Cardiovascular and respiratory actions of pituitary adenylate cyclase-activating polypeptides

Regulatory Peptides, 40 (1992) 29-39 © 1992 Elsevier Science Publishers B.V. All rights reserved 0167-0115/92/$05.00

29

REGPEP 01191

Cardiovascular and respiratory actions of pituitary adenylate cyclase-activating polypeptides Yoshihisa Ishizuka, Kazuhisa Kashimoto, Tohru Mochizuki, Keigo Sato, Keiich Ohshima and Noboru Yanaihara Laboratory of Bioorganic Chemistry, University of Shizuoka School of Pharmaceutical Science, Shizuoka (Japan) (Received 30 July 1991; revised version received 16 February 1992; accepted 20 March 1992)

Key words: Hypothalamic peptide; Pituitary adenylate cyclase-activating polypeptide; Vasoactive intestinal polypeptide (VIP); Femoral blood flow; Blood pressure; Heart rate

Summary Effects of pituitary adenylate cyclase-activating polypeptide (PACAP38) and PACAP27 on the cardiovascular and respiratory systems were examined and compared to those of vasoactive intestinal polypeptide (VIP) in anesthetized beagle dogs. Intravenous PACAP27 and PACAP38 produced a decrease in mean arterial blood pressure (MBP), and an increase in both femoral arterial blood flow (ABF) and in frequency of respiration (FR) with a dose-dependent relationship between 10 and 300 pmol/kg. PACAP27 produced a dose-dependent increase in heart rate (HR) between 10 and 300 pmol/kg while PACAP38 induced tachycardia which was not dosedependent. Administration of 300 pmol/kg PACAP38 and PACAP27 produced extreme hypertension after transient hypotension. PACAP38 produced severe bradycardia after transient tachycardia. The cardiovascular actions of PACAP38 were persistent compared to those of PACAP27. Intravenous injection of 10-300 pmol/kg VIP brought about hypotension, tachycardia and an increase in ABF and FR with a dose-dependent relationship. VIP, at 2000 pmol/kg, did not produce the biphasic response obtained by a large dose of PACAP38. The present studies demonstrate that PACAP partially possesses VIP-like cardiovascular and respiratory actions and that the C-terminal 11 amino acid residues of PACAP38 are presumably responsible for a prolongation of its actions.

Correspondence to: N. Yanaihara, Laboratory of Bioorganic Chemistry, University of Shizuoka School of Pharmaceutical Science, 52-1, Yada, Shizuoka, 422, Japan.

30 Introduction

Recently, a 38 amino acid hypothalamic peptide, named pituitary adenylate cyclaseactivating polypeptide (PACAP38) was discovered in extracts of ovine hypothalamic tissue [ 1]. PACAP27, corresponding to the N-terminal 27 amino acids of PACAP38, was also isolated during the purification of PACAP38 [2]. These peptides stimulate adenylate cyclase activity in rat anterior pituitary cell cultures. The N-terminal 28 amino acid sequence of PACAP38 shows a 68~o homology with the amino acid sequence of vasoactive intestinal polypeptide (VIP) [3]. VIP has several physiological activities including hypotension, tachycardia and bronchodilation in laboratory animals and man [4]. VIP displays a specific physiological action following activation of adenylate cyclase. The activation of adenylate cyclase by PACAP is about 1000-fold higher than that of VIP in rat anterior pituitary cells [2]. Intravenous administration of PACAP can produce a dose-dependent decrease in arterial blood pressure in the rat [2]. Immunohistochemical studies show the presence of PACAP nerve fibers in the central nervous system [5]. PACAP nerve fibers were observed surrounding the blood vessels in the ovine brain. Furthermore, specific binding sites of PACAP were detected in the bovine [6] and rat [7,8] brain, and in peripheral tissues [8-10] including lung, duodenum, liver and aorta. Lam et al. [7] have examined the regional distribution of PACAP receptors in the rat brain and reported that the specific binding sites for PACAP existed predominantly in the hypothalamus, brain stem, cerebellum, cerebral cortex and anterior pituitary. These reports suggested that PACAP may play a physiological role. The purpose of the present experiments was to determine whether PACAP has cardiovascular and respiratory actions. The effects of PACAP27 and PACAP38 on blood pressure, heart rate, blood flow and respiration were examined and compared to those of VIP in beagle dogs.

Materials and Methods

Peptide synthesis' Synthetic PACAP27 and PACAP38 were prepared by solid phase techniques using an automated peptide synthesizer (ABI model 430A). The peptide chain was elongated on a p-methylbenzhydrylamine-polystyrene-2~odivinylbenzene copolymer resin (0.72 mmol or 0.64 mmol NH2/g, 0.5 mmol, ABI) with use of appropriate N%Boc amino acid derivatives such as Boc-Leu, Boc-Val, Boc-Ala, Boc-Lys (C1-Z), Boc-Tyr (C12-Bzl), Boc-Met, Boc-Gln, Boc-Arg (Tos), Boc-Ser (Bzl), Boc-Thr (Bzl), Boc-Phe, Boc-Ile, Boc-Gly, Boc-Asp (OcHex), Boc-His (Dnp) and Boc-Asn. Starting with the resin, an appropriate N=-Boc amino acid derivatives was successively coupled via the HOBt active ester using a 4-fold excess amount in DMF. After construction of a desired peptide chain, the protected peptide resin was treated with anhydrous HF. The liberated peptide was extracted with 3 M acetic acid containing 0.02~o ethanethiol and lyophilized. The crude peptide was further purified by reverse phase HPLC on a preparative YMC Pack D-ODS-5 (2.0 x 25 cm) column in a solvent system of 0.01 M

31 HC1/CH3CN. Purities of synthetic PACAP27 and PACAP38 were confirmed by amino acid analysis of an acid hydrolysate and AP-M enzymatic digest, analytical HPLC and sequence analysis.

Surgical operation Female beagle dogs (9-10 kg) were used. The animals were anesthetized by intravenous injection of 20 mg/kg pentobarbital sodium (Dainihon Chemical Company). Surgery and experiments were done in rooms with controlled temperature (24-26 ° C) and humidity (50-60~o). A venula catheter (16G × 2" Top Corporation) filled with heparinized saline (10 U/ml) inserted 5 cm into left femoral artery and connected to a pressure transducer (model DX-300; Nihon-Kohden) to measure arterial blood pressure. The mean arterial blood pressure (MBP) was obtained by electronic damping of the pulsatile blood pressure signal. Heart rate (HR) was determined electronically from the pulse interval of blood pressure using a heart rate counter (model AT-601G; Nihon-Kohden). Frequency of respiration (FR) was counted by a respiration pick-up (model TR-602T; Nihon-Kohden) which recorded breathing movements of the chest. To measure arterial blood flow (ABF), an electromagnetic blood flowmeter probe (model MFV-3200; Nihon-Kohden) was placed on the right femoral artery. Each parameter monitored was simultaneously recorded on a thermal recorder (model RTA1300; Nihon-Kohden) connected to a Nihon-Kohden model RM-6000 polygraph system. Pentobarbital sodium (3-5 mg/kg per h) was infused by a digital infusion syringe pump (model 221W; Harvard Apparatus) through a polyethylene catheter (PE-50) inserted into the right femoral vein to maintain the anesthetized condition. Another polyethylene catheter (PE-50) inserted into the left femoral vein was used for administration of peptides.

Administration of peptides Stock solutions of peptides were prepared in distilled water. Appropriate dilutions were made with saline. Peptides were administered by bolus intravenous injection through an indwelling left femoral catheter. Multiple administration of peptides were started after cardiovascular parameters returned to baseline values.

Statistical analysis Results were subjected to analysis of variance (one- or two-way for appropriate groups). Newman-Keuls tests were done to differentiate group means if significant interactions were found by the analysis of variance.

Results

Resting values for MBP, HR, ABF and FR in beagle dogs are shown in Table I. Saline vehicle produced no changes in any parameters. Effects of VIP, PACAP27 and PACAP38, at 10 pmol/kg, on the MBP, HR, ABF and FR are shown in Fig. 1. All three peptides produced a decrease in the MBP and an increase in the HR, ABF and FR. Effects of PACAP38 on cardiovascular param-

32 TABLE I Resting values for cardiovascular and respiratory parameters in anesthetized beagle dogs Parameters

Resting values

Mean arterial blood pressure Heart rate Femoral arterial blood flow Frequency of respiration

156.2 + 5.3 mmHg 228.0 + 21.9 beat/rain 64.3 + 4.3 ml/min 16.8 + 1.9 counts/rain

Results are presented as mean _+S.E.M. from seven animals.

eters, especially the MBP, were persistent. The duration of the hypotensive action of these three peptides are shown in Table II. PACAP38-induced hypotension was significantly persistent when compared to other peptides. A

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33 TABLE II Lasting time of hypotension following peptides treatment in anesthetized beagle dogs Peptides

Lasting time

VIP PACAP27 PACAP38

1.04 + 0.27 1.66 + 0.26 2.80 _+0.29*

Results are presented as mean _+S.E.M. from 4-5 animals. *P< 0.01 and P < 0.05 compared to VIP-treated and PACAP27-treated groups, respectively.

The initial decrease in the MBP following injection of PACAP indicated a dosedependent relationship between 10 and 300 pmol/kg, but the effect was smaller than that of VIP (Fig. 2). PACAP27 and VIP produced a dose-dependent tachycardia. PACAP38, however, produced a slight increase in HR without a dose-dependent relationship (Fig. 2). Administration of 300 pmol/kg PACAP brought about an extreme hypertension after transient hypotension and a severe bradycardia following transient tachycardia (Fig. 3). VIP, at 2000 pmol/kg, did not show such a biphasic response (Fig. 4). The total of ABF indicated by the area under the response curve on the recording chart following intravenous injection of each peptide is shown in Fig. 5. PACAP38 produced a significant increase in ABF at doses of 10 and 30 pmol/kg when compared to other peptides. PACAP27, at 300 pmol/kg, produced an increase in ABF. PACAP as well as VIP produced a dose-dependent stimulation of respiration (Fig. 6). At 300 pmol/kg, the excitatory effect of VIP was more potent, when compared to PACAP.

Discussion

PACAP27 and PACAP38 are novel hypothalamic polypeptides which activate adenylate cyclase in rat pituitary cells [ 1]. The N-terminal 27 amino acid residues of these peptides exhibit a 71 ~o similarity with VIP. In the present study, effects of PACAP on MBP, HR, ABF and FR were examined in beagle dogs and compared to those of VIP. The present results demonstrate that PACAP partially possesses VIP-like cardiovascular and respiratory actions and that the C-terminal 11 amino acid residues of PACAP38 are responsible for a prolongation of its actions. The cardiovascular effects of PACAP27 that were observed following administration of 10-100 pmol/kg were similar to those of VIP. Depressor effects of PACAPs have been identified by Miyata et al. [2]. VIP-induced increase in blood flow and hypotension result mainly from the vasodilating activity of this peptide. PACAP27 presumably has a direct action on blood vessels since PACAP receptors exist in the aorta [ 10]. VIP might accelerate heart rate by its direct actions on heart [ 11 ] or dilation of coronary arteries [ 12]. Since there are no reports regarding PACAP receptors on cardiac

34

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tissue, it is not evident how PACAP exerts tachycardia. Therefore, further studies with isolated heart preparation should be undertaken to understand the mechanism of action of PACAP27. The cardiovascular effects of PACAP38 were obviously persistent when compared to those of PACAP27. Structural differences between PACAP27 and PACAP38 are due to the additional C-terminal 11 amino acid residues of PACAP38. The sequence of the C-terminal 11 amino acids is Gly-Lys-Arg-Tyr-Lys-Gln-Arg-Val-Lys-Asn-Lys amide. This sequence contains a large number of basic amino acid. The basic character of these additional amino acids is presumably responsible for the prolongation of PACAP38 actions. However, the binding affinity of PACAP38 to the PACAP receptor is similar to that of PACAP27 based on displacement studies for radioactive PACAP27 binding [6,7]. The prolongation of PACAP38 actions, therefore, may be due to its slow rate of degradation. Another difference in the action between PACAP27 and

36

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P A C A P 3 8 is observed in the heart rate. P A C A P 3 8 p r o d u c e d only a small increase in heart rate without a d o s e - d e p e n d e n t relationship. If the effect on the heart rate resulted from a peripheral action, P A C A P - i n d u c e d t a c h y c a r d i a might be mediated through the VIP receptor. It is possible that P A C A P 2 7 has a higher affinity for the VIP receptor

37

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than PACAP38, since the amino acid sequence of PACAP27 resembles that of VIP more closely when compared to PACAP38. It is noteworthy that in an in vivo cardiovascular experiment, interpretation of results is often complicated by counteracting vagal and baroreceptor reflexes. Consequently, further studies are necessary to understand the prolonged actions of PACAP38 on the cardiovascular system. Interestingly, the present study shows that a large dose of PACAP produced the reverse response following the initial change in blood pressure, heart rate and blood flow. Such a reversal was not observed in VIP-treated animals. The biphasic response produced by PACAPs is similar to the action of epinephrine, with the exception of the increase in peripheral blood flow in PACAP-treated animals. This unbalanced reaction seems likely to result from a large increase in cardiac output and/or some central actions of PACAPs. If the baroreceptor reflex or if the central responses to the baroreceptor were influenced by PACAPs, a precise regulation of the unusual blood pressure, cardiac output or peripheral resistance could not occur. In order to understand this biphasic response, it is necessary to examine cardiac output, the transport of PACAPs to the central nervous system through the blood-brain barrier and identification of their sites of action related to cardiovascular regulation. In conclusion, the present results demonstrate that PACAP has cardiovascular and respiratory actions, that the C-terminal 11 amino acid residues of PACAP38 are

38

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probably responsible for a prolongation of its actions, and that PACAP has novel in addition to its VIP-like activity.

References 1 Miyata, A., Arimura, A., Dahl, R. R., Minamino, N., Uehara, A., Jiang, L., Culler, M. D. and Coy. D. H., Isolation of a novel 38 residue-hypothalamic polypeptide which stimulates adenylate cyclase in pituitary cell, Biochem. Biophys. Res. Commun., 164 (1989) 567-574. 2 Miyata, A., Jiang, L., Dahl, R.D., Kitada, C., Kubo, K., Fujino, M., Minamino, N. and Arimura, A., Isolation of a neuropeptide corresponding to the N-terminal 27 residues of the pituitary adenylate cyclase activation polypeptide with 38 residues (PACAP38), Biochem. Biophys. Res. Commun., 170 (1990) 643-648. 3 Mutt, V. and Said, S.I., Structure of the porcine vasoactive intestinal octacosapeptide, Eur. J. Biochem., 42 (1974) 581-589. 4 Said, S.I., Vasoactive intestinal peptide, J. Endocrinol. Invest., 9 (1986) 191-200. 5 Koves, K., Arimura, A., Somogyvari-Vigh, A., Vigh, S. and Miller, J., Immunohistochemical demonstration of a novel hypothalamic peptide, pituitary adenylate cyclase-activating polypeptide, in the ovine hypothalamus, Endocrinology, 127 (1990) 264-271.

39 60htaki, T., Watanabe, T, Ishibashi, Y., Kitada, C., Tsuda, M., Gottschall, P. E., Arimura, A. and Fujino, M., Molecular identification of receptor for pituitary adenylate cyclase activating polypeptide, Biochem. Biophys. Res. Commun., 171 (1990) 838-844. 7 Tatsuno, I., Gottschall, P.E., Koves, K. and Arimura, A., Demonstration of specific binding sites for pituitary adenylate cyclase activating polypeptide (PACAP) in rat astrocytes, Biochem. Biophys. Res. Commun., 168 (1990) 1027-1033. 8 Gottschall, P. E., Tatsuno, I., Miyata, A. and Arimura, A., Characterization and distribution of binding sites for the hypothalamic peptide, pituitary adenylate cyclase-activating polypeptide, Endocrinology, 127 (1990) 272-277. 9 Robberecht, P., Gourlet, P., Cauvin, A., Buscail, L., Deneef, P., Arimura, A. and Christophe, J., PACAP and VIP receptors in rat liver membranes, Am. J. Physiol., 260 (1991) G97-G102. 10 Lain0 H-C., Takahashi, K., Ghatei, M.A., Kanse, S.M., Polak, J.M. and Bloom, S.R., Binding sites of a novel neuropeptide pituitary-adenylate-cyclase-activating polypeptide in the rat brain and lung, Eur. J. Biochem., 193 (1990) 725-729. 11 Rigel, D., Effects of neuropeptides on heart rate on dogs: comparison of VIP, PHI, NPY, CGRP and NT, Heart Circ. Physiol., 24 (1988) H311-H317. 12 Smitherman, T.C, Popma, J.J., Said, S.I.0 Krejs, G.J. and Dehmer, G.J., Coronary hemodynamic effects of intravenous vasoactive intestinal peptide in humans, Am. J. Physiol., 257 (1989) H1254-H 1262. 13 Said, S.I. and Mutt, V., Potent peripheral and splanchnic vasodilator peptide from normal gut, Nature, 225 (1970) 863-863.