Effects of pituitary adenylate cyclase activating polypeptide (PACAP) and vasoactive intestinal polypeptide (VIP) on the cardiovascular system in sheep

Peptides,Vol. 13, pp. 1029-1032, 1992

0196-9781/92 $5.00 + .00 Copyright © 1992 Pergamon Press Ltd.

Printed in the USA.

BRIEF COMMUNICATION

Effects of Pituitary Adenylate Cyclase Activating Polypeptide (PACAP) and Vasoactive Intestinal Polypeptide (VIP) on the Cardiovascular System in Sheep K. S A W A N G J A R O E N , *l C. R. D A L L E M A G N E , t

R. B. C R O S S * A N D J. D. C U R L E W I S *

*Department of Physiology and Pharmacology, The University of Queensland, Queensland 4072, Australia and ?School of Life Sciences, Queensland University of Technology, Brisbane 4000, Australia R e c e i v e d 7 F e b r u a r y 1992 SAWANGJAROEN, K., C. R. DALLEMAGNE, R. B. CROSS AND J. D. CURLEWIS. Effects of pituitary adenylate cyclase activating polypeptide (PACAP)and vasoactive intestinalpolypeptide (VIP) on the cardiovascularsystem in sheep. PEPTIDES 13(5) 1029-1032, 1992.--The cardiovascular effects of PACAP and VIP were studied in intact conscious sheep; PACAP (0.008, 0.04, 0.2, and 1.0 nmol/min) and VIP (0.07, 0.2, 0.6, and 1.8 nmol/min) were infused in conscious sheep for periods of 10 min. For each peptide there was a dose-dependent increase in heart rate. At the highest doses tested, pulse pressure and mean arterial pressure tended to increase and decrease, respectively. However, only the decrease in mean arterial pressure following the highest dose of VIP reached significance. At the highest doses tested, heart rate increased nearly threefold during the infusion while mean arterial pressure declined by 18.5%. In individual animals the decrease in blood pressure and increase in heart rate occurred simultaneously, so that we were unable to conclude whether the increase in heart rate was due to a baroreceptor reflex. Cardiovascular response Conscious sheep

Vasoactive intestinal polypeptide

P I T U I T A R Y adenylate cyclase activating polypeptide (PACAP) is a 38 amino acid peptide recently isolated from ovine hypothalami (9). While amino acids 29-38 of PACAP have no known homologues, the N-terminal sequence (1-28) exhibits 68% sequence identity with VIP. It is already known that PACAP is widely distributed within neurones of the hypothalamus (7,14) and specific PACAP binding sites are located centrally in regions including the hypothalamus and pituitary gland (5,6,8). In contrast, there is no published information on the distribution of PACAP in peripheral nerves and tissues. Nevertheless, high affinity PACAP binding sites do exist in such tissues, although at least in lung, liver, and blood vessels, these binding sites do not distinguish between PACAP and VIP (6,10). It seems, therefore, as with many neuropeptides, the role of P A C A P will be multifunctional, possibly as a hypophysiotropic hormone, neurotransmitter, or neuromodulator a n d / o r regulator within various tissues.

Requests for reprints should be addressed to K. Sawangjaroen.

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Pituitary adenylate cyclase activating polypeptide

Vasoactive intestinal polypeptide and related peptides are known to be vasoactive (15) and, because VIP and PACAP appear to act at the same peripheral receptor, it would seem likely that the two peptides may share similar actions on the cardiovascular system. This hypothesis was tested by infusion of various doses of PACAP and VIP into conscious sheep. METHOD

Animals and Experimental Protocol Experiments were conducted on 6 ewes, body weight 51.3 + 3.4 kg, in which the carotid artery had been surgically relocated into a loop of skin. The ewes were kept in a light-proof and temperature-controlled building and subjected to an artificial photoperiod ( 12L: 12D). A pair of ewes were selected at random and studied on consecutive days. On the first day, one ewe received the peptide at various doses while the other received the

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vehicle. Treatments were reversed on the following day. On the day of the experiment, each ewe was removed from its pen and placed in a trolley fitted with a sling where it remained for the entire experimental period. Two polyethylene cannulae were inserted into the carotid artery via the carotid loop under local anesthesia with 2% lidocaine (Xylocaine, Astra Pharmaceuticals Pty. Ltd.). One cannula was connected via a Statham pressure transducer to a model 7D Grass polygraph. Blood pressure, mean arterial pressure (electronically derived from blood pressure by the apparatus), and heart rate were continuously recorded for the duration of each experiment. The other cannula was connected to a syringe infusion pump so that the infusion was made towards the brain. After cannulation, the animals were allowed a quiet resting period of at least 2 h following which baseline cardiovascular parameters were recorded. Peptide or vehicle was then administered by intracarotid infusion (l.0 ml/min for 10 rain). In three separate experiments, the following doses of peptides were tested: PACAP, 1.0 nmol/min; PACAP, 0.008, 0.04, 0.2 nmol/min; VIP, 0.07, 0.2, 0.6, 1.8 nmol/min. Where the multiple doses were used, the order of the doses was in ascending strength. Each dose was tested in six ewes. For each peptide, the interval between doses was at least 1 h. In all experiments, the control ewes received the vehicle given at the same time as the peptide infusion.

Source and Preparation o/Peptides Both PACAP38 (Peptide Institute Inc., Osaka, Japan) and VIP (Auspep Pty. Ltd., Parkville, Australia) were dissolved in sterile water for injection and aliquoted to yield the appropriate amount for each experiment. The peptide solutions were then lyophilized and stored at -20°C. On the day of each experiment, peptides were dissolved in sterile water for injection and diluted to give the appropriate concentration.

Calculations and Statistics Heart rate and mean arterial pressure at 5, 2, 4, 6, 9, 15, 20, 25, and 40 min after the start of each infusion were calculated directly from the original tracing. Pulse pressure at the same time points was calculated as the difference between systolic and diastolic pressures. For analysis, the data for each variable from each peptide infusion was separated into three sets: before infusion, during infusion, and after infusion. The effect of treatment within each set was examined by analysis of variance (ANOVA) for repeated measurements with the degrees of freedom corrected by epsilon correction (16). Pairwise comparisons were then made using Duncan's multiple range test. Where the interaction between time and dose was significant, a one-way ANOVA was performed at each time point. RESULTS

Mean heart rate, pulse pressure, and arterial pressure at various time points before, during, and after infusion of PACAP and VIP are shown in Fig. 1. Both peptides markedly increased heart rate in a dose-dependent manner, with significant changes from the control at the dose 0.2 and 1.0 nmol/min for PACAP, F( 1.9, 9.4) = 12.43, p < 0.01, and 0.6 and 1.8 nmol/min of VIP, F( 1.5, 7.6) = 35.32, p < 0.01. At the highest doses tested (PACAP, 1.0 nmol/min; VIP, 1.8 nmol/min), heart rate began to increase within 1 rain of the start of infusion (results not shown) to reach a maximum of almost threefold the resting value at 5.0 + 0.6 and 4.9 +_ 0.4 min for PACAP and VIP, respectively. During the remainder of the infusion period and after the infusion had stopped, heart rate declined so that significant differences [PA-

CAP, F(1.5, 7) = 3.503, p > 0.05; VIP, F(1.3, 6.6) = 1.055, p > 0.05] from control recordings were not apparent by 5 min post-infusion. At the next lower dose (PACAP, 0.2 nmol/min: VIP, 0.6 nmol/min) heart rate tended to increase gradually through the entire infusion period. Mean arterial pressure tended to decrease during infusion of the highest doses of peptide, although at the various time points analyzed, this effect only reached significance for VIP when infused at 1.8 nmol/min, F(2.1, 10.5) - 22.26, p < 0.01. This decline in mean arterial pressure reached a minimum at 6.8 +_ 1.3 rain, which was later than that for maximum heart rate. At all time points examined, pulse pressure did not increase significantly during infusion of the peptide. In an additional experiment, we tested whether the hemodynamic effects of PACAP were influenced by the route of administration. In two ewes, PACAP at the dose 0.1 nmol/min was infused either intravenously via the jugular vein or via the intracarotid route. During infusion, heart rate increased to almost twofold the resting value irrespective of route of administration. while there was no apparent change in mean arterial pressure and pulse pressure. At the highest doses of peptide, we also observed skin flushing and increased nasal secretion in five out of six ewes receiving PACAP and in all ewes treated with VIP. DISCUSSION

The results of the present study clearly demonstrate that PACAP and VIP can markedly increase heart rate. What remains uncertain, however, is whether the increase in heart rate is in response to hypotension. Certainly, at the highest dose of each peptide, there was clear evidence of vasodilation, in the form of skin flushing and trends towards a decrease in mean arterial pressure and an increase in pulse pressure. However, only the decrease in mean arterial pressure during infusion of the highest dose of VIP was significant. In contrast, in the anesthetized rat, these peptides cause a rapid but transient dose-dependent hypotensive effect that is accompanied by an increase in heart rate due to reflex tachycardia (10). In our study of conscious sheep, it is quite clear, however, that the magnitude of the tachycardia was greater than would be expected from a baroreceptor activation. For example, when hypotension is induced by infusion of nitroprusside, a peripheral vasodilator, into conscious sheep, a 23.8% decrease in mean arterial pressure results in only a 53.9% increase in heart rate (Dallemagne, unpublished observation). By comparison, mean arterial pressure fell by 4.1 and 9.3% during infusion of 0.2 nmol PACAP/min and 0.6 nmol VIP/min but heart rate increased by 90.1 and 104.2%, respectively. It is therefore unlikely that the rapid tachycardia induced by PACAP and VIP is due solely to changes in blood pressure. Vasoactive intestinal polypeptide is a direct vasodilator in cerebral, pulmonary, coronary, and other systemic vessels (15). Vasoactive intestinal polypeptide exerts direct positive chronotropic effects on the heart in dog, monkey, and human (2,4,11 ), whereas it has only small effects in rat (Sawangjaroen, Brown, and Curlewis, unpublished results) and none in guinea pigs (12). These actions of VIP on heart rate correlate well with the ability of V1P to induce adenylate cyclase activity in heart membranes from these various species (1,13). Although there is no information about VIP receptors in the sheep heart, our results suggest that sheep are similar to the former group in which VIP has a direct stimulatory action on adenylate cyclase to increase cAMP and thus heart rate. Whether PACAP exists within the cardiovascular system or as a circulating hormone is currently unknown. Previous

EFFECTS O F PACAP A N D VIP O N SHEEP C A R D I O V A S C U L A R

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FIG. 1. Cardiovascular changes in response to PACAP and VIP infusion. The data shown are heart rate (A,B), mean arterial pressure (C,D), and pulse pressure (E,F) before, during, and after a 10-min infusion of either the vehicle (EEl--D, D---D) or PACAP at the doses 0.008 nmol/min (A--A), 0.04 nmol/min (11----II),0.2 nmol/min (A--A), or 1.0 nmol/min (X--X), or VIP at the doses 0.07 nmol/min (An-A), 0.2 nmol/min (An-A), 0.6 nmol/min (m---m), or 1.8 nmol/min (X---x). Values are the mean _+ SEM (n = 6). Some standard error bars are omitted for clarity. The stippled bars indicate the infusion period. Horizontal bars indicate the values used for statistical analysis. *p < 0.05: **p < 0.01; NS, nonsignificant as compared with the vehicle-infused control group.

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S A W A N G J A R O E N ET AL.

research has d e m o n s t r a t e d that VIP-containing fibers are widely distributed in vascular smooth muscle and throughout the heart (3). In view of the sequence identity between P A C A P and VIP, antibodies used previously to detect VIP could in fact have cross-reacted with PACAP. Therefore, the distribution of these two peptides in the cardiovascular system needs to be reevaluated. However, from a functional viewpoint, the overall similarity in cardiovascular responses to

either peptide tends to suggest a lack of functional diversity for these peptides. This is supported by recent observations that some populations of peripheral binding sites do not distinguish between P A C A P and VIP (6,10). ACKNOWLEDGEMENTS We wish to thank Mrs. Robyn Colcord, Miss Fiona Come, and Miss Denise Macgregor for their technical assistance.

REFERENCES I. Christophe, J.; Waelbroeck, M.; Chatelain, P.; Robberecht, P. Heart receptors for VIP, PHI and secretin are able to activate adenylate cyclase and to mediate inotropic and chronotropic effects. Species variations and physiopathology. Peptides 5:341-353; 1984. 2. De Neef, P.; Robberecht, P.; Chatelain, P.; Waelbroeck, M.; Christophe, J. The in vflro chronotropic and inotropic effects of vasoactive intestinal peptide (VIP) on the atria and ventricular papillary muscle from Cynomolgus monkey heart. Regul. Pept. 8:237-244; 1984. 3. Forssmann, W. G.; Triepel, J.; Daffner, C.; Heym, Ch.; Cuevas, P.; Noble, M. I. M.; Yanaihara, N. Vasoactive intestinal peptide in the heart. Ann. NY Acad. Sci. 527:405-420; 1988. 4. Franco-Cereceda, A.; Bengtsson, L.; Lundberg, J. M. lnotropic effects of calcitonin gene-related peptide, vasoactive intestinal polypeptide and somatostatin on the human fight atrium in vitro. Eur. J. Pharmacol. 134:69-76; 1987. 5. Gottschall, P. E.; Tatsuno, I.; Arimura, A. Hypothalamic binding sites for pituitary adenylate cyclase activating polypeptide: Characterization and molecular identification. FASEB J. 5:194-199; 1991. 6. Gottschall, P. E.; Tatsuno, I.: Miyata, A.; Arimura, A. Characterization and distribution of binding sites for the hypothalamic peptide, pituitary adenylate cyclase-activating polypeptide. Endocrinology 127:272-277; 1990. 7. Krves, K.; Arimura, A.; Somogyv~ri-Vigh, A.; Vigh, S.: Miller, J. Immunohistochemical demonstration of a novel hypothalamic peptide, pituitary adenylate cyclase-activating polypeptide, in the ovine hypothalamus. Endocrinology 127:264-271 ; 1990. 8. Masuo, Y.; Ohtaki, T.; Masuda, Y.: Nagai, Y.: Suno, M.; Tsuda, M.; Fujino, M. Autoradiographic distribution of pituitary adenylate cyclase activating polypeptide (PACAP) binding sites in the rat brain. Neurosci. Lett. 126:103-106; 1991.

9. Miyata, A.: Arimura, A.; Dahl, R. R.; Minamino, N.: Uehara, A.: Jiang, L.; Culler, M. D.; Coy, D. H. Isolation of a novel 38 residuehypothalamic polypeptide which stimulates adenylate cyclase in pituitary cells. Biochem. Biophys. Res. Commun. 164:567-574; 1989. 10. Nandha, K. A.; Benito-Orfila, M. A.; Smith, D. M.; Ghatei, M. A.: Bloom, S. R. 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. 129:69-73; 1991. 11. Rigel, D. F. Effects of neuropeptides on heart rate in dogs: Comparison of VIP, PHI, NPY, CGRP, and NT. Am. J. Physiol. 255: H311-H317: 1988. 12. Saito, A.; Kimura, S.; Goto, K. Calcitonin gene-related peptide as potential neurotransmitter in guinea pig fight atrium. Am. J. Physiol. 250:H693-H698; 1986. 13. Taton, G.: Chatelain, P.; Delhaye, M.: Camus, J. C.; De Neef, P.; Waelbroeck, M.; Tatemoto, K.; Robberecht, P.; Christophe, J. Vasoactive intestinal peptide (VIP) and peptide having N-terminal isoleucine amide (PHI) stimulate adenylate cyclase activity in human heart membranes. Peptides 3:897-900; 1982. 14. Vigh, S.: Arimura, A.; Krves, K.; Somogyv~iri-Vigh, A.; Sitton, J.: Fermin, C. D. Immunohistochemical localization of the neuropeptide, pituitary adenylate cyclase activating polypeptide (PACAP), in human and primate hypothalamus. Peptides 12:313-318: 1991. 15. Wharton, J.: Gulbenkian, S. Peptides in the mammalian cardiovascular system. In: Polak, J. M., ed. Regulatory peptides. Berlin: Birkh~iuser Verlag; 1989:292-316. 16. Winer, B. J. Statistical principles in experimental design. 2nd ed. New York: McGraw-Hill Book Company: 197 1:281-282.