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after irradiation and chemotherapy.VEGFR-1 ligand-activated hematopoieticstem cells could be used for stem-celltransplantation or organ restoration.However, problems could arise with sys-temic VEGFR-1 therapies. In some pa-tients with diabetes, atherosclerosis andtissue ischemia, VEGFR-1 stimulation ofneovascularization could also accelerateretinopathy, plaque formation or possi-bly tumor growth. Thus, a very locore-gional form of the therapy would bepreferable in such instances. On theother hand, systemic use of VEGFR-1 in-hibitors to inhibit tumor angiogenesiscould pose problems in patients withcompromised hematopoiesis and itcould inhibit collateral vessel develop-ment in patients with cardiovascular dis-eases. Thus, more extensive studies ofthese molecules in experimental diseasemodels are needed before these findingstranslate into clinical trials. However,this trio of studies will clearly revive andboost the attempts to design therapiesbased on angiogenesis research.1. Carmeliet, P. & Jain, R.K. Angiogenesis in cancer

and other diseases. Nature 407, 249–257 (2000).2. Ferrara, N. & Davis-Smyth, T. The biology of vas-

cular endothelial growth factor. Endocrine Rev. 18,4–25 (1997).

3. Ferrara, N. & Alitalo, K. Clinical applications of an-giogenic growth factors and their inhibitors.Nature Med. 5, 1359–1364 (1999).

4. Epstein, S.E., Kornowski, R., Fuchs, S. & Dvorak,H.F. Angiogenesis therapy: Amidst the hype, theneglected potential for serious side effects.Circulation 104, 115–119 (2001).

5. Luttun, A. et al. Revascularization of ischemic tis-sues by PlGF treatment and inhibition of tumorangiogenesis, arthritis and atherosclerosis by anti-Flt-1 antibody. Nature Med. 8, 831–840 (2002).

6. Hattori, K. et al. Placental growth factor reconsti-tutes hematopoiesis by recruiting VEGFR1+ stemcells from bone marrow microenviroment. NatureMed. 8, 841–849 (2002).

7. Gerber, H.-P. et al. VEGF regulates hematopoieticstem cell survival by an internal autocrine loopmechanism. Nature 417, 954–958 (2002).

8. Eriksson, U. & Alitalo, K. Structure, expression andreceptor-binding properties of novel vascular en-dothelial growth factors. Curr. Top. Microbiol.Immunol. 237, 41–57 (1999).

9. Aase, K. et al. Vascular endothelial growth factor-B-deficient mice display an atrial conduction de-fect. Circulation 104, 358–364 (2001).

10. Carmeliet, P. et al. Synergism between vascularendothelial growth factor and placental growthfactor contributes to angiogenesis and plasma ex-travasation in pathological conditions. NatureMed. 7, 575–583 (2001).

11. Matsumoto, T. & Claesson-Welsh, L. VEGF recep-tor signal transduction. Sci. STKE 2001, RE21(2001).

12. Shibuya, M. Structure and dual function of vascu-lar endothelial growth factor receptor-1 (Flt-1).Int. J. Biochem. Cell Biol. 33, 409–420 (2001).

13. Fong, G.H., Zhang, L., Bryce, D.M. & Peng, J.Increased hemangioblast commitment, not vascu-lar disorganization, is the primary defect in flt-1knock-out mice. Development 126, 3015–3025(1999).

14. Hiratsuka, S., Minowa, O., Kuno, J., Noda, T. &Shibuya, M. Flt-1 lacking the tyrosine kinase do-main is sufficient for normal development and an-giogenesis In mice. Proc. Natl. Acad. Sci. USA 95,9349–9354 (1998).

15. Cho, N.K. et al. Developmental control of bloodcell migration by the Drosophila VEGF pathway.Cell 108, 865–876 (2002).

16. Ziegler, B.L. et al. KDR receptor: A key markerdefining hematopoietic stem cells. Science 285,1553–1558 (1999).

17. Ziche, M. et al. Placenta growth factor-1 ischemotactic, mitogenic, and angiogenic. LabInvest. 76, 517–531 (1997).

18. Bergers, G. et al. Matrix metalloproteinase-9 trig-gers the angiogenic switch during carcinogenesis.Nature Cell Biol. 2, 737–744 (2000).

1Ludwig Institute for Cancer Research,Stockholm Branch, Stockholm, Sweden2Molecular/Cancer Biology Laboratory andLudwig Institute for Cancer Research,Haartman Institute and Biomedicum Helsinki, University of Helsinki, FinlandEmail: kari.alitalo@helsinki.fi

The compulsive abuse of alcohol andother drugs often continues in the

face of health, social and financialproblems caused by addiction. Thetremendous societal impact of alco-holism must be rooted in fundamentalneurobiological mechanisms and mustreflect a ‘rewiring’ of the neurocir-cuitry by chronic drug ingestion.Although this view is at least superfi-cially attractive, clear descriptions ofthe relevant brain circuitry and the ef-fects of alcohol have been slow toemerge. All drugs of abuse activatebrain dopaminergic systems and thisactivation of ‘reward’ pathways mayinitiate the pleasurable effects of thedrugs. But the simple idea of chemicalreward does not explain why a drugsuch as nicotine, usually not describedas pleasurable on first use, is at least asaddictive as cocaine, a drug that pro-duces strong initial rewarding effects.

A more attractive incarnation of the‘dopamine activation’ hypothesisholds that dopamine systems are criti-cal not just for inducing reward, but

for the more complex processes oflearning about reward and regulatingreward-related behaviors. Thus,dopamine-mediated changes in synap-tic function (that is, synaptic plastic-ity) would persist after termination ofthe initial reward and ‘reward memory’would underlie craving and other long-lasting aspects of drug addiction. Thisoutlook suggests that drugs of abuseprovide much stronger activation ofdopamine pathways than natural rein-forcers such as food or sex, and stimu-late an abnormal degree of synapticplasticity1. This idea is readily applica-ble to drugs such as cocaine ormethamphetamine, which act directlyon dopamine terminals and likelyflood the synapse with neurotransmit-ter. However, ethanol releasesdopamine only indirectly by increas-ing neuronal firing. Thus, the magni-tude of dopamine release by ethanol

appears to be much less than that pro-duced by stimulants.

For ethanol to be able to ‘hijack’ thereward memory machinery, as pro-posed for cocaine, a postsynapticmechanism to amplify the relativelyweak presynaptic actions of ethanolmight be required. In a recent report inCell, Yao, Diamond and colleagues pro-vide evidence for such a mechanism2.Their report describes a synergisticpostsynaptic interaction betweenethanol’s effects on synaptic levels ofdopamine and another important neu-romodulator, adenosine, and thus pro-vides an elegant example for such apostsynaptic amplification—one al-most as complex as the disease of alco-holism itself!

Yao et al. used a common immortal-ized neural culture system, NG-108-15cells, as a test bed for investigating howethanol can amplify the actions of amajor type of dopamine receptor, the D-2 dopamine receptor subtype (Fig. 1).Most investigators agree that ethanolweakly activates dopamine release and

Two-shot cocktail: Adenosine, dopamine and a twist of βγThe neurotransmitter dopamine underlies most addictive processes—but its faint response to ethanol has

puzzled researchers. New research suggests that ethanol interacts synergistically with adenosine and dopamine signaling to amplify the effect of drinking.

R. ADRON HARRIS & RICHARD A. MORRISETT

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numerous studies indicate that the D-2subtype communicates dopaminergicsignals in alcohol reinforcement.Additionally, Diamond’s group had pre-viously shown that ethanol inhibits aspecific type of equilibrative nucleosidetransporter (ENT-1). This inhibition in-creases extracellular levels of adenosine,resulting in increased stimulation of theA-2 adenosine receptor. Both D-2dopamine and A-2 adenosine receptorsinduce the release of βγsubunits from het-erotrimeric G-proteins,and βγ has beendemonstrated to acti-vate specific types ofadenylyl cyclase (ACIIand ACIV) and therebysynthesize cAMP. Thissecond messenger acti-vates a major effectorenzyme, protein kinaseA (known in its acti-vated form as PKA-Cα),which then translo-cates from cytoplasmicsites to nuclear siteswhere it induces longterm alterations in neu-ronal function. Thesealterations are thoughtto underlieplasticityprocesses related toethanol reinforcement.

Yao et al. provide fur-ther evidence of thefunctional effects of βγgeneration and subse-quent translocation ofPKA-Cα by analyzinggene expression ofcAMP-response ele-ments (CRE). Thisstudy showed that thecAMP–PKA–CRE sig-naling pathway couldbe induced in at least two ways: viaethanol inhibition of adenosine trans-porter function or by direct D-2 receptoractivation with a selective agonist, NPA(R(–)-2,10, 11-trihydroxy-N- propyl-no-raporphine HBr). However, the authors’most striking observation resulted fromthe application of ethanol together withthe D-2 agonist. This combination washighly synergistic for PKA-Cα transloca-tion and CRE-mediated gene expression.Indeed, concentrations of ethanol andD-2 agonist that alone resulted in barelydetectable changes in the activity of thecAMP–PKA–CRE signaling system re-

sulted in robust activation upon co-ap-plication. Because both receptors releaseβγ, the authors propose that the coinci-dent activation of these receptors byethanol induces a highly synergistic re-lease of this intracellular messenger.These findings are of great interest intheir own right. But Yao et al. went on touse a clever scheme involving transfec-tion of two minigenes that encode βγscavenger proteins. They showed that

this synergistic action was indeed de-pendent upon βγ production. In thepresence of ethanol and the D-2 agonist,expression of these minigenes com-pletely occluded translocation of PKA-Cα as well as expression of CRE.

How do these results relate to neuralmechanisms of compulsive drinking? Asa first step toward these critical questions,Yao et al. analyzed primary cultures ofhippocampal neurons. However, thischoice to assess synergism in a ‘real’ neu-ron was surprising. There is evidence thatENT-1 may not be a major regulator ofadenosine action in hippocampus3.

Moreover, although βγ production via D-2 receptor activation may induce ACII/IVactivation in NG108-15 cells, in the hip-pocampus, D-2 activation inhibits cAMPproduction (due to release of α i). In thehippocampus, D-2 activation is thereforeexpected to inhibit PKA-Cα transloca-tion. Nevertheless, synergy was found inhippocampal neurons when ethanol andthe D-2 agonist were co-applied, suggest-ing that activation of adenylyl cyclases by

βγ overides the in-hibitory effects thatmight be produced—perhaps on different cy-clases—by αi. However,direct measurement ofthe degree of synergy inthese neurons was notprovided, so there re-mains a question aboutthe quantitative congru-ence between resultsfrom cultured cells andneurons.

The elegant in vitrobiochemical approachraises the issue of the invivo significance ofethanol release of βγsubunits. Unfortunately,this was only addressedin one experiment. Yaoet al. used viral vectorsto deliver proteins (βγscavengers) that wereexpected to reduce βγfunction in nucleus ac-cumbens of rats, andthey reported reducedconsumption of anethanol/water solution.However, the baselinelevel of ethanol con-sumption was low, andit was not clear that therats achieved measur-

able blood ethanol levels even before in-jection of the viral constructs. Thus, thismodel must be considered one of respon-sible social drinking rather than alcoholdependence, and so there remain ques-tions on the role of βγ subunits in animalmodels of alcohol abuse. The biochemi-cal studies demonstrated notable effectswith ethanol concentrations of 25 and50 mM, and it would be of interest to usebehavior tests with blood ethanol levelsin the same range. Such studies wouldprovide exciting opportunities for be-havioral pharmacologists to link modu-lation of dopaminergic signaling with

D2-R A2-R

ADODA

ACII/IV-cAMP-PKA-CRE/Band/or Ion Channels

EtOH Reinforcement

EtOH

βγ

Fig. 1 A working model of ethanol mechanisms related to βγ production. Modest in-creases in dopamine (DA) release result in activation of dopamine D-2 receptors andrelease of βγ subunits from G proteins. Strong inhibition of adenosine (ADO) uptakeby ethanol results in increased extracellular ADO. Extracellular ADO activates A-2 re-ceptors and release of βγ from Gs. Thus, activation of D-2 receptor, with a selective ag-onist that mimics the effect of ethanol to induce dopamine release, along with anethanol-stimulated increase in extracellular adenosine results in the synergistic in-crease in the release of βγ. Exactly which effector systems are activated by βγ to inducealterations in ethanol reward and reinforcement processing remain unknown.

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behavioral actions of ethanol.Whatever the result of such studies, it

is certain to be complex, especially giventhat D-2 receptors are not the only sub-type implicated in ethanol reinforce-ment. Indeed, a recent reportdemonstrated a role for the D-1 receptorin regulation of ethanol sensitivity of theNMDA (N-methyl-D-aspartate) receptorin the nucleus accumbens4. Their reporthelped to explain how D-1 receptor andPKA-dependent processes may contributeto synaptic plasticity underlying ethanolreinforcement4. Another complicationthat emerges from the work of Yao et al.concerns the well-known function of βγsubunits in regulating key potassium andcalcium channels. Ethanol, for example,activates the G protein–dependent in-

ward-rectifier potassium channel (GIRK)and this action of ethanol may be medi-ated by βγ (refs. 5,6). Therefore, βγ maydetermine alcohol action via numeroustargets, including ion channels, kinasesand/or gene expression. Because of thesediverse implications, the work of Yao etal. is an key advance in unraveling thecomplex interplay of dopaminergic sig-naling processes implicated in ethanol re-inforcement. One tantalizing implicationof these developments is that novelagents that affect βγ subunits or their tar-gets may represent new avenues for thedevelopment of pharmacotherapies foralcoholism.

1. Berke, J.D. & Hyman, S.E. Addiction, dopamine,and the molecular mechanisms of memory.Neuron 25, 515–532 (2000).

2. Yao, L. et al. βγ dimers mediate synergy ofdopamine D2 and adenosine A2 receptor-stimu-lated PKA signaling and regulate ethanol con-sumption. Cell 109, 733–743 (2002).

3. Dunwiddie, T.V. & Diao, L. Regulation of extracel-lular adenosine in rat hippocampal slices istemeperature-dependent: Role of adenosinetransporters. Neuroscience 95, 81–88 (2000).

4. Maldve, R.E. et al. DARPP-32 and regulation of theethanol sensitivity of NMDA receptors in the nu-cleus accumbens. Nature Neurosci. 5, 641–648(2002).

5. Lewohl, J.M. et al. G-protein-coupled inwardlyrectifying potassium channels are targets of alco-hol action. Nature Neurosci. 2, 1084–1090 (1999).

6. Kobayashi, T. et al. Ethanol opens G-protein-acti-vated inwardly rectifying K+ channels. NatureNeurosci. 2, 1091–1097 (1999).

The Waggoner Center for Alcohol and Addiction ResearchUniversity of Texas, Austin, Texas, USAemail: ramorris@mail.utexas.edu

Obesity has few rivals in terms of itsadverse impact on human health1.

Among the most common medical dis-orders, its skyrocketing prevalence indeveloped countries1 has heightenedthe need for more effective treatmentoptions. Especially problematic for theobese patient is the tendency for lostweight to be regained, a phenomenondriven by a biological process. Termed‘energy homeostasis’, this process pro-motes the stability of body fuel storedin the form of fat by matching energyintake to energy expenditure overtime. Consequently, voluntary weightloss in both obese and lean individualsactivates compensatory responses (in-creased appetite; decreased energy ex-penditure) that favor its eventualrecovery2. It therefore follows that ef-fective strategies for sustained weightloss will be those that target this bio-logical system. In support of this view,Borowsky et al. report in this issue3 onthe development of a drug that antago-nizes the receptor for melanin-concen-trating hormone (MCH), a keyneuropeptide in brain pathways thatcontrol energy homeostasis. Not onlydoes this compound have potent anddurable anti-obesity effects in rodents,it has surprising antidepressant andanxiolytic (anxiety-reducing) proper-ties as well. Should such findings befaithfully translated into clinical prac-tice, the concept of a ‘wonder drug’

will surely have been redefined.The frustratingly slow pace of

progress towards effective obesitytreatments stands in sharp contrast tothe steady flow of remarkable new in-sights into the biology of energy home-ostasis. A landmark event was thediscovery in 1994 of the hormone lep-tin and the demonstration that geneticdeficiency of this molecule underliesthe severe obesity phenotype of ob/obmice4. Leptin is secreted from fat cellsin proportion to body-fat content andrecent changes in energy balance, thedifference between energy intake andenergy expenditure over time. Leptin ishypothesized to function as an ‘adipos-ity signal’ that conveys information tothe brain regarding the sufficiency ofbody energy stores (Fig. 1). As geneticleptin deficiency causes overeating andsevere obesity in humans5 as well asmice, and because leptin administra-tion reverses these effects, optimismgrew that leptin might revolutionizeobesity treatment. Unfortunately, thisinitial optimism was quashed by evi-dence that leptin resistance is commonamong the obese.

Like leptin, the pancreatic hormoneinsulin is also implicated as an afferentsignal to the brain that circulates in

proportion to body fat mass6. Inputfrom both leptin and insulin is trans-duced into adaptive responses that re-duce food intake, increase energyexpenditure and promote normal glu-cose homeostasis7 by neurons in thehypothalamic arcuate nucleus2. Withinthe arcuate nucleus are two distinctsubsets of neurons that exert opposingeffects on energy balance. Anabolicneuronal pathways contain moleculessuch as neuropeptide Y (NPY) andagouti-related protein (AGRP) thatstimulate food intake and weight gain,and are inhibited by insulin and lep-tin2. Opposing these effects are themelanocortins, catabolic peptides thatare encoded by the proopiome-lanocortin (POMC) gene and reducefood intake and promote weight loss8.Arcuate nucleus POMC neurons lie ad-jacent to NPY/AGRP neurons and arestimulated by insulin and leptin (Fig.1)2,9. By reducing plasma levels of in-sulin and leptin, diet-induced weightloss activates NPY/AGRP neurons whileinhibiting POMC neurons, a combina-tion that potently favors the recoveryof lost weight2,9. These neurons are alsotargets for the action of the gastric hor-mone ghrelin, which stimulates ap-petite10 and is implicated in mealtimehunger11. The arcuate nucleus, there-fore, has a key role in sensing, integrat-ing and responding to peripheralsignals that control food intake and

Rats lighten up with MCH antagonistA new anti-obesity drug that targets brain pathways controlling energy balance in rats also works

against depression and anxiety (pages 825–830).

MICHAEL W. SCHWARTZ &RICHARD W. GELLING

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