Involvement of the Sigma₁ Receptor in the Appetitive Effects of Cocaine

Cocaine induces in the brain a drastic plasticity affecting numerous neurotransmission systems. In turn, the drug provokes a complex pattern of behavioral responses, including locomotor hyperactivity, stereotyped behaviors, sensitization, and appetitive and reinforcing properties that rapidly generate the addictive process. The involvement of σ₁ receptors in cocaine effects has been suggested initially through the observation that σ₁ antagonists could attenuate several acute effects induced by the drug, such as hyperlocomotion, stereotypies, convulsions and lethality. We will describe thereafter the recent results showing that activation of the σ₁ receptor is also involved in the appetitive properties of cocaine, as measured using place preference conditioning in mice. Using selective σ₁ antagonists or an in vivo antisense strategy, the role of the σ₁ receptor in acquisition, expression and reactivation of conditioned place preference was demonstrated. The observation that repeated administration of cocaine rapidly provokes overexpression of the σ₁ receptor outlines its major role in these first psychological steps of addictive processes. The physiological interaction between cocaine and σ₁ receptors is detailed and the possibility that effective therapeutical strategies could target the σ₁ receptor is considered.

References

• 1 Akimoto K, Hamamura T, Otsuki S. Subchronic cocaine treatment enhances cocaine-induced dopamine efflux, studied by in vivo intracerebral dialysis.  Brain Res. 1989;  490 339-344
• 2 Alonso G, Phan V L, Guillemain I, Saunier M, Legrand A, Anoal M, Maurice T. Immunocytochemical localization of the σ₁ receptor in the adult rat central nervous system.  Neuroscience. 2000;  97 155-170
• 3 Ault D T, Werling L L. Differential modulation of NMDA-stimulated [³H]dopamine release from rat striatum by neuropeptide Y and sigma receptor ligands.  Brain Res. 1997;  760 210-217
• 4 Calcagnetti D J, Keck B J, Quatrella L A, Schechter M D. Blockade of cocaine-induced conditioned place preference: relevance to cocaine abuse therapeutics.  Life Sci. 1995;  56 475-483
• 5 Calligaro D O, Eldefrawi M E. High affinity stereospecific binding of [³H]cocaine in striatum and its relationship to the dopamine transporter.  Membr Biochem. 1988;  7 87-106
• 6 Contreras P C, Quirion R, O'Donohue T L. Autoradiographic distribution of phencyclidine receptors in the rat brain using [³H]1-(1-(2-thienyl)cyclohexyl)piperidine ([³H]TCP).  Neurosci Lett. 1986;  67 101-106
• 7 Curran T, Morgan J I. Fos: an immediate-early transcription factor in neurons.  J Neurobiol. 1995;  26 403-412
• 8 Debler E A, Hashim A, Lajtha A, Sershen H. Ascorbic acid and striatal transport of [³H]-1-methyl-4-phenylpyridine (MPP⁺) and [³H]dopamine.  Life Sci. 1988;  42 2553-2559
• 9 Ettenberg A, Geist T D. Animal model for investigating the anxiogenic effects of self-administered cocaine.  Psychopharmacology. 1991;  103 455-461
• 10 Ettenberg A, Raven M A, Danluck D A, Necessary B D. Evidence for opponent-process actions of intravenous cocaine.  Pharmacol Biochem Behav. 1999;  64 507-512
• 11 Gardner B, Zhu L X, Roth M D, Tashkin D P, Dubinett S M, Sharma S. Cocaine modulates cytokine and enhances tumor growth through sigma receptors.  J Neuroimmunol. 2004;  147 95-98
• 12 Gonzalez G M, Werling L L. Release of [³H]dopamine from guinea pig striatal slices is modulated by sigma1 receptor agonists.  Naunyn Schmiedebergs Arch Pharmacol. 1997;  356 455-461
• 13 Gonzalez-Alvear G M, Werling L L. regulation of [³H]dopamine release from rat striatal slices by sigma ligands.  J Pharmacol Exp Ther. 1994;  271 212-219
• 14 Gonzalez-Alvear G M, Werling L L. Sigma₁ receptors in rat striatum regulate NMDA-stimulated [³H]dopamine release via a presynaptic mechanism.  Eur J Pharmacol. 1995;  294 713-719
• 15 Graybiel A M, Besson M J, Weber E. Neuroleptic-sensitive binding sites in the nigrostriatal system: evidence for differential distribution of sigma sites in the substantia nigra, pars compacta of the cat.  J Neurosci. 1989;  9 326-338
• 16 Graybiel A M, Moratalla R, Robertson H A. Amphetamine and cocaine induce drug-specific activation of the c-fos gene in striosome-matrix compartments and limbic subdivisions of the striatum.  Proc Natl Acad Sci USA. 1990;  87 6912-6916
• 17 Gundlach A L, Largent B L, Snyder S H. Autoradiographic localization of sigma receptor binding sites in guinea pig and rat central nervous system with (+)³H-3-(3-hydroxyphenyl)-N-(1-propyl)piperidine.  J Neurosci. 1986;  6 1757-1770
• 18 Hanner M, Moebius F F, Flandorfer A, Knaus H G, Striessnig J, Kempner E, Glossman H. Purification, molecular cloning, and expression of the mammalian sigma₁-binding site.  Proc Natl Acad Sci USA. 1996;  93 8072-8077
• 19 Hayashi T, Maurice T, Su T P. Ca²⁺ signaling via sigma₁-receptors: Novel regulatory mechanism affecting intracellular Ca²⁺ concentration.  J Pharmacol Exp Ther. 2000;  293 788-798
• 20 Hayashi T, Su T P. Regulating ankyrin dynamics: Roles of sigma-1 receptors.  Proc Natl Acad Sci USA. 2001;  98 491-496
• 21 Hellewell S B, Bowen W B. A sigma-like binding site in rat pheochromocytoma (PC12) cells: decreased affinity for (+)-benzomorphans and lower molecular weight suggest a different sigma receptor form from that of guinea pig brain.  Brain Res. 1990;  527 244-253
• 22 Hellewell S B, Bruce A, Feinstein G, Orringer J, Williams W, Bowen W B. Rat liver and kidney contain high densities of σ₁ and σ₂ receptors: characterization by ligand binding and photoaffinity labeling.  Eur J Pharmacol. 1994;  268 9-18
• 23 Higgins S T. The influence of alternative reinforcers on cocaine use and abuse: A brief review.  Pharmacol Biochem Behav. 1997;  57 419-427
• 24 Hyttel J. Inhibition of [³H]dopamine accumulation in rat striatal synaptosomes by psychotropic drugs.  Biochem Pharmacol. 1978;  27 1063-1068
• 25 Itzhak Y, Alerhand S. Differential regulation of sigma and PCP receptors after chronic administration of haloperidol and phencyclidine in mice.  FASEB J. 1989;  3 1868-1872
• 26 Itzhak Y, Martin J L. Cocaine-induced conditioned place preference in mice: Induction, extinction and reinstatement by related psychostimulants.  Neuropsychopharmacology. 2002;  26 130-134
• 27 Itzhak Y, Stein I. Sigma binding sites in the brain; an emerging concept for multiple sites and their relevance for psychiatric disorders.  Life Sci. 1990;  47 1073-1081
• 28 Jansen K L, Faull R L, Dragunow M, Leslie R A. Distribution of excitatory and inhibitory amino acid, sigma, monoamine, catecholamine, acetylcholine, opioid, neurotensin, substance P, adenosine and neuropeptide Y receptors in human motor and somatosensory cortex.  Brain Res. 1991;  566 225-238
• 29 Jbilo O, Vidal H, Paul R, De Nys N, Bensaid M, Silve S, Carayon P, Davi D, Galiegue S, Bourrie B, Guillemot J C, Ferrara P, Loison G, Maffrand J P, Le Fur G, Casellas P. Purification and characterization of the human SR 31747A-binding protein. A nuclear membrane protein related to yeast sterol isomerase.  J Biol Chem. 1997;  272 27 107-27 115
• 30 Jenab S, Festa E D, Russo S J, Wu H BK, Inturrisi C E, Quinones-Jenab V. MK-801 attenuates cocaine induction of c-fos and preprodynorphin mRNA levels in Fischer rats.  Molecular Brain Res. 2003;  117 237-239
• 31 Kekuda R, Prasad P D, Fei Y J, Leibach F H, Ganapathy V. Cloning and functional expression of the human type 1 sigma receptor (hSigmaR1).  Biochem Biophys Res Commun. 1996;  229 553-558
• 32 Kitaichi K, Chabot J G, Moebius F F, Flandorfer A, Glossmann H, Quirion R. Expression of the purported sigma₁ (σ₁) receptor in the mammalian brain and its possible relevance in deficits induced by antagonism of the NMDA receptor complex as revealed using an antisense strategy.  J Chem Neuroanat. 2000;  20 375-387
• 33 Klein M, Cooper T B, Musacchio J M. Effects of haloperidol and reduced haloperidol on binding to sigma sites.  Eur J Pharmacol. 1994;  254 239-248
• 34 Koob G F. Drugs of abuse: anatomy, pharmacology and function of reward pathways.  Trends Pharmacol Sci. 1992;  13 177-184
• 35 Kuhar M J. Molecular pharmacology of cocaine: A dopamine hypothesis and its implications.  Ciba Found Symp. 1992;  166 81-89
• 36 Largent B L, Gundlach A L, Snyder S H. Pharmacological and autoradiographic discrimination of sigma and phencyclidine receptor binding sites in brain with (+)-[³H]SKF 10,047, (+)-[³H]-3-[3-hydroxyphenyl]-N-(1-propyl)piperidine and [³H]-1-[1-(2-thienyl)cyclohexyl] piperidine.  J Pharmacol Exp Ther. 1986;  238 739-748
• 37 Leshner A I. Addiction is a brain disease, and it matters.  Science. 1997;  278 45-47
• 38 Liu Y, Lerner M, Brackett D J, Howard E W, Matsumoto R R. Prevention of cocaine-induced changes in behavior and genes expression by a sigma receptor antagonist.  Int J Neuropsychopharmacol. 2002;  5 S153
• 39 Lu L, Zhang B, Liu Z, Zhang Z. Reactivation of cocaine conditioned place preference induced by stress is reversed by cholecystokinin-B receptors antagonist in rats.  Brain Res. 2002;  954 132-140
• 40 Martin W R, Eades C G, Thompson J A, Huppler R E, Gilbert P E. The effects of morphine - and nalorphine - like drugs in the nondependent and morphine-dependent chronic spinal dog.  J Pharmacol Exp Ther. 1976;  197 517-532
• 41 Matsumoto R R, Liu Y, Lerner M, Howard E W, Brackett D J. σ Receptors: potential medications development target for anti-cocaine agents.  Eur J Pharmacol. 2003;  469 1-12
• 42 Matsumoto R R, Mack A L. (±)-SM 21 attenuates the convulsive and locomotor stimulatory effects of cocaine in mice.  Eur J Pharmacol. 2001;  417 R1-R2
• 43 Matsumoto R R, McCracken K A, Pouw B, Miller J, Bowen W D, Williams W, De Costa B R. N-alkyl substituted analogs of the sigma receptor ligand BD1008 and traditional sigma receptor ligands affect cocaine-induced convulsions and lethality in mice.  Eur J Pharmacol. 2001;  411 261-273
• 44 Matsumoto R R, McCracken K A, Pouw B, Zhang Y, Bowen W D. Involvement of sigma receptors in the behavioral effects of cocaine: evidence from novel ligands and antisense oligodeoxynucleotides.  Neuropharmacology. 2002;  42 1043-1055
• 45 Maurice T, Lockhart B P. Neuroprotective and anti-amnesic potentials of sigma (σ) receptor ligands.  Prog Neuropsychopharmacol Biol Psychiatry. 1997;  21 69-102
• 46 Maurice T, Martin-Fardon R, Romieu P, Matsumoto R R. Selective sigma₁ (σ₁) receptor antagonists as a new promising strategy to prevent cocaine-induced behaviors and toxicity.  Neurosci Biobehav Res. 2002;  26 499-527
• 47 Maurice T, Phan V L, Urani A, Kamei H, Noda Y, Nabeshima T. Neuroactive neurosteroids as endogenous effector for the sigma₁ (σ₁) receptor: Pharmacological evidences and therapeutic opportunities.  Jpn J Pharmacol. 1999;  81 125-155
• 48 Maurice T, Urani A, Phan V L, Romieu P. The interaction between neuroactive steroids and the sigma₁ (σ₁) receptor function: behavioral consequences and therapeutic opportunities.  Brain Res Rev. 2001;  37 116-132
• 49 McCann D J, Su T P. Solubilization and characterization of haloperidol-sensitive (+)-[³H]SKF-10,047 binding sites (sigma sites) from rat liver membranes.  J Pharmacol Exp Ther. 1991;  257 547-554
• 50 McCracken K A, Bowen W D, de Costa B R, Matsumoto R R. Two novel sigma receptor ligands, BD1047 and LR172, attenuate cocaine-induced toxicity and locomotor activity.  Eur J Pharmacol. 1999;  370 225-232
• 51 McCracken K A, Bowen W D, Matsumoto R R. Novel σ receptor ligands attenuate the locomotor stimulatory effects of cocaine.  Eur J Pharmacol. 1999;  365 35-38
• 52 McLean S, Weber E. Autoradiographic visualization of haloperidol-sensitive sigma receptors in guinea-pig brain.  Neuroscience. 1988;  25 259-269
• 53 Mei J, Pasternak G W. Molecular cloning and pharmacological characterization of the rat sigma₁ receptor.  Biochem Pharmacol. 2001;  62 349-355
• 54 Menkel M, Terry M, Pontecorvo M, Katz J L, Witkin J M. Selective σ ligands block stimulant effects of cocaine.  Eur J Pharmacol. 1991;  201 251-252
• 55 Morgan J I, Curran T. Stimulus-transcription coupling in the nervous system: involvement of the inducible proto-oncogenes fos and jun.  Annu Rev Neurosci. 1991;  14 421-451
• 56 Morin-Surun M P, Collin T, Denavit-Saubié M, Baulieu E E, Monnet F P. Intracellular σ₁ receptor modulates phospholipase C and protein kinase C activation in the brain stem.  Proc Natl Acad Sci USA. 1999;  96 8196-8199
• 57 Mueller D, Stewart J. Cocaine-induced conditioned place preference: reinstatement by priming injections of cocaine after extinction.  Behav Brain Res. 2000;  115 39-47
• 58 Musacchio J M, Klein M, Santiago L J. High affinity dextromethorphan binding sites in guinea pig brain: Further characterization and allosteric interactions.  J Pharmacol Exp Ther. 1988;  247 424-431
• 59 O'Brien C P, McLellan A T. Myths about the treatment of addiction.  Lancet. 1996;  347 237-240
• 60 Okuyama S, Imagawa Y, Ogawa S, Araki H, Ajima A, Tanaka M, Muramatsu M, Nakazato A, Yamaguchi K, Yoshida M. NE-100, a novel sigma receptor ligand: in vivo tests.  Life Sci. 1993;  53 PL285-290
• 61 Pan W J, Hedaya M A. An animal model for simultaneous pharmacokinetic/pharmacodynamic investigations: application to cocaine.  J Pharmacol Toxicol Methods. 1998;  39 -8
• 62 Pan Y X, Mei J, Xu J, Wan B L, Zuckerman A, Pasternak G W. Cloning and characterization of a mouse σ₁ receptor.  J Neurochem. 1998;  70 2279-2285
• 63 Parsons L H, Weiss F, Koob G F. Serotonin_1B receptor stimulation enhances cocaine reinforcement.  J Neurosci. 1998;  18 10 078-10 089
• 64 Peeters M, Romieu P, Maurice T, Su T P, Maloteaux J M, Hermans E. Involvement of the sigma1 receptor in the modulation of dopaminergic transmission by amantadine. Eur J Neurosci 2004; in press
• 65 Phan V L, Urani A, Sandillon F, Privat A, Maurice T. Preserved sigma₁ (σ₁) receptor expression and behavioral efficacy in the aged C57BL/6 mouse.  Neurobiol Aging. 2003;  24 865-881
• 66 Pickens R, Thompson T. Cocaine-reinforced behavior in rats: effects of reinforcement magnitude and fixed-ratio size.  J Pharmacol Exp Ther. 1968;  161 122-129
• 67 Prasad P D, Li H W, Fei Y J, Ganapathy M E, Fujita T, Plumley L H, Yang-Feng T L, Leibach F H, Ganapathy V. Exon-intron structure, analysis of promoter region, and chromosomal localization of the human type 1 sigma receptor gene.  J Neurochem. 1998;  70 443-451
• 68 Quirion R, Bowen W D, Itzhak Y, Junien J L, Musacchio J M, Rothman R B, Su T P, Tam S W, Taylor D P. A proposal for the classification of sigma binding sites.  Trends Pharmacol Sci. 1992;  13 85-86
• 69 Quirion R, Chicheportiche R, Contreras P C, Johnson K M, Lodge D, Tam S W, Woods J H, Zukin S R. Classification and nomenclature of phencyclidine and sigma receptor sites.  Trends Neurosci. 1987;  10 444-446
• 70 Ramamoorthy J D, Ramamoorthy S, Mahesh V B, Leibach F H, Ganapathy V. Cocaine-sensitive σ-receptor and its interaction with steroid hormones in the human placental syncytiotrophoblast and in choriocarcinoma cells.  Endocrinology. 1995;  136 924-932
• 71 Reith M EA, Meisler B E, Sershen H, Lajtha A. Structural requirements for cocaine congeners to interact with dopamine and serotonin uptake sites in mouse brain and to induce stereotyped behavior.  Biochem Pharmacol. 1986;  35 1123-1129
• 72 Ritz M C, George F R. Cocaine-induced seizures and lethality appear to be associated with distinct central nervous system binding sites.  J Pharmacol Exp Ther. 1993;  264 1333-1343
• 73 Ritz M C, Lamb R J, Goldberg S R, Kuhar M J. Cocaine receptors on dopamine transporters are related to self-administration of cocaine.  Science. 1987;  237 1219-1223
• 74 Romieu P, Martin-Fardon R, Bowen W D, Maurice T. Sigma₁ (σ₁) receptor-related neuroactive steroids modulate cocaine-induced reward.  J Neurosci. 2003;  23 3572-3576
• 75 Romieu P, Martin-Fardon R, Maurice T. Involvement of the σ₁ receptor in the cocaine-induced conditioned place preference.  Neuroreport. 2000;  11 2885-2888
• 76 Romieu P, Meunier J, Garcia D, Zozime N, Martin-Fardon R, Bowen W D, Maurice T. The sigma₁ (τ₁) receptor activation is a key step for the reactivation of cocaine conditioned place preference by drug priming.  Psychopharmacology. 2004;  175 154-162
• 77 Romieu P, Phan V L, Martin-Fardon R, Maurice T. Involvement of the σ₁ receptor in cocaine-induced conditioned place preference: possible dependence on dopamine uptake blockade.  Neuropsychopharmacology. 2002;  26 444-455
• 78 Schechter M D, Calcagnetti D J. Continued trends in the conditioned place preference literature from 1992 to 1996, inclusive, with a cross-indexed bibliography.  Neurosci Biobehav Rev. 1998;  22 827-846
• 79 Schechter M D, Calcagnetti D J. Trends in place preference conditioning with a cross-indexed bibliography; 1957 - 1991.  Neurosci Biobehav Rev. 1993;  17 21-41
• 80 Seth P, Fei Y J, Li H W, Huang W, Leibach F H, Ganapathy V. Cloning and functional characterization of a sigma receptor from rat brain.  J Neurochem. 1998;  70 922-931
• 81 Seth P, Leibach F H, Ganapathy V. Cloning and structural analysis of the cDNA and the gene encoding the murine type 1 sigma receptor.  Biochem Biophys Res Commun. 1997;  241 535-540
• 82 Shaham Y, Shalev U, Lu L, de Wit H, Stewart J. The reinstatement model of drug relapse: history, methodology and major findings.  Psychopharmacology. 2003;  168 3-20
• 83 Shalev U, Grimm J W, Shaham Y. Neurobiology of relapse to heroin and cocaine seeking: a review.  Pharmacol Rev. 2002;  54 1-42
• 84 Sharkey J, Glen K A, Wolfe S, Kuhar M J. Cocaine binding at σ receptors.  Eur J Pharmacol. 1988;  149 171-174
• 85 Sora I, Hall F S, Andrews A M, Itokawa M, Li X F, Wei H B, Wichems C, Lesch K P, Murphy D L, Uhl G R. Molecular mechanisms of cocaine reward: Combined dopamine and serotonin transporter knockouts eliminate cocaine place preference.  Proc Natl Acad Sci USA. 2001;  98 5300-5305
• 86 Sora I, Wichems C, Takahashi N, Li X F, Zeng Z, Revay R, Lesch K P, Murphy D L, Uhl G R. Cocaine reward models: conditioned place preference can be established in dopamine- and in serotonin-transporter knockout mice.  Proc Natl Acad Sci USA. 1998;  95 7699-7704
• 87 Stolerman I. Drugs of abuse: Behavioural principles, methods and terms.  Trends Pharmacol Sci. 1992;  13 170-176
• 88 Su T P, Hayashi T. Understanding the molecular mechanism of sigma-1 receptors: towards a hypothesis that sigma-1 receptors are intracellular amplifiers for signal transduction.  Curr Med Chem. 2003;  10 2075-2082
• 89 Su T P. Evidence for sigma opioid receptor: Binding of [³H]SKF-10,047 to etorphine-inaccessible sites in guinea-pig brain.  J Pharmacol Exp Ther. 1982;  223 284-290
• 90 Takebayashi M, Hayashi T, Su T P. Nerve growth factor-induced neurite sprouting in PC12 cells involves sigma-1 receptors: implications for antidepressants.  J Pharmacol Exp Ther. 2002;  303 1227-1223
• 91 Tam S W, Cook L. Sigma opiates and certain antipsychotic drugs mutually inhibit (+)-[³H] SKF 10,047 and [³H]haloperidol binding in guinea pig brain membranes.  Proc Natl Acad Sci USA. 1984;  81 5618-5621
• 92 Taylor D P, Dekleva J. Potential antipsychotic BMY-14,802 selectively binds to sigma sites.  Drug Dev Res. 1987;  11 65-70
• 93 Taylor D P, Eison M S, Moon S L, Yocca F D. BMY-14,802: A potential antipsychotic with selective affinity for σ-binding sites.  Adv Neuropsychiat Psychopharmacology. 1991;  1 307-315
• 94 Tzschentke T M. Measuring reward with the conditioned place preference paradigm: a comprehensive review of drug effects, recent progress and new issues.  Prog Neurobiol. 1998;  56 613-672
• 95 Ujike H, Kuroda S, Otsuki S. σ Receptor antagonists block the development of sensitization to cocaine.  Eur J Pharmacol. 1996;  296 123-128
• 96 Ujike H, Tsuchida K, Akiyama K, Otsuki S. Supersensitivity of σ receptors after repeated administration of cocaine.  Life Sci. 1992;  51 PL31-36
• 97 Vaupel D B. Naltrexone fails to antagonize the sigma effects of PCP and SKF-10,047 in the dog.  Eur J Pharmacol. 1983;  92 269-274
• 98 Walker J M, Bowen W D, Walker F O, Matsumoto R R, De Costa B, Rice K C. Sigma receptors: Biology and function.  Pharmacol Rev. 1990;  42 355-402
• 99 Warner E A. Cocaine abuse.  Ann Intern Med. 1993;  119 226-235
• 100 Wilbert-Lampen U, Seliger C, Zilker T, Arendt R M. Cocaine increases the endothelial release of immunoreactive endothelin and its concentrations in human plasma and urine. Reversal by coincubation with σ-receptor antagonists.  Circulation. 1998;  98 385-390
• 101 Witkin J M, Terry M, Menkel M, Hickey P, Pontecorvo M, Ferkany J, Katz J L. Effects of the selective sigma receptor ligand 6-[6-(4-hydroxypiperidinyl)hexyloxy]-3-methylflavone (NPC 16377), on behavioral and toxic effects of cocaine.  J Pharmacol Exp Ther. 1993;  266 473-482
• 102 Woolverton W L, Johnson K M. Neurobiology of cocaine abuse.  Trends Pharmacol Sci. 1992;  13 193-200
• 103 Woolverton W L. Determinants of cocaine self-administration by laboratory animals.  Ciba Found Symp. 1992;  166 149-161
• 104 Young S T, Porrino L J, Iadarola M J. Cocaine induces striatal c-fos-immunoreactive proteins via dopaminergic D1 receptors.  Proc Natl Acad Sci USA. 1991;  88 291-1295
• 105 Zamanillo D, Andreu F, Ovalle S, Perez M P, Romero G, Farre A J, Guitart X. Up-regulation of sigma₁ receptor mRNA in rat brain by a putative atypical antipsychotic and sigma receptor ligand.  Neurosci Lett. 2000;  282 169-172