Vasoactive intestinal polypeptide receptor antagonism enhances the vagally induced increase in cardiac interval of the rat atrium in vitro

Exp Physiol 91.3 pp 641 646 641 Experimental Physiology Vasoactive intestinal polypeptide receptor antagonism enhances the vagally induced increase in cardiac interval of the rat atrium in vitro K. Hogan and F. Markos School of Pharmacy & Pharmaceutical Sciences, Trinity College Dublin, Dublin 2, Ireland The effect of vasoactive intestinal polypeptide (VIP) receptor antagonism on preganglionic vagal electrical stimulation and on vagal postganglionic activation using nicotine and 1,1-dimethyl- 4-phenylpiperazinium iodide on cardiac interval was evaluated in the isolated innervated rat right atrium. The vagus was stimulated at 4, 8, 16 and 32 Hz, pulse duration 1 ms, 2 V, for 3 s. All experiments were carried out in the presence of atenolol (4 μm). Vagal stimulation caused a frequency-dependent increase in cardiac interval which was amplified significantly at each frequency, except at 32 Hz, following application of the VIP receptor antagonist VIP(6 28) at 2 nm in 15 rats. Application of the ganglionic antagonist hexmethonium (28 μm, n = 7 rats) prior to 2 nm VIP(6 28) abolished this effect. Increasing the concentration of VIP(6 28) 1-fold to 2 nm did not result in a greater increase in cardiac interval than that obtained at 2 nm. Nicotine (.1,.3,.5, 1. and 2. mm) increased cardiac interval by direct activation of postganglionic vagal fibres, but 2 nm VIP(6 28) did not affect the nicotine concentration response (n = 6 rats). 1,1-Dimethyl-4-phenylpiperazinium iodide (25, 5, and μm; n = 6rats) was also used to induce an increase in cardiac interval; again it was not significantly altered by 2 nm VIP(6 28). Therefore, VIP receptor antagonism enhances the magnitude of vagally induced cardiac slowing, probably via an action at the preganglionic postganglionic synapse. (Received 14 February 6; accepted after revision 6 March 6; first published online 9 March 6) Corresponding author F. Markos: School of Pharmacy & Pharmaceutical Sciences, Trinity College Dublin, Dublin 2, Ireland. Email: hazmarkos@yahoo.com Vasoactive intestinal polypeptide (VIP) is a peptide transmitter closely associated with vagal cholinergic fibres in the rat heart which is thought to play role in cardiac function (Henning & Sawmiller, 1; Weihe et al. 1984). In contrast to acetylcholine, VIP is a positive chronotropic neurotransmitter which causes a large longlasting tachycardia when injected into the sinus node artery of the dog (Rigel, 1988). It is now generally accepted that VIP is the most likely mediator of the classical vagal tachycardia that occurs in response to high-frequency vagal stimulation in the presence of atropine (Hill et al. 1995). However, the VIP-mediated vagal tachycardia cannot be obtained in the absence of muscarinic receptor antagonism and only increases heart rate significantly in response to a very high level of vagal activity, which strongly suggests that the vagal tachycardia is probably not physiologically relevant (Markos & Snow, 6). It is more likely that the physiological function of VIP in the heart is not primarily related to its ability to increase heart rate by a direct action on the sinus node. Evidence in favour of this theory was obtained in a previous study carried out on chloralose-anaesthetized dogs, which showed that VIP antagonism causes a significant reduction in the magnitude of the normal sinus arrhythmia without affecting mean heart rate significantly (Markos & Snow, 1). This finding indicates that VIP is capable of altering/affecting cholinergic activity during normal cardiac vagal function and that the relationship between VIP and acetylcholine appears to be synergistic, at least with regard to maintaining sinus arrhythmia. Whether VIP modulates vagal chronotropic function in the rat is unknown; however, a recent study carried out on the isolated vagally innervated rat right atrium has shown that the vagal tachycardia is absent and that externally applied VIP does not increase heart rate significantly (Hogan & Markos, 6), which is in contrast to previous findings (Shvilkin et al. 1994). DOI: 1.1113/expphysiol.6.3365

642 K. Hogan and F. Markos Exp Physiol 91.3 pp 641 646 Therefore, our aim in this present study was to asses whether VIP receptor antagonism has an effect on vagal function in the isolated innervated rat right atrium. Cardiac interval (in ms), which is linearly related to an increase in right vagal frequency in the rat (Sweeney & Markos, 4), and not heart rate (in beats. min 1 ) was used as the index of sinus rhythm since it is more accurate (Daly, 1997). Methods Forty adult male Wistar rats with a mean weight of 396 g (range, 219 518 g), kept under standard laboratory conditions with access to water and standard pellet diet ad libitum, were used. Animals were killed humanely by a blow to the head followed by cervical dislocation, which was carried out by experienced personnel trained in this procedure according to local institutional guidelines. Right vagal atria preparation Twenty-eight rats were used in this series of experiments. The thorax was opened via a mid-line incision, and the heart, part of the trachea and right carotid artery with vagus attached were rapidly removed and placed in a jacketed organ bath and superfused with Hepesbuffered Tyrode salt solution containing atenolol (4 μm) at 37 C and bubbled continuously with % oxygen. The solution contained (mm): NaCl, 137; KCl, 2.7; MgCl 2,1; CaCl 2, 1.36; Na 2 HPO 4,.35; d-glucose, 5.5; and Hepes, 1; titrated to ph 7.4 using 5 m NaOH. A cannula, used to superfuse the preparation continuously with fresh Tyrode solution, was placed inside the right atrium. The right and left ventricles were dissected away, together with the oesophagus and lung, until the atria with right vagus attached and a portion of trachea remained. The trachea and the left atrial appendage were pinned out (with the tips of 25 gauge needles) on Sylgard in the jacketed organ bath. A hook, connected to a force transducer (FTO3 Grass Force Transducer), was attached to the right atrial appendage to measure atrial contractions. A Maclab/2e System (Maclab, AD Instruments Ltd) was used to display the cardiac contractions, and from these the cardiac interval (in ms) was measured. A glass suction electrode, the cathode (tip diameter, μm), was attached to either the thoracic vagus or its cardiac branch for electrical stimulation. was repeated after exposing the atria to Tyrode solution containing the VIP receptor antagonist VIP(6 28) at 2nm (n = 15 rats) or 2 nm (n = 6 rats) for 3 min. In seven rats, the preparation was pretreated with Tyrode solution containing hexamethonium (28 μm) for 3 min prior to administering Tyrode solution containing hexamethonium together with VIP(6 28) at 2 nm again for 3 min and the electrical stimulation protocol was repeated. Measurements were taken at the steady-state peak response by averaging data over 3 5 s. Isolated atria preparation Twelve rats were used in this series of experiments. As before, the thorax was opened via a mid-line incision, the heart and part of the trachea were rapidly removed, and the ventricles, most of the trachea and oesophagus were removed. A fine suture thread was attached to the right atrium. The left atrium was fixed to a tissue holder, and the preparation was placed into a 125 ml volume jacketed organ bath containing Hepes-buffered Tyrode solution containing atenolol (4 μm), to block β 1 -adrenergic receptors, and bubbled continuously with % oxygen at 37 C. The thread in the right atrium was then connected to a force transducer to measure atrial contractions, hence cardiac interval. Experimental protocol for isolated atria preparation The preparation was allowed to equilibrate for approximately 1 15 min before the onset of experiments. In order to stimulate vagal postganglionic fibres selectively, nicotine (n = 6 rats) was added to the bath cumulatively at 1 min intervals at the following concentrations:.1,.3,.5, 1. and 2. mm. The cardiac interval was recorded continuously, with one measurement being taken at the end of each 1 min drug application period. The nicotine concentration response protocol was repeated 3 min after pretreatment of the preparation with the VIP antagonist VIP(6 28) at 2 nm (n = 6). Solutions and drugs All solutions and drugs were obtained from Sigma-Aldrich Ireland Ltd (Dublin, Ireland) and were dissolved in Hepesbuffered Tyrode solution. Experimental protocol for vagus-attached preparation After a 1 15 min equilibration period the right thoracic vagus, or its cardiac branch, was stimulated electrically at increasing frequencies (4, 8, 16 and 32 Hz) with pulse duration of 1 ms at 2 V for 2 s, delivered at approximately 4 s intervals. The stimulation protocol Statistical analysis Two-factor analysis of variance (ANOVA) with repeated measures on both factors was used to compare the frequency response or the nicotine response curves, and Student s paired t test and one-way ANOVA were carried out where appropriate out using MS Excel (Office )

Exp Physiol 91.3 pp 641 646 VIP receptor antagonism enhances vagally induced increase in cardiac interval 643 and SPSS (SPSS Inc., Chicago, USA). P <.5 was considered significant. All values referring to the cardiac interval are given as means ± s.e.m. Results When recording began, the baseline cardiac interval, in the presence of atenolol (4 μm), was 311 ± 1 ms (range, 229 54 ms; n = 33). Effect of vagal stimulation on cardiac interval in the presence and absence of VIP(6 28) Figure 1A summarizes the data obtained from 15 rats showing the effect of vagal electrical stimulation at 4, 8, 16 and 32 Hz (pulse duration, 1 ms at 2 V) for 2 s on cardiac interval before and after application of VIP(6 A VIP(6-28) 2nM 4 8 12 16 2 24 28 32 Right vagus stimulation rate (Hz) B VIP(6-28) 2nM VIP(6-28) 2nM 4 8 12 16 2 24 28 32 Right vagus stimulation rate (Hz) Figure 1. The effect of vagal stimulation on cardiac interval before and after addition of 2 nm VIP(6 28) A, stimulation of the right vagus caused an increase in cardiac interval which was enhanced after application of VIP(6 28), except at 32 Hz. B shows that increasing the concentration of VIP(6 28) 1-fold to 2 nm did not enhance the vagally induced increase in cardiac interval. Data are plotted as means ± S.E.M. P <.5 two-factor ANOVA with repeated measures on both factors. 28) at 2 nm. Addition of VIP(6 28) at 2 nm resulted in a significant increase in cardiac interval following vagal stimulation (P =.1, two-factor ANOVA). Vasoactive intestinal polypeptide(6 28) (2 nm) caused an increase in cardiac interval from 319 ± 9 (range, 246 382 ms) to 339 ± 12 ms (range, 258 423 ms), which was statistically significant (P =.1, Student s paired t test, n = 15). As a consequence, the baseline heart rate for the VIP(6 28) experiment was significantly higher than the baseline, which was 297 ± 9 ms (range, 229 351 ms; P =.6, ANOVA). The concentration of VIP(6 28) was increased 1-fold to 2 nm in another six rats, and the results are shown in Fig. 1B. The data for the 2 nm VIP(6 28) in Fig. 2 is copied from Fig. 1. As can be seen in Fig. 1B, there was no further increase in cardiac interval as a result of the increase in VIP(6 28) concentration. The baseline cardiac interval for this experiment was 347 ± 22 ms (range, 292 424 ms), which was not significantly different from the baseline for the VIP(6 28) at 2 nm (P =.8, ANOVA). In a separate series of experiments, the ganglionic antagonist hexamethonium (28 μm, n = 7) was superfused for 3 min prior to application of 2 nm VIP(6 28) for 3 min. This abolished the enhancement of the vagally induced increase in cardiac interval observed following 2 nm VIP(6 28) alone (Fig. 2). There is no significant difference between the curve and the response obtained when the vagus was stimulated in the presence of hexamethonium and VIP(6 28). Vasoactive intestinal polypeptide(6 28) at 2 nm caused a small increase in baseline cardiac interval from 256 ± 16 (range, 221 347 ms) to 283 ± 17 ms (range, 222 33 ms), which was not significant (P =.26 Student s paired t test). The maximum increase in cardiac interval in this series of rats occurred in response to vagal stimulation at 32 Hz, hexmethonium 28 μm + VIP (6-28) 2nM 4 8 12 16 2 24 28 32 Right vagus stimulation rate (Hz) Figure 2. The effect of hexamethonium (28 μm) prior to 2 nm VIP(6 28) on the vagally induced increase in cardiac interval Ganglionic antagonism abolishes the vagally induced increase in cardiac interval following VIP receptor antagonism. Data are plotted as means ± S.E.M.

644 K. Hogan and F. Markos Exp Physiol 91.3 pp 641 646 which caused cardiac interval to increase to 44 ± 76 ms (range, 273 843 ms), which was reduced to an increase of 332 ± 42 ms (range, 232 563 ms) by pretreament with hexamethonium. Effect of nicotine and 1,1-dimethyl-4- phenylpiperazinium iodide (DMPP) on cardiac interval in the presence of 2 nm VIP(6 28) This series of experiments was conducted to assess whether VIP acts to attenuate vagal effects by an action at the postganglionic sinus node synapse. In order to stimulate vagal postganglionic fibres selectively, two nicotinic receptor agonists were applied separately in cumulative concentrations in the absence and presence of VIP(6 28). Figure 3A summarizes the data obtained from six rats showing the effect of nicotine on cardiac interval before and after VIP(6 28). There was no significant effect of VIP receptor antagonism on the nicotine-induced increase in cardiac interval. The baseline for the nicotine A B VIP (6-28) 2nM.2.4.6.8 1. 1.2 1.4 1.6 1.8 2. Nicotine (mm) VIP (6-28) 2 nm 5 15 DMPP (μm) Figure 3. The effect of VIP(6 28) on the nicotine- (A) or DMPP-induced increase in cardiac interval (B) Neither the nicotine- nor the DMPP-induced increase in cardiac interval was affected by VIP(6 28) at 2 nm. Data are plotted as means ± S.E.M. experiment was 39 ± 24 ms (range, 248 46 ms) and in the presence of VIP(6 28) it was 336 ± 15 ms (range, 28 367 ms), which was not statistically significant (P =.35; Student s paired t test). To confirm this observation, the same experiment was repeated in another six rats using DMPP (Fig. 3B), a specific agonist for neuronal nicotinic receptors. Again VIP(6 28) did not significantly affect the increase in cardiac interval caused by postganglionic vagal fibre stimulation by DMPP. The baseline cardiac interval for these experiments was 322 ± 38 ms (range, 249 54 ms), which was not different from the baseline cardiac interval for the VIP(6 28) protocol (P =.55; Student s paired t test), which was 348 ± 16 ms (range, 28 391 ms). Therefore, this series of experiments confirms the findings obtained using nicotine, indicating that VIP does not cause an attenuation of vagal cholinergic effects by an action at the postganglionic sinus node synapse. Discussion The results of this study show that VIP receptor antagonism enhances the magnitude of the increase in cardiac interval brought about by electrical vagal stimulation, indicating that VIP acts to attenuate vagal cholinergic effects on the sinus node. The concentration of the VIP antagonist VIP(6 28) used in this study has been previously evaluated and used in vivo (Markos & Snow, 1; Markos et al. 2), where it was found to be effective. Increasing the concentration 1-fold did not enhance the vagally induced increase in cardiac interval further. All currently available VIP receptor antagonists are peptide derivatives that include large sections of the original VIP. In the case of VIP(6 28), the first five amino acids have been cleaved, leaving a shortened VIP molecule that is thought to bind to the VIP receptors without eliciting an effect (Fishbein et al. 1994). Other versions, such as [d-p-cl-phe6, Leu17]-VIP, are substitute derivatives of VIP (Pandol et al. 1986). Consequently, there is no independent mechanism of action between any of the available VIP receptor antagonists, and two studies carried out in vivo have concluded that all the VIP antagonists used in these studies were equally effective, with a similar duration of action (Hill et al. 1995; Markos et al. 2). Another factor that must be considered when using currently available VIP receptor antagonists is that they do induce some partial agonism (Markos & Snow, 1). This is probably as a result of the internalization of the VIP receptor following agonist binding, and the receptor agonist complex is required to activate adenylyl cyclase (Svoboda et al. 1988). Therefore, once bound, the peptide VIP antagonists cannot be dislodged by an agonist, and so peptide VIP receptor antagonists cannot strictly be called competitive (Markos et al. 2). Vasoactive

Exp Physiol 91.3 pp 641 646 VIP receptor antagonism enhances vagally induced increase in cardiac interval 645 intestinal polypeptide receptor antagonism may account for the significant increase in the baseline cardiac interval observed in this study, but the exact site at which this potential effect is exerted cannot be deduced. It would be expected that the cholinergic enhancement described in this study in response to VIP receptor antagonism would be caused by prevention of the VIP released from nerves in the heart from binding to the receptors on the surface of the sinus node, since it has been shown that levels of VIP in the coronary perfusate are increased in response to high-frequency electrical stimulation of the vagus (Hill et al. 1995). This expectation appears to be contradicted by the second series of experiments, in which nicotine was used to activate vagal postganglionic fibres selectively (Sweeney & Markos, 4), which suggests that VIP probably exerts its cholinergic modulatory effect at the preganglionic postganglionic synapse, similar to neuronal nitric oxide (Sweeney & Markos, 4). The ganglion antagonist hexamethonium abolished the enhancement of vagal effects on cardiac interval, but it is difficult to conclude with any certainty from this whether VIP acts pre- or postganglionically, since hexamethonium would cause a general reduction in vagal activity. Also, a major limitation of using nicotine is that it is impossible to determine accurately whether nicotine activates nicotinic receptors on the ganglionic neurone or on ganglionic fibres leading away from the ganglia. The possibility that nicotine induces neurotransmitter release by a presynaptic action on the preganglionic fibre is countered by the finding that the nicotine-induced increase in cardiac interval is significantly inhibited by the ganglionic antagonist hexamethonium (Sweeney & Markos, 4). In addition, atropine also significantly reduces the bradycardia in response to exogenous nicotine. Taken together, this shows that nicotine activates cholinergic postganglionic fibres postsynaptically and not presynaptically. It is likely, however, that application of exogenous nicotine does not lead to the activation of the same modulatory interneurones activated by nerve stimulation, which again illustrates another limitation of this technique. Despite this, the same result was obtained using the specific neuronal nicotinic agonist DMPP to activate postganglionic vagal efferents. In support of a preganglionic site of action for VIP, it has been shown that VIP receptors do exist on vagal preganglionic nerves in the gut and can modulate vagal effects (Van Geldre & Lefebvre, 4). In addition, previous work has shown that VIP modulates rat ganglionic nicotinic, but not ganglionic muscarinic, receptors by increasing their open probability (Cuevas & Adams, 1996). A recent study in our laboratory has shown that externally applied VIP does not cause a significant increase in heart rate in the isolated rat right atrium (Hogan & Markos, 6), which also supports the finding that in the rat heart the role of VIP is primarily a vagal modulatory one. Whether VIP and acetylcholine inhabit the same nerve terminals in the rat heart has not been established conclusively, with one study carried out on whole-mount preparation intracardiac ganglia indicating that no VIP was found in the intracardiac ganglia of the rat heart (Richardson et al. 3). This same study, however, also provided immunohistochemical evidence that VIP immunoreactivity is in fact confined to the nerve fibres within cardiac ganglia (Richardson et al. 3), and therefore it makes it more likely that the effect of VIP antagonism observed in our study occurs at these interneurones. The cardiac ganglia possess a complex organization, with many potentially modulatory interneurones that together probably play an important role in integrating vagal neuronal activity independent of the CNS (Randall et al. 1995). Our results add some evidence in support for these findings, which indicate that the interaction between the preganglionic vagus and the postganglionic relay is probably more complex than a single synapse. In fact, there appears to be a higher level of interaction between the preganglionic vagus and postganglionic elements, with the probable participation of cardiac ganglia and interneurones, which modulate the vagal signal. In conclusion, VIP has a vagal cholinergic inhibitory effect in the rat heart. This effect is not exerted as would be expected at the level of the postganglionic sinus node synapse but probably at the preganglionic postganglionic synapse, similar to neuronal nitric oxide. References Cuevas J & Adams DJ (1996). Vasoactive intestinal polypeptide modulation of nicotinic ACh receptor channels in rat intracardiac neurones. J Physiol 493, 53 515. Daly M De B (1997). In Peripheral Arterial Chemoreceptors and Respiratory Cardiovascular Integration, Appendix I, pp. 597 64. Oxford University Press, Oxford. Fishbein VA, Coy DH, Hocart SJ, Jiang NY, Mrozinski JE Jr, Mantey SA & Jensen RT (1994). A chimeric VIP-PACAP analogue but not VIP pseudopeptides function as VIP receptor antagonists. Peptides 15, 95. Henning RJ & Sawmiller DR (1). Vasoactive intestinal peptide: cardiovascular effects. Cardiovasc Res 49, 27 37. Hill MR, Wallick DW, Martin PJ & Levy MN (1995). 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646 K. Hogan and F. Markos Exp Physiol 91.3 pp 641 646 Markos F & Snow HM (1). The potentiation of sinus arrhythmia by vasoactive intestinal polypeptide (VIP) in the anaesthetized dog. Neuropeptides 35, 238 243. Markos F & Snow HM (6). An investigation into the physiological relevance of the vagal tachycardia in the anaesthetized dog. Acta Physiol 186, 179 184. Pandol SJ, Dharmsathaphorn K, Schoeffield MS, Vale W & Rivier J (1986). Vasoactive intestinal peptide receptor antagonist [4Cl-d-Phe6, Leu17] VIP. Am J Physiol 25, G553 G557. Randall WC, Wurster RD, Randall DC & Xi-Moy SX (1996). From cardioaccelerator and inhibitory nerves to a heart brain : an evolution of concepts. In: Nervous Control of the Heart, ed. Shepherd JT & Vatner SF, pp. 173 199. Harwood Academic, Amsterdam. RichardsonRJ,GrkovicI&AndersonCR(3). Immunohistochemical analysis of intracardiac ganglia of the rat heart. Cell Tissue Res 314, 337 35. Rigel DF (1988). Effects of neuropeptides on heart rate in dogs: comparison of VIP, PHI, NPY, CGRP, and NT. Am J Physiol 255, H311 H317. Shvilkin A, Danilo P Jr, Chevalier P, Chang F, Cohen IS & Rosen MR (1994). Vagal release of vasoactive intestinal peptide can promote vagotonic tachycardia in the isolated innervated rat heart. Cardiovasc Res 28, 1769 1773. Svoboda M, De Neef P, Tastenoy M & Christophe J (1988). Molecular characteristics and evidence for internalization of vasoactive-intestinal-peptide (VIP) receptors in the tumoral rat-pancreatic acinar cell line AR 4-2 J. Eur J Biochem 176, 77 713. SweeneyC&MarkosF(4). The role of neuronal nitric oxide (NO) in the vagal of cardiac interval of the rat heart in vitro. Auton Neurosci 111, 11 115. Van Geldre LA & Lefebvre RA (4). Interaction of NO and VIP in gastrointestinal smooth muscle relaxation. Current Pharmaceut Design 1, 2483 2497. Weihe E, Reinecke M & Forssmann WG (1984). Distribution of vasoactive intestinal polypeptide-like immunoreactivity in the mammalian heart. Interrelation with neurotensin- and substance P-like immunoreactive nerves. Cell Tissue Res 236, 527 54. Acknowledgements We would like to thank Mr Brian Talbot and Ms Ann Hannan for their technical assistance.