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Neuropeptide Y-induced pressor responses: Activation of a non-adrenergic mechanism, potentiation by reserpine and blockade by nifedipine
European Journal of Pharmacology, 116 (1985) 33-39 Elsevier
33
N E U R O P E P T I D E Y-INDUCED P R E S S O R R E S P O N S E S : A C T I V A T I O N OF A N O N - A D R E N E R G I C M E C H A N I S M , P O T E N T I A T I O N BY R E S E R P I N E AND BLOCKADE BY N1FEDIPINE YAEKO MABE, KAZUHIKOTATEMOTO * and J. PABLO HUIDOBRO-TORO** Laboratory of Pharmacology, Department of Physiological Sciences, Faculty of Biological Sciences, Catholic University"of Chile, Casilla 114-D, Santiago I, Chile and * Nancy Pritzker Laboratory, Department of Psychiatry and Behavioral Sciences, Stanford University, School of Medicine, Palo Alto, CA, U.S.A.
Received 28 March 1985, revised MS received 12 June 1985, accepted 5 July 1985
Y. MABE, K. TATEMOTO and J.P. HUIDOBRO-TORO, Neuropeptide Y-induced pressor responses: activation of a non-adrenergic mechanism, potentiation by reserpine and blockade by nifedipine, European J. Pharmacol. 116 (1985) 33-39. Intravenous administration of neuropeptide Y (NPY) to pentobarbital anesthetized rats produced a short-lasting concentration-dependent increase in systolic and diastolic blood pressure. Pretreatment of rats with 2 mg/kg reserpine potentiated the NPY-induced pressor responses causing a leftward shift of the NPY concentration-response curve. In addition, reserpinization lengthened the duration of the NPY pressor effects. Reserpine also potentiated the noradrenaline-induced pressor effect but not that caused by angiotensin II. The NPY-induced increase in blood pressure was not antagonized by phenoxybenzamine. On the contrary, some degree Of potentiation was observed, particularly with the larger doses of NPY. The NPY pressor responses were reduced by nifedipine in control and in reserpinized rats. The results demonstrate that the NPY-induced pressor responses were not related to adrenergic mechanisms. NPY may activate calcium channels in the cardiovascular system to promote an influx of calcium, causing peripheral vasoconstriction. Phenoxybenzamine al-receptor blockade Neuropeptide Y-nifedipine interaction
Cardiovascular neuropeptide Y receptors Neuropeptide Y pressor responses
1. I n t r o d u c t i o n
Neuropeptide Y (NPY) is a 36 amino-acid peptide isolated from porcine brain and chemically characterized only 3 years ago (Tatemoto et al., 1982; Tatemoto, 1982). This peptide is now known to be present in the gut (Tatemoto et al., 1985) and in a variety of peripheral tissues including the heart, the spleen, reproductive organs, the peripheral vascular system, etc. (Lundberg et al., 1982; 1983; Gu et al., 1983; Furness et al., 1983). Furthermore, Emson et al. (1984) and Corder et al. (1984) purified and characterized NPY from hu** To whom all correspondence should be addressed at the Catholic University of Chile. 0014-2999/85/$03.30 © 1985 Elsevier Science Publishers B.V.
Reserpine treatment
man phaeochromocytoma tissues establishing the existence of the NPY in the human adrenal gland. The composition of human NPY is almost identical to that of N P Y derived from porcine materials. Thus, this compound constitutes another example of the growing family of gut-brain neuroregulatory peptides (Gregory, 1982; Iversen, 1984). A most interesting feature about this peptide is its apparent association with catecholamines. Lundberg et al. (1982) demonstrated that in the rat auricle, as well as in the vas deferens, NPY-like immunoreactivity decreased substantially following destruction of the noradrenergic nerve endings with 6-hydroxydopamine. A more direct approach using immunofluorescence histochemistry established that neurons in the human medulla ob-
34 longata contained both NPY-like immunoreactivity and tyrosine hydroxylase immunoreactivity (H6kfelt et al., 1983). In addition, Ekblad et al. (1984) used double immunohistochemical staining to demonstrate the co-existence of NPY-like immunoreactivity with dopamine /~-hydroxylase in perivascular nerve fibers. In view of the fact that NPY is distributed extensively along the lining of the peripheral vascular system, and that the local intra-arterial infusion of the peptide causes a reduction in the blood flow of the cat submandibular gland (Lundberg and Tatemoto, 1982: Lundberg and H6kfelt, 1983; Lundberg et al., 1984), we thought it of considerable physiological interest to explore whether NPY would modify systemic blood pressure. In addition, considering the close anatomical location of NPY and noradrenaline, we explored whether the NPY-induced pressor responses were related to the activation of adrenergic mechanisms. For this purpose, we examined the cardiovascular effects of i.v. bolus injections of NPY in reserpinized rats and in animals pretreated with phenoxybenzamine. The results demonstrated that NPY produced increases in systemic blood pressure via a non-adrenergic mechanism. The NPY-induced pressor responses were blocked by nifedipine suggesting that calcium channels are linked to the activation of the cardiovascular NPY receptors.
2. Materials and methods
2.1. Blood pressure monitoring Adult (250-300 g) male Sprague-Dawley rats from our University Reproduction Laboratories were anestetized with 40 m g / k g sodium pentobarbital i.p. The trachea was cannulated; the cervical vagi were severed bilaterally. The right-side carotid artery was cannulated and connected to a Statham gauge pressure transducer which was coupled t e a Grass polygraph. For further details see Huidobro-Toro and Musacchio (1981). A small diameter polyethylene catheter was placed in the femoral vein for i.v. bolus drug administrations. The carotid catheter and the strain gauge were filled with saline solution containing 5 U / m l sodium heparin.
2.2. Drug treatments 2.2.1. Reserpine Two doses of 1 m g / k g reserpine spaced 24 h apart were administrated i.p. 48 h prior to drug testing. The efficacy of the reserpine treatment was established by determining the lowering of the systemic blood pressure as compared to that in non-reserpinized rats and by the lack of tyramineinduced hypertensive responses upon bolus injection of 0.1-3 mg tyramine per rat. 2.2.2. Phenoxybenzamine Rats were injected i.p. with 1 m g / k g of the a-adrenoceptor blocker 48 h prior to testing the effect of drugs on the blood pressure. Prior to the i.v. injections of NPY all the phenoxybenzaminetreated rats were challenged with 0,3-3 ~g adrenaline per rat. 2.2.3. Nifedipine This calcium channel blocking agent was injected i.v. (100/~g per rat, 0.3 mg/kg) 5 min prior to testing the cardiovascular effect of NPY and other pressor drugs in control and reserpinized rats. 2.3. Drugs sources Natural porcine NPY was isolated and purified from brain by Dr. K. Tatemoto according to the methodology developed by Tatemoto (1982) and Tatemoto et al. (1982). Synthetic porcine NPY was purchased commercially from Sigma Chemical Co. (St. Louis, MO) and kindly donated to our laboratory by Drs. Holaday and Musacchio. Noradrenaline hydrochloride or adrenaline bitartrate were purchased from Sigma Chemical Co. Angiotensin II was obtained as a commercial lyophylizate from Ciba (Summit, N J). Reserpine was a kind donation from Laboratorios Chile (2.5 m g / m l ampules); nifedipine was obtained from Bayer Laboratories in Brazil. Phenoxybenzamine hydrochloride was a kind contribution of Prof. J.M. Musacchio (New York University). Peptides, catecholamines and phenoxybenzamine were dissolved in saline as 1 m g / m l stock solutions. Commercial reserpine ampules (2.5 m g / m l ) contained the alkaloid dis-
35 solved in polypropylene glycol. Nifedipine was solubilized in a mixture containing water, propylene glycol, ethyl alcohol (7 : 1.5 : 1.5). Other drugs were dissolved in saline shortly prior to administration.
2.4. Statistical analysis
3o0] 300
CONTROL
12 NPY
1700 TYR RESERPINE
200 100
The results are expressed as the mean + S.E. of the various group determinations. Student's t-test was used to evaluate statistical significance; a P value less than 0.05 was considered satisfactory for comparing results obtained in drug-treated and in control, non-treated groups of rats.
1.2 NPY
1700 TYR
lmin
Fig. 2. Potentiation of the neuropeptide Y (NPY) pressor responses by reserpine treatment. Representative tracings of the pressor responses produced by 1.2 nmol N P Y and 1700 nmol tyramine in control and reserpinized rats. The N P Y and the tyramine recordings were obtained from the same animal. At the left, blood pressure calibration in m m Hg.
3. Results
3.1. Neuropeptide Y (NPY) pressor responses The i.v. administration of NPY caused a concentration-dependent increase in systolic and diastolic blood pressure without significantly modifying the heart rate. At equimolar concentrations, NPY was about as potent as noradrenaline but significantly less potent than angiotensin II (fig. 1). The bolus injection of 1.2 nmol NPY (5/~g per rat) caused a rapid increase in the systolic blood pressure of 34.5 _+ 2.9 mm Hg, and 27 + 3.8 mm Hg in the diastolic blood pressure. The NPY-in-
E ~
8o
~
60
-g
40
"1" E 100
A11(15)
, L
"'/~A(15)
o 2O
g
~/'I
J
NPY (14)
o
4 LOG pMoI I.V./ rot Fig. 1. Pressor responses induced by neuropeptide Y (NPY) angiotensin II (All) and noradrenaline (NA). Each dose-response curve was obtained in a different group of rats. The basal blood pressure of each group was approximately the same. Symbols refer to the mean value and bars to the S.E. The n u m b e r of animals used to test each compound is shown in parentheses.
duced pressor response was short-lived, lasting for about 2-3 min (fig. 2). Natural porcine NPY or the different synthetic samples of the peptide proved equally effective in causing hypertension.
3.2. Reserpine treatment Pretreatment with reserpine induced a significant reduction in systolic and diastolic blood pressure. The mean systolic blood pressure in a group of 15 control rats was 161.6 +_ 3.7 and was lowered to 130.4 _+ 5.3 mm Hg in 11 reserpinized rats (P < 0.01). Likewise, diastolic blood pressure was reduced from 120.3 +_ 2.8 to 98.1 _+ 4.0 mm Hg (P < 0.01) after the reserpine treatment. Reserpine significantly potentiated the increase in blood pressure induced by NPY. Drug treatment not only augmented the mean increase in systolic or diastolic blood pressure but also lengthened the duration of the NPY pressor responses. In addition to these effects, the reserpinized rats exhibited a substantially increased differential pressure upon injection of NPY (fig. 2). The NPY concentration-response curve in the reserpinized rats was significantly displaced to the left, and upwards (fig. 3). A parallel series of experiments showed that reserpine potentiated the noradrenaline-induced pressor responses. The specificity of the reserpine-induced supersensitivity was established by the fact that the pressor potency of angiotensin II was not significantly altered by reserpine treatment (fig. 3).
36 140
--~- CONTROL
120
--o-- R E S E R P I N E T R E A T E D
-G T E E
100 80
60 0
0r I
40
,/ /-
o
20 <1
O 0 01 0.1 1 10 n moJ N PY
0.01 0.1 1 10 n mol Noradrenaline
1 10 100 1 0 0 0 p mol A ngioteDsin~
Fig. 3. Neuropeptide Y (NPY), noradrenaline and angiotensin II concentration-response curves in control and reserpinized rats. Systolic blood pressure was recorded continuously via a catheter in~,~'ed into the carotid artery; pressor agents were injected i.v. Five reserpinized rats were tested with NPY, 7 with noradrenalih . . . . u 6 with angiotensin II. Six control rats were used for each experimental group. Symbols express the mean change in systolic blood pressure and bars indicate the S.E.
In addition, tyramine had little or ~- pressor activity in the reserpinized rats. The ,. .,e often produced a decreased of systolic and diastolic blood pressure as is illustrated by the tracing shown in fig. 2.
and 3 nmol (fig. 4). The duration of the N P Y induced rise in blood pressure in this group of rats increased but not as markedly as in the reserpinized rats (fig. 4). Adrenaline caused mainly vasodilation in these rats as opposed to the classical pressor responses in non-treated, control rats (fig. 4).
3.3. Phenoxybenzamine treatment The NPY-induced pressor responses were not antagonized after non-equilibrium ~-adrenoceptor blockade by phenoxybenzamine. On the contrary, the NPY-induced pressor effects were significantly increased following drug treatment at doses of 1.2
oo[
A
100
3.4. Effect of nifedipine 3.4.1. Non-reserpinized rats The i.v. administration of 100/~g nifedipine per
PHENOXYBENZAMINE-TREATED I
E 100
E
phenoxybenzemine~._____(5) - ' - t 80
L
6 ADR
m
0.6 NPY
~L 6o 13.
~min'
B CONTROL
200[
0
u 20 • P< 0.05
# o
100 ~ o
6 ADR
0.6 NPY
4
0
1
r mol
2
NPYIv/rat
Fig. 4. Cardiovascular effects of neuropeptide Y (NPY) in control and phenoxybenzamine-treated,rats. Left panel: Representative tracings of the pressor responses caused by the i.v. injection of 6 nmol adrenaline (ADR) and 0.6 nmol NPY in an animal pretreated with phenoxybenzamine (A) or a control rat (B). Calibration in mm Hg. Right panel: NPY concentration-response curve in control and in phenoxybenzamine-treated rats. Symbols indicate the mean increase in systolic blood pressure; bars show the S.E.
37 NIFE DIPINE ( 0 . 3 m ~ / K ~ i.v.)
200-1~
1
1000 "
0.2
0.2
0.6 n mol NPY
EE 80
"1"
~ m
6o
/•ont
r ol (4)
N .-. -6 g
20
,~.]}, ,.} . . . . . . .
{- • N I F (4)
0 0
0.5 n tool
1 2 NPY i . v . / r a t
Fig. 5. Nifedipine blockade of the neuropeptide Y (NPY)-induced pressor responses. Upperpanel: Carotid blood pressure recording from a reserpinized rat showing the pressor effect of 0.2 nmol N P Y prior to and 5 min following 0.3 m g / k g nifedipine i.v. Lower panel: N P Y concentration-response curve prior to and 5 min after the i.v. administration of 0.3 m g / k g nifedipine i.v. Symbols refer to the mean changes in systolic blood pressure, bars to the S.E. All the NPY responses obtained after nifedipine treatment showed a P value less than 0.001.
rat, produced a short-lasting drop in systemic pressure that lasted no longer than 3 min. Once the blood pressure was stabilized at a slightly lower value, the pressor effect caused by 1.2 nmol NPY was substantially reduced, but not that produced by noradrenaline or angiotensin II (data not shown). 3.4.2. Reserpinized rats In a separate group of reserpinized rats, 100/~g nifedipine proved effective in blocking the pressor response induced by NPY (fig. 5). The nifedipine antagonism was non-competitive in nature as evidenced by the fact the NPY concentration-response curve was shifted to right in a non-parallel fashion (fig. 5).
4. Discussion Based on the discovery that NPY is co-stored with catecholamines in perivascular nerves (Lundberg et al., 1983; Furness et al., 1983; Ekblad et al., 1984), it was originally thought that the pressor
activity of NPY could involve activation of the adrenergic system. However, the finding that the pressor effects of NPY were not antagonized by effective reserpine treatment must be interpreted as indicating that the peptide does not act indirectly in the cardiovascular system causing the release of catecholamines. In addition, the fact that phenoxybenzamine does not reduce the NPY-induced increase in systemic blood pressure supports the notion that NPY does not activate the adrenergic receptors directly. Based on these findings it appears reasonable to postulate that the NPY pressor responses are due to the activation of a non-adrenergic mechanism. In this context, it is likely that the peptide occupies NPY receptors distributed on the vascular tree. Recent biochemical evidence documents the existence of selective NPY binding sites (Unden et al., 1984). Much to our surprise, the present results demonstrate a significant potentiation of the NPY cardiovascular responses after reserpinized or prolonged a-adrenergic blockade. The mechanism of the potentiation is not yet clear. It is well documented that the reserpine-induced catecholamines depletion of the noradrenergic vascular nerves caused smooth muscle supersensitivity, apparently due to increased a-adrenoceptor density (Weiner, 1980; Huidobro-Toro and Musacchio, 1981). Likewise, since reserpine also reduces NPY-like immunoreactivity of perivascular nerve fibers (Furness et al., 1983; Emson and De Quidt 1984) it is our hypothesis that the vascular membrane becomes supersensitive to NPY because of an increase in the number or the affinity of the NPY receptors. It is evident that the reserpine or phenoxybenzamine interaction is complex in nature, since in addition to the maximal NPY response being increased the duration of the NPY pressor effects is significantly prolonged. It is then possible that in addition to a change in receptor properties, reserpine and phenoxybenzamine may also alter NPY metabolism or modify a postsynaptic mechanism favoring muscle contractility as suggested by Carrier and Jurevics (1973). S i n c e NPY activity appears not to be adrenergic in nature, we explored next whether the NPY responses were sensitive to nifedipine, a blocker of the voltage-sensitive calcium channels. Edvinsson
38
et al. (1983) demonstrated that the NPY-induced vasoconstriction of isolated cerebral arteries was resistant to adrenergic or serotonergic blockers but antagonized by massive concentrations of diltiazero. The present results demonstrate that nifedipine is relatively potent to block the NPY pressor responses in both control and reserpinized rats. In addition, nifedipine showed a certain selectivity in blocking the NPY responses to a larger extent than those to noradrenaline or angiotensin II. This result is interpreted as linking the activation of the NPY receptors to the influx of calcium via channels sensitive to the dihydropyridine compounds. In the pithed rat, where the i.v. administration of NPY also causes pressor responses via a peripheral mechanism, the effect was also markedly antagonized by calcium channel blocking agents (C. DahlOf, personal communication). Part of the NPY-induced pressor responses may be explained from a physiological point of view by an increased peripheral vascular tone. In addition, NPY may also increase inotropism (Lundberg et al., 1984). The most interesting finding in terms of a physiological role of NPY in the control of circulation is derived from studies with isolated blood vessels. Although NPY produces vasoconstriction in vitro, indicating a peripheral role at the vascular level (Ekblad et al., 1984), the peptide substantially potentiated the vascular contractions induced by noradrenaline (Ekblad et al., 1984; Edvinsson et al., 1984). The basis of this synergism is not yet established, but gives new insight into the functinal aspects of co-transmission and on the understanding of the physiology of NPY in health and disease. However, matters are more complex, since in addition to a peripheral action, the intracerebral administration of NPY causes hypotension (Fuxe et al., 1983). In conclusion, NPY causes pressor responses via a non-adrenergic ,mechanism. Reserpine or phenoxybenzamine substantially potentiate NPY pressor activity. The increase in blood pressure is likely to be due in part to an increase in vascular resistance via the activation of NPY receptors apparently located at the effector level and linked to the activation of calcium channels.
Acknowledgements We are grateful to Mr. M. Aguirre from Laboratories Chile for the ampules of reserpine and to Drs. J. Holaday and J.M. Musacchio for providing us with the commercial samples of synthetic porcine NPY. Thanks are due to Mr. R. Miranda for artistic contributions and Mrs. M. Sagredo for secretarial help. This work was supported in part by grants from the Gildemeister Foundation (Santiago, Chile) and DIUC Grant 58/84 from the Catholic University of Chile.
References Carrier, O. and H.A. Jurevics, 1973, The role of calcium in 'non specific' supersensitivity of vascular muscle, J. Pharmacol. Exp. Ther. 184, 81. Corder, R., P.C. Emson and P.J. Lowry, 1984, Purification and characterization of human neuropeptide Y from adrenalmedullary phaeochromocytoma tissue, Biochem. J. 219, 699. Edvinsson, L., E. Ekblad, R. Hb.kanson and C. Wahlestedt, 1984, Neuropeptide Y potentiates the effect of various vasoconstrictor agents on rabbit blood vessels, Br. J. Pharmacol. 83, 519. Edvinsson, L., P. Emson, J. McCulloch, K. Tatemoto and R. Uddman, 1983, Neuropeptide Y: cerebrovascular innervation and vasomotor effects in the cat, Neurosci. Lett. 43, 79. Ekblad, E., L. Edvinsson, C. Wahlestedt, R. Uddman, R. HS.kanson and F. Sundler, 1984, Neuropeptide Y co-exists and co-operates with noradrenaline in perivascular nerve fibers, Reg. Pept. 8, 225. Emson, P.C., R. Corder and P.J. Lowry, 1984, Demonstration of a neuropeptide Y-like immunoreactivity in human phaeochromocytoma extracts. Reg. Pept. 8, 89. Emson, P.C. and M.E. De Quidt, 1984, NPY a new member of the pancreatic polypeptide family, Trends Neurosci. 7, 31. Furness, J.B., M. Costa, P.C. Emson, R. Hb,kanson. E. Moghimzadeh, F. Sundler, I.L. Taylor and R.E. Chance, 1983, Distribution, pathways and reactions to drug treatment of nerves to neuropeptide Y and pancreatic polypeptide-like immunoreactivity in the guinea-pig digestive tract, Cell Tissue Res. 234, 71. Fuxe, K., L. Agnati, A. Harfstrand, I. Zini, K. Tatemoto, E.M. Pich, T. HOkfelt, V. Mutt and L. Terenius, 1983, Central administration of NPY induces hypotension, bradypnea and EEG synchronization in the rat, Acta Physiol. Scand. 118, 189. Gregory, R.A.. 1982, Regulatory peptides of gut brain, Br. Med. Bull. 38, 219. Gu, J., T.E. Adrian, K. Tatemoto, J.M. Polak, J.M. Allen and S.R. Bloom, 1983, Neuropeptide tyrosine (NPY): A major cardiac neuropeptide, Lancet 1, 1008. HSkfelt, T., J.M. Lundberg, H: Lagercrantz, K. Tatemoto, V. Mutt, J. Lindberg, L. Terenius, B.J. Everitt, K. Fuxe, L. Agnati and M. Goldstein, 1983, Occurrence of neuroD
39 peptide Y (NPY)-like immunoreactivity in catecholamine neurons in the human medulla oblongata, Neurosci. Lett. 36, 217. Huidobro-Toro, J.P. and J.M. Musacchio, 1981, Naloxone reversal of insulin-induced hypotension in reserpine pretreated rats, Arch. Int. Pharmacodyn. Ther. 251,310. Iversen, L.L., 1984, Amino acids and peptides: fast and slow chemical signals in the nervous system?, Proc. Roy. Soc. London B 221,245. Lundberg, J.M. and T. HOkfelt, 1983, Co-existence of peptides and classical neurotransmitters, Trends Neurosci. 6. 325. Lundberg, J.M., X.Y. Hua and A. Franco-Cereceda, 1983, Effects of NPY on mechanical activity and neurotransmission in the heart, vas deferens and urinary bladder of the guinea pig, Acta Physiol. Scand. 121,325. Lundberg, J.M. and K. Tatemoto, 1982, Vascular effects of the peptides PYY and PHI: comparison with APP and VIP, European J. Pharmacol. 83, 143. Lundberg, J.M., L. Terenius, T. HOkfelt and M. Goldstein, 1983, High levels of neuropeptide Y in peripheral noradrenergic neurons in various mammals including man, Neurosci. Lett. 42, 167. Lundberg, J.M., L. Terenius, T. H6kfelt, C.R. Marling, K.
Tatemoto, V. Mutt, J.M. Polak, S.R. Bloom and M. Goldstein, 1982, Neuropeptide Y (NPY)-like immunoreactivity in peripheral noradrenergic neurons and effectors of NPY on sympathetic function, Acta Physiol. Scand. 166, 477. Tatemoto, K., 1982, Neuropeptide Y: complete amino acid sequence of the brain peptide. Proc. Natl. Acad. Sci. U.S.A. 79, 5485. Tatemoto, K., M. Carlquist and V. Mutt, 1982, Neuropeptide Y - a novel brain peptide with structural similarities to peptide YY and pancreatic polypeptide, Nature (London) 296, 659. Tatemoto, K., Siimesmaa, H. J6rnvall, J.M. Allen, J.M. Polak, S.R. Bloom and V. Mutt, 1985, Isolation and characterization of neuropeptide Y from porcine intestine, FEBS Lett. 179, 181. Unden, A., K. Tatemoto, V. Mutt and T. Bartfai, 1984, Neuropeptide Y receptor in rat brain, European J. Biochem. 145, 525. Weiner, N., 1980, Drugs that inhibit adrenergic nerves and block adrenergic receptors, in: The pharmacological Basis of Therapeutics, eds. A. Goodman Gilman, L.S. Goodman and A. Gilman (McMillan Publishing Co., New York) p. 176.
33
N E U R O P E P T I D E Y-INDUCED P R E S S O R R E S P O N S E S : A C T I V A T I O N OF A N O N - A D R E N E R G I C M E C H A N I S M , P O T E N T I A T I O N BY R E S E R P I N E AND BLOCKADE BY N1FEDIPINE YAEKO MABE, KAZUHIKOTATEMOTO * and J. PABLO HUIDOBRO-TORO** Laboratory of Pharmacology, Department of Physiological Sciences, Faculty of Biological Sciences, Catholic University"of Chile, Casilla 114-D, Santiago I, Chile and * Nancy Pritzker Laboratory, Department of Psychiatry and Behavioral Sciences, Stanford University, School of Medicine, Palo Alto, CA, U.S.A.
Received 28 March 1985, revised MS received 12 June 1985, accepted 5 July 1985
Y. MABE, K. TATEMOTO and J.P. HUIDOBRO-TORO, Neuropeptide Y-induced pressor responses: activation of a non-adrenergic mechanism, potentiation by reserpine and blockade by nifedipine, European J. Pharmacol. 116 (1985) 33-39. Intravenous administration of neuropeptide Y (NPY) to pentobarbital anesthetized rats produced a short-lasting concentration-dependent increase in systolic and diastolic blood pressure. Pretreatment of rats with 2 mg/kg reserpine potentiated the NPY-induced pressor responses causing a leftward shift of the NPY concentration-response curve. In addition, reserpinization lengthened the duration of the NPY pressor effects. Reserpine also potentiated the noradrenaline-induced pressor effect but not that caused by angiotensin II. The NPY-induced increase in blood pressure was not antagonized by phenoxybenzamine. On the contrary, some degree Of potentiation was observed, particularly with the larger doses of NPY. The NPY pressor responses were reduced by nifedipine in control and in reserpinized rats. The results demonstrate that the NPY-induced pressor responses were not related to adrenergic mechanisms. NPY may activate calcium channels in the cardiovascular system to promote an influx of calcium, causing peripheral vasoconstriction. Phenoxybenzamine al-receptor blockade Neuropeptide Y-nifedipine interaction
Cardiovascular neuropeptide Y receptors Neuropeptide Y pressor responses
1. I n t r o d u c t i o n
Neuropeptide Y (NPY) is a 36 amino-acid peptide isolated from porcine brain and chemically characterized only 3 years ago (Tatemoto et al., 1982; Tatemoto, 1982). This peptide is now known to be present in the gut (Tatemoto et al., 1985) and in a variety of peripheral tissues including the heart, the spleen, reproductive organs, the peripheral vascular system, etc. (Lundberg et al., 1982; 1983; Gu et al., 1983; Furness et al., 1983). Furthermore, Emson et al. (1984) and Corder et al. (1984) purified and characterized NPY from hu** To whom all correspondence should be addressed at the Catholic University of Chile. 0014-2999/85/$03.30 © 1985 Elsevier Science Publishers B.V.
Reserpine treatment
man phaeochromocytoma tissues establishing the existence of the NPY in the human adrenal gland. The composition of human NPY is almost identical to that of N P Y derived from porcine materials. Thus, this compound constitutes another example of the growing family of gut-brain neuroregulatory peptides (Gregory, 1982; Iversen, 1984). A most interesting feature about this peptide is its apparent association with catecholamines. Lundberg et al. (1982) demonstrated that in the rat auricle, as well as in the vas deferens, NPY-like immunoreactivity decreased substantially following destruction of the noradrenergic nerve endings with 6-hydroxydopamine. A more direct approach using immunofluorescence histochemistry established that neurons in the human medulla ob-
34 longata contained both NPY-like immunoreactivity and tyrosine hydroxylase immunoreactivity (H6kfelt et al., 1983). In addition, Ekblad et al. (1984) used double immunohistochemical staining to demonstrate the co-existence of NPY-like immunoreactivity with dopamine /~-hydroxylase in perivascular nerve fibers. In view of the fact that NPY is distributed extensively along the lining of the peripheral vascular system, and that the local intra-arterial infusion of the peptide causes a reduction in the blood flow of the cat submandibular gland (Lundberg and Tatemoto, 1982: Lundberg and H6kfelt, 1983; Lundberg et al., 1984), we thought it of considerable physiological interest to explore whether NPY would modify systemic blood pressure. In addition, considering the close anatomical location of NPY and noradrenaline, we explored whether the NPY-induced pressor responses were related to the activation of adrenergic mechanisms. For this purpose, we examined the cardiovascular effects of i.v. bolus injections of NPY in reserpinized rats and in animals pretreated with phenoxybenzamine. The results demonstrated that NPY produced increases in systemic blood pressure via a non-adrenergic mechanism. The NPY-induced pressor responses were blocked by nifedipine suggesting that calcium channels are linked to the activation of the cardiovascular NPY receptors.
2. Materials and methods
2.1. Blood pressure monitoring Adult (250-300 g) male Sprague-Dawley rats from our University Reproduction Laboratories were anestetized with 40 m g / k g sodium pentobarbital i.p. The trachea was cannulated; the cervical vagi were severed bilaterally. The right-side carotid artery was cannulated and connected to a Statham gauge pressure transducer which was coupled t e a Grass polygraph. For further details see Huidobro-Toro and Musacchio (1981). A small diameter polyethylene catheter was placed in the femoral vein for i.v. bolus drug administrations. The carotid catheter and the strain gauge were filled with saline solution containing 5 U / m l sodium heparin.
2.2. Drug treatments 2.2.1. Reserpine Two doses of 1 m g / k g reserpine spaced 24 h apart were administrated i.p. 48 h prior to drug testing. The efficacy of the reserpine treatment was established by determining the lowering of the systemic blood pressure as compared to that in non-reserpinized rats and by the lack of tyramineinduced hypertensive responses upon bolus injection of 0.1-3 mg tyramine per rat. 2.2.2. Phenoxybenzamine Rats were injected i.p. with 1 m g / k g of the a-adrenoceptor blocker 48 h prior to testing the effect of drugs on the blood pressure. Prior to the i.v. injections of NPY all the phenoxybenzaminetreated rats were challenged with 0,3-3 ~g adrenaline per rat. 2.2.3. Nifedipine This calcium channel blocking agent was injected i.v. (100/~g per rat, 0.3 mg/kg) 5 min prior to testing the cardiovascular effect of NPY and other pressor drugs in control and reserpinized rats. 2.3. Drugs sources Natural porcine NPY was isolated and purified from brain by Dr. K. Tatemoto according to the methodology developed by Tatemoto (1982) and Tatemoto et al. (1982). Synthetic porcine NPY was purchased commercially from Sigma Chemical Co. (St. Louis, MO) and kindly donated to our laboratory by Drs. Holaday and Musacchio. Noradrenaline hydrochloride or adrenaline bitartrate were purchased from Sigma Chemical Co. Angiotensin II was obtained as a commercial lyophylizate from Ciba (Summit, N J). Reserpine was a kind donation from Laboratorios Chile (2.5 m g / m l ampules); nifedipine was obtained from Bayer Laboratories in Brazil. Phenoxybenzamine hydrochloride was a kind contribution of Prof. J.M. Musacchio (New York University). Peptides, catecholamines and phenoxybenzamine were dissolved in saline as 1 m g / m l stock solutions. Commercial reserpine ampules (2.5 m g / m l ) contained the alkaloid dis-
35 solved in polypropylene glycol. Nifedipine was solubilized in a mixture containing water, propylene glycol, ethyl alcohol (7 : 1.5 : 1.5). Other drugs were dissolved in saline shortly prior to administration.
2.4. Statistical analysis
3o0] 300
CONTROL
12 NPY
1700 TYR RESERPINE
200 100
The results are expressed as the mean + S.E. of the various group determinations. Student's t-test was used to evaluate statistical significance; a P value less than 0.05 was considered satisfactory for comparing results obtained in drug-treated and in control, non-treated groups of rats.
1.2 NPY
1700 TYR
lmin
Fig. 2. Potentiation of the neuropeptide Y (NPY) pressor responses by reserpine treatment. Representative tracings of the pressor responses produced by 1.2 nmol N P Y and 1700 nmol tyramine in control and reserpinized rats. The N P Y and the tyramine recordings were obtained from the same animal. At the left, blood pressure calibration in m m Hg.
3. Results
3.1. Neuropeptide Y (NPY) pressor responses The i.v. administration of NPY caused a concentration-dependent increase in systolic and diastolic blood pressure without significantly modifying the heart rate. At equimolar concentrations, NPY was about as potent as noradrenaline but significantly less potent than angiotensin II (fig. 1). The bolus injection of 1.2 nmol NPY (5/~g per rat) caused a rapid increase in the systolic blood pressure of 34.5 _+ 2.9 mm Hg, and 27 + 3.8 mm Hg in the diastolic blood pressure. The NPY-in-
E ~
8o
~
60
-g
40
"1" E 100
A11(15)
, L
"'/~A(15)
o 2O
g
~/'I
J
NPY (14)
o
4 LOG pMoI I.V./ rot Fig. 1. Pressor responses induced by neuropeptide Y (NPY) angiotensin II (All) and noradrenaline (NA). Each dose-response curve was obtained in a different group of rats. The basal blood pressure of each group was approximately the same. Symbols refer to the mean value and bars to the S.E. The n u m b e r of animals used to test each compound is shown in parentheses.
duced pressor response was short-lived, lasting for about 2-3 min (fig. 2). Natural porcine NPY or the different synthetic samples of the peptide proved equally effective in causing hypertension.
3.2. Reserpine treatment Pretreatment with reserpine induced a significant reduction in systolic and diastolic blood pressure. The mean systolic blood pressure in a group of 15 control rats was 161.6 +_ 3.7 and was lowered to 130.4 _+ 5.3 mm Hg in 11 reserpinized rats (P < 0.01). Likewise, diastolic blood pressure was reduced from 120.3 +_ 2.8 to 98.1 _+ 4.0 mm Hg (P < 0.01) after the reserpine treatment. Reserpine significantly potentiated the increase in blood pressure induced by NPY. Drug treatment not only augmented the mean increase in systolic or diastolic blood pressure but also lengthened the duration of the NPY pressor responses. In addition to these effects, the reserpinized rats exhibited a substantially increased differential pressure upon injection of NPY (fig. 2). The NPY concentration-response curve in the reserpinized rats was significantly displaced to the left, and upwards (fig. 3). A parallel series of experiments showed that reserpine potentiated the noradrenaline-induced pressor responses. The specificity of the reserpine-induced supersensitivity was established by the fact that the pressor potency of angiotensin II was not significantly altered by reserpine treatment (fig. 3).
36 140
--~- CONTROL
120
--o-- R E S E R P I N E T R E A T E D
-G T E E
100 80
60 0
0r I
40
,/ /-
o
20 <1
O 0 01 0.1 1 10 n moJ N PY
0.01 0.1 1 10 n mol Noradrenaline
1 10 100 1 0 0 0 p mol A ngioteDsin~
Fig. 3. Neuropeptide Y (NPY), noradrenaline and angiotensin II concentration-response curves in control and reserpinized rats. Systolic blood pressure was recorded continuously via a catheter in~,~'ed into the carotid artery; pressor agents were injected i.v. Five reserpinized rats were tested with NPY, 7 with noradrenalih . . . . u 6 with angiotensin II. Six control rats were used for each experimental group. Symbols express the mean change in systolic blood pressure and bars indicate the S.E.
In addition, tyramine had little or ~- pressor activity in the reserpinized rats. The ,. .,e often produced a decreased of systolic and diastolic blood pressure as is illustrated by the tracing shown in fig. 2.
and 3 nmol (fig. 4). The duration of the N P Y induced rise in blood pressure in this group of rats increased but not as markedly as in the reserpinized rats (fig. 4). Adrenaline caused mainly vasodilation in these rats as opposed to the classical pressor responses in non-treated, control rats (fig. 4).
3.3. Phenoxybenzamine treatment The NPY-induced pressor responses were not antagonized after non-equilibrium ~-adrenoceptor blockade by phenoxybenzamine. On the contrary, the NPY-induced pressor effects were significantly increased following drug treatment at doses of 1.2
oo[
A
100
3.4. Effect of nifedipine 3.4.1. Non-reserpinized rats The i.v. administration of 100/~g nifedipine per
PHENOXYBENZAMINE-TREATED I
E 100
E
phenoxybenzemine~._____(5) - ' - t 80
L
6 ADR
m
0.6 NPY
~L 6o 13.
~min'
B CONTROL
200[
0
u 20 • P< 0.05
# o
100 ~ o
6 ADR
0.6 NPY
4
0
1
r mol
2
NPYIv/rat
Fig. 4. Cardiovascular effects of neuropeptide Y (NPY) in control and phenoxybenzamine-treated,rats. Left panel: Representative tracings of the pressor responses caused by the i.v. injection of 6 nmol adrenaline (ADR) and 0.6 nmol NPY in an animal pretreated with phenoxybenzamine (A) or a control rat (B). Calibration in mm Hg. Right panel: NPY concentration-response curve in control and in phenoxybenzamine-treated rats. Symbols indicate the mean increase in systolic blood pressure; bars show the S.E.
37 NIFE DIPINE ( 0 . 3 m ~ / K ~ i.v.)
200-1~
1
1000 "
0.2
0.2
0.6 n mol NPY
EE 80
"1"
~ m
6o
/•ont
r ol (4)
N .-. -6 g
20
,~.]}, ,.} . . . . . . .
{- • N I F (4)
0 0
0.5 n tool
1 2 NPY i . v . / r a t
Fig. 5. Nifedipine blockade of the neuropeptide Y (NPY)-induced pressor responses. Upperpanel: Carotid blood pressure recording from a reserpinized rat showing the pressor effect of 0.2 nmol N P Y prior to and 5 min following 0.3 m g / k g nifedipine i.v. Lower panel: N P Y concentration-response curve prior to and 5 min after the i.v. administration of 0.3 m g / k g nifedipine i.v. Symbols refer to the mean changes in systolic blood pressure, bars to the S.E. All the NPY responses obtained after nifedipine treatment showed a P value less than 0.001.
rat, produced a short-lasting drop in systemic pressure that lasted no longer than 3 min. Once the blood pressure was stabilized at a slightly lower value, the pressor effect caused by 1.2 nmol NPY was substantially reduced, but not that produced by noradrenaline or angiotensin II (data not shown). 3.4.2. Reserpinized rats In a separate group of reserpinized rats, 100/~g nifedipine proved effective in blocking the pressor response induced by NPY (fig. 5). The nifedipine antagonism was non-competitive in nature as evidenced by the fact the NPY concentration-response curve was shifted to right in a non-parallel fashion (fig. 5).
4. Discussion Based on the discovery that NPY is co-stored with catecholamines in perivascular nerves (Lundberg et al., 1983; Furness et al., 1983; Ekblad et al., 1984), it was originally thought that the pressor
activity of NPY could involve activation of the adrenergic system. However, the finding that the pressor effects of NPY were not antagonized by effective reserpine treatment must be interpreted as indicating that the peptide does not act indirectly in the cardiovascular system causing the release of catecholamines. In addition, the fact that phenoxybenzamine does not reduce the NPY-induced increase in systemic blood pressure supports the notion that NPY does not activate the adrenergic receptors directly. Based on these findings it appears reasonable to postulate that the NPY pressor responses are due to the activation of a non-adrenergic mechanism. In this context, it is likely that the peptide occupies NPY receptors distributed on the vascular tree. Recent biochemical evidence documents the existence of selective NPY binding sites (Unden et al., 1984). Much to our surprise, the present results demonstrate a significant potentiation of the NPY cardiovascular responses after reserpinized or prolonged a-adrenergic blockade. The mechanism of the potentiation is not yet clear. It is well documented that the reserpine-induced catecholamines depletion of the noradrenergic vascular nerves caused smooth muscle supersensitivity, apparently due to increased a-adrenoceptor density (Weiner, 1980; Huidobro-Toro and Musacchio, 1981). Likewise, since reserpine also reduces NPY-like immunoreactivity of perivascular nerve fibers (Furness et al., 1983; Emson and De Quidt 1984) it is our hypothesis that the vascular membrane becomes supersensitive to NPY because of an increase in the number or the affinity of the NPY receptors. It is evident that the reserpine or phenoxybenzamine interaction is complex in nature, since in addition to the maximal NPY response being increased the duration of the NPY pressor effects is significantly prolonged. It is then possible that in addition to a change in receptor properties, reserpine and phenoxybenzamine may also alter NPY metabolism or modify a postsynaptic mechanism favoring muscle contractility as suggested by Carrier and Jurevics (1973). S i n c e NPY activity appears not to be adrenergic in nature, we explored next whether the NPY responses were sensitive to nifedipine, a blocker of the voltage-sensitive calcium channels. Edvinsson
38
et al. (1983) demonstrated that the NPY-induced vasoconstriction of isolated cerebral arteries was resistant to adrenergic or serotonergic blockers but antagonized by massive concentrations of diltiazero. The present results demonstrate that nifedipine is relatively potent to block the NPY pressor responses in both control and reserpinized rats. In addition, nifedipine showed a certain selectivity in blocking the NPY responses to a larger extent than those to noradrenaline or angiotensin II. This result is interpreted as linking the activation of the NPY receptors to the influx of calcium via channels sensitive to the dihydropyridine compounds. In the pithed rat, where the i.v. administration of NPY also causes pressor responses via a peripheral mechanism, the effect was also markedly antagonized by calcium channel blocking agents (C. DahlOf, personal communication). Part of the NPY-induced pressor responses may be explained from a physiological point of view by an increased peripheral vascular tone. In addition, NPY may also increase inotropism (Lundberg et al., 1984). The most interesting finding in terms of a physiological role of NPY in the control of circulation is derived from studies with isolated blood vessels. Although NPY produces vasoconstriction in vitro, indicating a peripheral role at the vascular level (Ekblad et al., 1984), the peptide substantially potentiated the vascular contractions induced by noradrenaline (Ekblad et al., 1984; Edvinsson et al., 1984). The basis of this synergism is not yet established, but gives new insight into the functinal aspects of co-transmission and on the understanding of the physiology of NPY in health and disease. However, matters are more complex, since in addition to a peripheral action, the intracerebral administration of NPY causes hypotension (Fuxe et al., 1983). In conclusion, NPY causes pressor responses via a non-adrenergic ,mechanism. Reserpine or phenoxybenzamine substantially potentiate NPY pressor activity. The increase in blood pressure is likely to be due in part to an increase in vascular resistance via the activation of NPY receptors apparently located at the effector level and linked to the activation of calcium channels.
Acknowledgements We are grateful to Mr. M. Aguirre from Laboratories Chile for the ampules of reserpine and to Drs. J. Holaday and J.M. Musacchio for providing us with the commercial samples of synthetic porcine NPY. Thanks are due to Mr. R. Miranda for artistic contributions and Mrs. M. Sagredo for secretarial help. This work was supported in part by grants from the Gildemeister Foundation (Santiago, Chile) and DIUC Grant 58/84 from the Catholic University of Chile.
References Carrier, O. and H.A. Jurevics, 1973, The role of calcium in 'non specific' supersensitivity of vascular muscle, J. Pharmacol. Exp. Ther. 184, 81. Corder, R., P.C. Emson and P.J. Lowry, 1984, Purification and characterization of human neuropeptide Y from adrenalmedullary phaeochromocytoma tissue, Biochem. J. 219, 699. Edvinsson, L., E. Ekblad, R. Hb.kanson and C. Wahlestedt, 1984, Neuropeptide Y potentiates the effect of various vasoconstrictor agents on rabbit blood vessels, Br. J. Pharmacol. 83, 519. Edvinsson, L., P. Emson, J. McCulloch, K. Tatemoto and R. Uddman, 1983, Neuropeptide Y: cerebrovascular innervation and vasomotor effects in the cat, Neurosci. Lett. 43, 79. Ekblad, E., L. Edvinsson, C. Wahlestedt, R. Uddman, R. HS.kanson and F. Sundler, 1984, Neuropeptide Y co-exists and co-operates with noradrenaline in perivascular nerve fibers, Reg. Pept. 8, 225. Emson, P.C., R. Corder and P.J. Lowry, 1984, Demonstration of a neuropeptide Y-like immunoreactivity in human phaeochromocytoma extracts. Reg. Pept. 8, 89. Emson, P.C. and M.E. De Quidt, 1984, NPY a new member of the pancreatic polypeptide family, Trends Neurosci. 7, 31. Furness, J.B., M. Costa, P.C. Emson, R. Hb,kanson. E. Moghimzadeh, F. Sundler, I.L. Taylor and R.E. Chance, 1983, Distribution, pathways and reactions to drug treatment of nerves to neuropeptide Y and pancreatic polypeptide-like immunoreactivity in the guinea-pig digestive tract, Cell Tissue Res. 234, 71. Fuxe, K., L. Agnati, A. Harfstrand, I. Zini, K. Tatemoto, E.M. Pich, T. HOkfelt, V. Mutt and L. Terenius, 1983, Central administration of NPY induces hypotension, bradypnea and EEG synchronization in the rat, Acta Physiol. Scand. 118, 189. Gregory, R.A.. 1982, Regulatory peptides of gut brain, Br. Med. Bull. 38, 219. Gu, J., T.E. Adrian, K. Tatemoto, J.M. Polak, J.M. Allen and S.R. Bloom, 1983, Neuropeptide tyrosine (NPY): A major cardiac neuropeptide, Lancet 1, 1008. HSkfelt, T., J.M. Lundberg, H: Lagercrantz, K. Tatemoto, V. Mutt, J. Lindberg, L. Terenius, B.J. Everitt, K. Fuxe, L. Agnati and M. Goldstein, 1983, Occurrence of neuroD
39 peptide Y (NPY)-like immunoreactivity in catecholamine neurons in the human medulla oblongata, Neurosci. Lett. 36, 217. Huidobro-Toro, J.P. and J.M. Musacchio, 1981, Naloxone reversal of insulin-induced hypotension in reserpine pretreated rats, Arch. Int. Pharmacodyn. Ther. 251,310. Iversen, L.L., 1984, Amino acids and peptides: fast and slow chemical signals in the nervous system?, Proc. Roy. Soc. London B 221,245. Lundberg, J.M. and T. HOkfelt, 1983, Co-existence of peptides and classical neurotransmitters, Trends Neurosci. 6. 325. Lundberg, J.M., X.Y. Hua and A. Franco-Cereceda, 1983, Effects of NPY on mechanical activity and neurotransmission in the heart, vas deferens and urinary bladder of the guinea pig, Acta Physiol. Scand. 121,325. Lundberg, J.M. and K. Tatemoto, 1982, Vascular effects of the peptides PYY and PHI: comparison with APP and VIP, European J. Pharmacol. 83, 143. Lundberg, J.M., L. Terenius, T. HOkfelt and M. Goldstein, 1983, High levels of neuropeptide Y in peripheral noradrenergic neurons in various mammals including man, Neurosci. Lett. 42, 167. Lundberg, J.M., L. Terenius, T. H6kfelt, C.R. Marling, K.
Tatemoto, V. Mutt, J.M. Polak, S.R. Bloom and M. Goldstein, 1982, Neuropeptide Y (NPY)-like immunoreactivity in peripheral noradrenergic neurons and effectors of NPY on sympathetic function, Acta Physiol. Scand. 166, 477. Tatemoto, K., 1982, Neuropeptide Y: complete amino acid sequence of the brain peptide. Proc. Natl. Acad. Sci. U.S.A. 79, 5485. Tatemoto, K., M. Carlquist and V. Mutt, 1982, Neuropeptide Y - a novel brain peptide with structural similarities to peptide YY and pancreatic polypeptide, Nature (London) 296, 659. Tatemoto, K., Siimesmaa, H. J6rnvall, J.M. Allen, J.M. Polak, S.R. Bloom and V. Mutt, 1985, Isolation and characterization of neuropeptide Y from porcine intestine, FEBS Lett. 179, 181. Unden, A., K. Tatemoto, V. Mutt and T. Bartfai, 1984, Neuropeptide Y receptor in rat brain, European J. Biochem. 145, 525. Weiner, N., 1980, Drugs that inhibit adrenergic nerves and block adrenergic receptors, in: The pharmacological Basis of Therapeutics, eds. A. Goodman Gilman, L.S. Goodman and A. Gilman (McMillan Publishing Co., New York) p. 176.