Adenosine concentration in the porcine coronary artery wall and A2A receptor involvement in hypoxia-induced vasodilatation
1 Department of Cardiology, Center for Cardiovascular Research, Aalborg Hospital, Aarhus University Hospital, Denmark
2 Department of Pharmacology, University of Aarhus, Denmark
3 Institute of Physiology, Medical Faculty Carl Gustav Carus, Technical University Dresden, Germany
4 Department of Endocrinology M, Aarhus Sygehus, Aarhus University Hospital, Denmark
, http://www.100md.com 5 Wallenberg Laboratory, Sahlgrenska University Hospital, Gteborg, Sweden
Abstract
We tested whether hypoxia-induced coronary artery dilatation could be mediated by an increase in adenosine concentration within the coronary artery wall or by an increase in adenosine sensitivity. Porcine left anterior descendent coronary arteries, precontracted with prostaglandin F2 (10–5M), were mounted in a pressure myograph and microdialysis catheters were inserted into the tunica media. Dialysate adenosine concentrations were analysed by HPLC. Glucose, lactate and pyruvate were measured by an automated spectrophotometric kinetic enzymatic analyser. The exchange fraction of [14C]adenosine over the microdialysis membrane increased from 0.32 ± 0.02 to 0.46 ± 0.02 (n= 4, P < 0.01) during the study period. At baseline, interstitial adenosine was in the region of 10 nM which is significantly less than previously found myocardial concentrations. Hypoxia (PO2 30 mmHg for 60 min, n= 5) increased coronary diameters by 20.0 ± 2.6% (versus continuous oxygenation –3.1 ± 2.4%, n= 6, P < 0.001) but interstitial adenosine concentration fell. Blockade of adenosine deaminase (with erythro-9-(2-hydroxy-3-nonyl-)-adenine, 5 μM), adenosine kinase (with iodotubericidine, 10 μM) and adenosine transport (with n-nitrobenzylthioinosine, 1 μM) increased interstitial adenosine but the increase was unrelated to hypoxia or diameter. A coronary dilatation similar to that during hypoxia could be obtained with 30 μM of adenosine in the organ bath and the resulting interstitial adenosine concentrations (n= 5) were 20 times higher than the adenosine concentration measured during hypoxia. Adenosine concentration–response experiments showed vasodilatation to be more pronounced during hypoxia (n= 9) than during normoxia (n= 9, P < 0.001) and the A2A receptor antagonist ZM241385 (20 nM, n= 5), attenuated hypoxia-induced vasodilatation while the selective A2B receptor antagonist MRS1754 (20 nM, n= 4), had no effect. The lactate/pyruvate ratio was significantly increased in hypoxic arteries but did not correlate with adenosine concentration. We conclude that hypoxia-induced coronary artery dilatation is not mediated by increased adenosine produced within the artery wall but might be facilitated by increased adenosine sensitivity at the A2A receptor level.
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Introduction
Coronary artery dilatation to hypoxia is an important protective response that increases flow to endangered myocardium. The exact mechanisms causing the dilatation remain undefined but could be mediated by (1) local metabolites from the coronary artery endothelium or smooth muscle cells (Frbert et al. 2002), (2) metabolites released from the myocardium, (3) blood-borne metabolites, or (4) a direct effect of hypoxia on the membrane potential (Kobayashi et al. 1998) or the contractile apparatus (Frbert et al. 2005) of vascular smooth muscle cells.
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Adenosine is both a precursor and a metabolite of adenine nucleotides. The rate of adenine nucleotide degradation and cellular production and release of adenosine increase during myocardial ischaemia (Hall et al. 1995; Van Wylen, 1994). The potent vasodilator properties of adenosine have made it a candidate for hypoxia-induced vasodilatation. In addition, the very short half-life of adenosine in human blood plasma (Moser et al. 1989) suggests a highly local action of this purine at the site of production. Thus, adenosine meets the criteria for a local metabolic vasodilator. An extensive number of studies supports that adenosine could be one of the mediators coupling metabolic requirements with coronary blood flow (Merrill et al. 1986; Nakhostine & Lamontagne, 1993; Laxson et al. 1993; MacLean et al. 1998). However, experimental data are equivocal and others have found only a limited role for adenosine in hypoxia-induced (Gewirtz et al. 1987; Lee et al. 1992) and exercise-induced (Bache et al. 1988; Duncker et al. 1998) coronary dilatation. The finding that the vasorelaxation response to adenosine is virtually absent in hypoxic porcine carotid strips (Barron & Gu, 2000) further questions the importance of this metabolite in sustained hypoxic vasodilatation.
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Adenosine production may occur in the cytosol as well as in the extracellular region. Rapid enzymatic conversion of adenosine is provided by adenosine kinase and adenosine deaminase, keeping the cytosolic concentration in the nanomolar range (Deussen, 2000). Because of high intracellular rates of adenosine rephosphorylation and deamination the cytosolic concentration is normally below the extracellular concentration. This is the basis for the finding that adenosine plasma concentration increases during membrane transport blockade (Deussen, 2000). It is unknown whether adenosine acting on coronary artery smooth muscle cells and potentially evoking vasodilatation is derived from a vascular source or from adjacent myocardium. While for intramyocardial vessels both sites of adenosine production might be of physiological significance, in epicardial vessels only a vascular site of adenosine production can be imagined. To resolve this question, it is necessary to measure adenosine production in the vessel wall in response to a stimulus known to enhance adenosine production. A decrease in oxygen tension is a well documented stimulus of cardiac adenosine production (Deussen & Schrader, 1991). The present study was undertaken to test whether hypoxia increases coronary artery interstitial adenosine concentration sufficiently to induce vasodilatation. Thus, we measured coronary artery diameters while sampling interstitial adenosine by means of microdialysis in the arterial media layer. We also tested whether sensitivity to adenosine was changed during hypoxia compared with normoxia.
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This is the first report to measure adenosine within the wall of a coronary artery during changes in vessel diameter in response to hypoxia.
Methods
Hearts from Landrace-Yorkshire hogs were obtained at a local slaughterhouse. Immediately after killing the aorta was cannulated and the coronary circulation perfused with a physiological salt solution (PSS) containing 5.5 mM glucose, bubbled with 5% CO2 in O2 and buffered with Hepes. The hearts were bathed in PSS at 5°C for approximately 2 h until the start of the experiment. The left anterior descending coronary artery (LAD) was carefully dissected and the proximal 3–4 cm of the artery left intact. The external diameter of these vessel preparations ranged from 2.93 mm to 5.13 mm (mean 4.00 ± 0.08 mm, n= 38).
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Pressure myograph
Cylindrical arterial segments, 2 cm long, were mounted at both ends on stainless steel cannulae and fastened with sutures in PSS bubbled with 5% CO2 in O2 in a vessel chamber. The temperature was raised to 37°C. Before measurements the segments were stretched to the in situ length by operating a micrometer device (we have previously documented that length and distension are of importance for reactivity (Frbert et al. 1999) and we therefore measured the length of the arterial segment on the surface of the heart before dissection). A transmural pressure of 40 mmHg was applied for a 1-h stabilizing period and during experiments because we previously demonstrated this to be optimal in terms of coronary arterial response to an agonist in a pressure myograph (Frbert et al. 1999). The external diameter of the arterial segment was automatically determined by video imaging at a frequency of 20 Hz. The internal pressure was controlled by adjustment of two reservoirs containing PSS mounted on a pressure column and connected to the cannulae. Pressure transducers close to the ‘arterial’ end of each cannula measured the internal pressure. Transmural pressure, the outer diameter, and a video image of the arterial segment were continuously sampled and stored on computer (Myodaq software, version 2.03, DMT, Aarhus, Denmark). Before and after experiments the video dimension analyser was calibrated with a 3000 μm x 3000 μm phantom in the horizontal and vertical directions.
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Microdialysis
Two microdialysis catheters (CMA/7, CMA, Sweden) were placed in the smooth muscle interstitium of a coronary artery mounted in the pressure myograph as previously described (Frbert et al. 2002). In order to obtain enough dialysate for analysis we chose to use two microdialysis catheters – one for the analysis of adenosine and one for lactate, pyruvate and glucose. The microdialysis catheters have a 6 kDa molecular cut-off and an outer diameter of 0.24 mm. Perfusion of the catheters was started immediately after placement at a rate of 0.3 μl min–1 with isotonic saline. Pilot studies have demonstrated that catheter placement in the vessel wall does not influence contraction and dilatation of the coronary arteries. Sixty-minute samples of dialysate (18 μl sample–1) were collected. There was a delay of 12 min between the passage of the perfusate through the microdialysis catheter and collection in vials. This time delay was considered in the calculations and all data are presented in real time. All samples represented the entire time period of exposure (60 min of oxygenation, 60 min of hypoxia/sham or 60 min of reoxygenation).
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Procedures
After a stabilizing period in the organ bath artery tone was induced with prostaglandin F2 (PGF2; 10–5M). When a stable contraction was established, hypoxia was induced in a subset of experiments by adjusting the gas concentrations from 5% CO2 in O2 (organ bath PO2 > 650 mmHg, ISO2-D, WPI, FL, USA) to 5% CO2 in N2 (PO2 30 mmHg). The influence of 60 min of hypoxia was studied. Oxygenated conditions were re-established by switching back to PSS equilibrated with 5% CO2 in O2 and the arterial response during the following 60 min was recorded (reoxygenation period). Organ bath pH was constant at pH 7.4. Microdialysis samples were collected 60 min prior to hypoxia (2 samples, 1 per catheter), during hypoxia (2 samples) and during reoxygenation (2 samples).
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Various blockers were used to test the effects of increasing the endogenously released adenosine: iodotubericidine (ITU, 10 μM), a blocker of adenosine kinase; erythro-9-(2-hydroxy-3-nonyl-)-adenine (EHNA, 5 μM), a blocker of adenosine deaminase; and n-nitrobenzylthioinosine (NBTI, 1 μM), a blocker of adenosine membrane transport (Fig. 1). The blockers were applied both in the organ bath and in the perfusion fluid of the microdialysis catheter. EHNA and ITU were dissolved in saline and NBTI in dimethylsulfoxide (10–6M) (Deussen et al. 1999).
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ITU: iodotubericidine, a blocker of adenosine kinase. EHNA: erythro-9-(2-hydroxy-3-nonyl-)-adenine, a blocker of adenosine deaminase. NBTI: n-nitrobenzylthioinosine, a blocker of adenosine membrane transport.
In some experiments, adenosine concentration-response curves were constructed during oxygenation and during hypoxia with ITU (10 μM), EHNA (5 μM), and NBTI (1 μM) in the organ bath. In other experiments, the effect of adenosine A2A receptor blockade on hypoxia-induced dilatation was investigated by means of the selective non-xanthine A2A receptor antagonist ZM 241385 (4-(2-[7-amino-2-(2-furyl)[1,2,4]-triazolo[2,3-a][1,3,5] triazin-5-ylamino]ethyl)-phenol; 20 nM) or the selective A2B receptor antagonist MRS1754 (N-(4-cyano-phenyl)-2-[4-(2,6-dioxo-1,3-dipropyl-2,3,4,5,6,7-hexahydro-1H-purin-8-yl)-phenoxy]-acetamide; 20 nM) in addition to ITU (10 μM), EHNA (5 μM), and NBTI (1 μM) in the organ bath.
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Relative recovery
To determine the exchange fraction (called relative recovery, RR) of adenosine over the microdialysis membrane, a small amount of [14C]adenosine (1 μCi maximum, specific activity 50 mCi mmol–1) was included in the perfusate as an internal reference marker. The RR was measured in four arteries and calculated as ((perfusatedpm– dialysatedpm)/perfusatedpm) and interstitial adenosine concentration was calculated as ([adenosine]dialysate/RR), where [adenosine]dialysate is the concentration of adenosine in the dialysate. The RR increased throughout the study period: 0.32 ± 0.02 (before hypoxia), 0.44 ± 0.02 (0–60 min hypoxia) and 0.46 ± 0.02 (reoxygenation period). The values of RR for these time periods differed significantly from each other (P < 0.01, repeated measures ANOVA). The mean RR values were used for further calculations of adenosine concentration.
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We have recently determined RR for lactate in 11 vessel preparations under identical experimental conditions (Frbert et al. 2004) and these data were used in the present study for further calculations of lactate concentration: 0.29 ± 0.04 (before hypoxia), 0.37 ± 0.04 (0–60 min hypoxia) and 0.48 ± 0.05 (reoxygenation period).
Analyses of dialysates
Native adenosine was derivatized to etheno-adenosine (ado) following procedures described in the literature (Fenton & Dobson, 1987). In brief, 18 μl of a sample or an adenosine standard (5 pmol) was incubated with 182 μl of a derivatization mixture containing chloroazetaldehyde (62 mM), citrate phosphate buffer (76 mM; pH 4), and Krebs-Henseleit buffer (pH 7.4) in a volume ratio of 0.027, 0.28, and 0.69, respectively. Samples were incubated at 80°C for 40 min and then immediately cooled to 4°C until subjected to HPLC analysis on the same day. The HPLC system used was a Waters Alliance 2690 HPLC system, consisting of pumps, eluent degasser, mixing chamber and injection module. Details of the HPLC analysis have been published before in detail (Haink & Deussen, 2003). In brief, samples were injected (100 μl) onto an XTerra MS C18 column (4.6 x 50 mm, i.d. 3 mm, 5 μm particle size, 125 pore size; Waters Corp.) equipped with a precolumn (XTerra MS C18, 5 μm). Eluent flow-rate was 1.5 ml min–1 throughout the analysis. The eluents used were a tetrabutylammoniumhydrogensulphate (TBAS) buffer (5.7 mM TBAS, 30.5 mM KH2PO4 adjusted to pH 5.8 with 2 N KOH) and an acetonitrile buffer (acetonitrile: TBAS buffer, 2: 1). The starting condition was 90% TBAS buffer, which was linearly reduced to 60% within 1 min after sample injection. The TBAS buffer fraction was linearly returned from 60 to 90% between run time 2.4 and 2.5 min. The typical retention time of was 0.9 min. The eluent fluorescence was measured continuously with a Merck-Hitachi F 1050 fluorescence spectrophotometer (flow cell capacity 12 μl) set to an excitation wavelength of 280 nm and an emission wavelength of 410 nm and recorded via a SATIN-box (Waters Corp.) on a PC using Millenium 3.20 Software (Waters Corp.). The retention time and compound quantification were achieved using external standards of a known concentration (ado) as well as standards of native adenosine (5 pmol) subjected to the derivatization procedure. Glucose, lactate and pyruvate concentrations in the dialysate were measured by an automated spectrophotometric kinetic enzymatic analyser (CMA 600).
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Solutions
The PSS had the following composition (mM): NaCl 119, NaHCO3 25, KCl 4.7, MgSO4 1.2, CaCl2 1.5, glucose 5.5 and 2-[4-(2-hydroxyethyl)-1-piperazinyl]-ethansulphonic acid (Hepes) 20. In experiments where K-PSS was used, NaCl was exchanged for KCl on an equimolar basis to a final K+ concentration of 125 mM. All solutions were made using analytical grade chemicals and twice-distilled water. PGF2 (Dinoprost) was obtained from Upjohn, Germany, [14C]adenosine and ZM 241385 were from Sigma Chemical Co., St Louis, MO, USA, and adenosine (Adenocor) was from Sanofi-Synthelabo, Brndby, Denmark.
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Statistics
Values are presented as mean ±S.E.M. and number of vessels (one per pig). Because the coronary artery diameter varied between pigs the steady state diameter induced by PGF2 was used as an internal standard (0%). To test for differences at different time points (oxygenation, hypoxia 60 min, and reoxygenation) statistical comparisons were performed using repeated measures one-way analysis of variance with a Student-Newman-Keuls post hoc test. One-way analysis of variance was used to test for differences between groups. Differences were considered statistically significant when P < 0.05.
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Results
Diameter response
Data were obtained from 38 arteries (one per pig), all constricted by 10–5M prostaglandin F2. Sixty minutes of hypoxia (n= 5) increased normalized diameter by 20.0 ± 2.6% (P < 0.001 compared to continuous oxygenation: –3.1 ± 2.4%, n= 6). Addition of blockers of adenosine uptake and degradation evoked a transient vasodilatation in normoxic conditions (ITU and EHNA: 8.6 ± 2.1%, n= 14; ITU and EHNA: 4.7 ± 1.2%, n= 13) but before induction of hypoxia/sham diameters had returned to preblockade dimensions. During hypoxia diameter increase without addition of blockers (20.0 ± 2.6%, n= 5) was significantly greater compared with diameter increase after addition of ITU, EHNA and NBTI (9.1 ± 2.8%, n= 6, P < 0.05) (Fig. 2). During reoxygenation diameters were significantly greater with blockers (3.6 ± 2.9%) than without (–5.8 ± 1.2%, P < 0.05).
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P < 0.05 versus no blockade. #P < 0.05 versus corresponding condition during initial oxygen time period; n= 5–8 in all experiments. Error bars indicate S.E.M.
There were no differences in diameters between the three groups (no blockade; ITU and EHNA; ITU, EHNA and NBTI) not subjected to hypoxia.
Lactate, pyruvate and glucose
The lactate/pyruvate ratio is a sensitive measure of tissue hypoxia and this ratio was significantly increased in hypoxic arteries (Fig. 3). Addition of ITU and EHNA or ITU, EHNA and NBTI did not significantly alter the lactate/pyruvate ratio of the dialysate. Likewise, in arteries not subjected to hypoxia the lactate/pyruvate did not change (data not shown). At normoxia dialysate glucose concentration was 1.36 ± 0.18 mM and there were no significant changes during the experiments.
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Microdialysis catheters within the coronary artery wall sampled metabolites during the experiments. The lactate/pyruvate ratio increased during hypoxia. #P < 0.05 versus corresponding condition during initial oxygen time period. No blockade: n= 5; ITU, EHNA: n= 8; ITU, EHNA, NBTI: n= 6.
Dialysate adenosine
Dialysate adenosine concentration fell continuously during the course of the experiments independent of the protocol chosen (Fig. 4A). Control experiments (n= 5) without preconstriction with PGF2 (10–5M) demonstrated that preconstriction in itself did not affect adenosine concentrations compared to preconstricted arteries (0–60 min O2: 10.7 ± 0.6 nmol l–1versus preconstriction: 8.7 ± 2.2 nmol l–1; 60–120 min O2: 6.4 ± 0.7 nmol l–1versus preconstriction: 8.8 ± 2.0 nmol l–1; 120–180 min O2: 4.8 ± 0.5 nmol l–1versus preconstriction: 5.6 ± 1.8 nmol l–1, all not significant (n.s.)). The presence of ITU, EHNA and NBTI generally increased adenosine concentration significantly compared with no blockers (Fig. 4A). However, even during block of adenosine metabolism (ITU + EHNA) as well as in the additional presence of membrane transport block (NBTI), hypoxia did not affect the dialysate adenosine concentration (Fig. 4B). The presence of blockers of adenosine metabolism and adenosine membrane transport tended to increase the dialysate adenosine concentration also in experiments with continuous oxygenation without reaching the level of statistical significance (Fig. 4B). It should be noted that there were no statistically significant differences in the dialysate adenosine concentrations between the three sampling intervals in any of the groups studied with continuous oxygenation.
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A, adenosine concentration in microdialysates from the coronary artery wall during hypoxia and reoxygenation. Values are corrected for relative recovery (see text). P < 0.05 versus no blockade. #P < 0.05 versus corresponding condition during initial oxygen time period. No blockade: n= 5; ITU, EHNA: n= 8; ITU, EHNA, NBTI: n= 6. B, adenosine concentrations during continuous oxygenation. No blockade: n= 6; ITU, EHNA: n= 6; ITU, EHNA, NBTI: n= 7.
Adenosine concentration and diameter changes did not correlate during hypoxia (r2= 0.12, n.s.) or during continuous oxygenation (r2= 0.14, n.s.). Lactate/pyruvate ratio and adenosine concentration did not correlate during hypoxia (r2= 0.01, n.s.) or during continuous oxygenation (r2= 0.04, n.s.).
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Adenosine concentration–response experiments
We wanted to compare the dialysate adenosine concentration during adenosine-induced vasodilatation of similar magnitude to the 20% vasodilatation (Fig. 2) obtained during hypoxia. We therefore performed adenosine concentration–vasodilatation response experiments during microdialysis of the coronary artery medium (n= 5). A comparable degree of dilatation, 15.7 ± 1.6%, was obtained by an organ bath adenosine concentration of 30 μM. This yielded a dialysate adenosine concentration of 420 ± 190 nM– more than 20 times the maximal dialysate adenosine concentration measured during hypoxia.
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The finding of unchanged adenosine concentrations during hypoxia does not exclude a role for adenosine because hypoxia may sensitize the adenosine receptor or increase its expression. To investigate this we performed adenosine concentration–response experiments with ITU, EHNA and NBTI in the organ bath during normoxia (n= 9) and during hypoxia (n= 9) (Fig. 5A). Adenosine-induced vasodilatation was more pronounced during hypoxia than during normoxia (P < 0.001). We also studied the effect of the non-xanthine A2A receptor antagonist ZM241385 and the selective A2B receptor antagonist MRS1754 on hypoxia-induced vasodilatation (Fig. 5B). In contrast to the adenosine A2B receptor antagonist, MRS1754 (n= 5), the adenosine A2A receptor antagonist, ZM241385 (n= 4), significantly attenuated hypoxia-induced vasodilatation (ANOVA, P < 0.001) (Fig. 5B).
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A, adenosine concentration–response experiments in porcine coronary arteries with ITU, EHNA and NBTI in the organ bath. Vasodilatation was more pronounced during hypoxia (n= 9) than during normoxia (n= 9, P < 0.001). B, the effect of the non-xanthine A2A receptor antagonist, ZM241385 on hypoxia-induced vasodilatation. During 60 min of hypoxia ZM241385 (n= 5) attenuated hypoxia-induced vasodilatation compared to experiments without this antagonist (n= 5) or experiments with the selective A2B receptor antagonist MRS1754 (n= 4) (ANOVA, P < 0.001). In all experiments ITU, EHNA and NBTI were added to the organ bath.
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Discussion
The major results of the present study may be summarized as follows. (1) During hypoxia, coronary arteries dilated without an increase in interstitial adenosine concentration; rather, the dialysate adenosine concentration decreased. (2) A coronary dilatation similar to that during hypoxia could be obtained if high concentrations of adenosine were added to the organ bath. The resulting interstitial adenosine concentrations in these experiments were 20 times higher than the adenosine concentration measured during hypoxia. (3) Adenosine-induced vasodilatation was more pronounced during hypoxia than during normoxia. Furthermore, the A2A receptor antagonists ZM241385 decreased hypoxia-induced vasodilatation.
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Systemic hypoxia causes vasodilatation which may (Wang et al. 1994) or may not (Downey et al. 1982) be accompanied by myocardial adenosine increase. Adenosine is a potent vasodilator and it seems logical to assume that during systemic hypoxia this purine nucleoside could play a key role in stimulating vasodilatation. The sources of adenosine are adenine nucleotides, including AMP, cyclic AMP and ATP. Intracellular adenosine can be produced from S-adenosylhomocysteine by S-adenosylhomocysteine hydrolase and from 5'-AMP by cytosolic 5'nucleotidase. Extracellular 5'nucleotidase bound to the cell membrane produces adenosine (Deussen, 2000) (Fig. 1) although a previous report found little 5'nucleotidase in vascular smooth muscle (Rubio et al. 1973). Adenosine breakdown is brought about by adenosine deaminase and adenosine kinase.
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The question of adenosine involvement in hypoxic coronary vasodilatation has been addressed widely in the past. Busse et al. (1983) reported that a decrease in intraluminal PO2 to 40 mmHg dilated perfused arterial segments of rat and dog and the dilatation was not inhibited by theophylline. Mellemkjaer & Nielsen-Kudsk (1994) found that hypoxic dilatation (PO2 20 mmHg) of porcine coronary arteries was unaffected after application of theophylline. While investigations studying epicardial vessel preparations have generally failed to find evidence for the involvement for adenosine in hypoxic coronary vasodilatation, the evidence is rather ambiguous in studies in vivo (Merrill et al. 1986; Gewirtz et al. 1987; Lee et al. 1992; Stepp et al. 1996). Various differences between the experimental protocols used may account for contradictory results. However, a potential importance of the myocardial adenosine metabolism for hypoxic coronary vasodilatation is suggested by the study of Kerkhof et al. (2002). These authors studied vessel preparations with and without adjacent myocardium suspended in an organ chamber. Hypoxic vasorelaxation was considerably more pronounced in the presence of myocardial tissue. Also, in the presence of myocardial tissue the adenosine receptor blocker 8-phenyltheophylline reduced hypoxic coronary vessel relaxation.
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The adenosine hypothesis of metabolic flow regulation of the heart (Berne, 1963) states that a decrease of tissue PO2 induces enhanced adenosine production due to net ATP hydrolysis. The resulting increase of the adenosine concentration may cause coronary vasodilatation via action of this purine on vascular smooth muscle cells or endothelial cell adenosine receptors (Olsson & Pearson, 1990). In this feedback loop adenosine would act to relate vascular tone to tissue PO2. If it is assumed that this system is fine-tuned (high gain), minimal changes in tissue PO2 would affect adenosine production considerably. Then, in a situation in which coronary flow is controlled by adenosine, the block of adenosine receptors might be expected to result in a further augmentation of adenosine production, which could overcome the receptor blockade. Thus, studying the effects of application of adenosine receptor blockers on coronary hypoxic vasodilatation is not a critical test of the adenosine hypothesis. Rather, the adenosine concentration needs to be determined as well to investigate whether adenosine production, and hence the interstitial adenosine concentration, increases in response to the adenosine receptor blockade.
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In our study a coronary artery interstitial adenosine concentration of typically 10 nM was too low to cause vasodilatation in line with previous findings by Tune et al. (2000). Using a modelling strategy from measurements of coronary artery and venous adenosine in dogs Stepp et al. (1996) found an average estimated myocardial interstitial adenosine concentration of 92 nM. This contrasts with findings by the microdialysis technique. With no correction for recovery Wang et al. (1994) found canine myocardial dialysate concentrations of 490 nM at baseline and 790 nM during systemic hypoxia. The much higher values might be explained by the constant beating of the heart against the dialysis tubing leading to cell leakage. The relatively slow changes in coronary artery diameter render this problem negligible in our set-up. The coronary artery interstitial adenosine concentrations found in our study are, nevertheless, smaller than findings in the myocardium, and hypoxia did not increase adenosine concentration within the coronary artery wall. A likely explanation for this result is that oxygen demand per unit volume of endothelial or vascular smooth muscle cells is significantly lower than that of contracting cardiac muscle. Endothelial cells have been shown to remain well energized at PO2 levels above 3 mmHg (Mertens et al. 1990) and smooth muscle cells are known to have a much lower ATP turnover than cardiac muscle.
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While myocardial adenosine could play a role for hypoxia-induced dilatation in intramyocardial vessels it is probably less likely that adenosine in the blood stream could exert coronary dilatation during hypoxia. The concentration of adenosine (30 μM) used to produce vasodilatation in our study was supraphysiological and this is in line with a recent hypothesis that intravascular adenosine does not pass the endothelium, because of endothelial nucleoside transporter activity (Gamboa et al. 2003). Endothelium-dependent vasodilatation due to a direct effect of adenosine seems potentially possible, but in our opinion this concept is not sufficiently supported by experimental data (Stepp et al. 1996).
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We found unchanged or even decreased adenosine concentrations during hypoxia. However, this does not exclude a role for adenosine in hypoxia-induced vasodilatation because hypoxia may sensitize the adenosine receptor. This notion is supported by our finding that adenosine-induced vasodilatation was significantly more pronounced during hypoxia than during normoxia. Furthermore, ZM241385 decreased hypoxia-induced dilatation, which suggests that hypoxia increases sensitivity at the A2A receptor level or increases A2A receptor expression (Kobayashi & Millhorn, 1999). Both the A2A and A2B receptor subtypes have been demonstrated in coronary arteries (Kemp & Cocks, 1999; Flood & Headrick, 2001). Although in a majority of studies the A2A receptor subtype (in contrast to A2B) has been found to mediate vasorelaxation to adenosine this is most likely species dependent; in human small coronary arteries, adenosine mediates most of its vasodilator response via the A2B receptor (Flood & Headrick, 2001), while A2A adenosine receptors mediate dilatation in mouse coronary vessels, and A2B receptors in rat (Kemp & Cocks, 1999).
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The fall in interstitial adenosine during hypoxia and the non-significant fall during continuous oxygenation must be considered a possible methodological limitation. It is unlikely that a longer stabilizing period would have affected this. This can be seen from our testing of any effect on adenosine concentration from preconstriction (which we did not find) where the (non-significant) fall in adenosine continued for up to 3 h even without preconstriction, i.e. a situation comparable to a 3-h stabilizing period. Another possibility is that the total amount of adenosine (absolute recovery of adenosine) exhausted stores and production capacity. We consider this unlikely from the augmented adenosine concentrations after blockade with EHNA, ITU and NBTI. A physiological explanation might be that the 2-h period from the killing of the pig to the beginning of the experiment is an ischaemic preconditioning challenge for the coronary artery. Previously it was found that adenosine levels in phenylephrine-preconditioned rat cardiomyocytes decreased during ischaemia compared to non-preconditioned cells (Vasara et al. 2003).
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Although an NBMPR-sensitive mechanism plays a role for adenosine transport in rat aortic smooth muscle (Leung et al. 2005), in porcine coronary arteries NBTI inhibits adenosine entry (Rubin et al. 2000). The findings in the latter study suggest that an adenosine transporter with characteristics of an equilibrative, NBTI-sensitive subtype (ENT1) is the main transporter for adenosine in our preparation, but the presence of other nucleoside transporter subtypes cannot be excluded and thus other possibilities of adenosine entry into coronary smooth muscle also cannot be excluded.
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We found that the relative recovery of adenosine increased over the experimentation period. Such increase is a common finding with microdialysis in vitro in the model used in the present study (lactate) (Frbert et al. 2002), in living pigs (glucose) (Nielsen et al. 2005) and in humans (glycerol) (Djurhuus et al. 2004). The background for this change in recovery is likely to be multifactorial. Exchange across the microdialysis membrane is dependent on the physical properties of the fibre, the nature of the tissue under investigation, blood flow (or equivalent), analyte under investigation, etc. With the internal reference technique (radioactive adenosine) we do not believe that a change in recovery poses a threat for the interpretation of our data.
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Our microdialysis set-up requires open access to the organ bath, which limits the degree of hypoxia that can be achieved. However, the increase in lactate/pyruvate ratio provides excellent indirect evidence that our arterial tissue was in fact hypoxic. We chose to use oxygen supplementation in the organ bath during ‘normoxic’ conditions, thus reaching a PO2 of 650 mmHg. In our in vitro system, there is no flow through vasa vasorum and thus oxygen reaches the tissue only by diffusion through the medial layers. Previous in vitro experiments have shown zones of severe tissue hypoxia in the media of arteries incubated at 21% oxygen (Bjornheden et al. 1996). However, we cannot totally exclude the possibility that the tissue is slightly hyperoxic, since there is no good surrogate marker for hyperoxia.
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The physiological and pathophysiological implications of the present study are related to the epicardial circulation while the effect of hypoxia on organ blood flow is mainly determined by its effect on vasomotor tone in resistance vessels. However, the dilatory response to hypoxia is not confined to resistance arteries but is also present in conduit vessels (Wadsworth, 1994; Barron & Gu, 2000; Shimizu et al. 2000). Combined with the fact that coronary atherosclerosis is confined to epicardial arteries, even minor diameter changes could become highly important when a haemodynamically significant stenosis is present. Furthermore, information about adenosine metabolism in conductance coronary arteries is warranted because the leading methodology to assess the haemodynamic significance of a coronary stenosis is by inducing hyperaemia in the coronary circulation by means of adenosine, assessing coronary artery pressure before and after the stenosis and calculating the so-called fractional flow reserve (Pijls et al. 1996). Our findings of increased adenosine sensitivity during hypoxia may bridge some of the contradictions in previous studies of unchanged (Downey et al. 1982) and rising (Wang et al. 1994) adenosine concentrations to hypoxia that have made it difficult to link hypoxic vasodilatation to adenosine.
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In conclusion, we report the first measurement of adenosine within the wall of a coronary artery during hypoxia-induced dilatation. This dilatation was accompanied by a fall in interstitial adenosine and, although blockade of adenosine deaminase, adenosine kinase and adenosine transport increased interstitial adenosine, this increase was unrelated to hypoxia. More pronounced vasodilatation to adenosine during hypoxia compared with normoxia might be facilitated by increased adenosine sensitivity at A2A receptor level.
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Wang T, Sodhi J, Mentzer RM Jr & Van Wylen DG (1994). Changes in interstitial adenosine during hypoxia: relationship to oxygen supply: demand imbalance, and effects of adenosine deaminase. Cardiovasc Res 28, 1320–1325., 百拇医药(Ole Frbert, Gesine Haink, Ulf Simonsen, )
2 Department of Pharmacology, University of Aarhus, Denmark
3 Institute of Physiology, Medical Faculty Carl Gustav Carus, Technical University Dresden, Germany
4 Department of Endocrinology M, Aarhus Sygehus, Aarhus University Hospital, Denmark
, http://www.100md.com 5 Wallenberg Laboratory, Sahlgrenska University Hospital, Gteborg, Sweden
Abstract
We tested whether hypoxia-induced coronary artery dilatation could be mediated by an increase in adenosine concentration within the coronary artery wall or by an increase in adenosine sensitivity. Porcine left anterior descendent coronary arteries, precontracted with prostaglandin F2 (10–5M), were mounted in a pressure myograph and microdialysis catheters were inserted into the tunica media. Dialysate adenosine concentrations were analysed by HPLC. Glucose, lactate and pyruvate were measured by an automated spectrophotometric kinetic enzymatic analyser. The exchange fraction of [14C]adenosine over the microdialysis membrane increased from 0.32 ± 0.02 to 0.46 ± 0.02 (n= 4, P < 0.01) during the study period. At baseline, interstitial adenosine was in the region of 10 nM which is significantly less than previously found myocardial concentrations. Hypoxia (PO2 30 mmHg for 60 min, n= 5) increased coronary diameters by 20.0 ± 2.6% (versus continuous oxygenation –3.1 ± 2.4%, n= 6, P < 0.001) but interstitial adenosine concentration fell. Blockade of adenosine deaminase (with erythro-9-(2-hydroxy-3-nonyl-)-adenine, 5 μM), adenosine kinase (with iodotubericidine, 10 μM) and adenosine transport (with n-nitrobenzylthioinosine, 1 μM) increased interstitial adenosine but the increase was unrelated to hypoxia or diameter. A coronary dilatation similar to that during hypoxia could be obtained with 30 μM of adenosine in the organ bath and the resulting interstitial adenosine concentrations (n= 5) were 20 times higher than the adenosine concentration measured during hypoxia. Adenosine concentration–response experiments showed vasodilatation to be more pronounced during hypoxia (n= 9) than during normoxia (n= 9, P < 0.001) and the A2A receptor antagonist ZM241385 (20 nM, n= 5), attenuated hypoxia-induced vasodilatation while the selective A2B receptor antagonist MRS1754 (20 nM, n= 4), had no effect. The lactate/pyruvate ratio was significantly increased in hypoxic arteries but did not correlate with adenosine concentration. We conclude that hypoxia-induced coronary artery dilatation is not mediated by increased adenosine produced within the artery wall but might be facilitated by increased adenosine sensitivity at the A2A receptor level.
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Introduction
Coronary artery dilatation to hypoxia is an important protective response that increases flow to endangered myocardium. The exact mechanisms causing the dilatation remain undefined but could be mediated by (1) local metabolites from the coronary artery endothelium or smooth muscle cells (Frbert et al. 2002), (2) metabolites released from the myocardium, (3) blood-borne metabolites, or (4) a direct effect of hypoxia on the membrane potential (Kobayashi et al. 1998) or the contractile apparatus (Frbert et al. 2005) of vascular smooth muscle cells.
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Adenosine is both a precursor and a metabolite of adenine nucleotides. The rate of adenine nucleotide degradation and cellular production and release of adenosine increase during myocardial ischaemia (Hall et al. 1995; Van Wylen, 1994). The potent vasodilator properties of adenosine have made it a candidate for hypoxia-induced vasodilatation. In addition, the very short half-life of adenosine in human blood plasma (Moser et al. 1989) suggests a highly local action of this purine at the site of production. Thus, adenosine meets the criteria for a local metabolic vasodilator. An extensive number of studies supports that adenosine could be one of the mediators coupling metabolic requirements with coronary blood flow (Merrill et al. 1986; Nakhostine & Lamontagne, 1993; Laxson et al. 1993; MacLean et al. 1998). However, experimental data are equivocal and others have found only a limited role for adenosine in hypoxia-induced (Gewirtz et al. 1987; Lee et al. 1992) and exercise-induced (Bache et al. 1988; Duncker et al. 1998) coronary dilatation. The finding that the vasorelaxation response to adenosine is virtually absent in hypoxic porcine carotid strips (Barron & Gu, 2000) further questions the importance of this metabolite in sustained hypoxic vasodilatation.
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Adenosine production may occur in the cytosol as well as in the extracellular region. Rapid enzymatic conversion of adenosine is provided by adenosine kinase and adenosine deaminase, keeping the cytosolic concentration in the nanomolar range (Deussen, 2000). Because of high intracellular rates of adenosine rephosphorylation and deamination the cytosolic concentration is normally below the extracellular concentration. This is the basis for the finding that adenosine plasma concentration increases during membrane transport blockade (Deussen, 2000). It is unknown whether adenosine acting on coronary artery smooth muscle cells and potentially evoking vasodilatation is derived from a vascular source or from adjacent myocardium. While for intramyocardial vessels both sites of adenosine production might be of physiological significance, in epicardial vessels only a vascular site of adenosine production can be imagined. To resolve this question, it is necessary to measure adenosine production in the vessel wall in response to a stimulus known to enhance adenosine production. A decrease in oxygen tension is a well documented stimulus of cardiac adenosine production (Deussen & Schrader, 1991). The present study was undertaken to test whether hypoxia increases coronary artery interstitial adenosine concentration sufficiently to induce vasodilatation. Thus, we measured coronary artery diameters while sampling interstitial adenosine by means of microdialysis in the arterial media layer. We also tested whether sensitivity to adenosine was changed during hypoxia compared with normoxia.
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This is the first report to measure adenosine within the wall of a coronary artery during changes in vessel diameter in response to hypoxia.
Methods
Hearts from Landrace-Yorkshire hogs were obtained at a local slaughterhouse. Immediately after killing the aorta was cannulated and the coronary circulation perfused with a physiological salt solution (PSS) containing 5.5 mM glucose, bubbled with 5% CO2 in O2 and buffered with Hepes. The hearts were bathed in PSS at 5°C for approximately 2 h until the start of the experiment. The left anterior descending coronary artery (LAD) was carefully dissected and the proximal 3–4 cm of the artery left intact. The external diameter of these vessel preparations ranged from 2.93 mm to 5.13 mm (mean 4.00 ± 0.08 mm, n= 38).
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Pressure myograph
Cylindrical arterial segments, 2 cm long, were mounted at both ends on stainless steel cannulae and fastened with sutures in PSS bubbled with 5% CO2 in O2 in a vessel chamber. The temperature was raised to 37°C. Before measurements the segments were stretched to the in situ length by operating a micrometer device (we have previously documented that length and distension are of importance for reactivity (Frbert et al. 1999) and we therefore measured the length of the arterial segment on the surface of the heart before dissection). A transmural pressure of 40 mmHg was applied for a 1-h stabilizing period and during experiments because we previously demonstrated this to be optimal in terms of coronary arterial response to an agonist in a pressure myograph (Frbert et al. 1999). The external diameter of the arterial segment was automatically determined by video imaging at a frequency of 20 Hz. The internal pressure was controlled by adjustment of two reservoirs containing PSS mounted on a pressure column and connected to the cannulae. Pressure transducers close to the ‘arterial’ end of each cannula measured the internal pressure. Transmural pressure, the outer diameter, and a video image of the arterial segment were continuously sampled and stored on computer (Myodaq software, version 2.03, DMT, Aarhus, Denmark). Before and after experiments the video dimension analyser was calibrated with a 3000 μm x 3000 μm phantom in the horizontal and vertical directions.
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Microdialysis
Two microdialysis catheters (CMA/7, CMA, Sweden) were placed in the smooth muscle interstitium of a coronary artery mounted in the pressure myograph as previously described (Frbert et al. 2002). In order to obtain enough dialysate for analysis we chose to use two microdialysis catheters – one for the analysis of adenosine and one for lactate, pyruvate and glucose. The microdialysis catheters have a 6 kDa molecular cut-off and an outer diameter of 0.24 mm. Perfusion of the catheters was started immediately after placement at a rate of 0.3 μl min–1 with isotonic saline. Pilot studies have demonstrated that catheter placement in the vessel wall does not influence contraction and dilatation of the coronary arteries. Sixty-minute samples of dialysate (18 μl sample–1) were collected. There was a delay of 12 min between the passage of the perfusate through the microdialysis catheter and collection in vials. This time delay was considered in the calculations and all data are presented in real time. All samples represented the entire time period of exposure (60 min of oxygenation, 60 min of hypoxia/sham or 60 min of reoxygenation).
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Procedures
After a stabilizing period in the organ bath artery tone was induced with prostaglandin F2 (PGF2; 10–5M). When a stable contraction was established, hypoxia was induced in a subset of experiments by adjusting the gas concentrations from 5% CO2 in O2 (organ bath PO2 > 650 mmHg, ISO2-D, WPI, FL, USA) to 5% CO2 in N2 (PO2 30 mmHg). The influence of 60 min of hypoxia was studied. Oxygenated conditions were re-established by switching back to PSS equilibrated with 5% CO2 in O2 and the arterial response during the following 60 min was recorded (reoxygenation period). Organ bath pH was constant at pH 7.4. Microdialysis samples were collected 60 min prior to hypoxia (2 samples, 1 per catheter), during hypoxia (2 samples) and during reoxygenation (2 samples).
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Various blockers were used to test the effects of increasing the endogenously released adenosine: iodotubericidine (ITU, 10 μM), a blocker of adenosine kinase; erythro-9-(2-hydroxy-3-nonyl-)-adenine (EHNA, 5 μM), a blocker of adenosine deaminase; and n-nitrobenzylthioinosine (NBTI, 1 μM), a blocker of adenosine membrane transport (Fig. 1). The blockers were applied both in the organ bath and in the perfusion fluid of the microdialysis catheter. EHNA and ITU were dissolved in saline and NBTI in dimethylsulfoxide (10–6M) (Deussen et al. 1999).
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ITU: iodotubericidine, a blocker of adenosine kinase. EHNA: erythro-9-(2-hydroxy-3-nonyl-)-adenine, a blocker of adenosine deaminase. NBTI: n-nitrobenzylthioinosine, a blocker of adenosine membrane transport.
In some experiments, adenosine concentration-response curves were constructed during oxygenation and during hypoxia with ITU (10 μM), EHNA (5 μM), and NBTI (1 μM) in the organ bath. In other experiments, the effect of adenosine A2A receptor blockade on hypoxia-induced dilatation was investigated by means of the selective non-xanthine A2A receptor antagonist ZM 241385 (4-(2-[7-amino-2-(2-furyl)[1,2,4]-triazolo[2,3-a][1,3,5] triazin-5-ylamino]ethyl)-phenol; 20 nM) or the selective A2B receptor antagonist MRS1754 (N-(4-cyano-phenyl)-2-[4-(2,6-dioxo-1,3-dipropyl-2,3,4,5,6,7-hexahydro-1H-purin-8-yl)-phenoxy]-acetamide; 20 nM) in addition to ITU (10 μM), EHNA (5 μM), and NBTI (1 μM) in the organ bath.
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Relative recovery
To determine the exchange fraction (called relative recovery, RR) of adenosine over the microdialysis membrane, a small amount of [14C]adenosine (1 μCi maximum, specific activity 50 mCi mmol–1) was included in the perfusate as an internal reference marker. The RR was measured in four arteries and calculated as ((perfusatedpm– dialysatedpm)/perfusatedpm) and interstitial adenosine concentration was calculated as ([adenosine]dialysate/RR), where [adenosine]dialysate is the concentration of adenosine in the dialysate. The RR increased throughout the study period: 0.32 ± 0.02 (before hypoxia), 0.44 ± 0.02 (0–60 min hypoxia) and 0.46 ± 0.02 (reoxygenation period). The values of RR for these time periods differed significantly from each other (P < 0.01, repeated measures ANOVA). The mean RR values were used for further calculations of adenosine concentration.
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We have recently determined RR for lactate in 11 vessel preparations under identical experimental conditions (Frbert et al. 2004) and these data were used in the present study for further calculations of lactate concentration: 0.29 ± 0.04 (before hypoxia), 0.37 ± 0.04 (0–60 min hypoxia) and 0.48 ± 0.05 (reoxygenation period).
Analyses of dialysates
Native adenosine was derivatized to etheno-adenosine (ado) following procedures described in the literature (Fenton & Dobson, 1987). In brief, 18 μl of a sample or an adenosine standard (5 pmol) was incubated with 182 μl of a derivatization mixture containing chloroazetaldehyde (62 mM), citrate phosphate buffer (76 mM; pH 4), and Krebs-Henseleit buffer (pH 7.4) in a volume ratio of 0.027, 0.28, and 0.69, respectively. Samples were incubated at 80°C for 40 min and then immediately cooled to 4°C until subjected to HPLC analysis on the same day. The HPLC system used was a Waters Alliance 2690 HPLC system, consisting of pumps, eluent degasser, mixing chamber and injection module. Details of the HPLC analysis have been published before in detail (Haink & Deussen, 2003). In brief, samples were injected (100 μl) onto an XTerra MS C18 column (4.6 x 50 mm, i.d. 3 mm, 5 μm particle size, 125 pore size; Waters Corp.) equipped with a precolumn (XTerra MS C18, 5 μm). Eluent flow-rate was 1.5 ml min–1 throughout the analysis. The eluents used were a tetrabutylammoniumhydrogensulphate (TBAS) buffer (5.7 mM TBAS, 30.5 mM KH2PO4 adjusted to pH 5.8 with 2 N KOH) and an acetonitrile buffer (acetonitrile: TBAS buffer, 2: 1). The starting condition was 90% TBAS buffer, which was linearly reduced to 60% within 1 min after sample injection. The TBAS buffer fraction was linearly returned from 60 to 90% between run time 2.4 and 2.5 min. The typical retention time of was 0.9 min. The eluent fluorescence was measured continuously with a Merck-Hitachi F 1050 fluorescence spectrophotometer (flow cell capacity 12 μl) set to an excitation wavelength of 280 nm and an emission wavelength of 410 nm and recorded via a SATIN-box (Waters Corp.) on a PC using Millenium 3.20 Software (Waters Corp.). The retention time and compound quantification were achieved using external standards of a known concentration (ado) as well as standards of native adenosine (5 pmol) subjected to the derivatization procedure. Glucose, lactate and pyruvate concentrations in the dialysate were measured by an automated spectrophotometric kinetic enzymatic analyser (CMA 600).
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Solutions
The PSS had the following composition (mM): NaCl 119, NaHCO3 25, KCl 4.7, MgSO4 1.2, CaCl2 1.5, glucose 5.5 and 2-[4-(2-hydroxyethyl)-1-piperazinyl]-ethansulphonic acid (Hepes) 20. In experiments where K-PSS was used, NaCl was exchanged for KCl on an equimolar basis to a final K+ concentration of 125 mM. All solutions were made using analytical grade chemicals and twice-distilled water. PGF2 (Dinoprost) was obtained from Upjohn, Germany, [14C]adenosine and ZM 241385 were from Sigma Chemical Co., St Louis, MO, USA, and adenosine (Adenocor) was from Sanofi-Synthelabo, Brndby, Denmark.
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Statistics
Values are presented as mean ±S.E.M. and number of vessels (one per pig). Because the coronary artery diameter varied between pigs the steady state diameter induced by PGF2 was used as an internal standard (0%). To test for differences at different time points (oxygenation, hypoxia 60 min, and reoxygenation) statistical comparisons were performed using repeated measures one-way analysis of variance with a Student-Newman-Keuls post hoc test. One-way analysis of variance was used to test for differences between groups. Differences were considered statistically significant when P < 0.05.
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Results
Diameter response
Data were obtained from 38 arteries (one per pig), all constricted by 10–5M prostaglandin F2. Sixty minutes of hypoxia (n= 5) increased normalized diameter by 20.0 ± 2.6% (P < 0.001 compared to continuous oxygenation: –3.1 ± 2.4%, n= 6). Addition of blockers of adenosine uptake and degradation evoked a transient vasodilatation in normoxic conditions (ITU and EHNA: 8.6 ± 2.1%, n= 14; ITU and EHNA: 4.7 ± 1.2%, n= 13) but before induction of hypoxia/sham diameters had returned to preblockade dimensions. During hypoxia diameter increase without addition of blockers (20.0 ± 2.6%, n= 5) was significantly greater compared with diameter increase after addition of ITU, EHNA and NBTI (9.1 ± 2.8%, n= 6, P < 0.05) (Fig. 2). During reoxygenation diameters were significantly greater with blockers (3.6 ± 2.9%) than without (–5.8 ± 1.2%, P < 0.05).
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P < 0.05 versus no blockade. #P < 0.05 versus corresponding condition during initial oxygen time period; n= 5–8 in all experiments. Error bars indicate S.E.M.
There were no differences in diameters between the three groups (no blockade; ITU and EHNA; ITU, EHNA and NBTI) not subjected to hypoxia.
Lactate, pyruvate and glucose
The lactate/pyruvate ratio is a sensitive measure of tissue hypoxia and this ratio was significantly increased in hypoxic arteries (Fig. 3). Addition of ITU and EHNA or ITU, EHNA and NBTI did not significantly alter the lactate/pyruvate ratio of the dialysate. Likewise, in arteries not subjected to hypoxia the lactate/pyruvate did not change (data not shown). At normoxia dialysate glucose concentration was 1.36 ± 0.18 mM and there were no significant changes during the experiments.
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Microdialysis catheters within the coronary artery wall sampled metabolites during the experiments. The lactate/pyruvate ratio increased during hypoxia. #P < 0.05 versus corresponding condition during initial oxygen time period. No blockade: n= 5; ITU, EHNA: n= 8; ITU, EHNA, NBTI: n= 6.
Dialysate adenosine
Dialysate adenosine concentration fell continuously during the course of the experiments independent of the protocol chosen (Fig. 4A). Control experiments (n= 5) without preconstriction with PGF2 (10–5M) demonstrated that preconstriction in itself did not affect adenosine concentrations compared to preconstricted arteries (0–60 min O2: 10.7 ± 0.6 nmol l–1versus preconstriction: 8.7 ± 2.2 nmol l–1; 60–120 min O2: 6.4 ± 0.7 nmol l–1versus preconstriction: 8.8 ± 2.0 nmol l–1; 120–180 min O2: 4.8 ± 0.5 nmol l–1versus preconstriction: 5.6 ± 1.8 nmol l–1, all not significant (n.s.)). The presence of ITU, EHNA and NBTI generally increased adenosine concentration significantly compared with no blockers (Fig. 4A). However, even during block of adenosine metabolism (ITU + EHNA) as well as in the additional presence of membrane transport block (NBTI), hypoxia did not affect the dialysate adenosine concentration (Fig. 4B). The presence of blockers of adenosine metabolism and adenosine membrane transport tended to increase the dialysate adenosine concentration also in experiments with continuous oxygenation without reaching the level of statistical significance (Fig. 4B). It should be noted that there were no statistically significant differences in the dialysate adenosine concentrations between the three sampling intervals in any of the groups studied with continuous oxygenation.
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A, adenosine concentration in microdialysates from the coronary artery wall during hypoxia and reoxygenation. Values are corrected for relative recovery (see text). P < 0.05 versus no blockade. #P < 0.05 versus corresponding condition during initial oxygen time period. No blockade: n= 5; ITU, EHNA: n= 8; ITU, EHNA, NBTI: n= 6. B, adenosine concentrations during continuous oxygenation. No blockade: n= 6; ITU, EHNA: n= 6; ITU, EHNA, NBTI: n= 7.
Adenosine concentration and diameter changes did not correlate during hypoxia (r2= 0.12, n.s.) or during continuous oxygenation (r2= 0.14, n.s.). Lactate/pyruvate ratio and adenosine concentration did not correlate during hypoxia (r2= 0.01, n.s.) or during continuous oxygenation (r2= 0.04, n.s.).
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Adenosine concentration–response experiments
We wanted to compare the dialysate adenosine concentration during adenosine-induced vasodilatation of similar magnitude to the 20% vasodilatation (Fig. 2) obtained during hypoxia. We therefore performed adenosine concentration–vasodilatation response experiments during microdialysis of the coronary artery medium (n= 5). A comparable degree of dilatation, 15.7 ± 1.6%, was obtained by an organ bath adenosine concentration of 30 μM. This yielded a dialysate adenosine concentration of 420 ± 190 nM– more than 20 times the maximal dialysate adenosine concentration measured during hypoxia.
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The finding of unchanged adenosine concentrations during hypoxia does not exclude a role for adenosine because hypoxia may sensitize the adenosine receptor or increase its expression. To investigate this we performed adenosine concentration–response experiments with ITU, EHNA and NBTI in the organ bath during normoxia (n= 9) and during hypoxia (n= 9) (Fig. 5A). Adenosine-induced vasodilatation was more pronounced during hypoxia than during normoxia (P < 0.001). We also studied the effect of the non-xanthine A2A receptor antagonist ZM241385 and the selective A2B receptor antagonist MRS1754 on hypoxia-induced vasodilatation (Fig. 5B). In contrast to the adenosine A2B receptor antagonist, MRS1754 (n= 5), the adenosine A2A receptor antagonist, ZM241385 (n= 4), significantly attenuated hypoxia-induced vasodilatation (ANOVA, P < 0.001) (Fig. 5B).
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A, adenosine concentration–response experiments in porcine coronary arteries with ITU, EHNA and NBTI in the organ bath. Vasodilatation was more pronounced during hypoxia (n= 9) than during normoxia (n= 9, P < 0.001). B, the effect of the non-xanthine A2A receptor antagonist, ZM241385 on hypoxia-induced vasodilatation. During 60 min of hypoxia ZM241385 (n= 5) attenuated hypoxia-induced vasodilatation compared to experiments without this antagonist (n= 5) or experiments with the selective A2B receptor antagonist MRS1754 (n= 4) (ANOVA, P < 0.001). In all experiments ITU, EHNA and NBTI were added to the organ bath.
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Discussion
The major results of the present study may be summarized as follows. (1) During hypoxia, coronary arteries dilated without an increase in interstitial adenosine concentration; rather, the dialysate adenosine concentration decreased. (2) A coronary dilatation similar to that during hypoxia could be obtained if high concentrations of adenosine were added to the organ bath. The resulting interstitial adenosine concentrations in these experiments were 20 times higher than the adenosine concentration measured during hypoxia. (3) Adenosine-induced vasodilatation was more pronounced during hypoxia than during normoxia. Furthermore, the A2A receptor antagonists ZM241385 decreased hypoxia-induced vasodilatation.
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Systemic hypoxia causes vasodilatation which may (Wang et al. 1994) or may not (Downey et al. 1982) be accompanied by myocardial adenosine increase. Adenosine is a potent vasodilator and it seems logical to assume that during systemic hypoxia this purine nucleoside could play a key role in stimulating vasodilatation. The sources of adenosine are adenine nucleotides, including AMP, cyclic AMP and ATP. Intracellular adenosine can be produced from S-adenosylhomocysteine by S-adenosylhomocysteine hydrolase and from 5'-AMP by cytosolic 5'nucleotidase. Extracellular 5'nucleotidase bound to the cell membrane produces adenosine (Deussen, 2000) (Fig. 1) although a previous report found little 5'nucleotidase in vascular smooth muscle (Rubio et al. 1973). Adenosine breakdown is brought about by adenosine deaminase and adenosine kinase.
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The question of adenosine involvement in hypoxic coronary vasodilatation has been addressed widely in the past. Busse et al. (1983) reported that a decrease in intraluminal PO2 to 40 mmHg dilated perfused arterial segments of rat and dog and the dilatation was not inhibited by theophylline. Mellemkjaer & Nielsen-Kudsk (1994) found that hypoxic dilatation (PO2 20 mmHg) of porcine coronary arteries was unaffected after application of theophylline. While investigations studying epicardial vessel preparations have generally failed to find evidence for the involvement for adenosine in hypoxic coronary vasodilatation, the evidence is rather ambiguous in studies in vivo (Merrill et al. 1986; Gewirtz et al. 1987; Lee et al. 1992; Stepp et al. 1996). Various differences between the experimental protocols used may account for contradictory results. However, a potential importance of the myocardial adenosine metabolism for hypoxic coronary vasodilatation is suggested by the study of Kerkhof et al. (2002). These authors studied vessel preparations with and without adjacent myocardium suspended in an organ chamber. Hypoxic vasorelaxation was considerably more pronounced in the presence of myocardial tissue. Also, in the presence of myocardial tissue the adenosine receptor blocker 8-phenyltheophylline reduced hypoxic coronary vessel relaxation.
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The adenosine hypothesis of metabolic flow regulation of the heart (Berne, 1963) states that a decrease of tissue PO2 induces enhanced adenosine production due to net ATP hydrolysis. The resulting increase of the adenosine concentration may cause coronary vasodilatation via action of this purine on vascular smooth muscle cells or endothelial cell adenosine receptors (Olsson & Pearson, 1990). In this feedback loop adenosine would act to relate vascular tone to tissue PO2. If it is assumed that this system is fine-tuned (high gain), minimal changes in tissue PO2 would affect adenosine production considerably. Then, in a situation in which coronary flow is controlled by adenosine, the block of adenosine receptors might be expected to result in a further augmentation of adenosine production, which could overcome the receptor blockade. Thus, studying the effects of application of adenosine receptor blockers on coronary hypoxic vasodilatation is not a critical test of the adenosine hypothesis. Rather, the adenosine concentration needs to be determined as well to investigate whether adenosine production, and hence the interstitial adenosine concentration, increases in response to the adenosine receptor blockade.
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In our study a coronary artery interstitial adenosine concentration of typically 10 nM was too low to cause vasodilatation in line with previous findings by Tune et al. (2000). Using a modelling strategy from measurements of coronary artery and venous adenosine in dogs Stepp et al. (1996) found an average estimated myocardial interstitial adenosine concentration of 92 nM. This contrasts with findings by the microdialysis technique. With no correction for recovery Wang et al. (1994) found canine myocardial dialysate concentrations of 490 nM at baseline and 790 nM during systemic hypoxia. The much higher values might be explained by the constant beating of the heart against the dialysis tubing leading to cell leakage. The relatively slow changes in coronary artery diameter render this problem negligible in our set-up. The coronary artery interstitial adenosine concentrations found in our study are, nevertheless, smaller than findings in the myocardium, and hypoxia did not increase adenosine concentration within the coronary artery wall. A likely explanation for this result is that oxygen demand per unit volume of endothelial or vascular smooth muscle cells is significantly lower than that of contracting cardiac muscle. Endothelial cells have been shown to remain well energized at PO2 levels above 3 mmHg (Mertens et al. 1990) and smooth muscle cells are known to have a much lower ATP turnover than cardiac muscle.
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While myocardial adenosine could play a role for hypoxia-induced dilatation in intramyocardial vessels it is probably less likely that adenosine in the blood stream could exert coronary dilatation during hypoxia. The concentration of adenosine (30 μM) used to produce vasodilatation in our study was supraphysiological and this is in line with a recent hypothesis that intravascular adenosine does not pass the endothelium, because of endothelial nucleoside transporter activity (Gamboa et al. 2003). Endothelium-dependent vasodilatation due to a direct effect of adenosine seems potentially possible, but in our opinion this concept is not sufficiently supported by experimental data (Stepp et al. 1996).
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We found unchanged or even decreased adenosine concentrations during hypoxia. However, this does not exclude a role for adenosine in hypoxia-induced vasodilatation because hypoxia may sensitize the adenosine receptor. This notion is supported by our finding that adenosine-induced vasodilatation was significantly more pronounced during hypoxia than during normoxia. Furthermore, ZM241385 decreased hypoxia-induced dilatation, which suggests that hypoxia increases sensitivity at the A2A receptor level or increases A2A receptor expression (Kobayashi & Millhorn, 1999). Both the A2A and A2B receptor subtypes have been demonstrated in coronary arteries (Kemp & Cocks, 1999; Flood & Headrick, 2001). Although in a majority of studies the A2A receptor subtype (in contrast to A2B) has been found to mediate vasorelaxation to adenosine this is most likely species dependent; in human small coronary arteries, adenosine mediates most of its vasodilator response via the A2B receptor (Flood & Headrick, 2001), while A2A adenosine receptors mediate dilatation in mouse coronary vessels, and A2B receptors in rat (Kemp & Cocks, 1999).
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The fall in interstitial adenosine during hypoxia and the non-significant fall during continuous oxygenation must be considered a possible methodological limitation. It is unlikely that a longer stabilizing period would have affected this. This can be seen from our testing of any effect on adenosine concentration from preconstriction (which we did not find) where the (non-significant) fall in adenosine continued for up to 3 h even without preconstriction, i.e. a situation comparable to a 3-h stabilizing period. Another possibility is that the total amount of adenosine (absolute recovery of adenosine) exhausted stores and production capacity. We consider this unlikely from the augmented adenosine concentrations after blockade with EHNA, ITU and NBTI. A physiological explanation might be that the 2-h period from the killing of the pig to the beginning of the experiment is an ischaemic preconditioning challenge for the coronary artery. Previously it was found that adenosine levels in phenylephrine-preconditioned rat cardiomyocytes decreased during ischaemia compared to non-preconditioned cells (Vasara et al. 2003).
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Although an NBMPR-sensitive mechanism plays a role for adenosine transport in rat aortic smooth muscle (Leung et al. 2005), in porcine coronary arteries NBTI inhibits adenosine entry (Rubin et al. 2000). The findings in the latter study suggest that an adenosine transporter with characteristics of an equilibrative, NBTI-sensitive subtype (ENT1) is the main transporter for adenosine in our preparation, but the presence of other nucleoside transporter subtypes cannot be excluded and thus other possibilities of adenosine entry into coronary smooth muscle also cannot be excluded.
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We found that the relative recovery of adenosine increased over the experimentation period. Such increase is a common finding with microdialysis in vitro in the model used in the present study (lactate) (Frbert et al. 2002), in living pigs (glucose) (Nielsen et al. 2005) and in humans (glycerol) (Djurhuus et al. 2004). The background for this change in recovery is likely to be multifactorial. Exchange across the microdialysis membrane is dependent on the physical properties of the fibre, the nature of the tissue under investigation, blood flow (or equivalent), analyte under investigation, etc. With the internal reference technique (radioactive adenosine) we do not believe that a change in recovery poses a threat for the interpretation of our data.
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Our microdialysis set-up requires open access to the organ bath, which limits the degree of hypoxia that can be achieved. However, the increase in lactate/pyruvate ratio provides excellent indirect evidence that our arterial tissue was in fact hypoxic. We chose to use oxygen supplementation in the organ bath during ‘normoxic’ conditions, thus reaching a PO2 of 650 mmHg. In our in vitro system, there is no flow through vasa vasorum and thus oxygen reaches the tissue only by diffusion through the medial layers. Previous in vitro experiments have shown zones of severe tissue hypoxia in the media of arteries incubated at 21% oxygen (Bjornheden et al. 1996). However, we cannot totally exclude the possibility that the tissue is slightly hyperoxic, since there is no good surrogate marker for hyperoxia.
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The physiological and pathophysiological implications of the present study are related to the epicardial circulation while the effect of hypoxia on organ blood flow is mainly determined by its effect on vasomotor tone in resistance vessels. However, the dilatory response to hypoxia is not confined to resistance arteries but is also present in conduit vessels (Wadsworth, 1994; Barron & Gu, 2000; Shimizu et al. 2000). Combined with the fact that coronary atherosclerosis is confined to epicardial arteries, even minor diameter changes could become highly important when a haemodynamically significant stenosis is present. Furthermore, information about adenosine metabolism in conductance coronary arteries is warranted because the leading methodology to assess the haemodynamic significance of a coronary stenosis is by inducing hyperaemia in the coronary circulation by means of adenosine, assessing coronary artery pressure before and after the stenosis and calculating the so-called fractional flow reserve (Pijls et al. 1996). Our findings of increased adenosine sensitivity during hypoxia may bridge some of the contradictions in previous studies of unchanged (Downey et al. 1982) and rising (Wang et al. 1994) adenosine concentrations to hypoxia that have made it difficult to link hypoxic vasodilatation to adenosine.
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In conclusion, we report the first measurement of adenosine within the wall of a coronary artery during hypoxia-induced dilatation. This dilatation was accompanied by a fall in interstitial adenosine and, although blockade of adenosine deaminase, adenosine kinase and adenosine transport increased interstitial adenosine, this increase was unrelated to hypoxia. More pronounced vasodilatation to adenosine during hypoxia compared with normoxia might be facilitated by increased adenosine sensitivity at A2A receptor level.
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