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170

Cardiovascular Actions of Galanina

ERICA POTTERb

Prince of Wales Medical Research Institute, Prince of Wales Hospital, High Street, Randwick, 2031 Australia

n the dog, attenuation of the slowing of the heart evoked by vagal stimulation followingstimulation of the cardiac sympathetic nerve has been attributed to inhibition of acetyl-

choline release by neuropeptide Y (NPY) released from sympathetic nerves.1,2 NPY wasidentified as a sympathetic cotransmitter in 19823 and seemed an obvious candidate forthis effect which has a long duration of action, lasting many minutes beyond the effects ofnoradrenaline. The attenuation of cardiac vagal action also survives - and -adrenoceptorblockade, suggesting a neurotransmitter other than noradrenaline. However, since 1982,other neuropeptides have been identified in sympathetic nerves, and their role as modula-tors of autonomic neurotransmission has been systematically investigated. One of these,galanin (GAL), a 29 amino acid peptide (30 in humans), has been identified in sympatheticnerves in the cat4 and dog.5 In the Australian marsupial Trichosurus vulpecula, the thoracicsympathetic ganglia contain GAL but not NPY colocalized with noradrenaline.6 We haveexamined a possible role for GAL as a modulator of autonomic neurotransmission in sev-eral species, including the cat, dog, and Australian possum, Trichosurus vulpecula.

All animals were anesthetized with pentobarbitone sodium (Nembutal; Abbott Labora-tories, Sydney; 30 mg/kg). Anesthesia was induced in possums by intramuscular injectionof ketamine (Ketavet; Delta Veterinary Laboratories, Australia; 70 mg/kg) and xylazine(Rompun; Bayer; 10 mg/kg). All experiments were approved by the Institutional AnimalCare and Ethics Committee and with the appropriate licences. All animals were artificiallyventilated and held at constant temperature. Blood pressure and beat by beat pulse interval(PI, time between each heart beat) were measured. Both vagus nerves were cut and theright vagus stimulated supramaximally (1 ms, 4 Hz). The frequency of stimulation waschosen to increase pulse interval 100–300 ms, a submaximal effect for vagal slowing. Thestellate ganglion was identified after dissection which allowed forward reflection of theforeleg and removal of the second rib. The cardiac sympathetic nerve was identified byacceleration of the heart when stimulated electrically. The sympathetic nerve was stimu-lated supramaximally at high frequency (16–20 Hz) for 2–3 minutes.

In the cat, as in the dog, stimulation of the cardiac sympathetic nerve evokes a pro-longed attenuation of cardiac vagal action in the presence of effective -adrenoceptorblockade. FIGURE 1 is a polygraph record taken from an experiment on one anesthetized catin which prolonged attenuation of cardiac vagal action is clearly seen following sympa-thetic stimulation.

In a group of cats the magnitude of the attenuation of cardiac vagal action followingsympathetic stimulation was 41 ± 5%, and the time to half recovery of this effect was8 ± 1.5 min7 (n = 6). As in the dog, guanethidine abolished the inhibitory effect following

aThis work was supported by the National Health and Medical Research Council of Australia andthe National Heart Foundation of Australia.

bPhone, 61 2 9382 2683; fax, 61 2 93822722; e-mail, e.potter@unsw.edu.au

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POTTER: CARDIOVASCULAR ACTIONS OF GALANIN 171

sympathetic stimulation, suggesting that the effect was mediated by a sympathetic cotrans-mitter.

Intravenous injection of GAL (1.6–3.1 nmol/kg) mimicks the effect of sympatheticstimulation and an example from one cat is shown in FIGURE 2.

In the group of cats the maximum percentage of inhibition of cardiac vagal action fol-lowing injection of GAL was 41 ± 8% with a time to half recovery of 14 ± 3 minutes(n = 9). Intravenous injection of GAL evoked a decrease in BP of 10 ± 2 mm Hg, and thisdepressor effect returned to control levels in 8 minutes.7 The sequence of cat GAL is notknown, but GAL from several species has similar depressor effects. In a second study incats, porcine GAL showed a significantly greater depressor effect than did rat or humanGAL; 24 ± 3, 17 ± 3.5, and 12 ± 2 mm Hg, respectively. Recovery time was 3 ± 1, 6 ± 1,and 4 ± 0.5 minutes, respectively.8 In the cat, NPY does not cause attenuation of cardiacvagal action as it does in the dog, but it does increase blood pressure. In the dog, GALdoes not attenuate cardiac vagal action, but it does have a transient depressor response of 9± 4 mm Hg for 2 minutes for porcine GAL.

In the possum, the magnitude of the attenuation of cardiac vagal activity followingstimulation of the cardiac sympathetic nerve is 31 ± 10% with a time to half recovery of5 ± 1 minute (n = 8). Following exogenous GAL the magnitude of the response was

FIGURE 1. Record of pulse interval (PI) and blood pressure (BP) from an anesthetized cat, showingthe effects of stimulation of the cardiac sympathetic nerve. An increase in PI was evoked every 30seconds by stimulating the peripheral cut end of the right vagus. Sympathetic stimulation causedlong-lasting inhibition of vagal effects on PI.

FIGURE 2. Record of pulse interval (PI) and blood pressure (BP) from an anesthetized cat. Anincrease in PI was evoked every 30 seconds by stimulating the peripheral cut end of the right vagus.Intravenous injection of galanin evoked long-lasting inhibition of cardiac vagal action. Galanin alsohad a slight depressor action.

172 ANNALS NEW YORK ACADEMY OF SCIENCES

41 ± 4% with a time to half recovery of 13 minutes. GAL evoked a pressor response of57 ± 5 mm Hg. NPY had no effect on cardiac vagal action.9

Both the cat and the dog have NPY, GAL, and noradrenaline colocalized in sympa-thetic nerves. In the cat, GAL not NPY mimicks the attenuation of cardiac vagal actionfollowing sympathetic stimulation, whereas in the dog it is NPY not GAL that mimicksthe effect of sympathetic stimulation. In the possum, the sympathetic nerves contain GALbut not NPY, and consistent with this it is GAL, not NPY, that mimicks the effect of sym-pathetic stimulation.

Many GAL antagonists are now available. These are chimeric molecules of GAL andcan help define a physiological role for GAL.10,11 We have used three of these chimericGAL antagonists to look at the role of GAL in the cardiovascular system. We and othershave shown that the N-terminal of GAL is important for recognition of its receptor andsubsequent biological action.12,13 The chimeric antagonists have the N-terminal of GAL(usually GAL 1-13) linked to the C-terminal of other molecules. Galantide or M15 con-

FIGURE 3. Effects of GAL injection (left) and sympathetic stimulation (right) and after the admin-istration of the GAL antagonists M15, M32a, and C7 in ×10 or ×25 the molar dose of GAL. Topgraphs show maximum % inhibition of cardiac vagal action and the bottom graphs show the time tohalf recovery (** p < 0.01, *** p < 0.001).

POTTER: CARDIOVASCULAR ACTIONS OF GALANIN 173

sists of GAL 1–13 + substance P 5–11; C7 consists of GAL 1–13 + spantide (the substanceP antagonist); M32a consists of GAL 1–13 + NPY 24–36. Pretreatment with each of thesechimeric peptides has shown that the attenuation of cardiac vagal action following sympa-thetic stimulation or exogenous GAL is significantly reduced in both magnitude and dura-tion. These effects are summarized in FIGURE 3.

Interestingly, none of the chimeric antagonists had any effect on the decrease in bloodpressure evoked by intravenous injection of GAL.14 The chimeric antagonist M15 did notmodify the cardiac vagal inhibitory effects following sympathetic stimulation in the dog.15

GAL appears to have powerful modulating actions on the cardiovascular system whichare species specific. In the cat and possum it is proposed that GAL released from sympa-thetic nerves inhibits subsequent cardiac vagal action. In a study on human subjects, intra-venous injection of human GAL decreased sinus arrhythmia, an effect consistent withinhibition of vagal activity.16 The doses used in this study did not affect blood pressure. Inthe cat, however, GAL significantly decreased blood pressure. The vagus nerves and rightsympathetic nerve were cut in the animal studies, thus removing the major baroreceptorpathways, possibly unmasking a depressor effect on blood pressure which would havebeen buffered in the human study.

Three GAL receptors have now been cloned. One of these, GalR3, shows significantperipheral expression particularly in the heart.13 This receptor may mediate the inhibitoryeffects of GAL on cardiac vagal action following sympathetic stimulation. However, ourstudies suggest that there should be a second peripheral GAL receptor, on blood vessels,which mediates the depressor effects of GAL. In the studies using the chimeric antagoniststhe depressor action of galanin was not modified by any of the antagonists, even whenlarge doses were used.

The studies described here demonstrate that GAL has powerful actions in the cardio-vascular system. It is suggested that GAL released from sympathetic nerves in the cat,possum, and possibly human subjects inhibits acetylcholine release and subsequent slow-ing of the heart mediated by the GalR3 receptor. We also suggest that GAL has a directvasodilator action mediated by a possible GalR4 receptor.

ACKNOWLEDGMENTS

The help of my colleagues, Maureen Revington, Lesley Ulman, Gillian Courtice, andIan McCloskey, is acknowledged.

REFERENCES

1. POTTER, E.K. 1985. Prolonged non-adrenergic inhibition of cardiac vagal action following sym-pathetic stimulation. Neurosci. Lett. 54: 117–121.

2. POTTER, E.K. 1987. Presynaptic inhibition of cardiac vagal postganglionic nerves by neuropep-tide Y. Neurosci. Lett. 83: 101–106.

3. TATEMOTO, K. 1982. Neuropeptide Y: The complete amino acid sequence of the brain peptide.Proc. Natl. Acad.Sci. USA 79: 5485–5489.

4. KUMMER, W. 1987. Galanin- and neuropeptide Y-like immunoreactivities coexist in paraverte-bral sympathetic neurones of the cat. Neurosci. Lett. 78: 127–131.

5. MORIARTY, M., I.L. GIBBINS, E.K. POTTER & D.I. MCCLOSKEY. 1992. Comparison of the inhibitoryroles of neuropeptide Y and galanin on cardiac vagal action in the dog. Neurosci. Lett. 136:275–279.

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6. MORRIS,J.L., I.L. GIBBINS & R. MURPHY. 1986. Neuropeptide Y-like immunoreactivity is absentfrom most perivascular noradrenergic axons in a marsupial, the brush-tailed possum. Neuro-sci. Lett. 71: 264–270.

7. REVINGTON, M.L., E.K. POTTER & D.I. MCCLOSKEY. 1990. Prolonged inhibition of cardiac vagalaction following sympathetic stimulation and galanin in anaesthetised cats. J. Physiol. 431:495–503.

8. ULMAN. L.G., H.F. EVANS, T.P. IISMAA, E.K. POTTER, D.I. MCCLOSKEY & J. SHINE. 1992. Effects ofhuman, rat and porcine galanins on cardiac vagal action and blood pressure in the anaesthe-tised cat. Neurosci. Lett. 136: 105–108.

9. COURTICE, G.P., E.K. POTTER & D.I. MCCLOSKEY. 1993. Inhibition of cardiac vagal action bygalanin but not neuropeptide Y in the brush tailed possum Trichosurus vulpecula. J. Physiol.461: 379–386.

10. LINDSKOG, S., B. AHRÉN, T. LAND, U. LANGEL & T. BARTFAI. 1992. The novel high affinity antag-onist galantide blocks the galanin mediated inhibition of glucose-induced insulin secretion.Eur. J. Pharmacol. 210: 183–188.

11. CRAWLEY, J.N., J.K. ROBINSON, U. LANGEL & T. BARTFAI. 1993. Galanin receptor antagonistsM40 and C7 block galanin induced feeding. Brain Res. 600: 268–272.

12. ULMAN, L.G., E.K. POTTER & D.I. MCCLOSKEY. 1993. The effects of galanin and galanin frag-ments on cardiac vagal action and blood pressure in the anaesthetised cat. Reg. Pept. 44: 85–92.

13. WANG, S., C. HE, T. HASHEMI & M. BAYNE. 1997. Cloning and expressional characterisation of anovel galanin receptor. Identification of different pharmacophores within galanin for the threegalanin receptor subtypes. J. Biol. Chem. 272: 31949–31952.

14. ULMAN, L.G., E.K. POTTER & D.I. MCCLOSKEY. 1994. Functional effects of a family of galaninantagonists on the cardiovascular system in anaesthetised cats. Reg. Pept. 51: 17–23.

15. ULMAN, L.G., M. MORIARTY, E.K. POTTER & D.I. MCCLOSKEY. 1993. Galanin antagonist effectson cardiac vagal inhibitory actions of sympathetic stimulation in anaesthetised cats and dogs.J. Physiol. 464: 491–499.

16. CAREY, D.G., T.P. IISMAA, K.Y. HO, I.A. RAJKOVIC, J. KELLY, E.W. KRAEGEN, J. FERGUSON, A.S.INGLIS, J. SHINE & D.J. CHISHOLM. 1993. Potent effects of human galanin in man: Growth hor-mone secretion and vagal blockade. J. Clin. Endocrinol. Metab. 77: 90–93.