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(Circulation. 1999;99:2041-2047.)
© 1999 American Heart Association, Inc.
Basic Science Reports |
Quantification of Extracellular and Intracellular Adenosine Production
Understanding the Transmembranous Concentration Gradient
From the Institut für Physiologie, Medizinische Fakultät Carl Gustav Carus, TU Dresden (A.D., M.S.), and the Klinik für Kardiologie, Pneumologie, und Angiologie, Heinrich-Heine-Universität Düsseldorf (S.S., M.K.), Germany.
Correspondence to Dr Andreas Deussen, Professor and Chairman, Institut für Physiologie, Medizinische Fakultät Carl Gustav Carus, TU Dresden, Fetscherstraße 74, D-01307 Dresden, Germany.
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Abstract |
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 Top Abstract IntroductionMethodsResultsDiscussionReferences |
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Background—Inhibitors of adenosine membrane transport cause vasodilation and enhance the plasma adenosine concentration. However, it is unclear why the plasma adenosine concentration rises rather than falls when membrane transport is inhibited. We tested the hypothesis that the cytosolic adenosine concentration exceeds the interstitial concentration under well-oxygenated conditions.
Methods and Results—In isolated, isovolumically working guinea
pig hearts (n=50), the release rate of adenosine and
accumulation of S-adenosylhomocysteine (after 20 minutes
of 200 µmol/L homocysteine), a measure of the free cytosolic
adenosine concentration, were determined in the absence and
presence of specific and powerful blockers of adenosine
membrane transport (nitrobenzylthioinosine 1 µmol/L),
adenosine deaminase (erythro-9-hydroxy-nonyl-adenine 5
µmol/L), and adenosine kinase (iodotubericidine 10
µmol/L). Data analysis with a distributed multicompartment
model revealed a total cardiac adenosine production
rate of 2294 pmol · min-1 ·
g-1, of which 8% was produced in the extracellular
region. Because of a high rate of intracellular metabolism,
however, 70.3% of extracellularly produced adenosine was taken
up into cellular regions, an effect that was effectively eliminated by
membrane transport block. The resulting 
2.8-fold increase of the
interstitial adenosine concentration evoked
near-maximal coronary dilation.
Conclusions—We rejected the hypothesis that the cytosolic adenosine concentration exceeds the interstitial. Rather, there is significant extracellular production, and the parenchymal cell represents a sink, not a source, for adenosine under well-oxygenated conditions.
Key Words: adenosine • blood flow • inosine • S-adenosylhomocysteine
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Introduction |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Adenosine serves several important functions in the heart that are mediated via stimulation of cell surface receptors.1 However, because of rapid metabolism2 and a high degree of compartmentalization,3 4 the precise interstitial adenosine concentration that evokes these effects is unknown. Traditionally, it is assumed that the major site of adenosine production is the cytosol, from which the nucleoside diffuses into the interstitial space to finally reach the vascular region, from which a small fraction5 6 is washed out with the coronary flow. This concept, however, cannot explain the effects of membrane transport blockers, which increase the vascular adenosine concentration.2 An explanation for this discrepancy could be that these blockers inhibit only inward membrane transport in the heart. Alternatively, enhanced vascular concentrations seem possible if net uptake of adenosine supplied by extracellular dephosphorylation of adenine nucleotides7 8 is inhibited. A previous limitation in discerning between these alternative explanations has been the inability to measure the adenosine concentration on both sides of the membrane. However, because of the invention of the S-adenosylhomocysteine (SAH) technique9 and formulation of a related comprehensive mathematical model,10 it has become possible to quantify cytosolic adenosine concentrations. The present study was designed to determine the adenosine concentrations for the different tissue regions and thereby to assess the net transmembranous concentration gradient. Therefore, in a first experimental stage, we determined the coronary venous adenosine concentration as a measure of the intravascular concentration and the tissue SAH content in the presence of homocysteine as a measure of the free intracellular adenosine concentration. Measurements were made before and after blocking of the facilitative membrane transporter in the absence and presence of blockers of adenosine metabolism. In a second stage, the experimental results were analyzed with a mathematical model10 to calculate the adenosine production of different tissue regions and predict net transport between regions. The interstitial adenosine concentration was related to the measured changes of coronary flow.
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Methods |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Isolated Heart Experiments
Hearts from 50 guinea pigs (body mass, 250 to 350 g) were perfused with a modified Krebs buffer (perfusion pressure, 70 cm H2O; arterial PO2, >520 mm Hg; 37°C).9 Coronary flow (electromagnetic probe), perfusion pressure, isovolumic left ventricular pressure development, dP/dt, and heart rate (strain-gauge pressure transducers) were measured continuously. Hearts were included in the study only if (1) the maximum flow overshoot after a 20-second flow stop was at least twice the flow before occlusion, (2) spontaneous heart rate exceeded 210 bpm, and (3) left ventricular developed pressure exceeded 50 mm Hg. In 9 hearts, oxygen consumption was determined according to the Fick principle. For measurement of cardiac adenosine and inosine, release of the coronary effluent perfusate was collected over 1 to 2 minutes. Individual pathways of adenosine metabolism or membrane transport were inhibited by use of 5 µmol/L erythro-9-hydroxy-nonyl-adenine (EHNA) for adenosine deaminase, 10 µmol/L iodotubericidine (ITU) for adenosine kinase,6 11 and 1 µmol/L nitrobenzylthioinosine (NBTI), dipyridamole (DIP), or draflazine (DRA) for membrane transport.12 Blockers dissolved in physiological saline (except NBTI, which was dissolved in dimethylsulfoxide) were infused at rates of 0.1% (NBTI) or 1% (all others) of coronary flow. EHNA, ITU, and NBTI were from RBI; DIP was from Thomae Pharmaceuticals; and DRA was from Janssen Pharmaceuticals.
Rationale and Experimental Protocols
Global Cardiac Adenosine Production
Adenosine is metabolized via adenosine kinase
and adenosine deaminase. During complete inhibition of these
enzymes, the venous release rate of adenosine
represents a minimum estimate of the global production
rate. This approach rests on the assumption that a steady-state cardiac
adenosine concentration is reached during enzyme
inhibition.
Intracellular Versus Extracellular Adenosine Production
During continuous inhibition of adenosine kinase and
adenosine deaminase, membrane transport is blocked. If
adenosine is produced mainly in the cytosol, then membrane
transport block should decrease net cellular adenosine release
and consequently venous release. If, however, adenosine
production is mainly extracellular, no decrease but possibly
even an increase of venous adenosine release is to be
expected.
Intracellular adenosine production rate may also be estimated from tissue SAH content. During inhibition of adenosine metabolism and transport, L-homocysteine thiolactone was infused (intracoronary concentration, 200 µmol/L; 20 minutes) to drive the SAH-hydrolase reaction toward net production of SAH.9 The SAH content after homocysteine infusion represents the integral of the net reaction from adenosine to SAH, serving as an independent estimate of the minimum intracellular adenosine production rate. In control experiments, the SAH content was measured after 20 minutes of homocysteine alone. The implicit assumption in these experiments is that homocysteine thiolactone enters the cells by a pathway separate from that used by adenosine and that this process is unaffected by any of the blockers of transport or metabolism.
Transmembranous Concentration Gradient
The effects of adenosine transport blockers on
adenosine release are quantified under control conditions. If
the direction of the adenosine concentration gradient is from
intracellular to extracellular, then inhibition of adenosine
membrane transport should decrease the rate of adenosine
release into the coronary venous perfusate. However, if
adenosine is produced in the extracellular region and diffuses
into the cytosol to be further metabolized, then adenosine
release should rise under this intervention. In addition, SAH content
was determined after 20 minutes of infusion of
L-homocysteine thiolactone 200 µmol/L without and
with block of adenosine membrane transport.
Adenosine Deaminase Experiments
To test whether coronary flow changes were induced by
adenosine, purified6 adenosine deaminase
from calf intestinal mucosa (Boehringer Mannheim) was used. In
control experiments, adenosine deaminase was infused during
coronary dilation with bradykinin 0.1 µmol/L.
Sample Processing and Analytical Determinations
For measurement of adenosine and inosine,
coronary effluent samples were desalted and concentrated with
C-18 cartridges.4 For measurement of tissue SAH content,
hearts were freeze-clamped after 20 minutes of homocysteine infusion
and freeze-dried. Left ventricular samples were
homogenized in 0.5 mol/L perchloric acid.9
Compounds were quantified in 50- to 100-fold–concentrated samples of
effluent coronary perfusate or tissue
homogenate (0.1 g tissue dry mass/mL final extract) by
high-performance liquid chromatographic
analysis.9 Data reported are corrected for
analytical losses.
Statistics
Data are reported as mean±SD; significance levels are 2-sided.
The significance of differences among different experimental groups was
determined by use of 1-way ANOVA and post hoc t tests with
Bonferroni correction. Within-subject differences of purine release and
flow during additive blocking protocols were assessed with ANOVA for
repeated measures. If significant intraindividual effects were
discovered, conditions were tested against the next preceding condition
by Wilcoxon matched-pairs rank test. Linear correlation
coefficients were calculated from the individual data points.
Model Analysis
The axially distributed multicompartment model of
adenosine transport and metabolism has been
described in detail and validated against experimental
data.10 As a starting point for the present
analyses, the published parameter set was
used.10 The general modeling strategy was to fit measured
venous adenosine concentrations by constraining the flow term
by the measurements and using adenosine production
terms as free parameters. To obtain parameter
values that fitted the venous concentrations determined under various
experimental conditions equally well, several iterations of
parameter adjustment were necessary. Once this
parameter set had been obtained, it was used to fit the SAH
contents without any further adjustment.
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Results |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Experimental Measurements
Cardiac Dynamics and Oxygen Consumption
Control left ventricular pressure development was 84±15 mm Hg, left ventricular dP/dtmax 1440±330 mm Hg/s, heart rate 249±24 bpm, and coronary flow 6.7±1.2 mL · min-1 · g-1 (n=44). Arterial and coronary venous PO2 were 577±41 and 202±30 mm Hg, respectively, and myocardial oxygen consumption was 75±15 µL · min-1 · g-1 (n=9). The functional parameters, except flow and heart rate, were unaffected by the different enzyme or transport inhibitors. Changes in oxygen consumption after possible bradycardia during inhibition of adenosine kinase or membrane transport were prevented by pacing.
Global Cardiac Adenosine Production
In 14 hearts, control adenosine release was 53±24
pmol · min-1 ·
g-1, inosine release 489±239 pmol ·
min-1 · g-1, and
coronary flow 6.7±1.2 mL ·
min-1 · g-1.
During EHNA, adenosine release was 91±53 pmol ·
min-1 · g-1
(P=0.003 versus control), inosine release 416±235 pmol
· min-1 · g-1
(P=0.046 versus control), and flow 6.7±1.3 mL ·
min-1 · g-1
(P=0.422 versus control). During EHNA plus ITU,
adenosine release was 1511±406 pmol ·
min-1 · g-1
(P=0.001 versus EHNA), inosine release 475±231 pmol
· min-1 · g-1
(P=0.213 versus EHNA), and flow 14.3±3.1 mL ·
min-1 · g-1
(P=0.001 versus EHNA). Results are shown for the NBTI
subgroup in Figure 1
.
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Intracellular and Extracellular Adenosine Production
As shown in Figure 1
, additional infusion of NBTI during
EHNA plus ITU decreased adenosine release from 1744±518 to
297±106 pmol · min-1 ·
g-1 (P=0.028). Coronary flow
remained unaffected (12.8±3.5 versus 13.1±4.0 mL ·
min-1 · g-1).
Inosine release was 649±295 pmol ·
min-1 · g-1 before
and 584±225 pmol · min-1 ·
g-1 during additional infusion of NBTI
(P=NS). Results similar to those with NBTI (n=6) were
obtained with DIP (n=8). During inhibition of adenosine
deaminase and adenosine kinase, DIP decreased adenosine
release from 1337±183 to 429±135 pmol ·
min-1 · g-1
(P=0.012).
Control tissue contents of adenosine, inosine, and SAH were
1.6±0.7, 1.8±0.5, and 1.5±0.5 nmol/g, respectively (Figure 2). After 20 minutes of homocysteine
(n=7), SAH content was 7.9±1.6 nmol/g (P<0.05 versus
control). Tissue contents of adenosine and inosine remained
unchanged. During treatment with EHNA plus ITU plus NBTI (n=6),
the SAH content after 20 minutes of homocysteine was 23.7±2.8 nmol/g
(P<0.05 versus homocysteine). Similarly, the SAH content
was 17.5±5.9 nmol/g during EHNA plus ITU plus DIP treatment after 20
minutes of homocysteine (P<0.05 versus homocysteine,
P>0.05 versus EHNA plus ITU plus NBTI). During treatment
with EHNA plus ITU plus NBTI plus homocysteine, tissue
adenosine and inosine contents were 3.9±1.1 and 14.6±5.0
nmol/g, respectively (both P<0.05 versus homocysteine).
Tissue inosine content correlated linearly with duration (20 to 65
minutes) of membrane transport inhibition (r=0.886,
P=0.0001, n=19). Twenty minutes of NBTI increased the tissue
inosine content from 1.4±0.5 to 14.6±5.0 nmol/g and decreased the
coronary venous inosine concentration from 97±72 to 50±28
nmol/L (n=6). The tissue inosine content (nmol/g) was converted into a
concentration (µmol/L), assuming that tissue inosine was restricted
to intracellular regions, which constitute 0.60 mL/g
tissue.13 The respective tissue/venous
perfusate concentration ratio increased from 24.5 (control) to
488 after 20 minutes of NBTI (Figure 3).
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Transmembranous Concentration Gradient
In 16 hearts, control cardiac adenosine release was 46±22
pmol · min-1 ·
g-1 and coronary flow 6.6±1.3 mL
· min-1 · g-1
(Figure 4). NBTI enhanced
adenosine release to 179±72 pmol ·
min-1 · g-1 and
flow to 12.4±3.1 mL · min-1 ·
g-1 (both P=0.008). Similar effects
on adenosine release and flow were obtained with DIP and DRA.
Infusion of homocysteine resulted in SAH contents of 8.5±1.1 nmol/g
during NBTI plus EHNA (n=5), 7.7±2.0 nmol/g during DIP plus EHNA
(n=4), and 9.3±2.1 nmol/g during EHNA alone (n=3). These
concentrations were not significantly different from each other.
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Adenosine Deaminase Experiments
NBTI (n=2) or DIP (n=2) increased coronary flow 113% to
149% above control levels. Additional infusion of adenosine
deaminase (10 U/mL) blunted this response (flow, 0% to 23%
above control), and adenosine was undetectable in the
coronary venous perfusate. During bradykinin infusion
(0.1 µmol/L), flow rose by 90% to 136% (n=2) and remained
elevated (86% to 124% above control) during additional infusion of
adenosine deaminase.
Model Calculations
Starting with a previously published parameter
set,10 the effects of transport inhibition were simulated
by setting the parameter values of the membrane
permeability–surface area products to 2% of the starting
values10 (98% inhibition of transport at basal
adenosine concentrations) and flow rate to the measured value.
An interstitial adenosine production term
of 185 pmol · min-1 ·
g-1 fitted the mean measured release rate within
3%. This value was kept constant for all further simulations. Next,
the intracellular adenosine production rates were
estimated. Production rates of 1800 and 90 pmol ·
min-1 · g-1 for
the parenchymal and the endothelial-cell regions,
respectively, fitted the measurements of adenosine release made
during EHNA and ITU treatment, simulated by increasing the respective
enzyme Km values 100-fold, within 1%. With
transmethylation rates of 190 and 30 pmol ·
min-1 · g-1 in
parenchymal and endothelial cell
regions,10 14 respectively, total cardiac
production rate was 2295 pmol ·
min-1 · g-1 (Table 1).
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With these production rates, venous release rates calculated
for control conditions were slightly higher than those measured (90
versus 53±24 pmol · min-1 ·
g-1). This misfit was brought about by too-high
intracellular and/or interstitial concentration estimates,
which resulted in a too-high venous release rate. Likely explanations
for this moderate discrepancy were higher intracellular rates of
adenosine degradation and/or a lower
interendothelial gap permeability–surface area
product for adenosine than previously10
thought. Assuming a combination of both effects, the
parameter values for the Vmax of
adenosine kinase were enhanced by 50% (set to 150 and 56
nmol · min-1 ·
g-1 for the parenchymal and
endothelial-cell regions, respectively), and the
endothelial gap permeability–surface area product
was lowered by 20% (set to 2.0 mL ·
min-1 · g-1). With
these moderate adjustments, the model fitted the adenosine
release rates determined under the different conditions almost equally
well (Table 2). Furthermore, SAH contents
after 20-minute step inputs of 200 µmol/L homocysteine were
predicted reasonably well for all conditions without any further
adjustment of model parameters. This parameter
set also fitted the adenosine release rate and the tissue SAH
content of the previous study10 within 10%.
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Figure 5 shows measured coronary
flows plotted versus calculated adenosine concentrations of
capillary and interstitial regions, respectively. For a
control coronary venous adenosine concentration of 10.1
nmol/L, calculated capillary and interstitial
concentrations were 7.3 and 34 nmol/L, respectively. For membrane
transport block, capillary and interstitial concentrations
were 9.3 and 94 nmol/L, respectively. For simultaneous
inhibition of adenosine kinase and adenosine deaminase,
concentrations of 74 and 420 nmol/L, respectively, were calculated.
Simulation of concerted blocking of transport and
metabolism predicted that the capillary and
interstitial adenosine concentrations should
decrease but still remain above those calculated for inhibition of
adenosine membrane transport alone (11.3 and 121 nmol/L,
respectively). This explains the observation that NBTI in the presence
of EHNA plus ITU reduced venous adenosine release but left
coronary flow unchanged (Figure 1).
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, control;
, NBTI; 
, EHNA, ITU, NBTI; 
EHNA, ITU. |
Discussion |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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We tested the hypothesis that the cytosolic adenosine concentration exceeds the interstitial in the well-oxygenated heart. We found that nucleoside transport blockers effectively inhibit cellular efflux of adenosine when the cytosolic concentration is raised. However, during physiological conditions, nucleoside transport blockers raise the interstitial adenosine concentration by inhibiting net cellular uptake of extracellularly produced adenosine. The findings are consistent with the assumption of a bidirectional block of membrane transport and indicate that the parenchymal cell represents a sink rather than a source of adenosine under well-oxygenated conditions. The transmembranous concentration gradient from extracellular to intracellular is in contrast to our intuition that there is a concentration gradient from the parenchymal cell to the capillary region, because the largest production site is in the parenchymal cell.
Global Adenosine Production Rate
During EHNA plus ITU, adenosine release averaged 1511
pmol · min-1 ·
g-1 (Figure 1). This release rate is
lower than those of 2 previous studies5 6 that reported
3.3 and 3.48 nmol · min-1 ·
g-1, respectively, during the same
inhibitor protocol. Reasons for these discrepancies are not
entirely clear but are probably related to the quality of the isolated
heart preparation (see Methods, Isolated Heart Experiments). Modeling
adenosine production rates by fitting venous release
rates revealed an estimate of 2294 pmol ·
min-1 · g-1 (Table 1). This analysis accounted for incomplete enzyme
inhibition because of increased substrate concentrations. Global
production rate would have been underestimated by 31% if
inferred from direct measurement of the release rate (1511 pmol
· min-1 · g-1).
The present estimate of the global production rate is 67%
of those reported before,5 6 which did not take the
effects of increased substrate concentrations into account.
Intracellular Adenosine Production
During EHNA plus ITU, NBTI reduced coronary venous
adenosine release from 1744 to 297 pmol ·
min-1 · g-1
(Figure 1). Thus, NBTI blocked adenosine cell outward
transport effectively, and 
83% of the global adenosine
production originated from an intracellular site. This
conclusion is supported by measurements of SAH content after 20 minutes
of homocysteine. SAH content differed by 15.8 nmol/g in the
presence and absence, respectively, of concerted block of
adenosine metabolism and transport (Figure 2). Because the rate of SAH production is rather linear
during 20 minutes of homocysteine treatment under steady-state
conditions,9 a mean production rate of 790
pmol · min-1 ·
g-1 may be assumed. This value is 55% of the
decrease of venous adenosine release after NBTI (1447 pmol
· min-1 · g-1).
The difference is probably largely accounted for by a decreasing
effectiveness of enzyme inhibition at an increased cytosolic
adenosine concentration (Figure 2). If the measured SAH
content was fitted by the model, thereby accounting for incompleteness
of enzyme inhibition, a global production rate of 1623
pmol · min-1 ·
g-1 was obtained. This figure is 70.7% of that
estimated from fitting measurements of the venous adenosine
release rate (Table 1).
Effective block of outward membrane inosine transport by NBTI is
suggested by the reciprocal changes of tissue and effluent
perfusate concentrations (Figure 3). During
well-oxygenated conditions, inosine is derived largely from
inosine monophosphate, not adenosine. This is indicated
by the following lines of evidence. (1) At a concentration of 5
µmol/L EHNA, no residual activity of adenosine deaminase was
found in the guinea pig heart.6 (2) Although EHNA has only
small effects on coronary venous adenosine release
under well-oxygenated conditions, it doubles
adenosine release during tissue hypoxia14
when inosine production from adenosine is increased.
Thus, it is not a valid approach to determine adenosine
production under well-oxygenated conditions by
summing values of adenosine, inosine, (hypo)xanthine, and uric
acid.15
Extracellular Adenosine Production
Under control conditions, NBTI enhanced adenosine
release from 46 to 179 pmol · min-1
· g-1 (Figure 4), whereas SAH content
remained unchanged (8.5±1.1 versus 9.3±2.1 nmol/g). This suggested an
adenosine concentration gradient directed from extracellular to
intracellular. Because 1 µmol/L NBTI blocked adenosine
membrane transport sites nearly completely,12 the
adenosine release rate of 179 pmol ·
min-1 · g-1 is
probably a rather accurate figure of the extracellular
production rate in the isolated guinea pig heart (model
estimate, 185 pmol · min-1 ·
g-1). Compared with the above estimates of the
global adenosine production rate (1623 and 2294
pmol · min-1 ·
g-1, respectively), extracellular
production contributed 8% to 11% under
well-oxygenated conditions.
Adenosine produced in the interstitial region is
most likely derived from 5'-AMP. Membrane-bound 5'-nucleotidase has
been documented histochemically on the cell surface of
cardiomyocytes and vascular cells of guinea pig
heart.16 In this species, AMP is recovered from an
extracellular fluid region,7 and coronary venous
adenine nucleotide release is 40 pmol ·
min-1 · g-1 under
control conditions and 430 pmol ·
min-1 · g-1 during
ecto-5'-nucleotidase inhibition by 
,ß-methyleneadenosine
diphosphate.8 This release is quantitatively sufficient to
account for the extracellular adenosine production rate
estimated in the present experiments. Concerning possible sources
of extracellular nucleotides, our own experiments indicate
that endothelial cells11 and smooth muscle
cells17 need to be considered. Whether the multidrug
resistance (mdr1) gene product mediates cell
nucleotide release18 in cardiac tissues
remains to be shown.
Transmembranous Adenosine Concentration Gradient
Figure 6 summarizes interregional
adenosine flux rates calculated by model analysis from
experimental measurements (Figures 1 through 4). These net exchange rates are averages over the
entire capillary length. With an inflow adenosine concentration
of zero, regional concentrations increase monotonically along the
capillary length.10 Thus, concentrations lower than those
shown in Figure 6 would be expected near the
arterial inflow and higher concentrations near the venous
outflow. In extracapillary regions, this axial concentration gradient
is <20%.10 The present model calculations for
well-oxygenated conditions show the highest concentration
(34 nmol/L) in the interstitial region. The free
adenosine concentration of the parenchymal cell was slightly
lower and that of the endothelial cell notably lower
because of intracellular metabolism. The steeper
concentration gradient across the abluminal endothelial
cell membrane reflects the assumption of a 20-fold higher
endothelial adenosine deaminase activity per
milliliter of cell volume compared with the cardiomyocyte
region.19 Our analyses predict that of
extracellularly produced adenosine, 4.9% is taken up into
parenchymal cells, 65.4% is taken up into endothelial
cells, and 29.7% escapes from the interstitial region
through interendothelial clefts into the capillary
region.
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Similar model analyses conducted in a previous study on the
same experimental model20 revealed
interstitial adenosine concentrations of 6.8 nmol/L
under control conditions (our estimate, 34 nmol/L) and 191 nmol/L
during membrane transport block by dipyridamole (our
estimate, 94 nmol/L). Specific differences from the present study
were as follows. (1) Dipyridamole was used at a
concentration of 10 µmol/L, which has significant side effects
on adenosine metabolism21 22 and
therefore probably overestimated extracellular adenosine
production (496 versus 185 pmol ·
min-1 ·
g-1). (2) The previous study attributed
adenosine production exclusively to the
interstitial region. (3) The global adenosine
production rate (496 pmol ·
min-1 · g-1) was
smaller than that assessed in the present study (2295 pmol ·
min-1 · g-1, Table 1). For these reasons, that previous study most likely
underestimated the interstitial adenosine
concentration under control conditions and overestimated it during
membrane transport block.
Study Limitations
The conclusions that may be drawn from inhibitor
experiments depend on the potency and specificity of the
inhibitors. EHNA 5 µmol/L,6 ITU 1 to
10 µmol/L,6 11 and NBTI 0.1 to 1.0
µmol/L12 inhibit the respective enzymes or transporter
in guinea pig heart specifically and nearly completely. An
effectiveness of >90% was reported for DIP in the guinea pig heart at
concentrations of 1.0 to 5.0 µmol/L.12 Under our
experimental conditions, steady-state extraction of
[14C]adenosine before and during DIP
1 µmol/L was 56% and 3%, respectively (n=2). However, higher
concentrations may inhibit adenosine deaminase21
or phosphodiesterase.22 NBTI 10 µmol/L or DIP
10 µmol/L has no effect on in vitro SAH hydrolase activity
extracted from guinea pig liver (A.D., unpublished data). Although the
inhibitors were potent and specific at the concentrations
used, in intact tissue, block of 1 or several metabolic
pathways may enhance the concentration of unmetabolized substrate.
Therefore, estimates of adenosine production rates made
from outflow measurements must be taken as minimum figures or, even
better, should be subjected to a mathematical model analysis
that accounts for the effects of substrate accumulation.
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Acknowledgments |
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The support by grants De360/4-1 and De360/6-1 from the Deutsche Forschungsgemeinschaft and a grant from the Eberhard-Igler-Stiftung is gratefully acknowledged.
Received August 13, 1998; revision received November 19, 1998; accepted December 7, 1998.
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