BLOOD PRESSURE 1996; 5: 164- 169

Characterization of Neuropeptide Y Receptors Mediating Contraction, Potentiation and Inhibition of Relaxation

Nilsson T, You J, Sun X, Hedner T, Edvinsson L. ClinrLlctc'r.i=utiorl of' t i cwopept i t i~ Y recc~ptor.P tnc)(iilititig imtructioti , potentiiition rind inhihilion c?J'rc.lcr.\-rrtioti. Blood Pressure 1996; 5: 164- 169.

I n addition to its direct vasoconstrictive eflect, neuropeptide Y (NPY) potentiates noradrenaline- (NA) induced contraction and inhibits acetylcholine- (ACh) induced relaxation: The aim of the present study was to elucidate the NPY receptor subtypes responsible for mediating these three responses. NPY, peptide YY (PYY) and proj4NPY (a NPY Y , receptor agonist) induced equipotent and equally strong concentration-dependent contractions of guinea pig basilar arteries. NPY 13.3h (a NPY Y2 receptor agonist), however, caused only weak contraction with significantly lower potency. The NPY-induced contraction was significantly inhibited by the selective NPY Y I receptor antagonist BIBP3226 ( I pM). NPY, PYY and proj4NNPY but not NPYli.j6 significantly potentiated the NA-induced contraction in guinea pig mesenteric arteries. The potentiation was significantly inhibited by BIBP3226 ( I pM). In precontracted guinea pig basilar arteries, ACh induced a concentration-dependent relaxation which was significantly inhibited by NPY, PYY and NPY 13.3h but not by ~ ~ o ~ ~ N P Y . BIBP3226 had no significant effect on the NPY-induced inhibition of the relaxation. These results suggests that lhe NPY Y, receptors mediate the direct contraction and the potentiation of the NA-induced contraction but not the inhibition of the ACh-induced relaxation. This effect seems to be mediated by another NPY receptor subtype, presumably by the Y2 receptor, as judged from the agonist potency order. Key words: BIBP3226, cwchrul crrteries, guincw pig . tiiesrriteric cirteries, rieiiropeptide Y , p c y i t l e Y Y , p r o s 4 N P Y . N P Y1i.36.

INTRODUCTION Neuropeptide Y (NPY), a 36 amino acid peptide, is co-stored with noradrenaline (NA) in postganglionic nerves supplying arteries and veins and is released together with NA upon sympathetic nerve activation [I]. NPY contracts blood vessels in vitro [2] and in vivo [3]. Zukowska-Grojec and colleagues have recently provided the first evidence of a physiological role of NPY in regulation of vascular tone during stress in conscious rats [4].

Apart from direct vasoconstriction, NPY has the ability to potentiate the vasoconstriction evoked by NA at concentrations far below the concentrations required to evokc a direct vasoconstrictor response [5 ] . I n addition, NPY has been shown to inhibit an endothelium-dependent acetylcholine (ACh) induced relaxation [6].

NPY exerts its biological actions through at least three different receptor subtypes, Y , , Y2 and Y3 [7]. The Y I receptor has so far been cloned from rat and man [8,9]. This receptor is activated by NPY, PYY and the Y , agonist pro"4NPY [lo]. The recently cloned Y2 receptor [ I I] seems to be located mainly prejunc- tionally and is activated by NPY, PYY and N-terminal truncated fragments of NPY such as NPY13.36 [lo]. Even if bioactivity as well as binding studies indicate that the vascular NPY receptor is mainly of the Y ,

type there is also some evidence of a Yz mediated vasoconstriction [12- 141.

Furthermore, there seems to be a third receptor, Y,, which does not recognize PYY [7]. A pharmacological characterization of N P Y receptor subtypes has been difficult to perform because of the lack of potent and selective NPY antagonists [ 151. Recently, however, a non-peptide compound BIBP3226 has been developed and shown to behave as a competitive, specific and selective NPYl receptor antagonist [16, 171.

I n the present study, NPY, modified N P Y analogues and BIBP3226 were used in a n attempt to determine the NPY receptor subtypes that are responsible for the NPY-induced contraction, the potentiation of the NA-induced vasoconstriction and the inhibition of the ACh-induced vasodilatation.

METHODS Young male guinea pigs (body weight 200-250 g) were decapitated under pentobarbital anaesthesia (1 00 mg/kg). The mesenteric and basilar arteries were rapidly removed and placed in an ice-cold aerated buffer solution. The vessels were dissected free under a microscope and cut into cylindrical segments (1-2 m m long). The segments were mounted on two metal prongs, one of which was connected to a forcc

#(', 1996 Scandinavian University Press. I S S N ON03-705I RLOOD PRESSURE 1996

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displacement transducer (FT03C) and attached to a Macintosh Plus computer, and the other to a displace- ment device. The position of the holder could be changed by means of a movable unit allowing fine adjustments of vascular tension by varying the dis- tance between the metal prongs. The experiments were continuously recorded using the Macintosh software program ChartTM. The mounted specimens were immersed in temperature-controlled tissue baths con- taining a buffer solution of the following composition (mM): NaCl 119; NaHCOz 15; KCI 4.6; MgCI2 1.2; NaH2P04 1.2; CaCI2 1.5; glucose 1 1 . The buffer solu- tion was continuously gassed with 5”/0 C 0 2 in 02, giving a pH of 7.4. The mesenteric arteries were given an initial tension of 4 mN and the basilar arteries 2 mN and they were allowed to stabilize at this tension for 1 h. The contractile capacity of each vessel segment was examined by exposure to a potassium-rich (60 mM) buffer solution which had the same composi- tion as the standard solution except that some of the NaCl was exchanged for an equimolar concentration of KCI. These contractions served as internal stan- dards and were set as 100%. When two reproducible contractions had been achieved (variation less than 10%) the vessels were used for further studies.

Direct contraction was studied in the basilar artery. Concentration-response curves for NPY, PYY, proj4NPY and NPY 13.36 were obtained by adding the drug in a cumulative fashion. The NPY antagonist (BIBP3226 1 pM) was added 20min before NPY.

Potentiation experiments were performed on mesenteric arterial segments. Concentration-response curves for NA were obtained by adding the drug in a cumulative fashion. NPY, PYY, ~ ~ o ’ ~ N P Y or NPY13.36 was added 2min before NA. As controls, an equal number of matched vascular segments were used. These received NA without prior exposure to either peptide. The NPY antagonist (BIBP3226 1 pM) was added 20 min before NPY.

In order to study the relaxant activity, the basilar arterial segments were precontracted by 10 nM U46619 (a PGFt, derivate), causing a contraction which in control experiments remained stable for 45-60 min. When the precontraction was stable ACh was added in a cumulative fashion, resulting in con- centration-dependent relaxation. To study the inhibi- tion of the relaxation the peptides (NPY, PYY, ~ ~ o ~ ~ N P Y or NPY13.36, all at 30nM) were added directly before the precontraction. The NPY antago- nist BTBP3226 1 pM was added 20 min before NPY.

Results are given as a percentage of the potassium- induced contraction. Maximum effect of contraction or dilatation is expressed as E,,,,. The potency of the agonists was expressed as pD2 values (negative

logarithm of the molar concentration of agonist inducing half maximum response). The data were expressed as mean values Jr s.e. mean; n refers to the number of guinea pigs from which the vessels were collected.

Statistically significant differences were determined with Mann-Whitney U test (for unpaired observa- tions) and Wilcoxon signed-rank test (for paired obser- vations) using StatView I 1 on a Macintoch IIcx. P < 0.05 was considered significant.

The following drugs were used: NPY, PYY, ~ ~ o ~ ~ N P Y and NPY 13-16 (Auspep, Australia), BIBP3226 (a generous gift from Drs H. Doods and K . Rudolf, Dr Karl Thomae GmbH, Germany), NA, ACh and U46619 (9, I 1 -Dideoxy-1 Itr, 9tr-epoxymethano- prostaglandin F2[,) (Sigma, St. Luis, USA).

RESULTS Direct contraction NPY, PYY and ~ ~ o ~ ~ N P Y induced equipotent and equally strong concentration-dependent contractions in guinea pig basilar arteries (pDz 8.1 1 It 0.09, 8.15 Jr 0. I9 and 8.08 Jr 0.17, respectively), while NPY 13-36 only induced weak contractions with signifi- cantly (I) < 0.05) lower potency (pD2 7.25 Jr 0.35) (Fig. I ) . BIBP3226 ( 1 pM) potently inhibited the NPY-induced contraction and caused shift of the concentration-response cu

8o 1 - 1 60

a rightward ve for NPY

-10 -9 -8 -7

Peptide log (M) Fig. 1 . The contractile responses to NPY (0), PYY (O), ~ ~ o ’ ~ N P Y (B) and NPY,3.36 (0) in guinea pig basilar arteries. The results arc expressed as percentages of potas- sium-induced contraction, each point representing mean h s.e. mean of six experiments. * p < 0.05, **p < 0.01 (Mann- Whitney U-test).

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166 T. Nilsson et al.

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NA log (M)

Fig. 3 . The contractile res onse to NA in the absence (m) and

(0.1pM) (0). The results are expressed as percentages of potassium-induced contraction, each point representing mean f s.e. mean of 10 experiments. * p < 0.05, **p < 0.01 (Wilcoxon signed-rank test).

in the presence of pro3 B NPY (0.1 pM) (0) and NPY13.3h

l6O1 140

NA log (M)

Fig. 4. The contractile response to NA in the absence (m) and in the presence of NPY (0.1 pM) (0) and in the presence of both NPY (0.1 pM) and BIBP3226 ( I pM) (0). The results are expressed as percentages of potassium-induced contrac- tion, each point representing mean k s.e. mean of 6 to 10 experiments. **p < 0.01 (Wilcoxon signed-rank test).

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Neuropeptirle Y receptors in blood wsscls 161

Table I. The potentiating eflect o f N P Y , P Y Y , pro3‘NPY und NPY(3.36 (all in u concentration of’ 0.1 pA4) on the NA-induced contraction qf’guiriea pig mesmtcric urteries und the influence of’ BIBP3226 ( I p M ) on the potentiation

~ ~ _ _ _ ~ ~ - - ~~~~~~ ~~ ~ ~

PD? Ern,,, n _ _ _ ~ ~ ~ ~ ~ ~ ~ ~ _ _ _ ~ ~~ ~~. ~~ ~ _ _

NA 4.87 i 0.13 I I6 f 6 10 NA + NPY 5.12ItO.11** 127*6ns 10 NA + PYY 5.38 f O.lO** 129 5C 15 ns 8 NA + proj4NPY 5.09 3 0.16* I24 5 8 ns 10 NA + NPYl3-36 4 . 8 7 i 0 . 1 5 n s 121 f 8 n s 10 NA + NPY + BIBP3226 4.93 It 0.19 ns 1 I6 & 7 ns 6

Values represent mean 3 s.e. mean. * p < 0.05, **p < 0.01 and ns = non-significant vs NA alone (Wilcoxon signed- rank test).

potentiates NA-induced vasoconstriction and inhibits ACh-induced vasodilatation. The aim of the present study was to elucidate the NPY receptor subtypes through which NPY mediates these actions.

As has been shown before [IS], NPY and PYY induced strong concentration-dependent contractions in guinea pig basilar arteries. P ~ o ’ ~ N P Y , a NPY agonist which is supposed to retain full activity on YI receptors while being inactive at Y2 receptors [7], caused just as potent and strong contractions as NPY. NPY I an N-terminal truncated NPY agonist which potently activates the Y2 receptor but not the Y,

100 1 c k

80: 60 1 I . , . , . , . , . 1 . 1

-9 -8 -7 -6 -5 -4 -3

ACh log (M)

Fig. 5. The relaxant response to ACh in the absence (0) and in the presence of proj4NPY (30nM) (0) and NPY13.36 (30nM) (W). The results are expressed as percentages of the U466lY-induced contraction, each point representing mean 5C s.e. mean of 6 to 9 experiments. * p < 0.05 (Wilcoxon signed-rank test).

Table 11. The qfect of‘ N P Y , P Y Y , pro74NPY and NPY13.36 (all in N concentration qf 3OnM) on the ACh-inducrd reliisution q f guinea pig husilar arteries und the influence qfBIBP3226 (0.1 p M ) on the reluxu- tior1

~~~ ~~~ ~~~~~ - ~~~~~

PD2 Ernax I1

ACh 6.79 j, 0.21 Y l i 3 9 ACh + NPY 6.19iO.14* 8 7 f 4 n s 9 ACh + PYY 6.11 + 0.30* 86 f 3 ns 6 ACh + proj4NPY 6.71 i 0.20 ns 96 f 2 ns 6 ACh+NPY1j.j6 5.77 i O . 2 2 * 91 f 6ns 6 ACh+NPY +BIBP3226 6 .38f0 .18* 925211s 9

~ ~~~ ~~~

Values represent mean s.e. mean. * p < 0.05 and ns = non- significant vs ACh alone (Wilcoxon signed-rank test).

receptor [7], induced only weak contraction. Further- more, BIBP3226, a recently developed selective and highly potent NPY Y, receptor antagonist [17] potently inhibited the NPY-induced contraction. Pre- viously, BIBP3226 has shown selective binding affi- nity for NPY Y, receptors in human neuroblastoma cells, in rat cerebral cortex and has displayed highly antagonistic properties in virro against vasopressor responses evoked by NPY and Y , receptor agonists in isolated rat kidney and in vivo in pithed rats. In rat colon, a Y3 bioassay, and in rat vas deferens, a Y2 receptor bioassay, BIBP3226 failed to alter the

0 J , . I . , . , . , . , . ,

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ACh log (M)

Fig. 6. The relaxant response to ACh in the absence (m) and in the presence of NPY (30nM) (0) and in the presence of both NPY (30 nM) and BIBP3226 ( 1 p M ) (0). The results are expressed as percentages of the U46619-induced contraction, each point representing mean i s.e. mean of 9 experiments. * p < 0.05 (Wilcoxon signed-rank test).

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168 T. Nilssoii et al.

contractile effects of NPY [16, 19-21]. The result from the agonist as well as the antagonist experiments clearly indicate that the NPY Y , receptor is respon- sible for the direct contraction of cerebral arteries mediated by NPY. This agrees well with studies on human brain vessels [22,23].

As we have demonstrated previously [24], NPY significantly potentiated the NA-evoked constriction in guinea pig mesenteric arteries, causing a leftward shift of the concentration-response curve without change of maximum response. P ~ o ’ ~ N P Y and PYY mimicked the NPY response and caused a significant potentiation, while NPY 13.36 failed to potentiate the NA-induced contraction. The fact that ~ ~ o ’ ~ N P Y but not NPY 13.36 induced potentiation indicates that the potentiation is mediated by the NPY Y1 receptor. To further prove this, we used BIBP3226, which significantly inhibited the NPY-induced potentiation of the NA-induced contraction. Doods and colleagues have shown in the rat mesenteric bed that BIBP3226 antagonizes the NPY-mediated potentiation of the NA-elicted perfusion pressure [21]. This supports the view that the potentiation is mediated by the NPY Y, receptor. Furthermore, potentiation mediated by a Y3 receptor seems unlikely since PYY potentiated the NA-induced contraction as potent as NPY.

In concert with observations that some autonomic vasodilator neurons contain NPY [25], NPY has been found to inhibit vasodilatation induced by ACh, substance P and vasoactive intestinal peptide [6,26]. Thus, NPY may not only increase resistance in different vascular beds by direct effects, the increase in resistance may also be due to inhibition of the effects of the vasodilatory substances released from parasympathetic and sensory neurons. Our aim was to examine the NPY-induced inhibition of the ACh-evoked vasodilatation in guinea pig basilar arteries. As expected NPY significantly inhibited the relaxation, causing a rightward shift of the con- centration-response curve for ACh without change of maximum dilatory response. PYY and NPY 13.36

showed the same pattern, while ~ ~ o ~ ~ N P Y failed to inhibit the relaxation. These results, together with the fact that BIBP3226 was unable to inhibit the NPY-induced inhibition of the relaxation, clearly indicate that the inhibition of the ACh-induced relaxation is probably not mediated by the NPY Y, or Y3 receptors. This effect seems to be mediated by another NPY receptor, presumably the Y2 recep- tor.

In conclusion, the NPY-induced direct contraction and the potention of the NA-induced contraction seems to be mediated by the NPY Y I receptor, while the NPY-induced inhibition of the ACh-evoked

relaxation appears to be mediated by a different NPY receptor, possibly the Y2 receptor.

ACKNOWLEDGEMENTS We are grateful for the generous gift of BIBP3226 from Drs H. Doods and K. Rudolf, Dr Karl Thomae GmbH, Germany. The study was supported by the Swedish Medical Research Council (grant no. 5958) and the Medical Faculty. Lund University, Sweden.

REFERENCES 1. Fried G, Terenius L, Hokfelt T, Goldstein M. Evidence

for differential localization of noradrenaline and neuro- peptide Y (NPY) in neuronal storage vesicles isolated from rat vas deferens. J Neurosci 1985; 5: 450-5.

2. Edvinsson L, Hikanson R, Wahlestedt C, Uddman R. Effects of neuropeptide Y on the cardiovascular system. Trends Pharmacol Sci 1987; 8: 231-5.

3. Pernow J, Lundberg JM, Kaijser L. Vasoconstrictor effects in vivo and plasma disappearance rate of neuro- peptide Y in man. Life Sci 1987; 40: 47-54.

4. Zukowska-Grojec Z, Dayao EK, Karwatoska-Proko- pezuk E, Hauser GJ, Doods HN. Stress-induced mesenteric vasoconstriction in rats is mediated by neuro- peptide Y Y l receptors. Am J Physiol 1996. In press.

5. Edvinsson L, Ekblad E, Hikanson R, Wahlestedt C. Neuropeptide Y potentiates the effect of various vaso- constrictor agents on rabbit blood vessels. Br J Pharma-

6. Fallgren B, Ekblad E, Edvinsson L. Co-existence of neuropeptides and differential inhibition of vasodilator responses by neuropeptide Y in guinea pig uterine arteries. Neurosci Lett 1989; 100: 71-6.

7. Wahlestedt C, Reis DJ, Neuropeptide Y-related peptides and their receptors-are the receptors potential thera- peutic drug targets? Annu Rev Pharmacol Toxicol 1993;

8. Herzog H, Baumgartner M, Vivero C, Selbie LA, Bern- hard A, Shine J . Genomic organisation, localisation and allelic differences in the gene for the human neuropeptide Y Y I receptor. J Biol Chem 1993; 268: 7703-7.

9. Larhammar D, Blomquist AG, Yee F, Jazin E, Yo0 H, Wahlestedt C. Cloning and functional expression of human neuropeptide Y, peptide YY receptor of the Y , type. J Bio Chem 1992; 267: 10935-8.

10. Wahlestedt C, Yanaihara N, Hikanson R. Evidence for different pre- and postjunctional receptors for neuropep- tide Y and related peptides. Reg Pept 1986; 13: 307- 18.

1 1 . Rose PM, Fernandes P, Lynch JS, et al. Cloning and functional expression of a cDNA encoding a human type 2 Neuropeptide Y receptor. J Biol Chem 1995; 270:

12. Sheikh SP, Roach E, Fuhlendorff J, Williams JA. Loca- lization of Y l receptors for NPY and PYY on vascular smooth muscle cells in rat pancreas. Am J Physiol 1991;

13. Grundemar L, Morner S, Hogestiitt E, Wahlestedt C, Hikanson R. Characterization of vascular neuropeptide Y receptors. Br Pharmacol 1992; 105: 45-50.

14. Modin A, Pernow J, Lundberg JM. Evidence for two neuropeptide Y receptors mediating vasoconstriction. Eur J Pharmacol 1991; 203: 165 -71.

COI 1984; 83: 519-25.

32: 309-52.

2266 1 -4.

260: (3250-7.

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Neuropeptide Y receptors in blood vessels 169

15. Edvinsson L, Erlinge D, Sun XY, Hedner T. Neuropep- tide Y in sympathetic co-transmission: recent advances in the search for neuropeptide Y antagonists. Pharmacol Toxicol 1994; 74: 193-201.

16. Jacques D, Cadieux A, Dumont Y, Quirion R. Apparent affinity and potency of BIBP3226, a non-peptide neuro- peptide Y receptor antagonist, on purported neuropep- tide Y Y , , Y2 and Y3 receptors. Eur J Pharmacol 1995;

17. Rudolf K, Eberlein W, Engel W, et al. The first highly potent and selective non-peptide neuropeptide Y Y receptor antagonist: BIBP3226. Eur J Pharmacol 1994;

18. Fallgren B, Arlock P, Jansen I , Edvinsson L. Neuro- peptide Y in cerebrovascular function: comparison of membrane potential changes and vasomotor responses evoked by NPY and other vasoconstrictors in guinea pig basilar arteries. Neurosci Lett 1990; 114: 117-22.

19. Doods HN, Entzeroth M, Wienen W, et al. BIBP3226, a selective NPY YI receptor antagonist. Pharmacol Toxicol 1995; 76: suppl IV 76.

20. Wieland HA, Willim KD, Entzeroth M, et al. Subtype selectivity and antagonistic profile of the nonpeptide Y 1 receptor antagonist BIBP3226. J Pharmacol Exp Ther

21. Doods HN, Wienen W, Entzeroth M, et al. Pharmaco- logical characterization of the selective nonpeptide neuropeptid Y Y 1 receptor antagonist BIBP3226. J Pharmacol Exp Ther 1995; 275: 136-42.

22. Abounander R, Villemure J-G, Hamel E. Characteriza- tion of neuropeptide Y (NPY) receptors in human

278: R3-5.

271: 11-13.

1995; 275: 143-9.

cerebral arteries with selective agonists and the new Yl antagonist BIBP3226. Br J Pharmacol 1995; 116:

23. Nilsson T, Cantera L, Edvinsson L. Presence of neuro- peptide Y Y I receptors mediating vasoconstriction in human cerebral arteries. Neurosci Lett 1996; 203: 1-4.

24. Adamsson M, Fallgren B, Edvinsson L. Inhibition of neuropeptide Y-induced potentiation of norddrenahne- induced vasoconstriction by PP56 (D-myo-inositol 1,2,6- tris-phosphate). Br J Pharmacol 1992; 105: 93-6.

25. Leblanc GG, Trimmer BA, Landis SC. Neuropeptide Y- like immunoreactivity in rat cranial parasympathetic neurons: coexistence with vasoactive intestinal peptide and choline acetyltransferase. Proc Natl Acad Sci USA

26. Edvinsson L, Adamsson M. Neuropeptide Y inhibits relaxation of guinea pig cerebral, coronary, and uterine arteries: Blockade by D-myo-inosito~-I ,2,6-triphosfate. J Cardiovasc Pharmacol 1992; 20: 466-72.

2245 - 50.

1987; 84: 3511-5.

Submitted February 27, 1995: accepted August 17, 1995

Address for correspondence: Torun Nilsson Department of Cell Biology 1 EB blocket Lund University Hospital S-22185 Lund Sweden Phone: +46 46 173545 Fax: +46 46 137277

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