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Tachykinin receptors mediating airway macromolecular secretion
Life Sciences, Vol. 48, pp. 1609-1618 Printed in the U.S.A.
TACHYKININ
RECEPTORS
MEDIATING
Pergamon Press
AIRWAY
MACROROLECULAR
SECRETION
Steven E. Gentry Montefiore Hospital, University of Pittsburgh School of Medicine, 3459 Fifth Avenue, Pittsburgh, PA 15213 (Received in final form February 13, 1991) Summa r¥ Three tachykinin receptor types, termed NKI, NK2, and NK3, can be distinguished by the relative potency of various peptides in eliciting tissue responses. Airway macromolecular secretion is stimulated by the tachykinin substance P (SP). The purposes of this study were to determine the tachykinin receptor subtype responsible for this stimulation, and to examine the possible involvement of other neurotransmitters in mediating this effect. Ferret tracheal explants maintained in organ culture were labeled with 3H-glucosamine, a precursor of high molecular weight glycoconjugates (HMWG) which are released by airway secretory cells. Secretion of labeled HMWG then was determined in the absence and presence of the tachykinins SP, neurokinin A (NKA), neurokinin B (NKB), physalaemin (PHY), and eledoisin (ELE). All the tachykinins tested stimulated HMWG release to an approximately equal degree. Stimulation was concentration-related, with log concentrations giving halfmaximal effects (EC50) as follows: SP -9.47, NKA -7.37, NKB -5.98, P H Y - 8 . 0 8 , and ELE -7.68. This rank order of potency (SP > PHY _> ELE _> NKA > NKB) is most consistent with NKI receptors. To evaluate the possible contribution of other mediators, tachykinin stimulation was examined in the presence of several receptor blockers. The potency of SP was not diminished by pretreatment with atropine, propranolol, or chlorpheniramine, and atropine actually increased the magnitude of the secretc!y response. The SP receptor antagonist [D-Argl, D-Phe D-TrpV'9,Leu11]-SP blocked SP-induced secretion. These findings indicate that SP is a potent stimulus of airway m a c r o m o l e c u l a r secretion. This effect occurs through the action of NKI receptors, and is not dependent upon cholinergic, ~-adrenergic, or H-I histamine receptors. The facilitation by atropine of SP stimulation suggests the existence of a mechanism of cholinergic inhibition of SP-induced stimulation. Substance P (SP) is an undecapeptide which is widely distributed throughout the central and peripheral nervous system, and which belongs to a family of peptides called the tachykinins. Substance P has been implicated as a possible n e u r o t r a n s m i t t e r of the non-adrenergic, non-cholinergic innervation of the lung (i). In the lung, fibe[s containing SP have been d e m o n s t r a t e d in and 0024-3205/91 $3.00 +.00 Copyright (c) 1991 Pergamon Press plc
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around the surface epithelium, around blood vessels, and within airway smooth muscle (i). Functionally, SP has been shown to cause airway smooth muscle contraction, increased microvascular permeability, and tracheobronchial vasodilation (i). In addition, SP is a potent stimulant for the release of macromolecules from airway secretory cells (2-4). Recently, the presence of multiple types of receptors for tachykinins has been postulated (5,6). By comparing the relative potency of tachykinins in bioassay (7) and binding (8) studies, three receptor subtypes have been identified. These receptor subtypes, termed NKI, NK2, and NK3, respond p r e f e r e n t i a l l y to SP, neurokinin A (NKA), and neurokinin B (NKB), respectively. The purpose of this study was to determine the tachykinin receptor subtype mediating macromolecular secretion in the airways. Furthermore, the possible contribution of other n e u r o t r a n s m i t t e r s and mediators to tachykinin-induced secretion was examined. Methods and Materials Tissue preparation and culture Adult neutered female ferrets were killed by a lethal intraperitoneal injection of sodium pentobarbital. The trachea was removed aseptically, trimmed, opened along the posterior membranous portion, and pinned to a sterile dissecting tray filled with cold (4°C) p h o s p h a t e - b u f f e r e d saline. The trachea was cut into fullthickness explants (3x5 mm), which were transferred to siliconized 24-well culture plates, three to four explants per well. One ml of culture medium then was added to each well. The medium, termed D M H / B S A medium, was a modification of that used in previous studies (9), and consisted of a 50:50 mixture of Dulbecco's Modified Minimal Essential Medium and Ham's FI2 medium supplemented with ~-retinyl acetate (10-7M), gentamicin (50 ~g/ml), and bovine serum albumin (BSA, 1 mg/ml). The plates were incubated at 37°C on a rocker platform (4 cycles/min) in a humidified atmosphere of 95% air/5% CO . After a one-hour equilibration period the medium was removed a~d replaced with an equal volume of DMH/BSA medium containing 12 ~Ci/ml 3H-glucosamine, a precursor of mucous glycoproteins. The plates then were incubated as described for a further 18-hr labeling period. Stimulation with secretagogues After labeling, the medium was removed and the explants were washed for two 15 minute periods with medium of the composition described above, without BSA. After washing, the explants were incubated for three sequential 30-minute periods (termed P0, PI, and PII) in 1 ml of fresh DMH/BSA. Medium was removed and replaced at the end of each period. Period P0 served as an e q u i l i b r a t i o n phase, while period PI provided a measurement of baseline m a c r o m o l e c u l a r secretion. The agents of interest were added to the medium at the appropriate concentrations during period PII, and stimulated secretion was measured during this time. Previous studies have demonstrated that tachykinins are quickly degraded by proteolytic enzymes present in airway tissue (10). Accordingly, the protease inhibitors aprotinin (450 kallikrein inhibitor units/ml) (2,3) and bacitracin (i mg/ml) (Ii) also were added during periods PI and PII to minimize peptide degradation. The secretagogues used were the tachykinins substance P (SP), eledoisin (ELE), neurokinin A (NKA), neurokinin B (NKB), and physalaemin (PHY). SP stimulation was also examined in the presence of the cholinergic antagonist atropine (ATP), the 8-adrenergic antagonist propranolol (PRO), the H-I histamine antagonist chlorpheniramine
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(CHL), and the SP receptor antagonist [D-ArgI,D-PheL,D-Trp ~'9, Leu*1]-SP (SP-ANT). In these experiments, the antagonist of interest generally was added to the medium at a c o n c e n t r a t i o n of 10-5M during both periods PI and PII. Due to its expense and lability, however, SP-ANT was added only during PII. A s s a y of radiolabeled high molecular weight glycoconjugates M a c r o m o l e c u l a r secretion was measured by a m o d i f i c a t i o n of the method of Coles et al (2). Radiolabeled high molecular weight glycoconjugates (HMWG) were precipitated from harvested medium in 5% trichloroacetic acid (TCA) and 1% phosphotungstic acid (PTA). After washing, the precipitate was solubilized in 0.5 N NaOH and the incorporated radioactive label measured by liquid scintillation counting. The effect of peptides on HMWG secretion was q u a n t i f i e d using a secretory index calculated by dividing the radioactivity (CPM) in precipitated period PII medium by that in p r e c i p i t a t e d period PI medium. During each preparation several wells of control explants were maintained, to which no peptide was added during period PII. To minimize variation between preparations the secretory indices for all wells exposed to peptides were divided by the average control value for that preparation. In equation form, the expressed secretory index was calculated as: secretory index ~ ~peptide PII CPM)/(peptide (control PII CPM)/(control A secretory index greater than unity, an increase in secretion above baseline
therefore, levels.
PI CPM). PI CPM) is indicative
of
A s s a y of lactate dehydrogenase activity Lactate dehydrogenase (LDH) is a cytoplasmic enzyme which is released by injured or dying cells. An increase in LDH activity in the culture medium following addition of a pharmacologic agent therefore may indicate toxicity due to the agent. LDH activity in spent medium harvested from periods PI and PII was measured by the method of Wroblewski and LaDue (12) with minor m o d i f i c a t i o n s (9). In no instance did addition of an agent or antagonist cause a significant (>5 IU.hr-1.50 mg tissue -I) (2) increase in LDH release over baseline. This suggests that increased m a c r o m o l e c u l a r release following exposure to the agents was not due to toxicity. Statistics Tests of significance were made using one-sided, unpaired Student's t test, with P < .05 considered significant. All results are expressed as mean ± SEM. Dose-response curves were fitted to the data using a computerized non-linear least squares estimate. The potency of each peptide was quantified by the c o n c e n t r a t i o n giving half-maximal stimulation (EC50), determined from the fitted curve. Sources of Materials Ferrets were obtained from Fetter's Ferret Services, Pittsburgh, PA. Culture medium (DMEM/FvI2) was obtained from Gibco, Grand Island, NY. Glucosamine D-[6-SH] (sp act 25 Ci/mmole) was purchased from American Radiolabeled Chemicals, Inc., St. Louis, MO. Opti-Fluor scintillation fluid was obtained from Packard, Downer's Grove, IL. SP was purchased in p r e - w e i g h e d vials from Peninsula Laboratories, Belmont, CA. All other chemicals were obtained from Sigma Chemicals, St. Louis, MO.
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Results
Previous studies have shown that tachykinins are rapidly degraded by proteolytic enzymes present in airway tissues (10). Accordingly, the protease inhibitors aprotinin (2,3) and b a c i t r a c i n (Ii) were added to the medium whenever peptides were used. Experiments using the neutral endopeptidase inhibitor p h o s p h o r a m i d o n (10-SM) showed no greater facilitation of tachykinin stimulation than aprotinin and bacitracin (data not shown). This is in accord with other reports using the same concentration of aprotinin (3). DMH/BSA medium containing aprotinin and bacitracin (MED) did not stimulate HMWG release when compared to medium without the inhibitors (Figure I). However, all of the peptides studied did stimulate HMWG release into the medium (P < .01 in each case). At a concentration of 10 -6 M the peptides had approximately equal effects, causing an average 31.6 ± 3.2% increase in secretion over baseline. Stimulation by each of these agents was dose-dependent. Dose-response curves are shown in Figure 2. Notably, at a concentration of 10-9M, SP caused significantly greater stimulation than the other peptides (P < .01 in each case). This shift to the left of the dose-response curve of SP indicates a higher potency for this agent when compared to the other peptides. In order to quantitate this finding, dose-response curves for each peptide were mathematically fitted using a computerized non-linear regression estimate. From these fitted cureves, the log EC50 for each peptide was calculated (Table I). SP was the most potent tachykinin
1.60 X LLI C-~ 1.40 Z
>£Y"
0
1.20
I-I,I (.~ LLI (J')
1.00
0.80
MED
SP
NKA qKB PHY ELE FIG.
1
Effects of tachykinins (10-6M) on HMWG secretion by ferret tracheal explants. A secretory index greater than unity indicates stimulation of secretion. All the tachykinins tested stimulated secretion (P < .01 in each case), to approximately the same degree. Culture medium containing the protease inhibitors aprotinin and bacitracin (MED) did not alter secretion from baseline levels. Each bar represents the mean ± SEM of 4 to 8 separate experiments.
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Tachykinlns and Airway Secretion
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FIG.
1.8
SP
o
o
•
• NKA
A
Dose-response curves of tachykinin-induced HMWG secretion. All tachykinins stimul a t e d s e c r e t i o n in a dose-dependent manner. However, SP stimulated secretion at a lower concentration than a n y of the o t h e r p e p t i d e s (*, P < .01 in each case). Each point represents the m e a n ± SEM of 4 to 8 separate e x p e r i m e n t s . (top) Curves for SP, NKA, and NKB. (bottom) Curves for SP, PHY, and ELE.
/ ~
A NKB
1.4.
/
,
I
1.2 X I_kl
1.0
Z
>nO
0.8
1.6
bn I
C.) u_l u3
1.4
o
o SP
•
• PHY
[]
[] ELE
T /o
]" ~
2
1.2
1.0
0.8
I
,
-10
I
-8
,
I
i
-6
I
-4
LOG CONCENTRATION (moles/liter)
TABLE
Calculated Lo 9 EC50
EC50 V a l u e s Std.
I
for T a c h y k i n i n s Err.
EC50
SP
-9.47
.33
3.40
x 10 -I°
NKA
-7.37
.19
4.25 x 10 -8
NKB
-5.98
.39
1.05
x 10 -6
PHY
-8.08
.23
8.36
x 10 -9
ELE
-7.68
.22
2.08
x 10 -8
Values for log EC50 w e r e c a l c u l a t e d f r o m d o s e - r e s p o n s e curves fitted using non-linear least-squares regression estimates.
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tested, with an EC50 of 3.4 x 10-I°M. This value was s i g n i f i c a n t l y less than the EC50 of any other tachykinin tested (P < .005 in each case). The rank order of tachykinin potency was SP > PHY > ELE NKA > NKB. The effect of atroDine, propranolol, chlorpheniramine, and [D-Arg*,D-PheS,D-TrpT'9,Leun]~SP (10-5M) on the efficacy of SPinduced HMWG secretion (i0- M) is shown in Figure 3. Blockade of the SP receptor eliminated the stimulatory effect. However, none of the other receptor blockers tested inhibited stimulation. In fact, pre-treatment with atropine appeared to facilitate the tachykinin effect, increasing the secretory index of 10-6M SP from 1.25 to 1.49 (P < .01). The inhibitors in the absence of SP did not alter secretion from baseline levels. The effect of atropine, propranolol, and chlorpheniramine on the potency of SP is shown in Figure 4. Cholinergic, 8-adrenergic, or H-I histamine receptor blockade did not alter the potency of SP.
1.8 X Ld Z >_
+
1.6
1.4
0 I--- 1.2 I,I [3:::
+ T
Ld 1.0 0'3 0.8
SP
SP-ANT
ATP
+ SP
PRO
CHL
IY/7H - - S P
FIG.
3
SP stimulation of HMWG release, alone (open bar) and in the presence of receptor blockers (solid bars, 10-sM). A secretory index greater than unity indicates stimulation of secretion. The effects of SP (10-6M) were blocked by SP-ANT (*, P < .01). However, SP stimulation was not decreased by preincubation with PRO or CHL, and ATP increased the magnitude of stimulation (+, P < .01). Receptor blockers alone did not alter HMWG secretion from baseline (hatched bars). Each point represents the mean ± SEM of 4-6 separate experiments.
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Tachykinins and Airway Secretion
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Discussion These studies have examined the ability of a number of tachykinins to stimulate secretion of HMWG by ferret tracheal explants. All the tachykinins studied stimulated secretion in a d o s e - r e l a t e d manner, and stimulation by SP was blocked by a SP receptor antagonist. A l t h o u g h the efficacy of the tachykinins was a p p r o x i m a t e l y equal, SP was the most potent, with an EC50 of 3.4 x 10-I°M. Pretreatment of explants with cholinergic, ~-adrenergic, or H-I receptor antagonists did not alter the potency of SP. However, the efficacy of SP was increased in the presence of atropine, suggesting an inhibitory cholinergic modulation of tachykinin effects. Several previous studies have described increased macromolecular secretion in response to SP and other bioactive peptides. Baker, using canine tracheal explants in culture, demonstrated a 64% increase in the release of radiolabeled m a c r o m o l e c u l e s in response to 10-7M SP (13). This finding was confirmed by Coles et al, who also d e m o n s t r a t e d that stimulation was d o s e - d e p e n d e n t (2). These workers calculated the EC50 of SP to be 8.2 x 10-I°M, making this agent a p p r o x i m a t e l y one thousand fold more potent than the cholinergic agonist methacholine. More recently, Lundgren et al have d e m o n s t r a t e d the stimulatory effects of a number of tachykinins on glycoconjugate secretion by cat tracheal explants (3). A l t h o u g h these workers did not calculate EC50 values, SP stimulated secretion at a lower dose than the other tachykinins tested. They also d e m o n s t r a t e d t h e presence of SP receptors on submucosal glands,
1.6 X LLJ C~ 1.4 Z >rY 1.2 0 F-Ld rY rO 1.0 i,i GO 0.8
o
o
•
•
,",
z~ + PRO
•
,,
,
2
+ ATP
+
/
I"
C H L 6 _ ~ ° ~
I
-10
i
I
-8
i
I
-6
i
-4
LOG CONCENTRATION (moles/liter) FIG.4 Dose-respone curves of SP-induced HMWG secretion in the presence of receptor blockers. The potency of SP was not altered by p r e - i n c u b a t i o n with ATP, PRO, or CHL. However, the efficacy of SP was increased in the presence of ATP. Each point represents the mean ± SEM of 4 to 7 experiments.
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using autoradiographic techniques. Finally, Webber, using a preparation of whole ferret trachea in vitro, studied the effects of SP and NKA on smooth muscle contraction and on the secretion of several macromolecules (4). Although NKA was more potent at contracting tracheal smooth muscle, SP was more potent at stimulating m a c r o m o l e c u l a r release. The current studies have confirmed and expanded upon these findings. The blockade of SP stimulation by an SP receptor antagonist confirms that stimulation occurs through a specific receptor-mediated mechanism, rather than a non-specific toxic effect. The minimal LDH release and the classic d o s e - r e l a t e d nature of the tachykinin effect supports this conclusion. Furthermore, by constructing dose-response curves, the relative potency of the tachykinins can be determined. This is significant in light of the finding by Lee of multiple tachykinin receptor types (8). By comparing the potencies of tachykinins in several bioassay preparations and competitive binding studies (7,8,14), three receptor types have been distinguished. These receptors, termed NKI, NK2, and NK3, respond preferentially to SP, NKA, and NKB, respectively. An apparent fourth SP receptor type has also been identified by Rice and Singleton (15). Using isolated rat alveolar type II cells in culture, they found that SP inhibited secretion of phosphatidylcholine in a dose-dependent manner. This effect was not produced by the related peptides PHY and [pGlu s, MePheS,MeGly9]-SP[5-11]. On this basis, these authors suggested that another receptor, unrelated to those previously described, was responsible. In the current experiments, the rank order of potency of the tachykinins tested was SP > PHY ~ ELE ~ NKA > NKB. This fits well with the order of binding affinity described by Lee for NKI receptors, namely, SP > PHY > NKA > NKB > ELE (8). The efficacy of PHY, on the other hand, is inconsistent with the receptor type described by Rice and Singleton (15). It seems clear, then, that t a c h y k i n i n - i n d u c e d stimulation of airway macromolecular secretion is mediated through NKI receptors. This is in accord with the conclusions of Webber (4), and is compatible with the findings of Lundgren et al (3), although the latter did not specifically identify tachykinin receptor subtypes. Several investigators have suggested that tachykinin effects in some instances are mediated through other n e u r o t r a n s m i t t e r s or mediators (16-18). To evaluate this possibility, explants were pre-incubated with the cholinergic antagonist atropine, the 8-adrenergic antagonist propranolol, and the H-I histamine antagonist chlorpheniramine. These receptor blockers did not decrease the potency or the efficacy of SP in stimulating macromolecular secretion, on the contrary, atropine increased the efficacy of SP. These findings indicate that the stimulatory effects of SP on secretion do not occur through cholinergic, 8-adrenergic, or H-I mechanisms. However, they also suggest the presence of a cholinergic inhibitory mechanism which might serve as a "brake" on tachykinin-induced secretion. Such an inhibitory mechanism probably would operate indirectly on the airway submucosal glands. The submucosal glands are thought to be the primary source of secreted macromolecules in the airways, because of their greater volume relative to other airway secretory cells (19). Since receptors for SP have been identified on the submucosal glands, but not in the surface epithelium (3), tachykinininduced secretion most likely occurs by a direct effect of the
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Tachykinins and Airway Secretion
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peptide on the gland. Cholinergic agonists also act d i r e c t l y on submucosal glands, and cause an increase in secretion (20,21). Therefore, a cholinergic mechanism which inhibited secretion would necessarily have to occur at a site distant from the glands. Furthermore, since atropine alone did not alter secretion from baseline, cholinergic inhibition would only seem to occur in response to tachykinin exposure. A speculative m e c h a n i s m which could account for these findings might involve t a c h y k i n i n - i n d u c e d acetylcholine release, perhaps in an airway ganglion, which inhibits a stimulatory SP-containing nerve fiber. Such inhibitory cholinergic influence has not been described previously, although inhibitory presynaptic muscarinic cholinergic receptors have been reported (23). Investigators examining the effect of cholinergic blockade on tachykinin-induced secretion have found dissimilar results. Coles has reported that atropine does not inhibit SP stimulation of HMWG secretion in canine tracheal explants (22), but apparently he did not see facilitation of stimulation by cholinergic blockade. Conversely, Shimura, using isolated feline tracheal submucosal glands, found that atropine completely abolished the SP-induced secretory effect (16). Based on these findings, Shimura concluded that SP induces glandular secretion by a peripheral cholinergic mechanism. These results are confusing in light of the demonstration by Lundgren of SP receptors on cat tracheal submucosal glands (3), which presumably function to mediate glandular secretion. Clearly, further study is necessary to define the interactions between the cholinergic, adrenergic, and peptidergic components of the autonomic nervous system in the airways. Acknowledgments The author is grateful to Drs. James Dauber, Arthur Banner and John Yanos for their valuable discussion, and to Mrs. Anne Mainwaring and Mrs. Judith Hissom for their excellent technical assistance. This work was supported by a grant from the A m e r i c a n Lung Association. References i. 2.
3. 4. 5. 6. 7. 8. 9. i0.
P.J. BARNES, Am. Rev. Respir. Dis. 134 1289-1314 (1986). S.J. COLES, K.H. NEILL, and L.M. REID, J. Appl. Physiol. 57 1323-1327 (1984). J.D. LUNDGREN, C.J. WIEDERMANN, C. LOGUN, J. PLUTCHOK, M. KALINER, and J.H. SHELHAMER, Exper. Lung Res. 15 17-29 (1989). S.E. WEBBER, Br. J. Pharmacol, 98 1197-1206 (1989). S.H. BUCK and E. BURCHER, Trends Pharmacol. Sci. 7 65-68 (1986). S.P. WATSON, Life Sci. 25 797-808 (1984). D. REGOLI, G. DRAPEAU, S. DION, and P. D'ORLEANS-JUSTE, Life Sci 40 109-116 (1987). C.M. LEE, N.J. CAMPBELL, B.J. WILLIAMS, and L.L. IVERSEN, Eur. J. Pharmacol. 130 209-217 (1986). S.E. GENTRY, D.J. CULP, N.J. ROBERTS, JR., M.G. MARIN, R.L. SIMONS, and L.R. LATCHNEY, J. Virol. 62 1524-1529 (1988). D.B. BORSON, R. CORRALES, S. VARSANO, M. GOLD, N. VIRO, G. CAUGHEY, J. RAMACHANDRAN, and J.A. NADEL, Exper. Lung Res. 12 21-36 (1987).
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11 12 13 14 15 16 17 18 19 20 21 22.
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A.A. GASHI, D.B. BORSON, W.E. FINKBEINER, J.A. NADEL, and C.B. BASBAUM, Am. J. Physiol. 251 C 2 2 3 - C 2 2 9 (1986). F. W R O B L E W S K I and J.S. LADUE, Proc. Soc. Exp. Biol. Med. 90 2 1 0 - 2 1 3 (1955). A.P. BAKER, L.M. HILLEGASS, D.A. HOLDEN, and W.J. SMITH, Am. Rev. Respir. Dis. 115 811-817 (1977). C.M. LEE, L.L. IVERSEN, M.R. HANLEY, and G.E.B. S A N D B E R G , Nauyn-Schmiedebergs Arch. Pharmacol. 318 2 8 1 - 2 8 7 (1982). W.R. RICE and F.M. SINGLETON, Biochem. Biophys. Acta. 889 1 2 3 - 1 2 7 (1986). S. SHIMURA, T. SASAKI, H. OKAYAMA, H. SASAKI, and T. T A K I S H I M A , J. Appl. Physiol. 63 646-653 (1987). M.M. G R U N S T E I N , D.T. TANAKA, and J.S. G R U N S T E I N , J. Appl. Physiol. 57 1 2 3 8 - 1 2 4 6 (1984). G.F. JOOS~, R.A. PAUWELS, and M.E. V A N DER STRAETEN, Am. Rev. Respir. Dis. 137 1 0 3 8 - 1 0 4 4 (1988). N.P. R O B I N S O N , L. VENNING, H. KYLE and J.G. W I D D I C O M B E , J. Anat. 145 173-188 (1986). D.J. CULP and M.G. MARIN, J. Appl. Physiol. 61 1375-1382 (1986). S. SHIMURA, T. SASKAKI, H. SASAKI, and T. T A K I S H I M A , J. Appl. Physiol. 60 1 2 3 7 - 1 2 4 7 (1986). S.J. COLES, K.R. BHASKAR, D.D. O ' S U L L I V A N , K.H. NEILL, and L.M. REID, M u c u s and Mucosa, J. N u g e n t and M. O'Connor (eds), 40-60, P i t m a n Press, L o n d o n (1984) A.D. FRYER and J. M A C L A G A N , Br. J. Pharmacol. 83 973-978 (1984).
TACHYKININ
RECEPTORS
MEDIATING
Pergamon Press
AIRWAY
MACROROLECULAR
SECRETION
Steven E. Gentry Montefiore Hospital, University of Pittsburgh School of Medicine, 3459 Fifth Avenue, Pittsburgh, PA 15213 (Received in final form February 13, 1991) Summa r¥ Three tachykinin receptor types, termed NKI, NK2, and NK3, can be distinguished by the relative potency of various peptides in eliciting tissue responses. Airway macromolecular secretion is stimulated by the tachykinin substance P (SP). The purposes of this study were to determine the tachykinin receptor subtype responsible for this stimulation, and to examine the possible involvement of other neurotransmitters in mediating this effect. Ferret tracheal explants maintained in organ culture were labeled with 3H-glucosamine, a precursor of high molecular weight glycoconjugates (HMWG) which are released by airway secretory cells. Secretion of labeled HMWG then was determined in the absence and presence of the tachykinins SP, neurokinin A (NKA), neurokinin B (NKB), physalaemin (PHY), and eledoisin (ELE). All the tachykinins tested stimulated HMWG release to an approximately equal degree. Stimulation was concentration-related, with log concentrations giving halfmaximal effects (EC50) as follows: SP -9.47, NKA -7.37, NKB -5.98, P H Y - 8 . 0 8 , and ELE -7.68. This rank order of potency (SP > PHY _> ELE _> NKA > NKB) is most consistent with NKI receptors. To evaluate the possible contribution of other mediators, tachykinin stimulation was examined in the presence of several receptor blockers. The potency of SP was not diminished by pretreatment with atropine, propranolol, or chlorpheniramine, and atropine actually increased the magnitude of the secretc!y response. The SP receptor antagonist [D-Argl, D-Phe D-TrpV'9,Leu11]-SP blocked SP-induced secretion. These findings indicate that SP is a potent stimulus of airway m a c r o m o l e c u l a r secretion. This effect occurs through the action of NKI receptors, and is not dependent upon cholinergic, ~-adrenergic, or H-I histamine receptors. The facilitation by atropine of SP stimulation suggests the existence of a mechanism of cholinergic inhibition of SP-induced stimulation. Substance P (SP) is an undecapeptide which is widely distributed throughout the central and peripheral nervous system, and which belongs to a family of peptides called the tachykinins. Substance P has been implicated as a possible n e u r o t r a n s m i t t e r of the non-adrenergic, non-cholinergic innervation of the lung (i). In the lung, fibe[s containing SP have been d e m o n s t r a t e d in and 0024-3205/91 $3.00 +.00 Copyright (c) 1991 Pergamon Press plc
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around the surface epithelium, around blood vessels, and within airway smooth muscle (i). Functionally, SP has been shown to cause airway smooth muscle contraction, increased microvascular permeability, and tracheobronchial vasodilation (i). In addition, SP is a potent stimulant for the release of macromolecules from airway secretory cells (2-4). Recently, the presence of multiple types of receptors for tachykinins has been postulated (5,6). By comparing the relative potency of tachykinins in bioassay (7) and binding (8) studies, three receptor subtypes have been identified. These receptor subtypes, termed NKI, NK2, and NK3, respond p r e f e r e n t i a l l y to SP, neurokinin A (NKA), and neurokinin B (NKB), respectively. The purpose of this study was to determine the tachykinin receptor subtype mediating macromolecular secretion in the airways. Furthermore, the possible contribution of other n e u r o t r a n s m i t t e r s and mediators to tachykinin-induced secretion was examined. Methods and Materials Tissue preparation and culture Adult neutered female ferrets were killed by a lethal intraperitoneal injection of sodium pentobarbital. The trachea was removed aseptically, trimmed, opened along the posterior membranous portion, and pinned to a sterile dissecting tray filled with cold (4°C) p h o s p h a t e - b u f f e r e d saline. The trachea was cut into fullthickness explants (3x5 mm), which were transferred to siliconized 24-well culture plates, three to four explants per well. One ml of culture medium then was added to each well. The medium, termed D M H / B S A medium, was a modification of that used in previous studies (9), and consisted of a 50:50 mixture of Dulbecco's Modified Minimal Essential Medium and Ham's FI2 medium supplemented with ~-retinyl acetate (10-7M), gentamicin (50 ~g/ml), and bovine serum albumin (BSA, 1 mg/ml). The plates were incubated at 37°C on a rocker platform (4 cycles/min) in a humidified atmosphere of 95% air/5% CO . After a one-hour equilibration period the medium was removed a~d replaced with an equal volume of DMH/BSA medium containing 12 ~Ci/ml 3H-glucosamine, a precursor of mucous glycoproteins. The plates then were incubated as described for a further 18-hr labeling period. Stimulation with secretagogues After labeling, the medium was removed and the explants were washed for two 15 minute periods with medium of the composition described above, without BSA. After washing, the explants were incubated for three sequential 30-minute periods (termed P0, PI, and PII) in 1 ml of fresh DMH/BSA. Medium was removed and replaced at the end of each period. Period P0 served as an e q u i l i b r a t i o n phase, while period PI provided a measurement of baseline m a c r o m o l e c u l a r secretion. The agents of interest were added to the medium at the appropriate concentrations during period PII, and stimulated secretion was measured during this time. Previous studies have demonstrated that tachykinins are quickly degraded by proteolytic enzymes present in airway tissue (10). Accordingly, the protease inhibitors aprotinin (450 kallikrein inhibitor units/ml) (2,3) and bacitracin (i mg/ml) (Ii) also were added during periods PI and PII to minimize peptide degradation. The secretagogues used were the tachykinins substance P (SP), eledoisin (ELE), neurokinin A (NKA), neurokinin B (NKB), and physalaemin (PHY). SP stimulation was also examined in the presence of the cholinergic antagonist atropine (ATP), the 8-adrenergic antagonist propranolol (PRO), the H-I histamine antagonist chlorpheniramine
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Tachykinins and Airway Secretion
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(CHL), and the SP receptor antagonist [D-ArgI,D-PheL,D-Trp ~'9, Leu*1]-SP (SP-ANT). In these experiments, the antagonist of interest generally was added to the medium at a c o n c e n t r a t i o n of 10-5M during both periods PI and PII. Due to its expense and lability, however, SP-ANT was added only during PII. A s s a y of radiolabeled high molecular weight glycoconjugates M a c r o m o l e c u l a r secretion was measured by a m o d i f i c a t i o n of the method of Coles et al (2). Radiolabeled high molecular weight glycoconjugates (HMWG) were precipitated from harvested medium in 5% trichloroacetic acid (TCA) and 1% phosphotungstic acid (PTA). After washing, the precipitate was solubilized in 0.5 N NaOH and the incorporated radioactive label measured by liquid scintillation counting. The effect of peptides on HMWG secretion was q u a n t i f i e d using a secretory index calculated by dividing the radioactivity (CPM) in precipitated period PII medium by that in p r e c i p i t a t e d period PI medium. During each preparation several wells of control explants were maintained, to which no peptide was added during period PII. To minimize variation between preparations the secretory indices for all wells exposed to peptides were divided by the average control value for that preparation. In equation form, the expressed secretory index was calculated as: secretory index ~ ~peptide PII CPM)/(peptide (control PII CPM)/(control A secretory index greater than unity, an increase in secretion above baseline
therefore, levels.
PI CPM). PI CPM) is indicative
of
A s s a y of lactate dehydrogenase activity Lactate dehydrogenase (LDH) is a cytoplasmic enzyme which is released by injured or dying cells. An increase in LDH activity in the culture medium following addition of a pharmacologic agent therefore may indicate toxicity due to the agent. LDH activity in spent medium harvested from periods PI and PII was measured by the method of Wroblewski and LaDue (12) with minor m o d i f i c a t i o n s (9). In no instance did addition of an agent or antagonist cause a significant (>5 IU.hr-1.50 mg tissue -I) (2) increase in LDH release over baseline. This suggests that increased m a c r o m o l e c u l a r release following exposure to the agents was not due to toxicity. Statistics Tests of significance were made using one-sided, unpaired Student's t test, with P < .05 considered significant. All results are expressed as mean ± SEM. Dose-response curves were fitted to the data using a computerized non-linear least squares estimate. The potency of each peptide was quantified by the c o n c e n t r a t i o n giving half-maximal stimulation (EC50), determined from the fitted curve. Sources of Materials Ferrets were obtained from Fetter's Ferret Services, Pittsburgh, PA. Culture medium (DMEM/FvI2) was obtained from Gibco, Grand Island, NY. Glucosamine D-[6-SH] (sp act 25 Ci/mmole) was purchased from American Radiolabeled Chemicals, Inc., St. Louis, MO. Opti-Fluor scintillation fluid was obtained from Packard, Downer's Grove, IL. SP was purchased in p r e - w e i g h e d vials from Peninsula Laboratories, Belmont, CA. All other chemicals were obtained from Sigma Chemicals, St. Louis, MO.
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Vol. 48, No. 17, 1991
Results
Previous studies have shown that tachykinins are rapidly degraded by proteolytic enzymes present in airway tissues (10). Accordingly, the protease inhibitors aprotinin (2,3) and b a c i t r a c i n (Ii) were added to the medium whenever peptides were used. Experiments using the neutral endopeptidase inhibitor p h o s p h o r a m i d o n (10-SM) showed no greater facilitation of tachykinin stimulation than aprotinin and bacitracin (data not shown). This is in accord with other reports using the same concentration of aprotinin (3). DMH/BSA medium containing aprotinin and bacitracin (MED) did not stimulate HMWG release when compared to medium without the inhibitors (Figure I). However, all of the peptides studied did stimulate HMWG release into the medium (P < .01 in each case). At a concentration of 10 -6 M the peptides had approximately equal effects, causing an average 31.6 ± 3.2% increase in secretion over baseline. Stimulation by each of these agents was dose-dependent. Dose-response curves are shown in Figure 2. Notably, at a concentration of 10-9M, SP caused significantly greater stimulation than the other peptides (P < .01 in each case). This shift to the left of the dose-response curve of SP indicates a higher potency for this agent when compared to the other peptides. In order to quantitate this finding, dose-response curves for each peptide were mathematically fitted using a computerized non-linear regression estimate. From these fitted cureves, the log EC50 for each peptide was calculated (Table I). SP was the most potent tachykinin
1.60 X LLI C-~ 1.40 Z
>£Y"
0
1.20
I-I,I (.~ LLI (J')
1.00
0.80
MED
SP
NKA qKB PHY ELE FIG.
1
Effects of tachykinins (10-6M) on HMWG secretion by ferret tracheal explants. A secretory index greater than unity indicates stimulation of secretion. All the tachykinins tested stimulated secretion (P < .01 in each case), to approximately the same degree. Culture medium containing the protease inhibitors aprotinin and bacitracin (MED) did not alter secretion from baseline levels. Each bar represents the mean ± SEM of 4 to 8 separate experiments.
Vol. 48, No. 17, 1991
Tachykinlns and Airway Secretion
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FIG.
1.8
SP
o
o
•
• NKA
A
Dose-response curves of tachykinin-induced HMWG secretion. All tachykinins stimul a t e d s e c r e t i o n in a dose-dependent manner. However, SP stimulated secretion at a lower concentration than a n y of the o t h e r p e p t i d e s (*, P < .01 in each case). Each point represents the m e a n ± SEM of 4 to 8 separate e x p e r i m e n t s . (top) Curves for SP, NKA, and NKB. (bottom) Curves for SP, PHY, and ELE.
/ ~
A NKB
1.4.
/
,
I
1.2 X I_kl
1.0
Z
>nO
0.8
1.6
bn I
C.) u_l u3
1.4
o
o SP
•
• PHY
[]
[] ELE
T /o
]" ~
2
1.2
1.0
0.8
I
,
-10
I
-8
,
I
i
-6
I
-4
LOG CONCENTRATION (moles/liter)
TABLE
Calculated Lo 9 EC50
EC50 V a l u e s Std.
I
for T a c h y k i n i n s Err.
EC50
SP
-9.47
.33
3.40
x 10 -I°
NKA
-7.37
.19
4.25 x 10 -8
NKB
-5.98
.39
1.05
x 10 -6
PHY
-8.08
.23
8.36
x 10 -9
ELE
-7.68
.22
2.08
x 10 -8
Values for log EC50 w e r e c a l c u l a t e d f r o m d o s e - r e s p o n s e curves fitted using non-linear least-squares regression estimates.
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Tachykinins and Airway Secretion
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tested, with an EC50 of 3.4 x 10-I°M. This value was s i g n i f i c a n t l y less than the EC50 of any other tachykinin tested (P < .005 in each case). The rank order of tachykinin potency was SP > PHY > ELE NKA > NKB. The effect of atroDine, propranolol, chlorpheniramine, and [D-Arg*,D-PheS,D-TrpT'9,Leun]~SP (10-5M) on the efficacy of SPinduced HMWG secretion (i0- M) is shown in Figure 3. Blockade of the SP receptor eliminated the stimulatory effect. However, none of the other receptor blockers tested inhibited stimulation. In fact, pre-treatment with atropine appeared to facilitate the tachykinin effect, increasing the secretory index of 10-6M SP from 1.25 to 1.49 (P < .01). The inhibitors in the absence of SP did not alter secretion from baseline levels. The effect of atropine, propranolol, and chlorpheniramine on the potency of SP is shown in Figure 4. Cholinergic, 8-adrenergic, or H-I histamine receptor blockade did not alter the potency of SP.
1.8 X Ld Z >_
+
1.6
1.4
0 I--- 1.2 I,I [3:::
+ T
Ld 1.0 0'3 0.8
SP
SP-ANT
ATP
+ SP
PRO
CHL
IY/7H - - S P
FIG.
3
SP stimulation of HMWG release, alone (open bar) and in the presence of receptor blockers (solid bars, 10-sM). A secretory index greater than unity indicates stimulation of secretion. The effects of SP (10-6M) were blocked by SP-ANT (*, P < .01). However, SP stimulation was not decreased by preincubation with PRO or CHL, and ATP increased the magnitude of stimulation (+, P < .01). Receptor blockers alone did not alter HMWG secretion from baseline (hatched bars). Each point represents the mean ± SEM of 4-6 separate experiments.
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Tachykinins and Airway Secretion
1615
Discussion These studies have examined the ability of a number of tachykinins to stimulate secretion of HMWG by ferret tracheal explants. All the tachykinins studied stimulated secretion in a d o s e - r e l a t e d manner, and stimulation by SP was blocked by a SP receptor antagonist. A l t h o u g h the efficacy of the tachykinins was a p p r o x i m a t e l y equal, SP was the most potent, with an EC50 of 3.4 x 10-I°M. Pretreatment of explants with cholinergic, ~-adrenergic, or H-I receptor antagonists did not alter the potency of SP. However, the efficacy of SP was increased in the presence of atropine, suggesting an inhibitory cholinergic modulation of tachykinin effects. Several previous studies have described increased macromolecular secretion in response to SP and other bioactive peptides. Baker, using canine tracheal explants in culture, demonstrated a 64% increase in the release of radiolabeled m a c r o m o l e c u l e s in response to 10-7M SP (13). This finding was confirmed by Coles et al, who also d e m o n s t r a t e d that stimulation was d o s e - d e p e n d e n t (2). These workers calculated the EC50 of SP to be 8.2 x 10-I°M, making this agent a p p r o x i m a t e l y one thousand fold more potent than the cholinergic agonist methacholine. More recently, Lundgren et al have d e m o n s t r a t e d the stimulatory effects of a number of tachykinins on glycoconjugate secretion by cat tracheal explants (3). A l t h o u g h these workers did not calculate EC50 values, SP stimulated secretion at a lower dose than the other tachykinins tested. They also d e m o n s t r a t e d t h e presence of SP receptors on submucosal glands,
1.6 X LLJ C~ 1.4 Z >rY 1.2 0 F-Ld rY rO 1.0 i,i GO 0.8
o
o
•
•
,",
z~ + PRO
•
,,
,
2
+ ATP
+
/
I"
C H L 6 _ ~ ° ~
I
-10
i
I
-8
i
I
-6
i
-4
LOG CONCENTRATION (moles/liter) FIG.4 Dose-respone curves of SP-induced HMWG secretion in the presence of receptor blockers. The potency of SP was not altered by p r e - i n c u b a t i o n with ATP, PRO, or CHL. However, the efficacy of SP was increased in the presence of ATP. Each point represents the mean ± SEM of 4 to 7 experiments.
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Vol. 48, No. 17, 1991
using autoradiographic techniques. Finally, Webber, using a preparation of whole ferret trachea in vitro, studied the effects of SP and NKA on smooth muscle contraction and on the secretion of several macromolecules (4). Although NKA was more potent at contracting tracheal smooth muscle, SP was more potent at stimulating m a c r o m o l e c u l a r release. The current studies have confirmed and expanded upon these findings. The blockade of SP stimulation by an SP receptor antagonist confirms that stimulation occurs through a specific receptor-mediated mechanism, rather than a non-specific toxic effect. The minimal LDH release and the classic d o s e - r e l a t e d nature of the tachykinin effect supports this conclusion. Furthermore, by constructing dose-response curves, the relative potency of the tachykinins can be determined. This is significant in light of the finding by Lee of multiple tachykinin receptor types (8). By comparing the potencies of tachykinins in several bioassay preparations and competitive binding studies (7,8,14), three receptor types have been distinguished. These receptors, termed NKI, NK2, and NK3, respond preferentially to SP, NKA, and NKB, respectively. An apparent fourth SP receptor type has also been identified by Rice and Singleton (15). Using isolated rat alveolar type II cells in culture, they found that SP inhibited secretion of phosphatidylcholine in a dose-dependent manner. This effect was not produced by the related peptides PHY and [pGlu s, MePheS,MeGly9]-SP[5-11]. On this basis, these authors suggested that another receptor, unrelated to those previously described, was responsible. In the current experiments, the rank order of potency of the tachykinins tested was SP > PHY ~ ELE ~ NKA > NKB. This fits well with the order of binding affinity described by Lee for NKI receptors, namely, SP > PHY > NKA > NKB > ELE (8). The efficacy of PHY, on the other hand, is inconsistent with the receptor type described by Rice and Singleton (15). It seems clear, then, that t a c h y k i n i n - i n d u c e d stimulation of airway macromolecular secretion is mediated through NKI receptors. This is in accord with the conclusions of Webber (4), and is compatible with the findings of Lundgren et al (3), although the latter did not specifically identify tachykinin receptor subtypes. Several investigators have suggested that tachykinin effects in some instances are mediated through other n e u r o t r a n s m i t t e r s or mediators (16-18). To evaluate this possibility, explants were pre-incubated with the cholinergic antagonist atropine, the 8-adrenergic antagonist propranolol, and the H-I histamine antagonist chlorpheniramine. These receptor blockers did not decrease the potency or the efficacy of SP in stimulating macromolecular secretion, on the contrary, atropine increased the efficacy of SP. These findings indicate that the stimulatory effects of SP on secretion do not occur through cholinergic, 8-adrenergic, or H-I mechanisms. However, they also suggest the presence of a cholinergic inhibitory mechanism which might serve as a "brake" on tachykinin-induced secretion. Such an inhibitory mechanism probably would operate indirectly on the airway submucosal glands. The submucosal glands are thought to be the primary source of secreted macromolecules in the airways, because of their greater volume relative to other airway secretory cells (19). Since receptors for SP have been identified on the submucosal glands, but not in the surface epithelium (3), tachykinininduced secretion most likely occurs by a direct effect of the
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Tachykinins and Airway Secretion
1617
peptide on the gland. Cholinergic agonists also act d i r e c t l y on submucosal glands, and cause an increase in secretion (20,21). Therefore, a cholinergic mechanism which inhibited secretion would necessarily have to occur at a site distant from the glands. Furthermore, since atropine alone did not alter secretion from baseline, cholinergic inhibition would only seem to occur in response to tachykinin exposure. A speculative m e c h a n i s m which could account for these findings might involve t a c h y k i n i n - i n d u c e d acetylcholine release, perhaps in an airway ganglion, which inhibits a stimulatory SP-containing nerve fiber. Such inhibitory cholinergic influence has not been described previously, although inhibitory presynaptic muscarinic cholinergic receptors have been reported (23). Investigators examining the effect of cholinergic blockade on tachykinin-induced secretion have found dissimilar results. Coles has reported that atropine does not inhibit SP stimulation of HMWG secretion in canine tracheal explants (22), but apparently he did not see facilitation of stimulation by cholinergic blockade. Conversely, Shimura, using isolated feline tracheal submucosal glands, found that atropine completely abolished the SP-induced secretory effect (16). Based on these findings, Shimura concluded that SP induces glandular secretion by a peripheral cholinergic mechanism. These results are confusing in light of the demonstration by Lundgren of SP receptors on cat tracheal submucosal glands (3), which presumably function to mediate glandular secretion. Clearly, further study is necessary to define the interactions between the cholinergic, adrenergic, and peptidergic components of the autonomic nervous system in the airways. Acknowledgments The author is grateful to Drs. James Dauber, Arthur Banner and John Yanos for their valuable discussion, and to Mrs. Anne Mainwaring and Mrs. Judith Hissom for their excellent technical assistance. This work was supported by a grant from the A m e r i c a n Lung Association. References i. 2.
3. 4. 5. 6. 7. 8. 9. i0.
P.J. BARNES, Am. Rev. Respir. Dis. 134 1289-1314 (1986). S.J. COLES, K.H. NEILL, and L.M. REID, J. Appl. Physiol. 57 1323-1327 (1984). J.D. LUNDGREN, C.J. WIEDERMANN, C. LOGUN, J. PLUTCHOK, M. KALINER, and J.H. SHELHAMER, Exper. Lung Res. 15 17-29 (1989). S.E. WEBBER, Br. J. Pharmacol, 98 1197-1206 (1989). S.H. BUCK and E. BURCHER, Trends Pharmacol. Sci. 7 65-68 (1986). S.P. WATSON, Life Sci. 25 797-808 (1984). D. REGOLI, G. DRAPEAU, S. DION, and P. D'ORLEANS-JUSTE, Life Sci 40 109-116 (1987). C.M. LEE, N.J. CAMPBELL, B.J. WILLIAMS, and L.L. IVERSEN, Eur. J. Pharmacol. 130 209-217 (1986). S.E. GENTRY, D.J. CULP, N.J. ROBERTS, JR., M.G. MARIN, R.L. SIMONS, and L.R. LATCHNEY, J. Virol. 62 1524-1529 (1988). D.B. BORSON, R. CORRALES, S. VARSANO, M. GOLD, N. VIRO, G. CAUGHEY, J. RAMACHANDRAN, and J.A. NADEL, Exper. Lung Res. 12 21-36 (1987).
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11 12 13 14 15 16 17 18 19 20 21 22.
23.
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A.A. GASHI, D.B. BORSON, W.E. FINKBEINER, J.A. NADEL, and C.B. BASBAUM, Am. J. Physiol. 251 C 2 2 3 - C 2 2 9 (1986). F. W R O B L E W S K I and J.S. LADUE, Proc. Soc. Exp. Biol. Med. 90 2 1 0 - 2 1 3 (1955). A.P. BAKER, L.M. HILLEGASS, D.A. HOLDEN, and W.J. SMITH, Am. Rev. Respir. Dis. 115 811-817 (1977). C.M. LEE, L.L. IVERSEN, M.R. HANLEY, and G.E.B. S A N D B E R G , Nauyn-Schmiedebergs Arch. Pharmacol. 318 2 8 1 - 2 8 7 (1982). W.R. RICE and F.M. SINGLETON, Biochem. Biophys. Acta. 889 1 2 3 - 1 2 7 (1986). S. SHIMURA, T. SASAKI, H. OKAYAMA, H. SASAKI, and T. T A K I S H I M A , J. Appl. Physiol. 63 646-653 (1987). M.M. G R U N S T E I N , D.T. TANAKA, and J.S. G R U N S T E I N , J. Appl. Physiol. 57 1 2 3 8 - 1 2 4 6 (1984). G.F. JOOS~, R.A. PAUWELS, and M.E. V A N DER STRAETEN, Am. Rev. Respir. Dis. 137 1 0 3 8 - 1 0 4 4 (1988). N.P. R O B I N S O N , L. VENNING, H. KYLE and J.G. W I D D I C O M B E , J. Anat. 145 173-188 (1986). D.J. CULP and M.G. MARIN, J. Appl. Physiol. 61 1375-1382 (1986). S. SHIMURA, T. SASKAKI, H. SASAKI, and T. T A K I S H I M A , J. Appl. Physiol. 60 1 2 3 7 - 1 2 4 7 (1986). S.J. COLES, K.R. BHASKAR, D.D. O ' S U L L I V A N , K.H. NEILL, and L.M. REID, M u c u s and Mucosa, J. N u g e n t and M. O'Connor (eds), 40-60, P i t m a n Press, L o n d o n (1984) A.D. FRYER and J. M A C L A G A N , Br. J. Pharmacol. 83 973-978 (1984).