: 1663-1666,199O. 28. Takuwa, Y., Y. Kasuya, N. Takuwa, M. Kudo, M. Yanagisawa, K. Goto, T. Masaki, and K. Yamashita. Endothelin receptor is coupled to phospholipase C via a pertussis toxininsensitive guanine nucleotide-binding regulatory protein in vascular smooth muscle cells. J. CZin. Inuest. 85: 653-658, 1990. 29. Turner, N. C., R. F. Power, J. M. Polak, S. R. Bloom, and C. T. Dollery. Endothelin-induced contraction of tracheal smooth muscle and identification of specific endothelin binding sites in the trachea of the rat. Br. J. Phurmucol. 98: 361-366, 1989. 30. Uchida, Y., H. Ninomiya, M. Saotome, A. Nomura, M. Ohtsuka, M. Yanagisawa, K. Goto, T. Masaki, and S. Hasegawa. Endothelin, a novel vasoconstrictor, as potent bronchoconstrictor. Eur. J. Phurmucol. 154: 227-228, 1988. 31. Umemura, S., D. D. Smyth, and W. P. Pettinger. cu2-Adrenoceptor stimulation and cellular CAMP levels in microdissected rat glomeruli. Am. J. Physiol. 250 (Renal Fluid Electrolyte Physiol. 19): F103-F108,1986. 32. Widdicombe, J. H. Use of cultured airway epithelial cells in studies of ion transport. Am. J. Physiol. 258 (Lung Cell. Mol. Physiol. 2): L13-L18, 1990. 33. Word, R. A., K. E. Kamm, J. T. Stull, and M. L. Casey. Endothelin increases cytoplasmic calcium and myosin phosphorylation in human myometrium. Am. J. Obstet. Gynecol. 162: 11031108,199O. 34. Yanagisawa, M., H. Kurihara, S. Kimura, Y. Tomobe, M. Kobayashi, Y. Mitsui, Y. Yazaki, K. Goto, and T. Msaki. A novel potent vasoconstrictor peptide produced by vascular endothelial cells. Nature Lond. 332: 411-415, 1988.
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secretion
S. SHIMURA, H. ISHIHARA, M. SATOH, T. MASUDA, N. NAGAKI, H. SASAKI, AND T. TAKISHIMA First Department of Internal Medicine, Tohoku University School of Medicine, Sendai 980, Japan Shimura, S., H. Ishihara, M. Satoh, T. Masuda, N. Nagaki, H. Sasaki, and T. Takishima. Endothelin regulation of mucus glycoprotein secretion from feline tracheal submucosal glands. Am. J. Physiol. 262 (Lung Cell. Mol. Physiol. 6): L208-L213, 1992.-We examined the effects of endothelin on both the trichloroacetic acid precipitable 3H-labeled glycoconjugate release and intracellular Ca2+ concentration ( [Ca”+]i]) measured by the usage of fura- in submucosal glands isolated from feline trachea. Endothelin-1 produced a significant increase in glycoconjugate release from the isolated glands in a dose-dependent fashion, reaching a response of 161% of the control at 10m6 M. Atropine, propranolol, phentolamine, or indomethacin did not produce any significant alterations in the ET-l-evoked glycoconjugate secretion from the isolated glands. In contrast, in tracheal explants which contained epithelium, ET-l produced a significant reduction in the glycoconjugate secretion in a dose-dependent fashion, reaching a response of 59% of the control at low6 M. In the presence of cultured epithelial cells, ET-l also produced a significant reduction in the glycoconjugate secretion from isolated glands. In isolated glands, ET- 1 produced a sustained increase in the [ Ca”‘] i which was abolished by the removal of Ca2+ from the medium or by the presence of cultured epithelial cells. Pretreatment with indomethacin failed to alter the epithelial inhibitory action evoked by ET-l in both the glycoconjugate secretion and the [Ca2+]; in isolated glands. ET-2 and ET-3 failed to produce significant alterations in the glycoconjugate secretion or [Ca2+];. These findings indicate 1) that ET-l induces mucus glycoprotein secretion via a Ca2+ influx and 2) that it possibly augments the an epithelial action inhibitory to the mucus glycoprotein secretion from airway submucosal glands. endothelin-I, tration
feline trachea,
intracellular
calcium
ion concen-
is a newly discovered polypeptide secreted from the vessel wall endothelium in response to various stimuli such as hypoxemia (17,34). Further, the mRNAs for endothelin have been shown by in situ hybridation in the airway epithelial cells (10). Endothelin-1 and human endothelin-3 are released from cultured canine, porcine, and human tracheobronchial epithelial cells (1, 12). Since airway mucosa consists of a large number of small vessels, smooth muscles, submucosal glands, connective tissue, and epithelium, it is possible that endothelin is locally produced in the airway mucosa and affects smooth muscle, glandular, and epithelial functions. Recently, endothelin was found to constrict guinea pig, ferret, and rat airway smooth muscle (7, 8, 13, 21, 29, 30) and is implied to act via the release of cyclooxygenase products of arachidonates or by the activation of the calcium channel. Thus endothelin may play some role in the pathophysiological aspects of various respiratory diseasesincluding bronchial asthma (14, 26). Nomura et al. (14) have reported that a patient showed bronchial washing fluid with a raised endothelin-immu-
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noreactive level during status asthmatics, suggesting that endothelin plays a part in bronchoconstriction in bronchial asthma. Further, Ninomiya et al. (13) have observed that endothelin stimulates the mast cells to release histamine via a Ca2+ influx and plays a role in guinea pig tracheal constriction. Although both mucus hypersecretion and bronchoconstriction are characteristic of asthma, as well as of other pulmonary diseases, to our knowledge, the effect of endothelin on airway mucus secretion has not been examined. We have been successful in isolating submucosal glands from the trachea (22, 25), which play a principal role in mucus secretion (16). The purpose of the present study is to examine the effects of endothelin both on the mucus glycoprotein secretion and on the intracellular Ca2+ concentration ([Ca”‘];) of acinar cells in isolated feline tracheal submucosal glands, since a number of experiments have implicated [Ca2+]i as a second messenger in other tissues stimulated by endothelin (6, 8, 27, 28, 30, 33). METHODS Isolation of submucosal glands. The tracheae were removed from 20 adult cats (3.0-4.5 kg of body wt) under an anesthesia of intramuscular ketamine hydrochloride and intravenous thiopental sodium (each 30 mg/kg body wt). Each trachea was cut into rings 3-4 cm long and fixed with pins, posterior (membranous) wall side up, in Krebs-Ringer bicarbonate (KRB) solution at 4°C. Fresh unstained submucosal glands were mechanically isolated under a stereomicroscope using two pairs of sharpened tweezers and microscissors as described previously (22, 25). The entire procedure was performed in a KRB solution which contained (in mM) 125 NaCl, 5 KCl, 1.2 MgC12, 1.0 CaC12, 25 NaHC03, 1.2 NaH2P04, and 11 glucose, gassed with 5% CO,95% 02, pH 7.4, at 20°C. Measurement of 3H-labeled glycoconjugate release. Measurement of 3H-labeled mucus glycoconjugate release from isolated submucosal glands or from tracheal explants was made by a previously reported method (12, 13, 14). Membranous portions of the trachea were fragmented into 3 X 3 mm explants which contained surface epithelium and 5-10 submucosal glands (22). Five to 10 isolated glands or one tracheal explant were incubated for 16 h with 2 ml of medium 199 which contained D-[6“Hlglucosamine hydrochloride (2.5 &i/ml, specific activity 2740 &i/mmol, Amersham Japan, Tokyo), penicillin (100 U/ml) and streptomycin (100 pg/ml) in a controlled atmosphere chamber. The chamber was constantly gassed with 5% C02-55% N240%02 at 37°C. After being washed with the culture medium, samples were allowed to further incubate at 37°C for two successive 2-h periods (periods I and II) in 2 ml of the culture medium without a radiolabeled precursor. At the end of each period, the culture media was harvested and precipitated with 10% trichloroacetic acid (TCA) and 1% phosphotungstic acid (PTA) at 4°C overnight. The precipitated radioactivity was measured by a liquid scintillation counter (LSC-1000, Aloka, Tokyo). The effect of endothelin or other drugs on the release
0 1992 the American
Physiological
Society
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of 3H-labeled mucus glycoconjugate was determined by adding these to the culture medium at the beginning of period II. Each antagonist was added to the medium 15 min before the addition of the agonist. The ratio of precipitated radioactivity (dpm) from period II to that from period I was determined for each sample and was taken as the secretory index. The effects of the pharmacological agents were determined by comparing the secretory indices of the manipulated samples with those of the matched, unmanipulated control samples. [Ca2+/i measurement. The [Ca2+]; of acinar cells in isolated submucosal glands was measured by a method previously reported (5). The isolated submucosal glands were incubated at room temperature for 2-4 h in a loading solution, which contained 5 PM acetoxymethyl ester of fura(fura-2/AM) and 0.2% of the detergent, Cremophor EL, to increase the solubility of the fura-2/AM. Cremophor (0.2%) itself does not alter the secretory response of isolated glands (5). After the fura- loading, the glands were rinsed with KRB three times at intervals of 5 min (for IO-20 min) to wash out any fura-2/AM attached to the glands. The fura- fluorescence was determined by an apparatus consisting of an inverted microscope (TMD, Nikon, Tokyo) with an objective lens (Flour, x40, Nikon), a xenon lamp, and a photomultiplier for measurement of the furafluorescence of live tissue. The time course of the fura- signal was continuously recorded with a fluorimeter (CAM-220, Japan Spectroscopic, Tokyo) at 37°C in KRB solution. After furaloading, each gland was put in a Rose chamber (0.25 ml in volume, RKI, Ikemoto, Tokyo) on the stage of the inverted microscope and was perfused with warmed (37°C) KRB solution gassed with 5% C02-95% O2 at a constant rate. The microscopic field was adjusted to the acinar portion of the gland at a magnification of x400. The excitation light was automatically alternated between 340 and 380 nm at intervals of 100 Hz, and the fluorescence from the glands was collected by the photomultiplier through a 510 nm filter. The ratio of the fluorescence intensity due to 340 nm (F340) and to that due to 380 nm (F380) was calculated and recorded as a function of time. We used different gland samples for each measurement of the [Ca”+];, since the decrease in the fluorescence intensity was too large to repeat the measurement in the same sample (5). The ratio was then converted to [Ca”+]; using the method of Grynkiewicz et al. (4) and Scanlon et al. (20), i.e., using the equation [Ca2+]; = (R - R,in)/(R,,x - R) & S where R is the experimentally determined minimal and maximal fura- fluorescence ratio (F340/F380), & is the dissociation constant, and S is the ratio of the fluorescence at 380 nm in a Ca2+-free solution to that in a Ca2+-containing solution in the presence of ionomycin. The & was assumed to be 224 nM (4). R,,, and I&in were determined by the addition of 10s5 M ionomycin and 5 mM ethylene glycol-bis(P-aminoethyl ether)-N,N,N’,N’-tetraacetic acid (EGTA), respectively. Adenosine 3’,5’-cyclic monophosphate (CAMP) measurements. After a lo-min preincubation, isolated glands were incubated at 20°C with 10v5 M isoproterenol or low6 M endothelin-1 for 30 min in a KRB solution (pH 7.4). The KRB solution contained 1% bovine serum albumin (BSA) and 0.5 mM 3isobutyl-1-methylxanthine (IBMX) as a CAMP phosphodiesterase inhibitor. The reaction was terminated by the addition of 10% TCA for measurement of CAMP concentration (3, 31). For each measurement, the solution from 20 to 30 glands was extracted with water-saturated ether. The aqueous phase was dried and kept at -20°C until the CAMP assay. Before the assay, sodium acetate buffer (pH 6.2) was added to each sample and acetylated to enhance the sensitivity for the immunoassay. The amount of CAMP was determined in duplicate by the kits for 125I radioimmunoassay (Yamasa Shoyu, Ltd., Tokyo). The precipitates were dissolved in 1 N NaOH and assayed for protein by the method of Lowry et al. (9). Results are expressed
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as picomoles of CAMP per milligram of protein. The effects of isoproterenol and endothelin-1, were compared with the control values obtained by using tissues from the same animals. Isolation of epithelial cells. Immediately after removal, the tracheae were immersed and washed in KRB solution at 4°C with penicillin (50 IU/ml), streptomycin (50 IU/ml), and gentamycin (50 pg/ml). Cartilaginous portions were fixed by pins in KRB solution at 4°C and bubbled with 5% C02-95% 02. Shallow longitudinal incisions on each specimen were made by sharpened scissors at l-mm intervals, and the epithelium was peeled by microforceps. The strips thus obtained were immersed in 4 ml of cold protease (Sigma protease XIV, 0.1% in Eagles minimal essential medium, 4°C) overnight. The digestion was stopped by the addition of fetal bovine serum (FBS, 10% vol/vol) and then filtered through a stainless mesh with holes 100 ,ccrn in diameter. The epithelial cells released by the protease were collected by centrifugation (500 g, 5 min) and again washed with Ham’s F-12 with hormone supplement. Trypan blue exclusion test showed that viability was over 95%. The epithelial cells (1 x IO”) were seeded on collagen-coated plastic Petri dishes (35 mm in diameter) and cultured in Ham’s F-12 nutrient mixture supplemented with penicillin (50 IU/ ml), streptomycin (50 IU/ml), and gentamycin (50 pg/ml), containing 10% FBS, hydrocortisone (0.36 pg/ml), transferrin insulin (IO ,pg/ml), and endothelial (O-5 dml), T3 (0.2 kdml), cell growth supplement (ECGS, 7.5 pg/ml). The epithelial cells were then incubated in a 95% air-5% CO2 humidified incubator at 37°C. Epithelial cells in a dish showed a confluent appearance 3 or 4 days after the seeding and each dish contained 5 x lo6 epithelial cells. Epithelial cells showing a confluent appearance were fixed with glutaraldehyde, embedded in Epon 812 and cut into l-pm sections. A light-microscopic examination showed that the cultured epithelial cells consisted of basal cells and a few ciliated cells with a monolayer appearance and were not contaminated with fibroblasts. We used the dishes that contained cultured epithelial cells with a confluent appearance in period II and examined the effect of epithelial cells on the glycoconjugate secretion from isolated submucosal glands. Reagents. Drugs used in the present study were endothelin1 (human/porcine), endothelin-2 (human), endothelin-3 (human/rat) (Peptide Institute, Osaka, Japan), acetyl-P-methylcholine chloride (methacholine), propranolol, phentolamine, EGTA, (Wako Pure Chemicals, Osaka), fura-2/AM (Dojin, Kumamoto, Japan), Cremophor EL (Nakarai Chemicals, Kyoto, Japan), ionomycin (Calbiochem, La Jolla, CA), FBS, Eagle’s MEM, -Ham’s F-12 (GIBCO, Grand Island, NY), atropine sulfate, protease XIV, Eagle’s MEM, hydrocortisone, transferrin, T3, insulin, ECGS, IBMX, BSA, fraction V (Sigma Chemical, St. Louis, MO), medium 199 (Flow, McLean, VA), and Aquasol 2 (New England Nuclear, Boston, MA). Prostaglandins E1, E2 and 12-analogue OP-41483 were generous gifts from Ono Pharmaceutical, Tokyo, Japan. Statistical analysis. Data are means t SE. For mean comparisons, the two-tailed paired or unpaired Student’s t test was used, and the Cochran Cox t test was used when Bartlett’s test for uniformity of variance showed it to be nonuniform. A P < 0.05 was considered statistically significant. RESULTS
Glycoconjugate secretion. Endothelin-1 produced an increase in the TCA-precipitable glycoconjugate secretion from isolated glands in a dose-dependent fashion as shown in Fig. 1. Endothelin-1 produced the responses of 104, 136, 141, 150, and 161% of control at 10-l’, lo-‘, lo-s, 10s7, and 10Y6 M, respectively. A mixture of atropine (10B6 M), propranolol (10s5 M), and phentolamine
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18Q-
** sQEI
z
MO-
%
-P.Eg
140-
i!i Q) ‘co
&
120-
lOOA
10’‘OM 10-W ET-l ET-l
10’“M ET-1
10’‘M ET-l
10’“M ET-l
10’“M Et-1
!A:
PHE
10’“M ET-1
IND
Fig. 1. Endothelin-1 (ET-l)-induced glycoconjugate secretion from feline tracheal isolated glands. ET-l produced an increase in glycoconjugate secretion in a dose-dependent fashion, which was not altered by treatment with 3 antagonists or with indomethacin. ATR, lOA M atropine; PRP, lo-” M propranolol; PHE, low5 M phentolamine, IND, lOA” M indomethacin. Means t SE of 5-10 experiments. * P < 0.05, ** P < 0.01, compared with controls.
10""M ET-l
i
10'"M ET-l
10'"M ET-l
10"M ET-l
10'"M ET-l
10’“M ET-l t IND
*
Fig. 2. Effect of endothelin-1 (ET-l) on glycoconjugate secretion from feline tracheal explants. In contrast to isolated glands, ET-1 induced a reduction in glycoconjugate secretion from tracheal explants, which was partially inhibited by indomethacin. Mean k SE of 5-9 experiments. tt P < 0.01, compared with isolated glands simulated by 10m6 M ET-l. §P < 0.05, compared with explants stimulated by 10V6 M ET1. Other symbols as in Fig. 1.
( 10m5M) did not alter the endothelin-1 ( 10s6 M)-evoked glycoconjugate secretion (163 t 8% of control, n = 6) (Fig. 1). Pretreatment with indomethacin (10V5 M) did not significantly alter endothelin-1 (10B6M) evoked glycoconjugate secretion (158 t 4% of control, n = 5) from isolated glands, as in our previous report (18). In contrast, endothelin-1 produced a significant decrease in glycoconjugate secretion from the tracheal explants in a dose-dependent fashion, reaching a response of 59 t 8% of control at 10s6 M (n = 5, P < O.Ol), as shown in Fig. 2. Pretreatment with 10m5M indomethacin partially inhibited the endothelin-1 ( 10B6M)-evoked decreases in glycoconjugate secretion from tracheal explants. This is, indomethacin significantly increased the endothelin-l-evoked response from 59 & 8% of control to 81 t 4% of control by endothelin-1 alone (P < 0.05) (Fig. 2). However, the secretory response to endothelin1 in indomethacin-treated explants was still much smaller than that evoked by endothelin-1 in isolated glands (P < 0.01) (Figs. 1 and 2), indicating that indomethacin failed to abolish the inhibition action. Since indomethacin ( l.0m5M) itself reduced glycoconjugate secretion from tracheal explant (88 t 6% of control, n = 4, P < 0.05), we used explants treated with indomethacin
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as the control for this experiment. The use of the epithelial cell-containing dish in period II produced secretory indices of 0.68 t 0.06 (n = 7), which were similar to 0.70 t 0.01 (n = 56) from corresponding samples, as was the case in our previous report (18). Hence, in the experiment of cultured epithelial cells, we used a dish with no epithelial cells in the period II as the corresponding control sample. Dry weight of isolated glands represents the amount of secretory cells in isolated glands and correlates negatively with the secretory index under the definite number of epithelial cells (11, 23). To avoid any variations due to differing sizes of glands, the secretory index in each sample was divided by the dry weight of the isolated glands (23) and compared with that of a corresponding control in the following experiment of epithelial inhibition. Endothelin-1 produced an inhibition of the glycoconjugate secretion from isolated glands in the presence of cultured epithelial cells (5 x 106) in a dose-dependent fashion, and endothelin-1 (10B6M) produced a response of 70 t 4% of corresponding control containing cultured epithelial cells (n = 9, P c O.Ol), as shown in Fig. 3. Pretreatment with 10B5 M indomethacin did not significantly alter the epithelial inhibitory action evoked by 10B6M endothelin-1 in the glycoconjugate secretion from isolated glands (74 t 6% of control, n = 4) (Fig. 3). Glycoconjugate secretion from indomethacin ( 10B5M) -treated isolated glands with cultured epithelial cells in response to endothelin-1 was still significantly smaller than that from isolated glands in response to endothelin-1 (Fig. 3). Indomethacin (10B5M) itself did not alter the glycoconjugate secretion from isolated glands with cultured epithelial cells (98 t 6% of control, n = 4). To examine the effect of extracellular Ca ions, Ca2’free KRB solution with 0.2 mM EGTA was used instead of medium 199 in periods I and II. In Ca2+-containing KRB solution, endothelin-1 (10m6M) produced a significant increase in the glycoconjugate secretion from isolated glands (143 t 8% of control, n = 5). In Ca2+-free KRB, however, there was no significant stimulation of the glycoconjugate secretion by endothelin-1 in isolated glands by 10s6M endothelin-1 (110 t 15% of control, n
- 5)
Neither endothelin-2 nor endothelin-3 significantly altered glycoconjugate secretion from isolated glands in the absence or in the presence of cultured epithelial cells. For example, endothelin-2 and endothelin-3 (10B6 M 10""M 10'"M 10'"M ET-l ET-l ET-l
10''M ET-l
10’8M ET-l
10'"M ET-l
t
IND
Fig. 3. Effect of endothelin-1 (ET-l) on glycoconjugate secretion from feline isolated glands in presence of cultured epithelial cells. ET-l produced a reduction in glycoconjugate secretion from isolated glands with culture epithelial cells in a dose-dependent fashion, and reduction was not altered even after treatment with indomethacin. Means k SE of 4-8 experiments. tt P < 0.01 compared with isolated glands alone stimulated by ET-l (10m6 M). Other symbols as in Fig. 1.
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each) induced responses of 99 t 6% (n = 5) and 103 t 180 (n = 6) of control in isolated glands, respectively, and responses of 103 t 5% (n = 5) and 98 t 7% of control (n = 5) in tracheal explants. As shown in Fig. 4, prostaglandin Fzcuproduced a significant increase in the glycoconjugate secretion from isolated glands (122 t 7% of control at lOa M, n = 5, P < 0.05), and prostaglandin 1, analogue, OP-41483, produced a significant reduction in the glycoconjugate secretion from isolated glands (86 t 4% of control at 10m5 M, n = 5, P < 0.05). Prostaglandin E1 (10m4M, n = 5) or E, (10B4 M, n = 5) did not produce any significant 80 J h I I I I I 1 alteration in the glycoconjugate secretion from isolated 0123 5 7 10min glands (Fig. 4). Fig. 6. Time courses of the [Ca2+]i in acinar cells of isolated glands [Ca2+/ in acinar cells. Endothelin-1 produced signifi(ET-l) in absence (0) and presence cant increases in [Ca2+]i in the acinar cells of isolated stimulated by 10V6 M endothelin-1 of cultured epithelial cells (0). Mean k SE of 5 experiments. ET-l glands. The time course of the [Ca2+]i rise evoked by produced an increase in [Ca2+]i without any initial transient increase, endothelin-1 showed a prolonged increase without any which was abolished in the presence of cultured epithelial cells. * P < initial transient increase. A representative example is 0.05, ** P < 0.01, compared with baseline values. t P < 0.05, t"f P < shown in Fig. 5A. At low6 M, [Ca2+]; was 120, 131, 146, 0.01, compared with those in absence of cultured epithelial cells. 158, 158, and 160% of the prior baseline value at 1, 2, 3, 180, 5, 7, and 10 min after stimulation, respectively (Fig. 6). ** Because the resting levels of [Ca2+]; varied greatly from 2s z MO68 to 210 nM among samples as in our previous experi> ment (5), the plateau level (5-10 min after stimulation) z as a percentage of the prior baseline level was used in 3 1401 the following analysis of [Ca2+]i data. As shown in Fig. ES + 4%
140
s
g120YV
*
g h zr20120 oo= 8s g gp100 cJ&
loo&
80 I
T
PGFna
klr'
IL1
PGE,
PGEz
A *
PGlz
Fig. 4. Effects of prostaglandins Fza (PGF2,, 10m4 M)), E, (PGE1, 10m5 M), E2 (PGE,, 10m5 M), and prostaglandin 12 analogue (OP-41483, 10m4 M) on glycoconjugate secretion from feline isolated glands. PGF2, produced a slight increase in the glycoconjugate secretion from isolated glands, whereas PGIz produced a slight but significant decrease. Means t SE of 5 experiments. P < 0.05, compared with controls.
[Ca++li
A
(nM)
-y+--
1::: 50
t
200 150 100
50
t lmin
Fig. 5. Representative examples of time course of [Ca2+]; in acinar cells of isolated glands stimulated by 10V6 M endothelin-1. Endothelin1 produced an increase in [Ca2+]; without the initial transient rises as shown in A. In contrast, in presence of cultured epithelial cells, it failed to produce an increase in [Ca2+]; as shown in B. Arrows, beginning of stimulation by endothelin1.
10""M ET-1
10"M ET-1
10'"M ET-1
10"M ET-1
10'"M ET-l
1O'"M 10'"M ET-1 ET-1 t t ATR IND PRP PHE Fig. 7. Endothelin-1 (ET-l) induced [Ca2+]i rise in acinar cells of isolated glands. [Ca2+]i rise is expressed as a % plateau level of before baseline levels in each sample. Mean k SE of 4-8 experiments. ET-l produced an increase in the [Ca”‘] in a dose-dependent fashion, which was not affected bY treatment with indomethacin (lO-5 M). Symbols as in Fig. 1.
7, the endothelin-l-evoked [Ca2+]i rise was dose dependent at concentrations of 10-l’ to 10B6 M. Removal of Ca2+ from the medium abolished the endothelin-1 (lOa M)-evoked increases in [Ca2+]i (98 t 10% of the baseline value, n = 5). To understand the effect of epithelial cells on the [Ca2+]i of acinar cells in isolated glands, we measured the [Ca2+]i in isolated glands 0.3 x 0.5 mm in size placed in the center of the Rose chamber containing cultured epithelial cells on collagen-coated plastic disk (4 x 106/ chamber). Endothelin-1 did not produce any significant alterations in [Ca”‘] of acinar cells in isolated glands in the presence of cultured epithelial cells, and 10B6 M endothelin-1 produced a response of 96 t 5% of the baseline value in isolated glands in the presence of cultured epithelial cells (n = 5) (Figs. 5A and 6). CAMP concentration of isolated glands. Isoproterenol (10Y5 M) produced significant increases in the CAMP concentration of isolated glands (46 t 5 pM/mg protein, n = 5), compared with untreated control glands (8 t 2 pM/mg protein, n = 3, P < 0.01). However, 10B5 M
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endothelin-1 failed to produce any significant increases in the CAMP concentration of isolated glands (10 t 6 pM/mg protein, n = 5), compared with controls. DISCUSSION
Our previous experiment (23) revealed that the TCAprecipitable glycoconjugates from isolated submucosal glands represent mucus glycoprotein. Therefore, the present study indicates that endothelin induces mucus glycoprotein secretion from feline tracheal isolated submucosal glands, inducing a [Ca2+]i rise in the acinar cells, whereas endothelin inhibits mucus glycoprotein secretion from submucosal glands in the presence of epithelial cells. In the present study, we chose 2 h for the collection periods as in our previous experiments (18, 23, 24), and it is possible that transient increases induced by endothelin-1 were averaged out. Endothelin may produce a glandular contraction and a resultant initial and transient mucin secretion as do methacholine and substance P (24, 25). However, since it is known that epithelium does not have the inhibitory action on the glandular contraction (23)) the increase in glycoprotein secretion for the 2-h collection period represents mainly the secretion from secretory cells in the submucosal glands. Muscarinic, a- and ,&adrenergic antagonists, and indomethacin all failed to alter the endothelin-evoked mucus glycoprotein secretion from isolated glands. These findings indicate that endothelin stimulates its own receptors of secretory cells inducing a mucus glycoprotein secretion, although some effects of endothelin on the bronchopulmonary system are known to be due to cyclooxygenase products (7, 21). In ferret airways, endothelin evokes smooth muscle contraction through the activation of its own receptors (8), and endothelin-1 receptors have been found in human and rat bronchial smooth muscle cells (12, 29). The present study suggests that stimulus-secretion coupling in an endothelin-evoked mucus glycoprotein secretion involves Ca ions as shown in other tissues (6, 8, 27, 28, 30, 33), since it produced a rise in the [ Ca2+]; of acinar cells and failed to alter the [cAMP]i levels of isolated glands. The removal of Ca2+ from the medium abolished both the endothelin-evoked mucus glycoprotein secretion and the [Ca”‘] rise in the present study, indicating that the endothelin-evoked rise in [Ca2+]i is mainly due to a Ca2+ influx from the extracellular solution. The Ca2+ influx is though to be more important in the mucus glycoprotein secretion from airway submucosal glands than the Ca2+ release from intracellular storages (5), as in other exocrine cells (15, 19). In contrast, in the tracheal explants or isolated glands with cultured epithelial cells, endothelin produced significant reductions in the glycoconjugate secretion from submucosal glands. Although the cultured epithelial cells consisted mainly of basal cells (different from normal epithelium), they showed fundamentally a similar action in submucosal gland secretion to that in airway explants as well as in the case of epithelial ion transport (32). Since airway epithelium has been implied to have an inhibitory action on mucus secretion evoked by secretagogues from submucosal glands (18), it is possible that the decreases in the mucus glycoprotein secretion are
GLAND
SECRETION
due to an epithelial inhibitory action stimulated by endothelin. Indomethacin partially inhibited the endothelin-induced reductions in the glycoconjugate secretion from tracheal explants but did not do so in the isolated glands with cultured epithelial cells, suggesting that cyclooxygenase products of arachidonates from sources other than epithelium have, in part, a role in the inhibitory action in the tracheal explants. Presumably, prostacyclin released from the vascular endothelial cells plays a role in the reduction of the glycoconjugate secretion, since OP-41483, a prostaglandin I2 analogue, induced a slight but significant reduction in the glycoconjugate secretion from isolated glands and also since endothelin is known to stimulate prostacyclin release from vascular endothelium in other tissues (2). On the other hand, since epithelial cells have an inhibitory action on the mucus glycoprotein secretion from isolated submucosal glands even after treatment with indomethacin, epithelium secretes an inhibitory factor other than prostaglandins. Our previous experiment (11, 18) implied the presence of a factor inhibitory to mucus secretion, which is derived from epithelium but differs from the products of arachidonates. The present experiment, which showed the diverse secretory responses to endothelin-1 in the presence or absence of epithelial cells, confirmed the presence of an epithelial factor inhibitory to the mucus glycoprotein secretion from airway submucosal glands. Such an inhibitory factor release evoked by endothelin may represent an autoregulation because epithelium can secrete endothelin (1, 10,12). Since the definite chemical nature of this factor has not yet been determined, the possibility that a metabolite of endothelin produced by the epithelial cells was responsible for the inhibition of mucin secretion from isolated glands remained unresolved. From the present findings, however, one can speculate that endothelin does inhibit the mucus secretion from submucosal glands in normal and intact airways, whereas it does induce mucus hypersecretion in diseased airways with decreased, impaired or absent epithelial cells. In conclusion, the present study showed that endothelin produces a mucus glycoprotein secretion from airway submucosal glands, mediating intracellular Ca ions as a second messenger. Further, it is possible that endothelin stimulates an inhibitory factor release from airway epithelial cells. We acknowledge Dr. Ronald Scott for suggestions concerning English and K. Shibuya for typing the manuscript. This study was supported by Grant-in-Aid (No. 01570422) from the Ministry of Education, Science, and Culture, Japan. Address for reprint requests: T. Takishima, First Dept. of Internal Medicine, Tohoku Univ. School of Medicine, l-1 Seiryo-machi, Aobaku, Sendai 980, Japan. Received
1 May
1991; accepted
in final
form
12 September
1991.
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19. Sato, K., and F. Sato. Role of calcium in cholinergic and adrenergic mechanisms of eccrine sweat secretion. Am. J. Physiol. 241: (Cell Physiol. 10): Cll3-C120, 1981. 20. Scanlon, M., D. A. Williams, and F. S. Fay. A Ca2+-insensitive form of Furaassociated with polymorphonuclear leukocytes. J. BioZ. Chem. 262: 6308-6312, 1987. 21. Schumacher, W. A., T. E. Steinbacher, G. T. Allen, and M. L. Ogletree. Role of thromboxane receptor activation in the bronchospastic response to endothelin. Prostuglundins 40: 71-79, 1990. 22. Shimura, S. Methods for the morphological study of tracheal and bronchial glands. In: Models of Lung Disease. Microscopy and Structural Methods, edited by J. Gil. New York: Dekker, 1990, p. 309-358. (Lung Biol. Health Dis. Ser.) 23. Shimura, S., T. Sasaki, K. Ikeda, K. Yamauchi, H. Sasaki, and T. Takishima. Direct inhibitory action of glucocorticoid on glycoconjugate secretion from airway submucosal glands. Am. Reu. Respir. Dis. 141: 1044-1049, 1990. 24. Shimura, S., T. Sasaki, H. Okayama, H. Sasaki, and T. Takishima. Effect of substance P on mucus secretion of isolated submucosal gland from feline trachea. J. Appl. Physiol. 63: 646653, 1987. 25. Shimura, S., T. Sasaki, H. Sasaki, and T. Takishima. Contractility of isolated single submucosal gland from trachea. J. Appl. PhrysioZ. 60: 1237-1247, 1986. 26. Springall, D. R., P. H. Howarth, H. Counihan, R. Djukanovic, S. T. Holgate, and J. M. Polak. Endothelin immunoreactivity of airway epithelium in asthmatic patients. Luncet 337: 697-701, 1991. 27. Stojilkovic, S. S., F. Merelli, T. Iida, L. Z. Krsmanovic, and K. J. Catt. Endothelin stimulation of cytosolic calcium and gonadotropin secretion in anterior pituitary cells. Science Wash. DC 248: 1663-1666,199O. 28. Takuwa, Y., Y. Kasuya, N. Takuwa, M. Kudo, M. Yanagisawa, K. Goto, T. Masaki, and K. Yamashita. Endothelin receptor is coupled to phospholipase C via a pertussis toxininsensitive guanine nucleotide-binding regulatory protein in vascular smooth muscle cells. J. CZin. Inuest. 85: 653-658, 1990. 29. Turner, N. C., R. F. Power, J. M. Polak, S. R. Bloom, and C. T. Dollery. Endothelin-induced contraction of tracheal smooth muscle and identification of specific endothelin binding sites in the trachea of the rat. Br. J. Phurmucol. 98: 361-366, 1989. 30. Uchida, Y., H. Ninomiya, M. Saotome, A. Nomura, M. Ohtsuka, M. Yanagisawa, K. Goto, T. Masaki, and S. Hasegawa. Endothelin, a novel vasoconstrictor, as potent bronchoconstrictor. Eur. J. Phurmucol. 154: 227-228, 1988. 31. Umemura, S., D. D. Smyth, and W. P. Pettinger. cu2-Adrenoceptor stimulation and cellular CAMP levels in microdissected rat glomeruli. Am. J. Physiol. 250 (Renal Fluid Electrolyte Physiol. 19): F103-F108,1986. 32. Widdicombe, J. H. Use of cultured airway epithelial cells in studies of ion transport. Am. J. Physiol. 258 (Lung Cell. Mol. Physiol. 2): L13-L18, 1990. 33. Word, R. A., K. E. Kamm, J. T. Stull, and M. L. Casey. Endothelin increases cytoplasmic calcium and myosin phosphorylation in human myometrium. Am. J. Obstet. Gynecol. 162: 11031108,199O. 34. Yanagisawa, M., H. Kurihara, S. Kimura, Y. Tomobe, M. Kobayashi, Y. Mitsui, Y. Yazaki, K. Goto, and T. Msaki. A novel potent vasoconstrictor peptide produced by vascular endothelial cells. Nature Lond. 332: 411-415, 1988.
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