European Journal of Pharmacology, 238 (1993) 297-301
© 1993 Elsevier Science Publishers B.V. All rights reserved 0014-2999/93/$06.00
Scratching behavior induced by bombesin-related peptides. Comparison of bombesin, gastrin-releasing peptide and phyllolitorins A k i r a M a s u i a, N o b u m a s a K a t o
a, T a k a h i r o
I t o s h i m a a Koichi T s u n a s h i m a and Noboru Yanaihara d
b, T e r u m i
a Department of Psychiatry, Shiga University of Medical Science, Otsu 520-21, Japan b Musashi Hospital, National Center of Neurology and Psychiatry, Tokyo187, Japan c Faculty of Pharmaceutical Sciences, Universityof Tokyo, Tokyo 113, Japan d Faculty of Pharmaceutical Sciences, Shizuoka Prefectural University, Shizuoka 420, Japan Received 16 February 1993, revised MS received 23 April 1993, accepted 27 April 1993
Bombesin and 10 bombesin-related peptides were administered intracerebroventricularly to conscious and freely moving rats. All peptides tested were found to elicit excessive grooming, especially scratching behavior. Bombesin itself had the most potent and long-lasting activity in eliciting scratching behavior. Naturally occurring peptides such as neuromedin B and gastrin-releasing peptide (GRP)-(18-27) were short-acting compared with exogenous peptides such as bombesin and synthesized analogs. Two phyllolitorins, a new bombesin subfamily, were also examined in this study. [LeuS]phyllolitorin induced more scratching than [PheS]phyllolitorin and proved to be virtually equipotent to bombesin. Corticotropin-releasing factor (CRF) and substance P induced considerable excessive grooming, but both peptides were strikingly weak in inducing scratching behavior. It is suggested that (1) scratching represents a specific behavior commonly induced by bombesin-related peptides and (2) the relative potency to induce scratching behavior reflects the metabolic stability of the peptide, e.g. endogenous versus exogenous, shorter versus longer sequences, or chemical protection of N-terminus. Bombesin; Gastrin-releasing peptide (GRP); Phyllolitorin; Scratching behaviour; Structure-activity relationship; (Rat)
Bombesin, a tetradecapeptide originally isolated from amphibian skin (Anastasi et al., 1971), has potent biological effects such as thermoregulation, contraction of smooth muscle, gastrointestinal hormone secretion, anorexia and excessive grooming. The excessive grooming induced by bombesin is different from that induced by substance P (Van Wimersma Greidanus and Maigret, 1988) and corticotropin-releasing factor (CRF) (Sutton et al., 1982). The grooming repertory induced by bombesin mainly consists of scratching (Van Wimersma Greidanus et al., 1985b), whereas all grooming elements are equally enhanced by other peptides (Van Wimersma Greidanus et al., 1985a). Thus changes in scratching behavior can indicate relative bombesin-like activity.
Correspondence to: A. Masui, Department of Psychiatry, Shiga University of Medical Science, Seta Tsukinowacho, Otsu 520-21, Japan. Tel. + 81-775-48-2292, fax + 81-775-43-9698.
Many bombesin-like peptides, which share common amino acid sequences in the C-terminus, have been demonstrated to exist in many species. Gastrin-releasing peptide (GRP), known as a bombesin counterpart in mammals and first isolated from porcine gastrointestinal tract (McDonald et al., 1979), may be present in various forms of different molecular size (Falconieri Erspamer et al., 1988; Cowan, 1988): GRP-(1-27), GRP-(14-27), and GRP-(18-27), also named GRP-10 or neuromedin C (Minamino et al., 1984a). In neuromedin B (Minamino et al., 1983), which is one of the ranatensin family, valine is substituted for threonine at position 6 and leucine for phenylalanine at position 9 as compared with GRP-(18-27). Neuromedin B is reported to be present in the pituitary in high amounts and to be present in higher concentrations than GRP(18-27) in every brain region (Minamino et al., 1984b). Phyllolitorins (Yasuhara et al., 1983), nonapeptide, are the newest member of the family and have a histidine substituted for serine at position 7 (fig. 1). As expected from the similarities in the amino acid structure, these peptides share similar bioactivities with
bombesin. The structure-activity relationship between bombesin/GRP-like peptides has been investigated in vivo and in vitro (Broccardo et al., 1975; Rivier and Brown, 1978; Mukai et al., 1987). Previous reports have dealt with the hormonal, thermoregulatory or gastrointestinal activities of these peptides. Excessive grooming or more literally scratching behavior has never been adopted as an index to compare the relative potencies of peptides of the bombesin family. We evaluated whether phylIolitorins and analogs possess bombesinlike properties.
bombesin receptor antagonist (Heinz-Erian et al., 1987).
2.2. Operation Wistar male rats, weighing 250-300 g, were stereotaxically implanted with polyethylene cannulas into the 3rd ventricle at least 3 days before the behavioral study. The rats were housed individually under a 12-h light/dark cycle (light on 8:00) and allowed food and water ad libitum.
2.3. Behavioral study 2. Materials and methods
2.1. Peptides The peptides listed in fig. 1 were purchased from Peninsula Laboratories, Inc., Belmont, CA. U.S.A., except phyllolitorins and [des-Trp3]phyllolitorins, which were synthesized in the laboratory. Phyllolitorins exist as two subtypes: [Leu8]phyllolitorin and [Phe8]phyllo litorin. The behavioral activity of two synthetic analogs in which tryptophan at position 3 was deleted was also examined ([des-Trp3,LeuS]phyllolitorin and [desTrp3,PheS]phyllolitorin, fig. 1). [D-Phea2,Leu]4] bombesin has been proposed to be a candidate
Rats were allocated randomly to different treatment groups, given peptide intracerebroventricularly (i.c.v.) and placed in observation cages immediately after the i.c.v, injection (1 /xg/5 /~1 per min). Behavior was measured as total time spent scratching or grooming every 5 min for a period of 30 min, starting immediately after the i.c.v, injection. In the present study, grooming indicated head washing, paw licking, bodily grooming and anogenital grooming, according to the strategies reported by Van Wimersma Greidanus et al. (1985b), but scratching was scored separately. Bombesin and GRP-(1-27) were administered i.c.v, in doses from 0.1, 0.3, 1, 3, to 1 0 / z g / 5 / z l per min to determine the dose dependence of the scratching behavior elicited.
I Amino acid sequences o f bornbesin-related peptides ]
~ eua)phyBolitotm ~h~.~hyl~Norm Ac-GRP(20-271 GRP(18-2 7) GRP(14-2 7)
I)Glu-Leu pGlu-L eu-
A c -Hi~ i ~
VaJ ~S~Leu-Met-NH 2
Gly-Asn-HL~ i ~ VaJ ~ L e U - M e t - N H = ii!ii!i!iiiiiiiliiiiill ... Met- Tyr-Pro-Arg-Gly-Asn-His. ::::::::::::::::::::::
(des Tr~, Leua)phyflolitorin (des Trp3, PheSj~hylk)liLorin
A la ~- -Me t- Tyr-Pro-Arg-Gly-Asn-His ,
pGlu-L eu. .,.,.......,......,. ::::::::::::i::::~/a# i ~ ............
pGlu-L eu. ; :::::::::::::::::::::::
¢a~ iiiii~Phe-Met-NH2 :'X..............
tab ~ . ~ .
-L eu-L eu-NH2
Fig. 1. Amino acid sequences of bombesin and bombesin-related peptides. Common amino acid sequences are shown in the C-terminal heptapeptide. Neuromedin B, GRP-(18-27), GRP-(14-27) and GRP-(1-27) have been reported to occur in mammals. Phyllolitorins are the newest member of the family. Acetyl-GRP-(20-27), an artificial analog, is acetylated in the N-terminal histidine residue of the octapeptide (Ac, acetyl).
t~edin B ~euS]ohy~l~o~~a,~hyllolitorke
t~ 0 0.1
0.3 1 d o s e (~g)
Fig. 2. Dose-response curve for scratching induced by bombesin (hatched bar) and GRP-(1-27) (open bar). Data are expressed as mean scratching times ( s / 3 0 min)+S.E.M. Number of rats is expressed in each column. * P < 0.05, ** P < 0.01 as compared with GRP-(1-27).
2.4. Statistics ,
The data were expressed as the means + S.E.M. Statistic significance was determined with a two-tailed Student's t-test.
The dose-response relationship for scratching behavior is shown in fig. 2. An increase in scratching behavior was obvious at all doses of bombesin and GRP-(1-27) compared to saline treatment (1.25 + 1.0 s / 3 0 min). The dose-response curve of bombesin seemed to have an inverted-U shape, namely the potency was found to be maximum at a dose of 1-3/~g, whereas the potency of GRP-(1-27) was almost equal over the doses 0.3-10 /zg. The scratching-inducing effect of bombesin was more prominent than that of
10 20 30 0 10 20 30 0 10 20 30 0 10 20 30 time (rain] Fig. 3. Time course of scratching induced by eight active bombesinrelated peptides. Peptides of the G R P family are listed in the lower part and placed in order from lower to higher molecular weight. Exogenous or artificial peptides are expressed in hatched columns and naturally occurring peptides in open columns. Data are given as mean scratching times (s/5 min) 5: S.E.M. Ac, acetyl.
GRP-(1-27) at a dose of 1 /xg (P < 0.01) and 3 ~ g (P < 0.05). In the time-course experiment, the scratching behavior induced by each peptide (1 /xg) was determined repeatedly at 5-rain intervals for 30 min after injection, as shown in fig. 3. Two distinct features were noted in the time course: one was prolonged, such as with bombesin, [LeuS]phyllolitorin and acetyl-GRP-(20-27),
TABLE 1 The list of bombesin-related peptides used in this study. All peptides were injected i.c.v, at a dose of 1 /.,g/5/xl. Total time spent scratching is expressed as a mean time ( s / 3 0 min):l:S.E.M. Relative potency was calculated on a weight basis. Name
Number of animals
Scratching ( s / 3 0 min)
Relative potency (bombesin = 100)
Bombesin Neuromedin B [Leu8]phyllolitorin [PheS]phyllolitorin Acetyl-GRP-(20-27) GRP-(18-27) GRP-(14-27) GRP-(1-27) [des-Trp 3,Leu8 ]phyllolitorin [des-Trp 3,PheS]phyllolitorin [D-PhetZ,LeuZ4]bombesin Vehicle
1621 1 132 988 1020 990 1120 1667 2803 802 834 1611 -
6 6 8 4 6 8 7 5 4 6 4 4
750 _+36 246 + 24 654 + 42 a 372 + 42 414 + 12 243 5:39 183 + 15 330 5:33 N.D. N.D. N.D. 1.25 + 1.08
100 32 88 50 54 32 24 44 -
a p < 0.01 as compared with [PheS]phyllolitorin. M.W., molecular weight; N.D., not detected.
GRP-(18-27)(15) substance P(4)
i. . . . . . . i. . . . .
time (s/30 rain) Fig. 4. The behavior displayed by rats treated with GRP-(18-27) compared to that of rats treated with substance P and with CRF at the same dose of 1 ~g/5 ~1. The behavior of vehicle-treated rats is shown as reference. The mean groomingtime is shown as an open bar and scratching time as a hatched bar. Number of rats is expressed in parentheses.
and the other was rapidly attenuated, such as with neuromedin B, GRP-(14-27) and GRP-(18-27). GRP(1-27) and [PheS]phyllolitorin exhibited an intermediate effect. The total time spent scratching after each peptide in a 30-min period is shown in table 1. The relative potency (calculated on a weight basis) is also listed in table 1. All bombesin-related peptides, which share common amino acid sequences in the C-terminus, were active in inducing scratching behavior. Among these peptides, bombesin was the most potent and elicited intense scratching behavior. GRP-(1-27), GRP-(14-27) and GRP-(18-27) seemed less potent than bombesin. Acetyl-GRP-(20-27), the shortest analog in the study, retained bombesin-like bioactivity. Both phyllolitorins were active, with [Leu8]phyllolitorin having significantly higher activity than [PheS]phyllolitorin (P < 0.01). Removal of tryptophan at position 3 ([des-Trp3]phyllolitorins) resulted in a complete loss of bioactivity. Neither [D -Phe12,Leula]bombesin nor saline induced scratching behavior: As shown in fig. 4, substance P (1 /~g) also induced excessive grooming. The total time spent scratching was only 8.5 +8.1 s/30 min, the main component being grooming behavior. CRF (1 /zg) appeared to be active in inducing grooming behavior (360 ± 90 s/30 min) and strikingly weak in inducing scratching behavior (2.1 ± 1.5), as compared to GR'P-(18-27) (135 + 63).
Bombesin and related peptides have a broad spectrum of biological activities. These peptides of the bombesin family have a similar amino acid sequence at the C terminus, as shown in fig. 1. It has been reported that the minimum fragment for biological activity is the C terminal heptapeptide (Rivier and Brown, 1978) or
octapeptide (Broccardo et al., 1975), in terms of smooth muscle contraction and hypothermia, respectively. In both studies, tryptophan at position 7 and histidine at position 3 from the C terminus were considered to be essential for bombesin-like activity. This assumption was partially invalidated by the discovery of phyllolitorins (Yasuhara et al., 1983), in which the histidine is replaced by serine. In the present study, we assessed the relative potency of naturally occurring bombesin-related peptides. Phyllolitorins were found to induce scratching behavior: [LeuS]phyllolitorin possessed a potency almost identical to that of bombesin and the potency of [Phe8]peptide was 60% of that of [LeuS]peptide (table 1). To our knowledge, the behavioral effect of the phyllolitorin subfamily has not been described. The present results completely differ from those of previous studies in which the activities of phyllolitorins were evaluated as a function of hypothermia and muscle contraction. Broccardo and Cardamone (1985) found that [Phe8]phyllolitorin failed to alter rat body temperature, while [LeuS]phyllolitorin elicited a 5-6% hypothermic response as compared to bombesin. Falconieri Erspamer et al. (1988) have shown that the phyllolitorins have very weak potency in several smooth muscle preparations except rat urinary bladder and chicken intestine. In the rat urinary bladder, both peptides are equiactive, whereas [Phe8]phyllolitorin displays a 5-fold higher potency than [LeuS]phyllo litorin in the chicken intestine. The present results indicate that the replacement of histidine by serine brings only a minimal change with respect to the induction of scratching behavior. These potent behavioral effects of the two phyllolitorins are in contrast to previous results obtained with a smooth muscle preparation, which reflected peripheral-type bioactivities. The substitution of histidine for the D-isomer of phenylalanine drastically affected the potency, as manifested by the fact that [D-Phe~2,Leula]bombesin had no behavioral effect. The [des-Trp3]analog of phyllolitorin was inactive, as expected from the results of a previous study on hypothermic effects (Rivier and Brown, 1978). The results for the time course of scratching behavior shown in fig. 2 suggest that bombesin-related peptides can be distinguished into two categories: one includes bombesin, [Leu8]phyllolitorin, acetyl-GRP(20-27) with a prolonged activity, and another is characterized by a rapid inactivation, such as observed with neuromedin B, GRP-Th18-27) and GRP-(14-27). This distinction might reflect the metabolic stability of each peptide in vivo. This implies that (1) shorter fragments are degraded more rapidly (GRP-(1-27)> GRP-(1427) =."GRP-(18-27); bombesin > neuromedin B); and (2) the protection of the N-terminus by acetylation or a pyloric ring confers resistance to endopeptidase (bombesin > GRP-(18-27); acetyl-GRP-(20-27)). It can
301 be assumed that endogenously produced peptides are cleaved more rapidly than exogenous peptides. Neur o m e d i n B a n d G R P a n a l o g s have b e e n r e p o r t e d to exist in t h e c e n t r a l n e r v o u s system in t h e rat ( M i n a m i n o et al., 1984b), w h e r e a s b o m b e s i n is virtually u n d e t e c t a b l e in t h e rat b r a i n w h e n a s s a y e d with a specific a n t i s e r u m a g a i n s t b o m b e s i n ( Y a n a i h a r a , C. et al., 1980; Y a n a i h a r a , N. et al., 1981). In this r e g a r d , no s t u d y has r e p o r t e d o n t h e p r e s e n c e o f p h y l l o l i t o r i n s in t h e rat brain. [Phe8]phyllolitorin was i n a c t i v a t e d m o r e r a p i d l y t h a n [Leu8]phyllolitorin, It is thus t e m p t i n g to s p e c u l a t e t h a t t h e p r e s e n t results p r o v i d e e v i d e n c e for t h e existence o f [PheS]phyllolitorin, b u t n o t [LeuS]phyllo litorin, in t h e r a t brain. Comparison of GRP-(18-27), substance P and CRF in t e r m s o f t h e g r o o m i n g / s c r a t c h i n g e l i c i t e d f u r t h e r e m p h a s i z e d t h e specificity o f b o m b e s i n - r e l a t e d p e p tides in eliciting scratching. If we n o t e t h e initial 5 - m i n r e s p o n s e (fig. 3), every active b o m b e s i n - r e l a t e d p e p t i d e caused a considerable amount of scratching behavior. In c o n t r a s t , g r o o m i n g b e h a v i o r can b e e l i c i t e d by a broad spectrum of peptides including substance P and C R F . W e e x a m i n e d t h e effect o f [Leu13-O-CH2NH Leu14]bombesin, a m o r e r e c e n t l y d e v e l o p e d b o m b e s i n a n t a g o n i s t ( C o y et al., 1988), a n d f o u n d t h a t t h e p e p t i d e e l i c i t e d significant g r o o m i n g w i t h o u t c h a n g i n g s c r a t c h i n g b e h a v i o r ( d a t a n o t shown). It is s u g g e s t e d t h a t a s t e r e o t y p e d a b n o r m a l b e h a v i o r such as scratching is m e d i a t e d by c e n t r a l m e c h a n i s m s in w h i c h p e p t i d e s o f t h e b o m b e s i n family a r e involved, w h e r e a s g r o o m i n g c a n b e d r i v e n by a m o r e n o n - s p e c i f i c m e c h a nism. T h i s relatively u n i f o r m p o t e n c y of t h e b o m b e s i n family to i n d u c e s c r a t c h i n g differs f r o m t h e i r p o t e n c y to elicit s m o o t h m u s c l e c o n t r a c t i o n ( F a l c o n i e r i E r s p a m e r et al., 1988). M u k a i et al. (1987) d e m o n s t r a t e d t h a t n e u r o m e d i n B was m o r e t h a n 10 t i m e s less p o t e n t t h a n G R P - ( 1 8 - 2 7 ) in s t i m u l a t i n g t h e r e l e a s e o f g a s t r o p a n c r e a t i c h o r m o n e s . It s h o u l d b e i n v e s t i g a t e d w h e t h e r t h e s e d i s c r e p a n c i e s r e p r e s e n t d i f f e r e n c e s in r e c e p t o r subtypes, n a m e l y , s c r a t c h i n g - i n d u c i n g versus grooming-inducing and central versus peripheral.
References Anastasi, A., V. Erspamer and M. Bucci, 1971, Isolation and structure of bombesin and alytensin, two analogous active peptides from the skin of the European amphibians Bombina and Alytes, Experientia 27, 166. Broccardo, M. and A. Cardamone, 1985, The effect of a new amphibian peptide, LeuSphyllolitorin, on thermoreguration in the rat, Peptides 6, 99.
Broccardo, M., G. Falconieri Erspamer, P. Melchiorri and L. Negri, 1975, Relative potency of bombesin-like peptides, Br. J. Pharmacol. 55, 221. Cowan, A., 1988, New bombesin antagonist shown to have 'encouraging profile', Trends Pharmacol. Sci. 9, 1. Coy, D.H., P. Heinz-Erian, N.-Y. Jiang, Y. Sasaki, J. Taylor, J.-P. Moreau, W.T. Wolfrey, J.D. Gardner and R.T. Jensen, 1988, Probing peptide backbone function in bombesin. A reduced peptide bond analogue with potent and specific receptor antagonist activity, Biol. Chem. 263, 5056. Falconieri Erspamer, G., C. Severini, V. Erspamer, P. Melchiorri, G.D. Fave and T. Nakajima, 1988, Parallel bioassay of 27 bombesin-like peptides on 9 smooth muscle preparations. Structure-activity relationships and bombesin receptor subtypes, Reg. Pept. 21, 1. Heinz-Erian, P., D.H. Coy, N. Tamura, S.W. Tones, J.D. Gardner and R.T. Jensen, 1987, [D-Phe12].bombesin analogues: a new class of bombesin receptor antagonists, Am. J. Physiol. 252, G439. McDonald, T.J., H. Jornvall, L. Niisson, M. Vange, M. Ghatei, S.R. Bloom and V. Mutt, 1979, Characterization of a gastrin releasing peptide from porcine non-antral gastric tissue, Biochem. Biophys. Res. Commun. 90, 227. Minamino, N., K. Kangawa and H. Matsuo, 1983, Neuromedin B: A novel bombesin-like peptide identified in porcine spinal cord, Biochem. Biophys. Res. Commun. 114, 541. Minamino, N., K. Kangawa and H. Matsuo, 1984a, Neuromedin C: A bombesin-like peptide identified in porcine spinal cord, Biochem. Biophys. Res. Commun. 119, 14. Minamino, N., K. Kangawa and H. Matsuo, 1984b, Neuromedin B is a major bombesin-like peptide in rat brain: regional distribution of neuromedin B and neuromedin C in rat brain, pituitary and spinal cord, Biochem. Biophys. Res. Commun. 124, 925. Mukai, H., K. Kawai, Y. Suzuki, K. Yamashita and E. Munekata, 1987, Stimulation of dog gastropancreatic hormone release by neuromedin B and its analogues, Am. J. Physiol. 252, E765. Rivier, J.E. and M.R. Brown, 1978, Bombesin, bombesin analogues, and related peptides: effects on thermoregulation, Biochemistry 17, 1766. Sutton, R.E., G.F. Koob, M. Le Moal, J. Rivier and W. Vale, 1982, Corticotropin releasing factor produces behavioral activation in rats, Nature 297, 331. Van Wimersma Greidanus, Tj.B., D.K. Donker, F.F.M. Van Z!nnicq Bergmann, R. Bekenkamp, C, Maigret and B. Spruijt, 1985a, Comparison between excessive grooming induced by bombesin or by ACTH: the differential elements of grooming and development of tolerance, Peptides 6, 369. Van Wimersma Greidanus, Tj.B., D.K. Donker, R. Walhof, J.C.A. Van Grafhorst, De Vries, S.J. Van Schaik, C. Maigret, B.M. Spruijt and D.L. Colbern, 1985b, The effect of neurotensin, naloxone and haloperidol on elements of excessive grooming behavior induced by bombesin, Peptides 6, 1179. Van Wimersma Greidanus, Tj.B. and C. Maigret, 1988, Grooming behavior induced by substance P, Eur. J. Pharmacol. 154, 217. Yanaihara, C., A. Inoue, T. Mochizuki, J. Ozaki, H. Sato and N. Yanaihara, 1980, Bombesin-like immunoreactivity in mammalian tissues, Biomed. Res. 1, 96. Yanaihara, N., C. Yanaihara, T. Mochizuki, K. Iwahara, T. Fujita and T. Iwanaga, 1981, Immunoreactive GRP, Peptides 2 (Suppl. 2), 185. Yasuhara, T., T. Nakajima, K. Nikihara, C. Yanaihara, N. Yanaihara, V. Erspamer and G..Falconieri Erspamer, 1983, Two new frog skin peptides, phyllolitorins, of the bombesin-ranatensin family from Phyliomedusa sauvagei, Biomed. Res. 4, 407.