Galanin and somatostatin inhibition of neurokinin A and B induced airway mucus secretion in the rat

Life seiencts, vol. 57, No. 3, pp. Bw89,1995 Copyright 0 1995 E?kwicr Science Ltd Printed in the USA. All rights resmvcd 0024-3205/95 $950 t .oo

Pergamon

GALANIN

AND SOMATOSTATIN INHIBITION OF NEUROKININ INDUCED AIRWAY MUCUS SECRETION IN THE RAT

U. Wagnerl,

H.C. Fehmann1>3,

D. Bredenbriikerl,

F. Yul, P. J.Barth2

A AND B

and

P. von Wichertl Departments of Internal Medicine 1, Pathology2 Endocrinology3,

and Clinical Research Unit for Gastrointestinal

Philipps-University

35033-Marburg,

of Marburg,

Germany

(Received in final form April 243,1995)

Neurokinin A and B are present in neurons

situated in lung and NK- 1 receptors have been described on tracheal submucosal gland cells. In the present study we compared the ability of substance P (SP), neurokinin A (NKA) and neurokinin B (NKB) to stimulate airway mucus secretion. Furthermore, we characterized the interaction of NKA and NKB with galanin and somatostatin. The rank order of the tachykinins to stimulate airway mucus secretion was SP > NKA > NKB suggesting that NK-1 receptors mediate these effects(ECm:SP: 50 nmobl. NKA: 200 nmohl, NKB: 400 nmol/l). Galanin and somatostatin were equally potent to inhibit NK-A and NK-B stimulated airway mucus release. These results suggest that NK-A and NK-B are potent stimulators of airway macromolecule secretion. Galanin and somatostatin potently inhibit these actions of the tachykinins. Therefore, airway mucus secretion is controlled by a complex network of several different mediators. Key Words: g&&n, somatostatin, neurokinin A and B, airway mucus In the lung several neuropeptides are contained in a heterologous collection of fibres with connections to the central nervous system as well as connections to the upper and lower respiratory tract ( 1). These peptides serve as neurotransmitters, hormones and paracrine mediators (2). In addition, these peptides influence inflammatory cell function by modulating their secretory and chemotacticresponse (3). Thus, these peptides were suggested as mediators involved in the pathophysiology of asthma (4, 5). Airway mucus secretion is controlled by a complex network of neural and endocrine factors, and peptidergic mediators play an important role in the regulation of tracheal macromolecule release (6, 7). For example, mucus secretion is stimulated by VIP, glucagon-like peptide- and amylin (810). Neurokinin A and B are members of the tachykinin-family of peptides, and they are present in neurons situated in lung (11). Recently, it was shown that substance P stimulates airway mucus secretion (10, 12,13). Somatostatin and galanin inhibit substance P induced increase of mucus release (10). In this study we characterized the effects of NK A and NK B on airway mucus secretion from isolated rat trachea and the interaction of both peptides with somatostatin and galanin. __________-________ Correspondence: Dr.HC Fehmann, Department of Internal Medicine, Philipps-University of Marburg. 35033-Marburg, Germany

2&l

GaIanin & Somatostatin Inhibit Tachykinins

Vol. 57, No. 3, 1995

Material and Methods

Substance P, neurokinin A, neurokinin B. somatostatin- 14 and rat galanin were from Bissendorf Peptides (Hannover, Germany). Tissue culture media were from Gibco (Eggenstein, FRG), Na235S04 was from Amersham (Braunschweig, FRG), cellulose dialysis tubings were from Serva (Heidelberg, FRG). Animals Male Sprague-Dawley rats (Zentralinstitutftir Versuchstierzucht, Hannover, FRG) were kept in a light and temperature controlled room. They had free access to water and a rat standard diet (Altromin, Lage, FRG).

Studies on tracheal mucin secretion Rats (average body weight: 4oog) were anesthesized by an intraperitoneal injection of 70 mg/kg b.w. pentobarbitone (Nembutal). The trachea (cranially from the larynx, caudally from bifurcation) was excised from the animals after midline collar incision and median sternotomy and immediately transferred into ice cold culture medium (M-199). The connective tissue was removed and the trachea was opened by an incision along the paries membranaceus. The opened trachea was then mounted between the two halves of a plastic chamber (modified Ussingchamber; ref.14) and 7 ml of medium M-199 in Earl’s balanced salt solution equilibrated with carbogen at 370C (pH 7.4) was added at the mucosal side and submucosal side, respectively. -50 PCi of Na235S04 was added to the submucosal side and was allowed to equilibrate with the tissues for the duration of the experiment. The luminal solution was collected every 15 min and was replaced with fresh unlabeled medium. The samples to analyze mucus secretion were collected as explained in “experimental protocols”. The average value of the initial three collections (min 15, 30, 45) was defined as “basal” secretion for each individual preparation. Samples were transferred into cellulose dialysis tubings (cut off 12000-14000 molecular mass) and dialyzed against destilled water which contained unlabeled SO4 to remove unincorporated Na235S04. Sodium azide ( 10 mg/l) was added to prevent bacterial degradation. The radioactivity associated with the samples was then determined in a liquid scintillation counter. The counts of labeled macromolecules represents the mucus secretion rate. Experimental protocols All experiments were performed on different organ preparations. 1. Studies on the effect of substance P, NK A and NK B on airway mucus secretion: after the determination of basal airway mucus secretion (three fractions every 15 min) substance P, NK A and NK B were applied at the mucosal side at a concentration of I pmol/l and removed with the following sample 15 min later. Subsequently a luminal stimulation with 1 mM acetylcholine was performed as quality control of the bioassay system. 2. Mucosal stimulation was done with 1 pmol/l NK A plus 1 pmol/l somatostatin or l~mol/l galanin, 3. with l~mol/l NK B plus 1 pmol/l somatostatin or 1 pmol/l galanin , 4. with 1 ymolll galanin or 1 pmol/l somatostatin. Our model does not allow longer experiments that would be necessary to study even more complicated protocols (wash-out effects, preincubations).

Statistics The effects are presented relatively to basal secretion (% of basal). Data are presented as +/S.E.M. Statistical analysis was performed with Student’s t-test for unpaired samples. 4 15 experiments for each experimental protocol were performed.

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Gala& & Somatostatin Inhibit Tachykinins

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TABLE I

Stimulatory effects of substance P, neurokinin

A and neurokinin

B on mucus secretion from

isolated rat trachea (% above basal ).

Concentration

Substance P

Neurokinin

A

Neurokinin

101+5

102*3

1

nmolll

120+7

106+3

103+2

10

nmol/l

1.54*19

llti2

IO&3

B

120+7

100 nmolll

Results First, we compared the ability of the different tachykinins to stimulate airway mucus secretion. Substance P stimulated airway macromolecule output maximal at 1 ymol/l (276 + 16 %; tab. I). At this concentration both neurokinin A and B were less potent than substance P (NKA: 185 f 9 %; NKB: 175 + 14 %). Further experiments demonstrated that this is also true for lower peptide concentrations (tab. I). The EC9 for substance P, neurokinin A and neurokinin to stimulate mucus secretion from isolated rat trachea were 50 nmol/l, 200 nmol/l and 400 nmol/l, resp. Both galanin ( 1 pmol/l) and somatostatin (I pmolll) inhibited NKA stimulated mucus secretion from isolated rat trachea (NKA: 1% + 13 %, NKA + galanin: 127 + 6 %, p < 0.05, NKA + somatostatin: 126 + 14 %; p < 0.05). Both peptides inhibited also the NKB induced increase of airway mucus release(NKB: 174 + 14 %. NKB + galanin: 151 + 6%, p < 0.05, NKB + somatostatin: 139 + 14%, p
Discussion The tachykinins NKA and NKB are present in rat lung in sensory nerves and thus they are part of the non adrenergic non cholinergic nervous system (NANC). Recently, it was shown that the tachykinins influence pulmonal blood flow ( 15). Furthermore, the tachykinins are able to activate inflammatory cell function. Thus, they are possibly involved in the pathophysiology of asthma. Results of this study agree to the concept that tachykinin receptor antagonists might be useful in the treatment of asthma. In good agreement to previous results this study indicates that the tachykinins are also involved in the regulaton of an-way mucus secretion. interestingly, NK-I receptors were Iocalizedon tracheal submucosal gland cells ( 15, 16).

Galanin & Somatostatin Inhibit Tachykinins

286

0

Basal

NKA 10dmoVl

Galanin inhibits N&I-stimulated,

T

0

GAL

I Odmol/l

NKAIGAL I Odmol/l

Vol. 57, No. 3, 199.5

ACh I 0.‘mol/l

Fig. 1 but not basal mucus secretion from isolated rat trachea

=

SEM

Basal

NKA I Odmovl

Somatostatin inhibits NKA-stimulated, isolated rat trachea.

SOM 1Odmol/l

NKAISOM 1OdmoUl

ACh

I O.‘moVI

Fig.2 but not basal airway mucus secretion from

Galanin & Somatostatin Inhibit Tachykinins

Vol. 57, No. 3, 1995

“F

0

T

=SEM

Basal

Galanin inhibits NKB-stimulated,

NKB

GAL

NKBIGAL

lO%WVl

1OdmoVl

10dmoVl

ACh I 0.‘moUI

Fig.3 but not basal mucus secretion from isolated rat trachea.

0

Basal

NKB

SOM

NKBlSOM

1OdmoUl

I O”mol/l

I O~“mol/I

Somatostatin inhibits NKEktimulated, trachea.

ACh

1O“mol/I

Fig.4 but not basal mucus secretion from isolated rat

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GaIanin & Somatostatin Inhibit Tachykinins

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Neurokinin-1 receptors are characterized by their ability to bind several ligands with different affinities (17). Substance P is the peptide with the highest affinity to this receptor while NKA and NKB are weaker ligands at the naturally occuring and at the recombinantly expressed NK- 1 receptor. Recently, it was shown that NK-1 receptors are expressed in lung (15, 16). Our data agree to these findings since we found a clear rank order of the tachykinins to stimulate mucus secretion from isolated rat trachea: substance P > NKA > NKB. In several tissues the NK-1 receptor is coupled to the IP3/PKC pathway of signal transduction ( 17). So far, tracheal submucosal gland cells expressing the NK- 1 receptor have not been isolated and their intracellularcoupling is still unknown in this tissue. Interestingly, we could demonstrate in this study that the stimulatory action of NKA and NKB on airway mucus secretion can be inhibited by galanin and somatostatin. The interaction does not occur at the receptor level, since both peptides do not bind to the NK-1 receptor. Since the galanin and somatostatin receptors are also characterized by a high ligand specificity (18, 19) we suggest that the interaction of these peptides occurs at an intracellular post-receptor level. The galanin receptor is able to activate several intracellular effector systems (20). It decreases adenylate cyclase activity as well as galanin activates K+channels which leads to a intracellular Ca2+ levels. Furthermore, hyperpolarization of the plasma membrane but whether this is also the case in mucus secreting cells is unknown (2 1). Several somatostatin receptor subtypes have been isolated by molecular cloning. They are characterized by a distinct organ expression pattern and different affinities to somatostatin -14 and -28 and some somatostatin analogs, respectively (22). Furthermore, they are able to couple to different intracellulareffectors. At least three somatostatin receptors were shown to be expressed in lung (23, 24). In future studies we will address the question which somatostatin receptor subtype is expressed in mucus secreting cells of airways. This will also allow to characterize the coupling of these receptors in this tissue in greater detail. In previous studies we have shown that mucus secretion from airways is regulated by several peptides. Glucagon-like peptide- 1, a product of the intestinal proglucagon processing, amylin, cosecreted with insulin, and substance P increase mucus secretion (8-10, 12, 13). On the other hand, somatostatin and galanin are negative regulators of stimulated mucus release (12). Thus, mucus secretion from airways is regulated by a complex network of mediators with stimulatory and inhibitory effects. The imbalance of these factors or their receptors may contribute to the pathophysiology of airway disease.

Acknowledgement We thank Sabine Koch and Friederike Schwarz for invaluable technical assistance. was supported by the Deutsche Forschungsgemeinschaft (Wa 844/2- 1,844/3- 1).

This study

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Somatostatin Inhibit Tachykinins

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