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Pharmacological evidence for the release of galanin from rat stomach
Neuropeptides (1993) 25,47-50 (0 Longman Group UK Ltd 1993
Pharmacological Evidence for the Release of Galanin from Rat Stomach U. HOLZER-PETSCHE and R. L. MOSER Department of Experimental and Clinical Pharmacology, Karl-Franzens-Univeritfit, 4, A-8010 Graz, Austria (Reprint teque@ to UHP)
Univefsit&splatz
Abstract-The neuropeptide galanin has been shown to occur in nerve fibres in the circular muscle layer of the rat stomach. The present experiments aimed at demonstrating a functional correlate for this observation by testing the motor effects of galanin on circular strips of the rat gastric corpus in vitro. Exogenous galanin elicited only small contractions of the smooth muscle which were dose-related but did not show a clear sigmoid dose-response relationship. These responses were resistant to atropine plus guanethidine or TTX. When the muscle strips were electrically stimulated, they showed pronounced rebound contractions after the end of the stimulus. These rebound contractions were significantly reduced by either desensitizing the strips to galanin or by addition of spantide. It is concluded that galanin is released from the myenteric plexus in the stomach and acts to modulate gastric contractions either postsynaptically or by modifying the release of tachykinins.
Introduction The neuropeptide galanin has been shown to occur in the rat stomach. A dense innervation with galaninirnmunoreactive fibres haa been demonstrated in the myenteric plexus and circular smooth muscle of the gastric corpus with only few galanin-immunoreactive cell bodies, mainly in the myenteric plexus.1-3 Kirchgessner & Gershon4 identified galanininnnunoreactive efferent fibres in the vagus nerves. Galanin released from such nerve fibres might be involved in the motor control of the rat stomach, because the gastric smooth muscle expresses specific binding sites for the peptide. Tatemoto et al6
have already shown that galanin contracts the longitudinal muscle strip of the rat gastric fundus. The action of galanin on this preparation has been studied in detail by Katsoulis et a1,7demonstrating contraction of the rat fundus by galanin via receptors on the smooth muscle. The present experiments were undertaken to elucidate a role for galanin in the circular muscle of the rat corpus, thus defining a f&ctional correlate for the above mentioned histological observations.
Materials and methods Sprague-Dawley rats (Institut f%r Versuchstierkunde, Himberg, Austria) of either sex weighing 250-350 g were fasted overnight with free access
Date received 20 November 1992 Date accepted 22 January 1993
47
48 to tap water and killed by a blow on the head. The stomach was rapidly removed and circular, mucosafree strips were dissected from the gastric corpus. The strips were suspended in 2 ml organ baths containing Krebs solution gassed with 5% C02-95% 02 at 37’C for measurement of isometric contractions.s The muscle strips were equilibrated for 60 min under a basal tension of 10 mN, however, for the experiments with field stimulation in the presence of atropine and guanethidine initial basal tension was 20 mN. Sequential concentration-response curves for galanin were constructed with intervals of 60 min between increasing doses in order to avoid desensitization.v Tetrodotoxin (TTX) was added at a concentration of 1 pM 10 min before galanin. When atropine (0.6 PM) and guanethidine (5 pM) were used, they were present throughout the experiment. Electrical field stimulation (EFS) was performed by means of platinum ring electrodes through which the strip of tissue was threaded. Trains of pulses of 0.3 ms duration, 20 Hz (in normal Krebs) or 10 Hz (in Krebs containing atropine and guanethidine), and supramaximal voltage were delivered for 45 s from a Grass S44 stimulator. The stimulation fiequency was chosen so as to give optimal motor responses of the strips both during stimulation as well as after the end of the stimulus (rebound contraction). The motor responses considered optimal were submaximal; increasing one component of the response by a change in stimulation frequency, however, decreased the other component. The motor responses of the muscle strips to EFS were abolished by 1 l.tM TTX. Desensitization to galanin was obtained by incubating the strips with a galanin concentration of 500 nM for 5 min and delivering the next stimulus without a previous wash. At this time, addition of 50 nM galanin had no effect. Spantide was used at a concentration of 100 PM, which does not discriminate between NKl and MG receptors.‘O Since spantide led to a small contraction of the muscle strips, it was left in contact with the tissue for 5 min, by which time the contraction had subsided. Only then the test stimulus (EFS or peptide) was delivered. Statistical comparisons were made using the Kruskal-Wallis H-test or the Wilcoxon test for paired comparisons, as appropriate. P < 0.05 was considered statistically significant.
NELJFCOPEPTIDES
-
-
-
15
5
50
-
-
150
5OOIlMGAL
Typicaltracingsof the responsesto galaninof isolated circularmusclestripsof rat gastriccorpusin vitro. Fig. 1
Rat galanin was obtained from Bachem (Bubendorf, Switzerland), Substance. P from Cambridge Research Biochemicals (Northwich, UK), and spantide ([D-Arg1,D-Trp7+‘,Leu11] Substance P) was generously supplied by Dr. S. Leander (Fening Pharmaceuticals, Mahno, Sweden). Galanin was dissolved in 0.1 M acetic acid and aliquots were immediately freeze-dried. For each experiment an aliquot was freshly dissolved in isotonic NaCl containing 1% (w/v) bovine serum albumin. Substance P (1 mM) was dissolved in isotonic NaCI, spantide was dissolved in 0.01 M acetic acid at a concentration of 10 mM. All further dilutions were made with isotonic NaCl. Atropine (Merck, Darmstadt, FRG), guanethidine (Ismelina, CibaGeigy, Basel, Switzerland), and tetrodotoxin
a -
f‘ E -6-
z .a :
+J 4.-c a al :20
6
GAL (log
M)
Fig. 2 Concentration-response curves for galaninon isolated circularmuscle stripsof rat gastriccorpus.0 normalKrebssolution (n = 6); 0 Krebs solution with 0.6 FM atropiue and 5 PM guanethidiue (n = 9); 0 Krebs solution with 1 GM TTX (n = 7).
PHARMACOLOGICAL
EVIDENCE
FOR 173~ RELEASE
OF GALANIN
FROM
49
RAT STOMACH
(Sigma, Deisenhofen, FRG) were dissolved in, and diluted with, isotonic NaCI. The volumes added never exceeded 3% of the bath volume. It was verified that the vehicles alone had no effect on the motility of the smooth muscle strips. Results Galanin contracted circular muscle strips of rat gastric corpus in a concentration-dependent manner, although no sigmoid dose-response curve could be established (Figs 1 & 2). The magnitude of the maximal response was rather small, amounting only to 8 f 2% of the response to bethanechol in control preparations (n = 6). These responses were not significantly changed by TTX or atropine plus guanethidine (Fig. 2). EFS led to tonic contractions of the muscle strips during the duration of the stimulus, some strips
0 A
5 mN 1
-22
0
B
1 al
t
before
5
after
cieamdutlofltoGhL Fig. 3
Responses of isolated circulsr muscle strips of rat gastric corpus in vitro to EFS ( 5). (A) normal Krebs solution; (B) Krebs solution with 0.6 ruM atrooine and 5 ,uM oar. ouanethidine. 1 ~~~ ~~.~~_.
dasenmlti2atlon
to GAL
apantlda
mpmtide + deaena. to GAL
Fig. 4
Responses of rat gastric corpus strips to electrical field stimulation before and after desensitization to galanin (A & B), 100 FM spantide (C), and desensitization to galanin plus spantide (D). (A) in normal Krebs solution; (B, C, &D) in Krebs containing atropine plus guanethidine. 0 response during EFS before galanin desensitization or spantide (n = 9); m rebound contraction before galanin desensitization or spantide (n = 8); mresponse during EFS after galanin desensitization or spantide (n = 9); mrebound contraction after galanin desensitization or spantide (n = 8); * P < 0.05 vs response before galanin desensitization or spantide.
showed a biphasic response consisting of a small contraction followed by a small relaxation. Immediately after the end of the stimulation a fast and strong rebound contraction occurred (Fig. 3A & 4A). In the presence of atropine plus guanethidine the tonic response during the stimulus was relaxation only, while the rebound contraction was unaltered (Fig. 3B & 4B). Both in the absence and in the presence of atropine plus guanethidine desensitization to galanin significantly reduced the rebound contraction by 32 k 12% (n = 9) and 20 f 8% (n = 8) respectively without influencing the motor response during stimulation (Fig. 4A & B). A reduction of the rebound contraction by 54 k 12% (n = 9) was also observed in the presence of spantide (Fig. 4C). The combination of galanin desensitization and spantide had no additive effect compared with either treatment alone (reduction by 32 + 6%, n = 9; Fig. 4D). It was verified that galanin desensitization did not influence the motor responses of the muscle strips to substance P (n = 3) and that spantide did not change the responses to galanin (n = 5). Discussion As in other gastrointestinal tissues of the rat,“z7,9JlJ2 galanin exerts a contractile effect on the circular muscle of the gastric corpus. There was, however,
50
NEUROPEPTIDES
no sigmoid dose-response relationship, and the direct motor effect of galanin was very small cornpared with that of bethanechol or of other neuropeptides (e.g. tachykinins; see ref 8). In contrast, the same solutions that induced only small contractions of the gastric corpus evoked responses of the rat ileum similar to those published by Muramatsug (n = 4). Therefore, the small size bf the gastric response is considered to be inherent to the preparation.
Whereas the contraction induced by electrical field stimulation is purely cholinergic in nature, a strong non-cholinergic rebound contraction occurs at the end of the stimulus. Hunt et alI3 have shown that this rebound contraction is caused by the release of tachykinins, since it is abolished by desensitization to substance P. In line with these findings is the ability of spantide to reduce the rebound contraction in the present study. However, the rebound contraction involves also the action of gala&, since it is reduced by galanin desensitization. This can be taken as evidence for a release of galanin at the end of the electrical stimulus. The lack of influence of TTX or atropine and guanethidine as well as the presence of specific binding sites on rat gastric muscle membranes5 point to a site of action of galanin directly on the smooth muscle. In view of the littleness of the response, however, it appears unlikely that galanin plays a major role in the direct contraction of the smooth muscle. Rather, a modulator role is proposed, in that galanin, released from nerve endings in the vicinity of smooth muscle cells, depolarizes the muscle membrane so as to facilitate the generation of action potentials by other excitatory transmitters. An alternative mechanism might be a presynaptic action of galanin: galanin might facilitate presynaptically the release of tachvkinins. which in turn act via recentors on the smooth muscle. This latter mechanism of action would also agree with the lack of an addid
tive effect
I
of cralanin desensitization
and suantide
in
reducing the rebound contraction: when the postsynaptic ulation
action
of the tachykinins
of their
become evident.
presynaptic
_
is blocked,
release
would
a modno more
-
Taken together, these results provide evidence for the release of galanin by electrical field stimulation
and indicate a role for galanin in the modulation of gastric motility. Acknowledgements The work was supported by the Austrian Scientific Research Fund, grant no. P7858, and by the Franz Lanyar Foundation.
References 1. Ekblad, E., Rakaeus, A., H&anson, R. and Sundler, F. (1985). Galanin nerve fibers in the rat gut: distribution. origin id projections. Neuroscience 16: 155-363. 2. Melander, T., H&felt, T., Riikaeus, A, Fabrenkrug, J., Tatemoto, K. and Mutt, V. (1985). Distribution of galaninlike immunoreactivity in the gastro-intestinal tract of several mammalian species. Cell Tissue Research 239: 253-270. 3. Bishop, A. E., Polak, J. M., Bauer, F. E., Christofides, N. D., Carlei, F. and Bloom, S. R. (1986). Occurrence and distribution of a newly discovered peptide, galanin, in the mammalian enteric nervous system. Gut 27: 849-857. 4. Kirchgessner, A. L. and Gershon, M. D. (1989). Identification of vagal efferent fibers and putative target neurons in the enteric nervous system of the rat. Journal of Comparative Neurology 285: 38-53. 5. Rossowski, W. J., Rossowski, T. M., Zacharia, S., Ertan, A. and Coy, D. H. (1990). Galanin binding sites in rat gastric and jejunal smooth muscle membrane preparations. Peptides 11: 333-33s. 6. Tatemoto, K., Rakaeus, A., Jiimvall, H., McDonald, T. J. and Mutt, V. (1983). Galanin - a novel biologically active peptide from porcine intestine. FEBS Letters 164: 124-128. 7. Katsoulis, S., Schmidt, W. E., Schwiirer, H. and Creutzfeldt, W. (1990). Effects of galanin, its analogues and fragments on rat isolated fundus strips. British Journal ofPharmacology 101: 297-300. 8. Holzer-Petsche, U., Lembeck, F. and Se&z, H. (1987). Contractile effects of substance P and neurokinin A on the rat stomach in vivo and in vitro. British Journal of Pharmacology 90: 273-279. 9. Muramatsu, I. and Yanaihara, N. (1988). Contribution of galanin to non-cholinergic, non-adrenergic transmission in rat ileum. British Journal of Pharmacology 94: 1241-1249. 10. Maggi, C. A., Patacchini, R., Feng, D. M. and Folkers, K. (1991). Activity of spantide- and spantideat various tachykinin receptors and NK-2 tachykininreceptor subtypes. Eur. J. Pharmacol. 199: 127-129. 11. Ekblad, E., H&anson, R., Sundler, F. and Wahlestedt, C. (1985). Galanin: neurombdulatory and direct contractile effects on smooth muscle preparations. British Journal of Pharmacology 86: 241-246. 12. Botella, A., Delvaux, M., Frexinos, J. and B&no, L. (1992). Comparative effects of galanin on isolated smooth muscle cells from ileum in five mammalian species. Life Sciences 50: 1253-1261. 13. Hunt, W. B., O’Hagan, D. T. and Wilkinson, J. (1983). Inhibition of the rebound contraction of the rat gastric corpus strip to field stimulation following substance P (SP) desensitization. Journal of Physiology (London) 342: 26P-27P.
Pharmacological Evidence for the Release of Galanin from Rat Stomach U. HOLZER-PETSCHE and R. L. MOSER Department of Experimental and Clinical Pharmacology, Karl-Franzens-Univeritfit, 4, A-8010 Graz, Austria (Reprint teque@ to UHP)
Univefsit&splatz
Abstract-The neuropeptide galanin has been shown to occur in nerve fibres in the circular muscle layer of the rat stomach. The present experiments aimed at demonstrating a functional correlate for this observation by testing the motor effects of galanin on circular strips of the rat gastric corpus in vitro. Exogenous galanin elicited only small contractions of the smooth muscle which were dose-related but did not show a clear sigmoid dose-response relationship. These responses were resistant to atropine plus guanethidine or TTX. When the muscle strips were electrically stimulated, they showed pronounced rebound contractions after the end of the stimulus. These rebound contractions were significantly reduced by either desensitizing the strips to galanin or by addition of spantide. It is concluded that galanin is released from the myenteric plexus in the stomach and acts to modulate gastric contractions either postsynaptically or by modifying the release of tachykinins.
Introduction The neuropeptide galanin has been shown to occur in the rat stomach. A dense innervation with galaninirnmunoreactive fibres haa been demonstrated in the myenteric plexus and circular smooth muscle of the gastric corpus with only few galanin-immunoreactive cell bodies, mainly in the myenteric plexus.1-3 Kirchgessner & Gershon4 identified galanininnnunoreactive efferent fibres in the vagus nerves. Galanin released from such nerve fibres might be involved in the motor control of the rat stomach, because the gastric smooth muscle expresses specific binding sites for the peptide. Tatemoto et al6
have already shown that galanin contracts the longitudinal muscle strip of the rat gastric fundus. The action of galanin on this preparation has been studied in detail by Katsoulis et a1,7demonstrating contraction of the rat fundus by galanin via receptors on the smooth muscle. The present experiments were undertaken to elucidate a role for galanin in the circular muscle of the rat corpus, thus defining a f&ctional correlate for the above mentioned histological observations.
Materials and methods Sprague-Dawley rats (Institut f%r Versuchstierkunde, Himberg, Austria) of either sex weighing 250-350 g were fasted overnight with free access
Date received 20 November 1992 Date accepted 22 January 1993
47
48 to tap water and killed by a blow on the head. The stomach was rapidly removed and circular, mucosafree strips were dissected from the gastric corpus. The strips were suspended in 2 ml organ baths containing Krebs solution gassed with 5% C02-95% 02 at 37’C for measurement of isometric contractions.s The muscle strips were equilibrated for 60 min under a basal tension of 10 mN, however, for the experiments with field stimulation in the presence of atropine and guanethidine initial basal tension was 20 mN. Sequential concentration-response curves for galanin were constructed with intervals of 60 min between increasing doses in order to avoid desensitization.v Tetrodotoxin (TTX) was added at a concentration of 1 pM 10 min before galanin. When atropine (0.6 PM) and guanethidine (5 pM) were used, they were present throughout the experiment. Electrical field stimulation (EFS) was performed by means of platinum ring electrodes through which the strip of tissue was threaded. Trains of pulses of 0.3 ms duration, 20 Hz (in normal Krebs) or 10 Hz (in Krebs containing atropine and guanethidine), and supramaximal voltage were delivered for 45 s from a Grass S44 stimulator. The stimulation fiequency was chosen so as to give optimal motor responses of the strips both during stimulation as well as after the end of the stimulus (rebound contraction). The motor responses considered optimal were submaximal; increasing one component of the response by a change in stimulation frequency, however, decreased the other component. The motor responses of the muscle strips to EFS were abolished by 1 l.tM TTX. Desensitization to galanin was obtained by incubating the strips with a galanin concentration of 500 nM for 5 min and delivering the next stimulus without a previous wash. At this time, addition of 50 nM galanin had no effect. Spantide was used at a concentration of 100 PM, which does not discriminate between NKl and MG receptors.‘O Since spantide led to a small contraction of the muscle strips, it was left in contact with the tissue for 5 min, by which time the contraction had subsided. Only then the test stimulus (EFS or peptide) was delivered. Statistical comparisons were made using the Kruskal-Wallis H-test or the Wilcoxon test for paired comparisons, as appropriate. P < 0.05 was considered statistically significant.
NELJFCOPEPTIDES
-
-
-
15
5
50
-
-
150
5OOIlMGAL
Typicaltracingsof the responsesto galaninof isolated circularmusclestripsof rat gastriccorpusin vitro. Fig. 1
Rat galanin was obtained from Bachem (Bubendorf, Switzerland), Substance. P from Cambridge Research Biochemicals (Northwich, UK), and spantide ([D-Arg1,D-Trp7+‘,Leu11] Substance P) was generously supplied by Dr. S. Leander (Fening Pharmaceuticals, Mahno, Sweden). Galanin was dissolved in 0.1 M acetic acid and aliquots were immediately freeze-dried. For each experiment an aliquot was freshly dissolved in isotonic NaCl containing 1% (w/v) bovine serum albumin. Substance P (1 mM) was dissolved in isotonic NaCI, spantide was dissolved in 0.01 M acetic acid at a concentration of 10 mM. All further dilutions were made with isotonic NaCl. Atropine (Merck, Darmstadt, FRG), guanethidine (Ismelina, CibaGeigy, Basel, Switzerland), and tetrodotoxin
a -
f‘ E -6-
z .a :
+J 4.-c a al :20
6
GAL (log
M)
Fig. 2 Concentration-response curves for galaninon isolated circularmuscle stripsof rat gastriccorpus.0 normalKrebssolution (n = 6); 0 Krebs solution with 0.6 FM atropiue and 5 PM guanethidiue (n = 9); 0 Krebs solution with 1 GM TTX (n = 7).
PHARMACOLOGICAL
EVIDENCE
FOR 173~ RELEASE
OF GALANIN
FROM
49
RAT STOMACH
(Sigma, Deisenhofen, FRG) were dissolved in, and diluted with, isotonic NaCI. The volumes added never exceeded 3% of the bath volume. It was verified that the vehicles alone had no effect on the motility of the smooth muscle strips. Results Galanin contracted circular muscle strips of rat gastric corpus in a concentration-dependent manner, although no sigmoid dose-response curve could be established (Figs 1 & 2). The magnitude of the maximal response was rather small, amounting only to 8 f 2% of the response to bethanechol in control preparations (n = 6). These responses were not significantly changed by TTX or atropine plus guanethidine (Fig. 2). EFS led to tonic contractions of the muscle strips during the duration of the stimulus, some strips
0 A
5 mN 1
-22
0
B
1 al
t
before
5
after
cieamdutlofltoGhL Fig. 3
Responses of isolated circulsr muscle strips of rat gastric corpus in vitro to EFS ( 5). (A) normal Krebs solution; (B) Krebs solution with 0.6 ruM atrooine and 5 ,uM oar. ouanethidine. 1 ~~~ ~~.~~_.
dasenmlti2atlon
to GAL
apantlda
mpmtide + deaena. to GAL
Fig. 4
Responses of rat gastric corpus strips to electrical field stimulation before and after desensitization to galanin (A & B), 100 FM spantide (C), and desensitization to galanin plus spantide (D). (A) in normal Krebs solution; (B, C, &D) in Krebs containing atropine plus guanethidine. 0 response during EFS before galanin desensitization or spantide (n = 9); m rebound contraction before galanin desensitization or spantide (n = 8); mresponse during EFS after galanin desensitization or spantide (n = 9); mrebound contraction after galanin desensitization or spantide (n = 8); * P < 0.05 vs response before galanin desensitization or spantide.
showed a biphasic response consisting of a small contraction followed by a small relaxation. Immediately after the end of the stimulation a fast and strong rebound contraction occurred (Fig. 3A & 4A). In the presence of atropine plus guanethidine the tonic response during the stimulus was relaxation only, while the rebound contraction was unaltered (Fig. 3B & 4B). Both in the absence and in the presence of atropine plus guanethidine desensitization to galanin significantly reduced the rebound contraction by 32 k 12% (n = 9) and 20 f 8% (n = 8) respectively without influencing the motor response during stimulation (Fig. 4A & B). A reduction of the rebound contraction by 54 k 12% (n = 9) was also observed in the presence of spantide (Fig. 4C). The combination of galanin desensitization and spantide had no additive effect compared with either treatment alone (reduction by 32 + 6%, n = 9; Fig. 4D). It was verified that galanin desensitization did not influence the motor responses of the muscle strips to substance P (n = 3) and that spantide did not change the responses to galanin (n = 5). Discussion As in other gastrointestinal tissues of the rat,“z7,9JlJ2 galanin exerts a contractile effect on the circular muscle of the gastric corpus. There was, however,
50
NEUROPEPTIDES
no sigmoid dose-response relationship, and the direct motor effect of galanin was very small cornpared with that of bethanechol or of other neuropeptides (e.g. tachykinins; see ref 8). In contrast, the same solutions that induced only small contractions of the gastric corpus evoked responses of the rat ileum similar to those published by Muramatsug (n = 4). Therefore, the small size bf the gastric response is considered to be inherent to the preparation.
Whereas the contraction induced by electrical field stimulation is purely cholinergic in nature, a strong non-cholinergic rebound contraction occurs at the end of the stimulus. Hunt et alI3 have shown that this rebound contraction is caused by the release of tachykinins, since it is abolished by desensitization to substance P. In line with these findings is the ability of spantide to reduce the rebound contraction in the present study. However, the rebound contraction involves also the action of gala&, since it is reduced by galanin desensitization. This can be taken as evidence for a release of galanin at the end of the electrical stimulus. The lack of influence of TTX or atropine and guanethidine as well as the presence of specific binding sites on rat gastric muscle membranes5 point to a site of action of galanin directly on the smooth muscle. In view of the littleness of the response, however, it appears unlikely that galanin plays a major role in the direct contraction of the smooth muscle. Rather, a modulator role is proposed, in that galanin, released from nerve endings in the vicinity of smooth muscle cells, depolarizes the muscle membrane so as to facilitate the generation of action potentials by other excitatory transmitters. An alternative mechanism might be a presynaptic action of galanin: galanin might facilitate presynaptically the release of tachvkinins. which in turn act via recentors on the smooth muscle. This latter mechanism of action would also agree with the lack of an addid
tive effect
I
of cralanin desensitization
and suantide
in
reducing the rebound contraction: when the postsynaptic ulation
action
of the tachykinins
of their
become evident.
presynaptic
_
is blocked,
release
would
a modno more
-
Taken together, these results provide evidence for the release of galanin by electrical field stimulation
and indicate a role for galanin in the modulation of gastric motility. Acknowledgements The work was supported by the Austrian Scientific Research Fund, grant no. P7858, and by the Franz Lanyar Foundation.
References 1. Ekblad, E., Rakaeus, A., H&anson, R. and Sundler, F. (1985). Galanin nerve fibers in the rat gut: distribution. origin id projections. Neuroscience 16: 155-363. 2. Melander, T., H&felt, T., Riikaeus, A, Fabrenkrug, J., Tatemoto, K. and Mutt, V. (1985). Distribution of galaninlike immunoreactivity in the gastro-intestinal tract of several mammalian species. Cell Tissue Research 239: 253-270. 3. Bishop, A. E., Polak, J. M., Bauer, F. E., Christofides, N. D., Carlei, F. and Bloom, S. R. (1986). Occurrence and distribution of a newly discovered peptide, galanin, in the mammalian enteric nervous system. Gut 27: 849-857. 4. Kirchgessner, A. L. and Gershon, M. D. (1989). Identification of vagal efferent fibers and putative target neurons in the enteric nervous system of the rat. Journal of Comparative Neurology 285: 38-53. 5. Rossowski, W. J., Rossowski, T. M., Zacharia, S., Ertan, A. and Coy, D. H. (1990). Galanin binding sites in rat gastric and jejunal smooth muscle membrane preparations. Peptides 11: 333-33s. 6. Tatemoto, K., Rakaeus, A., Jiimvall, H., McDonald, T. J. and Mutt, V. (1983). Galanin - a novel biologically active peptide from porcine intestine. FEBS Letters 164: 124-128. 7. Katsoulis, S., Schmidt, W. E., Schwiirer, H. and Creutzfeldt, W. (1990). Effects of galanin, its analogues and fragments on rat isolated fundus strips. British Journal ofPharmacology 101: 297-300. 8. Holzer-Petsche, U., Lembeck, F. and Se&z, H. (1987). Contractile effects of substance P and neurokinin A on the rat stomach in vivo and in vitro. British Journal of Pharmacology 90: 273-279. 9. Muramatsu, I. and Yanaihara, N. (1988). Contribution of galanin to non-cholinergic, non-adrenergic transmission in rat ileum. British Journal of Pharmacology 94: 1241-1249. 10. Maggi, C. A., Patacchini, R., Feng, D. M. and Folkers, K. (1991). Activity of spantide- and spantideat various tachykinin receptors and NK-2 tachykininreceptor subtypes. Eur. J. Pharmacol. 199: 127-129. 11. Ekblad, E., H&anson, R., Sundler, F. and Wahlestedt, C. (1985). Galanin: neurombdulatory and direct contractile effects on smooth muscle preparations. British Journal of Pharmacology 86: 241-246. 12. Botella, A., Delvaux, M., Frexinos, J. and B&no, L. (1992). Comparative effects of galanin on isolated smooth muscle cells from ileum in five mammalian species. Life Sciences 50: 1253-1261. 13. Hunt, W. B., O’Hagan, D. T. and Wilkinson, J. (1983). Inhibition of the rebound contraction of the rat gastric corpus strip to field stimulation following substance P (SP) desensitization. Journal of Physiology (London) 342: 26P-27P.