European Journal of Pharmacology, 216 (1992) 1l 3- l 17 © 1992 Elsevier Science Publishers B.V. All rights reserved 0014-2999/92/$05.00
113
EJP 52466
Relaxant effect of pituitary adenylate cyclase-activating polypeptide on guinea-pig tracheal smooth muscle Nobuyasu Araki and Kenzo Takagi Second Department of buernal Medicine, Nagoya Unil'ersity School of Medicine, Nagoya, Japan Received 7 November 1991, revised MS received 3 March 1992, accepted 10 March 1992
We investigated the relaxant effect of the pituitary adenylate cyclase-activating polypeptide with 27 residues (PACAP27) and with 38 residues (PACAP38) on guinea-pig tracheal smooth muscle. Both forms of PACAP showed dose-dependent relaxant effects. The ECs~~ of PACAP27 was 8.7 _+ 1.9 x 10 -8 M and that of PACAP38 was 6.8 _+ 1.0 × 10 s M. Both increased cyclic AMP levels dose dependently and the elevation of cyclic AMP preceded the relaxation of tracheal smooth muscle. There was a marked difference in the duration of action of the two peptides. PACAP38 showed a longer-lasting relaxation compared to PACAP27. Furthermore PACAP38 maintained significantly higher levels of cyclic AMP, with cyclic AMP levels at 60 min after a 5-min exposure to PACAPs (10 6 M) being 14.0_+ 1.4 p M / m g protein for PACAP27 and 35.9_+ 2.4 p M / m g protein for PACAP38. These results suggest that PACAP27 and PACAP38 may be novel potent relaxants in tracheal smooth muscle and their relaxant effect might be mediated by cyclic AMP. However PACAP38 had a longer-lasting action on relaxation of tracheal smooth muscle and production of tissue cyclic AMP than PACAP27. PACAP ( pituitary adenylate cyclase-activating polypeptide); Trachea (guinea-pig); Smooth muscle; cAMP
1. Introduction N o v e l n e u r o p e p t i d e s which s t i m u l a t e a d e n y l a t e cyclase activity in rat a n t e r i o r p i t u i t a r y cell c u l t u r e s w e r e r e c e n t l y i s o l a t e d f r o m ovine h y p o t h a l a m i c tissues a n d d e s i g n a t e d as P A C A P 3 8 ( p i t u i t a r y a d e n y l a t e cyclaseactivating p o l y p e p t i d e with 38 r e s i d u e s ) a n d P A C A P 2 7 ( p i t u i t a r y a d e n y l a t e c y c l a s e - a c t i v a t i n g p o l y p e p t i d e with 27 r e s i d u e s ) ( M i y a t a et al., 1989, 1990). C l o n i n g o f P A C A P 3 8 c D N A s c o n f i r m e d t h e e x p r e s s i o n of the c o r r e s p o n d i n g m R N A s a n d the p r e s e n c e o f this neur o p e p t i d e in ovine h y p o t h a l a m u s a n d in h u m a n testis ( K i m u r a et al., 1990). P A C A P 2 7 b i n d i n g sites with very high specificity w e r e o b s e r v e d in rat b r a i n a n d lung ( G o t t s c h a l l et al., 1990; L a m et al., 1990). T h o u g h b o t h forms o f P A C A P c a u s e d a h y p o t e n s i v e effect in t h e a n a e s t h e t i z e d rat ( N a n d h a et al. , 1990) a n d h a d a v a s o d i l a t o r action on r a b b i t a o r t a ( W a r r e n et al., 1991), t h e r e a r e n o p u b l i s h e d d a t a c o n c e r n i n g the effect of
Correspondence to: K. Takagi, Second Department of Internal Medicine, Nagoya University School of Medicine, Tsuruma-cho, Showa-ku, Nagoya 466 Japan. Tel. 81.52.741 2111 ext. 2209, fax 81.52.733 8126.
P A C A P on airway s m o o t h muscle. T h e p u r p o s e o f this study was to e l u c i d a t e the r e l a x a n t effects of b o t h forms o f P A C A P on t r a c h e a l s m o o t h muscle a n d the m e c h a n i s m of the relaxation.
2. Materials and methods 2.1. Tissue preparation and tracheal smooth muscle relaxation Male Hartley guinea-pigs weighing 200-400 g were s t u n n e d a n d bled, a n d the t r a c h e a s w e r e r e m o v e d . O n e t r a c h e a l ring was cut f r o m t h e p h a r y n g e a l e n d o f each t r a c h e a . T h e ring was o p e n e d by c u t t i n g t h r o u g h the cartilaginous region diametrically opposite the tracheal s m o o t h muscle, a n d t h e e n d s o f t h e c a r t i l a g i n o u s region w e r e ligated. T h e p r e p a r a t i o n was p l a c e d in a 1-ml o r g a n bath. T e n s i o n was m e a s u r e d with an isom e t r i c t r a n s d u c e r ( M i n e b e a Co., Ltd., U L 10GR, J a p a n ) , a n d t e n s i o n c h a n g e s w e r e r e c o r d e d with a p e n r e c o r d e r ( T O A E l e c t r o n i c s Ltd., F B R - 2 5 2 A , J a p a n ) . U s i n g t h e m e t h o d p r e v i o u s l y d e s c r i b e d ( B a b a et al., 1986), t h e p r e p a r a t i o n was p e r f u s e d at a c o n s t a n t flow r a t e of 2.0 m l / m i n at 37°C in K r e b s solution of t h e
114
following composition (mM): NaC1 137, K H C O 3 5.9, CaCI 2 2.4, MgC12 1.2 and glucose 11.8. Relaxation was produced with isoprenaline (2 × 10 -s M) and a tension of about 0.5 g was applied to the preparation. The preparation was again perfused at 37°C in Krebs solution and resting tone was allowed to develop. After the tension had stabilized, the preparation was then perfused for 20 min in the presence of either form of P A C A P at various concentrations. The complete relaxation occurring on exposure of the tissue to Ca2+-free medium containing 0.01 m M E G T A was defined as 100%, and the relaxation caused by P A C A P was calculated in terms of this standard.
0
g 50-
IO0 10 9
3x10 9
10 e
3x10-8
10 7
[PEPTIDE]
3x10-7
lO-S
M
Fig. 1. Dose-dependent effects of PACAP27 (e) and PACAP38 (©) on resting tone in guinea-pig tracheal smooth muscle. The results are expressed as % relaxation induced by Ca2+-free medium. The data points represent m e a n s with S.E.M. shown by vertical bars (n = 8).
2.2. Measurement of tissue cyclic A M P leuels We measured tissue cyclic A M P levels and evaluated the effect of both forms of P A C A P on cyclic A M P accumulation in tracheal smooth muscle. We resected the m e m b r a n o u s portion from the trachea and obtained a strip by carefully removing the adjacent tissue from the m e m b r a n o u s portion. The preparation, containing epithelium, was equilibrated in a bath with 10 ml Krebs solution for 30 min. The solution was kept at 37°C. The preparation was exposed to P A C A P in various concentrations for the times indicated, then immediately frozen with liquid nitrogen to stop the reaction. We then homogenized the preparation in 2.0 ml icecold 0.1 N HCI, using a Potter homogenizer, and centrifuged the homogenate at 1500 x g for 30 min at 4°C. Cyclic A M P levels in the supernatant fluid were measured by radioimmunoassay ( H o n m a et al., 1977). The protein concentration of the homogenate was determined by the method of Lowry et al. (1951) with bovine albumin as a standard. All biochemical analyses were performed in duplicate.
2.3. Materials PACAP27 and PACAP38 were obtained from Peptide Institute, Inc. (Osaka, Japan). Isoprenaline and E G T A were purchased from Sigma Chemical Co. (St. Louis, MO, U.S.A.). 125I-Succinyi cyclic A M P was purchased from Yamasa Syoyu Co. (Tokyo, Japan). All other chemicals were reagent grade, or the best commercially available grade.
2.4. Statistics
This study was performed in accordance with the guidelines for animal experimentation set by Nagoya University.
3. Results
3.1. Relaxation by PACAP Figure 1 shows the concentration-response curves for the effect of PACAP27 and PACAP38 on resting tone in tracheal smooth muscle. The dose-dependent relaxant effects were observed at concentrations of 10 -9, 3 x 10 -9, 10 8, 3 x 10 -8, 10 -7, 3 X 10 - 7 M and almost complete relaxation was observed at 10 -6 M. The ECs0 values for the relaxant effects of PACAP27 and PACAP38 on resting tone were 8.7 _+ 1.9 × 10 _.8 and 6.8 +_ 1.0 x 10 -8 M, respectively (means with S.E.M., n = 8), both had similar relaxant potency.
3.2. Tissue cyclic AMP levels Figure 2 shows that significant dose-dependent increases in tissue levels of cyclic A M P were observed for
2OO
~E
IOO
0
,j *
o
*
Io ~
Io -~
*
,
*
(O -~
lo 6
(eEP11O£~ M
The data are expressed as means with S.E.M. We used the non-paired Student's t-test for statistical analysis. P values of less than 0.05 were considered statistically significant.
Fig. 2. Dose-dependent effects of PACAP27 (o) and PACAP38 ( o ) on tissue levels of cyclic A M P in guinea-pig tracheal smooth muscle. The preparation was exposed to each P A C A P for 3 rain at each concentration. The data points represent m e a n s with S.E.M. shown by vertical bars (n = 6). (* P < 0.001 vs. control).
115
AO-
-LF PACAP27
150 .=
4,
CaZ+-free sol.
.
,i,
-100
IOmin
50.
PACAP36 50
.ca
1'5
Fig. 4. The upper trace shows the force generated by guinea-pig tracheal smooth muscle exposed for 5 min to PACAP27 (10 ~' M) on resting tone, followed by exposure to Krebs solution. The lower trace shows the force with PACAP38 (10 6 M), superfused with CaZ+-free medium containing 0.01 mM E G T A at 280 rain.
Time (min)
8 O-
-300
- 2OO ~ ~
50-
v
~:
~
-100 .,c
1O0 -
CaZ+- free sol.
~
.0
100
~
1"
5
1'0
{5
ministration while relaxation of tracheal smooth muscle approached a plateau at 5 min after administration. With PACAP38 (fig. 3B), even 15 min after administration, the tissue cyclic AMP levels were increasing slowly and tracheal smooth muscle was relaxing gradually.
0
3.4. Duration of relaxant effect
Time (rain)
Fig. 3. Time course of the relaxant (el and the cyclic AMP-increasing ( o ) effects of a maximum effective concentration (10 -6 M) of PACAP27 (A) and PACAP38 (B). After exposure of the muscle to PACAP for 0, 1, 3, 5 and 15 min, the tissue levels of cyclic AMP and the isometric force were measured. The data points for % relaxation (n = 7) and cyclic AMP (n = 6) represent means with S.E.M. shown by vertical bars (*, P < 0.001 vs. control).
Figure 4 shows typical traces for the duration of the relaxant effect of PACAP27 and PACAP38 on tracheal smooth muscle. Figure 5 shows the curves for the correlation between time and relaxation. The relaxant effect decreased rapidly 5 min after exposure to PACAP27, but PACAP38 had a considerably more prolonged duration of action. The percent relaxation
both forms of PACAP with similar potency (15.2 + 3.1 in control, 120.0+5.9 p M / m g protein at 10 -6 M PACAP27 and 15.3 + 2.1 in control, 159.6 + 16.3 p M / m g protein at 10 -6 M PACAP38).
3.3. Time course relationship between relaxation and elevation of cyclic AMP levels Figure 3 shows that the elevation of tissue cyclic AMP preceded the relaxation of tracheal smooth muscle. Both forms of PACAP slightly relaxed smooth muscle but the tissue cyclic AMP was greatly increased 1 min after the addition of 10 -6 M of either form of PACAP. With PACAP27 (fig. 3A) the tissue cyclic AMP levels approached a plateau at 3 min after ad-
50-
106-
,
o
6'o
,~o
Time (rain)
Fig. 5. Effects of 5-min superfusion of PACAP27 (el and PACAP38 ( o ) on resting tone followed by exposure to Krebs solution. The results are expressed as % relaxation induced by Cae+-free medium. The data points for PACAP27 (n = 10) and PACAP38 (n = 7) represent means with S.E.M. shown by vertical bars.
TABLE 1 The mean level of cyclic AMP generated in tracheal smooth muscle from guinea-pigs immediately after, 20 min after and 60 min after superfusion with Krebs solution following 5-min exposure to PACAP27 (10 -6 M) and PACAP38 (10 -6 M), compared with the control. The data represent means with S.E.M. Cyclic AMP ( p M / m g protein)
PACAP27 (n = 6) PACAP38 (n = 6) a p < 0.001 vs. control.
Control
0 min
20 min
60 min
16.6_+5.6 15.3_+2.1
125.2_+ 18.2 a 186.1 _+ 9.2 a
29.8_+4.0 44.9+3.8 a
14.0_+ 1.4 35.9_+2.4 a
116
20 min after superfusion with PACAP27 and PACAP38 was 22.9 _+ 2.3% (n = 10) and 88.6 + 3.5% (n = 7), respectively (means with S.E.M.).
3.5. Relationship between tissue cyclic A M P and longlasting action Table 1 shows that tissue cyclic AMP levels decreased drastically after termination of the exposure of tracheal smooth muscle to PACAP27, although they remained at significantly higher levels even 20 min and 60 min after termination of superfusion with PACAP38. PACAP38 thus had a longer-lasting action on production of tissue cyclic AMP and relaxation of tracheal smooth muscle.
4. Discussion
Since its discovery and characterization, PACAP has attracted much attention. Two forms of PACAP were discovered in hypothalamic tissues by monitoring pituitary adenylate cyclase-stimulating activity (Miyata et al., 1989, 1990). Receptor studies showed PACAP binding sites to be present in high density in the hypothalamus and lung in addition to the pituitary (Gottshall et al., 1990). On the other hand a very small number of PACAP binding sites were found in the aorta (Lam et al., 1990), but Warren et al. (1991) demonstrated that both forms of PACAP were potent, long-lasting, and endothelium-independent vasodilators. Little has been reported regarding the effects of these peptides on smooth muscle preparations other than vascular ones. Our present studies have both shown an effect of PACAP on a non-vascular smooth muscle preparation, i.e. tracheal smooth muscle, and demonstrated the mechanism of their action, through increased cyclic AMP synthesis. PACAP27 and PACAP38 caused nearly equipotent relaxant effects on resting tone in guinea-pig tracheal smooth muscle. The relaxant ECs0 values might represent the high sensitivity of this preparation to PACAP similar to that reported for atrial natriuretic peptide (ECs0 = 1 . 2 ~ 5 . 2 × 10 -7 M, Watanabe et al., 1988), forskolin ( E C 5 0 = 2 . 0 × 10 -s M, Tsukawaki et al., 1987), and sodium nitroprusside (ECs0 = 2.2 × 10 -7 M, Suzuki et al., 1986). Both forms of PACAP increased cyclic AMP levels under the same conditions as those under which relaxation was observed. The relaxant effects of these peptides seem to parallel the extent of the elevation of the level of cyclic AMP. In addition, we observed that an increase in cyclic AMP levels preceded the relaxation of the tracheal smooth muscle. This observation is compatible with the principle that adenylate cyclase-
stimulating agonists induce cyclic AMP accumulation, resulting in relaxation of smooth muscle. The main difference in effects we observed between PACAP27 and PACAP38 was the duration for which they could relax the smooth muscle, with the latter having the longer-lasted relaxant action. Moreover, we demonstrated that PACAP38 maintained significantly higher levels of tissue cyclic AMP concurrent with almost complete relaxation of tracheal smooth muscle for 60 rain, even after its superfusion was stopped. It is possible that PACAP38 remained at the binding sites and continued to stimulate adenylate cyclase. Considering that the actual percentage decreases of cyclic AMP with time were quite similar in both peptides, the mechanism of the longer-lasting relaxation by PACAP38 may be attributed to the initially higher level of cyclic AMP, which might facilitate relaxation of the tracheal smooth muscle, and the subsequent significantly higher levels of cyclic AMP, although lower than the initial peak, which might maintain relaxation of the tracheal smooth muscle. Other mechanisms might probably participate in their different action, which could be related to the difference in their structure. In the case of rabbit aorta, Warren et al. (1991) demonstrated that the relaxant action of both forms of PACAP was surprisingly long-lasted, which could be explained by tight binding to the PACAP receptor. This difference between trachea and aorta might result from tissue and species differences. There might be some subtypes of PACAP receptors. Two classes of PACAP27-binding sites were found in rat lung membranes (Lam et al., 1990). The coexistence of three subtypes of receptors interacting with PACAP has been reported for rat liver membrane, namely high-affinity vasoactive intestinal polypeptide (VIP), low-affinity VIP, and specific PACAP receptors (Robberecht et al., 1991). The presence of high-affinity PACAP receptors in human neuroblastoma cell line NB-OK (Cauvin et al., 1990), in the rat pancreatic carcinoma cell line A R 4-2J (Buscail et al., 1990), and also in rat astrocyte membranes (Tatsuno et al., 1990) was observed. The role of PACAP receptors in the mechanism of long-lasting relaxant action obviously needs to be studied. The N-terminal portion 1-28 of PACAP38 shows 68% homology with VIP (Miyata et al., 1989). VIP is a neurotransmitter which produces dose-dependent relaxation of guinea-pig airways by increasing the tissue cyclic AMP content (Hand et al., 1984, Kitamura et al., 1980). Because PACAP, demonstrated in the hypothalamus (K6ves et al., 1990), was previously suggested to function as a neurotransmitter and as a potent airway smooth muscle relaxant by raising cyclic AMP levels dose dependently as shown by our data, it might act physiologically as a bronchodilator neuropeptide similar to VIP. In normal individuals, there is a balance
117
between the bronchodilator (/3-adrenergic, peptide histidine methionine and VIP) and bronchoconstricting (o~-adrenergic, cholinergic, substance P, neurokinin, and calcitonin gene-related peptide) mechanisms. In subjects with asthma, however, there is an imbalance toward the bronchoconstricting mechanism (Thomas, 1991). Accordingly drug-inducible PACAP or PACAP itself might be useful for the treatment of bronchial asthma by correcting the imbalance toward bronchoconstriction. We showed that both forms of PACAP tested may act as novel potent relaxants on tracheal smooth muscle by raising tissue cyclic AMP levels. PACAP38 had a longer-lasting action on relaxation of tracheal smooth muscle and production of tissue cyclic AMP than PACAP27. Other mechanisms involved in the different action in PACAPs besides cyclic AMP synthesis remain to be investigated.
Acknowledgements The authors thank Dr. Kiyoshi Suzuki and Mrs. Takako Watanabe for their cooperation and express their gratitude to Mr. M. Bodman, language consultant of our department, for reading the previous draft and making suggestions on language and style. This study was supported in part by a grant-in-aid for scientific research from the Ministry of Education, Science and Culture of Japan.
References Baba, K., T. Satake, K. Takagi and T. Tomita, 1986, Effects of verapamil on the response of the guinea-pig tracheal muscle to carbachol, Br. J. Pharmacol., 88, 441. Buscail, L., P. Gourlet, A. Cauvin, P.D. Neef, D. Gossen, A. Arimura, A. Miyata, D.H. Coy, P. Robberecht and J. Christophe, 1990, Presence of highly selective receptors for PACAP (pituitary adenylate cyclase activating peptide) in membranes from the rat pancreatic acinar cell line AR 4-2J, Fed. Eur. Biochem. Soc, 262, 77. Cauvin, A., L. Buscail, P. Gourlet, P.D. Neef, D. Gossen, A. Arimura, A. Miyata, D.H. Coy, P. Robberecht and J. Christophe, 1990, The novel VlP-like hypothalamic polypeptide PACAP interacts with high affinity receptors in the human neuroblastoma cell line NB-OK, Peptides l I, 773. Gottschall, P.E., I. Tatsuno, A. Miyata and A. Arimura, 1990, Characterization and distribution of binding sites for the hypothalamic peptide, pituitary adenylate cyclase-activating polypeptide, Endocrinology 127, 272. Hand, J.M., R.B. Laravuso and J.A. Will, 1984, Relaxation of isolated guinea pig trachea, bronchi and pulmonary arteries produced by vasoactive intestinal peptide (VIP), Eur. J. Pharmacol. 98, 279. Honma, M., T. Satoh, J. Takezawa and M. Ui, 1977, An ultrasensirive method for the simultaneous determination of cyclic AMP
and cyclic GMP in small-volume samples from blood and tissue, Biochem. Med. 18, 257. Kimura, C., S. Ohkubo, K. Ogi, M. Hosoya, Y. Itoh, H. Onda, A. Miyata, L. Jiang, R.R. Dahl, H.H. Stibbs, A. Arimura and M. Fujino, 1990, A novel peptide which stimulates adenylate cyclase: molecular cloning and characterization of the ovine and human cDNAs, Biochem. Biophys. Res. Commun. 166, 81. Kitamura, S., Y. Ishihara and S.I. Said, 1980, Effect of VIP, phenoxybenzamine and prednisolone on cyclic nucleotide content of isolated guinea-pig lung and trachea, Eur. J. Pharmacol. 67, 219. K6ves, K., A. Arimura, A. Somogyv~.ri-vigh, S. Vigh and J. Miller, 1990, Immunohistochemical demonstration of a novel hypothalamic peptide, pituitary adenylate cyclase-activating polypeptide, in the ovine hypothalamus, Endocrinology 127, 264. Lam, H.C., K. Takahashi, M.A. Ghatei, S.M. Kanse, J.M. Polak and S.R. Bloom, 1990, Binding sites of a novel neuropeptide pituitary-adenylate-cyclase-activating polypeptide in the rat brain and lung, Eur. J. Biochem. 193, 725. Lowry, O.H., N.J. Rosebrough, A.L. Farr and R.J. Randall, 1951, Protein measurement with the Folin phenol reagent, J. Biol. Chem. 193, 265. Miyata, A., A. Arimura, R.R. Dahl, N. Minamino, A. Uehara, L. Jiang, M.D. Celler and D.H. Coy, 1989, Isolation of a novel 38 residue-hypothalamic polypeptide which stimulates adenylate cyclase in pituitary cells, Biochem. Biophys. Res. Commun. 164, 567. Miyata,A., L. Jiang, R.D. Dahl, C. Kitada, K. Kubo, M. Fuiino, N. Minamino and A. Arimura, 1990, Isolation of a neuropeptide corresponding to the N-terminal 27 residues of the pituitary adenylate cyclase activating polypeptide with 38 residues (PACAP38), Biochem. Biophys. Res. Commun. 17//, 643. Nandha, K.A,, M.A. Benito-Orfila, D.M. Smith, M.A. Ghatei and S.R. Bloom, 1990, Action of pituitary adenylate cyclase-activating polypeptide and vasoactive intestinal polypeptide on the rat vascular system: effects on blood pressure and receptor binding, J. Endocrinol. 129, 69. Robberecht, P., P. Gourlet, A. Cauvin, L. Buscail, P.D. Neef, A. Arimura and J. Christophe, 1991, PACAP and VIP receptors in rat liver membranes, Am. J. Physiol. 26/I, G97. Suzuki, K., K. Takagi, T. Satake, S. Sugiyama and T. Ozawa, 1986, The relationship between tissue levels of cyclic GMP and tracheal smooth muscle relaxation in the guinea-pig, Clin. Exp. Pharmacol. Physiol. 13, 39. Tatsuno, I., P.E. Gottschall, K. K6ves and A. Arimura, 1990, Demonstration of specific binding sites for pituitary adenylate cyclase activating polypeptide (PACAP) in rat astrocytes, Biochem. Biophys. Res. Commun. 168, 1027. Thomas, B.C., 1991, Neuropeptides and tile lung, J. Allergy Clin. Immunol. 88, 1. Tsukawaki, M., K. Suzuki, R. Suzuki, K. Takagi and T. Satake, 1987, Relaxant effects of forskolin on guinea pig tracheal smooth muscle, Lung 165, 225. Warren, J.B., L.E. Donnelly, S. Cullen, B.E. Robertson, M.A. Ghatei, S.R. Bloom and J. MacDermot, 1991, Pituitary adenylate cyclase-activating polypeptide: a novel, long-lasting, endothelium-independent vasorelaxant, Eur.J. Pharmacol. 197, 131. Watanabe, H., K. Takagi and T. Satake, 1988, Relaxant effects of atrial natriuretic polypeptide on guinea pig tracheal smooth muscle, Prog. Biochem. Pharmacol. 23, 136.
113
EJP 52466
Relaxant effect of pituitary adenylate cyclase-activating polypeptide on guinea-pig tracheal smooth muscle Nobuyasu Araki and Kenzo Takagi Second Department of buernal Medicine, Nagoya Unil'ersity School of Medicine, Nagoya, Japan Received 7 November 1991, revised MS received 3 March 1992, accepted 10 March 1992
We investigated the relaxant effect of the pituitary adenylate cyclase-activating polypeptide with 27 residues (PACAP27) and with 38 residues (PACAP38) on guinea-pig tracheal smooth muscle. Both forms of PACAP showed dose-dependent relaxant effects. The ECs~~ of PACAP27 was 8.7 _+ 1.9 x 10 -8 M and that of PACAP38 was 6.8 _+ 1.0 × 10 s M. Both increased cyclic AMP levels dose dependently and the elevation of cyclic AMP preceded the relaxation of tracheal smooth muscle. There was a marked difference in the duration of action of the two peptides. PACAP38 showed a longer-lasting relaxation compared to PACAP27. Furthermore PACAP38 maintained significantly higher levels of cyclic AMP, with cyclic AMP levels at 60 min after a 5-min exposure to PACAPs (10 6 M) being 14.0_+ 1.4 p M / m g protein for PACAP27 and 35.9_+ 2.4 p M / m g protein for PACAP38. These results suggest that PACAP27 and PACAP38 may be novel potent relaxants in tracheal smooth muscle and their relaxant effect might be mediated by cyclic AMP. However PACAP38 had a longer-lasting action on relaxation of tracheal smooth muscle and production of tissue cyclic AMP than PACAP27. PACAP ( pituitary adenylate cyclase-activating polypeptide); Trachea (guinea-pig); Smooth muscle; cAMP
1. Introduction N o v e l n e u r o p e p t i d e s which s t i m u l a t e a d e n y l a t e cyclase activity in rat a n t e r i o r p i t u i t a r y cell c u l t u r e s w e r e r e c e n t l y i s o l a t e d f r o m ovine h y p o t h a l a m i c tissues a n d d e s i g n a t e d as P A C A P 3 8 ( p i t u i t a r y a d e n y l a t e cyclaseactivating p o l y p e p t i d e with 38 r e s i d u e s ) a n d P A C A P 2 7 ( p i t u i t a r y a d e n y l a t e c y c l a s e - a c t i v a t i n g p o l y p e p t i d e with 27 r e s i d u e s ) ( M i y a t a et al., 1989, 1990). C l o n i n g o f P A C A P 3 8 c D N A s c o n f i r m e d t h e e x p r e s s i o n of the c o r r e s p o n d i n g m R N A s a n d the p r e s e n c e o f this neur o p e p t i d e in ovine h y p o t h a l a m u s a n d in h u m a n testis ( K i m u r a et al., 1990). P A C A P 2 7 b i n d i n g sites with very high specificity w e r e o b s e r v e d in rat b r a i n a n d lung ( G o t t s c h a l l et al., 1990; L a m et al., 1990). T h o u g h b o t h forms o f P A C A P c a u s e d a h y p o t e n s i v e effect in t h e a n a e s t h e t i z e d rat ( N a n d h a et al. , 1990) a n d h a d a v a s o d i l a t o r action on r a b b i t a o r t a ( W a r r e n et al., 1991), t h e r e a r e n o p u b l i s h e d d a t a c o n c e r n i n g the effect of
Correspondence to: K. Takagi, Second Department of Internal Medicine, Nagoya University School of Medicine, Tsuruma-cho, Showa-ku, Nagoya 466 Japan. Tel. 81.52.741 2111 ext. 2209, fax 81.52.733 8126.
P A C A P on airway s m o o t h muscle. T h e p u r p o s e o f this study was to e l u c i d a t e the r e l a x a n t effects of b o t h forms o f P A C A P on t r a c h e a l s m o o t h muscle a n d the m e c h a n i s m of the relaxation.
2. Materials and methods 2.1. Tissue preparation and tracheal smooth muscle relaxation Male Hartley guinea-pigs weighing 200-400 g were s t u n n e d a n d bled, a n d the t r a c h e a s w e r e r e m o v e d . O n e t r a c h e a l ring was cut f r o m t h e p h a r y n g e a l e n d o f each t r a c h e a . T h e ring was o p e n e d by c u t t i n g t h r o u g h the cartilaginous region diametrically opposite the tracheal s m o o t h muscle, a n d t h e e n d s o f t h e c a r t i l a g i n o u s region w e r e ligated. T h e p r e p a r a t i o n was p l a c e d in a 1-ml o r g a n bath. T e n s i o n was m e a s u r e d with an isom e t r i c t r a n s d u c e r ( M i n e b e a Co., Ltd., U L 10GR, J a p a n ) , a n d t e n s i o n c h a n g e s w e r e r e c o r d e d with a p e n r e c o r d e r ( T O A E l e c t r o n i c s Ltd., F B R - 2 5 2 A , J a p a n ) . U s i n g t h e m e t h o d p r e v i o u s l y d e s c r i b e d ( B a b a et al., 1986), t h e p r e p a r a t i o n was p e r f u s e d at a c o n s t a n t flow r a t e of 2.0 m l / m i n at 37°C in K r e b s solution of t h e
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following composition (mM): NaC1 137, K H C O 3 5.9, CaCI 2 2.4, MgC12 1.2 and glucose 11.8. Relaxation was produced with isoprenaline (2 × 10 -s M) and a tension of about 0.5 g was applied to the preparation. The preparation was again perfused at 37°C in Krebs solution and resting tone was allowed to develop. After the tension had stabilized, the preparation was then perfused for 20 min in the presence of either form of P A C A P at various concentrations. The complete relaxation occurring on exposure of the tissue to Ca2+-free medium containing 0.01 m M E G T A was defined as 100%, and the relaxation caused by P A C A P was calculated in terms of this standard.
0
g 50-
IO0 10 9
3x10 9
10 e
3x10-8
10 7
[PEPTIDE]
3x10-7
lO-S
M
Fig. 1. Dose-dependent effects of PACAP27 (e) and PACAP38 (©) on resting tone in guinea-pig tracheal smooth muscle. The results are expressed as % relaxation induced by Ca2+-free medium. The data points represent m e a n s with S.E.M. shown by vertical bars (n = 8).
2.2. Measurement of tissue cyclic A M P leuels We measured tissue cyclic A M P levels and evaluated the effect of both forms of P A C A P on cyclic A M P accumulation in tracheal smooth muscle. We resected the m e m b r a n o u s portion from the trachea and obtained a strip by carefully removing the adjacent tissue from the m e m b r a n o u s portion. The preparation, containing epithelium, was equilibrated in a bath with 10 ml Krebs solution for 30 min. The solution was kept at 37°C. The preparation was exposed to P A C A P in various concentrations for the times indicated, then immediately frozen with liquid nitrogen to stop the reaction. We then homogenized the preparation in 2.0 ml icecold 0.1 N HCI, using a Potter homogenizer, and centrifuged the homogenate at 1500 x g for 30 min at 4°C. Cyclic A M P levels in the supernatant fluid were measured by radioimmunoassay ( H o n m a et al., 1977). The protein concentration of the homogenate was determined by the method of Lowry et al. (1951) with bovine albumin as a standard. All biochemical analyses were performed in duplicate.
2.3. Materials PACAP27 and PACAP38 were obtained from Peptide Institute, Inc. (Osaka, Japan). Isoprenaline and E G T A were purchased from Sigma Chemical Co. (St. Louis, MO, U.S.A.). 125I-Succinyi cyclic A M P was purchased from Yamasa Syoyu Co. (Tokyo, Japan). All other chemicals were reagent grade, or the best commercially available grade.
2.4. Statistics
This study was performed in accordance with the guidelines for animal experimentation set by Nagoya University.
3. Results
3.1. Relaxation by PACAP Figure 1 shows the concentration-response curves for the effect of PACAP27 and PACAP38 on resting tone in tracheal smooth muscle. The dose-dependent relaxant effects were observed at concentrations of 10 -9, 3 x 10 -9, 10 8, 3 x 10 -8, 10 -7, 3 X 10 - 7 M and almost complete relaxation was observed at 10 -6 M. The ECs0 values for the relaxant effects of PACAP27 and PACAP38 on resting tone were 8.7 _+ 1.9 × 10 _.8 and 6.8 +_ 1.0 x 10 -8 M, respectively (means with S.E.M., n = 8), both had similar relaxant potency.
3.2. Tissue cyclic AMP levels Figure 2 shows that significant dose-dependent increases in tissue levels of cyclic A M P were observed for
2OO
~E
IOO
0
,j *
o
*
Io ~
Io -~
*
,
*
(O -~
lo 6
(eEP11O£~ M
The data are expressed as means with S.E.M. We used the non-paired Student's t-test for statistical analysis. P values of less than 0.05 were considered statistically significant.
Fig. 2. Dose-dependent effects of PACAP27 (o) and PACAP38 ( o ) on tissue levels of cyclic A M P in guinea-pig tracheal smooth muscle. The preparation was exposed to each P A C A P for 3 rain at each concentration. The data points represent m e a n s with S.E.M. shown by vertical bars (n = 6). (* P < 0.001 vs. control).
115
AO-
-LF PACAP27
150 .=
4,
CaZ+-free sol.
.
,i,
-100
IOmin
50.
PACAP36 50
.ca
1'5
Fig. 4. The upper trace shows the force generated by guinea-pig tracheal smooth muscle exposed for 5 min to PACAP27 (10 ~' M) on resting tone, followed by exposure to Krebs solution. The lower trace shows the force with PACAP38 (10 6 M), superfused with CaZ+-free medium containing 0.01 mM E G T A at 280 rain.
Time (min)
8 O-
-300
- 2OO ~ ~
50-
v
~:
~
-100 .,c
1O0 -
CaZ+- free sol.
~
.0
100
~
1"
5
1'0
{5
ministration while relaxation of tracheal smooth muscle approached a plateau at 5 min after administration. With PACAP38 (fig. 3B), even 15 min after administration, the tissue cyclic AMP levels were increasing slowly and tracheal smooth muscle was relaxing gradually.
0
3.4. Duration of relaxant effect
Time (rain)
Fig. 3. Time course of the relaxant (el and the cyclic AMP-increasing ( o ) effects of a maximum effective concentration (10 -6 M) of PACAP27 (A) and PACAP38 (B). After exposure of the muscle to PACAP for 0, 1, 3, 5 and 15 min, the tissue levels of cyclic AMP and the isometric force were measured. The data points for % relaxation (n = 7) and cyclic AMP (n = 6) represent means with S.E.M. shown by vertical bars (*, P < 0.001 vs. control).
Figure 4 shows typical traces for the duration of the relaxant effect of PACAP27 and PACAP38 on tracheal smooth muscle. Figure 5 shows the curves for the correlation between time and relaxation. The relaxant effect decreased rapidly 5 min after exposure to PACAP27, but PACAP38 had a considerably more prolonged duration of action. The percent relaxation
both forms of PACAP with similar potency (15.2 + 3.1 in control, 120.0+5.9 p M / m g protein at 10 -6 M PACAP27 and 15.3 + 2.1 in control, 159.6 + 16.3 p M / m g protein at 10 -6 M PACAP38).
3.3. Time course relationship between relaxation and elevation of cyclic AMP levels Figure 3 shows that the elevation of tissue cyclic AMP preceded the relaxation of tracheal smooth muscle. Both forms of PACAP slightly relaxed smooth muscle but the tissue cyclic AMP was greatly increased 1 min after the addition of 10 -6 M of either form of PACAP. With PACAP27 (fig. 3A) the tissue cyclic AMP levels approached a plateau at 3 min after ad-
50-
106-
,
o
6'o
,~o
Time (rain)
Fig. 5. Effects of 5-min superfusion of PACAP27 (el and PACAP38 ( o ) on resting tone followed by exposure to Krebs solution. The results are expressed as % relaxation induced by Cae+-free medium. The data points for PACAP27 (n = 10) and PACAP38 (n = 7) represent means with S.E.M. shown by vertical bars.
TABLE 1 The mean level of cyclic AMP generated in tracheal smooth muscle from guinea-pigs immediately after, 20 min after and 60 min after superfusion with Krebs solution following 5-min exposure to PACAP27 (10 -6 M) and PACAP38 (10 -6 M), compared with the control. The data represent means with S.E.M. Cyclic AMP ( p M / m g protein)
PACAP27 (n = 6) PACAP38 (n = 6) a p < 0.001 vs. control.
Control
0 min
20 min
60 min
16.6_+5.6 15.3_+2.1
125.2_+ 18.2 a 186.1 _+ 9.2 a
29.8_+4.0 44.9+3.8 a
14.0_+ 1.4 35.9_+2.4 a
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20 min after superfusion with PACAP27 and PACAP38 was 22.9 _+ 2.3% (n = 10) and 88.6 + 3.5% (n = 7), respectively (means with S.E.M.).
3.5. Relationship between tissue cyclic A M P and longlasting action Table 1 shows that tissue cyclic AMP levels decreased drastically after termination of the exposure of tracheal smooth muscle to PACAP27, although they remained at significantly higher levels even 20 min and 60 min after termination of superfusion with PACAP38. PACAP38 thus had a longer-lasting action on production of tissue cyclic AMP and relaxation of tracheal smooth muscle.
4. Discussion
Since its discovery and characterization, PACAP has attracted much attention. Two forms of PACAP were discovered in hypothalamic tissues by monitoring pituitary adenylate cyclase-stimulating activity (Miyata et al., 1989, 1990). Receptor studies showed PACAP binding sites to be present in high density in the hypothalamus and lung in addition to the pituitary (Gottshall et al., 1990). On the other hand a very small number of PACAP binding sites were found in the aorta (Lam et al., 1990), but Warren et al. (1991) demonstrated that both forms of PACAP were potent, long-lasting, and endothelium-independent vasodilators. Little has been reported regarding the effects of these peptides on smooth muscle preparations other than vascular ones. Our present studies have both shown an effect of PACAP on a non-vascular smooth muscle preparation, i.e. tracheal smooth muscle, and demonstrated the mechanism of their action, through increased cyclic AMP synthesis. PACAP27 and PACAP38 caused nearly equipotent relaxant effects on resting tone in guinea-pig tracheal smooth muscle. The relaxant ECs0 values might represent the high sensitivity of this preparation to PACAP similar to that reported for atrial natriuretic peptide (ECs0 = 1 . 2 ~ 5 . 2 × 10 -7 M, Watanabe et al., 1988), forskolin ( E C 5 0 = 2 . 0 × 10 -s M, Tsukawaki et al., 1987), and sodium nitroprusside (ECs0 = 2.2 × 10 -7 M, Suzuki et al., 1986). Both forms of PACAP increased cyclic AMP levels under the same conditions as those under which relaxation was observed. The relaxant effects of these peptides seem to parallel the extent of the elevation of the level of cyclic AMP. In addition, we observed that an increase in cyclic AMP levels preceded the relaxation of the tracheal smooth muscle. This observation is compatible with the principle that adenylate cyclase-
stimulating agonists induce cyclic AMP accumulation, resulting in relaxation of smooth muscle. The main difference in effects we observed between PACAP27 and PACAP38 was the duration for which they could relax the smooth muscle, with the latter having the longer-lasted relaxant action. Moreover, we demonstrated that PACAP38 maintained significantly higher levels of tissue cyclic AMP concurrent with almost complete relaxation of tracheal smooth muscle for 60 rain, even after its superfusion was stopped. It is possible that PACAP38 remained at the binding sites and continued to stimulate adenylate cyclase. Considering that the actual percentage decreases of cyclic AMP with time were quite similar in both peptides, the mechanism of the longer-lasting relaxation by PACAP38 may be attributed to the initially higher level of cyclic AMP, which might facilitate relaxation of the tracheal smooth muscle, and the subsequent significantly higher levels of cyclic AMP, although lower than the initial peak, which might maintain relaxation of the tracheal smooth muscle. Other mechanisms might probably participate in their different action, which could be related to the difference in their structure. In the case of rabbit aorta, Warren et al. (1991) demonstrated that the relaxant action of both forms of PACAP was surprisingly long-lasted, which could be explained by tight binding to the PACAP receptor. This difference between trachea and aorta might result from tissue and species differences. There might be some subtypes of PACAP receptors. Two classes of PACAP27-binding sites were found in rat lung membranes (Lam et al., 1990). The coexistence of three subtypes of receptors interacting with PACAP has been reported for rat liver membrane, namely high-affinity vasoactive intestinal polypeptide (VIP), low-affinity VIP, and specific PACAP receptors (Robberecht et al., 1991). The presence of high-affinity PACAP receptors in human neuroblastoma cell line NB-OK (Cauvin et al., 1990), in the rat pancreatic carcinoma cell line A R 4-2J (Buscail et al., 1990), and also in rat astrocyte membranes (Tatsuno et al., 1990) was observed. The role of PACAP receptors in the mechanism of long-lasting relaxant action obviously needs to be studied. The N-terminal portion 1-28 of PACAP38 shows 68% homology with VIP (Miyata et al., 1989). VIP is a neurotransmitter which produces dose-dependent relaxation of guinea-pig airways by increasing the tissue cyclic AMP content (Hand et al., 1984, Kitamura et al., 1980). Because PACAP, demonstrated in the hypothalamus (K6ves et al., 1990), was previously suggested to function as a neurotransmitter and as a potent airway smooth muscle relaxant by raising cyclic AMP levels dose dependently as shown by our data, it might act physiologically as a bronchodilator neuropeptide similar to VIP. In normal individuals, there is a balance
117
between the bronchodilator (/3-adrenergic, peptide histidine methionine and VIP) and bronchoconstricting (o~-adrenergic, cholinergic, substance P, neurokinin, and calcitonin gene-related peptide) mechanisms. In subjects with asthma, however, there is an imbalance toward the bronchoconstricting mechanism (Thomas, 1991). Accordingly drug-inducible PACAP or PACAP itself might be useful for the treatment of bronchial asthma by correcting the imbalance toward bronchoconstriction. We showed that both forms of PACAP tested may act as novel potent relaxants on tracheal smooth muscle by raising tissue cyclic AMP levels. PACAP38 had a longer-lasting action on relaxation of tracheal smooth muscle and production of tissue cyclic AMP than PACAP27. Other mechanisms involved in the different action in PACAPs besides cyclic AMP synthesis remain to be investigated.
Acknowledgements The authors thank Dr. Kiyoshi Suzuki and Mrs. Takako Watanabe for their cooperation and express their gratitude to Mr. M. Bodman, language consultant of our department, for reading the previous draft and making suggestions on language and style. This study was supported in part by a grant-in-aid for scientific research from the Ministry of Education, Science and Culture of Japan.
References Baba, K., T. Satake, K. Takagi and T. Tomita, 1986, Effects of verapamil on the response of the guinea-pig tracheal muscle to carbachol, Br. J. Pharmacol., 88, 441. Buscail, L., P. Gourlet, A. Cauvin, P.D. Neef, D. Gossen, A. Arimura, A. Miyata, D.H. Coy, P. Robberecht and J. Christophe, 1990, Presence of highly selective receptors for PACAP (pituitary adenylate cyclase activating peptide) in membranes from the rat pancreatic acinar cell line AR 4-2J, Fed. Eur. Biochem. Soc, 262, 77. Cauvin, A., L. Buscail, P. Gourlet, P.D. Neef, D. Gossen, A. Arimura, A. Miyata, D.H. Coy, P. Robberecht and J. Christophe, 1990, The novel VlP-like hypothalamic polypeptide PACAP interacts with high affinity receptors in the human neuroblastoma cell line NB-OK, Peptides l I, 773. Gottschall, P.E., I. Tatsuno, A. Miyata and A. Arimura, 1990, Characterization and distribution of binding sites for the hypothalamic peptide, pituitary adenylate cyclase-activating polypeptide, Endocrinology 127, 272. Hand, J.M., R.B. Laravuso and J.A. Will, 1984, Relaxation of isolated guinea pig trachea, bronchi and pulmonary arteries produced by vasoactive intestinal peptide (VIP), Eur. J. Pharmacol. 98, 279. Honma, M., T. Satoh, J. Takezawa and M. Ui, 1977, An ultrasensirive method for the simultaneous determination of cyclic AMP
and cyclic GMP in small-volume samples from blood and tissue, Biochem. Med. 18, 257. Kimura, C., S. Ohkubo, K. Ogi, M. Hosoya, Y. Itoh, H. Onda, A. Miyata, L. Jiang, R.R. Dahl, H.H. Stibbs, A. Arimura and M. Fujino, 1990, A novel peptide which stimulates adenylate cyclase: molecular cloning and characterization of the ovine and human cDNAs, Biochem. Biophys. Res. Commun. 166, 81. Kitamura, S., Y. Ishihara and S.I. Said, 1980, Effect of VIP, phenoxybenzamine and prednisolone on cyclic nucleotide content of isolated guinea-pig lung and trachea, Eur. J. Pharmacol. 67, 219. K6ves, K., A. Arimura, A. Somogyv~.ri-vigh, S. Vigh and J. Miller, 1990, Immunohistochemical demonstration of a novel hypothalamic peptide, pituitary adenylate cyclase-activating polypeptide, in the ovine hypothalamus, Endocrinology 127, 264. Lam, H.C., K. Takahashi, M.A. Ghatei, S.M. Kanse, J.M. Polak and S.R. Bloom, 1990, Binding sites of a novel neuropeptide pituitary-adenylate-cyclase-activating polypeptide in the rat brain and lung, Eur. J. Biochem. 193, 725. Lowry, O.H., N.J. Rosebrough, A.L. Farr and R.J. Randall, 1951, Protein measurement with the Folin phenol reagent, J. Biol. Chem. 193, 265. Miyata, A., A. Arimura, R.R. Dahl, N. Minamino, A. Uehara, L. Jiang, M.D. Celler and D.H. Coy, 1989, Isolation of a novel 38 residue-hypothalamic polypeptide which stimulates adenylate cyclase in pituitary cells, Biochem. Biophys. Res. Commun. 164, 567. Miyata,A., L. Jiang, R.D. Dahl, C. Kitada, K. Kubo, M. Fuiino, N. Minamino and A. Arimura, 1990, Isolation of a neuropeptide corresponding to the N-terminal 27 residues of the pituitary adenylate cyclase activating polypeptide with 38 residues (PACAP38), Biochem. Biophys. Res. Commun. 17//, 643. Nandha, K.A,, M.A. Benito-Orfila, D.M. Smith, M.A. Ghatei and S.R. Bloom, 1990, Action of pituitary adenylate cyclase-activating polypeptide and vasoactive intestinal polypeptide on the rat vascular system: effects on blood pressure and receptor binding, J. Endocrinol. 129, 69. Robberecht, P., P. Gourlet, A. Cauvin, L. Buscail, P.D. Neef, A. Arimura and J. Christophe, 1991, PACAP and VIP receptors in rat liver membranes, Am. J. Physiol. 26/I, G97. Suzuki, K., K. Takagi, T. Satake, S. Sugiyama and T. Ozawa, 1986, The relationship between tissue levels of cyclic GMP and tracheal smooth muscle relaxation in the guinea-pig, Clin. Exp. Pharmacol. Physiol. 13, 39. Tatsuno, I., P.E. Gottschall, K. K6ves and A. Arimura, 1990, Demonstration of specific binding sites for pituitary adenylate cyclase activating polypeptide (PACAP) in rat astrocytes, Biochem. Biophys. Res. Commun. 168, 1027. Thomas, B.C., 1991, Neuropeptides and tile lung, J. Allergy Clin. Immunol. 88, 1. Tsukawaki, M., K. Suzuki, R. Suzuki, K. Takagi and T. Satake, 1987, Relaxant effects of forskolin on guinea pig tracheal smooth muscle, Lung 165, 225. Warren, J.B., L.E. Donnelly, S. Cullen, B.E. Robertson, M.A. Ghatei, S.R. Bloom and J. MacDermot, 1991, Pituitary adenylate cyclase-activating polypeptide: a novel, long-lasting, endothelium-independent vasorelaxant, Eur.J. Pharmacol. 197, 131. Watanabe, H., K. Takagi and T. Satake, 1988, Relaxant effects of atrial natriuretic polypeptide on guinea pig tracheal smooth muscle, Prog. Biochem. Pharmacol. 23, 136.
