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ACTH-(11-24) antagonizes ACTH-(1-24)-induced behavioral syndrome
Neuropeptides (1994) 26,24 l-244 0 Longman Group UK Ltd 1994
ACTH-( 1 l-24) Antagonizes ACTH-( I-24)-induced Behavioral Syndrome S. GENEDANI and M. BERNARDI Institute of Pharmacology, (Reprint requests to SG)
University of Modena, via Campi 287, I4 1100 Modena, Italy
Abstract-The possible involvement of the ACTH-(11-24) fragment on the stretchings, yawnings and penile erections induced by the intracerebroventricular injection of ACTH(I-24) in rats, was studied. The results indicate that this C-terminal fragment is devoid of any behavioral activity, but inhibits the behavioral syndrome induced by ACTH-(l-24). This suggests that the fragment-(1 l-24) of the ACTH molecule may contain or represent an address sequence for brain ACTH receptors and may be involved in the termination of the behavioral response to melanocortins.
Introduction Adrenocorticotropic hormone (ACTH) induces a variety of central and peripheral responses, the maximum active sequence being -(l-24).’ Structure-activity studies on ACTH-( l-24) have shown that consecutive sequences codify different types of information.2 The N-terminal part of the molecule has behavioral, corticotropic, melanotropic and lipolytic activities, with different minimum structural requirements.3 On the other hand, the C-terminal sequence -(l l-24) has only some residual activity on avoidance behavior, but is unable to induce any other behavioral response.3,4 As far as hormonal effects are concerned, contradictory results have been published regarding the intkence of ACTH-(1 l-24) on lipolysis and on
Date received 26 July 1993 Date accepted 27 September 1993
adrenocortical steroidogenesis. Opmeer et al5 reported that ACTH-( 1 l-24) has lipolytic activity, while Elliott et al6 reported that it stimulates magnesium accumulation in rat adipocyte membrane vescicle, but not lipolysis. Some authors7,8have described that ACTH-( 1 l-24) has no effect on basal steroidogenesis, and that it inhibits ACTH-(1-39)Gnduced steroidogenesis in trypsinized adrenocortical cells;9 otherslo have shown that ACTH-( 1l-24) has a potent steroidogenic activity and potentiates the effect of ACTH-( l-39) on collagenase-digested adrenocortical cells. Finally, Li et al” have found that ACTH(1 l-24) elicits only a fraction of ACTH-(1-24) steroidogenic activity and inhibits ACTH-( 1-24)induced cortisol release in bovine adrenal cells. With the aim to further investigate the involvement of the C-terminal sequence in the activity of ACTH, we studied the effect of ACTH-( 1 l-24) on the typical behavioral syndrome characterized by recurrent episodes of stretching, yawning and penile
241
242
NEUROPEPTIDES
erection induced by the i.c.v. injection of ACTH(l-24), in rats.1zJ3 Materials and methods Animals and treatments Adult male rats of a Wistar strain (Charles River, Calco, Como, Italy) weighting 220-240 g were used.The animals were caged in groups of 4-6 in climatized colony rooms (21 f 1°C; 60% humidity) with standard laboratory food and water ad libitum, on a controlled light-dark cycle (light on 07:00-19:OO). Stainless-steel cannulae (Plastic Products Co., Roanoke, VA, USA) were stereotaxitally imaplanted into a brain lateral ventricle under Ketamine (110 mg/kg i.p.; Farmaceutici Gellini, Aprilia, Italy) plus Xilazine (1.8 mg/kg i.p.; Bayer Italia, Milan, Italy) anesthesia. ACTH-( l-24) (Ciba Geigy, Basle, Switzerland)
and ACTH-( 1 l-24) (Sigma Chemical Co., St Louis, MO, USA), freshly dissolved in saline, were i.c.v. injected at equimolecular doses [ACTH-(1-24) 3 ug; ACTH-( 11-24) 1.69 pg] in a volume of 5 ~1 at the rate of 1 ul/20 s via an internal cannula connected by polyethylene tubing to a 10 &Hamilton syringe driven by a micrometric screw. The rats were randomly divided into 4 groups and i.c.v. treated as follows: group 1-saline, 5 ~1,followed 7 min later by saline, 5 ~1; group 2-saline, 5 ul, followed 7 min later by ACTH-( l-24), 3 ugg; group 3ACTH-( 1l-24), 1.69 ug followed 7 min later by saline, 5 ~11;group 4-ACTH-(1 l-24), 1.69 ug, followed 7 min later by ACTH-( l-24), 3 ug. Behavioral studies All behavioral experiments were performed between 08:OO and- 14:O0. The observers were
30 8
20 10 0 R/T= 10HS
20/21
w20
16120
R/T=18/1@
14/21
l6/20
lW20
hnibefwtbn3
R/T- 14/I@
6/21
0/20
2120
Influence of ACTH-( 1I-24) on the behavioral syndrome induced by ACTH-( l-24) in adult male rats [(m = ACTH-( l-24); = ACTH-( 1l-24); q = ACTH-(1 l-24) + ACTH-( l-24); n = saline)], Data are means f SEM of data obtained from all animals in each group. R/T = number of responders/number of treated rats. +p < 0.05 vs ACTH-(l-24) (ANOVA followed by Bonferroni test). Fig. 1
ACTH-( 1 l-24) ANTAGONIZES
ACTH-( l-24)-INDUCED
BEHAVIORAL
unaware of the treatment. Immediately after ACTH(l-24) or saline treatment, the rats were placed in glass boxes (80 x 60 x 40 cm), 5 per box, and observed for 2 h, latency to onset of the behavioral syndrome was measured and stretching, yawning and penile erection episodes were scored. At the end of the experiments, toluidine blue dye was injected through the camutla, the rats were killed by decapitation (under ethyl ether anesthesia) and the brains removed and visually inspected to ascertain the correct position of the cannula tip into a lateral ventricle. Only those animals that were found to have the tip of the cannula correctly positioned were used for the statistical evaluation of data. Statistical analysis Statistical analysis of data was performed by ANOVA followed by the Bonferroni multiple comparison test.
Results As shown in Figure 1, ACTH-( 1l-24) per se did not induce the behavioral syndrome, but significantly antagonized stretchings, yawnings and penile erections induced by the i.c.v. injection of ACTH(l-24), without affecting the percentage of animals with the syndrome. The latency to the appearance of the syndrome (i.e. the time between ACTH-(1-24) treatment I-a@rW b the WnWme
(mid
40
20
0 Fig. 2 Influence ofACTH-( 1l-24) on the latency to the appearance of the behavioral syndrome induced by ACTH-( l-24) in adult male rats [(W = ACTH-(1-24); = ACTH-(1 l-24); q = ACTH-( 1l-24) + ACTH-(l-24)]. Data are means f SEM of the latencies, in minutes to the appearance of the first sign of the syndrome. *p -z 0.05 vs ACTH-(l-24) (ANOVA followed by Bonferroni test).
SYNDROME
243
and the appearance of the fnst sign of the typical behavioral picture) was significantly prolonged by ACTH-( 1 l-24) pretreatment (Fig. 2). Discussion The present results show that ACTH-( 1l-24) is devoid of any behavioral activity, but inhibits the behavioral syndrome induced by ACTH-(1-24) in adult male rats. The lack of behavioral activity of this C-terminal sequence of ACTH is in agreement with previous findings which showed that this ACTH fragment has no central effects, with the exception of some residual activity on avoidance behavior.4 Moreover, studies on the peripheral effects of ACTH-(1 l-24) [lipolysis5 and steroidogenesis’O>“] have produced contradictory results.7-9*1* In Schwyzer’s sychnologic information organization theory, the sequence -( 1 l-24) of the ACTH molecule is proposed to contain an address sequence which shares affinity sites without triggering steroidogenic response and a message sequence for a subtype of receptor on the rat adipocyte.14 Owing to its steroidogenic activity, it has been suggested that the C-sequence of ACTH-( l-24) may contain message elements in addition to affinity sites for adrenal cells.5JoJ1 Structure-activity studies on the ACTH molecule indicate that whatever receptor is involved in behavioral activity it is quite distinct from that involved in steroidogenic activity.lS However, our data suggest, in common with Schwyzer’s sychnologic theory,14 that the C-terminal fragment of ACTH-(1-24) may contain an address sequence which has aflinity sites for central receptors involved in behavior without triggering any behavioral response. Thus, ACTH-( 1l-24) may bind receptor-sites and prevent the binding and behavioral activities of the whole molecule whose N-terminal part contains the message sequence. It would seem that activation of adenylate cyclase is mainly responsible for the behavioral effects of melanocortins13 and it has been shown that the ACTH-( 1 l-24) peripheral activity is not linked to CAMP formation;” this supports the hypothesis of the absence of a message sequence for behavioral activity in this C-terminal part of ACTH molecule. Neuropeptides are enzymatically cleaved to shorter active or inactive fragments; our results,
244 which show that ACTH-( 1 l-24) inhibits ACTH(1-24)-induced behavioral syndrome, suggest that the C-terminal sequence of ACTH may be involved in the termination of the behavioral response to melanocortins.
References 1. De Wied, D. and Jolles, J. Neuropeptides derived from proopiomelanocortin behavioral, physiological and neurochemical effects. Physiol. Rev. 1982; 62: 976-1059. 2. Hechter, 0. and Braun, T. Peptide hormone-receptor interaction: an informational transaction. In: Margoulies, M. and Greenwood, F. C., eds. Structure-activity relationships of protein and polypeptide hormones, part 1, Excerpta Med., Amsterdam, 1971; 212-227. 3. De Wied, D. and Wolterink, G. Structure-activity studies on the neuroactive and neurotropic effects of neuropeptides related to ACTH. Ann. N.Y. Acad. Sci. 1988; 525: 130-140. 4. Greven, H. M. and De Wied, D. Influence of peptides strutturally related to ACTH and MSH on active avoidance behaviour in rats. In: Van Wimersma Greidanus, T. B. ed. Frontiers of hormone research, vol. 4. Basel: Karger, 1977; 140-152. 5. Opmeer, F. A., Van Ree, J. M. and De Wied, D. ACTHinduced lipolysis in rat adipocytes: structure-activity relationshio. Arch. Pharmacol. 1978: 302: 31-36. 6. Elliou,*D. A., Draper, M. W. and Rizack, M. A. Evidence for separate peptide sequences related to the lipolytic and mag-
NEUROPEPTIDES
7.
8.
9.
10.
11.
12.
13.
14. 15.
nesium-accumulating activities of ACTH. Analogy with adrenergic receptors. J. Med. Chem. 1977; 20: 584-586. Seelig, S. and Sayer, G. Isolated adrenal cortex cells: ACTH agonist, partial agonist, cyclic AMP and corticosterone production. Arch. Biochem. Biophys. 1973; 154: 23&239. Finn, F. M., Johns, P. A., Niski, N. and Hofmann, K. Differential response to adrenocorticotropic hormone analogs of bovine adrenal plasma membranes and cells. J. Biol. Chem. 1976; 251: 3576-3585. Seelig, S., Sayers, G., Schwyzer, R. and Schiller, P. Isolated adrenal cells: ACTH-(11-24). a competitive antagonist of ACTH-(l-39) and ACTH-(I-10). FEBS Lea. 1971; 19: 232-234. Szalay, K. S., De Wied, D. and Stark, E. Effects of ACTH(1 l-24) on the corticosteroid production of isolated adrenocortical cells. J. Steroid. Biochem. 1989; 32: 259-262. Li, Z. G., Park, D. and Labella, F. S. Adrenocorticotropin(l-10) and -( 1l-24) promote adrenal steroidogenesis by different mechanisms. Endocrinology 1989; 125: 592-596. Ferrari, W. Behavioral changes in animals after intracisternal injection with adrenocorticotrophic hormone and melanocytestimulating hormone. Nature 1958; 18 1: 925-926. Bertolini, A., Vergoni, W., Gessa, G. L. and Ferrari, W. Induction of sexual excitement by the action of adrenocorticotrophic hormone in brain. Nature 1969; 221: 667669. Schwyzer, R. Organization and transduction of peptide information. Trends Pharmacol. Sci. 1980; 1: 327-331. Wiegant, V. M., Jolles, J., Colbern, D. L., Zimmerman, E. and Gispen, W. H. Intracerebroventricular ACTH activates the pituitary-adrenal system: dissociation from a behavioral response. Life Sci. 1979; 25: 1791-1796.
ACTH-( 1 l-24) Antagonizes ACTH-( I-24)-induced Behavioral Syndrome S. GENEDANI and M. BERNARDI Institute of Pharmacology, (Reprint requests to SG)
University of Modena, via Campi 287, I4 1100 Modena, Italy
Abstract-The possible involvement of the ACTH-(11-24) fragment on the stretchings, yawnings and penile erections induced by the intracerebroventricular injection of ACTH(I-24) in rats, was studied. The results indicate that this C-terminal fragment is devoid of any behavioral activity, but inhibits the behavioral syndrome induced by ACTH-(l-24). This suggests that the fragment-(1 l-24) of the ACTH molecule may contain or represent an address sequence for brain ACTH receptors and may be involved in the termination of the behavioral response to melanocortins.
Introduction Adrenocorticotropic hormone (ACTH) induces a variety of central and peripheral responses, the maximum active sequence being -(l-24).’ Structure-activity studies on ACTH-( l-24) have shown that consecutive sequences codify different types of information.2 The N-terminal part of the molecule has behavioral, corticotropic, melanotropic and lipolytic activities, with different minimum structural requirements.3 On the other hand, the C-terminal sequence -(l l-24) has only some residual activity on avoidance behavior, but is unable to induce any other behavioral response.3,4 As far as hormonal effects are concerned, contradictory results have been published regarding the intkence of ACTH-(1 l-24) on lipolysis and on
Date received 26 July 1993 Date accepted 27 September 1993
adrenocortical steroidogenesis. Opmeer et al5 reported that ACTH-( 1 l-24) has lipolytic activity, while Elliott et al6 reported that it stimulates magnesium accumulation in rat adipocyte membrane vescicle, but not lipolysis. Some authors7,8have described that ACTH-( 1 l-24) has no effect on basal steroidogenesis, and that it inhibits ACTH-(1-39)Gnduced steroidogenesis in trypsinized adrenocortical cells;9 otherslo have shown that ACTH-( 1l-24) has a potent steroidogenic activity and potentiates the effect of ACTH-( l-39) on collagenase-digested adrenocortical cells. Finally, Li et al” have found that ACTH(1 l-24) elicits only a fraction of ACTH-(1-24) steroidogenic activity and inhibits ACTH-( 1-24)induced cortisol release in bovine adrenal cells. With the aim to further investigate the involvement of the C-terminal sequence in the activity of ACTH, we studied the effect of ACTH-( 1 l-24) on the typical behavioral syndrome characterized by recurrent episodes of stretching, yawning and penile
241
242
NEUROPEPTIDES
erection induced by the i.c.v. injection of ACTH(l-24), in rats.1zJ3 Materials and methods Animals and treatments Adult male rats of a Wistar strain (Charles River, Calco, Como, Italy) weighting 220-240 g were used.The animals were caged in groups of 4-6 in climatized colony rooms (21 f 1°C; 60% humidity) with standard laboratory food and water ad libitum, on a controlled light-dark cycle (light on 07:00-19:OO). Stainless-steel cannulae (Plastic Products Co., Roanoke, VA, USA) were stereotaxitally imaplanted into a brain lateral ventricle under Ketamine (110 mg/kg i.p.; Farmaceutici Gellini, Aprilia, Italy) plus Xilazine (1.8 mg/kg i.p.; Bayer Italia, Milan, Italy) anesthesia. ACTH-( l-24) (Ciba Geigy, Basle, Switzerland)
and ACTH-( 1 l-24) (Sigma Chemical Co., St Louis, MO, USA), freshly dissolved in saline, were i.c.v. injected at equimolecular doses [ACTH-(1-24) 3 ug; ACTH-( 11-24) 1.69 pg] in a volume of 5 ~1 at the rate of 1 ul/20 s via an internal cannula connected by polyethylene tubing to a 10 &Hamilton syringe driven by a micrometric screw. The rats were randomly divided into 4 groups and i.c.v. treated as follows: group 1-saline, 5 ~1,followed 7 min later by saline, 5 ~1; group 2-saline, 5 ul, followed 7 min later by ACTH-( l-24), 3 ugg; group 3ACTH-( 1l-24), 1.69 ug followed 7 min later by saline, 5 ~11;group 4-ACTH-(1 l-24), 1.69 ug, followed 7 min later by ACTH-( l-24), 3 ug. Behavioral studies All behavioral experiments were performed between 08:OO and- 14:O0. The observers were
30 8
20 10 0 R/T= 10HS
20/21
w20
16120
R/T=18/1@
14/21
l6/20
lW20
hnibefwtbn3
R/T- 14/I@
6/21
0/20
2120
Influence of ACTH-( 1I-24) on the behavioral syndrome induced by ACTH-( l-24) in adult male rats [(m = ACTH-( l-24); = ACTH-( 1l-24); q = ACTH-(1 l-24) + ACTH-( l-24); n = saline)], Data are means f SEM of data obtained from all animals in each group. R/T = number of responders/number of treated rats. +p < 0.05 vs ACTH-(l-24) (ANOVA followed by Bonferroni test). Fig. 1
ACTH-( 1 l-24) ANTAGONIZES
ACTH-( l-24)-INDUCED
BEHAVIORAL
unaware of the treatment. Immediately after ACTH(l-24) or saline treatment, the rats were placed in glass boxes (80 x 60 x 40 cm), 5 per box, and observed for 2 h, latency to onset of the behavioral syndrome was measured and stretching, yawning and penile erection episodes were scored. At the end of the experiments, toluidine blue dye was injected through the camutla, the rats were killed by decapitation (under ethyl ether anesthesia) and the brains removed and visually inspected to ascertain the correct position of the cannula tip into a lateral ventricle. Only those animals that were found to have the tip of the cannula correctly positioned were used for the statistical evaluation of data. Statistical analysis Statistical analysis of data was performed by ANOVA followed by the Bonferroni multiple comparison test.
Results As shown in Figure 1, ACTH-( 1l-24) per se did not induce the behavioral syndrome, but significantly antagonized stretchings, yawnings and penile erections induced by the i.c.v. injection of ACTH(l-24), without affecting the percentage of animals with the syndrome. The latency to the appearance of the syndrome (i.e. the time between ACTH-(1-24) treatment I-a@rW b the WnWme
(mid
40
20
0 Fig. 2 Influence ofACTH-( 1l-24) on the latency to the appearance of the behavioral syndrome induced by ACTH-( l-24) in adult male rats [(W = ACTH-(1-24); = ACTH-(1 l-24); q = ACTH-( 1l-24) + ACTH-(l-24)]. Data are means f SEM of the latencies, in minutes to the appearance of the first sign of the syndrome. *p -z 0.05 vs ACTH-(l-24) (ANOVA followed by Bonferroni test).
SYNDROME
243
and the appearance of the fnst sign of the typical behavioral picture) was significantly prolonged by ACTH-( 1 l-24) pretreatment (Fig. 2). Discussion The present results show that ACTH-( 1l-24) is devoid of any behavioral activity, but inhibits the behavioral syndrome induced by ACTH-(1-24) in adult male rats. The lack of behavioral activity of this C-terminal sequence of ACTH is in agreement with previous findings which showed that this ACTH fragment has no central effects, with the exception of some residual activity on avoidance behavior.4 Moreover, studies on the peripheral effects of ACTH-(1 l-24) [lipolysis5 and steroidogenesis’O>“] have produced contradictory results.7-9*1* In Schwyzer’s sychnologic information organization theory, the sequence -( 1 l-24) of the ACTH molecule is proposed to contain an address sequence which shares affinity sites without triggering steroidogenic response and a message sequence for a subtype of receptor on the rat adipocyte.14 Owing to its steroidogenic activity, it has been suggested that the C-sequence of ACTH-( l-24) may contain message elements in addition to affinity sites for adrenal cells.5JoJ1 Structure-activity studies on the ACTH molecule indicate that whatever receptor is involved in behavioral activity it is quite distinct from that involved in steroidogenic activity.lS However, our data suggest, in common with Schwyzer’s sychnologic theory,14 that the C-terminal fragment of ACTH-(1-24) may contain an address sequence which has aflinity sites for central receptors involved in behavior without triggering any behavioral response. Thus, ACTH-( 1l-24) may bind receptor-sites and prevent the binding and behavioral activities of the whole molecule whose N-terminal part contains the message sequence. It would seem that activation of adenylate cyclase is mainly responsible for the behavioral effects of melanocortins13 and it has been shown that the ACTH-( 1 l-24) peripheral activity is not linked to CAMP formation;” this supports the hypothesis of the absence of a message sequence for behavioral activity in this C-terminal part of ACTH molecule. Neuropeptides are enzymatically cleaved to shorter active or inactive fragments; our results,
244 which show that ACTH-( 1 l-24) inhibits ACTH(1-24)-induced behavioral syndrome, suggest that the C-terminal sequence of ACTH may be involved in the termination of the behavioral response to melanocortins.
References 1. De Wied, D. and Jolles, J. Neuropeptides derived from proopiomelanocortin behavioral, physiological and neurochemical effects. Physiol. Rev. 1982; 62: 976-1059. 2. Hechter, 0. and Braun, T. Peptide hormone-receptor interaction: an informational transaction. In: Margoulies, M. and Greenwood, F. C., eds. Structure-activity relationships of protein and polypeptide hormones, part 1, Excerpta Med., Amsterdam, 1971; 212-227. 3. De Wied, D. and Wolterink, G. Structure-activity studies on the neuroactive and neurotropic effects of neuropeptides related to ACTH. Ann. N.Y. Acad. Sci. 1988; 525: 130-140. 4. Greven, H. M. and De Wied, D. Influence of peptides strutturally related to ACTH and MSH on active avoidance behaviour in rats. In: Van Wimersma Greidanus, T. B. ed. Frontiers of hormone research, vol. 4. Basel: Karger, 1977; 140-152. 5. Opmeer, F. A., Van Ree, J. M. and De Wied, D. ACTHinduced lipolysis in rat adipocytes: structure-activity relationshio. Arch. Pharmacol. 1978: 302: 31-36. 6. Elliou,*D. A., Draper, M. W. and Rizack, M. A. Evidence for separate peptide sequences related to the lipolytic and mag-
NEUROPEPTIDES
7.
8.
9.
10.
11.
12.
13.
14. 15.
nesium-accumulating activities of ACTH. Analogy with adrenergic receptors. J. Med. Chem. 1977; 20: 584-586. Seelig, S. and Sayer, G. Isolated adrenal cortex cells: ACTH agonist, partial agonist, cyclic AMP and corticosterone production. Arch. Biochem. Biophys. 1973; 154: 23&239. Finn, F. M., Johns, P. A., Niski, N. and Hofmann, K. Differential response to adrenocorticotropic hormone analogs of bovine adrenal plasma membranes and cells. J. Biol. Chem. 1976; 251: 3576-3585. Seelig, S., Sayers, G., Schwyzer, R. and Schiller, P. Isolated adrenal cells: ACTH-(11-24). a competitive antagonist of ACTH-(l-39) and ACTH-(I-10). FEBS Lea. 1971; 19: 232-234. Szalay, K. S., De Wied, D. and Stark, E. Effects of ACTH(1 l-24) on the corticosteroid production of isolated adrenocortical cells. J. Steroid. Biochem. 1989; 32: 259-262. Li, Z. G., Park, D. and Labella, F. S. Adrenocorticotropin(l-10) and -( 1l-24) promote adrenal steroidogenesis by different mechanisms. Endocrinology 1989; 125: 592-596. Ferrari, W. Behavioral changes in animals after intracisternal injection with adrenocorticotrophic hormone and melanocytestimulating hormone. Nature 1958; 18 1: 925-926. Bertolini, A., Vergoni, W., Gessa, G. L. and Ferrari, W. Induction of sexual excitement by the action of adrenocorticotrophic hormone in brain. Nature 1969; 221: 667669. Schwyzer, R. Organization and transduction of peptide information. Trends Pharmacol. Sci. 1980; 1: 327-331. Wiegant, V. M., Jolles, J., Colbern, D. L., Zimmerman, E. and Gispen, W. H. Intracerebroventricular ACTH activates the pituitary-adrenal system: dissociation from a behavioral response. Life Sci. 1979; 25: 1791-1796.