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Effect of three galanin antagonists on the pressor response to galanin in the Cane toad, Bufo marinus
Regulatory Peptides 67 Ž1996. 163–168
Effect of three galanin antagonists on the pressor response to galanin in the Cane toad, Bufo marinus David A. Mahns 1, Gillian P. Courtice
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School of Physiology and Pharmacology, UniÕersity of New South Wales, Sydney, NSW 2052, Australia Received 15 December 1995; revised 22 August 1996; accepted 23 August 1996
Abstract Galanin is a neuropeptide that causes a marked pressor response in several non-mammalian vertebrate species, and some marsupials. In this study, the effect of three galanin antagonists were tested on the pressor response to an intravenous dose Ž6.3 nmolrkg. of porcine galanin in anaesthetised Cane toads, Bufo marinus. Antagonists were injected at either 20 or 50 times the molar dose Ž=MD. of galanin. The antagonist, C7 ŽGalanin 1–13-spantide. reduced the pressor effect of galanin by 32.2 " 6.0% when delivered at 20 = MD Ž n s 4. and by 42.9 " 15.7% when delivered at 50 = MD Ž n s 4. of galanin, the response recovering within 30 min. A second antagonist, M32a ŽGalanin 1–13-NPY 24–36. had no effect on the pressor response to galanin at 20 = MD Ž n s 4., but significantly reduced the pressor effect by 54.8 " 6.4% at 50 = MD Ž n s 5., which also recovered within 30 min. Administration of a third antagonist, galantide or M15 ŽGalanin 1–13-Substance P 5–11., resulted in a profound drop in blood pressure, and did not affect the response to galanin at either dose. In conclusion, C7 and M32a are effective, short-term antagonists of the blood pressure effects of galanin in the toad. Keywords: Neuropeptide; C7; M32a; Galantide; M15; Vasoconstriction
1. Introduction Galanin, a 29 amino acid peptide, was first isolated from porcine intestine in 1983 w1x. Immunohistochemical studies and receptor binding studies have demonstrated a wide distribution of galanin and its receptors in both the central nervous system and the periphery in several vertebrate species w2–5x. It has been shown to have diverse physiological effects in several organ systems w6–9x, including effects in the cardiovascular system. Within the cardiovascular system, galanin has been shown to inhibit the action of the vagus on the heart in some species, and have effects on blood pressure w10–13x. Although in placental mammals, intravenous injection of galanin results in either a slight vasodepressor effect or no effect on blood pressure ŽCat: w10,11x; Dog: w14x. in many other vertebrate species, galanin causes vasoconstriction and an increase in blood pressure ŽToad: w12x; O’possum: w15x; Possum: w13,16x; Sharks: w2x.. In studies on isolated )
Corresponding author. Curlew Biological Services, P.O. Box 67, Pacific Palms, NSW 2428. Australia. Tel. and Fax: Ž61-65. 540-547. 1 Present address: Prince of Wales Medical Research Institute, High Street, Randwick, Sydney. NSW 2031. Australia.
vessels too, galanin has either no effect or causes vasodilatation in mammalian arterial and venous preparations w17,18x, but causes contraction of arterial strips from the intestine of the Atlantic cod w3x and segments of mesenteric arteries from toads ŽMcManus and Courtice, unpublished data. and elasmobranchs w2x. Further elucidation of the physiological role of galanin in the cardiovascular system of various vertebrate species may be aided by the development of effective galanin antagonists. Recently, a number of putative galanin antagonists have become available w9,19,20x and it is the purpose of this paper to report their effects on the pressor response to galanin in a non-mammalian vertebrate, the Cane toad, Bufo marinus. Since the pressor effect of galanin is very marked in this species, it was of interest to know if these effects were reduced by known galanin antagonists. Three putative galanin antagonists known as C7, M32a and Galantide Žor M15. ŽTable 1. were tested. These same three antagonists have been shown to have no effect on the blood pressure response Žvasodepressor. to galanin in anaesthetised cats w21x, although they were all potent at reducing the prolonged cardiac vagal inhibition attributed to galanin release from cardiac sympathetic nerves in cats w21x.
0167-0115r96r$15.00 Copyright q 1996 Elsevier Science B.V. All rights reserved. PII S 0 1 6 7 - 0 1 1 5 Ž 9 6 . 0 0 1 3 3 - 4
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2. Methods Twenty-five toads ranging in weight from 50 to 300 g, were anaesthetised by immersion in a 0.15–0.3% solution of tricaine methanesulphonate ŽMS222, Thomson& Joseph, UK.. Anaesthesia was maintained throughout the experimental period by covering the skin with swabs soaked in MS222. Adequate oxygenation of the animal was maintained by passing a slow stream of moist oxygen through a catheter introduced into the base of the left lung. Gas was allowed to escape via a T-piece in the catheter that was occluded at regular intervals to inflate the lung with oxygen. All experiments were carried out at room temperature Ž22–258C.. Prior to experimentation toads were held in the laboratory at 20–258C for up to 6 weeks. Arterial blood pressure was monitored by the introduction of a catheter into the right femoral artery attached to a Statham ŽP23. blood pressure transducer, and the signal was monitored on a Grass polygraph ŽModel 79.. The blood pressure signal was used to trigger either a linear display unit ŽNeurolog 701. for continuous measurement of pulse interval or a Grass tachograph for measurement of heart rate. To eliminate reflex chronotropic effects on the heart both vagosympathetic trunks were cut high in the neck above the point where the nerves divide into the cardiac, pulmonary, oesophageal and gastric branches. At least 1 h prior to experimentation, an injection of bretylium tosylate ŽBretylate, Wellcome: 10–20 mgrkg. w22x was administered to block sympathetic effects on the heart and blood vessels. For the administration of drugs and peptides, catheters were introduced into both the left and right femoral veins. In order to assess the ability of the three putative antagonists, M15, C7 and M32a ŽAuspep, Australia w21x. to antagonise the pressor response to porcine galanin in the toad, the change in blood pressure to a standard intravenous dose of porcine galanin ŽPeninsula, USA; 6.3 nmolrkg. was recorded. The change in blood pressure was measured as the difference between a steady mean blood
pressure immediately prior to injection of galanin, and the maximal mean blood pressure achieved after injection. The duration of the response was determined as the time taken for blood pressure to return to pre-injection levels. When blood pressure had returned to control levels, one of the antagonists was administered at 20 times the molar dose of porcine galanin Ž126 nmolrkg. or 50 times the molar dose of porcine galanin Ž315 nmolrkg.. Only one dose of one antagonist was tested in any one toad. Four toads were tested for each antagonist at 20 times the molar dose of galanin. In these toads, any changes in blood pressure in response to the antagonist were recorded, and blood pressure allowed to recover fully to pre-injection levels before a second dose of porcine galanin was administered. The maximum increase in blood pressure and duration of the pressor response to the second dose of porcine galanin were compared with the response to the initial galanin injection by a paired t-test. In a second series of toads, the pressor response to an initial dose of porcine galanin Ž6.3 nmolrkg. was assessed prior to the administration of an antagonist at 50 times the molar dose of galanin Ž315 nmolrkg.. In these experiments, a second and a third dose of porcine galanin were given at 10 and 30 min, respectively, after administration of an antagonist. The vascular responses to all three doses of galanin were compared by analysis of variance, followed by a Student-Newman-Keuls multiple comparisons test. M15 and C7 were tested in 4 toads each, and M32a in 5 toads. All results are presented as mean " standard error. In statistical comparisons, a 5% level of significance was accepted.
3. Results 3.1. C7: Galanin 1–13-spantide Administration of C7 at both 20 times and 50 times the molar dose of galanin, significantly inhibited the vascular
Fig. 1. Blood pressure and heart rate traces from an anaesthetised toad showing the vascular response to intravenous injection of porcine galanin before Žleft-hand panel., 10 min after Žmiddle panel. and 30 min after Žright-hand panel. the administration of C7 at 50 times the molar dose of galanin. Ten minutes after administration of C7 the pressor response to galanin was reduced, but recovered by 30 min.
D.A. Mahns, G.P. Courticer Regulatory Peptides 67 (1996) 163–168
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initial injections of porcine galanin caused an elevation of blood pressure of 8.5 " 0.5 mmHg, lasting 19.2 " 3.0 min. The second injection of porcine galanin was administered after full recovery from the small pressor response to C7, and caused an elevation of blood pressure of 5.2 " 0.8 mmHg, which was significantly reduced compared to the initial injection Žpaired t-test P s 0.027.. The duration of the response was not significantly affected Ž15.3 " 0.7 min.. C7 administered at 50 times the molar dose of porcine galanin also significantly reduced the pressor activity of porcine galanin ŽFig. 2A.. At 10 min after the injection of C7, the change in blood pressure response to galanin was significantly reduced Ž3.9 " 1.2 mmHg, lasting 10.3 " 0.3 min. compared with the control dose Ž6.2 " 0.5 mmHg, lasting 13.5 " 0.8 min. ŽANOVA: P s 0.049.. At 30 min the response to the third injection of porcine galanin had recovered Ž6.2 " 0.8, lasting 15.0 " 0.2 min. such that it was not significantly different from the initial response to porcine galanin. 3.2. M32a: Galanin 1–13-NPY 24–36
Fig. 2. Mean changes in arterial blood pressure in response to the administration of porcine galanin Ž6.3 nmolrkg. before Žshaded bars. and after Žopen bars. the administration of ŽA. C7 and ŽB. M32a. Administration of C7, at both 20 times Ž ns 4. and 50 times Ž ns 4. the molar dose significantly reduced the pressor response to galanin. Administration of M32a at 50 times the molar dose of galanin Ž ns 5. significantly reduced the pressor response to galanin after 10 min. The response recovered after 30 min Ž ) P - 0.05..
response to porcine galanin ŽFig. 1.. C7 caused small, direct effects on blood pressure, increasing it by 2.6 " 1.3 mmHg lasting 12.0 " 1.1 min Ž20 = MD., and 3.0 " 0.9 mmHg Ž50 = MD.. Administration of C7 at 20 times the molar dose of porcine galanin significantly reduced the blood pressure effect in response to galanin ŽFig. 2A., but had no effect on the duration of the pressure response. In four animals with resting mean blood pressure of 19.9 " 3.6 mmHg,
Administration of M32a to four toads at 20 times the molar dose of porcine galanin did not affect the pressor activity of porcine galanin ŽFig. 2B.. In this group of animals the mean resting arterial blood pressure was 26.2 " 2.5 mmHg, and administration of M32a caused an increase in blood pressure of 4.3 " 0.5 mmHg, which took more than 20 min to return to control levels in all animals. Galanin Ž6.3 nmolrkg. raised blood pressure by 5.5 " 0.6 mmHg, lasting 22.4 " 0.4 min before the antagonist and 5.7 " 0.6 mmHg, lasting 22.7 " 0.7 min after the antagonist. Administration of M32a at 50 times the molar dose of porcine galanin significantly reduced the pressor effect of galanin tested after 10 min ŽANOVA: P s 0.02. ŽFig. 2B.. Resting mean arterial blood pressure was 19.7 " 3.2 mmHg, and M32a caused a rise in blood pressure of 5.1 " 0.7 mmHg. Injection of galanin Ž6.3 nmolrkg. resulted in an elevation of blood pressure of 6.6 " 1.1 mmHg, lasting 16.3 " 2.8 min during the control period. Ten minutes after the injection of M32a a second galanin injection raised pressure by 2.7 " 0.2 mmHg. Thirty minutes after the administration of M32a galanin raised blood
Fig. 3. Blood pressure trace from an anaesthetised toad in response to two injections of galanin Ž6.3 nmolrkg., before and 10 min after the administration of M15 at 50 times the molar dose Ž315 nmolrkg. of galanin. Injection of M15 produced a marked fall in blood pressure. Ten minutes after injection of M15, galanin caused an increase in blood pressure during the recovery period of the vasodepressor actions of M15.
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pressure by 6.5 " 1.3 mmHg, which was not significantly different from the response to the first galanin injection. 3.3. M15: Galanin 1–13-substance P 5–11 Administration of M15 at 126 nmolrkg Ž20 times the molar dose of porcine galanin. caused a large fall in blood pressure. In four toads the resting mean blood pressure was 24.8 " 1.3 mmHg which fell by 10.3 " 1.1 mmHg after M15. Prior to the administration of M15, porcine galanin Ž6.3 nmolrkg. rasied blood pressure by 6.9 " 1.2 mmHg lasting for 21.4 " 0.5 min. After full recovery of blood pressure to pre-injection levels Ž16.1 " 2.3 min., a second dose of porcine galanin resulted in an elevation of blood pressure of 5.9 " 1.2 mmHg. There was no significant difference between the vascular responses to the first and second injections of porcine galanin Žpaired t-test: maximal change P s 0.58; duration P s 0.41, n s 4.. In a further four animals M15 was administered at 50 times the molar dose which caused a fall in blood pressure of 9.6 " 0.3 mmHg ŽFig. 3.. Galanin Ž6.3 nmolrkg. caused an elevation of blood pressure of 5.9 " 0.9 mmHg lasting 22.3 " 0.5 min before the administrations of M15 Žresting mean blood pressures 19.7 " 2.8 mmHg. and 3.7 " 1.3 mmHg 10 min after the administration of M15. 30 min after administration of M15 galanin raised blood pressure by 5.3 " 1.7 mmHg, lasting 16.1 " 12.3 min. The vascular responses to the three doses of porcine galanin were not significantly different from each other ŽANOVA, P s 0.67.
4. Discussion Of the three putative antagonists to galanin tested here, both M32a and C7 significantly inhibited the blood pressure response to galanin. This is the first demonstration of galanin antagonists affecting the blood pressure responses to galanin. All three antagonists produced direct vascular actions; both M32a and C7 produced a small hypertension, while the third peptide M15 produced a potent and prolonged hypotension. The antagonist known as C7 inhibited the blood pressure response to galanin when administered at both 20 times and 50 times the molar dose of galanin. At 20 times the molar dose, C7 inhibited the maximal blood pressure response by 32.2 " 6.0%, and by 42.9 " 15.7% at 50 times the molar dose. Since the blood pressure response to galanin in the toad is not desensitised with sequential doses of galanin ŽPreston and Courtice, unpublished observations., the response described here is most likely to be directly due to presence of the antagonist. This antagonism was short lived, and the effects of galanin on blood pressure returned within a 30-min period. These results are in contrast to those from the cat, where C7 was shown to cause a slight rise in blood pressure, as it did in the toad, but did not antagonise the blood pressure response Ždepres-
sor. to galanin w21x. However, the prolonged inhibition of cardiac slowing by the vagus in the cat, which has been attributed to galanin, was reduced by C7 w21x. In other physiological systems also, C7 has been shown to antagonise effects attributed to galanin. For example, C7 has been shown to inhibit both the galanin stimulated feeding behaviour of the rat w23,24x as well as the facilatatory role of galanin in the spinal flexor reflex w19x. The second antagonist tested, M32a was derived from an antagonist known as M32 w25x which was made up of galanin 1–13 and NPY 25–36. M32a includes the 24th amino acid of NPY in the NPY fragment 24–36 w21x and has been shown to be potent at antagonising the prolonged cardiac vagal inhibition attributed to galanin in the cat. In the toad M32a itself caused a small increase in blood pressure when administered at either 20 times or 50 times the molar dose. It is likely that this increase is due to the galanin 1–13 portion of M32a ŽTable 1., rather than the NPY 24-36 portion, since NPY 24–36 has been shown to be a potent agonist at the Y2 NPY receptor, but not at the Y1 receptor on the blood vessels in mammals w26x. M32a was effective as an antagonist of the pressor effect of galanin at 50 times the molar dose, but not at 20 times the molar dose. In the latter case, galanin may have been tested after the effective period of M32a, as administration of galanin was delayed until after the direct effects of M32a on blood pressure had subsided, in the order of 20 min. In anesthetised cats also, M32a at 20 times the molar dose failed to inhibit the vascular effects Ždepressor. of galanin w21,27x. It did, however, at the same dose, antagonise the effects of galanin on cardiac vagal action in the cat w21,27x. In the toad, 10 min after the administration of M32a at 50 times the molar dose of galanin, the pressor response to porcine galanin was significantly inhibited by 54.8 " 6.4%. In these experiments, the response recovered within 30 min. Both C7 and M32a, exhibit useful antagonist properties, but have a short duration of action in the toad. Useful application of these tools require tests to be carried out within 10 min of administration. Alternatively, continuous infusion may produce longer periods of efficacy, though this was not tested here. One of the first galanin antagonists to be synthesised was M15, a combination of galanin 1–13 and substance P 5–11 ŽTable 1.. Within other physiological systems, M15 has been shown to cause widespread antagonism to the actions of galanin. It inhibits the release of acetylcholine from the rat hippocampus w28x and striatum w29x and reduces the response to galanin in the hypothalamus w30x. In the periphery, M15 has been shown to block the effect of galanin on glucose-induced insulin secretion using isolated cells from the pancreas, and displaces w 125 Ixgalanin from the membranes of insulin producing Rinm5F cells w31x. In the cardiovascular system of the cat, M15 has been shown to reverse the prolonged cardiac vagal inhibition attributed to galanin w27x.
D.A. Mahns, G.P. Courticer Regulatory Peptides 67 (1996) 163–168
The use of M15 as an antagonist of the hypertensive actions of galanin in the toad was hampered by the prolonged hypotensive actions of M15 itself Žsee Fig. 3.. Marked hypotension was still evident 10 min after administration of M15, the time that other antagonists were shown to be effective. By the time the hypotensive actions of M15 had subsided, the concentration of M15 in the animal may have degraded and become insufficient to affect the response to galanin. A similar hypotensive action of M15 was observed in the cat, which led Ulman and co-workers w27x to propose that M15 exhibited a partial agonist activity, possibly resulting from both the galanin 1–13 portion of the molecule, and the substance P portion. In the cat, the full galanin molecule itself produces a slight hypotensive action w10,11x, and substance P is a potent vasodilator in most mammalian species w32x. In the toad also, substance P has been shown to produce potent vasodilatation w33x, and it is likely that the observed drop in blood pressure in response to M15 in the present study is the result of agonistic activity of the substance P portion of the molecule. Another possible explanation for the lack of antagonistic activity of M15 in the toad, is that M15 contains the amino acid methionine, which may be oxidised in vivo, thus possibly reducing its activity. Whatever the mechanism, M15 was not useful as an antagonist of galanin induced pressor effects in the toad. In conclusion, C7 was found to be a consistent antagonist of the pressor response to galanin, inhibiting the vascular response by 32.2 " 6.0% Ž20 times the molar dose. and 42.9 " 15.7% Ž50 times the molar dose.. M32a proved to be an effective antagonist when administered at 50 times the molar dose, inhibiting vascular activity by 54.8 " 6.4%. M15 is unlikely to be a useful antagonist in whole animal experiments due to its prolonged, direct hypotensive actions.
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Acknowledgements
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This study was supported by the Australian Research Council. We are grateful to Professor Erica Potter for supplying us with the antagonists, and to Clare McManus for expert assistance in the laboratory.
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Effect of three galanin antagonists on the pressor response to galanin in the Cane toad, Bufo marinus David A. Mahns 1, Gillian P. Courtice
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School of Physiology and Pharmacology, UniÕersity of New South Wales, Sydney, NSW 2052, Australia Received 15 December 1995; revised 22 August 1996; accepted 23 August 1996
Abstract Galanin is a neuropeptide that causes a marked pressor response in several non-mammalian vertebrate species, and some marsupials. In this study, the effect of three galanin antagonists were tested on the pressor response to an intravenous dose Ž6.3 nmolrkg. of porcine galanin in anaesthetised Cane toads, Bufo marinus. Antagonists were injected at either 20 or 50 times the molar dose Ž=MD. of galanin. The antagonist, C7 ŽGalanin 1–13-spantide. reduced the pressor effect of galanin by 32.2 " 6.0% when delivered at 20 = MD Ž n s 4. and by 42.9 " 15.7% when delivered at 50 = MD Ž n s 4. of galanin, the response recovering within 30 min. A second antagonist, M32a ŽGalanin 1–13-NPY 24–36. had no effect on the pressor response to galanin at 20 = MD Ž n s 4., but significantly reduced the pressor effect by 54.8 " 6.4% at 50 = MD Ž n s 5., which also recovered within 30 min. Administration of a third antagonist, galantide or M15 ŽGalanin 1–13-Substance P 5–11., resulted in a profound drop in blood pressure, and did not affect the response to galanin at either dose. In conclusion, C7 and M32a are effective, short-term antagonists of the blood pressure effects of galanin in the toad. Keywords: Neuropeptide; C7; M32a; Galantide; M15; Vasoconstriction
1. Introduction Galanin, a 29 amino acid peptide, was first isolated from porcine intestine in 1983 w1x. Immunohistochemical studies and receptor binding studies have demonstrated a wide distribution of galanin and its receptors in both the central nervous system and the periphery in several vertebrate species w2–5x. It has been shown to have diverse physiological effects in several organ systems w6–9x, including effects in the cardiovascular system. Within the cardiovascular system, galanin has been shown to inhibit the action of the vagus on the heart in some species, and have effects on blood pressure w10–13x. Although in placental mammals, intravenous injection of galanin results in either a slight vasodepressor effect or no effect on blood pressure ŽCat: w10,11x; Dog: w14x. in many other vertebrate species, galanin causes vasoconstriction and an increase in blood pressure ŽToad: w12x; O’possum: w15x; Possum: w13,16x; Sharks: w2x.. In studies on isolated )
Corresponding author. Curlew Biological Services, P.O. Box 67, Pacific Palms, NSW 2428. Australia. Tel. and Fax: Ž61-65. 540-547. 1 Present address: Prince of Wales Medical Research Institute, High Street, Randwick, Sydney. NSW 2031. Australia.
vessels too, galanin has either no effect or causes vasodilatation in mammalian arterial and venous preparations w17,18x, but causes contraction of arterial strips from the intestine of the Atlantic cod w3x and segments of mesenteric arteries from toads ŽMcManus and Courtice, unpublished data. and elasmobranchs w2x. Further elucidation of the physiological role of galanin in the cardiovascular system of various vertebrate species may be aided by the development of effective galanin antagonists. Recently, a number of putative galanin antagonists have become available w9,19,20x and it is the purpose of this paper to report their effects on the pressor response to galanin in a non-mammalian vertebrate, the Cane toad, Bufo marinus. Since the pressor effect of galanin is very marked in this species, it was of interest to know if these effects were reduced by known galanin antagonists. Three putative galanin antagonists known as C7, M32a and Galantide Žor M15. ŽTable 1. were tested. These same three antagonists have been shown to have no effect on the blood pressure response Žvasodepressor. to galanin in anaesthetised cats w21x, although they were all potent at reducing the prolonged cardiac vagal inhibition attributed to galanin release from cardiac sympathetic nerves in cats w21x.
0167-0115r96r$15.00 Copyright q 1996 Elsevier Science B.V. All rights reserved. PII S 0 1 6 7 - 0 1 1 5 Ž 9 6 . 0 0 1 3 3 - 4
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2. Methods Twenty-five toads ranging in weight from 50 to 300 g, were anaesthetised by immersion in a 0.15–0.3% solution of tricaine methanesulphonate ŽMS222, Thomson& Joseph, UK.. Anaesthesia was maintained throughout the experimental period by covering the skin with swabs soaked in MS222. Adequate oxygenation of the animal was maintained by passing a slow stream of moist oxygen through a catheter introduced into the base of the left lung. Gas was allowed to escape via a T-piece in the catheter that was occluded at regular intervals to inflate the lung with oxygen. All experiments were carried out at room temperature Ž22–258C.. Prior to experimentation toads were held in the laboratory at 20–258C for up to 6 weeks. Arterial blood pressure was monitored by the introduction of a catheter into the right femoral artery attached to a Statham ŽP23. blood pressure transducer, and the signal was monitored on a Grass polygraph ŽModel 79.. The blood pressure signal was used to trigger either a linear display unit ŽNeurolog 701. for continuous measurement of pulse interval or a Grass tachograph for measurement of heart rate. To eliminate reflex chronotropic effects on the heart both vagosympathetic trunks were cut high in the neck above the point where the nerves divide into the cardiac, pulmonary, oesophageal and gastric branches. At least 1 h prior to experimentation, an injection of bretylium tosylate ŽBretylate, Wellcome: 10–20 mgrkg. w22x was administered to block sympathetic effects on the heart and blood vessels. For the administration of drugs and peptides, catheters were introduced into both the left and right femoral veins. In order to assess the ability of the three putative antagonists, M15, C7 and M32a ŽAuspep, Australia w21x. to antagonise the pressor response to porcine galanin in the toad, the change in blood pressure to a standard intravenous dose of porcine galanin ŽPeninsula, USA; 6.3 nmolrkg. was recorded. The change in blood pressure was measured as the difference between a steady mean blood
pressure immediately prior to injection of galanin, and the maximal mean blood pressure achieved after injection. The duration of the response was determined as the time taken for blood pressure to return to pre-injection levels. When blood pressure had returned to control levels, one of the antagonists was administered at 20 times the molar dose of porcine galanin Ž126 nmolrkg. or 50 times the molar dose of porcine galanin Ž315 nmolrkg.. Only one dose of one antagonist was tested in any one toad. Four toads were tested for each antagonist at 20 times the molar dose of galanin. In these toads, any changes in blood pressure in response to the antagonist were recorded, and blood pressure allowed to recover fully to pre-injection levels before a second dose of porcine galanin was administered. The maximum increase in blood pressure and duration of the pressor response to the second dose of porcine galanin were compared with the response to the initial galanin injection by a paired t-test. In a second series of toads, the pressor response to an initial dose of porcine galanin Ž6.3 nmolrkg. was assessed prior to the administration of an antagonist at 50 times the molar dose of galanin Ž315 nmolrkg.. In these experiments, a second and a third dose of porcine galanin were given at 10 and 30 min, respectively, after administration of an antagonist. The vascular responses to all three doses of galanin were compared by analysis of variance, followed by a Student-Newman-Keuls multiple comparisons test. M15 and C7 were tested in 4 toads each, and M32a in 5 toads. All results are presented as mean " standard error. In statistical comparisons, a 5% level of significance was accepted.
3. Results 3.1. C7: Galanin 1–13-spantide Administration of C7 at both 20 times and 50 times the molar dose of galanin, significantly inhibited the vascular
Fig. 1. Blood pressure and heart rate traces from an anaesthetised toad showing the vascular response to intravenous injection of porcine galanin before Žleft-hand panel., 10 min after Žmiddle panel. and 30 min after Žright-hand panel. the administration of C7 at 50 times the molar dose of galanin. Ten minutes after administration of C7 the pressor response to galanin was reduced, but recovered by 30 min.
D.A. Mahns, G.P. Courticer Regulatory Peptides 67 (1996) 163–168
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initial injections of porcine galanin caused an elevation of blood pressure of 8.5 " 0.5 mmHg, lasting 19.2 " 3.0 min. The second injection of porcine galanin was administered after full recovery from the small pressor response to C7, and caused an elevation of blood pressure of 5.2 " 0.8 mmHg, which was significantly reduced compared to the initial injection Žpaired t-test P s 0.027.. The duration of the response was not significantly affected Ž15.3 " 0.7 min.. C7 administered at 50 times the molar dose of porcine galanin also significantly reduced the pressor activity of porcine galanin ŽFig. 2A.. At 10 min after the injection of C7, the change in blood pressure response to galanin was significantly reduced Ž3.9 " 1.2 mmHg, lasting 10.3 " 0.3 min. compared with the control dose Ž6.2 " 0.5 mmHg, lasting 13.5 " 0.8 min. ŽANOVA: P s 0.049.. At 30 min the response to the third injection of porcine galanin had recovered Ž6.2 " 0.8, lasting 15.0 " 0.2 min. such that it was not significantly different from the initial response to porcine galanin. 3.2. M32a: Galanin 1–13-NPY 24–36
Fig. 2. Mean changes in arterial blood pressure in response to the administration of porcine galanin Ž6.3 nmolrkg. before Žshaded bars. and after Žopen bars. the administration of ŽA. C7 and ŽB. M32a. Administration of C7, at both 20 times Ž ns 4. and 50 times Ž ns 4. the molar dose significantly reduced the pressor response to galanin. Administration of M32a at 50 times the molar dose of galanin Ž ns 5. significantly reduced the pressor response to galanin after 10 min. The response recovered after 30 min Ž ) P - 0.05..
response to porcine galanin ŽFig. 1.. C7 caused small, direct effects on blood pressure, increasing it by 2.6 " 1.3 mmHg lasting 12.0 " 1.1 min Ž20 = MD., and 3.0 " 0.9 mmHg Ž50 = MD.. Administration of C7 at 20 times the molar dose of porcine galanin significantly reduced the blood pressure effect in response to galanin ŽFig. 2A., but had no effect on the duration of the pressure response. In four animals with resting mean blood pressure of 19.9 " 3.6 mmHg,
Administration of M32a to four toads at 20 times the molar dose of porcine galanin did not affect the pressor activity of porcine galanin ŽFig. 2B.. In this group of animals the mean resting arterial blood pressure was 26.2 " 2.5 mmHg, and administration of M32a caused an increase in blood pressure of 4.3 " 0.5 mmHg, which took more than 20 min to return to control levels in all animals. Galanin Ž6.3 nmolrkg. raised blood pressure by 5.5 " 0.6 mmHg, lasting 22.4 " 0.4 min before the antagonist and 5.7 " 0.6 mmHg, lasting 22.7 " 0.7 min after the antagonist. Administration of M32a at 50 times the molar dose of porcine galanin significantly reduced the pressor effect of galanin tested after 10 min ŽANOVA: P s 0.02. ŽFig. 2B.. Resting mean arterial blood pressure was 19.7 " 3.2 mmHg, and M32a caused a rise in blood pressure of 5.1 " 0.7 mmHg. Injection of galanin Ž6.3 nmolrkg. resulted in an elevation of blood pressure of 6.6 " 1.1 mmHg, lasting 16.3 " 2.8 min during the control period. Ten minutes after the injection of M32a a second galanin injection raised pressure by 2.7 " 0.2 mmHg. Thirty minutes after the administration of M32a galanin raised blood
Fig. 3. Blood pressure trace from an anaesthetised toad in response to two injections of galanin Ž6.3 nmolrkg., before and 10 min after the administration of M15 at 50 times the molar dose Ž315 nmolrkg. of galanin. Injection of M15 produced a marked fall in blood pressure. Ten minutes after injection of M15, galanin caused an increase in blood pressure during the recovery period of the vasodepressor actions of M15.
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pressure by 6.5 " 1.3 mmHg, which was not significantly different from the response to the first galanin injection. 3.3. M15: Galanin 1–13-substance P 5–11 Administration of M15 at 126 nmolrkg Ž20 times the molar dose of porcine galanin. caused a large fall in blood pressure. In four toads the resting mean blood pressure was 24.8 " 1.3 mmHg which fell by 10.3 " 1.1 mmHg after M15. Prior to the administration of M15, porcine galanin Ž6.3 nmolrkg. rasied blood pressure by 6.9 " 1.2 mmHg lasting for 21.4 " 0.5 min. After full recovery of blood pressure to pre-injection levels Ž16.1 " 2.3 min., a second dose of porcine galanin resulted in an elevation of blood pressure of 5.9 " 1.2 mmHg. There was no significant difference between the vascular responses to the first and second injections of porcine galanin Žpaired t-test: maximal change P s 0.58; duration P s 0.41, n s 4.. In a further four animals M15 was administered at 50 times the molar dose which caused a fall in blood pressure of 9.6 " 0.3 mmHg ŽFig. 3.. Galanin Ž6.3 nmolrkg. caused an elevation of blood pressure of 5.9 " 0.9 mmHg lasting 22.3 " 0.5 min before the administrations of M15 Žresting mean blood pressures 19.7 " 2.8 mmHg. and 3.7 " 1.3 mmHg 10 min after the administration of M15. 30 min after administration of M15 galanin raised blood pressure by 5.3 " 1.7 mmHg, lasting 16.1 " 12.3 min. The vascular responses to the three doses of porcine galanin were not significantly different from each other ŽANOVA, P s 0.67.
4. Discussion Of the three putative antagonists to galanin tested here, both M32a and C7 significantly inhibited the blood pressure response to galanin. This is the first demonstration of galanin antagonists affecting the blood pressure responses to galanin. All three antagonists produced direct vascular actions; both M32a and C7 produced a small hypertension, while the third peptide M15 produced a potent and prolonged hypotension. The antagonist known as C7 inhibited the blood pressure response to galanin when administered at both 20 times and 50 times the molar dose of galanin. At 20 times the molar dose, C7 inhibited the maximal blood pressure response by 32.2 " 6.0%, and by 42.9 " 15.7% at 50 times the molar dose. Since the blood pressure response to galanin in the toad is not desensitised with sequential doses of galanin ŽPreston and Courtice, unpublished observations., the response described here is most likely to be directly due to presence of the antagonist. This antagonism was short lived, and the effects of galanin on blood pressure returned within a 30-min period. These results are in contrast to those from the cat, where C7 was shown to cause a slight rise in blood pressure, as it did in the toad, but did not antagonise the blood pressure response Ždepres-
sor. to galanin w21x. However, the prolonged inhibition of cardiac slowing by the vagus in the cat, which has been attributed to galanin, was reduced by C7 w21x. In other physiological systems also, C7 has been shown to antagonise effects attributed to galanin. For example, C7 has been shown to inhibit both the galanin stimulated feeding behaviour of the rat w23,24x as well as the facilatatory role of galanin in the spinal flexor reflex w19x. The second antagonist tested, M32a was derived from an antagonist known as M32 w25x which was made up of galanin 1–13 and NPY 25–36. M32a includes the 24th amino acid of NPY in the NPY fragment 24–36 w21x and has been shown to be potent at antagonising the prolonged cardiac vagal inhibition attributed to galanin in the cat. In the toad M32a itself caused a small increase in blood pressure when administered at either 20 times or 50 times the molar dose. It is likely that this increase is due to the galanin 1–13 portion of M32a ŽTable 1., rather than the NPY 24-36 portion, since NPY 24–36 has been shown to be a potent agonist at the Y2 NPY receptor, but not at the Y1 receptor on the blood vessels in mammals w26x. M32a was effective as an antagonist of the pressor effect of galanin at 50 times the molar dose, but not at 20 times the molar dose. In the latter case, galanin may have been tested after the effective period of M32a, as administration of galanin was delayed until after the direct effects of M32a on blood pressure had subsided, in the order of 20 min. In anesthetised cats also, M32a at 20 times the molar dose failed to inhibit the vascular effects Ždepressor. of galanin w21,27x. It did, however, at the same dose, antagonise the effects of galanin on cardiac vagal action in the cat w21,27x. In the toad, 10 min after the administration of M32a at 50 times the molar dose of galanin, the pressor response to porcine galanin was significantly inhibited by 54.8 " 6.4%. In these experiments, the response recovered within 30 min. Both C7 and M32a, exhibit useful antagonist properties, but have a short duration of action in the toad. Useful application of these tools require tests to be carried out within 10 min of administration. Alternatively, continuous infusion may produce longer periods of efficacy, though this was not tested here. One of the first galanin antagonists to be synthesised was M15, a combination of galanin 1–13 and substance P 5–11 ŽTable 1.. Within other physiological systems, M15 has been shown to cause widespread antagonism to the actions of galanin. It inhibits the release of acetylcholine from the rat hippocampus w28x and striatum w29x and reduces the response to galanin in the hypothalamus w30x. In the periphery, M15 has been shown to block the effect of galanin on glucose-induced insulin secretion using isolated cells from the pancreas, and displaces w 125 Ixgalanin from the membranes of insulin producing Rinm5F cells w31x. In the cardiovascular system of the cat, M15 has been shown to reverse the prolonged cardiac vagal inhibition attributed to galanin w27x.
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The use of M15 as an antagonist of the hypertensive actions of galanin in the toad was hampered by the prolonged hypotensive actions of M15 itself Žsee Fig. 3.. Marked hypotension was still evident 10 min after administration of M15, the time that other antagonists were shown to be effective. By the time the hypotensive actions of M15 had subsided, the concentration of M15 in the animal may have degraded and become insufficient to affect the response to galanin. A similar hypotensive action of M15 was observed in the cat, which led Ulman and co-workers w27x to propose that M15 exhibited a partial agonist activity, possibly resulting from both the galanin 1–13 portion of the molecule, and the substance P portion. In the cat, the full galanin molecule itself produces a slight hypotensive action w10,11x, and substance P is a potent vasodilator in most mammalian species w32x. In the toad also, substance P has been shown to produce potent vasodilatation w33x, and it is likely that the observed drop in blood pressure in response to M15 in the present study is the result of agonistic activity of the substance P portion of the molecule. Another possible explanation for the lack of antagonistic activity of M15 in the toad, is that M15 contains the amino acid methionine, which may be oxidised in vivo, thus possibly reducing its activity. Whatever the mechanism, M15 was not useful as an antagonist of galanin induced pressor effects in the toad. In conclusion, C7 was found to be a consistent antagonist of the pressor response to galanin, inhibiting the vascular response by 32.2 " 6.0% Ž20 times the molar dose. and 42.9 " 15.7% Ž50 times the molar dose.. M32a proved to be an effective antagonist when administered at 50 times the molar dose, inhibiting vascular activity by 54.8 " 6.4%. M15 is unlikely to be a useful antagonist in whole animal experiments due to its prolonged, direct hypotensive actions.
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Acknowledgements
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This study was supported by the Australian Research Council. We are grateful to Professor Erica Potter for supplying us with the antagonists, and to Clare McManus for expert assistance in the laboratory.
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