neuropeptide y stimulates dna synthesis in human vascular smooth muscle cells through neuropeptide y...

BRIEF REPORT / RAPPORT BREF

Neuropeptide Y stimulates DNA synthesis inhuman vascular smooth muscle cells throughneuropeptide Y Y1 receptors

T. Nilsson and L. Edvinsson

Abstract: We investigated the mitogenic effect, measured as [3H]thymidine incorporation, of neuropeptide Y (NPY) onsmooth muscle cells (SMCs) from human subcutaneous arteries (diameter: 0.4 mm). NPY stimulated DNA synthesis ina concentration-dependent manner, Emax 32 ± 5% relative to control. The effect was potently antagonised by the NPYY1 receptor antagonist BIBP3226 ((R)-N2-(diphenylacetyl)-N-[(4-hydroxy-phenyl)methyl]-D-arginine-amide), indicatingthe effect to be mediated via the NPY Y1 receptor. Noradrenaline (NA) also induced mitogenesis, Emax 35 ± 10% rela-tive to control. When added together, NPY and NA potentiated the [3H]thymidine incorporation, Emax 109 ± 38% rela-tive to control. Also, this effect seems to be mediated by the NPY Y1 receptor, since BIBP3226 blocked the effect(44 ± 9% relative to control). The mitogenic effect of NPY and NA, two important transmitters of the sympatheticnervous system, might have clinical consequences on conditions with elevated sympathetic nerve activity.

Key words: BIBP3226, mitogenesis, neuropeptide Y, vascular smooth muscle cells.

Résumé : Nous avons examiné l’effet mitogénique, mesuré par l’incorporation de [3H]thymidine, du neuropeptide Y(NPY) sur les cellules musculaires lisses (CML) d’artères sous-cutanées d’humains (diamètre : 0,4 mm). Le NPY astimulé la synthèse de l’ADN de manière concentration-dépendante (Emax 32 ± 5% de la valeur témoin). L’effet a étéfortement inhibé par l’antagoniste du récepteur Y1 du NPY, BIBP3226 ((R)-N2-(diphénylacétyl)-N-[(4-hydroxy-phényl)méthyl]-D-arginine-amide), ce qui indique qu’il a été véhiculé par le récepteur Y1 du NPY. La noradrénaline(NA) a aussi induit une mitogenèse (Emax 35 ± 10% de la valeur témoin). L’addition concomitante de NPY et de NA apotentialisé l’incorporation de [3H]thymidine (Emax 109 ± 38% de la valeur témoin). Cet effet semble aussi véhiculépar le récepteur Y1 du NPY, puisqu’il a été bloqué par BIBP3226 (44 ± 9% de la valeur témoin). L’effet mitogéniquedu NPY et de la NA, deux importants transmetteurs du système nerveux sympathique, pourrait avoir une importanceclinique pour les cas d’hyperactivité nerveuse sympathique.

Mots clés : BIBP3226, mitogènése, neuropeptide Y, cellules du muscle lisse vasculaire.

[Traduit par la Rédaction] Brief Report / Rapport bref 259

Introduction

Neuropeptide Y (NPY), first purified from porcine brain,in 1982, by Tatemoto and colleagues (Tatemoto et al. 1982)was named NPY since it begins and ends with the aminoacid tyrosine (Y). NPY is the most prominent neuropeptidein the sympathetic nervous system, and is usually present insympathetic neurones innervating blood vessels, but not insympathetic neurones to exocrine glands and fat (Cannonet al. 1986; Lundberg et al. 1982). NPY is co-stored with

noradrenaline (NA) in large dense core vesicles in the sym-pathetic ganglion cells, in perivascular sympathetic nervesand is released together with NA upon sympathetic nerve ac-tivation (Edvinsson et al. 1987; Fried et al. 1985; Lundberget al. 1985).

NPY has been shown to be a potent constrictor of humanblood vessels in vitro and in vivo and has, in addition, beenshown, at subthreshold concentrations, to potentiate contrac-tions induced by NA (Bergdahl et al. 1996; Nilsson et al.1996a; Pernow et al. 1987). Furthermore, NPY has beendemonstrated to have mitogenic effects on human vascularsmooth muscle and endothelial cells, and has also beenshown to potentiate the mitogenesis induced by NA (Erlingeet al. 1994; Zukowska-Grojec et al. 1998).

So far five distinct NPY receptors have been cloned (forreview see Michel et al. 1998). Binding studies as well aspharmacological studies with selective NPY Y1 antagonistsindicate that the predominant vascular NPY receptor is ofthe Y1 type, but there is also some evidence of a Y2-mediated

Can. J. Physiol. Pharmacol. 78: 256–259 (2000) © 2000 NRC Canada

256

Received June 14, 1999.

T. Nilsson and L. Edvinsson.1 Division of ExperimentalVascular Research, Department of Medicine, Lund UniversityHospital, Lund, Sweden.

1Author for correspondence at the Department ofInternal Medicine, EB blocket, Lund University Hospital,S-22185 Lund, Sweden (e-mail: [email protected]).

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vascular effect (Bergdahl et al. 1996; Modin et al. 1991;Nilsson et al. 1996a, 1996b; Sheikh et al. 1991). The NPY re-ceptor responsible for inducing mitogenesis has not yet beenestablished.

The aims of the present study were to investigate, onsmooth muscle cells (SMCs) from human subcutaneous ar-teries, whether NPY mediates mitogenesis and potentiationof NA-induced mitogenesis and whether these effects aremediated via the NPY Y1 receptor.

Materials and methods

Cell culturePrimary cultures of vascular smooth muscle cells (VSMCs)

were prepared as described by Meyer-Lehnert and Schrier (1989).Briefly, human subcutaneous arteries (diameter 0.4 mm) were re-moved from six patients, free from known cardiovascular diseases,during abdominal surgery and placed in Dulbecco’s modified Eaglemedium (DMEM) containing streptomycin (10 000 mg/mL) andpenicillin (10 000 U/mL).

Using a binocular microscope, the adventitia and the outer layerwere stripped off. The vessels were opened by a longitudinal cut,and the intima was removed by scraping. The vessels were placedinto fresh DMEM, minced, and incubated with collagenase for 2 hat 37°C. After incubation, the tissues were flushed through 16- and18-gauge needles and centrifuged gently, and the cells were resus-pended in collagenase-free DMEM supplemented with streptomy-cin (10 000 mg/mL), penicillin (10 000 U/mL), and 10% fetal calfserum. The resulting suspension was then plated into 25-cm2 flasksand cultured at 37°C in a humidified 5% CO2 – 95% air atmo-sphere until the cells reached confluence (7–10 days). VSMCswere passaged 3 times before use. After 6 or 7 passages the cellsstopped dividing.

Determination of DNA synthesisDNA synthesis was measured using tritiated [3H]thymidine in-

corporation. VSMC cultures were suspended by trypsinization,counted, and replated into 24-well plates at the density of 30 000cells/well in media (described above) containing 10% fetal calf se-rum (FCS). After 48 h the cells were starved in 0.5% FCS for 48 hto decrease proliferation and induce quiescence. All the studiedsubstances were then added and present for 19 h except forBIBP3226, which was added 20 min earlier. The cells were incu-bated with [3H]thymidine (1 µCi/mL; 1 Ci = 37 GBq) during thelast 4 h. The medium was then aspirated, and the cells werewashed three times with PBS and twice with ice-cold 10%trichloroacetic acid. The fixed cellular material was solubilized in1 mL 0.2 M NaOH for 2 h. All the NaOH from each well (1 mL)was mixed with 4 mL of OptiPhase “HiSafe” 3 liquid scintillationcocktail. The amount of [3H]thymidine taken up by the cells wasestimated by counting in a Wallac 1410 liquid scintillation counter.

MaterialNeuropeptide Y was from Auspep, Australia; BIBP3226 was a

generous gift from Dr. H. Doods, Preclinical Research Department,Boehringer-Ingelheim, 88397 Biberach, Germany; noradrenalinewas from Sigma Co., U.S.A.; [3H]thymidine was from Amersham,U.K.; and fetal calf serum was from Gibco, U.S.A.

Statistical analysesValues are means ± standard error of the mean (SEM). n refers

to the number of wells examined. Statistically significant differ-ences between groups were determined with the Mann–Whitney Utest. A probability (P) level of < 0.05 was considered significant.Maximal induced DNA synthesis is expressed as Emax.

EthicsThe study was approved by the Human Ethics Committee of the

Lund University Hospital. All subjects were informed and hadagreed to participate in the study.

Results

NPY induced a concentration-dependent increase in DNAsynthesis in human SMCs from subcutaneous arteries, Emax32 ± 5% relative to control (0 ± 6%, 2001 ± 115 dpm).BIBP3226 (1 µM) reduced the NPY-induced DNA synthesisfrom 32 ± 5% for NPY alone to 19 ± 5%, a reduction by15% (Fig. 1). BIBP3226 did not, per se, cause any signifi-cant increase in [3H]thymidine incorporation (12 ± 5% ofcontrol).

NA (10 µM) stimulated the mitogenesis in human SMCsfrom subcutaneous arteries, Emax 35 ± 10% relative to con-trol. NPY (0.1 µM) significantly potentiated the effect medi-ated by NA (Emax 109 ± 38%). This effect was inhibited byBIBP3226 1 µM (Emax 44 ± 9%) (Fig. 2).

Fetal calf serum (FCS 10%) stimulated DNA synthesis by650 ± 30% relative to control. The mitogenic effects of NPY(0.1, 1.0 µM) and NA (10 µM) were approximately 2.5, 5,and 5%, respectively, as compared with the FCS 10%, butwhen NPY and NA were added together, the mitogenic ef-fect was potentiated to approximately 17%.

Discussion

In the present study we have investigated the mitogeniceffect of NPY in SMCs from human subcutaneous arteries.These arteries are tonically influenced by the sympatheticnervous system and participate in the regulation of bloodflow and blood pressure.

© 2000 NRC Canada

Brief Report / Rapport bref 257

Fig. 1. Effect of NPY on [3H]thymidine incorporation on humanSMCs from subcutaneous arteries, expressed as percentage ofcontrol. Values are means ± SEM. NPY 0.1 µM, n = 28; NPY 1µM, n = 52; NPY 1 µM with BIBP3226 1 µM, n = 38. Control,0 ± 6%; 2001 ± 115 dpm. *P < 0.05, **P < 0.01.



NPY simulated DNA synthesis in a concentration-dependent manner and with approximately the same magni-tude as has been shown before on human SMCs from subcu-taneous arteries and on porcine aortic SMCs (Erlinge et al.1994; Shigeri and Fujimoto 1993). To elucidate if it is theNPY Y1 receptor subtype that mediates the mitogenic effectwe used the potent and selective NPY Y1 receptor antagonistBIBP3226 (Rudolf et al. 1994). In contrast with Zukowska-Grojec and colleagues (1998), who found that BIBP3226displayed an intrinsic stimulatory effect on formation of cap-illary-like tubes by human umbilical vein endothelial cells(HUVECs), we observed that BIBP3226 itself did not stimu-late mitogenesis in human SMCs from subcutaneous arter-ies. On the other hand, BIBP3226 potently antagonized themitogenic effect of NPY, indicating the effect to be NPY Y1receptor mediated. This agrees well with previous studiesusing truncated NPY analogues, where the NPY Y1 receptoragonist, Pro34-NPY, but not the NPY Y2 receptor agonist,NPY(13–36), stimulated [3H]thymidine incorporation to thesame degree as NPY (Erlinge et al. 1994; Shigeri andFujimoto 1993). In addition, we have previously observed,with RT-PCR, the presence of mRNA encoding the humanNPY Y1 receptor in SMCs from human subcutaneous arter-ies (Erlinge 1994). However, the NPY Y1 antagonist,BIBP3226, did not totally abolish the mitogenic effect ofNPY, indicating the possible involvement of other NPY orNPY-like receptors.

As has been shown before, NA stimulated DNA synthesisin human SMCs from subcutaneous arteries (Erlinge et al.1994). It is well established that NPY, at subthreshold con-centrations, potentiates the NA-induced vasoconstriction.

The potentiation is thought to be mediated via the NPY Y1receptor (Bergdahl et al. 1996; Edvinsson et al. 1984). In thepresent study we have shown that NPY also potentiates theNA-induced stimulation of [3H]thymidine incorporation inSMCs from human subcutaneous arteries. Also, thispotentiation seems to be mediated by the NPY Y1 receptor,since BIBP3226 potently reduced the effect.

In conclusion, we have demonstrated that NPY stimulatesDNA synthesis and potentiation of NA-induced mitogenesisand that the effects are mediated by the NPY Y1 receptor.

Since both NPY and NA are important transmittors of thesympathetic nervous system, the mitogenic effect may be ofimportance in pathological conditions that involve elevatedsympathetic nerve activity, where their mitogenic effect maylead to vascular hypertrophy and participate in vascular wallthickening (Folkow 1982).

Acknowledgement

The study was supported by the Swedish Medical Re-search Council (grant 5859).

References

Bergdahl, A., Nilsson, T., Cantera, L., Nilsson, L., Sun, X.-Y.,Hedner, T., Erlinge, D., Valdemarsson, H., S., and Edvinsson, L.1996. Neuropeptide Y potentiates noradrenaline-induced con-traction through the neuropeptide Y Y1 receptor. Eur. J.Pharmacol. 316: 59–64.

Cannon, B., Nedergaard, J., Lundberg, J.M., Hökfelt, T.,Terenius, L., and Goldstein, M. 1986. Neuropeptide tyrosine (Y)is co-stored with noradrenaline in vascular but not parenchymalsympathetic nerves of brown adipose tissue. Exp. Cell Res. 164:546–550.

Edvinsson, L., Ekblad, E., Håkanson, R., and Wahlestedt, C. 1984.Neuropeptide Y potentiates the effect of various vasoconstrictoragents on rabbit blood vessels. Br. J. Pharmacol. 83: 519–525.

Edvinsson, L., Copeland, J.R., Emson, P.C., McCulloch, J., andUddman, R. 1987. Nerve fibers containing neuropeptide Y inthe cerebrovascular bed: immunocytochemistry, radio im-munoassay and vasomotor effects. J. Cerebral Blood FlowMetab. 7: 45–57.

Erlinge, D. 1994. The sympathetic cotransmitters neuropeptide Yand ATP in the regulation of the vascular smooth muscle cell.Thesis, Lund University, Lund, Sweden.

Erlinge, D., Brunkwall, J., and Edvinsson, L. 1994. NeuropeptideY stimulates proliferation of human vascular smooth musclecells: co-operation with noradrenaline and ATP. Regul. Pept. 50:259–265.

Folkow, B. 1982. Physiological aspects of primary hypertension.Physiol. Rev. 62: 347–504.

Fried, G., Terenius, L., Hökfelt, T., and Goldstein, M. 1985. Evi-dence for differential localisation of noradrenaline andneuropeptide Y (NPY) in neuronal storage vesicles isolatedfrom rat vas deferens. J. Neurosci. 5: 450–485.

Lundberg, J.M., Terenius, L., Hökfelt, T., Martling, C.-R.,Tatemoto, K., Mutt, V., Polak, J., Bloom, S., and Goldstein, M.1982. Neuropeptide Y (NPY)-like immunoreactivity in periph-eral noradrenergic neurones and effects of NPY on sympatheticfunctions. Acta Physiol. Scand. 116: 477–480.

Lundberg, J.M., Martinson, A., Hemsén, A., Theodorsson-Norheim, A., Svedenhag, J., Ekblom, B., and Hjelmdahl, P.1985. Corelease of neuropeptide Y and catecholamines during

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Fig. 2. The potentiating effect of NPY on the NA-induced[3H]thymidine incorporation on human SMCs from subcutaneousarteries, expressed as percentage of control. Values are means ±SEM. NA 10 µM, n = 15; NPY 0.1 µM, n = 28; NPY 0.1 µMand NA 10 µM, n = 6; NPY 0.1 µM and NA 10 µM andBIBP3226 1 µM, n = 6. Control, 0 ± 6%; 2001 ± 115 dpm.*P < 0.05.



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physical exercise in man. Biochem. Biophys. Res. Commun.133: 30–36.

Meyer-Lehnert, H., and Schrier, R.W. 1989. Potential mechanismof cyclosporine A-induced vascular muscle contraction. Hyper-tension, 13: 352–360.

Michel, M.C., Beck-Sickinger, A., Cox, H., Doods, H.N., Herzog,H., Larhammar, D., Quirion, R., Schwartz, T., and Westfall, T.1998. XVI. International Union of Pharmacology recommenda-tions for the nomenclature of neuropeptide Y, peptide YY, andpancreatic polypeptide receptors. Pharmacol. Rev. 50: 143–150.

Modin, A., Pernow, J., and Lundberg, J.M. 1991. Evidence for twoneuropeptide Y receptors mediating vasoconstriction. Eur. J.Pharmacol. 203: 165–171.

Nilsson, T., Erlinge, D., Cantera, L., and Edvinsson, L. 1996a.Contractile effects of neuropeptide Y in human subcutaneous re-sistance arteries are mediated by Y1 receptors. J. Cardiovasc.Pharmacol. 28: 764–768.

Nilsson, T., Junping, Y., Sun, X.-Y., Hedner, T., and Edvinsson, L.1996b. Characterisation of neuropeptide Y receptors mediatingcontraction, potentiation and inhibition of relaxation. BloodPressure, 5: 164–169.

Pernow, J., Lundberg, J.M., and Kaijser, L. 1987. Vasoconstriction

effects in vivo and plasma disappearance rate of neuropeptide Yin man. Life Sci. 40: 47–54.

Rudolf, K., Eberlein, W., Engel, W., Wieland, H.A., Willim, K.D.,Entzeroth, M., Wienen, W., Beck-Sickinger, A.G., and Doods,H.N. 1994. The first highly potent and selective non-peptideneuropeptide Y Y1 receptor antagonist: BIBP 3226. Eur. J.Pharmacol. 271: 11–13.

Sheikh, S.P., Roach, E., Fuhlendorff, J., and Williams, J.A. 1991.Localisation of Y1 receptors for neuropeptide and PYY on vas-cular smooth muscle cells in rat pancreas. Am. J. Physiol. 260:G250–G257.

Shigeri, Y., and Fujimoto, M. 1993. Neuropeptide Y stimulatesDNA synthesis in vascular smooth muscle cells. Neurosci. Lett.149: 19–22.

Tatemoto, K., Carlquist, M., and Mutt, V. 1982. Neuropeptide Y—a novel brain peptide with structural similarities to peptide YYand pancreatic polypeptide. Nature (London), 296: 659–660.

Zukowska-Grojec, Z., Karwatowska-Prokopczuk, E., Rose, W.,Rone, J., Movafagh, S., Ji, H., Yeh, Y., Chen, W.-T., Kleinman,K., Grouzmann, E., and Grant, D.S. 1998. Neuropeptide Y; anovel angiogenic factor from the sympathetic nerves and endo-thelium. Circ. Res. 83: 187–195.

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