Neuropeptides and Airway Submucosal Gland Secretion 1 - J SANAE SHIMURA, TSUKASA SASAKI, KAOKO IKEDA, HIROSHI ISHIHARA, MASATOSHI SATO, HIDETADA SASAKI, and TAMOTSU TAKISHIMA Introduction Various neuropeptide-containing nerveshave been demonstrated not only in smooth muscle but also in submucosal glands of the large airways in guinea pig, rat, dog, cat, and human tissues by immunocytochemical analyses (1-5). The neuropeptides include vasoactive intestinal peptide (VIP), peptide histidine isoleucine (PHI), peptide histidine methionine (PHM), substance P (SP), calcitonin gene-related peptide, neurokinins A and B, neuropeptide Y, galanin, gastrin-releasing peptides, cholecytonin and somatostatin. Further, localization of the receptors for neuropeptides by autoradiography has been reported in lungs, showing a high density of binding over the submucosal glands of bronchi (6, 7). Thus, neuropeptides have been implicated in the control of airway submucosal gland secretion. Among these neuropeptides, to date, effects of SP, VIP, and bombesin on airway submucosal gland secretion have been investigated (8-15). These investigators used airway explants containing surface epithelium, submucosal tissues, and sometimes cartilage in addition to submucosal glands. Recently, we have been successful in isolating submucosal glands from trachea, which has enabled us to examine submucosal gland secretion in a well-defined condition, excluding the potential effects of the surrounding tissues (5, 16-18). Such effects may include an epithelial inhibitory action on submucosal gland secretion (19), tissue peptidases released during experiments (11, 20, 21),and difficulty in the penetration of such high molecular weight peptides as neuropeptides to the effector site of the submucosal gland. It is possible that differences in the environments of submucosal glands in different tissues may account for the observed variations in glandular secretory responses. From data on isolated submucosal glands, we have postulated that airway submucosal gland secretion consists of two actions; mucus discharge from secretory cells and ejection of mucus from the duct by glandular contraction. The glandular contraction through myoepithelial cell activity represents an initial and short-time (1 to 2 min) secretory response and is mainly related to reflexly increased secretion in vivo in airwaysubmucosal gland (16, 17). It is important to distinguish mucus squeezing, the short-time response, from secretions in secretory cells for understanding secretory responses to various stimuli in airway submucosal glands. Methods for Glandular Contraction and Glycoprotein Secretion from Secretory Cells The methods used are described in detail in
SUMMARY Our study using feline tracheal isolated submucosal gland preparation has revealed that substance P (SP) produces an increase in submucosal gland secretion through the actions of both mucus ejection by glandular contraction and macromolecule secretion from secretory cells, and that the two actions are both mediated by a peripheral cholinergic action. In contrast, SP has no significant effect on macromolecule secretion from secretory cells in tracheal explants, probably because of epithelial suppression. Our study using an isolated gland preparation has also indicated that VIP potentiates mucous glycoprotein secretion induced by cholinergic stimulation through an interaction between muscarinic and VIP receptors in secretory cells. However, VIP failed to Induce any significant glandular contraction or relaxation, indicating a lack of VIP receptors or a difference in the subtypes of muscarinic receptors in myoepithelial cells in submucosa glands. AM REV RESPIR DIS 1991; 143:S25-S27
our previous reports (5, 16-19) and will be outlined only here. Tracheas were removed from adult cats under anesthesia and fixed in Krebs-Ringer bicarbonate solution. Fresh, unstained submucosal glands were mechanically isolated from the membranous portion of the trachea using two pairs of sharpened tweezers and microscissors (16). For measuring the glandular contraction, isolated single glands wereheld by two sharp glass hooks in an experimental chamber in which warmed (37 0 C) Krebs Ringer bicarbonate solution was circulated (5, 16, 17).The upper hook was connected to a strain transducer for continuous recording of isometric tension. The isolated gland was stimulated with various drugs by superfusing the drug through a micropipette placed near the upper portion of the gland. Measurement of radiolabeled mucus glycoconjugate release was made using the method of Marom and coworkers (22) as modified by us (5, 18, 19).After 16h of incubation with ['Hlglucosamine, the isolated glands or tracheal explants were allowed to further incubate for two successive 2-h periods (Periods I and II). The secretory index was defined as the ratio of radioactivity collected in Period II to that in Period I. The effects of pharmacologic agents were determined by comparing the secretory indices of the drugtreated samples with those of matched, control samples. SP-induced Glandular Contraction SP (10- 12to 10-4M) produced dose-dependent increasesin the contractile response, and maximal tension induced by SP was 70070 of the response to methacholine in the corresponding concentrations. SP-induced contraction is blocked completely by atropine and augmented by neostigmine. Pretreatment with hemicholinium 3, an acetylcholine synthesis inhibitor, inhibited the contractile response to SP. Pretreatment with tetrodotoxin did not inhibit the contractile response to SP (5) (figure 1). Capsaicin induced tension of a magnitude similar to that of SP. These findings
indicate that SP induces glandular contraction, which is related to the squeezing of mucus in ducts and secretory tubules and that this action is mediated by peripheral cholinergic mechanism. Borson and coworkers (9) and Coles and colleagues (10) have reported the short-time response to SP of 14C-Iabeled mucous glycoprotein secretion from explants of canine tracheal mucosa in vitro. Because SP-evoked secretion is followed by a period (10to 20 min after stimulation) of apparent secretion inhibition, they have speculated that SP acts to increase the rate of clearance of mucus from the ducts, probably by induced contraction of secretory tubules and ducts. Our study revealed that SP induces contraction of isolated submucosal glands from feline trachea in a dose-dependent fashion, confirming the speculation of Coles and colleagues (10). SP-inducedGlycoprotein Secretion SP (10-7 M) produced a significant increase (74070 above control) in radiolabeled glycoconjugate release from isolated glands, whereas SP had no significant effects on glycoconjugate release from tracheal explants, probably because of epithelial suppression (5, 19). Atropine abolished SP-evokedglycoconjugate release in isolated glands (figure 2). These findings indicate that SP stimulates radiolabeled glycoconjugate release in isolated submucosal gland, probably involving mucus synthesis and/or cellular secretion, and that this action is also mediated by a peripheral
I From the First Department of Internal Medicine, Tohoku University School of Medicine, Sendai, Japan. 2 Supported by Grants-in-Aid for General Scientific ResearchNo. 6057032and No. 6257043 from the Ministry of Education, Science, and Culture of Japan. J Correspondence and requests for reprints should be addressed to Tamotsu Takishima, M.D., First Department of Internal Medicine, Tohoku University School of Medicine, 1-1 Seiryo-Machi, Sendai 980, Japan.
S25
526
SHIMURA, SASAKI, IKEDA, ISHIHARA, SATO, SASAKI, AND TAKISHIMA
(O,o)
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(0/0)
350
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200
8 150
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Fig. 1. Effects of tetrodotoxin (TTX, 10-7 M), atropine (ATR, 10-6 M), neostigmine (NED, 10-5 M), hemicholinium-3 (HC-3, 10-4 M), and substance P antagonist (SPA, 10-7 M), oPr0 2 , oTrp7.9)-SP or (oPr0 2 , oTrp7, oTrp9)SP on SP-induced contractions in isolated glands. TTX and SP-A did not alter SP-induced contraction. Atropine and HC-3 abolished SP-induced contraction, and neostigmine augmented response by 329% of control values. Single asterisk indicates p < 0.05; double asterisks indicate p < 0,01;NS = not significant. (From reference 5 with permission.)
cholinergic mechanism, the same mechanism as that in SP-evoked myoepithelial contraction described above. Although the present study has not demonstrated directly secretory cells secretion, there is some indirect evidence showing that the SP· induced increase in glycoconjugate release from isolated glands reflects the increase in cellular secretion and/or mucus synthesis. Using electron microscopy, Gashi and coworkers (13)have reported that high doses of SP stimulate acinar cell degranulation in ferret tracheal submucosal gland. Because glandular contraction (mucus squeezing) is complete within 1 min after stimulation, periods of incubation as long as 2 h may involve cell secretion in isolated submucosal glands. Epithelial suppression of cellular secretion (19) and SP-induced glandular contraction without epithelial suppression (5) may be consistent with the short-time response to SP reported by Coles and colleagues (10) and Borson and coworkers (9) using normal tracheal explants, in which mucus squeezing caused by glandular contraction is thought to play the main role.
I
SP
SP
SP
SP-A
ATR
(n=6)
(n=S)
(n=4)
ATR
SP
(n=4)
(n=s)
Fig. 2. 3H-labeled glycoconjugate release from isolated glands (open columns) and tracheal explants (shaded column). Substance P (SP, 10-7 M) induced significant increase in glycoconjugate release from isolated glands (174% of control). SP antagonist (SP-A, 10-7 M), oPr0 2 , oTrp7.9)-SP' did not inhibit SP-induced glycoconjugate release, which did not differ significantly from SPtreated samples. Atropine (ATR, 10-6 M) abolished SPinduced release compared with SP-treated samples. Atropine itself did not alter glycoconjugate release. In tracheal explants, however, SP failed to induce a significant increase in glycoconjugate release compared with control values. Single asterisk indicates p < 0.05; double asterisks indicate p < 0,01; NS = not significant. (From reference 5 with permission.)
isopropylfluorophosphate, cysteine, and ioadoacetic acid. In the present study, a spectrum of peptidase inhibitors and reagents all failed to augment significantly the secretory effect of VIP in tracheal explants (18). These results suggest that local enzymatic degradation may not be a primary route for inactivation of VIP in tracheal explants in the present study, differing from the case of SP-induced mucus secretion (11). Recently,Altiere and Diamond (23) have also reported a lack of influence of various peptidase inhibitors on relaxation induced by VIP in bronchial smooth muscle. VIP Augmentation of Cholinergicinduced Glycoprotein Secretion VIP, at a low concentration that did not pro-
duce any significant increases over control values, produced a 2.4- to 5-fold augmentation of the glycoconjugate release induced by 10-9 to 10-7 M methacholine (18) (figure 4). Atropine or VIP antiserum abolished the augmentation. VIP did not produce any alteration in isoproterenol- nor phenylephrineevoked glycoconjugate secretion. VIP (as much as 10-5 M) did not produce any alteration in the tension, even when the gland had been contracted with methacholine, nor any augmentation of contraction induced by methacholine. These results indicate that VIP induces mucous glycoprotein release from secretory cells and also that it potentiates the secretion induced by cholinergic stimulation. In the present study, the Hill coefficient was calculated to be less than 1 for the doseresponse curve of methacholine alone. Some investigators have suggested that Hill coefficient values for agonists that are significantly less than 1 arise from heterogeneity in the affinity of the muscarinic acetylcholine receptor population (24, 25). Furthermore, the addition of VIP to methacholine produces an increase in the Hill coefficient with a decrease in EC so) . These facts support the idea that VIP enhances glycoconjugate secretion by switching muscarinic receptors to the highaffinity conformer from the low-affinity one in airwaysubmucosal glands, as demonstrated in salivary gland (26). Although VIP is a physiologically important tracheal smooth muscle relaxant, in our study VIP failed to induce any significant glandular contraction or relaxation or to produce any augmentation of methacholineinduced contraction. The lack of effect of VIP on the myoepithelial cells could be due either to a lack of VIP receptors or to a different muscarinic receptor subtype. Recent studies (27)suggest a difference in distribution of the subtypes of muscarinic receptors between tracheal glands and smooth muscles. Such a difference in innervation and receptors in myoepithelial cells and secretory cells is also suggested in the case of adrenergic innerva-
Secretory Index
(% of control)
VIP-induced Glycoprotein Secretion In isolated glands VIP (10- 10 to 10-6 M) produced a dose-dependent increase in 3H-Iabeled glycoconjugate release of as much as 300070 of the control value, which was inhibited by VIP antiserum and not inhibited by atropine, propranolol, or phentolamine. By contrast, in tracheal explants, a much smaller secretory response to VIP than that in isolated glands was observed. Compared with control values, no significant increases in glycoconjugate secretion by tracheal explants were found (18)(figure 3). To examine why VIP is active in isolated glands but not in explants, four peptidase inhibitors and three reagents were added to VIP for both isolated glands and explants. These included bacitracin, phosphoramidon, pepstatin, bestatin, di-
300
~/I I . (6V
250
(4)
200
ttt
*** (5 )
15O
NS
I
I
1/
---
(6) NS
100
0 ....
I
10-. 0 VIP(M)
~~
_6'/
(4) NS
J
Fig. 3. 3H-labeled glycoconjugate secretion induced by VIP from isolated glands (closed circles) and tracheal explants (open circles) expressed as a percentage of control in the secretory index. In isolated glands, VIP produced an increase in a dose-dependent fashion, and significant increases compared with control values were observed at concentrations of 10-8,10-7, and 10-6 M, whereas no significant increase, compared with control values, was observed in tracheal explants. NS = not significant; single asterisk indicates p < 0.05; double asterisks indicate p < 0,01;triple asterisks indicate p < 0.001 compared with control values; single dagger indicates p < 0.05; triple daggers indicate p < 0.001,differences between isolated gland and explant preparation. Number of experiments is shown in parenthesis; bars represent SEM. Mean ± SEM. (From reference 18 with permission.)
527
NEUROPEPTIDES AND AIRWAY SUBMUCOSAL GLAND SECRETION
Fig. 4. Dose-response curves to methacholine (MCh) only and MCh in addition to a low concentration of VIP (10-9 M), which itself does not produce any significant increase over control values in 3H-labeled glycoconjugate release from isolated glands. VIP augmented MCh-evoked secretion by a 2.4to 5.1-fold increment at concentrations of 10-9to 10-6 M MCh. NS = not significant; single asterisk indicates p < 0.05; double asterisks indicate p < 0.Q1; triple asterisks indicate p < 0.001 compared with control values; single dagger indicates p < 0.05; double daggers indicate p < 0.01; triple daggers indicate p < 0.001, differences between the response to MCh alone and that to MCh plus VIP. Number of experiments is shown in parentheses. Bars represent SEM. Open circles = MCh alone; closed circles = MCh + VIP 10-9 M. Mean ± SEM. (From reference 18 with permission.)
Secretory Index
(% of control)
400
350
300
250
200
150
100
tion and ~-adrenergic receptors in airway submucosal glands (5, 16, 17).
Acknowledgment The writers gratefully acknowledge Ms. Reiko Haryu for typing the manuscript.
References 1. Polak JM, Bloom SR. Regulatory peptides and neuronspecific enolase in the respiratory tracts of man and other animals. Exp Lung Res 1982; 3:313-28. 2. Wharton J, Polak JM, Bloom SR, Will JA, Brown MR, Pearse AGE. Substance P-like immunoreactive nervesin mammalian lung. InvestCell Pathol 1979; 2:3-10. 3. Dey RD, Shannon WA Jr, Said SI. Localization of VIP-immunoreactive nerves in airways and pulmonary vessels of dogs, cats, and human subjects. Cell Tissue Res 1981; 220:231-8. 4. Lazarus SC, Basbaum CB, Barnes PJ, Gold WM. cAMP immunocytochemistry provides evidence for functional VIP receptors in trachea. Am J Physiol 1986; 251:C115-9. 5. Shimura S, Sasaki T, Okayama H, Sasaki H, Takishima T. Effect of substance P on mucus secretion of isolated submucosal gland from feline trachea. J Appl Physiol 1987; 63:646-53.
Methacholine (M)
6. Carstairs JR, Barnes PJ. Visualization of vasoactive intestinal peptide receptors in human and guinea pig lung. J Pharmacol Exp Ther 1986; 239:249-55. 7. LeyK, Morice AH, Madonna 0, Sever PS. Autoradiographic localization of VIP receptors in human lung. FEBS Lett 1986; 199:198-202. 8. Baker AP, Hillegass LM, Holden DA, Smith WJ. Effect of kallidin, substance-P, and other basic polypeptides on the production of respiratory macromolecules. Am Rev Respir Dis 1977; 115:811-7. 9. Borson DB, Corrales RJ, Viro N, Nadel JA. Substance P (SP) regulation of 35S04-macromolecule secretion from ferret trachea (abstract). Am Rev Respir Dis 1985; 131:A27. 10. Coles SJ, Neil KH, Reid LM. Potent stimulation of glycoprotein secretion in canine trachea by substance P. J Appl Physiol 1984; 57:1323-7. 11. Borson DB, Corrales R, Varsano S, et al. Enkephalinase inhibitors potentiate substance P-induced secretion of 35S04-macromolecules from ferret trachea. Exp Lung Res 1986; 12:21-36. 12. Coles SJ, Said SI, Reid LM. Inhibition by vasoactive intestinal peptide of glycoconjugate and lysozyme secretion by human airway in vitro. Am Rev Respir Dis 1981; 124:531-6. 13. Gashi AA, Borson DB, Finkbeiner WE, Nadel JA, Basbaum CB. Neuropeptides degranulate se-
rous cells of ferret tracheal glands. Am J Physiol 1986; 251:C223-9. 14. PeatfieldAC, Barnes PJ, Bratcher C, Nadel JA, Davis B. Vasoactiveintestinal peptide stimulatestracheal submucosal gland secretion in ferret. Am Rev Respir Dis 1983; 128:89-93. 15. Webber SE, Widdicombe JG. The effect of vasoactiveintestinal peptide on smooth muscletone and mucus secretion from the ferret trachea. Br J Pharmacol 1987; 91:139-48. 16. Shimura S, Sasaki T, Sasaki H, Takishima T. Contractility of isolated single submucosal gland from trachea. J Appl Physiol 1986; 60:1237-47. 17. Shimura S, Sasaki T, Okayama H, Sasaki H, Takishima T. Neural control of contraction in isolated submucosal gland from feline trachea. J Appl Physiol 1987; 62:2404-9. 18. Shimura S, Sasaki T, Ikeda K, Sasaki H, Takishima T. VIP augments cholinergic-induced glycoconjugate secretion in tracheal submucosal glands. J Appl Physiol 1988; 65:2537-44. 19. Sasaki T, Shimura S, Sasaki H, Takishima T. Effect of epithelium on mucus secretion from feline tracheal submucosal glands. J Appl Physiol 1989; 66:764-70. 20. Bunnett NW. Airway neuropeptides. 2. Release and breakdown. Postsecretory metabolism of peptides. Am Rev Respir Dis 1987;136(Suppl:S27-34). 21. Keltz TN, Straus E, Yalow RS. Degradation of vasoactive intestinal polypeptide by tissue homogenates. Biochem BiophysResCommun 1980; 92:669-74. 22. Marom Z, Shelhamer JH, Kaliner M. Effects of arachidonic acid, monohydroxyeicosatetraenoic acid and prostaglandins on the release of mucus glycoproteins from human airways in vitro. J Clin Invest 1981; 67:1695-702. 23. Altiere RJ, Diamond L. Relaxation of cat tracheobronchial and pulmonary arterial smooth muscle by vasoactiveintestinal peptide: lack of influence by peptidase inhibitors. Br J Pharmacol 1984; 82:321-8. 24. Halvorsesn SW, Nathanson NM. In vivo regulation of muscarinic acetylcholine receptor number and function in embryonic chick heart. J Biol Chern 1981; 256:7941-8. 25. Schmerlik MI, Searles RP. Ligand interactions with membrane-bound porcine atrial muscarinic receptors. Biochemistry 1980; 19:3407-13. 26. Lundberg JM, Hedlund B, Bartfai T. Vasoactive intestinal polypeptide enhances muscarinic ligand binding in cat submandibular salivarygland. Nature 1982; 295:147-9. 27. YangCM, Farley JM, Dwyer TM. Muscarinic stimulation of submucosal glands in swine trachea. J Appl Physiol 1988; 64:200-9.
SUMMARY Our study using feline tracheal isolated submucosal gland preparation has revealed that substance P (SP) produces an increase in submucosal gland secretion through the actions of both mucus ejection by glandular contraction and macromolecule secretion from secretory cells, and that the two actions are both mediated by a peripheral cholinergic action. In contrast, SP has no significant effect on macromolecule secretion from secretory cells in tracheal explants, probably because of epithelial suppression. Our study using an isolated gland preparation has also indicated that VIP potentiates mucous glycoprotein secretion induced by cholinergic stimulation through an interaction between muscarinic and VIP receptors in secretory cells. However, VIP failed to Induce any significant glandular contraction or relaxation, indicating a lack of VIP receptors or a difference in the subtypes of muscarinic receptors in myoepithelial cells in submucosa glands. AM REV RESPIR DIS 1991; 143:S25-S27
our previous reports (5, 16-19) and will be outlined only here. Tracheas were removed from adult cats under anesthesia and fixed in Krebs-Ringer bicarbonate solution. Fresh, unstained submucosal glands were mechanically isolated from the membranous portion of the trachea using two pairs of sharpened tweezers and microscissors (16). For measuring the glandular contraction, isolated single glands wereheld by two sharp glass hooks in an experimental chamber in which warmed (37 0 C) Krebs Ringer bicarbonate solution was circulated (5, 16, 17).The upper hook was connected to a strain transducer for continuous recording of isometric tension. The isolated gland was stimulated with various drugs by superfusing the drug through a micropipette placed near the upper portion of the gland. Measurement of radiolabeled mucus glycoconjugate release was made using the method of Marom and coworkers (22) as modified by us (5, 18, 19).After 16h of incubation with ['Hlglucosamine, the isolated glands or tracheal explants were allowed to further incubate for two successive 2-h periods (Periods I and II). The secretory index was defined as the ratio of radioactivity collected in Period II to that in Period I. The effects of pharmacologic agents were determined by comparing the secretory indices of the drugtreated samples with those of matched, control samples. SP-induced Glandular Contraction SP (10- 12to 10-4M) produced dose-dependent increasesin the contractile response, and maximal tension induced by SP was 70070 of the response to methacholine in the corresponding concentrations. SP-induced contraction is blocked completely by atropine and augmented by neostigmine. Pretreatment with hemicholinium 3, an acetylcholine synthesis inhibitor, inhibited the contractile response to SP. Pretreatment with tetrodotoxin did not inhibit the contractile response to SP (5) (figure 1). Capsaicin induced tension of a magnitude similar to that of SP. These findings
indicate that SP induces glandular contraction, which is related to the squeezing of mucus in ducts and secretory tubules and that this action is mediated by peripheral cholinergic mechanism. Borson and coworkers (9) and Coles and colleagues (10) have reported the short-time response to SP of 14C-Iabeled mucous glycoprotein secretion from explants of canine tracheal mucosa in vitro. Because SP-evoked secretion is followed by a period (10to 20 min after stimulation) of apparent secretion inhibition, they have speculated that SP acts to increase the rate of clearance of mucus from the ducts, probably by induced contraction of secretory tubules and ducts. Our study revealed that SP induces contraction of isolated submucosal glands from feline trachea in a dose-dependent fashion, confirming the speculation of Coles and colleagues (10). SP-inducedGlycoprotein Secretion SP (10-7 M) produced a significant increase (74070 above control) in radiolabeled glycoconjugate release from isolated glands, whereas SP had no significant effects on glycoconjugate release from tracheal explants, probably because of epithelial suppression (5, 19). Atropine abolished SP-evokedglycoconjugate release in isolated glands (figure 2). These findings indicate that SP stimulates radiolabeled glycoconjugate release in isolated submucosal gland, probably involving mucus synthesis and/or cellular secretion, and that this action is also mediated by a peripheral
I From the First Department of Internal Medicine, Tohoku University School of Medicine, Sendai, Japan. 2 Supported by Grants-in-Aid for General Scientific ResearchNo. 6057032and No. 6257043 from the Ministry of Education, Science, and Culture of Japan. J Correspondence and requests for reprints should be addressed to Tamotsu Takishima, M.D., First Department of Internal Medicine, Tohoku University School of Medicine, 1-1 Seiryo-Machi, Sendai 980, Japan.
S25
526
SHIMURA, SASAKI, IKEDA, ISHIHARA, SATO, SASAKI, AND TAKISHIMA
(O,o)
.-------** ------, ,--**---,
(0/0)
350
*
ec 300
ec
o
200
8 150
u
NS-,
;-1
~
IT
'0200
§ 100
NS
NS
NS
NS
NS
.~
~
o
NEO (n=5)
He-3
**
SP-A
(n=3)
(n=3)
Fig. 1. Effects of tetrodotoxin (TTX, 10-7 M), atropine (ATR, 10-6 M), neostigmine (NED, 10-5 M), hemicholinium-3 (HC-3, 10-4 M), and substance P antagonist (SPA, 10-7 M), oPr0 2 , oTrp7.9)-SP or (oPr0 2 , oTrp7, oTrp9)SP on SP-induced contractions in isolated glands. TTX and SP-A did not alter SP-induced contraction. Atropine and HC-3 abolished SP-induced contraction, and neostigmine augmented response by 329% of control values. Single asterisk indicates p < 0.05; double asterisks indicate p < 0,01;NS = not significant. (From reference 5 with permission.)
cholinergic mechanism, the same mechanism as that in SP-evoked myoepithelial contraction described above. Although the present study has not demonstrated directly secretory cells secretion, there is some indirect evidence showing that the SP· induced increase in glycoconjugate release from isolated glands reflects the increase in cellular secretion and/or mucus synthesis. Using electron microscopy, Gashi and coworkers (13)have reported that high doses of SP stimulate acinar cell degranulation in ferret tracheal submucosal gland. Because glandular contraction (mucus squeezing) is complete within 1 min after stimulation, periods of incubation as long as 2 h may involve cell secretion in isolated submucosal glands. Epithelial suppression of cellular secretion (19) and SP-induced glandular contraction without epithelial suppression (5) may be consistent with the short-time response to SP reported by Coles and colleagues (10) and Borson and coworkers (9) using normal tracheal explants, in which mucus squeezing caused by glandular contraction is thought to play the main role.
I
SP
SP
SP
SP-A
ATR
(n=6)
(n=S)
(n=4)
ATR
SP
(n=4)
(n=s)
Fig. 2. 3H-labeled glycoconjugate release from isolated glands (open columns) and tracheal explants (shaded column). Substance P (SP, 10-7 M) induced significant increase in glycoconjugate release from isolated glands (174% of control). SP antagonist (SP-A, 10-7 M), oPr0 2 , oTrp7.9)-SP' did not inhibit SP-induced glycoconjugate release, which did not differ significantly from SPtreated samples. Atropine (ATR, 10-6 M) abolished SPinduced release compared with SP-treated samples. Atropine itself did not alter glycoconjugate release. In tracheal explants, however, SP failed to induce a significant increase in glycoconjugate release compared with control values. Single asterisk indicates p < 0.05; double asterisks indicate p < 0,01; NS = not significant. (From reference 5 with permission.)
isopropylfluorophosphate, cysteine, and ioadoacetic acid. In the present study, a spectrum of peptidase inhibitors and reagents all failed to augment significantly the secretory effect of VIP in tracheal explants (18). These results suggest that local enzymatic degradation may not be a primary route for inactivation of VIP in tracheal explants in the present study, differing from the case of SP-induced mucus secretion (11). Recently,Altiere and Diamond (23) have also reported a lack of influence of various peptidase inhibitors on relaxation induced by VIP in bronchial smooth muscle. VIP Augmentation of Cholinergicinduced Glycoprotein Secretion VIP, at a low concentration that did not pro-
duce any significant increases over control values, produced a 2.4- to 5-fold augmentation of the glycoconjugate release induced by 10-9 to 10-7 M methacholine (18) (figure 4). Atropine or VIP antiserum abolished the augmentation. VIP did not produce any alteration in isoproterenol- nor phenylephrineevoked glycoconjugate secretion. VIP (as much as 10-5 M) did not produce any alteration in the tension, even when the gland had been contracted with methacholine, nor any augmentation of contraction induced by methacholine. These results indicate that VIP induces mucous glycoprotein release from secretory cells and also that it potentiates the secretion induced by cholinergic stimulation. In the present study, the Hill coefficient was calculated to be less than 1 for the doseresponse curve of methacholine alone. Some investigators have suggested that Hill coefficient values for agonists that are significantly less than 1 arise from heterogeneity in the affinity of the muscarinic acetylcholine receptor population (24, 25). Furthermore, the addition of VIP to methacholine produces an increase in the Hill coefficient with a decrease in EC so) . These facts support the idea that VIP enhances glycoconjugate secretion by switching muscarinic receptors to the highaffinity conformer from the low-affinity one in airwaysubmucosal glands, as demonstrated in salivary gland (26). Although VIP is a physiologically important tracheal smooth muscle relaxant, in our study VIP failed to induce any significant glandular contraction or relaxation or to produce any augmentation of methacholineinduced contraction. The lack of effect of VIP on the myoepithelial cells could be due either to a lack of VIP receptors or to a different muscarinic receptor subtype. Recent studies (27)suggest a difference in distribution of the subtypes of muscarinic receptors between tracheal glands and smooth muscles. Such a difference in innervation and receptors in myoepithelial cells and secretory cells is also suggested in the case of adrenergic innerva-
Secretory Index
(% of control)
VIP-induced Glycoprotein Secretion In isolated glands VIP (10- 10 to 10-6 M) produced a dose-dependent increase in 3H-Iabeled glycoconjugate release of as much as 300070 of the control value, which was inhibited by VIP antiserum and not inhibited by atropine, propranolol, or phentolamine. By contrast, in tracheal explants, a much smaller secretory response to VIP than that in isolated glands was observed. Compared with control values, no significant increases in glycoconjugate secretion by tracheal explants were found (18)(figure 3). To examine why VIP is active in isolated glands but not in explants, four peptidase inhibitors and three reagents were added to VIP for both isolated glands and explants. These included bacitracin, phosphoramidon, pepstatin, bestatin, di-
300
~/I I . (6V
250
(4)
200
ttt
*** (5 )
15O
NS
I
I
1/
---
(6) NS
100
0 ....
I
10-. 0 VIP(M)
~~
_6'/
(4) NS
J
Fig. 3. 3H-labeled glycoconjugate secretion induced by VIP from isolated glands (closed circles) and tracheal explants (open circles) expressed as a percentage of control in the secretory index. In isolated glands, VIP produced an increase in a dose-dependent fashion, and significant increases compared with control values were observed at concentrations of 10-8,10-7, and 10-6 M, whereas no significant increase, compared with control values, was observed in tracheal explants. NS = not significant; single asterisk indicates p < 0.05; double asterisks indicate p < 0,01;triple asterisks indicate p < 0.001 compared with control values; single dagger indicates p < 0.05; triple daggers indicate p < 0.001,differences between isolated gland and explant preparation. Number of experiments is shown in parenthesis; bars represent SEM. Mean ± SEM. (From reference 18 with permission.)
527
NEUROPEPTIDES AND AIRWAY SUBMUCOSAL GLAND SECRETION
Fig. 4. Dose-response curves to methacholine (MCh) only and MCh in addition to a low concentration of VIP (10-9 M), which itself does not produce any significant increase over control values in 3H-labeled glycoconjugate release from isolated glands. VIP augmented MCh-evoked secretion by a 2.4to 5.1-fold increment at concentrations of 10-9to 10-6 M MCh. NS = not significant; single asterisk indicates p < 0.05; double asterisks indicate p < 0.Q1; triple asterisks indicate p < 0.001 compared with control values; single dagger indicates p < 0.05; double daggers indicate p < 0.01; triple daggers indicate p < 0.001, differences between the response to MCh alone and that to MCh plus VIP. Number of experiments is shown in parentheses. Bars represent SEM. Open circles = MCh alone; closed circles = MCh + VIP 10-9 M. Mean ± SEM. (From reference 18 with permission.)
Secretory Index
(% of control)
400
350
300
250
200
150
100
tion and ~-adrenergic receptors in airway submucosal glands (5, 16, 17).
Acknowledgment The writers gratefully acknowledge Ms. Reiko Haryu for typing the manuscript.
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Methacholine (M)
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