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Enhanced Cellular Adenosine Uptake Limits AdenosineReceptor Stimulation in PatientsWith Hyperhomocysteinemia
Niels P.Riksen,Gerard A.Rongen,Godfried H.J.Boers,Henk J.Blom,Petra H.H.van den Broek,Paul Smits
Objective—Endogenous adenosine has several cardioprotective effects. We postulate that in patients with hyperhomocys-teinemia increased intracellular formation of S-adenosylhomocysteine decreases free intracellular adenosine.Subse-quently,facilitated diffusion ofextracellularadenosine into cellsthrough dipyridamole-sensitive transportersisenhanced, limiting adenosine receptor stimulation. We tested this hypothesis in patients with classical homocystinuria(n⫽9,plasma homocysteine 93.1⫾24.7mol/L) and matched controls (n⫽8,homocysteine 9.1⫾1.0).
Methods and Results—Infusion of adenosine (0.5, 1.5, 5.0, and 15.0g/min/dL forearm) into the brachial artery increasedforearm blood flow, as measured with venous occlusion plethysmography, to 2.9⫾0.4, 4.3⫾0.5, 5.6⫾1.1, and 9.6⫾2.1in the patientsand to 2.8⫾0.6,4.4⫾1.0,9.0⫾1.7,and 17.0⫾3.1 mL/min/dL in controls(P⬍0.05).However,adenosine-induced vasodilation in the presence of dipyridamole (100g/min/dL) was similar in both groups (P⫽0.9).Additionally,in isolated erythrocytes,adenosine uptake was accelerated by incubation with homocysteine (half-time6.4⫾0.3 versus8.1⫾0.5 minutes,P⬍0.001) associatedwith increasedintracellularformationofS-adenosylhomocysteine (P⬍0.0001).
Conclusions—In hyperhomocysteinemia,adenosine-induced vasodilation is impaired butis restored by dipyridamole.Accelerated cellular adenosine uptake probably accounts for these observations.These impaired actions of adenosinecould well contribute to the cardiovascular complications of hyperhomocysteinemia. (Arterioscler Thromb Vasc Biol.2005;25:109-114.)
Key Words: adenosine hyperhomocysteinemia dipyridamole forearm nucleoside transport
H yperhomocysteinemia is an independent risk factor foratherosclerosis and thromboembolism.Itis poorly un-
derstood which mechanism is responsible for these cardio-vascular complications.Recently,we and others have drawn attention to a new
hypothesis,focusing on the influence of homocysteine onthe metabolism of the endogenous nucleoside adenosine.1,2
According to this hypothesis, a homocysteine-induced fallin extracellular adenosine contributes to the cardiovascularsequelae of hyperhomocysteinemia.Fundamental to this is thereversibility of the reaction in which S-adenosylhomocysteine(AdoHcy) is hydrolyzed to form homocysteine and adenosine.3
Although theequilibrium constantof thisreaction favorsAdoHcy synthesis,under physiologicalconditions AdoHcy ishydrolyzed to homocysteine and adenosine, because both reac-tion products are rapidly metabolized.In hyperhomocysteine-mia, the reaction shifts toward AdoHcy synthesis at the expenseof free intracellular adenosine.Subsequently,facilitated diffu-sion of extracellular adenosine into the cells through the dipyr-
sine induces severaleffects,which could protectagainstthe developmentof atherosclerosisand thrombosisandagainst ischemia-reperfusion injury.1,4Particularly in situ-ations of hypoxia or ischemia,when the concentration ofadenosine increases rapidly,these effects work in concertto protectthe affected tissue.5 Previousanimalstudiessuggestthatthe effectof intracellular AdoHcy formationon thetransmembranousadenosinegradientand thusdiffusion of extracellular adenosine into the cells is mostpronounced in these very situations of high concentrationsof adenosine,thus limiting adenosine receptor stimulationwhen mostneeded.6,7 Therefore,we speculatethatinhyperhomocysteinemia,decreased extracellular adenosineconcentrations contribute to the developmentof the asso-ciated cardiovascular problems.
Original received June 12,2004; final version accepted November 1,2004.From the Departments of Pharmacology-Toxicology (N.P.R.,G.A.R.,P.H.H.v.d.B.,P.S.),InternalMedicine (N.P.R.,G.A.R.,G.H.J.B.,P.S.),and
Pediatrics (H.J.B.),University Medical Centre Nijmegen,the Netherlands.Correspondence to Niels P. Riksen, MD, Department of Pharmacology-Toxicology 233, University Medical Centre Nijmegen, Geert Grooteplein 21,
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In the presentstudy,we addressed this issue in patientswith severe hyperhomocysteinemia.We estimated basalin-travascular and muscle interstitial adenosine concentration bymicrodialysis.Secondly,we measured adenosine-inducedforearm vasodilation.According to our hypothesis,acceler-ated cellular adenosine uptake would decrease the amount offree extracellular adenosine able to stimulate adenosine re-ceptorsand,consequently,would attenuateadenosine-induced vasodilation in thispatientgroup.Inhibition ofcellular adenosine uptake by dipyridamole should restore thisdiminished response. Additionally, we aimed to more directlydemonstratehomocysteine-inducedincreasedintracellularformation of AdoHcy and subsequent accelerated adenosineuptake in isolated human erythrocytes.
MethodsSubjectsAfterapprovalofthe localethics committee,adultpatients withhyperhomocysteinemia due to homozygouscystathionine–syn-thase deficiency from our outpatient clinic were asked to participateif their fasting total plasma homocysteine was more than 20mol/Ldespite treatment. Exclusion criteria were mental retardation, previ-ous vascularevents,asthma,and oralanticoagulation.Twelvepatients were considered eligible, and nine agreed to participate andsigned informed consent. Their homocysteine-lowering therapy con-sisted of vitamin B6 (250 to 750 mg, n⫽9), folic acid (5 mg, n⫽9),vitamin B12 (10g orally each day or 1 mg intramuscularly eachtwo months,n⫽7),and betaine anhydricum (6 g,n⫽7).Furthertreatment consisted only of acetylsalicylic acid (80 mg in one patient;stopped one week before the experiment) and alendronic acid (70 mgweekly in 2 patients).A controlgroup of eighthealthy volunteerswas composed with similarage and body massindex asthepatient group.
InstrumentationAll tests were performed in the morning in a temperature-controlledlaboratory(23°C)afteran overnightfastand 24 hoursofcaffeine abstinence.Afterlocalanesthesia(xylocaine2%),a microdialysisprobe
(CMA/70 microdialysis catheter,Microdialysis AB)was insertedinto the flexor digitorum superficialis muscle of the nondominantarm,guided by a 14-gauge venflon cannula.An identicalmicrodi-alysis probe was inserted into a deep antecubitalvein of the samearm. Each probe was connected to a microdialysis pump (CMA/107microdialysis pump,Microdialysis AB) and continuously perfusedwith isotonic saline at2 L/min.The effluentwas collected at
15-minute intervals to obtain 30-L samples (dialysate).Sampleswere stored at ⫺20°C until analysis.Subsequently,the brachialartery ofthe nondominantarm was
cannulated and forearm blood flow (FBF) was measured in each armusing mercury-in-silastic venous occlusion plethysmography as de-scribed previously.8 Each drug dosage was infused for 5 minutes.In the first hour of the study, blood was drawn for determination
of plasmatotalhomocysteine,AdoHcy,S-adenosylmethionine(AdoMet),vitamin B6,folate,vitamin B12,and cholesterol.
Experimental ProtocolImmediately afterinsertion ofthe microdialysis probes,dialysatesampling was started. Two hours after insertion, both microdialysisprobes were removed and suspended in isotonic saline for in vitrocalibration,as previously described.9
Subsequently,baseline FBF wasmeasured during infusion ofsaline followed by infusion of increasing dosages of adenosine (0.5,1.5, 5, and 15g/min/dL forearm). After 30 minutes of equilibration,baseline FBF measurementwas repeated,followed by infusion ofdipyridamole (100g/min/dL) and increasing dosages of adenosine(0.15,0.5,and 1.5g/min/dL) on top of dipyridamole infusion.Incombination with dipyridamole,we used lowerconcentrations ofadenosine because of the well-known potentiating effectof dipyri-damole on adenosine-induced vasodilation.10 Finally,maximalva-sodilation was measured during postocclusive reactive hyperemia totest for possible structural vascular changes in the patient group, asdescribed previously.8
Adenosine Uptake in Isolated ErythrocytesIn 6 additional healthy volunteers, erythrocytes were isolated for invitro experiments.In hyperhomocysteinemia,erythrocytes are rele-vant in the regulation of circulating endogenous adenosine becauseadenosine is efficiently taken up by erythrocytes through the dipyridam-ole-sensitive transporter and because homocysteine and AdoHcy areincreased in erythrocytes of patients with hyperhomocysteinemia.11–13
Freshly isolated erythrocytes were resuspended in MOPS buffer toobtain a 2% solution. Fifty-L portions were incubated at 37°C withL-homocysteine (100mol/L) in DTT and with DTT alone for 10minutes (paired experiments). Subsequently, adenosine was added ina final concentration of 1mol/L. After 0, 3, 6, 10, and 15 minutes,adenosine uptake and deamination were completely blocked withhigh-dose dipyridamole (10mol/L) and erythro-9-(2-hydroxynon-3-yl)-adenine (8mol/L),respectively.Subsequently,after centrif-ugation through a dibutylphtalate layer, the adenosine concentrationin the supernatant and the AdoHcy concentration in the erythrocyteswere determined.The effectof homocysteine on adenosine uptakewas maximal after 6 minutes of uptake. To investigate the adenosineconcentration dependency of this homocysteine effect, an additionalseries of experiments was conducted with 6 minutes of adenosineuptake butwith a variable adenosine concentration ranging from0.125mol/L to 2mol/L (n⫽4).Finally,we determined theinhibiting effect of dipyridamole (0.2mol/L, 5-minute incubation)on theaccelerating effectof homocysteinewith 6 minutesofadenosine uptake (n⫽2).
Drugs and SolutionsSolutions of adenosine (Adenocor,Sanofi-Synthelabo) and dipyri-damole (Persantin,BoehringerIngelheim)were freshly preparedwith NaCl0.9% as solvent.L-homocysteine was freshly preparedimmediately before each in vitro experimentfromL-homocysteinethiolactone as previously described.14
Analytical ProceduresDialysate adenosine concentration was determined by high-perfor-mance liquid chromatography (HPLC)with reversed-phase ion-pairing separation and UV detection.Plasma homocysteine wasdetermined by reverse phase (RP)-HPLC as described previously.15
Plasma AdoHcy and AdoMetwere determined by tandem massspectrometry,based on the work of Struys et al.16
Figure 1. Simplified representation of the biochemicalreactionsrelevant to our hypothesis. Under normalconditions, adenosineis continuously produced by the hydrolysis of AdoHcy (left). Inhyperhomocysteinemia, adenosine production from AdoHcyhydrolysis is hampered or the reaction is even reversed, andAdoHcy willaccumulate at the expense of free adenosine (right).Dipyridamole inhibits cellular adenosine uptake by blockade ofthe ENT. AMP indicates adenosine mono-phosphate; 5⬘-NT,5⬘-nucleotidase; ADA, adenosine deaminase; AK, adenosinekinase; Ino, inosine; Met, methionine; ENT, equilibrative nucleo-side transporter.
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StatisticsValues are expressed as mean⫾SE unless otherwise stated. P⬍0.05is considered statistically significant. Because plasma concentrationsof homocysteine,AdoHcy,AdoMet,and vitamins did notshow aGaussian distribution (P⬎0.1; Shapiro–Wilk test for normality), theMann–Whitney testwas used to compare groups.Otherbaselineparameters were normally distributed, and consequently a Student ttest was used.To compareadenosine-induced vasodilation between thetwo
groups,a repeated measures ANOVA was used.Finally,for eachsubject,the area underthe curves (AUC)ofchange in FBF wascalculated for the adenosine-induced vasodilation with and withoutdipyridamole.The ratio ofthe AUC (with dipyridamole)and theAUC (withoutdipyridamole)was used to quantify the effectofdipyridamole. Because these ratios were not normally distributed, aMann–Whitney test was used to compare groups.In the in vitro experiments, the decrease of extracellular adenosine
in timewas fittedaccordingto one phaseexponentialdecay(GraphPad Prism version 4.00 for Windows,GraphPad Software),and half times were compared with paired Student t test.
ResultsBaseline CharacteristicsDemographic data are shown in Table 1.Plasma concentra-tionsof totalhomocysteine,AdoHcy,and AdoMetwerehigherin thepatientgroup (P⬍0.005).Treatmentwithhigh-dose folic acid and pyridoxine resulted in higher plasmaconcentrations of folate and vitamin B6 in the patient group(P⬍0.005).In one patient,we were notable to inserttheintravenous probe and intraarterial cannula, and consequentlyonly data on interstitial adenosine were available.
Microdialysis ExperimentsImmediately afterintramuscularinsertion,dialysate adeno-sine concentration is known to be high because of myocytedamage and decreases to baseline level within one hour.9 To
estimate basalinterstitialadenosine concentration,we aver-aged thedialysateconcentration ofthetwo consecutivedialysate samplestaken 1.5 hoursafterinsertion.In thepatientgroup,basaladenosine concentration was96⫾21nmol/L (n⫽7; 2 patients were excluded because of extremelylow concentration of creatine and phosphocreatine in the firstmicrodialysis sample, indicating misplacement of the probe),whereas in the controlgroup basaladenosine concentrationwas 73⫾9 nmol/L (n⫽8,P⫽0.3).The dialysate adenosine concentration from the intravas-
cular probe was atsteady-state immediately after insertion.Therefore, we averaged the values of all dialysate samples toone value.In the patientgroup this baseline concentrationyielded 142⫾33 nmol/L (n⫽8), whereas in the control groupit yielded 135⫾25 nmol/L (n⫽8,P⫽0.7).Adenosine recov-ery of the intramuscular and intravascular probes was 48⫾4%and 47⫾5% for patients and 47⫾4% and 39⫾2% for con-trols,respectively (P⫽0.9 and 0.2,respectively).
Plethysmography ExperimentsBaseline FBF in the infused arm was 2.0⫾0.2 and 2.5⫾0.5mL/min/dL forpatientsand controls,respectively (n⫽8;P⫽0.4).During infusion ofincreasing adenosine dosages,FBF in the patient group was 2.9⫾0.4, 4.3⫾0.5, 5.6⫾1.1, and9.6⫾2.1 mL/min/dL,respectively (Figure 2).In the controlgroup,FBF was 2.8⫾0.6,4.4⫾1.0,9.0⫾1.7,and 17.0⫾3.1mL/min/dL,respectively.This adenosine-induced vasodila-tion was attenuated in the patient group (P⬍0.05).After 30-minute equilibration,baseline FBF was 2.7⫾0.3
and 3.1⫾0.7 mL/min/dL in the patientgroup and controlgroup,respectively (n⫽8; P⫽0.6).Infusion of dipyridamoleincreased FBF to 4.3⫾0.5 mL/min/dL in patientsand to4.7⫾1.0 in controls (P⫽0.7 between groups).Subsequentinfusion of adenosine on top dipyridamole increased FBF to10.9⫾1.6,16.3⫾2.6,and 27.2⫾2.9 mL/min/dL in patientsand to 8.8⫾1.4, 14.8⫾2.4, and 24.4⫾4.0 in controls, respec-tively (P⫽0.9). The ratio of the AUC for adenosine-inducedvasodilation with dipyridamole and withoutdipyridamolewas 0.40⫾0.08 in the patientgroup and 0.16⫾0.03 in thecontrolgroup (P⬍0.05).Moreover,with both experimentalgroupstaken together,therewas a negativecorrelationbetween totalplasma homocysteine concentration and the
TABLE 1.Demographic Characteristics of the Study GroupsPatients Controls
sion.‡P⬍0.005 compared to controlgroup.§Upper limit of detection 270 nmol/L.
Figure 2. Adenosine-induced vasodilation without dipyridamole(squares) and during coinfusion with dipyridamole (triangles) inpatients (open symbols; mean⫾SE, n⫽8) and controls (filledsymbols; n⫽8).
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AUC for adenosine-inducedvasodilation(Spearmanr⫽⫺0.53, P⫽0.035)and a positivecorrelation betweenplasma homocysteine and the effectof dipyridamole ex-pressed asthe ratio ofthe AUC’sforadenosine-inducedvasodilation with and without concomitant infusion of dipyr-idamole (Spearman r⫽0.59,P⫽0.015).During theexperiment,neithermean arterialpressure
(MAP) nor the FBF in the control arm differed between thepatientgroup and the controlgroup (P⬎0.1;ANOVA forrepeated measures).The effectsofadenosine infusion onheart rate and blood pressure are shown in Table 2. Concom-itant infusion of adenosine with dipyridamole was associatedwith an increase in heartrate in both groups (ANOVA forrepeated measures,P⬍0.001).Also,infusion ofadenosinewithout dipyridamole was associated with an increase in heartrate in the patientgroup (P⬍0.05).Blood pressure was notinfluenced by adenosine infusion. There were no differencesbetween both groups.Finally,minimalforearm vascular resistance (MAP/FBF)
during postocclusive reactive hyperemia was 2.1⫾0.2 and2.4⫾0.2 AU for patients and controls, respectively (P⫽0.3).
Adenosine Uptake in Isolated ErythrocytesPlasma homocysteine of the 6 subjects in this study averaged9.1⫾0.9mol/L. Adenosine uptake into the erythrocytes andsubsequent metabolism results in a decrease of extracellularadenosine in time (Figure 3a).Adenosine uptake was accel-erated by incubation with homocysteine (half time 6.4⫾0.3versus 8.1⫾0.5 minutes,P⬍0.001).Intracellularly,AdoHcyincreased in time in cells incubated with homocysteine,butnotin controlsamples (Figure 3b;P⬍0.0001,ANOVA forrepeated measures). At 6 minutes, adenosine in the superna-tantwas lowerin the homocysteine-incubated cells.Thisabsolute homocysteine-induced difference in adenosine con-centration significantly increased when the initial adenosineconcentration was varied from 0.125mol/L to 2.0mol/L(Figure 3c and 3d; P⬍0.05, ANOVA for repeated measures).Finally, dipyridamole significantly inhibited the accelerat-
ing effectof homocysteine on cellularadenosine uptake.Homocysteinedecreased thefreeextracellularadenosine
concentration after6 minutesof adenosineuptakewith14.5⫾0.5% in the absence and with 2.9⫾0.5% in the pres-ence of dipyridamole (Figure 4; P⬍0.05,n⫽2).
DiscussionIn the presentstudy,we show for the firsttime thatadeno-sine-induced forearm vasodilation is attenuated in patientswith hyperhomocysteinemia due to classical homocystinuria,and that this attenuation is completely restored by coinfusionof the adenosine uptake inhibitor dipyridamole.The association between hyperhomocysteinemia and ath-
erosclerosis and thrombosis was first established in patientswith classicalhomocystinuria.17 Withouttreatment,50% ofsuchlike patients experience a vascular event before the ageof 30 years.18Treatment is aimed at minimizing the biochem-icalabnormalities and consists ofpyridoxine (vitamin B6)and folic acid and,ifnecessary,betaine anhydricum andvitamin B12.19This treatment regimen significantly improvesvascular outcome.20 Our patients continued to have elevatedplasma homocysteine levels despite treatment. Because of thedefective degradation of homocysteine to cystathionine,ho-mocysteine can only be remethylated to methionine or con-verted to AdoHcy. This altered metabolic profile is illustratedby theincreased plasmaconcentrationsof AdoHcy and
TABLE 2.Systemic Effects of Adenosine InfusionHeart Rate Mean ArterialPressure
†baseline 2 (P⬍0.001).No differences between groups.
Figure 3. Time-course of adenosine concentration in the super-natant (a) and the intracellular AdoHcy concentration (b) afteraddition of 1mol/L of adenosine to erythrocytes. Also shownis the adenosine concentration in the supernatant 6 minutesafter the addition of variable concentrations of adenosine (c;n⫽4) and the absolute homocysteine-induced difference inadenosine concentration after 6 minutes of uptake (d; n⫽4).Open squares indicate incubation with homocysteine; filledsquares, controlgroup.
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AdoMetin our patientgroup.Previous in vivo experimentson vascularfunction in this patientgroup are scarce anddemonstrated impaired flow-mediated dilation and carotidartery wall hypertrophy.21–23
In the present study, adenosine-induced forearm vasodila-tion wasreduced in patientswith hyperhomocysteinemia.Based on the observations thatdipyridamole restores thisimpaired vasodilation and that in erythrocytes homocysteineaccelerates cellular adenosine uptake,we conclude thatthisimpaired adenosine-induced vasodilation is caused by accel-erated cellular adenosine uptake. Adenosine-induced vasodi-lation ispartly endothelium-dependent.24 We did notusealternative endothelium-dependentvasodilatorsto testforendothelial function in our patient group. However, as shownby previous studies in patients with classical homocystinuria,endothelialdysfunction could wellalso be presentin ourpatients. Could endothelial dysfunction or structural vasculardamage,both previously described in a comparable patientgroup, challenge our conclusion? In our opinion this is not thecase.The observation that dipyridamole restored adenosine-induced vasodilation suggeststhataccelerated adenosineuptake through the equilibrative nucleoside transporter, ratherthan endothelialdysfunction,accountsfortheattenuatedvasodilation.Itneedsto be realized thatdipyridamole isproposed to have alternative mechanisms of actions besidesinhibition of nucleoside transport.25 Phosphodiesterase inhi-bition could theoretically potentiate endothelium-dependentcGMP-mediated dilation,thusimproving thisportion ofadenosine-induced vasodilation.However,in an identicalexperimentalmodelas in the presentstudy,our group haspreviously shown thatdipyridamole-induced localvasculareffects are indeed solely caused by adenosine uptake inhibi-tion.Dipyridamole potentiated the vasodilatorresponse toadenosine,10and dipyridamole-induced vasodilation (100g/min/dL) was inhibited by the adenosine receptor antagonisttheophylline.26Moreover, we showed in isolated erythrocytesthathomocysteine indeed accelerates cellular adenosine up-take,which is counteracted by dipyridamole.Although the
effects of homocysteine in this model are rather modest, thesein vitro observations provide additional evidence that accel-erated cellular adenosine uptake mightaccountfor the ob-served impaired adenosine-induced vasodilation.Minimalforearm vascular resistance was similar in both
groups. This parameter accurately reflects structural arteriolarstatus,27 indicating thatin our patientgroup,atleastin theforearm vascularbed,no structuralvascularchanges werepresent.Itshould be mentioned thatreactive hyperemia isshown to be partially adenosine-dependent.28This portion ofthe hyperemia in our study would theoretically be diminishedin the patients with hyperhomocysteinemia.However,be-cause ofthe maximalstimulusforvasodilation after13minutes of ischemia,other mediators probably compensatefor this decrease in adenosine-induced vasodilation.27
In previousexperiments,itwas shown in guineapighearts29,30and ratbrain31 thatperfusion with homocysteine(thiolactone)decreased organ releaseof adenosine.Theresults from ourin vitro experiments showed thatalso inhuman erythrocytes, homocysteine accelerates cellular uptakeof adenosine. Moreover, we demonstrated that this is indeedassociated with increased intracellular formation of AdoHcy.Finally,this effectof homocysteine is more pronounced athigherconcentration ofadenosine (Figures 2 and 3).Thisobservation could wellexplain why baseline endogenousadenosine, as estimated by microdialysis, was not reduced inour patient group.This is in strong contrast to the study byChen etal,which demonstrated an ⬇50% reduction inendogenousbaselineadenosineconcentration induced bymild elevation ofplasma homocysteine from 6.7⫾0.4 to14.7⫾0.5mol/L.2 This discrepancy could be caused by thedifferences between man and rat concerning protein-bindingof homocysteine,6 AdoHcy hydrolase activity,3 and charac-teristics ofthe equilibrative nucleoside transporter.32 Also,the experimental methionine-induced hyperhomocysteinemiain the ratmodeldiffers from the hyperhomocysteinemia inourpatientgroup,possibly affecting tissueand cellulardistribution ofhomocysteine.But most importantly,theresults from ourin vitro studies suggestthatthe effectofAdoHcy synthesis on the rate of cellular adenosine uptake islimited to situations of high extracellular adenosine concen-trations, as for example in ischemia, when myocardial inter-stitial adenosine concentration can increase up to 40-fold inpigs.7 A recent microdialysis study on the pig heart showedthatlocalapplication ofhomocysteinereduced dialysateadenosineconcentrationduringhypoxia,butnotduringnormoxia.7 Likewise, Kloor et al concluded from a rat studythathomocysteine isthe rate-limiting factorforAdoHcysynthesis in hypoxic conditions when tissue levels of adeno-sine are elevated, whereas in normoxia the availability of freeadenosine is rate-limiting.6This suggests that also in vivo, theeffectof homocysteine on adenosine concentration is morepronounced and therefore more easily detected in situationsofhigh concentrations ofadenosine than undernormoxicbaseline conditions.Unfortunately,for practicalreasons wewere not able to collect microdialysis samples during infusionof adenosine or during ischemia.We have shown that the vasodilating effect of adenosine,
which wasreduced in hyperhomocysteinemia,was com-
Figure 4. Extracellular concentration of adenosine after 0 and 6minutes of adenosine uptake by isolated human erythrocytes inthe absence (open bars) and presence (filled bars) of homocys-teine. Dipyridamole significantly reduces the accelerating effectof homocysteine on adenosine uptake (P⬍0.05, n⫽2).
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pletely restored by the nucleoside uptake inhibitor dipyridam-ole. Extrapolating this finding, dipyridamole would be bene-ficialin patients with hyperhomocysteinemia by preservingthe protective cardiovascular effects of adenosine in hypoxiaor ischemia. To our best knowledge, dipyridamole has neverbeen tested systematically in such patients in clinical trials.Severallimitations ofthe presentstudy need to be dis-
cussed.First,we used a group ofpatientswith classicalhomocystinuria,using high dosesof vitamins,includingvitamin B6 and folicacid.Itremainsto beestablishedwhether changes in adenosine metabolism are also importantin patients with mild hyperhomocysteinemia. Considering thevitamin therapy in our patient group, we are not aware of anyactions of these vitamins on nucleoside transport.Secondly,considering the large interindividual variation,microdialysismay lack sufficient sensitivity to exclude subtle differences inbaselineadenosineconcentration between thetwo studygroups.Moreover,the valuesobtained with microdialysismightnotreflectthe adenosine concentrations in specificmicroenvironments,such as near the endothelial lining.In conclusion, in patients with severe hyperhomocysteine-
mia,adenosine-induced effects are impaired,which couldcontribute to the cardiovascular complications of this disease.
AcknowledgmentsN.P.R. is a MD– clinical research trainee financially supported by theNetherlands Organization for Scientific Research (ZonMw). G.A.R.is a fellow of the Royal Netherlands Academy of Arts and Sciences.H.J.B.isan established investigatorof theNetherlandsHeartFoundation (D97.021). The contribution of P.H.H.v.d.B. was finan-cially supported by the European Union (project number QLK1-CT-2000-00069).The authors thank D.Oppenraaijfor technicalassis-tance in the determinations of plasma homocysteine,AdoHcy andAdoMet and E. van Balenforher contributionto theinvitro experiments.
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