Mitogenic actions of neuropeptide Y in vascular smooth muscle cells: synergetic interactions with...

Mitogenic actions of neuropeptide Y in vascularsmooth muscle cells: synergetic interactions withthe �-adrenergic system1,2

Jennifer Pons, Joanna Kitlinska, Hong Ji, Edward W. Lee, and Zofia Zukowska

Abstract: Neuropeptide Y (NPY), a sympathetic cotransmitter and vasoconstrictor, also stimulates vascular smoothmuscle cell (VSMC) growth, but which of its Y1–Y5 receptors are involved remains unclear. In quiescent rat VSMCs,NPY receptor mRNAs were undetectable (reverse transcription – polymerase chain reaction), but Y1, Y2, and Y5 ex-pression were upregulated or induced following NPY treatment. Concomitantly, NPY increased up to twofold[3H]thymidine incorporation and cell number bimodally, with a high-affinity peak at pM and low affinity peak at nMconcentrations. The Y1 or Y5 (not Y2) antagonist alone did not change the high-affinity peak but decreased the low af-finity peak by 50% and fully blocked NPY’s response when combined. In VSMCs lacking NPY receptors and respon-siveness, transient Y1 cDNA transfection restored their mitogenic response (blocked by the Y1 antagonist). In VSMCswith low or no NPY responsiveness, pre-exposure to β-adrenergic receptor agonist (isoproterenol), forskolin, or dibu-tyryl cAMP augmented NPY’s mitogenic effect, while upregulating Y1, Y2, and Y5 receptor expression (isoproterenolonly). Thus, NPY is a potent vascular mitogen acting via Y1 and Y5 receptors. However, since their expression is lowin nonproliferating cells, amplification of NPY’s mitogenic responses requires upregulation of at least the Y1 receptorby NPY itself or β-adrenergic, cAMP-dependent activation.

Key words: neuropeptide Y, NPY receptors, beta adrenergic receptor, cyclic AMP, vascular smooth muscle cells.

Résumé : Le neuropeptide Y (NPY), vasoconstricteur et co-transmetteur sympathique, stimule aussi la croissance descellules du muscle lisse vasculaire (CMLV); toutefois, le rôle de ses récepteurs Y1-Y5 n’a pas encore été déterminé.Dans les CMLV quiescentes de rats, les ARNm des récepteurs du NPY n’ont pu être détectés (technique de transcrip-tion inverse suivie de réaction en chaîne de la polymérase), mais l’expression des récepteurs Y1, Y2 et Y5 a augmentéaprès un traitement avec le NPY. Le NPY a augmenté jusqu’à un facteur deux l’incorporation de [3H]thymidine et lenombre de cellules de manière bimodale avec une crête de haute affinité aux concentrations pM et une crête de faibleaffinité aux concentrations nM. L’antagoniste du récepteur Y1 ou Y5 (pas Y2) seul n’a pas modifier la crête de hauteaffinité, mais il a diminué la crête de faible affinité de 50 % et a bloqué totalement la réponse au NPY lorsqu’utiliséen combinaison. Dans les CMLV dépourvues de récepteurs du NPY et de réactivité, la transfection transitoire del’ADNc du récepteur Y1 a rétabli leur réponse mitogène (bloquée par l’antagoniste de Y1). Dans les CMLV montrantune réactivité faible ou nulle au NPY, une pré-exposition à l’agoniste du récepteur β-adrénergique (isoprotérénol), à laforskoline ou au dibutyryl AMPc a augmenté l’effet mitogène du NPY et stimulé l’expression des récepteurs Y1, Y2 etY5 (isoprotérénol seulement). Ainsi, le NPY est un puissant mitogène vasculaire agissant par l’intermédiaire des récep-teurs Y1 et Y5. Toutefois, leur expression étant faible dans les cellules non proliférantes, l’amplification des réponsesmitogènes au NPY nécessite une augmentation du récepteur Y1 par le NPY même ou par l’activation dépendante del’AMPc et du récepteur β-adrénergique.

Mots clés : neuropeptide Y, récepteur NPY, récepteur bêta-adrénergique, AMP cyclique, cellules du muscle lissevasculaire.

[Traduit par la Rédaction] Pons et al. 185

Introduction

Neuropeptide Y (NPY) is a 36-amino-acid, amidated pep-tide that is ubiquitously present in the mammalian nervous

system and highly conserved throughout evolution. It be-longs to a family of peptides, which includes the gut hor-mones peptide YY (PYY) and pancreatic polypeptide (PP)(Grundemar 1997). In the cardiovascular system, NPY is

Can. J. Physiol. Pharmacol. 81: 177–185 (2003) doi: 10.1139/Y02-166 © 2003 NRC Canada

177

Received 14 May 2002. Published on the NRC Research Press Web site://cjpp.nrc.ca on 18 February 2003.

J. Pons, J. Kitlinska, H.Ji, E.W. Lee, and Z. Zukowska.3 Department of Physiology and Biophysics, Georgetown UniversityMedical Center, Washington, DC 20057, U.S.A.

1This paper has undergone the journal’s usual peer review process.2Presented at the Satellite Meeting of the XVII World Congress of the International Society for Heart Research in theCardiovascular System and Inflammatory Mediators, Montréal, Que., 12–14 July 2001.

3Corresponding author (e-mail: [email protected]).

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colocalized and coreleased with norepinephrine and, hence,increased NPY plasma levels are found in various conditionsof sympathetic nerve hyperactivity such as stress, hyperten-sion, and congestive heart failure (Zukowska-Grojec andWahlestedt 1993; Walker et al. 1991; Michel and Rascher1995; Feng et al. 2000). NPY is a potent vasoconstrictor,acting directly and indirectly by amplifying the actions ofcatecholamines (Zukowska-Grojec and Wahlestedt 1993;Walker et al. 1991; Wahlestedt et al. 1990). In addition, itexerts a vast range of physiological effects, which includethe stimulation of food intake, anxiolysis, and the regulationof hormone secretion (Turton et al. 1997). Recently, our(Zukowska-Grojec et al. 1998a,b; Kim et al. 1998) and other(Shigeri and Fujimoto 1993; Goldberg et al. 1998) studieshave shown that NPY, at low physiological concentrations,has growth-promoting effects on VSMCs (Zukowska-Grojecet al. 1998a; Kim et al. 1998; Shigeri and Fujimoto 1993)and cardiomyocytes (Goldberg et al. 1998) and trophic andangiogenic effects on endothelial cells (Zukowska-Grojec etal. 1998b).

There are six known NPY receptor subtypes, Y1–Y6 (allexcept Y3 are cloned) which are differentially activated byNPY, PYY, and (or) PP (Michel et al. 1998), as well as byNPY and (or) PYY-derived fragments resulting from pro-teolytic cleavage (Zukowska-Grojec 1997). The major prod-uct is formed by dipeptidyl peptidase IV (DPPIV), a highlyspecific endothelial peptidase, which cleaves off proline inthe penultimate position and generates NPY3-36, a bioactiveand circulating fragment with altered receptor specificity(Mentlein et al. 1993a). NPY receptors are Gi/o-coupled,seven transmembrane receptors, which are linked to the inhi-bition of adenylyl cyclase (Herzog et al. 1992) and an in-crease in intracellular calcium levels (Soares Lemos et al.1997). The Y1 receptor is the main vascular receptor, medi-ating vasoconstriction particularly of small resistance vessels(Malstrom and Lundberg 1997; Zukowska-Grojec et al.1996; Nilsson et al. 1996) and activated only by the full-length NPY1-36 (Grundemar 1997). The Y2 receptor, whichis the main neuroinhibitory receptor (Grundemar 1997), andthe Y5 receptor, which is involved in feeding regulation (Huet al. 1996), are both activated by NPY1-36 and its N-terminally truncated form, NPY3-36 (Michel et al. 1998). Thevascular functions of the Y2 receptor, which is also found inblood vessels, and of the Y5 receptor, which is believed to belocalized to the brain only (Borowsky et al. 1998), arelargely unknown. Hence, which of the multiple NPY recep-tor(s) is (are) involved in promoting VSMC growth remainsundefined.

Little is known about the regulation of NPY receptors. Assympathetic cotransmitters, NPY and catecholamines cross-regulate each other’s release and actions (Zukowska-Grojecand Wahlestedt 1993). Presynaptically, NPY suppressesnorepinephrine release via Y2 receptors, while postsynapti-cally, the interaction between NPY Y1 and α1-adrenergic re-ceptors augments vasoconstriction (Zukowska-Grojec andWahlestedt 1993; Michalkiewicz et al. 2001). In addition, we(Zukowska-Grojec 1997) and others (Michel et al. 1992)have also shown that prolonged exposure to catecholaminesaugments NPY-induced vasoconstriction in rats (Wahlestedtet al. 1990) and enhances NPY-stimulated intracellular Ca2+

mobilization in VSMCs (Zukowska-Grojec 1997) and SK-

N-MC neuroblastoma cells (Michel et al. 1992) via β-adrenergic and not α-adrenergic receptors. NPY andnorepinephrine were also found to potentiate each other’smitogenic responses (Nilsson and Edvinsson 2000).

The purpose of this study was to characterize which NPYreceptor subtypes mediate its vascular growth-promoting ef-fects and to determine the mechanisms and receptor sub-types involved in the interactions between NPY andcatecholamines in the regulation of cell growth.

Materials and methods

VSMC cultureA10 rat aortic VSMCs (Kimes and Brandt 1976) (ATCC,

Manassas, Va.) at passages 16–25 (up to passage 30 for thetransfection studies) and primary rat VSMCs (Biowhittaker,Inc., Walkersville, Md.) at passages 4–8 were used. Thecells were grown in a 5% CO2 humidified incubator inDulbecco’s modified essential medium with 4.5 g/L glucosesupplemented with 100 units/mL penicillin, 100 µg/mLstreptomycin, 2 mmol/L glutamine, 25 mmol/L HEPES (allfrom Biosource International, Camarillo, Calif.), and 10%fetal bovine serum (FBS) for A10 cells or SmBM-2 bulletkit (Biowhittaker, Inc.) for primary VSMCs. Cells weresubcultured at 90–95% confluency. Initial studies were per-formed on A10 cells and later with primary rat VSMCs, asnoted, because A10 cells at lower passages were no longeravailable from the vendor.

Mitogenic assays

DNA synthesis and cell countingVSMCs were plated onto six-well plates (5 × 104 cells/well),

grown until 50–70% confluent, starved in 0.5% FBS-containing medium for 24 h to induce quiescence, and sub-sequently, treated with NPY (diluted in 0.5% bovine serumalbumin, BSA) in 0.5% FBS-containing medium for another48–72 h. Four hours into the treatment period, 8 µCi (1 Ci =37 Gbq)[3H]thymidine was added to each well. The treat-ment reactions were stopped by trypsinization and twoaliquots of 10 µL were taken per well to count the number ofcells on a hemocytometer. Then, six 50-µL aliquots weretransferred to a 96-well plate. Another two 10-µL aliquotswere taken per well to count the number of cells on thehemocytometer again. Distilled water (100 µL) was added tothe 96-well plates which were then frozen at –20°C. Thesamples were then harvested onto filter mats (Wallac, Turku,Finland) using a Tomec Harvester 96 Mach II (Orange,Conn.) and [3H]thymidine uptake (cpm/well) was measuredusing a β-plate liquid scintillation counter.

Blocking NPY’s effect with specific receptor antagonistsA10 cells were plated onto 96-well plates (2 × 103

cells/well), grown until they reached 50–70% confluency,starved in 0.5% FBS-containing medium for 24 h, andtreated with NPY (in 0.5% BSA) in 0.5% FBS-containingmedium, with or without receptor antagonists added at thesame time for 24 h. Four hours into the treatment period,each well was pulsed with 0.5 µCi [3H]thymidine. Treatmentreactions were stopped by trypsinization, 100 µL distilledwater was added to each well, and the cells were frozen, har-vested, and counted as previously mentioned.

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Sensitization to NPY’s effect by pre-exposure to �-adrenergic activation or cAMP increase

Primary VSMCs were plated onto 96-well plates (1.2 ×103 cells/well), grown until they reached 50–70% conflu-ency, and starved in serum-free smooth muscle basalmedium (SmBM) for 24 h. Then, they were incubated(primed) with 10–5 M propranolol or prazosin for 8 h or10–6 M forskolin, 10–4 M dibutyryl cAMP (dbcAMP), or10–6 M isoproterenol in serum-free SmBM for 6 h prior totreatment (concentration and time of isoproterenol pretreat-ment used according to data from Zukowska-Grojec (1997)and Michel et al. (1992)), and later NPY (in 0.5% BSA) wasadded to 0.25% FBS media for another 24 h. The treatmentreactions were stopped by trypsinization, 100 µL distilledwater was added to each well, and the cells were frozen, har-vested, and counted as previously mentioned.

RNA isolation and RT–PCR analysisIn parallel to mitogenic assays, cells from the same pas-

sage were plated onto 100-mm culture dishes (3 × 105

cells/dish), grown until 50–70% confluent, starved, andprimed or treated identically as described above. They werelysed and the RNA was isolated using Tri-Reagent (MolecularResearch Center, Inc., Cincinnati, Ohio). First strand cDNAwas obtained by reverse transcription (MMLV Reverse Tran-scriptase, Stratagene, La Jolla, Calif.). The cDNA was am-plified by PCR (Taq DNA Polymerase, Promega, Madison,Wis.) for 35 cycles (denaturing at 94°C for 1 min, annealingat 50°C for 1 min, and extension at 72°C for 1.5 min) usingspecific primers for the Y1, Y2, Y4, and Y5 receptors. Thefollowing primer sequences were used: GenBank accessionnumber z11504 (forward-bases +195 to +215, reverse-bases+1038 to +1024) for the Y1 receptor; GenBank accessionnumber U32500 (forward-bases +415 to +435, reverse-bases+1244 to +1224) for the Y2 receptor; GenBank accessionnumber U84145 (forward-bases +267 to +286, reverse-bases+918 to +898) for the Y4 receptor; and GenBank accessionnumber U66274 (forward-bases +242 to +263, reverse-bases+1571 to +1551) for the Y5 receptor. The 18S rRNA (prim-ers from Ambion, Inc., Austin, Tex.) was amplified for 20cycles, serving as the PCR control. The samples were run ona 2% agarose gel at 120 V and stained with ethidium bro-mide. The gels were visualized and photographed under ul-traviolet light.

Y1 receptor cDNA transfectionA10 cells with undetectable NPY receptor expression

were plated onto 100-mm culture dishes (3 × 105 cells/dish)for RT–PCR and, in parallel, onto 24-well plates (7.5 × 103)for mitogenic studies and grown until 50–70% confluent.They were then transiently transfected with Y1 receptorcDNA using the calcium phosphate method (Gibco BRL,Gaithersburg, Md.). The cells were placed in 0.5% FBS me-dium overnight and treated with NPY, with or without the Y1receptor antagonist, for 24 h. For the mitogenic assay,2.5 µCi/well of [3H]thymidine was added 4 h into the treat-ment time. The reactions were stopped by trypsinization(150 µL/well) and deionized water (300 µL/well) was added.Three 150 µL aliquots were then transferred to a 96-wellplate. Data represent 3 wells per treatment with 3 counts perwell.

Peptides and drugsPorcine NPY1-36 was purchased from Peninsula

Laboratories (San Carlos, Calif.). Isoproterenol, propranolol,prazosin, forskolin, and dbcAMP were from Sigma (St.Louis, Mo.). The Y1 receptor antagonist H409/22 acetate(Malmstrom et al. 2000), was a gift from AstraZeneca R&D(Mölndal, Sweden). The Y2 receptor antagonist BIIE0246(Doods et al. 1999) was from Boehringer-Ingelheim Pharma(Biberach, Germany). Y5 receptor antagonist, CGP71683A(Rueeger et al. 2000), (Norvartis International AG, Basel,Switzerland) was obtained from AstraZeneca R&D.

Statistical analysis of dataData are presented as means ± SE, absolute values or per-

cent of control, and analyzed by one-way repeated-measuresanalysis of variance (one-way RM ANOVA) followed byDunnett’s t test or Friedman repeated-measures analysis ofvariance (RM ANOVA) on ranks, as indicated, usingSigmaStat 2.03 software (SPSS Science, Chicago, Ill.). P <0.05 was considered statistically significant for the indicatedn per group.

Results

Bimodal mitogenic response to NPY in A10 VSMCsTreatment of quiescent A10 cells with NPY in 0.5% FBS-

containing medium for 48 and 72 h caused similar bimodalincreases in [3H]thymidine uptake and cell number (Fig. 1).At 48 h, cells treated with 10–12 M and 10–8 M NPY in-creased [3H]thymidine incorporation from 6962 ± 1802(0.5% FBS-containing medium) to 11 861 ± 1509 (P < 0.05)and 12 154 ± 2379 (P < 0.05), respectively (Fig. 1C). Theparallel cell counts from the same wells showed increases in

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Pons et al. 179

Fig. 1. Bimodal mitogenic effects of neuropeptide Y (NPY) in0.5% FBS-containing medium on A10 rat aortic VSMCs after48 h (A and C) and 72 h (B and D). [3H]Thymidine uptake (Cand D) and cell number (A and B) were measured from thesame wells. *, P < 0.05 as compared with 0.5% FBS alone (one-way RM ANOVA followed by Dunnett’s t test; n = 6 wells).

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cell number at 48 h from 10 609 ± 2780 (0.5% FBS) to28 109 ± 4926 (P < 0.05) and 24 609 ± 2575 (P < 0.05), re-spectively (Fig. 1A). There were corresponding bimodalgrowth peaks at lower (10–12 M) and higher (10–9 M) con-centrations of NPY for both cell number and [3H]thymidineincorporation at 72 h (Figs. 1B and 1D).

NPY-induced mitogenesis and NPY receptor mRNAexpression

A10 cells grown in 0.5% FBS-containing medium aloneshowed no detectable expression of any NPY receptors(Fig. 2B, upper panels). When NPY was added to 0.5%FBS-containing medium, DNA synthesis levels increased upto twofold as compared with unstimulated cells after 24 h(P < 0.05, Figs. 2A and 2B). The corresponding RT–PCR(Fig. 2B, upper panel) revealed an upregulation of Y1, Y2,and Y5 receptor mRNA expression by 10–10 M NPY at 24 h.In cells that showed no detectable expression of Y1, Y2, andY5 receptors following peptide treatment (although Y4mRNA was present), there was a loss of the mitogenic re-sponse to NPY (Figs. 2A and 2B).

Effects of NPY receptor antagonists on NPY-inducedmitogenesis and receptor expression

The NPY receptor antagonists H409/22 acetate (Y1 antag-onist), BIIE0246 (Y2 antagonist), or CGP71683A (Y5 antag-onist) (10–7 M each), either as a single treatment orcombined, did not have any significant effects on[3H]thymidine uptake of 0.5% FBS-treated VSMCs in theabsence of NPY (Fig. 3). Also, when the Y1, Y2, or Y5 an-tagonists were added, one at a time, simultaneously withNPY to VSMCs for 24 h, there was no effect on NPY-mediated DNA synthesis at a lower concentration of NPY(10–12 M; Figs. 3A–3C). However, at a higher concentrationof NPY (10–8 M), the Y1 antagonist significantly decreasedDNA synthesis from 211 ± 8 (NPY alone) to 144 ± 26%(with Y1 antagonist, P < 0.05; Fig. 3A). The Y5 antagonisttended to attenuate NPY-induced DNA synthesis (from211 ± 8 to 176 ± 18%; Fig. 3C), while the Y2 antagonist hadno effect (Fig. 3B). The combination of the Y1 and Y5 antag-onists (Fig. 3D) significantly decreased NPY-mediated DNAsynthesis at both doses of NPY (i.e., from 172 ± 10 to 119 ±23% (NPY 10–12 M, P < 0.05) and from 211 ± 8 to 125 ±22% (NPY 10–8 M, P < 0.05)).

Transient transfection of Y1 receptor cDNAIn A10 cells, which were resistant to NPY and devoid of

any detectable NPY receptor mRNAs (except Y4), transienttransfection with Y1 receptor cDNA rendered them respon-sive to NPY with a significant increase in [3H]thymidine up-take from 30 976 ± 4633 (0.5% FBS only) to 53 701 ± 2073at 10–14 M NPY (P < 0.05) and 59 977 ± 3014 at 10–8 MNPY (P < 0.05; Fig. 4C). The induction of an NPY mito-genic effect was associated with Y1 receptor mRNA expres-sion (Fig. 4C). Conversely, cells, which were not transfectedor transfected without Y1 cDNA (Figs. 4A and 4B) showedno mitogenic response to NPY and no NPY receptor mRNAexpression. Moreover, the Y1 antagonist (10–7 M) signifi-cantly blocked 10–8 M NPY’s mitogenic effect in the Y1 re-ceptor cDNA transfected A10 cells (Fig. 4C) but had no

effect in cells, which were not transfected or transfectedwithout Y1 cDNA (Figs. 4A and 4B).

Effects of isoproterenol, forskolin, or dbcAMP on NPY-induced mitogenesis and NPY receptor expression

Primary VSMCs, which were treated with NPY (10–14–10–8 M), showed a similar pattern of mitogenic response asA10 cells with increases in DNA synthesis levels of up to

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Fig. 2. (A) Rat aortic A10 cells, responsive and nonresponsive toNPY in 0.5% FBS-containing medium after 24 hr treatment.(B) A representative mitogenic and parallel RT–PCR assay show-ing NPY’s effect after 24 h, which corresponded to Y1, Y2, andY5 receptor expression (10–10 M) in parallel wells (B, leftgraph). Loss of the NPY-mediated response and Y1, Y2, and Y5

receptor expression in A10 cells (B, right graph). *, P < 0.05 ascompared with cells in 0.5% FBS-containing medium (median of42 170, mean of 52 444 = 100%; Friedman RM ANOVA onranks; n = 3 different cultures); †, P < 0.05 as compared withcells in 0.5% FBS-containing medium (100%) (one-way RMANOVA followed by Dunnett’s t test; n = 6 wells).

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50% (P < 0.05; Fig. 5A). Pre-exposure of VSMCs to 10–6 Misoproterenol for 6 h had no effect on basal [3H]thymidineuptake of cells treated with 0.25% FBS alone (Fig. 5B).However, isoproterenol pretreatment increased the effect ofNPY on DNA synthesis, as compared with that of NPYalone, from 121 ± 7 to 163 ± 11% (NPY 10–14 M; P < 0.05),from 120 ± 11 to 161 ± 7% (NPY 10–12 M; P < 0.05) andfrom 135 ± 9 to 175 ± 8% (NPY 10–10 M; P < 0.05;Fig. 5B). Corresponding RT–PCR results (Fig. 5D) showedthat pre-exposure of these cells to 10–6 M isoproterenol(with or without NPY) upregulated NPY Y1, Y2, and Y5 re-ceptor expression from previously undetectable levels inVSMCs treated with NPY alone. Isoproterenol’s effect on10–10 M NPY’s mitogenic response was blocked by the β-adrenergic receptor antagonist, propranolol (10–5 M, given2 h prior to isoproterenol) (from 36 ± 6 to 6 ± 7%) but notby the α1-adrenergic receptor antagonist, prazosin (10–5 M,Fig. 5C). Both antagonists alone had no effect on controlDNA synthesis levels (Fig. 5C).

Six-hour pretreatment of VSMCs with forskolin (10–6 M)increased NPY’s effect on DNA synthesis, as compared withthe effect of 10–10 M NPY alone, by 23 ± 6% (P < 0.05;Fig. 5C). Also, pre-exposure of VSMCs to dbcAMP (10–4 M)increased 10–10 M NPY-stimulated DNA synthesis by 20 ±5% (P < 0.05; Fig. 5C).

Discussion

Since its discovery in 1982, NPY’s role in the cardiovas-cular system has centered on its vasoconstrictive effects(Zukowska-Grojec and Wahlestedt 1993; Walker et al. 1991;Wahlestedt et al. 1990). However, recent studies have shownthat NPY influences cardiovascular cells in other ways aswell, that is, promoting VSMC proliferation (Zukowska-

Grojec et al. 1998a; Kim et al. 1998; Shigeri and Fujimoto1993), cardiomyocyte hypertrophy (Goldberg et al. 1998),and angiogenesis (Zukowska-Grojec et al. 1998b). Here, weshow that NPY is a direct mitogen increasing DNA synthe-sis and VSMC proliferation by up to twofold. The NPY-mediated mitogenic response appears to be bimodal andhave two peaks, one at picomolar and the other at nanomolarconcentrations of the peptide, although the pattern of themitogenic response slightly varies between VSMCssubcultured from the rat aortic A10 cell line or primary cul-ture. For the A10 cell line, this unusual dose-response curvewas identical whether we used [3H]thymidine uptake or cellcounts as measures of cell proliferation. The parallel in-creases in DNA synthesis and cell number indicate that NPYpromotes hyperplasia of VSMCs.

Previously, we have reported that the mitogenic effect ofNPY shows a unique pharmacological profile (Zukowska-Grojec et al. 1998a). Since this profile did not correspond toany specific receptor subtype, we have suggested that morethan one receptor subtype is involved. We now show thatNPY-mediated cell growth includes Y1 and Y5 receptor sub-type activation. These receptors, however, are not alwaysconstitutively expressed or are expressed at very low levelsin VSMCs and, thus, for NPY to exert its growth-promotingeffect, receptor induction or upregulation is required. Usingthe A10 cell line, which has undetectable levels of NPY re-ceptors when quiescent, we determined that NPY (10–10 M)itself induces or upregulates Y1, Y2, Y4, and Y5 receptormRNA expression (RT–PCR). The receptor upregulationcorrelated with the ability of these cells to respond to NPYwith mitogenesis. Interestingly, NPY lost its ability to stimu-late DNA synthesis in cells of higher passages that have alsolost the expression of NPY receptor mRNA except for Y4.Thus, we inferred that of the four receptors, Y1, Y2, and Y5could potentially be NPY’s proliferative receptors.

Why some VSMCs respond to NPY and express its recep-tors while others show no mitogenic response or receptor ex-pression may be due to the phenotypic heterogeneity ofVSMCs. Both rat and human VSMCs have been shown toexhibit within-the-population heterogeneity in terms of theirphenotype and responses to several factors or agonists, andthis, in turn, has been linked to alterations in the cells’ over-all responsiveness (Lemire et al. 1994; Bochaton-Piallat etal. 1996; Li et al. 2001). For instance, both Fas-resistant and-sensitive cells have been found in VSMCs (Chan et al.2000), while A7R5 cells show a heterogenic calcium re-sponse to endothelin (Suzuki et al. 1991).

To determine the specific receptor subtypes whose activa-tion is required for NPY’s mitogenic response, we then usedthe Y1, Y2, and Y5 antagonists (10–7 M each), alone or incombination. At lower concentrations of NPY (subpicomolarto picomolar), the Y1 and Y5 antagonists by themselves didnot affect NPY-induced mitogenesis. However, each of theseantagonists inhibited the second NPY-mediated growth peakat the nanomolar concentration of the peptide. In contrast,the Y2 antagonist had no effect on NPY’s response at eitherdose. The combination of the Y1 and Y5 antagonists abol-ished the NPY-induced mitogenic effect at both low andhigh concentrations of the peptide. These data support thenotion that more than one receptor is involved in NPY-induced mitogenesis, specifically, the Y1 and Y5 receptors.

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Fig. 3. Effects of Y1 (A), Y2 (B), and Y5 (C) antagonists(10–7 M each), alone and combined (D) on NPY-inducedmitogenic responses in rat aortic A10 cells in 0.5% FBS-containing medium (mean of 36 822 = 100%). *, P < 0.05 ascompared with NPY alone (one-way RM ANOVA followed byDunnett’s t test; n = 6 wells).

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Previously, our (Zukowska-Grojec et al. 1998a) and other(Nilsson and Edvinsson 2000) studies examining the effectsof NPY at a limited range of nanomolar concentrations,suggested that Y1 is the main mitogenic receptor. However, amultireceptor mode of action was already reported in otherbiological systems — for example, in NPY’s control of feed-ing, which was initially thought to be mediated by a singlereceptor, either Y1 or Y5, but now is believed to involve bothreceptors (Flynn et al. 1999). Thus, we propose that inVSMCs, which express Y1 and Y5 receptors, constitutive orinducible (e.g. by NPY itself), NPY stimulates cell prolifera-tion by activating both receptors.

Interestingly, the ability of the Y1 receptor alone to sup-port NPY-induced VSMC proliferation is clearly indicatedby our transfection studies. In VSMCs transiently trans-fected with Y1 receptor cDNA, NPY increased DNA synthe-sis, an effect which was blocked by the Y1 antagonist.Whether or not single Y5 receptor transfection rendersVSMCs previously resistant to NPY responsive to itsmitogenic effect, could not have been determined, owing tothe failure of transfection. Difficulties with Y5 receptortransfection in cells lacking the endogenous Y1 receptor, aswell as the absence of Y5 expression in tissues without Y1receptors (Naveilhan et al. 1998), have been previously re-ported and may be related to the shared regulatory elementsof the Y1 and Y5 genes (Herzog et al. 1997).

While NPY’s vasoconstrictive effects require high physio-logical concentrations (nM–µM) (Zukowska-Grojec andWahlestedt 1993), its growth promoting activities begin withconcentrations that are below circulating plasma levels,which are in the nanomolar range in rats (Zukowska-Grojec1997). In addition to the high affinity peak at picomolar con-centrations, the NPY-mediated stimulation of growth wasalso present at nanomolar concentrations of the peptide, in-dicating the presence of low affinity mitogenic responsive-

ness. Our observation has posed an interesting dilemma, sincethe affinities for all known NPY receptors were reportedwithin low nanomolar concentrations (Michel et al. 1998).Little is known about the affinities of NPY receptors inVSMCs, where use of radioligand binding methods has beenhindered by extremely low levels of NPY receptor expres-sion. Recently, however, studies by Jacques et al. (2000)confirmed the presence in human VSMCs of high affinityNPY receptor(s), in large part of the Y1 type, by demonstratingthat NPY increases cytosolic and nuclear calcium signalingin these cells, starting at subpicomolar concentrations. To-gether, these findings indicate that VSMCs may express theY1 and Y5 receptors as either two subtypes or two differentreceptor states, with either high (pM) or low (nM) affinity.

The existence of an atypical, high-affinity response toNPY has been also reported by others. A dichotomous re-sponse was found for NPY and PYY (70% sequencehomology to NPY). Low doses (fM) of NPY and PYY sup-press gastric function via Y2 receptors, whereas higher doses(pM) activate gastric function via Y1 receptors (Chen et al.1997a,b). Our findings also suggest that in VSMCs, NPYmay exert similar differential effects which lead to twoproliferative peaks (at pM and nM) through the Y1 and Y5receptors. The possibility of an antiproliferative response inbetween the two proliferative peaks, mediated by anantimitogenic receptor, cannot be excluded. The Y4 receptormay be suspected to play such a role, since its expressionwas found in cells resistant to NPY’s mitogenesis. However,the presence of the peaks and valleys in NPY’s mitogeniccurve may also have alternative explanations (see below).

Upon agonist stimulation, many G protein coupled recep-tors (GPCRs) show a decreased sensitivity to further activa-tion, or desensitization, which occurs through receptorphosphorylation or endocytosis (Bohm et al. 1997). The Y1receptor has recently been reported to undergo rapid

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Fig. 4. The mitogenic effect of NPY on A10 cells transiently transfected with Y1 receptor cDNA (C) as compared with control cells,not transfected (A) and transfected without cDNA (B), and its blockade by the Y1 receptor antagonist (10–7 M). The upregulation ofY1 receptor mRNA expression is shown in C (panel insert). *, P < 0.05 (one-way RM ANOVA followed by Dunnett’s t test; n = 6wells).

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internalization and recycling through clathrin-coated pits intransfected HEK293 cells which is accompanied by de-creased adenylyl cyclase and calcium responses to NPY(Gicquiaux et al. 2001). Such a rapid internalization of highaffinity NPY receptors upon stimulation by picomolar con-centrations of the peptide in VSMCs may also explainNPY’s bimodal mitogenic response observed in our study. Ifso, desensitization and disappearance of the high affinity re-ceptors from the cell membrane would lead to a decreasedresponse to NPY at 10–10 M. Then, while the high affinityreceptors become internalized, increasing NPY concentra-tions to nanomolar levels would begin to activate the low af-finity receptors and produce the second peak of NPY’sproliferative activity.

Additionally, the full NPY molecule (NPY1-36) is a potentsubstrate for enzymatic cleavage by DPPIV, a highly specificprotease found on endothelial cells (Lojda 1979) and lym-phocytes (Mentlein et al. 1984) and in serum (Mentlein et al.1993b), forming the N-terminally truncated peptide, NPY3-

36, which binds to Y2 and Y5 but not Y1 (Mentlein et al.1993a). Moreover, we have found that in primary VMSCsNPY may induce expression of DPPIV mRNA by RT–PCR(Kitlinska et al. 2001). Thus, the presence of DPPIV maycontribute to NPY’s mitogenic response by affecting the ag-

onist affinities for different NPY receptor subtypes and theirsubsequent activation and functions. As we have shown inthis study, NPY’s growth response involves Y1 and Y5 acti-vation not Y2. Thus, increased DPPIV activity at concentra-tions in between the two proliferative peaks may cause adecrease in the cells’ response at the Y1 receptor to NPYstimulation, contributing to its bimodal proliferative effect.

Bimodal dose–response curves have also been found inother systems. In porcine ciliary nonpigmented epithelialmembranes, prostaglandin E2 receptor binding assays haverevealed a higher affinity subtype, which inhibited adenylatecyclase, and a lower affinity subtype, which activatedadenylate cyclase (Sano and Shichi 1993). Since it is notknown if NPY receptor subtypes couple to other G proteinsbesides Gi, it cannot be ruled out that they exhibit similardifferential G-protein receptor coupling which may lead toits bimodal VSMC mitogenic response.

To determine if high-affinity NPY mitogenic responsive-ness is inducible in VSMCs, we studied the interactions ofNPY with its cotransmitters, catecholamines. Previously, we(Zukowska-Grojec 1997), using VSMCs, and others (Michelet al. 1992), using SK-N-MC cells, have observed that cellscan be rendered highly responsive to NPY following prior,prolonged exposure to catecholamines. Furthermore, this

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Pons et al. 183

Fig. 5. Effects of NPY in 0.25%-FBS containing medium on DNA synthesis in subcultured primary rat aortic VSMCs after 24 h (A).Potentiating effects of VSMC pre-exposure (6–8 h) to isoproterenol (ISO, 10–6 M) in the absence (B; n = 18 wells) or presence ofpropranolol (PROP, 10–5 M; n = 24 wells) or prazosin (PRAZ, 10–5 M; n = 6 wells), forskolin (10–6 M; n = 12 wells) or dbcAMP(10–4 M; n = 12 wells) on mitogenesis mediated by NPY (10–10 M) (C) and on NPY receptor expression by RT–PCR (with and with-out NPY 10–8 M, D). *P < 0.05 as compared with cells in 0.25% FBS-containing medium (mean of 24 922 = 100%; one-way RMANOVA followed by Dunnett’s t-test; n = 3 separate cultures); †, P < 0.05 as compared with NPY alone (one-way RM ANOVA fol-lowed by Dunnett’s t test).

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potentiation appeared to be dependent on β-, and not α-,adrenergic receptor activation (Zukowska-Grojec 1997) andnew protein synthesis (Michel et al. 1992). In the presentstudy, we showed that the pre-exposure of VSMCs toisoproterenol increased NPY’s mitogenic response, as wellas upregulated Y1, Y2, Y4, and Y5 receptor mRNA expres-sion, and the former effect was blocked by propranolol. Thisindicates that the priming of cells with β-adrenergic activa-tion enhances NPY’s effects by upregulating NPY receptorgene expression and, consequently, their signaling.

Recently, a growing number of studies have shown thatGPCRs from different receptor families can formheterodimers, altering the properties and functions of the re-spective receptors (Devi 2001; Jordan et al. 2001). Forexample, the Gs-coupled, β2-adrenergic receptor forms oligo-meric complexes with Gi-coupled, δ and κ opioid receptorspromoting different receptor trafficking properties intransfected CHO cells (Jordan et al. 2001). Whether or notoligomerization of β2-adrenergic and NPY receptors leads tothe formation of a higher affinity NPY proliferative receptorstate and (or) augments, NPY’s mitogenic responses, re-quires further studies. In the present study, the interaction ofthe β-adrenergic and NPY systems in promoting VSMCgrowth was found to include cAMP-dependent signalingpathways since forskolin and dbcAMP also increased NPY’smitogenic response. How, specifically, cAMP acts to pro-mote NPY-mediated VSMC proliferation is unknown butthis action may involve protein kinase A (PKA) - mediatedβ-adrenergic receptor desensitization, which leads to thephosphorylation of the receptor, its uncoupling from Gs pro-teins, and coupling it to Gi proteins (Daaka et al. 1997). Al-ternatively, it may be mediated by interactions of β-adrenergic receptors with calcium channels which have beenshown in the heart and brain (Davare et al. 2001).

In summary, NPY is a potent VSMC mitogen, active atphysiological plasma concentrations. Its mitogenic effect in-volves multiple, high and low affinity receptors of the Y1and Y5 types, which are either constitutively expressed or in-duced or upregulated by NPY itself and by β-adrenergic –cAMP-dependent signaling. Due to the later synergistic in-teraction, NPY’s growth-promoting activity may be particu-larly augmented in pathophysiological conditions withchronic β-adrenergic activation, such as hypertension andcongestive heart failure.

Acknowledgements

This work was supported by grants awarded to ZofiaZukowska from National Institutes of Health (HL55310) andfrom AstraZeneca R&D, Sweden.

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