Nitric Oxide Production and Nitric Oxide Synthase Expression in Platelets from Heroin Abusers before and after Ultrarapid Detoxification

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Ann. N.Y. Acad. Sci. 965: 479–486 (2002). © 2002 New York Academy of Sciences.

Nitric Oxide Production and Nitric Oxide Synthase Expression in Platelets from Heroin Abusers before and after Ultrarapid Detoxification

ANA BATISTA,a TICE MACEDO,a PAULA TAVARES,a CARLOS FONTES RIBEIRO,a JOÃO RELVAS,b PIEDADE GOMES,c CARLOS RAMALHEIRA,b ISABEL BOTTO,b LUISA VALE,b LUÍS FERREIRA,b

ORLANDO GÜETE,b AND GUADALUPE RUIZb aInstitute of Pharmacology and Experimental Therapeutics, Faculty of Medicine, University of Coimbra, 3004-504, Coimbra, PortugalbDepartment of Psychiatry, Coimbra University Hospital, 3049 Coimbra, PortugalcDepartment of Anesthesiology, Coimbra University Hospital, 3049 Coimbra, Portugal

ABSTRACT: Prolonged heroin abuse has been associated with neurotoxicity.Thus, the involvement of nitric oxide (NO) in heroin-induced dopaminergicneurotoxicity could be a reasonable explanation for heroin-induced changes inbrain. Enzymatically derived NO has been implicated in numerous physiolog-ical and pathological processes in the brain. Whereas during development NOparticipates in growing and maturation processes, excess NO production in theadult in response to inflammation, injury, or trauma, participates in both celldeath and repair. The expression and activity of the inducible isoform of NOsynthase (iNOS) play a pivotal role in sustained and elevated NO release. Re-cent evidence suggests that neurons can respond to proinflammatory stimuliand take part in brain inflammation. The effect of heroin abuse on platelet NOproduction and on expression of iNOS in drug addicts submitted to an ul-trarapid detoxification was studied. The NO production was estimated fromthe nitrite concentration, and nitric oxide synthase was determined by Westernblotting analysis. Results showed no difference in nitrite content of restingplatelets between heroin abuser and control groups. However, after plateletstimulation, heroin abusers showed significantly lower nitrite values. TheWestern blotting analysis reinforced these results. After ultrarapid detoxifica-tion, platelet nitrite production in heroin abusers showed no differences com-pared to control subjects. Our results suggest that heroin consumptiondecreases the iNOS synthase expression and platelet NO production. Detoxifi-cation treatment restores these changes.

KEYWORDS: neurotoxicity; nitric oxide; nitric oxide synthase; platelets; heroin;ultrarapid detoxification

Address for correspondence: Tice Macedo, Institute of Pharmacology and Experimental Ther-apeutics, Faculty of Medicine, University of Coimbra, Rua Larga, 3004-504, Coimbra, Portugal.Voice: +351-239-857720; fax: +351-239-823236.

[email protected]

480 ANNALS NEW YORK ACADEMY OF SCIENCES

INTRODUCTION

Reinforcing properties of drugs of abuse have been partly ascribed to an increasein extracellular dopamine (DA) levels in the mesolimbic system, as observed in an-imal models.1 In addition, deamination of DA by monoamine oxidase-B with gener-ation of reactive oxygen species may damage dopaminergic neurons.2 Otherneurotransmitters than DA have been implicated as well.3 Moreover, damage due toexcess glutamate, which changes the permeability of cells to Ca2+ by acting at N-methyl-D-aspartate (NMDA) receptors, is regarded as a major mechanism of neuro-degeneration that occurs during severe hypoxia or ischemia.4,5 Calcium influx, pro-moted by the activation of NMDA receptors, induces the stimulation of nitric oxidesynthase (NOS),6 thus increasing cellular oxidation reactions through the formationof peroxynitrite, which is believed to be the major mediator of NO cytotoxic ef-fects.7,8 In drug addiction, a similar neuronal damage may occur because mesolim-bic DA neurons receive rich glutaminergic inputs from both the neocortex and thesubthalamic nucleus. Although there is no clear evidence that human drug addictssuffer from similar neurotoxic pathways, it is reasonable to conceive it.

The human platelet has been proposed as a peripheral model for the study ofdopamine neurons of the central nervous system, because it stores, releases, and me-tabolizes DA like a dopamine neuronal cell.9 Furthermore, it synthesizes NO and re-active oxygen species, allowing the ex vivo study of the NO system and oxidativestress. Abnormal platelet NOS expression, either inducible NOS (iNOS) or consti-tutive NOS (cNOS), and abnormal NO production by drug addicts may have severalresulting effects. Also, alteration of NO levels may unbalance the release of otherplatelet-derived factors, contributing to endothelial and vascular dysfunction.

The aim of this work was to verify the effects of heroin consumption in parame-ters such as production of NO and expression of iNOS in human platelets. We havetaken the platelet as a neuronal model, with the goal of using the results to make in-ferences about neurotoxicity mechanisms.

MATERIALS AND METHODS

Subjects and Blood Sampling

Venous blood samples were obtained from 16 heroin addicts (14 males and 2 fe-males; mean age 31 ± 8, range 18 to 46 years) attending an ultrarapid detoxificationtherapy with opioid receptor antagonists naloxone and naltrexone (FIG. 1).10−12

Mean heroin abuse duration was 11 ± 7, range 4 to 23 years, while mean daily con-sumption was 1.12 ± 0.5, range 0.25 to 3 g. The study was approved by the EthicsCommittee of Coimbra University Hospital, and the samples collected with in-formed consent. Blood work was done twice, before (time T1) and shortly after (timeT2) detoxification treatment, always between the 10:00 A.M. and 12:30 P.M. period.A control group of 17 healthy volunteers (12 males and 5 females; mean age 30 ±11, range 20 to 56 years) was sampled in a similar fashion.

Blood (10 mL) was collected into tubes containing 3.8% ACD as anticoagulant,and within 1 h centrifuged at 160 × g to separate platelet-rich plasma (PRP) from the

481BATISTA et al.: NITRIC OXIDE AND HEROIN ABUSE

erythrocyte fraction. Platelets were further isolated from PRP by centrifugation at730 × g. Samples were used for NO estimation and for determination of iNOSexpression.

NO Production

Isolated platelets were resuspended in physiological saline buffer containing 145mM NaCl, 5 mM KCl, 1 mM MgSO4.7H2O, 10 mM D-glucose, plus 10 mM HEPESadjusted to pH 7.40; and incubated in the presence or absence of an iNOS stimuluswith interleukin-1-β (IL1-β) (4 ng/mL) (Roche) plus bacterial lipopolysaccharide(LPS) (200 µg/mL) (Sigma) at 37°C for 2 h.

Nitric oxide production was evaluated from the determination of stable end-products nitrite and nitrate. The total nitrite amount was quantified by the Griess di-azotization reaction,13 following the reduction of nitrate to nitrite by treating thesamples with 20 mU/mL nitrate reductase (Sigma) in the presence of 1.44 mMNADH (Sigma), at 37°C for 1 h.14 Total nitrite amount was expressed as the sum ofextracellular and intraplatelet nitrite contents, determined in the supernatants beforeand after lysing cells, respectively.

iNOS Expression

Platelets were lysed by incubation with buffer containing 140 mM NaCl, 15 mMEDTA, 20 mM Tris, 2 mM PMSF, 10% glycerol, 1% NP40, 0.1% Triton X-100, pH8.0, at 4°C for 15 min. Supernatants were stored at –70°C until experimental proce-dures were undertaken. Proteins (80 µg/lane) were size-fractionated electrophoreti-cally in a 8% polyacrilamide gel. Prestained molecular weight marker standards(Bio-Rad) were run in parallel. After completion, proteins were transferred to nitro-cellulose membranes (Schleicher & Schuell) and blocked overnight at 4°C. Mem-branes were incubated with primary polyclonal antibody against iNOS

FIGURE 1. Schematic representation of standard ultrarapid opioid detoxification pro-tocol. Blood samples were collected at time T1, before the beginning of therapy, and at timeT2, immediately after completion.


(Transduction Laboratories) at 1:10,000 dilution for 1 h. Goat anti-rabbit antibodyconjugated to horseradish peroxidase (Bio-Rad) was used as a secondary antibodyat a 1:3,000 dilution, for another 1-h incubation period. Specific protein bands weredetected by enhanced chemiluminescence (ECL) (Amersham) on special autorad-iography film (Amersham).

Statistics

Statistically significant differences with respect to control values were analyzedby one-way analysis of variance and Sheffé’s multiple-comparisons test. Significantdifferences were set at p < 0.05.

RESULTS AND DISCUSSION

Results in FIGURE 2 show the basal levels of platelet NO, as given by the nitrate/nitrite concentration, determined in control group and heroin group at time T1 andtime T2 of detoxification treatment. Experiments were conducted in the absence ofcalcium in the medium. Since platelets have both constitutive and inducible NOSisoforms,15 this feature restricts the functioning of the constitutive Ca2+/calmodulin-dependent enzyme, and allows to study the inducible synthase. Basal NO concentra-tion was significantly lower in drug addicts than in control subjects. However, afteriNOS stimulation with IL1-β plus LPS, NO was still significantly lower during theinfluence of heroin; it increased, however, to control levels after naloxone/naltrex-one administration.

FIGURE 2. NO production by platelets of healthy subjects and of heroin addicts sub-mitted to detoxification with naloxone/naltrexone. Times T1 and T2 refer to determinationsmade prior and after treatment, respectively. Induction of iNOS was achieved with IL1-β(4 ng/mL) plus LPS (200 µg/mL). Results are expressed as mean ± SEM of NO nmol per109 platelets; ∗p < 0.05, significantly different from control group.


The net NO production, relative to the induction of iNOS, was estimated by sub-tracting data obtained in the absence of stimulus from data collected under stimula-tion. As depicted in FIGURE 3, there was a trend to decreased NO production underheroin consumption but a significant NO increase after drug withdrawal. This resultwas further stressed by expressing variation in NO levels in terms of percentage, asshown in FIGURE 4. Following detoxification, a strong increase in iNOS activity wasobserved.

To examine whether the small NO production, observed following iNOS stimu-lation in samples from subjects under the effect of heroin, was due to lower expres-sion of the enzyme or to expression of a dysfunctional enzyme, Western blottinganalysis of iNOS from T1 blood samples was performed. The results of this experi-ment are shown in FIGURE 5. Immunoblotting revealed a band corresponding toiNOS with estimated molecular weight of approximately 200 kDa, which is in agree-ment with earlier reports on the isolation and characterization of the platelet-inducibe NOS isoform.15 Densitometry analysis of the bands confirmed a lower ex-pression of iNOS in heroin addicts compared to normal subjects.

Overall, data from the present study suggest that heroin consumption decreasesboth the iNOS synthase expression and platelet NO production, and that detoxifica-tion with opioid antagonists restores these changes. These results are in very goodagreement with those reported by Lysle and How,16 who found that heroin adminis-tration to rats induced a pronounced reduction of iNOS expression in the spleen,lung, and liver, along with a reduction in NO plasma levels. Furthermore, naltrexonewas observed to inhibit the heroin-induced effects. Also, data agree with those re-ported by Sullivan et al.,17 who observed an increase in NO formation during mor-phine withdrawal in rats. However, it differs from those reported by Zhou et al.,18

FIGURE 3. Production of NO due to induction of platelet iNOS with IL1-β (4 ng/mL)plus LPS (200 µg/mL). Times T1 and T2 refer to determinations made prior and after heroindetoxification, respectively. Results are expressed as mean ± SEM of NO nmol per 109 plate-lets; ∗p < 0.05, significantly different from T1 group.


FIGURE 4. Percentage of increase of NO production after stimulation of iNOS activitywith IL1-β (4 ng/mL) plus LPS (200 µg/mL). Times T1 and T2 refer to determinations madeprior and after heroin detoxification, respectively. Data are expressed as mean ± SEM of per-centage of NO increase relative to basal conditions; ∗p < 0.05 and #p < 0.05, significantlydifferent from control and T1 groups, respectively.

FIGURE 5. Western blotting analysis of iNOS extracted from platelets of control andheroin addicts subjects. Inset graph represents relative band intensities. Data are expressed asmean ± SEM of arbitrary density units; ∗p < 0.05, significantly different from control group.


who verified an increase in human NO plasma levels with prolonged heroin abuseand increased drug daily consumption.

When conceiving this study, we aimed to address whether a link would exist be-tween dopaminergic neurotoxicity and prolonged heroin abuse. The hypothesis re-lating NO to neurodegeneration relied on the fact that DA neurons, throughpromoting glutamate/Ca2+ influx pathways, would unbalance NOS normal regula-tion, raising NO levels, thus triggering a cascade of cytotoxic effects. Our data, how-ever, suggest that heroin abuse decreases both iNOS expression and NO production.One first pertinent question is whether any of the substances administered along withnaloxone and naltrexone has effects on the platelet NO metabolism. If so, results areconditioned by its use during detoxification. Second, only a small amount of litera-ture is available regarding human studies and heroin, the bulk of it concerning ani-mal studies using morphine. Whether humans/experimental animals and heroin/morphine exert similar effects on NO is yet to be determined.

The progressive nature of drug addiction coupled with the slow and protacted de-generation of mesolimbic neurons may present an opportunity for therapeutic inter-vention. Accessing the NO platelet pathway was a first attempt to provide insightsinto the neuronal pathway. We feel this is an important open field requiring furtherinvestigation. We intend to extend future studies to the effects of heroin and mor-phine on animal and cellular lines models, as well as to the monitoring of other sig-nificant molecules, such as peroxynitrite and 3-nitrotyrosine.

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Nitric Oxide Production and Nitric Oxide Synthase Expression in Platelets from Heroin Abusers before and after Ultrarapid Detoxification