Neuropeptide Y and Sympathetic Neurotransmission" JAN M. LUNDBERG, ANDERS FRANCO-CERECEDA, JEAN-SILVAIN LACROIX, AND JOHN PERNOW Department of Pharmacology Karolinska lnstitutet S-104 01 Stockholm, Sweden
INTRODUCTION Neuropeptide Y (NPY) is a peptide with 36-amino acid residues which was originally isolated from the porcine brain' and probably represents the earlier described neuronal pancreatic polypeptide-like immunoreactivity. A major portion of the NPY-immunoreactive nerves in peripheral organs represents classical sympathetic fibres where NPY co-exists with noradrenaline (NA).3v4NPY is thus present in perivascular sympathetic fibres as well as in the noradrenergic nerves to the muscle of the heart, spleen, and vas deferens. Vasoconstrictor Actions of NPY Exogenous NPY causes potent, long-lasting reduction in local blood flow in many organs of experimental (FIG. I ) as well as in the human forearm.' In vitro, NPY contracts small isolated blood vessels such as cerebral,* splenic,' renal and skeletal muscle arteries'' with threshold effects in the nM concentration range. The NPY effect is mediated via activation of specific nonadrenergic receptor mechanisms and the vasoconstriction is characterized by a slow onset and a long duration. The vasoconstrictor effect of NPY is independent of the vascular endothelium suggesting an action directly on vascular smooth muscle cells.' In the human forearm local i.a. infusion of NPY causes both reduction in blood flow and increase in venous tone suggesting constriction of both resistance and capacitance vessels' in analogy with data from pig nasal mucosa. I '
NPY Receptors and Intmcellular Messengers Calcium antagonists like nifedipine inhibit the vasoconstrictor response to NPY on renal and skeletal muscle arteries but not on mesenteric veins." The NPY effect was largely uninfluenced by changes in extracellular Ca2+ concentrations suggesting that NPY rather acts via changes in intracellular Ca2+ (see REF. 10). NPY does not stimulate inositol phosphate turnover in blood vessels per seI2 while the forskolin-stimulated cyclic AMP formation in vascular smooth muscle" and heart muscle'3 is inhibited. High affinity-binding sites for NPY with receptor characteristics have been demonstrated in both
"The present paper summarizes data obtained with support from the Swedish Medical Research Council (14X-6554), the Swedish Tobacco Company, the American Council for Tobacco Research and the Heart & Lung Foundation. 166
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blood vessels and the capsule of the pig spleen.'2*'4*'5A large amidated C-terminal portion of NPY is a prerequisite for receptor binding, inhibition of forskolin-stimulated cyclic AMP formation and vasoconstrictor effects. I2,l4 The structurally related peptide YY (1-36) binds to NPY receptors with similar or larger affinity than NPY and also evokes potent vasoconstricti~n.'~'~~'~.'~ Supersensitivity to the vasoconstrictor effects of NPY after sympathetic denervation has been observed in the rat tail artery in vitro'" and in the pig nasal mucosa in vivo" but not in the pig spleen.I4 NPY and "Nonadrenergic" Vasoconshiction In the submandibular salivary gland of the cat it was demonstrated' that NPY mimicked the slowly developing and long-lasting decrease in blood flow evoked by sympathetic nerve stimulation using a high frequency in the presence of a-and P-adrenoceptor
FIGURE 1. Effects of electrical sympathetic nerve stimulation (NS, bar) and local i.a. infusion of NPY (2 nmol/min) on vascular tone as revealed by perfusion pressure (PP, mmHg) in (a) cat spleen (10 Hz for 2 min) and (b) dog gracilis muscle (6.9 Hz 30 sec). The nerve stimulation evoked vasoconstriction (increase in perfusion pressure) in controls is markedly prolonged in the animals where reserpine pretreatment ( 1 mg/kg i . v . ) was combined with preganglionic decentralization to maintain tissue NPY levels and to deplete NA by 99%. This long-lasting nerve response is mimicked 23,24. by NPY infusion. For details see REFERENCES
antagonists. Treatment with reserpine in order to deplete (by 99%)the tissue stores of NA may represent an alternative approach to study nonadrenergic transmission since it cannot be excluded that the doses of adrenoceptor antagonists used are not high enough to eliminate all the effects of the released NA. It is well established, however, that only minor vasoconstrictor responses to nerve stimulation in, e.g., the spleen remain in reserpine treatment animals, apparently leaving a minor or no role for nonadrenergic mechanisms. Interestingly, recent studies have shown that reserpine treatment is associated with a reversible depletion not only of NA but also of NPY from sympathetic nerve fibres in a variety of tissues including heart, skeletal muscle and Several characteristic features separate the effects of reserpine pretreatment on NA storage from that on NPY. Thus, higher doses of reserpine are required to deplete NPY than NA. The depletion of NPY is slower in onset and less pronounced than that of NA in terminal regions.'9.2' Furthermore, the NPY content of sympathetic axons and in terminals of some tissues like the vas deferens is not reduced after reserpine suggesting an action which is not directly related to storage mechanisms.20,2' Most strikingly, the
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reserpine-induced reduction of tissue NPY levels is, in contrast to the effect on NA, entirely dependent on an intact nerve activity since either pretreatment with a nicotinic receptor blocking agent such as chlorisondamine, surgical transection at the pre- or postganglionic levels or clonidine, which reduces sympathetic discharge, impairs the reserpine effect on NPY but not on NA level^.^'-^^ The reserpine-induced depletion of NPY is therefore likely to be caused by an increased release of the peptide in excess of synthesis and resupply capacity by axonal transport (see REF. 25). It is known that reserpine pretreatment is associated with a rapid increase in firing rate of postganglionic sympathetic nerves.26 Treatment with reserpine can therefore be combined with pharmacological agents or surgical procedures in order to prevent the increased neuronal activity to the organ studied which after 24 h results in a situation where NA is depleted by 98-99%, while the tissue content of NPY is preserved. Stimulation of the sympathetic nerves in the spleen with high frequency then evoked a marked long-lasting vasoconstrictor response which was mimicked by the action of exogenous NPYZ3(FIG. 1). This experimental approach has now been tested in a variety of preparations in vivo in addition to the cat spleen,23 i.e., the pig spleen,28 dog skeletal muscle,24 pig nasal mucosa'l and pig kidneyz9 with similar results. The high correlation ( r = 0.79-0.91) between the vasoconstrictor effects and the detectable overflow of NPY into the local venous effluent, the characteristic time course of the response being slowly developing and long-lasting and fatigue of the response upon repeated stimulation in these reserpinized preparations further favour a peptide like NPY as the mediator. Although release of NPY seems to be facilitated by high frequency stimulation, NPY outflow is detected both from spleen" and kidney29 already at stimulation with a low frequency like 0.5 Hz. Furthermore, in reserpinized pigs, nasal vasoconstriction is observed even in response to single impulses' ' suggesting that exocytosis of the NPY content from large dense cored vesicles can occur also under such conditions although to a lesser extent than at high frequency stimulation. Following reserpine treatment the levels of NPY in sympathetic ganglia are elevated and an increased accumulation of peptide occurs above an axonal ligation suggesting both enhanced synthesis and subsequent anterograde axonal transport. 2o Accordingly, recent data have shown that expression of specific NPY messenger RNA in sympathetic ganglion cells is increased following reserpine admini~tration.~~ The observation that subchronic pretreatment with chlorisondamine reduces the NPY content in sympathetic ganglion cellsz2 further supports the concept of a nicotinic receptor-stimulated regulation of NPY synthesis.
Co-Release of NPY and NA upon Sympathetic Activation Release of NPY, as revealed by either overflow into the local venous effluent from organs of experimental animaIs28.29*3'*32 or human h e a d 3 as well as increases in systemic plasma levels in man34*35*36 occurs upon sympathetic activation. The ratio between the overflow of NPY and that of NA increases with the frequency of stimulationz8 and the correlation between the NPY and NA overflow represents a sigmoid curve,37 suggesting that NPY release is preferentially facilitated by high stimulation (FIG.2). The local plasma concentration of endogenous NPY-LI in the pig splenic or renal venous effluent upon high frequency stimulation in reserpinized animals is in the nM range, i.e., similar to where exogenous NPY evokes vasocor~striction.~~~~~~~ Since further characterization by high performance liquid chromatography suggests that the NPY-LI detected by the antiserum (Nl) used represents NPY ( 1-36);28 this suggests that endogenous NPY concentrations which are likely to be much higher close to release sites are more than sufficient to activate receptors on vascular smooth muscle cells and to contribute to the functional response. fJpon prolonged stimulation (evoked by either increased sympathetic
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discharge as for reserpine or electrical stimulation), NPY release cannot be maintained and tissue content of NPY is then reduced, however.28 The mechanisms underlying the frequency-dependent differential secretion of NPY may be related to the partly separate vesicular storage of these two agents in the sympathetic nerve terminals, whereby NPY seems to be exclusively present in the large densecored vesiclesz8 (FIG.3). Pretreatment with guanethidine inhibits the stimulation-evoked release of both NPY and NA," while tyramine evokes NA secretion without influencing NPY indicating that separate release can occur.32 After Q -adrenoceptor antagonists the NPY and NA overflow is enhanced in parallel, h~weve?'.~' (FIG.3). The plasma levels of NPY in healthy human subjects are mainly elevated upon heavy physical exercise (FIG.3) or other situations with a strong sympathetic activation such as after h y p o ~ i aor~ adrenoceptor ~ b l ~ k a d e . ' ~ - ~Therefore, " also in man the secretion of NPY as detected by elevated systemic plasma levels seems to be enhanced at high degrees of sympathetic activation compared to that of NA. Most species including man have
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FIGURE 2. Correlation between (a) output of NPY (pmol) and NA (pmol) from the blood perfused pig spleen upon sympathetic nerve stimulation with different frequencies ( r = 0.96) and (b) levels (pM) of NPY and NA in human venous plasma upon graded physical exercise ( r = 0.87). Note the similar relative increase in NPY at high NA levels both from pig spleen and humans. For details see REFERENCES 28, 34, 36.
relatively low resting plasma levels of NPY suggesting that the basal rate of NPY release is low, if any.25 In the rat, however, very high plasma NPY levels are present also during basal conditions which most likely is related to the occurrence of NPY in trombocytes of this species.38 Therefore, NPY levels in rat blood may not be an appropriate indicator of sympathoadrenal activity26 but mainly reflect the degree of trombocyte degran~lation,~' especially since plasma NPY in the rat is very sensitive to vinblastinea treatment, which markedly reduces trombocyte numbers in peripheral blood.
NPY as Pre- and Postjunctional Modulator of Sympathetic Transmission NPY or PYY exerts prejunctional actions on release of transmitter from sympathetic nerves as revealed by inhibition of nerve-evoked contractions3 and stimulation-evoked , ~ ~ overflow from perivascular nerves endogenous NA and NPY output in v ~ v o ~or' 'H-NA
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of rat and man in vitr~'~,~' (FIG.3). Experimental data studying contractile responses or analysis of transmitter overflow suggest that NPY also inhibits the release of both acetylcholine and NA in the heart.44-46 In the mouse vas deferens NPY induced a potentiation of the contractile effects of NA and adenosine-5-triphosphate (ATP).47 Furthermore, NPY reduced the stimulation-
+
Release
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I
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I
I
NA
NPY
FIGURE 3. Schematical illustration of a varicosity of a sympathetic nerve containing both NA and NPY in large dense-cored vesicles and only NA in the small vesicles. At low frequency stimulation mainly small vesicles secrete their content of NA acting on postjunctional a,-adrenocepton to evoke vasoconstriction or P,-adrenoceptors to increase cardiac contractility. NA also activates prejunctional a,-adrenoceptors inhibiting release of itself and of NPY from the large vesicles. NPY is secreted upon stronger stimulation and induces vasoconstriction as well as inhibits release of NA and itself. There is some evidence that the vasoconstriction and the prejunctional effect of NPY is mediated via separate receptor mechanisms (designed NPY- 1 and NPY-2, respectively). Nicotine increases basal NA and NPY secretion while angiotensin U (AT 11) facilitates nerve stimulation-evoked release of NA and NPY. Due to the complex interplay between NA and NPY both at the pre- and postjunctional levels a variety of drugs which interfere with NA mechanisms will also change especially NPY release.
evoked contraction and secretion of 'H-NA and selectively depressed the stimulus evoked but not the spontaneously occurring excitatory junction potentials in smooth muscle cells. Possibly this action of NPY, similarly to a,-agonists is dependent on an inhibitory effect in part via a target upstream of the v a r i c ~ s i t i e s . ~ ~ NPY not only evokes vasoconstriction per se but also enhances the contractions of
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blood vessels to a variety of agents including NA in ~ i t r o .The ~ . enhancing ~~ effect of NPY is not unique but shared by other vasoconstrictors like serotonin and characterized by being more pronounced on larger vessels where NPY exerts minor or no contractions per se.
NPY Release in Human Cardiovascular Disease The release of NPY upon sympathetic activation in healthy volunteers as revealed by increase in systemic plasma level^^^.^^ or elevated levels in the coronary sinus upon h y p o ~ i combined a~~ with the potent, long-lasting vasoconstrictor effects of NPY in man’ raise the possibility that NPY may be involved in the pathophysiology of human cardiovascular disorders. NPY is a potent constrictor of small human coronary vessels in ~ i f r - 0 , ~ ~ and elevated plasma levels of NPY are present in patients with heart disease such as acute myocardial infarction, angina pectoris and especially severe left heart failure.5o The possible functional role of NPY under physiological conditions with intact noradrenergic mechanisms, as well as involvement in human vascular disorders, remains to be established with the use of specific NPY antagonists at the pre- and postjunctional levels. It seems clear, however, that many drugs commonly used in experimental studies on cardiovascular control or in treatment of hypertensive or vasopastic disorders in humans also influence NPY mechanisms.
SUMMARY The coexistence of neuropeptide Y (NPY) with noradrenaline (NA) in perivascular nerves as well as in sympathetic nerves to muscle in the heart, spleen and vas deferens suggests a role for NPY in autonomic transmission. Sympathetic nerve stimulation or reflexogenic activation in experimental animals or man are associated with NPY release as revealed by overflow mainly upon strong activation. This difference between NPY and NA secretion may be related to the partly separate subcellular storage whereby NPY seems to be exclusively present in the large dense-cored vesicles. The NPY secretion is likely to be regulated by the local biophase concentrations of NA acting on prejunctional alpha-2-adrenoceptors since alpha-2 agonists inhibit and antagonists enhance NPY overflow, respectively. Furthermore, after NA has been depleted by reserpine, the nerve stimulation-evoked release of NPY is enhanced leading to a progressive depletion of tissue content of NPY. Exogenous NPY binds to both pre- and postjunctional receptors, inhibits NA and NPY release, enhances NA-evoked vasoconstriction and induces vasoconstriction per se. The prejunctional action of NPY which is especially noticeable in the vas deferens may serve to reduce transmitter secretion upon excessive stimulation. The long-lasting vasoconstriction evoked by sympathetic stimulation in several tissues including skeletal muscle, nasal mucosa and spleen, which remains in animals pretreated with reserpine (to deplete NA) combined with preganglionic denervation (to prevent the concomitant excessive NPY release and depletion), is mimicked by NPY and highly correlated to NPY release. Under these circumstances the NPY content in the local venous effluent reaches levels at which exogenous NPY evokes vasoconstriction. REFERENCES 1.
TATEMOTO, K. 1982. Neuropeptide Y:the complete amino-acid sequence of the brain peptide. Roc. Natl. Acad. Sci. USA 79: 5485-5489.
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2. LUNDBERG, J . M., L. TERENIUS, T. H ~ K F E L&T K. TATEMOTO. 1984. Comparative immunohistochemical and biochemical analysis of pancreatic polypeptide-like peptides with special reference to presence of neuropeptide Y in central and peripheral neurons. J. Neurosci. 45: 2376-2386. 3. LUNDBERG, J . M., L. TERENIUS, T. H ~ K F E L C-R. T , MARTLING, K. TATEMOTO, V. MUTT,J. POLAK,S. R. BLOOM& M. GOLDSTEIN. 1982. Neuropeptide Y (NPY)-like immunoreactivity in peripheral noradrenergic neurons and effects of NPY on sympathetic function. Acta Physiol. Scand. 116: 477-480. EKBLAD,E., L. EDVINSSON, C. WAHLESTEDT, R. UDDMAN, R. HAKANSSON & F. SUNDLER. 1984. Neuropeptide Y co-exists and co-operates with noradrenaline in perivascular nerve fibres. Regul. Pept. 8: 225-235. LUNDBERG, 1. M. & K. TAKEMOTO. 1982. Pancreatic polypeptide family (APP, BPP, NPY and PYY) in relation to alpha-adrenoceptor-resistantsympathetic vasoconstriction. Acta Physiol. &and. 116: 393-402. & J. M. LUNDBERG. 1987. Release RUDEHILL, A,, M. OLCBN,A. SOLLEVI,B. HAMBERGER of neuropeptide Y upon hemorrhagic hypovolemia in relation to vasoconstrictor effects in the pig. Acta Physiol. Scand. 131: 517-523. 7. PERNOW,J., J . M. LUNDBERG & L. KAIISER.1987. Vasoconstrictor effects in vivo and plasma disappearance rate of neuropeptide Y in man. Life Sci. 40: 47-54. L., P.C. EMSON,J. MCCULLOCH, K. TATEMOTO & R. UDDMAN.1983. Neuro8. EDVINSSON, peptide Y, cerebrovascular innervation and vasomotor effects in the cat. Neurosci. Lett. 43: 79-84. 9. PERNOW,J. & J . M. LUNDBERG. 1988. Neuropeptide Y induces potent contraction of arterial vascular smooth muscle via an endothelium-independent mechanism. Acta. Physiol. Scand. 134: 157-158. 10. PERNOW, J . , T. SVENBERG & J . M. LUNDBERG. 1987. Actions of calcium antagonists on preand postjunctional effects of neuropeptide Y on human peripheral blood vessels in v i m . Eur. J. Pharmacol. 136: 207-218. 11. LACROIX, J . S., P. STlARNE, A. A N G G ~&DJ. M. LUNDBERG. 1988. Sympathetic vascular control of the pig nasal mucosa. (11): reserpine-resistant, non-adrenergic nervous responses in relation to neuropeptide Y and ATP. Acta Physiol. Scand. 133: 183-197. J . M., A. HEMSBN,0. LARSSON, A. RUDEHILL, A. SARIA& B. B. FREDHOLM. 12. LUNDBERG, 1988. Neuropeptide Y receptor in pig spleen: binding characteristics, reduction of cyclic AMP formation and calcium antagonist inhibition of vasoconstriction. Eur. J. Pharmacol. 145: 21-29. 13. MILLAR,B. C., H. M. PIPER& B. J. MCDERMOTT.1988. The antiadrenergic effect of neuropeptide Y on the ventricular cardiomyocyte. Naunyn-Schmiedeberg’s Arch. Pharmacol. 338: 426-429. 14. LUNDBERG, J. M., A. HEMSBN,A. RUDEHILL, A. HARFSTRAND, 0. LARSSON, A. SOLLEVI, A. SARIA,T. H ~ K F E L T K., FUXE& B. B. FREDHOLM. 1988. Neuropeptide Y- and alphaadrenergic receptors in pig spleen: localization, binding characteristics, cyclic AMP effects and functional response in control and denervated animals. Neuroscience 24: 659-672. 15. MACKERREL, A. D. JR., A. HEMSBN,J. LACROIX & J. M. LUNDBERG. 1989. Analysis of structure-function relationships of neuropeptide Y using molecular dynamics simulations and pharmacological activity and binding measurements. Regul. Pept. 25: 295-3 18. 16. NEILD,T. 0. 1987. Actions of neuropeptide Y on innervated and denervated rat tail arteries. J. Physiol. 386: 19-30. J. S. & J. M. LUNDBERG. 1989. Adrenergic and neuropeptide Y supersensitivity in 17. LACROIX, denervated nasal mucosa vasculature of the pig. Europ. J . Pharmacol. 169 125-136. L. X., E. BARNES,S. Z. LANCER& N. WEINER.1974. Release of norepinephrine 18. CUBEDDU, and dopamine-P-hydroxylase by nerve stimulation. I. Role of neuronal and extraneuronal uptake and of alpha-presynaptic,yeceptors.J. Pharmacol. Exp. Ther. 190: 43 1-450. J . M., A. SARIA,A. A N G G ~ D T.. H ~ K F E L&TL. TERENIUS. 1984. Neuropeptide 19. LUNDBERG, Y and noradrenaline interaction in peripheral cardiovascular control. Clin. Exp. Theory Pract. A6: 1961-1972. J . M., A. SARIA.A. FRANCO-CERECEDA, T. H ~ K F E LL. T , TERENIUS & M. GOLD20. LUNDBERG, STEIN. 1985. Differential effects of reserpine and 6-hydroxydopamine on neuropeptide Y and
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noradrenaline in peripheral neurons. Naunyn-Schmiedeberg’s Arch. Pharmacol. 328: 33 1340. NAGATA,M., A. FRANCO-CERECEDA, A. SARIA,R. AMMAN& J. M. LUNDBERG. 1987. Reserpine-induced depletion of neuropeptide Y in the guinea-pig: tissue-specific effects and mechanism of action. J. Aut. Nerv. Syst. 20: 257-263. LUNDBERG, J. M., A. SARIA,A. FRANCO-CERECEDA & E. THEOWRSSON-NORHEIM. 1985. Treatment with sympatholytic drugs changes tissue content of neuropeptide Y in cardiovascular nerves and adrenal gland. Acta Physiol. Scand. 124: 603-61 I . LUNDBERG, J. M., G . FRIED,J. PERNOW,E. THEOWRSSON-NORHEIM & A. A N G G ~ R D 1986. . NPY-a mediator of reserpine-resistant, non-adrenergic vasoconstriction in cat spleen after preganglionic denervation? Acta Physiol. Scand. 126: 151-152. PERNOW, J., T. KAHAN& J. M. LUNDBERG. 1988. Neuropeptide Y and reserpine-resistant vasoconstriction evoked by sympathetic nerve stimulation in dog skeletal muscle. Br. I. Pharmacol. 94: 952-960. LUNDBERG, J. M., J. PERNOW,A. FRANCO-CERECEDA & A. RUDEHILL.1987. Effects of antihypertensive drugs on sympathetic vascular control in relation to neuropeptide Y. J. Cardiovasc. Pharmacol. 12 (Suppl. 10) 551-568. PERNOW, J., P. THORBN,B-I. MILLBERG & J. M. LUNDBERG. 1988. Renal sympathetic nerve activation in relation to reserpine-induced depletion of neuropeptide Y in the kidney of the rat. Acta Physiol. Scand. 1Jq: 53-59. FRANCO-CERECEDA, A,, M. NAGATA, T. H. SVENSSON & J. M. LUNDBERG. 1987. Differential effects of clonidine and reserpine treatment on neuropeptide Y content in some sympathetically innervated tissues of the guinea-pig. Eur. 1. Pharmacol. 142: 267-273. LUNDBERG, J. M., A. RUDEHILL, A. SOLLEVI, G. FRIED& G. WALLIN.1989. Co-release of neuropeptide Y and noradrenaline from pig spleen in vivo: importance of subcellular storage, nerve impulse frequency and pattern, feedback regulation and resupply by axonal transport. Neuroscience 28: 475-486. PERNOW, J. & J. M. LUNDBERG. 1989. Release and vasoconstrictor effects of neuropeptide Y in relation to non-adrenergic sympathetic control of renal blood flow in the pig. Acta Physiol. Scand. 136: 507-517. SCHALLING, M., A. FRANCO-CERECEDA, T. HOKFELT,H. PERSON & J. M. LUNDBERG. 1988. Increased neuropeptide Y messenger RNA and peptide in sympathetic ganglia after reserpine pretreatment. Eur. J. Pharmacol. 156: 419-420. LUNDBERG, J. M., A. ANGG~RD, E. THEOWRSSON-NORHEIM & J. PERNOW. 1984. Guanethidine-sensitive release of NPY-like immunoreactivity by sympathetic nerve stimulation. Neurosci. Lett. 52: 175-180. LUNDBERG, J. M., A. RUDEHILL, A. SOLLEVI & B. HAMBERGER. 1989. Evidence for cotransmitter role of neuropeptide Y in pig spleen. Br. J. Pharmacol. 96: 675-687. KAIJSER, L., J. PERNOW, B. BERGLUND & J. M. LUNDBERG. 1990. Neuropeptide Y is released together with noradrenaline from the human heart during exercise and hypoxia. Clin. Physiol. 1 0 179-188. LUNDBERG, I . M., A. MARTINSSON, A. HEMSBN,E. THEOWRSSON-NORHEIM, J. SVEDENHAG, E. EKBLOM & P. HJEMDAHL. 1985. Co-release of neuropeptide Y and catecholamines during physical exercise in man. Biochem. Biophys. Res. Commun. 133: 30-36. PERNOW.J., J. M. LUNDBERG, L. KAISER, P. HJEMDAHL, E. THEOWRSSON-NORHEIM, E. MARTINSSON & B. PERNOW.1986. Plasma neuropeptide Y-like immunoreactivity and catecholamines during various degrees of sympathetic activation in man. Clin. Physiol. 6 561578. PERNOW,J., J. M. LUNDBERG & L. KAIJSER.1988. a-Adrenoceptor influence on plasma levels of neuropeptide Y-like immunoreactivity and catecholamines during rest and sympathoadrenal activation in humans. J. Cardiovasc. Pharmacol. 12: 593-599. LUNDBERG, J. M., J. PERNOW & J . S. LACROIX.1989. Neuropeptide Y: sympathetic cotransmitter and modulator? NIPS 4: 13-17. ERICSSON, A,, M. SCHALLING, K. MCINTYRE, J. M. LUNDBERG, D. LARHAMMAR, K. SEROO C Y , K. HOKFELT& H. PERSON. 1987. Detection of neuropeptide Y and its mRNA in megacaryocytes: enhanced levels in certain autoimmune mice. Proc. Natl. Acad. Sci. USA 84: 5585-5590.
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C. A. VAZ, H.R. KEISER& Z. ZUKOWSKA-GROJIC. 1988. 39. MYERS,K. M., M. Y. FARKAT, Release of immunoreactiveneuropeptide Y by rat platelets. Biochem. Biophys. Res. Comm. 155: 118-122. C., SODERBLOM 40. HEMSBN.A., M. STENSDOTTER, & J. M. LUNDBERG. 1989. Effects of vinblastine, reserpine and capsaicin on neuropeptide Y in the sympathoadrenal system and trombocytes of the rat. XXXl IUPS. Helsinki. P4297. 41. PERNOW, 3. & 1. M. LUNDBERG. 1989. Modulation of noradrenaline and neuropeptide Y (NPY) release in the pig kidney in vivo: involvement of alpha,, NPY and angiotensin I1 receptors. Naunyn-Schmiedeberg’sArch. Pharmacol. 340: 379-385. 42. LUNDBERG, 1. M., A. RUDEHILL& A. SOLLEVI. 1989. Pharmacological characterization of neuropeptide Y and noradrenaline mechanisms in sympathetic control of pig spleen. Eur. J. Pharmacol. 163: 103-113. 43. PERNOW, J., A. SARIA& J. M. LUNDBERG. 1986. Mechanisms underlying pre- and postjunctional effects of neuropeptide Y in sympathetic vascular control. Acta Physiol. Scand. 1M: 239-249. A., J. M. LUNDBERG 44. FRANCO-CERECEDA, & C. DAHLOF.1985. Neuropeptide Y and sympathetic control of heart contractility and coronary vascular tone. Acta Physiol. Scand. 129: 361-370. J. M., X-Y. HUA& A. FRANCO-CERECEDA. 1984. Effects of neuropeptide Y 45. LUNDBERG, (NPY) on mechanical activity and neurotransmission in the heart, vas deferens and urinary bladder of the guinea pig. Acta Physiol. Scand. 121: 325-332. 46. POTTER,E. K. 1985. Prolonged non-adrenergic inhibition of cardiac vagal action following sympathetic stimulation: neuromodulationby neuropeptide Y? Neurosci. Lett. 54: 117-121. 47. STJARNE, & P. ASTRAND. 1986. Neuropeptide Y-a cotransmitter with L., J. M. LUNDBERG noradrenaline and adenosine-5-triphosphatein the sympathetic nerves of the mouse vas deferens? A biochemical, physiological and electropharmacological study. Neuroscience 1 8 151-166. 48. STJARNE.L., E. STJXRNE,M. MSHGINA & J. M. LUNDBERG. 1988. Neuropeptide Y may inhibit sympathetic transmitter secretion by an effect upstream of varicosities. Acta Physiol. Scand. 134: 577-578. 49. FRANCO-CERECEDA, A. & J. M. LUNDBERG. 1987. Potent effects of neuropeptide Y and calcitonin gene-related peptide on human coronary vascular tone in v i m . Acta Physiol. stand. 131: 159-160. J., A. SOLLEVI, B. ULLMAN,A. FRANCO-CERECEDA & J. M. LUNDBERG. 1990. 50. HULTING, Plasma neuropeptide Y on admission to a coronary care unit. Elevated NPY levels in a subgroup of patients with heart failure. Cardiovasc. Res. 24: 102-108.
INTRODUCTION Neuropeptide Y (NPY) is a peptide with 36-amino acid residues which was originally isolated from the porcine brain' and probably represents the earlier described neuronal pancreatic polypeptide-like immunoreactivity. A major portion of the NPY-immunoreactive nerves in peripheral organs represents classical sympathetic fibres where NPY co-exists with noradrenaline (NA).3v4NPY is thus present in perivascular sympathetic fibres as well as in the noradrenergic nerves to the muscle of the heart, spleen, and vas deferens. Vasoconstrictor Actions of NPY Exogenous NPY causes potent, long-lasting reduction in local blood flow in many organs of experimental (FIG. I ) as well as in the human forearm.' In vitro, NPY contracts small isolated blood vessels such as cerebral,* splenic,' renal and skeletal muscle arteries'' with threshold effects in the nM concentration range. The NPY effect is mediated via activation of specific nonadrenergic receptor mechanisms and the vasoconstriction is characterized by a slow onset and a long duration. The vasoconstrictor effect of NPY is independent of the vascular endothelium suggesting an action directly on vascular smooth muscle cells.' In the human forearm local i.a. infusion of NPY causes both reduction in blood flow and increase in venous tone suggesting constriction of both resistance and capacitance vessels' in analogy with data from pig nasal mucosa. I '
NPY Receptors and Intmcellular Messengers Calcium antagonists like nifedipine inhibit the vasoconstrictor response to NPY on renal and skeletal muscle arteries but not on mesenteric veins." The NPY effect was largely uninfluenced by changes in extracellular Ca2+ concentrations suggesting that NPY rather acts via changes in intracellular Ca2+ (see REF. 10). NPY does not stimulate inositol phosphate turnover in blood vessels per seI2 while the forskolin-stimulated cyclic AMP formation in vascular smooth muscle" and heart muscle'3 is inhibited. High affinity-binding sites for NPY with receptor characteristics have been demonstrated in both
"The present paper summarizes data obtained with support from the Swedish Medical Research Council (14X-6554), the Swedish Tobacco Company, the American Council for Tobacco Research and the Heart & Lung Foundation. 166
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blood vessels and the capsule of the pig spleen.'2*'4*'5A large amidated C-terminal portion of NPY is a prerequisite for receptor binding, inhibition of forskolin-stimulated cyclic AMP formation and vasoconstrictor effects. I2,l4 The structurally related peptide YY (1-36) binds to NPY receptors with similar or larger affinity than NPY and also evokes potent vasoconstricti~n.'~'~~'~.'~ Supersensitivity to the vasoconstrictor effects of NPY after sympathetic denervation has been observed in the rat tail artery in vitro'" and in the pig nasal mucosa in vivo" but not in the pig spleen.I4 NPY and "Nonadrenergic" Vasoconshiction In the submandibular salivary gland of the cat it was demonstrated' that NPY mimicked the slowly developing and long-lasting decrease in blood flow evoked by sympathetic nerve stimulation using a high frequency in the presence of a-and P-adrenoceptor
FIGURE 1. Effects of electrical sympathetic nerve stimulation (NS, bar) and local i.a. infusion of NPY (2 nmol/min) on vascular tone as revealed by perfusion pressure (PP, mmHg) in (a) cat spleen (10 Hz for 2 min) and (b) dog gracilis muscle (6.9 Hz 30 sec). The nerve stimulation evoked vasoconstriction (increase in perfusion pressure) in controls is markedly prolonged in the animals where reserpine pretreatment ( 1 mg/kg i . v . ) was combined with preganglionic decentralization to maintain tissue NPY levels and to deplete NA by 99%. This long-lasting nerve response is mimicked 23,24. by NPY infusion. For details see REFERENCES
antagonists. Treatment with reserpine in order to deplete (by 99%)the tissue stores of NA may represent an alternative approach to study nonadrenergic transmission since it cannot be excluded that the doses of adrenoceptor antagonists used are not high enough to eliminate all the effects of the released NA. It is well established, however, that only minor vasoconstrictor responses to nerve stimulation in, e.g., the spleen remain in reserpine treatment animals, apparently leaving a minor or no role for nonadrenergic mechanisms. Interestingly, recent studies have shown that reserpine treatment is associated with a reversible depletion not only of NA but also of NPY from sympathetic nerve fibres in a variety of tissues including heart, skeletal muscle and Several characteristic features separate the effects of reserpine pretreatment on NA storage from that on NPY. Thus, higher doses of reserpine are required to deplete NPY than NA. The depletion of NPY is slower in onset and less pronounced than that of NA in terminal regions.'9.2' Furthermore, the NPY content of sympathetic axons and in terminals of some tissues like the vas deferens is not reduced after reserpine suggesting an action which is not directly related to storage mechanisms.20,2' Most strikingly, the
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reserpine-induced reduction of tissue NPY levels is, in contrast to the effect on NA, entirely dependent on an intact nerve activity since either pretreatment with a nicotinic receptor blocking agent such as chlorisondamine, surgical transection at the pre- or postganglionic levels or clonidine, which reduces sympathetic discharge, impairs the reserpine effect on NPY but not on NA level^.^'-^^ The reserpine-induced depletion of NPY is therefore likely to be caused by an increased release of the peptide in excess of synthesis and resupply capacity by axonal transport (see REF. 25). It is known that reserpine pretreatment is associated with a rapid increase in firing rate of postganglionic sympathetic nerves.26 Treatment with reserpine can therefore be combined with pharmacological agents or surgical procedures in order to prevent the increased neuronal activity to the organ studied which after 24 h results in a situation where NA is depleted by 98-99%, while the tissue content of NPY is preserved. Stimulation of the sympathetic nerves in the spleen with high frequency then evoked a marked long-lasting vasoconstrictor response which was mimicked by the action of exogenous NPYZ3(FIG. 1). This experimental approach has now been tested in a variety of preparations in vivo in addition to the cat spleen,23 i.e., the pig spleen,28 dog skeletal muscle,24 pig nasal mucosa'l and pig kidneyz9 with similar results. The high correlation ( r = 0.79-0.91) between the vasoconstrictor effects and the detectable overflow of NPY into the local venous effluent, the characteristic time course of the response being slowly developing and long-lasting and fatigue of the response upon repeated stimulation in these reserpinized preparations further favour a peptide like NPY as the mediator. Although release of NPY seems to be facilitated by high frequency stimulation, NPY outflow is detected both from spleen" and kidney29 already at stimulation with a low frequency like 0.5 Hz. Furthermore, in reserpinized pigs, nasal vasoconstriction is observed even in response to single impulses' ' suggesting that exocytosis of the NPY content from large dense cored vesicles can occur also under such conditions although to a lesser extent than at high frequency stimulation. Following reserpine treatment the levels of NPY in sympathetic ganglia are elevated and an increased accumulation of peptide occurs above an axonal ligation suggesting both enhanced synthesis and subsequent anterograde axonal transport. 2o Accordingly, recent data have shown that expression of specific NPY messenger RNA in sympathetic ganglion cells is increased following reserpine admini~tration.~~ The observation that subchronic pretreatment with chlorisondamine reduces the NPY content in sympathetic ganglion cellsz2 further supports the concept of a nicotinic receptor-stimulated regulation of NPY synthesis.
Co-Release of NPY and NA upon Sympathetic Activation Release of NPY, as revealed by either overflow into the local venous effluent from organs of experimental animaIs28.29*3'*32 or human h e a d 3 as well as increases in systemic plasma levels in man34*35*36 occurs upon sympathetic activation. The ratio between the overflow of NPY and that of NA increases with the frequency of stimulationz8 and the correlation between the NPY and NA overflow represents a sigmoid curve,37 suggesting that NPY release is preferentially facilitated by high stimulation (FIG.2). The local plasma concentration of endogenous NPY-LI in the pig splenic or renal venous effluent upon high frequency stimulation in reserpinized animals is in the nM range, i.e., similar to where exogenous NPY evokes vasocor~striction.~~~~~~~ Since further characterization by high performance liquid chromatography suggests that the NPY-LI detected by the antiserum (Nl) used represents NPY ( 1-36);28 this suggests that endogenous NPY concentrations which are likely to be much higher close to release sites are more than sufficient to activate receptors on vascular smooth muscle cells and to contribute to the functional response. fJpon prolonged stimulation (evoked by either increased sympathetic
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discharge as for reserpine or electrical stimulation), NPY release cannot be maintained and tissue content of NPY is then reduced, however.28 The mechanisms underlying the frequency-dependent differential secretion of NPY may be related to the partly separate vesicular storage of these two agents in the sympathetic nerve terminals, whereby NPY seems to be exclusively present in the large densecored vesiclesz8 (FIG.3). Pretreatment with guanethidine inhibits the stimulation-evoked release of both NPY and NA," while tyramine evokes NA secretion without influencing NPY indicating that separate release can occur.32 After Q -adrenoceptor antagonists the NPY and NA overflow is enhanced in parallel, h~weve?'.~' (FIG.3). The plasma levels of NPY in healthy human subjects are mainly elevated upon heavy physical exercise (FIG.3) or other situations with a strong sympathetic activation such as after h y p o ~ i aor~ adrenoceptor ~ b l ~ k a d e . ' ~ - ~Therefore, " also in man the secretion of NPY as detected by elevated systemic plasma levels seems to be enhanced at high degrees of sympathetic activation compared to that of NA. Most species including man have
(b)
305
I.l*u//, 200]
~0.96
r10.87
0
/
150
'
z t
50
0
0 1000
NA
2000 (pmol)
3000
e3" 0
10
20
30
40
NA (nM)
FIGURE 2. Correlation between (a) output of NPY (pmol) and NA (pmol) from the blood perfused pig spleen upon sympathetic nerve stimulation with different frequencies ( r = 0.96) and (b) levels (pM) of NPY and NA in human venous plasma upon graded physical exercise ( r = 0.87). Note the similar relative increase in NPY at high NA levels both from pig spleen and humans. For details see REFERENCES 28, 34, 36.
relatively low resting plasma levels of NPY suggesting that the basal rate of NPY release is low, if any.25 In the rat, however, very high plasma NPY levels are present also during basal conditions which most likely is related to the occurrence of NPY in trombocytes of this species.38 Therefore, NPY levels in rat blood may not be an appropriate indicator of sympathoadrenal activity26 but mainly reflect the degree of trombocyte degran~lation,~' especially since plasma NPY in the rat is very sensitive to vinblastinea treatment, which markedly reduces trombocyte numbers in peripheral blood.
NPY as Pre- and Postjunctional Modulator of Sympathetic Transmission NPY or PYY exerts prejunctional actions on release of transmitter from sympathetic nerves as revealed by inhibition of nerve-evoked contractions3 and stimulation-evoked , ~ ~ overflow from perivascular nerves endogenous NA and NPY output in v ~ v o ~or' 'H-NA
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of rat and man in vitr~'~,~' (FIG.3). Experimental data studying contractile responses or analysis of transmitter overflow suggest that NPY also inhibits the release of both acetylcholine and NA in the heart.44-46 In the mouse vas deferens NPY induced a potentiation of the contractile effects of NA and adenosine-5-triphosphate (ATP).47 Furthermore, NPY reduced the stimulation-
+
Release
I
I
-
I
I
NA
NPY
FIGURE 3. Schematical illustration of a varicosity of a sympathetic nerve containing both NA and NPY in large dense-cored vesicles and only NA in the small vesicles. At low frequency stimulation mainly small vesicles secrete their content of NA acting on postjunctional a,-adrenocepton to evoke vasoconstriction or P,-adrenoceptors to increase cardiac contractility. NA also activates prejunctional a,-adrenoceptors inhibiting release of itself and of NPY from the large vesicles. NPY is secreted upon stronger stimulation and induces vasoconstriction as well as inhibits release of NA and itself. There is some evidence that the vasoconstriction and the prejunctional effect of NPY is mediated via separate receptor mechanisms (designed NPY- 1 and NPY-2, respectively). Nicotine increases basal NA and NPY secretion while angiotensin U (AT 11) facilitates nerve stimulation-evoked release of NA and NPY. Due to the complex interplay between NA and NPY both at the pre- and postjunctional levels a variety of drugs which interfere with NA mechanisms will also change especially NPY release.
evoked contraction and secretion of 'H-NA and selectively depressed the stimulus evoked but not the spontaneously occurring excitatory junction potentials in smooth muscle cells. Possibly this action of NPY, similarly to a,-agonists is dependent on an inhibitory effect in part via a target upstream of the v a r i c ~ s i t i e s . ~ ~ NPY not only evokes vasoconstriction per se but also enhances the contractions of
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blood vessels to a variety of agents including NA in ~ i t r o .The ~ . enhancing ~~ effect of NPY is not unique but shared by other vasoconstrictors like serotonin and characterized by being more pronounced on larger vessels where NPY exerts minor or no contractions per se.
NPY Release in Human Cardiovascular Disease The release of NPY upon sympathetic activation in healthy volunteers as revealed by increase in systemic plasma level^^^.^^ or elevated levels in the coronary sinus upon h y p o ~ i combined a~~ with the potent, long-lasting vasoconstrictor effects of NPY in man’ raise the possibility that NPY may be involved in the pathophysiology of human cardiovascular disorders. NPY is a potent constrictor of small human coronary vessels in ~ i f r - 0 , ~ ~ and elevated plasma levels of NPY are present in patients with heart disease such as acute myocardial infarction, angina pectoris and especially severe left heart failure.5o The possible functional role of NPY under physiological conditions with intact noradrenergic mechanisms, as well as involvement in human vascular disorders, remains to be established with the use of specific NPY antagonists at the pre- and postjunctional levels. It seems clear, however, that many drugs commonly used in experimental studies on cardiovascular control or in treatment of hypertensive or vasopastic disorders in humans also influence NPY mechanisms.
SUMMARY The coexistence of neuropeptide Y (NPY) with noradrenaline (NA) in perivascular nerves as well as in sympathetic nerves to muscle in the heart, spleen and vas deferens suggests a role for NPY in autonomic transmission. Sympathetic nerve stimulation or reflexogenic activation in experimental animals or man are associated with NPY release as revealed by overflow mainly upon strong activation. This difference between NPY and NA secretion may be related to the partly separate subcellular storage whereby NPY seems to be exclusively present in the large dense-cored vesicles. The NPY secretion is likely to be regulated by the local biophase concentrations of NA acting on prejunctional alpha-2-adrenoceptors since alpha-2 agonists inhibit and antagonists enhance NPY overflow, respectively. Furthermore, after NA has been depleted by reserpine, the nerve stimulation-evoked release of NPY is enhanced leading to a progressive depletion of tissue content of NPY. Exogenous NPY binds to both pre- and postjunctional receptors, inhibits NA and NPY release, enhances NA-evoked vasoconstriction and induces vasoconstriction per se. The prejunctional action of NPY which is especially noticeable in the vas deferens may serve to reduce transmitter secretion upon excessive stimulation. The long-lasting vasoconstriction evoked by sympathetic stimulation in several tissues including skeletal muscle, nasal mucosa and spleen, which remains in animals pretreated with reserpine (to deplete NA) combined with preganglionic denervation (to prevent the concomitant excessive NPY release and depletion), is mimicked by NPY and highly correlated to NPY release. Under these circumstances the NPY content in the local venous effluent reaches levels at which exogenous NPY evokes vasoconstriction. REFERENCES 1.
TATEMOTO, K. 1982. Neuropeptide Y:the complete amino-acid sequence of the brain peptide. Roc. Natl. Acad. Sci. USA 79: 5485-5489.
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2. LUNDBERG, J . M., L. TERENIUS, T. H ~ K F E L&T K. TATEMOTO. 1984. Comparative immunohistochemical and biochemical analysis of pancreatic polypeptide-like peptides with special reference to presence of neuropeptide Y in central and peripheral neurons. J. Neurosci. 45: 2376-2386. 3. LUNDBERG, J . M., L. TERENIUS, T. H ~ K F E L C-R. T , MARTLING, K. TATEMOTO, V. MUTT,J. POLAK,S. R. BLOOM& M. GOLDSTEIN. 1982. Neuropeptide Y (NPY)-like immunoreactivity in peripheral noradrenergic neurons and effects of NPY on sympathetic function. Acta Physiol. Scand. 116: 477-480. EKBLAD,E., L. EDVINSSON, C. WAHLESTEDT, R. UDDMAN, R. HAKANSSON & F. SUNDLER. 1984. Neuropeptide Y co-exists and co-operates with noradrenaline in perivascular nerve fibres. Regul. Pept. 8: 225-235. LUNDBERG, 1. M. & K. TAKEMOTO. 1982. Pancreatic polypeptide family (APP, BPP, NPY and PYY) in relation to alpha-adrenoceptor-resistantsympathetic vasoconstriction. Acta Physiol. &and. 116: 393-402. & J. M. LUNDBERG. 1987. Release RUDEHILL, A,, M. OLCBN,A. SOLLEVI,B. HAMBERGER of neuropeptide Y upon hemorrhagic hypovolemia in relation to vasoconstrictor effects in the pig. Acta Physiol. Scand. 131: 517-523. 7. PERNOW,J., J . M. LUNDBERG & L. KAIISER.1987. Vasoconstrictor effects in vivo and plasma disappearance rate of neuropeptide Y in man. Life Sci. 40: 47-54. L., P.C. EMSON,J. MCCULLOCH, K. TATEMOTO & R. UDDMAN.1983. Neuro8. EDVINSSON, peptide Y, cerebrovascular innervation and vasomotor effects in the cat. Neurosci. Lett. 43: 79-84. 9. PERNOW,J. & J . M. LUNDBERG. 1988. Neuropeptide Y induces potent contraction of arterial vascular smooth muscle via an endothelium-independent mechanism. Acta. Physiol. Scand. 134: 157-158. 10. PERNOW, J . , T. SVENBERG & J . M. LUNDBERG. 1987. Actions of calcium antagonists on preand postjunctional effects of neuropeptide Y on human peripheral blood vessels in v i m . Eur. J. Pharmacol. 136: 207-218. 11. LACROIX, J . S., P. STlARNE, A. A N G G ~&DJ. M. LUNDBERG. 1988. Sympathetic vascular control of the pig nasal mucosa. (11): reserpine-resistant, non-adrenergic nervous responses in relation to neuropeptide Y and ATP. Acta Physiol. Scand. 133: 183-197. J . M., A. HEMSBN,0. LARSSON, A. RUDEHILL, A. SARIA& B. B. FREDHOLM. 12. LUNDBERG, 1988. Neuropeptide Y receptor in pig spleen: binding characteristics, reduction of cyclic AMP formation and calcium antagonist inhibition of vasoconstriction. Eur. J. Pharmacol. 145: 21-29. 13. MILLAR,B. C., H. M. PIPER& B. J. MCDERMOTT.1988. The antiadrenergic effect of neuropeptide Y on the ventricular cardiomyocyte. Naunyn-Schmiedeberg’s Arch. Pharmacol. 338: 426-429. 14. LUNDBERG, J. M., A. HEMSBN,A. RUDEHILL, A. HARFSTRAND, 0. LARSSON, A. SOLLEVI, A. SARIA,T. H ~ K F E L T K., FUXE& B. B. FREDHOLM. 1988. Neuropeptide Y- and alphaadrenergic receptors in pig spleen: localization, binding characteristics, cyclic AMP effects and functional response in control and denervated animals. Neuroscience 24: 659-672. 15. MACKERREL, A. D. JR., A. HEMSBN,J. LACROIX & J. M. LUNDBERG. 1989. Analysis of structure-function relationships of neuropeptide Y using molecular dynamics simulations and pharmacological activity and binding measurements. Regul. Pept. 25: 295-3 18. 16. NEILD,T. 0. 1987. Actions of neuropeptide Y on innervated and denervated rat tail arteries. J. Physiol. 386: 19-30. J. S. & J. M. LUNDBERG. 1989. Adrenergic and neuropeptide Y supersensitivity in 17. LACROIX, denervated nasal mucosa vasculature of the pig. Europ. J . Pharmacol. 169 125-136. L. X., E. BARNES,S. Z. LANCER& N. WEINER.1974. Release of norepinephrine 18. CUBEDDU, and dopamine-P-hydroxylase by nerve stimulation. I. Role of neuronal and extraneuronal uptake and of alpha-presynaptic,yeceptors.J. Pharmacol. Exp. Ther. 190: 43 1-450. J . M., A. SARIA,A. A N G G ~ D T.. H ~ K F E L&TL. TERENIUS. 1984. Neuropeptide 19. LUNDBERG, Y and noradrenaline interaction in peripheral cardiovascular control. Clin. Exp. Theory Pract. A6: 1961-1972. J . M., A. SARIA.A. FRANCO-CERECEDA, T. H ~ K F E LL. T , TERENIUS & M. GOLD20. LUNDBERG, STEIN. 1985. Differential effects of reserpine and 6-hydroxydopamine on neuropeptide Y and
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noradrenaline in peripheral neurons. Naunyn-Schmiedeberg’s Arch. Pharmacol. 328: 33 1340. NAGATA,M., A. FRANCO-CERECEDA, A. SARIA,R. AMMAN& J. M. LUNDBERG. 1987. Reserpine-induced depletion of neuropeptide Y in the guinea-pig: tissue-specific effects and mechanism of action. J. Aut. Nerv. Syst. 20: 257-263. LUNDBERG, J. M., A. SARIA,A. FRANCO-CERECEDA & E. THEOWRSSON-NORHEIM. 1985. Treatment with sympatholytic drugs changes tissue content of neuropeptide Y in cardiovascular nerves and adrenal gland. Acta Physiol. Scand. 124: 603-61 I . LUNDBERG, J. M., G . FRIED,J. PERNOW,E. THEOWRSSON-NORHEIM & A. A N G G ~ R D 1986. . NPY-a mediator of reserpine-resistant, non-adrenergic vasoconstriction in cat spleen after preganglionic denervation? Acta Physiol. Scand. 126: 151-152. PERNOW, J., T. KAHAN& J. M. LUNDBERG. 1988. Neuropeptide Y and reserpine-resistant vasoconstriction evoked by sympathetic nerve stimulation in dog skeletal muscle. Br. I. Pharmacol. 94: 952-960. LUNDBERG, J. M., J. PERNOW,A. FRANCO-CERECEDA & A. RUDEHILL.1987. Effects of antihypertensive drugs on sympathetic vascular control in relation to neuropeptide Y. J. Cardiovasc. Pharmacol. 12 (Suppl. 10) 551-568. PERNOW, J., P. THORBN,B-I. MILLBERG & J. M. LUNDBERG. 1988. Renal sympathetic nerve activation in relation to reserpine-induced depletion of neuropeptide Y in the kidney of the rat. Acta Physiol. Scand. 1Jq: 53-59. FRANCO-CERECEDA, A,, M. NAGATA, T. H. SVENSSON & J. M. LUNDBERG. 1987. Differential effects of clonidine and reserpine treatment on neuropeptide Y content in some sympathetically innervated tissues of the guinea-pig. Eur. 1. Pharmacol. 142: 267-273. LUNDBERG, J. M., A. RUDEHILL, A. SOLLEVI, G. FRIED& G. WALLIN.1989. Co-release of neuropeptide Y and noradrenaline from pig spleen in vivo: importance of subcellular storage, nerve impulse frequency and pattern, feedback regulation and resupply by axonal transport. Neuroscience 28: 475-486. PERNOW, J. & J. M. LUNDBERG. 1989. Release and vasoconstrictor effects of neuropeptide Y in relation to non-adrenergic sympathetic control of renal blood flow in the pig. Acta Physiol. Scand. 136: 507-517. SCHALLING, M., A. FRANCO-CERECEDA, T. HOKFELT,H. PERSON & J. M. LUNDBERG. 1988. Increased neuropeptide Y messenger RNA and peptide in sympathetic ganglia after reserpine pretreatment. Eur. J. Pharmacol. 156: 419-420. LUNDBERG, J. M., A. ANGG~RD, E. THEOWRSSON-NORHEIM & J. PERNOW. 1984. Guanethidine-sensitive release of NPY-like immunoreactivity by sympathetic nerve stimulation. Neurosci. Lett. 52: 175-180. LUNDBERG, J. M., A. RUDEHILL, A. SOLLEVI & B. HAMBERGER. 1989. Evidence for cotransmitter role of neuropeptide Y in pig spleen. Br. J. Pharmacol. 96: 675-687. KAIJSER, L., J. PERNOW, B. BERGLUND & J. M. LUNDBERG. 1990. Neuropeptide Y is released together with noradrenaline from the human heart during exercise and hypoxia. Clin. Physiol. 1 0 179-188. LUNDBERG, I . M., A. MARTINSSON, A. HEMSBN,E. THEOWRSSON-NORHEIM, J. SVEDENHAG, E. EKBLOM & P. HJEMDAHL. 1985. Co-release of neuropeptide Y and catecholamines during physical exercise in man. Biochem. Biophys. Res. Commun. 133: 30-36. PERNOW.J., J. M. LUNDBERG, L. KAISER, P. HJEMDAHL, E. THEOWRSSON-NORHEIM, E. MARTINSSON & B. PERNOW.1986. Plasma neuropeptide Y-like immunoreactivity and catecholamines during various degrees of sympathetic activation in man. Clin. Physiol. 6 561578. PERNOW,J., J. M. LUNDBERG & L. KAIJSER.1988. a-Adrenoceptor influence on plasma levels of neuropeptide Y-like immunoreactivity and catecholamines during rest and sympathoadrenal activation in humans. J. Cardiovasc. Pharmacol. 12: 593-599. LUNDBERG, J. M., J. PERNOW & J . S. LACROIX.1989. Neuropeptide Y: sympathetic cotransmitter and modulator? NIPS 4: 13-17. ERICSSON, A,, M. SCHALLING, K. MCINTYRE, J. M. LUNDBERG, D. LARHAMMAR, K. SEROO C Y , K. HOKFELT& H. PERSON. 1987. Detection of neuropeptide Y and its mRNA in megacaryocytes: enhanced levels in certain autoimmune mice. Proc. Natl. Acad. Sci. USA 84: 5585-5590.
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C. A. VAZ, H.R. KEISER& Z. ZUKOWSKA-GROJIC. 1988. 39. MYERS,K. M., M. Y. FARKAT, Release of immunoreactiveneuropeptide Y by rat platelets. Biochem. Biophys. Res. Comm. 155: 118-122. C., SODERBLOM 40. HEMSBN.A., M. STENSDOTTER, & J. M. LUNDBERG. 1989. Effects of vinblastine, reserpine and capsaicin on neuropeptide Y in the sympathoadrenal system and trombocytes of the rat. XXXl IUPS. Helsinki. P4297. 41. PERNOW, 3. & 1. M. LUNDBERG. 1989. Modulation of noradrenaline and neuropeptide Y (NPY) release in the pig kidney in vivo: involvement of alpha,, NPY and angiotensin I1 receptors. Naunyn-Schmiedeberg’sArch. Pharmacol. 340: 379-385. 42. LUNDBERG, 1. M., A. RUDEHILL& A. SOLLEVI. 1989. Pharmacological characterization of neuropeptide Y and noradrenaline mechanisms in sympathetic control of pig spleen. Eur. J. Pharmacol. 163: 103-113. 43. PERNOW, J., A. SARIA& J. M. LUNDBERG. 1986. Mechanisms underlying pre- and postjunctional effects of neuropeptide Y in sympathetic vascular control. Acta Physiol. Scand. 1M: 239-249. A., J. M. LUNDBERG 44. FRANCO-CERECEDA, & C. DAHLOF.1985. Neuropeptide Y and sympathetic control of heart contractility and coronary vascular tone. Acta Physiol. Scand. 129: 361-370. J. M., X-Y. HUA& A. FRANCO-CERECEDA. 1984. Effects of neuropeptide Y 45. LUNDBERG, (NPY) on mechanical activity and neurotransmission in the heart, vas deferens and urinary bladder of the guinea pig. Acta Physiol. Scand. 121: 325-332. 46. POTTER,E. K. 1985. Prolonged non-adrenergic inhibition of cardiac vagal action following sympathetic stimulation: neuromodulationby neuropeptide Y? Neurosci. Lett. 54: 117-121. 47. STJARNE, & P. ASTRAND. 1986. Neuropeptide Y-a cotransmitter with L., J. M. LUNDBERG noradrenaline and adenosine-5-triphosphatein the sympathetic nerves of the mouse vas deferens? A biochemical, physiological and electropharmacological study. Neuroscience 1 8 151-166. 48. STJARNE.L., E. STJXRNE,M. MSHGINA & J. M. LUNDBERG. 1988. Neuropeptide Y may inhibit sympathetic transmitter secretion by an effect upstream of varicosities. Acta Physiol. Scand. 134: 577-578. 49. FRANCO-CERECEDA, A. & J. M. LUNDBERG. 1987. Potent effects of neuropeptide Y and calcitonin gene-related peptide on human coronary vascular tone in v i m . Acta Physiol. stand. 131: 159-160. J., A. SOLLEVI, B. ULLMAN,A. FRANCO-CERECEDA & J. M. LUNDBERG. 1990. 50. HULTING, Plasma neuropeptide Y on admission to a coronary care unit. Elevated NPY levels in a subgroup of patients with heart failure. Cardiovasc. Res. 24: 102-108.
