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Neuropeptide Y interaction with the adrenergic transmission line: A study of its effect on alpha-2 adrenergic receptors
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NEUROPEPTIDE Y INTERACTION WITH THE ADRENERGIC TRANSMISSION LINE: A S T U D Y O F I T S EFFECT ON ALPHA-2 ADRENERGIC RECEPTORS M. MARTIRE and G. PISTRITTO
Istituto di Farmacologia, Facoltgl di Medicina e Chirurgia, Universitgl Cattolica del Sacro Cuore, Largo Vito 1, 00168 Rome, Italy (Received in final form 27 September 1991)
SUMMARY Neuropeptide Y (NPY), first isolated in 1982, is widely distributed among the neurons of the central and peripheral nervous systems, often in close association with catecholamines. Because of its wide distribution and concentrations in selected areas of the brain, NPY is considered a putative neurotransmitter with several possible physiological effects including modulation of blood pressure, food intake and pituitary hormone release at a central level. Peripherally, the peptide seems to be involved, via direct and indirect mechanisms, in noradrenaline (NA)-mediated vasoconstriction. The ability of NPY to interact with the catecholamine transmission line may underly a possible modulatory influence of NPY on catecholamine receptor characteristics. We recently observed interaction between alpha-2 adrenergic receptors and those for NPY at the presynaptic level. Additional data from our studies in spontaneously hypertensive rats suggest that impairment of these interactions may contribute to the hypertension in this strain. KEY WORDS:neuropeptide Y-alpha-2-adrenoceptors interaction.
INTRODUCTION Research in recent years has led to the identification of a surprisingly large number of new bioactive substances. The physiological roles of many of these peptides, especially those discovered during the last decade, are now emerging from studies carried out long after they were first isolated and sequenced. One such protein, neuropeptide Y (NPY), was first isolated using a novel chemical approach for identifying peptides with C-terminal amide residues [1]. It was later purified from porcine brain tissue [2]. The peptide consists of 36 amino acids and is characterized by N-terminal tyrosine and C-terminal tyrosine amide residues [3]. These latter features led to its designation as neuropeptide 'Y', which, in single-letter amino acid nomenclature, stands for tyrosine. Correspondence to: Dr Maria Martire. 1043-6618/92/030203-13/$03.00/0
© 1992 The Italian Pharmacological Society
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There is a remarkable degree of sequence homology between NPY and members of the pancreatic peptide (PP) family. Avian pancreatic polypeptide, the first peptide of this group to be discovered, was isolated in 1975 from chicken pancreas as a by-product of insulin purification [4]. Similar peptides were later found in the islets of Langerhans of numerous mammalian species as well [5]. Several peptides initially linked to the gastrointestinal system have also been identified in brain tissue, and the search was thus begun for peptides resembling those of the pancreatic family in the CNS of mammals. Using antisera raised against different forms of PP, primarily avian and bovine, several investigators reported immunostaining of a PP-like peptide in the central and peripheral nervous systems of several mammalian species including man [6-8]. However, the features of this immunostaining suggested that the peptide was not identical t6 that found within the pancreas. In addition, true PP could not be detected within neural structures in radioimmunoassays, and it became clear that the antisera were crossreacting with an as yet unknown peptide that was structurally related to PP. This peptide was later identified as NPY. After porcine NPY had become available for the development of radioimmunoassays, NPY-like immunoreactivity began to be reported in various regions, both central and peripheral, of the nervous systems of numerous mammalian species including man [9-17]. These findings indicate that NPY is one of the most prevalent peptides in the brain with quantities that range from 500 to 1500 pmol/g tissue field in those areas of highest concentration, depending on the extraction method used [18]. High-affinity binding sites for NPY have also been identified with autoradiographic techniques in various areas of the brain and spinal cord [19-21]. Attempts to characterize NPY receptors as far as their distribution and their responses to physiological stimuli are concerned have been hampered, however, by the lack of a specific antagonist for this peptide. Many of the discrepancies that now exist regarding the distribution of these receptors [22, 24] might be resolved if such an antagonist could be found, and studies aimed at identifying the physiological role(s) of this putative neurotransmitter would certainly be facilitated. It has recently been shown that there are two types of NPY binding sites which may represent two types of physiological receptors for this peptide. These binding sites, designated Y1 and Y2 and differing in both their affinities and specificities, have been characterized in studies using mono-iodinated radioligands of both intact NPY and of a long C-terminal fragment, NPY 13-36 [25, 26]. TheY2 sites seem to predominate in the CNS [27] and, on the basis of its inhibitory effects on noradrenaline (NA) release in the peripheral nervous system, it has been classified as a prejunctional receptor [28-30].
CO-LOCALIZATION WITH CATECHOLAMINES Numerous studies have demonstrated the existence of co-localization of NPY-like immunoreactivity and catecholamines [31-33]. This association has been revealed in immunohistochemical studies using antisera raised against the transmitters
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themselves, transmitter-synthesizing enzymes and NPY. The distribution of NPYlike immunoreactivity within the central and peripheral nervous systems seems to be quite similar to that of NA-neuron markers such as the catecholaminesynthesizing enzymes dopamine beta-hydroxylase (DBH) and tyrosine hydroxylase (TH). Additional evidence of this co-localization is furnished by the observation that injection of the neurotoxin 6-hydroxydopamine results in the disappearance of not only DBH or TH but also of NPY [34, 35]. Both NA and NPY are also depleted by reserpine treatment [35, 36], and the nerve stimulation-evoked release of NPY is inhibited in the presence of the adrenergic blocking agent guanethidine [37]. Electron microscopy and fractionation studies have revealed the presence of at least two types of organelles within nerve endings containing NA and NPY [38]. In the rat vas deferens, NPY seems to be located exclusively in large, dense-core vesicles [39, 40], whereas the catecholamines are also found in the smaller synaptic vesicles. Studies have shown that the two transmitters are selectively released from the nerve ending depending on the frequency of the action potential [41]. Another factor that determines whether NA or NPY will be released is availability. After depletion, the nerve ending must be resupplied with NPY that has been synthesized in the cell body and transported to the synapse along the axon [35, 39]. Although the co-localization of NA and NPY has been amply demonstrated in immunohistochemical studies, its functional significance is somewhat less clear. Experimental evidence emerging primarily from studies on the peripheral nervous system suggest that the two substances may interact in a cooperative manner on effector cells [44]. This association within the post-ganglionic sympathetic nervous system has been studied rather extensively [42, 43]. Sympathetic neurons containing NPY have been found to innervate heart, gut, spleen, respiratory and urogenital tracts [45]. Current research is being focused on the densely innervated vascular smooth musculature where NPY seems to have at least three effects. At the prejunctional level it causes suppression of NA release. Postjunctionally, NPY potentiates the NA-evoked response but also evokes a direct response that is not mediated by the adrenoceptors [46]. Centrally, the function of NPY in catecholamine neurons is far from clear. The peptide is believed to be involved primarily in the regulation of blood pressure, food intake and neuroendocrine processes [47-50]. Intracisternal or intraventricular administration of sub-nanomolar doses of NPY to alphachloralose-anesthetized or conscious, unrestrained male rats has been shown to produce marked, prolonged bradycardia and vasodepression [51]. Hypotension was also produced by local injection of the peptide into the nucleus of the solitary tract of this animal [52]. This effect is thought to be similar to that of adrenaline, which is the principal vasodepressive transmitter within the medulla oblongata [53]. Infusion of NPY into the paraventricular nucleus of the hypothalamus has been found to increase both food intake and drinking [55, 56]. The peptide has also been found to increase anterior pituitary hormone secretion not only in intact rats but also those in which the anterior pituitary has been rendered inactive [5O, 57, 58].
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I N T E R A C T I O N BETWEEN N P Y A N D A D R E N O C E P T O R S ON CENTRAL C A T E C H O L A M I N E NEURONS: POSSIBLE P A T H O P H Y S I O L O G I C A L IMPLICATIONS NPY displays a wide variety of functional activities depending on its location and coexistence with catecholamines. It can reasonably be assumed that interaction occurs between the peptide and amines at a receptor level as well. Such interaction has been reported for coexisting neurotransmitters such as vasoactive intestinal peptide and acetylcholine in the submandibular gland [59], dopamine and cholecystokinin octapeptide (sulphated), dopamine and neurotensin, and 5hydroxytryptamine and substance P in the CNS [60]. These interactions are manifested by changes in the affinity of receptors or in the number of receptors available for the classical neurotransmitters observed in the presence of the neuropeptides. It has been shown that presynaptic receptors for a given transmitter can regulate the release of all other transmitters coexisting within the same nerve ending. In addition, the number of autoreceptor types contained in a nerve ending is equal to that number of different neurotransmitters it releases [41]. Using a system of superfused synaptosomes, we analyzed the effects of interaction between NPY receptors and alpha-2 autoreceptors on depolarizationevoked NA release from nerve terminals isolated from the medulla oblongata, hypothalamus and frontoparietal cortex of rats. We found that NPY increased the inhibition of potassium-evoked 3H-NA release produced by the specific alpha-2 receptor agonist, clonidine. These findings indicate that NPY receptors may exist on NA presynaptic membranes and that they can inhibit NA release by enhancing the function of the transmitter's autoreceptors (Fig. 1). The NPY receptor is probably high-affinity since maximum inhibition was achieved with application of only 1 n~ [63]. Prejunctional effects are also reportedly involved in sympathetic vasoregulation. Neuropeptide Y produces a concentration-dependent inhibition of electrically-induced secretion of 3H-NA in isolated rat vas deferens [64]. Some investigators have observed NPY modulation of electrically-induced 3HNA release in hypothalamic slice models [66], but others have failed to confirm this finding [65]. Electrophysiological in vitro studies have also shown that excitatory neurotransmission in rat hippocampal slices is inhibited by NPY [67] possibly by reducing the presynaptic Ca 2÷ influx [68]. In locus ceruleus cells, the peptide lowers action potentials probably by increasing membrane permeability to potassium ions through activation of a G protein. This effect is much less marked in the presence of alpha-2 adrenoceptor antagonists [69]. The interaction between presynaptic NPY receptors and alpha-2 receptors in the medulla oblongata and hypothalamus probably occurs at the synaptic level since both NPY and NA are present in these regions. In contrast, the receptor-receptor interaction that takes place in the cerebral cortex is probably a local-circuit phenomenon: cortical NA nerve terminal networks do not, in fact, contain NPY but the peptide is present in scattered interneurons of the cortical layers [70]. It is possible that the latter control alpha-autoreceptor function in the NA afferents arising from the brain stem and especially from the locus ceruleus. The observed
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reduction in turnover in both adrenaline and NA in several regions of the medulla and hypothalamus following intraventricular injection of low doses of NPY also suggests the latter's capacity to enhance the function of alpha autoreceptors in NA nerve terminals [71, 72]. We extended our study to include NPY's modulatory effects on the alpha-2 receptors controlling 5-hydroxytryptamine (5HT) release in the cerebral cortex, hypothalamus and medulla [62]. There is evidence to suggest that the alpha-2 adrenoceptors located on 5HT terminals are stereochemically different from those found on NA nerve endings [73, 74]. The alpha-2 heteroreceptors are postsynaptic to the catecholamine nerve terminals and, for this reason, any receptor-receptor interactions in which they are involved would be especially interesting. Unfortunately we did not find any modulation of the heteroreceptors that inhibit 3H-5HT release by NPY when administered in nanomolar doses [62]. It would thus seem that NPY has different effects on pre- and postsynaptic alpha-2 adrenoceptors in rat cerebral membrane preparations. Low concentrations of NPY have been found to reduce the affinity of alpha-2 adrenergic agonist binding sites leading us to assume that the peptide can also reduce the sensitivity of the postsynaptic receptors to alpha-2 adrenaline itself [75]. Autoradiographic studies show that the adrenoceptor agonist, clonidine, exerts a similar effect on 125I-NPY binding sites in the nucleus of the solitary tract of the rat [76]. At the postsynaptic membrane level, then, the interaction between alphaadrenergic receptors and those for NPY seems to be an antagonistic one. In contrast, the principal effect of NPY receptor activation on alpha-2 autoreceptors presynaptically is that of increased binding affinity. The net result of these interactions is a decrease in adrenergic transmission: at the presynaptic membrane there is a reduction in NA release and any that is released is bound less efficiently
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Fig. 1. Dose-dependent inhibition of potassium-evoked release of -~H-noradrenaline from synaptosomes induced by clonidine and clonidine plus 1 nM neuropeptide Y. The percentage of inhibition, calculated by subtracting baseline from total release, is plotted against various concentrations of clonidine. Data are expressed as means+sEM from 6-8 experiments performed in triplicate. The effects of clonidine plus NPY were significantly different (P<0.01) from those of clonidine alone.
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Fig. 2. Effects of various concentrations of clonidine with and without neuropeptide Y (NPY) on potassium-evoked 3H-noradrenaline release by medulla oblongata synaptosomes from spontaneously hypertensive rats (SHR). Data are expressed as means+_SEM from 3-5 experiments performed in triplicate.
to its receptors at the post-synaptic membrane. The reduction in both demand and release may improve the economy of the monoamine synapse. In light of the potent vasodepressor effects observed after intracisternal or intraventricular injections of NPY, we extended our study of the receptor-receptor interactions in a spontaneously hypertensive (SH) rat model. The peptide's enhancement of clonidine-induced inhibition of potassium-evoked 3H-NA release in synaptosomes from the medulla oblongata was less marked in these animals than that seen in preparations from normotensive Wistar Kyoto rats [77]. With respect to the quantity used in the latter model, a 100-fold increase in NPY concentration was necessary before significantly enhanced inhibition could be observed in the preparations from the hypertensive rats (Fig. 2). These findings may reflect a marked loss of affinity by the presynaptic NPY binding sites on NA nerve terminals in the SH rat. There may also be a decrease in the function of the alpha-adrenergic autoreceptors in these animals [77]. These findings are consistent with those of previous studies on membrane preparations from the medulla oblongata of SH rats: 10 nM of NPY failed to reduce the affinity of alpha-adrenergic agonist binding [78]. The resistance of the preand postsynaptic alpha-adrenergic receptors to NPY modulation in this animal is also reflected in the fact that the EDso value for the hypotensive response to intracisternal NPY is 10 times higher in the hypertensive strain than it is in normotensive Wistar Kyoto rats [54].
CONCLUSIONS The existence of neuropeptide receptors on axon terminals of monoamine neurons can be postulated on the basis of findings demonstrating the coexistence of these
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peptides with catecholamines in many areas of the CNS. It is still not clear, however, how these co-localized transmitters interact with one another or with the receptors. We have demonstrated that co-localized NA and NPY act synergistically to stimulate the alpha-2 autoreceptors for NA in synaptosomes isolated from brain tissue. These findings suggest that the binding sites for the peptide and those for the monoamine are located side by side on the same cell membrane. The interaction we have noted may involve the direct induction of conformational changes in the NA receptor proteins or an indirect effect mediated by regulatory proteins [79, 80]. Like the alpha-2 agonist clonidine, NPY inhibits basal and forskolin-stimulated adenylate cyclase activity in various tissues [81-83]. There is strong evidence to suggest that this effect, in both the central and peripheral nervous systems, is mediated by a G-protein (probably Gi) that is sensitive to both pertussis toxin and N-ethylmaleimide [83-85]. Neuropeptide-Y binding is also reduced in the presence of guanine nucleotides suggesting that a guanine nucleotide-binding
cAMP ATP
Fig. 3. Hypothetical stimulatory (+) and inhibitory (-) mechanisms for the NA/NPY costoring neurons and the postsynaptic receptor-receptor interaction between NPY and the alpha2 adrenoceptors is indicated. The postsynaptic interaction may take place either between the recognition sites or via the inhibitory GTP-dependent coupling unit (G protein). The presynaptic NPY receptor may affect the presynaptic alpha-2 adrenoceptors as well.
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protein may be coupled with the NPY receptors [86, 87]. The role of an inhibitory G protein has also been demonstrated in in vivo studies: treatment with pertussis toxin has been found to counteract the reduced affinity of alpha-2 agonist binding sites caused by activation of NPY receptors [88] as well as the central cardiovascular effects produced by NPY and clonidine [89]. It is conceivable that coupling of receptors to their regulatory proteins activates intramembrane feedback mechanisms which can modulate receptor-binding capacity. The synergistic effects that we observed betwen NPY and NA may, thus, be the result of changes in the affinity or density of the NA receptors stemming from NPY-induced increases in the coupling of receptors and regulatory proteins (Fig. 3). It is also possible that the receptors for co-localized transmitters communicate with one another by means of reactions used for intracellular communication [90]. The fact that NPY's modulation of alpha-2 adrenergic receptors is much less efficient in SH rats indicates that this intramembranal integrative mechanism might be altered in these animals. Such impairment might well contribute to the animal's state of hypertension. Demonstration of the existence of co-localized receptor interactions opens a new field for drug development. It may one day be possible to modulate the transmission line without resorting to use of agonist and antagonist agents upon which many of today's medical treatments are based. While there is evidence to suggest that NPY acts as either a neurotransmitter or neuromodulator, further study is needed to determine its overall significance. The physiological importance of the peptide's co-localization with NA and other transmitters, thus far observed primarily through histochemical techniques, is one of the most interesting questions that needs to be clarified.
ACKNOWLEDGEMENTS This study was supported by grants from the CNR Target Project 'Biotechnology and Bioinstrumentation' 89.00206.70 and 90.0087.PF70. The authors wish to thank Ms Marian Kent for her editorial assistance in preparing the manuscript.
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B, eds. Coexistence of neuronal messengers: a new principle in chemical transmission. Progress in brain research, vol. 68. Amsterdam: Elsevier, 1986; 279-87. 47. Emson PC, De Quidt ME. NPY: a new member of the pancreatic polypeptide family. Trends Neurosci 1984; 7: 31. 48. Allen JM, Bloom SR. Neuropeptide Y: a putative neurotransmitter. Neurochem Int 1986; 8(1): 1-8. 49. Gray TS, Morley JE. Neuropeptide Y: anatomical distribution and possible function in mammalian nervous system. Life Sci 1986; 38: 389-401. 50. McDonald JK. NPY and related substances. CRC Crit Rev Neurobiol 1988; 4(1): 97-135. 51. Fuke K, Agnati LF, Harfstrand A, et al. Central administration of neuropeptide Y induces hypotension, bradyphnea and EEG synchronization in the rat. Acta Physiol Scand 1983; 118: 189-92. 52. Carter DA, Vallejo M, Leightman SL. Cardiovascular effects of neuropeptide Y in the nucleus tractus solitarius of the rat: relationship with noradrenaline and vasopressin. Peptides 1985; 6(3): 421-5. 53. Fuxe K, Bohme P, Agnati LF, et al. On the role of central adrenaline neurons in central cardiovascular regulation. In: Fuxe K, Goldstein M, Hokfelt B, Hokfelt Teds. Central adrenaline neurons. Basic aspects and their role in cardiovascular functions. WennerGren Int Symp Set, vol. 33. New York: Plenum Press, 1980: 161-82. 54. Harfstrand A, Fuxe K, Agnati LF, et al. Studies on neuropeptide Y catecholamine interactions in central cardiovascular regulation in the [3-chloralose anaesthetized rat. Evidence for a possible new way of activating the c~2-adrenergic transmission line. Clin Exp Hypertens Theory Pract 1984; A6: 1947-50. 55. Levine AS, Morley JE. Neuropeptide Y: a potent inducer of consummatory behaviour in rats. Peptides 1984; 5: 1025-9. 56. Clark JT, Kabra PS, Crowley WR, Kabra SP. Neuropeptide Y and human pancreatic polypeptide stimulate feeding behavior in rats. Endocrinology 1984; 115: 427-9. 57. Kerkerian L, Guy J, Lefevre G, Pelletier G. Effect of neuropeptide Y (NPY) on the release of anterior pituitary hormones in the rat. Peptides 1985; 6: 1201--4. 58. Chabot JG, Enjalbert A, Pelletier G, Dubois PM, Morel G. Evidence for a direct action of neuropeptide Y in the rat pituitary gland. Neuroendocrinology 1988; 47:511-7. 59. Lundberg JM, Hedlund B, Bartfai T. Vasoactive intestinal polypeptide enhances muscarinic ligand binding in cat submandibular salivary gland. Nature 1982; 295: 147-9. 60. Fuxe K, Agnati LF. Receptor-receptor interactions in the central nervous system. A new integrative mechanism in synapses. Med Res Rev 1985; 5(4): 441-82. 61. Martire M, Fuxe K, Pistritto G, Preziosi P, Agnati LF. Neuropeptide Y enhances the inhibitory effects of clonidine on 3H-noradrenaline release in synaptosomes isolated from the medulla oblongata of the male rat. JNeural Transm 1986; 67:113-24. 62. Martire M, Fuxe K, Pistritto G, Preziosi P, Agnati LF. Neuropeptide Y increases the inhibitory effects of clonidine on potassium-evoked SH-noradrenaline but not 3H-5hydroxytryptamine release from synaptosornes of the hypothalamus and the frontoparietal cortex of the male Sprague-Dawley rat. J Neural Transm 1989; 78:61-72. 63. Fuxe K, Agnati LF, Benfenati F, et al. Evidence for the existence of receptor-receptor interactions in the central nervous system. Studies on the regulation of monoamine receptors by neuropeptides. JNeural Transm 1983; 16 (Suppl): 165-79. 64. Lundberg JM, Stjarne L. Neuropeptide Y (NPY) depresses the secretion of 3Hnoradrenaline and the contractile response evoked by field stimulation in rat vas deferens. Acta Physiol Scand 1984; 120: 477-9. 65. Serfozo P, Barffai T, Vizi ES. Presynaptic effects of neuropeptide Y on 3H-noradrenaline and 3H-acetylcholine release. Regul Peptides 1986; 16:117-23. 66. Yokoo H, Schlesinger DH, Goldstein M. The effect of neuropeptide Y on stimulation evoked release of 3H-noradrenaline from rat hypothalamic and cerebral cortical slices. Eur J Pharmacol 1987; 143: 283-6.
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67. Colmers WF, Lukowiak K, Pittman QJ. Presynaptic action of neuropeptide Y in area CA1 of the rat hippocampal slice. J Physiol 1987; 383: 285-~99. 68. Colmers WF, Lukowiak K, Pittman QJ. Neuropeptide Y action in the rat hippocampal slice: site and mechanism of presynaptic inhibition. J Neurosci 1988; 8(10): 3827-37. 69. Illes P, Regenold JT. Interaction between neuropeptide Y and noradrenaline on central catecholamine neurons. Nature 1990; 344: 62-3. 70. Fuxe K, Agnati LF, Narfstrand A, et al, Morphofunctional studies on the neuropeptide Y/adrenaline costoring terminal systems in the dorsal cardiovascular region of the medulla oblongata. Focus on receptor-receptor interactions in cotransmission. In: Hokfelt T, Fuxe K, Pernow B, eds. Progress in brain research, vol. 68. Amsterdam: Elsevier, 1986; 303-20. 71. Harfstrand A, Eneroth P, Agnati LF, Fuxe K. Further studies on the effects of central administration of neuropeptide Y on neuroendocrine function in the male rat: relationship to hypothalamic catecholamines. Regul Pept 1987; 17: 167-9. 72. Shimizu H, Bray GA. Effects of neuropeptide Y on norepinephrine and serotonin metabolism in rat hypothalamus in vivo. Brain Res Bull 1989; 22(6): 945-50. 73. Gothert M, Huth H, Schlicker E. Characterization of the receptor subtypes involved in c~adrenoceptor-mediated modulation of serotonin release from rat brain cortex slices. Naunyn Sch Arch Pharmacol 1981; 317: 199-203. 74. Raiteri M, Maura G, Versace P. Functional evidence for stereochemically different c~adrenoceptors regulating central norepinephrine and serotonin release. J Pharmacol Exp Ther 1983; 224: 679-84. 75. Agnati LF, Fuxe K, Benfenati F, et al. Neuropeptide Y in vitro selectively increases the number of ~2-adrenergic binding sites in membranes of the medulla oblongata of the rat. Acta Physiol Scand 1983; 118: 293-5. 76. Harfstrand A, Fuxe K, Agnati LF, Fredholm BB. Reciprocal interactions between c~adrenoceptor agonist and neuropeptide Y binding sites in the nucleus tractus solitarius of the rat. A biochemic and autoradiographic analysis. J Neural Transm 1989; 75: 83-99. 77. Martire M, Fuxe K, Pistritto G, Preziosi P, Agnati LF. Reduced inhibitory effects of clonidine and fieuropeptide Y on 3H-noradrenaline release from synaptosomes of the medulla oblongata of the spontaneously hypertensive rat. J Neural Transm 1989; 76(3): 181-9. 78. Agnati LF, Fuxe K, Benfenati F, et al. Failure of neuropeptide Y in vitro to increase the number of binding sites in membrane" of the medulla oblongata of the spontaneously hypertensive rat. Acta Physiol Scand 1983; 119: 309-12. 79. Rodbell R. The role of hormone receptors and GTP-regulatory proteins in membrane transduction. Nature 1980; 284: 17-22. 80. Worley PF, Barabon JM, Snyder SH. Beyond receptors: multiple second-messenger systems in brain. Ann Neurol 1986; 21(3): 217-29. 81. Fredholm BB, Jansen I, Edvinsson L. Neuropeptide Y is a potent inhibitor of cAMP accumulation in feline cerebral vessels. Acta Physiol Scand 1985; 124: 467-9. 82. Westlind-Danielsson A, Andell S, Abens J, Bartfai T. Neuropeptide Y and peptide YY inhibit adenylate cyclase activity in the rat striatum. Acta Physiol Scand 1988; 132: 425-30, 83. Fredh01m BB, Haggblad J, Harfstrand A, Larsson O. Tissue differences in the effects of neuropeptide Y, adenosine and noradrenaline at the second messenger level. In: Mutt V, Fuxe K, Hokfelt T, Lundberg JM, eds. Neuropeptide Y. Nobel Conf Ser, New York: Raven Press, 1989; 181-9. 84. Kassis S, Olasmaa M, Terenius L, Fishman PH. Neuropeptide Y inhibits cardiac adenylate cyclase through a pertussis toxin-sensitive G protein. J Biol Chem 1987; 262: 3429-31. 85. Harfstrand A, Fredholm BB, Fuxe K. Inhibitory effects on cyclic AMP accumulation in slices of the nucleus tractus solitarius. Neurosci Lett 1987; 76: 185-90. 86. Unden A, Bartfai T. Regulation of neuropeptide Y (NPY) binding by guanine nucleotides in the rat cerebral cortex. FEBS Lett 1984; 177: 125-7.
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87. Gilman AG. G proteins: transducers of receptor-generated signals. Ann Rev Biochem 1987; 56: 615-50. 88. Von Euler G, Fuxe K, Von der Ploeg I, Fredholm BB, Agnati LF. Pertussis toxin treatment counteracts intramembrane interactions between neuropeptide Y receptors and c~-adrenoceptors. Eur J Pharmacol 1989; 172z 435-41. 89. Fuxe K, Von Euler G, Von der Ploeg I, Fredholm BB, Agnati LF. Pertussis toxin treatment counteracts the cardiovascular effects of neuropeptide and clonidine in the awake unrestrained rat. Neurosci Lett t989; 101: 337-41. 90. Hollemberg MD. Receptor regulation on receptor-receptor communication. In: Fuxe K, Agnati LF, eds. Receptor-receptor interactions. Wenner-Gren Int Syrup Ser, vol. 48. New York: Plenum Press, 1987: 546-54.
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NEUROPEPTIDE Y INTERACTION WITH THE ADRENERGIC TRANSMISSION LINE: A S T U D Y O F I T S EFFECT ON ALPHA-2 ADRENERGIC RECEPTORS M. MARTIRE and G. PISTRITTO
Istituto di Farmacologia, Facoltgl di Medicina e Chirurgia, Universitgl Cattolica del Sacro Cuore, Largo Vito 1, 00168 Rome, Italy (Received in final form 27 September 1991)
SUMMARY Neuropeptide Y (NPY), first isolated in 1982, is widely distributed among the neurons of the central and peripheral nervous systems, often in close association with catecholamines. Because of its wide distribution and concentrations in selected areas of the brain, NPY is considered a putative neurotransmitter with several possible physiological effects including modulation of blood pressure, food intake and pituitary hormone release at a central level. Peripherally, the peptide seems to be involved, via direct and indirect mechanisms, in noradrenaline (NA)-mediated vasoconstriction. The ability of NPY to interact with the catecholamine transmission line may underly a possible modulatory influence of NPY on catecholamine receptor characteristics. We recently observed interaction between alpha-2 adrenergic receptors and those for NPY at the presynaptic level. Additional data from our studies in spontaneously hypertensive rats suggest that impairment of these interactions may contribute to the hypertension in this strain. KEY WORDS:neuropeptide Y-alpha-2-adrenoceptors interaction.
INTRODUCTION Research in recent years has led to the identification of a surprisingly large number of new bioactive substances. The physiological roles of many of these peptides, especially those discovered during the last decade, are now emerging from studies carried out long after they were first isolated and sequenced. One such protein, neuropeptide Y (NPY), was first isolated using a novel chemical approach for identifying peptides with C-terminal amide residues [1]. It was later purified from porcine brain tissue [2]. The peptide consists of 36 amino acids and is characterized by N-terminal tyrosine and C-terminal tyrosine amide residues [3]. These latter features led to its designation as neuropeptide 'Y', which, in single-letter amino acid nomenclature, stands for tyrosine. Correspondence to: Dr Maria Martire. 1043-6618/92/030203-13/$03.00/0
© 1992 The Italian Pharmacological Society
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There is a remarkable degree of sequence homology between NPY and members of the pancreatic peptide (PP) family. Avian pancreatic polypeptide, the first peptide of this group to be discovered, was isolated in 1975 from chicken pancreas as a by-product of insulin purification [4]. Similar peptides were later found in the islets of Langerhans of numerous mammalian species as well [5]. Several peptides initially linked to the gastrointestinal system have also been identified in brain tissue, and the search was thus begun for peptides resembling those of the pancreatic family in the CNS of mammals. Using antisera raised against different forms of PP, primarily avian and bovine, several investigators reported immunostaining of a PP-like peptide in the central and peripheral nervous systems of several mammalian species including man [6-8]. However, the features of this immunostaining suggested that the peptide was not identical t6 that found within the pancreas. In addition, true PP could not be detected within neural structures in radioimmunoassays, and it became clear that the antisera were crossreacting with an as yet unknown peptide that was structurally related to PP. This peptide was later identified as NPY. After porcine NPY had become available for the development of radioimmunoassays, NPY-like immunoreactivity began to be reported in various regions, both central and peripheral, of the nervous systems of numerous mammalian species including man [9-17]. These findings indicate that NPY is one of the most prevalent peptides in the brain with quantities that range from 500 to 1500 pmol/g tissue field in those areas of highest concentration, depending on the extraction method used [18]. High-affinity binding sites for NPY have also been identified with autoradiographic techniques in various areas of the brain and spinal cord [19-21]. Attempts to characterize NPY receptors as far as their distribution and their responses to physiological stimuli are concerned have been hampered, however, by the lack of a specific antagonist for this peptide. Many of the discrepancies that now exist regarding the distribution of these receptors [22, 24] might be resolved if such an antagonist could be found, and studies aimed at identifying the physiological role(s) of this putative neurotransmitter would certainly be facilitated. It has recently been shown that there are two types of NPY binding sites which may represent two types of physiological receptors for this peptide. These binding sites, designated Y1 and Y2 and differing in both their affinities and specificities, have been characterized in studies using mono-iodinated radioligands of both intact NPY and of a long C-terminal fragment, NPY 13-36 [25, 26]. TheY2 sites seem to predominate in the CNS [27] and, on the basis of its inhibitory effects on noradrenaline (NA) release in the peripheral nervous system, it has been classified as a prejunctional receptor [28-30].
CO-LOCALIZATION WITH CATECHOLAMINES Numerous studies have demonstrated the existence of co-localization of NPY-like immunoreactivity and catecholamines [31-33]. This association has been revealed in immunohistochemical studies using antisera raised against the transmitters
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themselves, transmitter-synthesizing enzymes and NPY. The distribution of NPYlike immunoreactivity within the central and peripheral nervous systems seems to be quite similar to that of NA-neuron markers such as the catecholaminesynthesizing enzymes dopamine beta-hydroxylase (DBH) and tyrosine hydroxylase (TH). Additional evidence of this co-localization is furnished by the observation that injection of the neurotoxin 6-hydroxydopamine results in the disappearance of not only DBH or TH but also of NPY [34, 35]. Both NA and NPY are also depleted by reserpine treatment [35, 36], and the nerve stimulation-evoked release of NPY is inhibited in the presence of the adrenergic blocking agent guanethidine [37]. Electron microscopy and fractionation studies have revealed the presence of at least two types of organelles within nerve endings containing NA and NPY [38]. In the rat vas deferens, NPY seems to be located exclusively in large, dense-core vesicles [39, 40], whereas the catecholamines are also found in the smaller synaptic vesicles. Studies have shown that the two transmitters are selectively released from the nerve ending depending on the frequency of the action potential [41]. Another factor that determines whether NA or NPY will be released is availability. After depletion, the nerve ending must be resupplied with NPY that has been synthesized in the cell body and transported to the synapse along the axon [35, 39]. Although the co-localization of NA and NPY has been amply demonstrated in immunohistochemical studies, its functional significance is somewhat less clear. Experimental evidence emerging primarily from studies on the peripheral nervous system suggest that the two substances may interact in a cooperative manner on effector cells [44]. This association within the post-ganglionic sympathetic nervous system has been studied rather extensively [42, 43]. Sympathetic neurons containing NPY have been found to innervate heart, gut, spleen, respiratory and urogenital tracts [45]. Current research is being focused on the densely innervated vascular smooth musculature where NPY seems to have at least three effects. At the prejunctional level it causes suppression of NA release. Postjunctionally, NPY potentiates the NA-evoked response but also evokes a direct response that is not mediated by the adrenoceptors [46]. Centrally, the function of NPY in catecholamine neurons is far from clear. The peptide is believed to be involved primarily in the regulation of blood pressure, food intake and neuroendocrine processes [47-50]. Intracisternal or intraventricular administration of sub-nanomolar doses of NPY to alphachloralose-anesthetized or conscious, unrestrained male rats has been shown to produce marked, prolonged bradycardia and vasodepression [51]. Hypotension was also produced by local injection of the peptide into the nucleus of the solitary tract of this animal [52]. This effect is thought to be similar to that of adrenaline, which is the principal vasodepressive transmitter within the medulla oblongata [53]. Infusion of NPY into the paraventricular nucleus of the hypothalamus has been found to increase both food intake and drinking [55, 56]. The peptide has also been found to increase anterior pituitary hormone secretion not only in intact rats but also those in which the anterior pituitary has been rendered inactive [5O, 57, 58].
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I N T E R A C T I O N BETWEEN N P Y A N D A D R E N O C E P T O R S ON CENTRAL C A T E C H O L A M I N E NEURONS: POSSIBLE P A T H O P H Y S I O L O G I C A L IMPLICATIONS NPY displays a wide variety of functional activities depending on its location and coexistence with catecholamines. It can reasonably be assumed that interaction occurs between the peptide and amines at a receptor level as well. Such interaction has been reported for coexisting neurotransmitters such as vasoactive intestinal peptide and acetylcholine in the submandibular gland [59], dopamine and cholecystokinin octapeptide (sulphated), dopamine and neurotensin, and 5hydroxytryptamine and substance P in the CNS [60]. These interactions are manifested by changes in the affinity of receptors or in the number of receptors available for the classical neurotransmitters observed in the presence of the neuropeptides. It has been shown that presynaptic receptors for a given transmitter can regulate the release of all other transmitters coexisting within the same nerve ending. In addition, the number of autoreceptor types contained in a nerve ending is equal to that number of different neurotransmitters it releases [41]. Using a system of superfused synaptosomes, we analyzed the effects of interaction between NPY receptors and alpha-2 autoreceptors on depolarizationevoked NA release from nerve terminals isolated from the medulla oblongata, hypothalamus and frontoparietal cortex of rats. We found that NPY increased the inhibition of potassium-evoked 3H-NA release produced by the specific alpha-2 receptor agonist, clonidine. These findings indicate that NPY receptors may exist on NA presynaptic membranes and that they can inhibit NA release by enhancing the function of the transmitter's autoreceptors (Fig. 1). The NPY receptor is probably high-affinity since maximum inhibition was achieved with application of only 1 n~ [63]. Prejunctional effects are also reportedly involved in sympathetic vasoregulation. Neuropeptide Y produces a concentration-dependent inhibition of electrically-induced secretion of 3H-NA in isolated rat vas deferens [64]. Some investigators have observed NPY modulation of electrically-induced 3HNA release in hypothalamic slice models [66], but others have failed to confirm this finding [65]. Electrophysiological in vitro studies have also shown that excitatory neurotransmission in rat hippocampal slices is inhibited by NPY [67] possibly by reducing the presynaptic Ca 2÷ influx [68]. In locus ceruleus cells, the peptide lowers action potentials probably by increasing membrane permeability to potassium ions through activation of a G protein. This effect is much less marked in the presence of alpha-2 adrenoceptor antagonists [69]. The interaction between presynaptic NPY receptors and alpha-2 receptors in the medulla oblongata and hypothalamus probably occurs at the synaptic level since both NPY and NA are present in these regions. In contrast, the receptor-receptor interaction that takes place in the cerebral cortex is probably a local-circuit phenomenon: cortical NA nerve terminal networks do not, in fact, contain NPY but the peptide is present in scattered interneurons of the cortical layers [70]. It is possible that the latter control alpha-autoreceptor function in the NA afferents arising from the brain stem and especially from the locus ceruleus. The observed
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reduction in turnover in both adrenaline and NA in several regions of the medulla and hypothalamus following intraventricular injection of low doses of NPY also suggests the latter's capacity to enhance the function of alpha autoreceptors in NA nerve terminals [71, 72]. We extended our study to include NPY's modulatory effects on the alpha-2 receptors controlling 5-hydroxytryptamine (5HT) release in the cerebral cortex, hypothalamus and medulla [62]. There is evidence to suggest that the alpha-2 adrenoceptors located on 5HT terminals are stereochemically different from those found on NA nerve endings [73, 74]. The alpha-2 heteroreceptors are postsynaptic to the catecholamine nerve terminals and, for this reason, any receptor-receptor interactions in which they are involved would be especially interesting. Unfortunately we did not find any modulation of the heteroreceptors that inhibit 3H-5HT release by NPY when administered in nanomolar doses [62]. It would thus seem that NPY has different effects on pre- and postsynaptic alpha-2 adrenoceptors in rat cerebral membrane preparations. Low concentrations of NPY have been found to reduce the affinity of alpha-2 adrenergic agonist binding sites leading us to assume that the peptide can also reduce the sensitivity of the postsynaptic receptors to alpha-2 adrenaline itself [75]. Autoradiographic studies show that the adrenoceptor agonist, clonidine, exerts a similar effect on 125I-NPY binding sites in the nucleus of the solitary tract of the rat [76]. At the postsynaptic membrane level, then, the interaction between alphaadrenergic receptors and those for NPY seems to be an antagonistic one. In contrast, the principal effect of NPY receptor activation on alpha-2 autoreceptors presynaptically is that of increased binding affinity. The net result of these interactions is a decrease in adrenergic transmission: at the presynaptic membrane there is a reduction in NA release and any that is released is bound less efficiently
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Fig. 1. Dose-dependent inhibition of potassium-evoked release of -~H-noradrenaline from synaptosomes induced by clonidine and clonidine plus 1 nM neuropeptide Y. The percentage of inhibition, calculated by subtracting baseline from total release, is plotted against various concentrations of clonidine. Data are expressed as means+sEM from 6-8 experiments performed in triplicate. The effects of clonidine plus NPY were significantly different (P<0.01) from those of clonidine alone.
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Fig. 2. Effects of various concentrations of clonidine with and without neuropeptide Y (NPY) on potassium-evoked 3H-noradrenaline release by medulla oblongata synaptosomes from spontaneously hypertensive rats (SHR). Data are expressed as means+_SEM from 3-5 experiments performed in triplicate.
to its receptors at the post-synaptic membrane. The reduction in both demand and release may improve the economy of the monoamine synapse. In light of the potent vasodepressor effects observed after intracisternal or intraventricular injections of NPY, we extended our study of the receptor-receptor interactions in a spontaneously hypertensive (SH) rat model. The peptide's enhancement of clonidine-induced inhibition of potassium-evoked 3H-NA release in synaptosomes from the medulla oblongata was less marked in these animals than that seen in preparations from normotensive Wistar Kyoto rats [77]. With respect to the quantity used in the latter model, a 100-fold increase in NPY concentration was necessary before significantly enhanced inhibition could be observed in the preparations from the hypertensive rats (Fig. 2). These findings may reflect a marked loss of affinity by the presynaptic NPY binding sites on NA nerve terminals in the SH rat. There may also be a decrease in the function of the alpha-adrenergic autoreceptors in these animals [77]. These findings are consistent with those of previous studies on membrane preparations from the medulla oblongata of SH rats: 10 nM of NPY failed to reduce the affinity of alpha-adrenergic agonist binding [78]. The resistance of the preand postsynaptic alpha-adrenergic receptors to NPY modulation in this animal is also reflected in the fact that the EDso value for the hypotensive response to intracisternal NPY is 10 times higher in the hypertensive strain than it is in normotensive Wistar Kyoto rats [54].
CONCLUSIONS The existence of neuropeptide receptors on axon terminals of monoamine neurons can be postulated on the basis of findings demonstrating the coexistence of these
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peptides with catecholamines in many areas of the CNS. It is still not clear, however, how these co-localized transmitters interact with one another or with the receptors. We have demonstrated that co-localized NA and NPY act synergistically to stimulate the alpha-2 autoreceptors for NA in synaptosomes isolated from brain tissue. These findings suggest that the binding sites for the peptide and those for the monoamine are located side by side on the same cell membrane. The interaction we have noted may involve the direct induction of conformational changes in the NA receptor proteins or an indirect effect mediated by regulatory proteins [79, 80]. Like the alpha-2 agonist clonidine, NPY inhibits basal and forskolin-stimulated adenylate cyclase activity in various tissues [81-83]. There is strong evidence to suggest that this effect, in both the central and peripheral nervous systems, is mediated by a G-protein (probably Gi) that is sensitive to both pertussis toxin and N-ethylmaleimide [83-85]. Neuropeptide-Y binding is also reduced in the presence of guanine nucleotides suggesting that a guanine nucleotide-binding
cAMP ATP
Fig. 3. Hypothetical stimulatory (+) and inhibitory (-) mechanisms for the NA/NPY costoring neurons and the postsynaptic receptor-receptor interaction between NPY and the alpha2 adrenoceptors is indicated. The postsynaptic interaction may take place either between the recognition sites or via the inhibitory GTP-dependent coupling unit (G protein). The presynaptic NPY receptor may affect the presynaptic alpha-2 adrenoceptors as well.
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protein may be coupled with the NPY receptors [86, 87]. The role of an inhibitory G protein has also been demonstrated in in vivo studies: treatment with pertussis toxin has been found to counteract the reduced affinity of alpha-2 agonist binding sites caused by activation of NPY receptors [88] as well as the central cardiovascular effects produced by NPY and clonidine [89]. It is conceivable that coupling of receptors to their regulatory proteins activates intramembrane feedback mechanisms which can modulate receptor-binding capacity. The synergistic effects that we observed betwen NPY and NA may, thus, be the result of changes in the affinity or density of the NA receptors stemming from NPY-induced increases in the coupling of receptors and regulatory proteins (Fig. 3). It is also possible that the receptors for co-localized transmitters communicate with one another by means of reactions used for intracellular communication [90]. The fact that NPY's modulation of alpha-2 adrenergic receptors is much less efficient in SH rats indicates that this intramembranal integrative mechanism might be altered in these animals. Such impairment might well contribute to the animal's state of hypertension. Demonstration of the existence of co-localized receptor interactions opens a new field for drug development. It may one day be possible to modulate the transmission line without resorting to use of agonist and antagonist agents upon which many of today's medical treatments are based. While there is evidence to suggest that NPY acts as either a neurotransmitter or neuromodulator, further study is needed to determine its overall significance. The physiological importance of the peptide's co-localization with NA and other transmitters, thus far observed primarily through histochemical techniques, is one of the most interesting questions that needs to be clarified.
ACKNOWLEDGEMENTS This study was supported by grants from the CNR Target Project 'Biotechnology and Bioinstrumentation' 89.00206.70 and 90.0087.PF70. The authors wish to thank Ms Marian Kent for her editorial assistance in preparing the manuscript.
REFERENCES
1. Tatemoto K, Mutt V. Isolation of two novel candidate hormones using a chemical method for finding naturally occurring peptides. Nature 1980; 285" 417-8. 2. Tatemoto K, Carlquist M, Mutt V. Neuropeptide Y--a novel brain peptide with structural similarities to peptide YY and pancreatic polypeptide. Nature 1982; 296: 659-60. 3. Tatemoto K. Neuropeptide Y: Complete aminoacid sequence of the brain peptide. Proc Natl Acad Sci USA 1982; 79: 5485-9. 4. Kimmel JR, Hayden LJ, Pollock HG. Isolation and characterization of a new pancreatic polypeptide hormone. J Biol Chem 1975; 250: 9369-76. 5. Lin TM, Chance RE. Gastrointestinal actions of a new bovine pancreatic polypeptide (BPP). In: Chey WY, Brooks FP, eds. Endocrinology of the gut. New Jersey: CR Slack, 1974: 143-5.
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