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Antiinflammatory activity of adrenomedullin in the acetic acid peritonitis in rats
ELSEVIER
PII SOO24-3205(99)00406-3
Life Sciences Vol. 65, No. 15, pp. PL 203-208, 1999 Copyright 0 1999 Elsevia Science Inc. printed in the USA. All rights reserved 0024-3205/99/S-see hnt matter
PHARU4COLOGY LElTERS Accelerated Communication
ANTIINFLAMMATORY ACTIVITY OF ADRENOMEDULLIN IN THE ACETIC ACID PERITONITIS IN RATS
Giuseppe Clementi, Antonina Caruso, Vincenza Maria Catena Cutuli, Agatina Prato, Nunzio Guide Mangano and Matilde Amico-Roxas.
Institute of Pharmacology, University of Catania, School of Medicine, Viale A. Doria 6, Catania, Italy. (Submitted March 1, 1999;accepted April 19,1999; received in final form June 9, 1999)
Abstract. The antiinflammatory effect of ADM was studied in different models of inflammation and compared to the one of CGRP. Peptides were active against acetic acid-induced peritonitis in the rats. ADM and CGRP exerted the antiinflammatory effect at different doses, 400 and 20 ng/kg respectively, but with different efficacy (ADM >CGRP). This effect was blocked by pretreatment with CGRP (8-37) fragment or with L-NAME. No antiinflammatory activity was evidenced against serotonin - or carrageenin - induced rat paw edema. Our data suggest that ADM exerts antiinflammatory activity in the model characterized by a vascular component. This effect involves CGRP receptors and appears to be mediated by nitric oxide system. 0 1999ElsevierScience Inc. Key Words: adrenomedullin, CGRP, inflammation
lntroductiin Adrenomedullin (ADM) is a 52 amino-acid peptide, originally isolated from a human adrenal pheochromocytoma by using a detection system based on its ability to elevate platelet CAMP levels (1, 2). It is considered to be a member of the calcitonin gene-related peptide (CGRP) superfamily, including CGRP, amylin and calcitonin 13). lmmunoreactive ADM has been detected by RIA in many tissues, including normal adrenal medulla, heart, kidney, pancreas, intestine and plasma (1, 4). Vasodilation seems to be the main physiological effect of ADM (2) and its hypotensive activity is second only to CGRP (5, 6). Other effects of ADM include bronchodilation (7) inhibition of angiotensin - induced aldosterone secretion (8) and inhibition of gastric emptying in rats (9). Moreover, we have recently shown the gastroprotective effect of ADM in reserpine - induced gastric lesions and have suggested that this effect depends on improvement of the blood microcirculation in gastric mucosal through an involvement of CGRP receptors (10).
Corresponding Author: dr. Giuseppe Clementi, Institute of Pharmacology, University of Catania, School of Medicine, viale Andrea Doria 6,95125 Catania (Italy). Fax: +39-095-333219.
PL-204
Adrenomednllin and Inflammation
It has been also suggested that ADM may play an important airway inflammation via a CAMP-dependent mechanism (11).
Vol. 65, No. 15, 1999
role in the regulation
of
On the other hand we have shown the antiinflammatory effect of CGRP and AMY in some experimental models which are characterized by a vascular component (12). Considering that the biological activity profiles in the cardiovascular system of CGRP and of ADM are similar (3, 13), in the present study we have verified the possible antiinflammatory activity of the ADM in some experimental models studying the possible mechanisms involved.
Materials and methods Animals: Male Sprague-Dawley (230-240 g) were starved for 48 h before use but were allowed free access to tap water, which was removed 1 h before the experiment. Room temperature was maintained at 22 +l’C; 65 +- 5% relative humidity; 12-h light/dark. Acetic acid peritonitis test: Groups of five rats were tested by the method of ArrigoniMartelli (14). Peritonitis was produced by i.p. injection of 10 ml/kg of a 0.5% acetic acid solution. Thirty minutes later, the rats were killed by ether inhalation and the peritonitis exudate collected and measured. Human ADM (50 to 600 ng/kg) or CGRP (0.01 to 0.06 pg/kg) were administered i.p. in 5 ml/kg saline, 5 min before acetic acid challenge; control animals received the same amount of saline. To evaluate the time-course of the antiexudative activity of ADM, the dose used was 400 ng/kg while the times of administration varied (0, 15, 60 and 120 min before irritant agent). A group of rats were injected with CGRP (8-37) (lug/kg i.p.) 5 min before ADM (400 @kg) or CGRP (20 ng/kg). Another group of rats received 50 mg/kg of NG-nitro-L-arginine methyl ester (L-NAME) 5 min before the administration of the peptides. Carrageenin-induced rat paw edema: Groups of five rats were used. Edema was produced in the right hind paw by subplantar of 0.1 ml of 1% carrageenin solution. The volume was measured by a mercury plethysmometer prior to the injection of carrageenin and 3 h later. The increase in volume of the paw 3 h after injection of carrageenin was adopted as a measure of edema. Swelling in treated animals was calculated as the percentage of inhibition in comparision to the controls. Serotonin-induced rat paw edema: Groups of five rats were used. Edema was produced in the right hind paw by subplantar injection of 0.1 ml 0.005% serotonin in sterile saline. The volume of the paw was measured by a mercury plethysmometer before and 45 min after serotonin challenge; edema was determined by difference in volume. ADM or CGRP were injected at the doses of 400 or 20 nglkg S.C. respectively, 15 min before serotonin. Chemical and drugs: Human ADM, CGRP and CGRP 8-37 fragment were purchased from Peninsula Laboratories Europe Ldt. (St. Helens Merseyside); serotonin, carrageenin, acetic acid and L-NAME were purchased from Sigma (Italy). Statistical analysis: Statistical analysis was performed using Dunnett’s test for single and multiple comparitions. The level of significance was set at ~~0.05.
PL-205
Vol. 65, No. 15, 1999
Results In the acetic acid-induced peritonitis test ADM exerts the maximal activity (69%) at the dose of 400 ng/kg (table I) TABLE I Antiexudative effect of ADM or CGRP on acetic acid-induced peritonitis in rats (n=5). Treatment Saline ADM
Dose Wkg __ 50 100 200 400 600
Exudate volume ml (mean f S.E.) 1.88 f 0.06 1.52 i 1.30 f 1.02 f 0.58 f 1.04 f
0.10 0.03 0.06 0.04 0.05
Percent Inhibition __
(Dunn& test) _-
19
n.s.
31 46 69 44
co.05 (0.05 co.05 co.05
25 10 co.05 1.40 f 0.03 39 20 co.05 1.14 * 0.04 26 40 co.05 1.36 f 0.05 n.s. 12 60 1.66 f 0.13 ADM or CGRP or saline (2 ml/kg s.c.) were injected 15 min before 0.5% acetic acid (10 ml/kg i.p.). The volume of peritoneal exudate was evaluated 30 min after acetic acid injectiin. CGRP
and when it is administered 15 min before of the acetic acid. However a significant antiinflammatory activity is also present when the peptide is administered simultaneously (55%) or 60 min (48%) before of the acetic acid (table II). TABLE II Tieme-course of antiexudative effect of ADM and CGRP on acetic acid-induced peritonitis in rats (n--5).
Saline
Time (min) 0
ADM CGRP
0 0
Treatment
Saline
15
ADM CGRP
15 15
Saline
60
ADM CGRP
60 60
Exudate volume ml (mean f S.E.) 1.90 f 0.09 0.66 f 0.05 1.40 f 0.04 1.68 f 0.06 0.56 f 0.04 1.14 f 0.04 1.90 f 0.06 0.98 f 0.06 I .42 f 0.05
Percent Inhibition
(Dunn& test) -_
55 26
eo.05 co.05
69 39
co.05 co.05
__
__ 48 25
X0.05 X0.05
__ -_ 120 Saline 1.92 f 0.11 n.s. 19 120 ADM 1.56* 0.08 n.s. 7 120 CGRP 1.78 f 0.09 ADM (400 @kg s.c.), CGRP (20 ng/kg s.c.) or saline (2 ml/kg s.c.) were enjected at the fuced time before 0.5% acetic acid (10 ml/kg i.p.). The volume of peritoneal exudate was evaluated 30 min after acetii acid injedon.
PL-206
Adrenomedullin and Iufhmmation
Vol. 65, No. 15, 1999
The maximal antiinflammatory activity (39%) of CGRP is present at the dose of 20 nglkg. At this dose the peptide causes about 57% of exudation more than ADM. Pretreatment with CGRP (8-37), at the dose of 1 uglkg, blocks the antiiflammatory effect of both peptides. Pretreatment with L-NAME, at the dose of 50 mg/kg, also prevents the antiinflammatory activity of ADM and CGRP (fig.1). No protective effect is observed either in serotonin - or carrageenin induced paw edema.
I I.‘,‘I.“‘I.‘.‘I.“‘I”“I‘“‘I”‘.I”.’I”””””
0
10
20
30
40
Xl
60
70
60
90
100
% bbibition Fig.1
ADM, CGRP or saline (2 ml/kg s.c.) were injected 15 min before 0.5 acetic acid (10 mlikg i.p.). CGRP (8-37) (l@kg) or NAME (50 mg!kg were administered i.p. 5 min before ADM, CGRP or saline. The volume of peritoneal exudate was evaluated 30 min after acetic acid injection. In the control group the volume of exudate was 1.92 f 0.08 ml. * ~0.05 vs CGFU’ ** p
Discussion The present results show the antiinflammatory activity of ADM in an acetic acid peritonitis test and indicate that this peptide exerts its antiinflammatory effect through CGRP receptors and the NO system. Our study suggests that ADM exerts the maximal antiinflammatory effect at the dose of 400; higher doses produce lower effects, exhibiting a bell-shaped curve. This behavior might be explained as the effect of the interaction with two or more receptors, involving opposite actions (15) or by the existence of multiplestate cell, signaling pathways (16). A better characterization of ADM receptors is necessary to allow a better understanding of this effect.
Vol. 65, No. 15, 1999
Adrenomedullin and Inflammation
PL-207
Our data suggest an involvement of CGRP receptors since the antiinflammatory effect of both peptides is blocked by human CGRP (8-37). It agrees with cardiovascular activities of ADM inhibited after administration of the CGRP antagonist, CGRP (8-37) (5). So it is possible that ADM acts at the sites of CGRP, determining a relaxation of endothelian cells and reduction of formation of endothelian gaps and thus interfering with the activities of proinflammatory mediators. The possibility that the antiinflammatory effect of ADM depends, at least in part, on its vascular activity is supported by the fact that the peptide exerts protective effect in inflammation induced by acetic acid, which has been claimed to be primarily a vascular irritant. No antiinflammatory effect is present in inflammatory models mostly due to histamine, serotonin and bradykinin release, like carragenin - and serotonin induced plasma extravasation. On the other hand it is possible that different mechanisms can be involved in the antiinflammatory effect of ADM; in fact there is evidence that this peptide inhibits the secretion of cytokine-induced neutrophil chemoattractant, a member of interleukine-8 family, from lypopolysaccharide - stimulation rat alveolar macrofages “in vitro” (11). This study confirms the protective effect of CGRP in the same test and suggests that its antiinflammatory activity is obtained at a dose 20 times lower than ADM. Also in this case higher doses produce lower effects, exhibiting bell-sharped curve. This evidence agrees with the results of Raud et al. (17) that suggest an antiinflammatory role of CGRP, but it is in contrast with other results that show an proinflammatory role of the peptide (18, 19). We think that these discordant findings may be due to different doses and experimental models used. Even though CGRP shows an antiinflammatory activity at lower doses than ADM, it determines a lower pharmacological efficacy. We think that it is due to the greater vasodilation activity of CGRP, in fact this effect can interfere negatively with the blood microcirculation. Our results also suggest that the antiinflammatory effects of ADM and CGRP involve the NO system; in fact pretreatment with L-NAME, a selective inhibitor of NO-synthesis, inhibits the gastroprotective effect of both peptides. This evidence agrees with the studies of Holzer et al. (20) where CGRP exerts its vasodilation at the gastrointestinal level involving the nitric oxide (NO) system. In conclusion, we show that ADM exerts antiinflammatory effect in the acetic acid induced peritonitis test. It is present only in the inflammatory model characterized by a vascular component. We suggest that ADM acts on the same CGRP receptors, but we cannot exclude the possibility that the peptide can also act on separate receptors. Moreover, we suggest that the antiinflammatory effect of the peptide involves the NOsystem.
References 1. K. KITAMURA, J. SAKATA, K. KANGAWA, M. KOJIMA, H. MATSUO and T. ETO, Biochem. Biophys. Res. Commun. 194 720-725 (1993). 2. K. KITAMURA, K KANGAWA, M. KAWAMOTO, Y. ICHIKI, S. NAKAMURA and H. MATSUO, Biochem. Biophys. Res. Commun. 192 553-560 (1993). 3. S.J. WIMALAWANSA, Crit. Rev. Neurobiol. 11 (2-3) 167-239 (1997).
PL-208
Adrenomedullin and Inhmmation
Vol. 65, No. 15, 1999
4. Y. ICHIKI, K. KITAMURA, K. KANAGAWA, M. KAWAMOTO, H. MATSUO and T. ETO, Febs Lett. 338 6-10 (1994). 5. C. NUKI, H. KAWASAKI, K. KITAMURA, M. TAKENAGA, K. KANGAWA, T. ET0 and A. WADA, Biochem. Biophys. Res. Commun. 196 245251 (1993). 6. M. FUKUHARA, T. TSUCHIHASAHI, I. ABE and M. FUJISHIMA, Am. J. Phisyol. 269 R1289-R1294 (1995). 7. H. KANAZAWA, N. KURIHARA, K. HIRATA, S. KUDOH, T. KAWAGUCHI and T. TAKEDA, Biochem. Biophys. Res. Commun. 205 251-254 (1994). 8. T. YAMAGUCHI, K. BABA, Y. DOI and K. YANO, Life Sci. 56 379-387 (1988). 9. V. MARTINEZ, F. CUlTITTA and Y. TACHE’, Endocrinology 138 3749-3755 (1997). 10. G. CLEMENTI, A. CARUSO, V.M.C. CUTULI, E. DE BERNARDIS, A. PRATO, N.G. MANGANO and M.A. ROXAS, Europ. J. Pharmacol. 360 51-54 (1998). ll.H. KAMOI, H. KANAZAWA, K. HIRATA, N. KURIHARA, Y. YANO and S. OTANI, Biochem. Biophys. Res. Commun. 26 1031-1035 (1995). 12. G. CLEMENTI, A. CARUSO, V.M.C. CUTULI, A. PRATO, E. DE BERNARDIS, C. E. FIORE and M. AMICO-ROXAS, Life Sci. 57 193-197 (1995). 13. S. J. WIMAlAWANSA, Endocr. Rev. 17 533-585 (1996) 14.E. ARRIGO-MARTELLI, Boll. Chim. Farm. 107 29-42 (1968). 15.G.E. ROVATI and S. NICOSIA, Trends Pharmacol. Sci. 15 140-144 (1994) 16.V. PLISKA, Trends Pharmacol. Sci. 15 178-181 (1994) 17.J. RAUD, T. LUNDEBERG, G. BRODDA-JANSEN, E. THEODERSSON and P. HEDQPVIST, Biochem. Biophys. Res. Commun. 180 1429-1435 (1991) 18.S.D. BRAIN and T.J. WILLIAMS, Br. J. Pharmacol. 86 855-860 (1985) 19.T.L. BUCKLEY S.D. BRAIN, M. RAMPART and T.J. WILLIAMS, Br. J. Pharmacol. 103 1515-1519 (1991) 20.P. HOLZER, Rev. Physiol. Biochem. Pharmacol. 121 50-146 (1992).
PII SOO24-3205(99)00406-3
Life Sciences Vol. 65, No. 15, pp. PL 203-208, 1999 Copyright 0 1999 Elsevia Science Inc. printed in the USA. All rights reserved 0024-3205/99/S-see hnt matter
PHARU4COLOGY LElTERS Accelerated Communication
ANTIINFLAMMATORY ACTIVITY OF ADRENOMEDULLIN IN THE ACETIC ACID PERITONITIS IN RATS
Giuseppe Clementi, Antonina Caruso, Vincenza Maria Catena Cutuli, Agatina Prato, Nunzio Guide Mangano and Matilde Amico-Roxas.
Institute of Pharmacology, University of Catania, School of Medicine, Viale A. Doria 6, Catania, Italy. (Submitted March 1, 1999;accepted April 19,1999; received in final form June 9, 1999)
Abstract. The antiinflammatory effect of ADM was studied in different models of inflammation and compared to the one of CGRP. Peptides were active against acetic acid-induced peritonitis in the rats. ADM and CGRP exerted the antiinflammatory effect at different doses, 400 and 20 ng/kg respectively, but with different efficacy (ADM >CGRP). This effect was blocked by pretreatment with CGRP (8-37) fragment or with L-NAME. No antiinflammatory activity was evidenced against serotonin - or carrageenin - induced rat paw edema. Our data suggest that ADM exerts antiinflammatory activity in the model characterized by a vascular component. This effect involves CGRP receptors and appears to be mediated by nitric oxide system. 0 1999ElsevierScience Inc. Key Words: adrenomedullin, CGRP, inflammation
lntroductiin Adrenomedullin (ADM) is a 52 amino-acid peptide, originally isolated from a human adrenal pheochromocytoma by using a detection system based on its ability to elevate platelet CAMP levels (1, 2). It is considered to be a member of the calcitonin gene-related peptide (CGRP) superfamily, including CGRP, amylin and calcitonin 13). lmmunoreactive ADM has been detected by RIA in many tissues, including normal adrenal medulla, heart, kidney, pancreas, intestine and plasma (1, 4). Vasodilation seems to be the main physiological effect of ADM (2) and its hypotensive activity is second only to CGRP (5, 6). Other effects of ADM include bronchodilation (7) inhibition of angiotensin - induced aldosterone secretion (8) and inhibition of gastric emptying in rats (9). Moreover, we have recently shown the gastroprotective effect of ADM in reserpine - induced gastric lesions and have suggested that this effect depends on improvement of the blood microcirculation in gastric mucosal through an involvement of CGRP receptors (10).
Corresponding Author: dr. Giuseppe Clementi, Institute of Pharmacology, University of Catania, School of Medicine, viale Andrea Doria 6,95125 Catania (Italy). Fax: +39-095-333219.
PL-204
Adrenomednllin and Inflammation
It has been also suggested that ADM may play an important airway inflammation via a CAMP-dependent mechanism (11).
Vol. 65, No. 15, 1999
role in the regulation
of
On the other hand we have shown the antiinflammatory effect of CGRP and AMY in some experimental models which are characterized by a vascular component (12). Considering that the biological activity profiles in the cardiovascular system of CGRP and of ADM are similar (3, 13), in the present study we have verified the possible antiinflammatory activity of the ADM in some experimental models studying the possible mechanisms involved.
Materials and methods Animals: Male Sprague-Dawley (230-240 g) were starved for 48 h before use but were allowed free access to tap water, which was removed 1 h before the experiment. Room temperature was maintained at 22 +l’C; 65 +- 5% relative humidity; 12-h light/dark. Acetic acid peritonitis test: Groups of five rats were tested by the method of ArrigoniMartelli (14). Peritonitis was produced by i.p. injection of 10 ml/kg of a 0.5% acetic acid solution. Thirty minutes later, the rats were killed by ether inhalation and the peritonitis exudate collected and measured. Human ADM (50 to 600 ng/kg) or CGRP (0.01 to 0.06 pg/kg) were administered i.p. in 5 ml/kg saline, 5 min before acetic acid challenge; control animals received the same amount of saline. To evaluate the time-course of the antiexudative activity of ADM, the dose used was 400 ng/kg while the times of administration varied (0, 15, 60 and 120 min before irritant agent). A group of rats were injected with CGRP (8-37) (lug/kg i.p.) 5 min before ADM (400 @kg) or CGRP (20 ng/kg). Another group of rats received 50 mg/kg of NG-nitro-L-arginine methyl ester (L-NAME) 5 min before the administration of the peptides. Carrageenin-induced rat paw edema: Groups of five rats were used. Edema was produced in the right hind paw by subplantar of 0.1 ml of 1% carrageenin solution. The volume was measured by a mercury plethysmometer prior to the injection of carrageenin and 3 h later. The increase in volume of the paw 3 h after injection of carrageenin was adopted as a measure of edema. Swelling in treated animals was calculated as the percentage of inhibition in comparision to the controls. Serotonin-induced rat paw edema: Groups of five rats were used. Edema was produced in the right hind paw by subplantar injection of 0.1 ml 0.005% serotonin in sterile saline. The volume of the paw was measured by a mercury plethysmometer before and 45 min after serotonin challenge; edema was determined by difference in volume. ADM or CGRP were injected at the doses of 400 or 20 nglkg S.C. respectively, 15 min before serotonin. Chemical and drugs: Human ADM, CGRP and CGRP 8-37 fragment were purchased from Peninsula Laboratories Europe Ldt. (St. Helens Merseyside); serotonin, carrageenin, acetic acid and L-NAME were purchased from Sigma (Italy). Statistical analysis: Statistical analysis was performed using Dunnett’s test for single and multiple comparitions. The level of significance was set at ~~0.05.
PL-205
Vol. 65, No. 15, 1999
Results In the acetic acid-induced peritonitis test ADM exerts the maximal activity (69%) at the dose of 400 ng/kg (table I) TABLE I Antiexudative effect of ADM or CGRP on acetic acid-induced peritonitis in rats (n=5). Treatment Saline ADM
Dose Wkg __ 50 100 200 400 600
Exudate volume ml (mean f S.E.) 1.88 f 0.06 1.52 i 1.30 f 1.02 f 0.58 f 1.04 f
0.10 0.03 0.06 0.04 0.05
Percent Inhibition __
(Dunn& test) _-
19
n.s.
31 46 69 44
co.05 (0.05 co.05 co.05
25 10 co.05 1.40 f 0.03 39 20 co.05 1.14 * 0.04 26 40 co.05 1.36 f 0.05 n.s. 12 60 1.66 f 0.13 ADM or CGRP or saline (2 ml/kg s.c.) were injected 15 min before 0.5% acetic acid (10 ml/kg i.p.). The volume of peritoneal exudate was evaluated 30 min after acetic acid injectiin. CGRP
and when it is administered 15 min before of the acetic acid. However a significant antiinflammatory activity is also present when the peptide is administered simultaneously (55%) or 60 min (48%) before of the acetic acid (table II). TABLE II Tieme-course of antiexudative effect of ADM and CGRP on acetic acid-induced peritonitis in rats (n--5).
Saline
Time (min) 0
ADM CGRP
0 0
Treatment
Saline
15
ADM CGRP
15 15
Saline
60
ADM CGRP
60 60
Exudate volume ml (mean f S.E.) 1.90 f 0.09 0.66 f 0.05 1.40 f 0.04 1.68 f 0.06 0.56 f 0.04 1.14 f 0.04 1.90 f 0.06 0.98 f 0.06 I .42 f 0.05
Percent Inhibition
(Dunn& test) -_
55 26
eo.05 co.05
69 39
co.05 co.05
__
__ 48 25
X0.05 X0.05
__ -_ 120 Saline 1.92 f 0.11 n.s. 19 120 ADM 1.56* 0.08 n.s. 7 120 CGRP 1.78 f 0.09 ADM (400 @kg s.c.), CGRP (20 ng/kg s.c.) or saline (2 ml/kg s.c.) were enjected at the fuced time before 0.5% acetic acid (10 ml/kg i.p.). The volume of peritoneal exudate was evaluated 30 min after acetii acid injedon.
PL-206
Adrenomedullin and Iufhmmation
Vol. 65, No. 15, 1999
The maximal antiinflammatory activity (39%) of CGRP is present at the dose of 20 nglkg. At this dose the peptide causes about 57% of exudation more than ADM. Pretreatment with CGRP (8-37), at the dose of 1 uglkg, blocks the antiiflammatory effect of both peptides. Pretreatment with L-NAME, at the dose of 50 mg/kg, also prevents the antiinflammatory activity of ADM and CGRP (fig.1). No protective effect is observed either in serotonin - or carrageenin induced paw edema.
I I.‘,‘I.“‘I.‘.‘I.“‘I”“I‘“‘I”‘.I”.’I”””””
0
10
20
30
40
Xl
60
70
60
90
100
% bbibition Fig.1
ADM, CGRP or saline (2 ml/kg s.c.) were injected 15 min before 0.5 acetic acid (10 mlikg i.p.). CGRP (8-37) (l@kg) or NAME (50 mg!kg were administered i.p. 5 min before ADM, CGRP or saline. The volume of peritoneal exudate was evaluated 30 min after acetic acid injection. In the control group the volume of exudate was 1.92 f 0.08 ml. * ~0.05 vs CGFU’ ** p
Discussion The present results show the antiinflammatory activity of ADM in an acetic acid peritonitis test and indicate that this peptide exerts its antiinflammatory effect through CGRP receptors and the NO system. Our study suggests that ADM exerts the maximal antiinflammatory effect at the dose of 400; higher doses produce lower effects, exhibiting a bell-shaped curve. This behavior might be explained as the effect of the interaction with two or more receptors, involving opposite actions (15) or by the existence of multiplestate cell, signaling pathways (16). A better characterization of ADM receptors is necessary to allow a better understanding of this effect.
Vol. 65, No. 15, 1999
Adrenomedullin and Inflammation
PL-207
Our data suggest an involvement of CGRP receptors since the antiinflammatory effect of both peptides is blocked by human CGRP (8-37). It agrees with cardiovascular activities of ADM inhibited after administration of the CGRP antagonist, CGRP (8-37) (5). So it is possible that ADM acts at the sites of CGRP, determining a relaxation of endothelian cells and reduction of formation of endothelian gaps and thus interfering with the activities of proinflammatory mediators. The possibility that the antiinflammatory effect of ADM depends, at least in part, on its vascular activity is supported by the fact that the peptide exerts protective effect in inflammation induced by acetic acid, which has been claimed to be primarily a vascular irritant. No antiinflammatory effect is present in inflammatory models mostly due to histamine, serotonin and bradykinin release, like carragenin - and serotonin induced plasma extravasation. On the other hand it is possible that different mechanisms can be involved in the antiinflammatory effect of ADM; in fact there is evidence that this peptide inhibits the secretion of cytokine-induced neutrophil chemoattractant, a member of interleukine-8 family, from lypopolysaccharide - stimulation rat alveolar macrofages “in vitro” (11). This study confirms the protective effect of CGRP in the same test and suggests that its antiinflammatory activity is obtained at a dose 20 times lower than ADM. Also in this case higher doses produce lower effects, exhibiting bell-sharped curve. This evidence agrees with the results of Raud et al. (17) that suggest an antiinflammatory role of CGRP, but it is in contrast with other results that show an proinflammatory role of the peptide (18, 19). We think that these discordant findings may be due to different doses and experimental models used. Even though CGRP shows an antiinflammatory activity at lower doses than ADM, it determines a lower pharmacological efficacy. We think that it is due to the greater vasodilation activity of CGRP, in fact this effect can interfere negatively with the blood microcirculation. Our results also suggest that the antiinflammatory effects of ADM and CGRP involve the NO system; in fact pretreatment with L-NAME, a selective inhibitor of NO-synthesis, inhibits the gastroprotective effect of both peptides. This evidence agrees with the studies of Holzer et al. (20) where CGRP exerts its vasodilation at the gastrointestinal level involving the nitric oxide (NO) system. In conclusion, we show that ADM exerts antiinflammatory effect in the acetic acid induced peritonitis test. It is present only in the inflammatory model characterized by a vascular component. We suggest that ADM acts on the same CGRP receptors, but we cannot exclude the possibility that the peptide can also act on separate receptors. Moreover, we suggest that the antiinflammatory effect of the peptide involves the NOsystem.
References 1. K. KITAMURA, J. SAKATA, K. KANGAWA, M. KOJIMA, H. MATSUO and T. ETO, Biochem. Biophys. Res. Commun. 194 720-725 (1993). 2. K. KITAMURA, K KANGAWA, M. KAWAMOTO, Y. ICHIKI, S. NAKAMURA and H. MATSUO, Biochem. Biophys. Res. Commun. 192 553-560 (1993). 3. S.J. WIMALAWANSA, Crit. Rev. Neurobiol. 11 (2-3) 167-239 (1997).
PL-208
Adrenomedullin and Inhmmation
Vol. 65, No. 15, 1999
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