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(Received for publication, November 27, 1995; and in revised form, January 23, 1996) From the
In the presence of its substrates hypoxanthine and xanthine,
xanthine oxidase generates oxygen free radicals that cause postischemic
injury. Recently, it has been demonstrated that the burst of xanthine
oxidase-mediated free radical generation in the reperfused heart is
triggered by a large increase in substrate formation, which occurs
secondary to the degradation of adenine nucleotides during ischemia. It
is not known, however, whether blocking this substrate formation is
sufficient to prevent radical generation and functional injury.
Therefore, studies were performed in isolated rat hearts in which
xanthine oxidase substrate formation was blocked with the adenosine
deaminase inhibitor erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA), and
measurements of contractile function and free radical generation were
performed. Chromatographic measurements of the intracellular adenine
nucleotide pool showed that preischemic administration of EHNA blocked
postischemic hypoxanthine, xanthine, and inosine formation. Electron
paramagnetic resonance spin trapping measurements of free radical
generation showed that inhibition of adenosine deaminase with EHNA
blocked free radical generation and that it also increased the recovery
of contractile function by more than 2-fold. Exogenous infusion of
hypoxanthine and xanthine totally reversed the protective effects of
EHNA. These results demonstrate that blockade of xanthine oxidase
substrate formation by adenosine deaminase inhibition can prevent free
radical generation and contractile dysfunction in the postischemic
heart.
Oxygen free radical generation has been shown to be an important
mechanism of cellular injury in ischemic and reperfused
tissues(1, 2) . Studies in a variety of tissues
including heart, lung, kidney, and brain have demonstrated that
intravascular administration of antioxidant enzymes or free
radical-scavenging drugs can prevent reperfusion damage and improve
postischemic function(3, 4) . These studies have
provided indirect evidence of free radical generation in postischemic
tissues. More recently, free radical generation has been measured in
ischemic tissues with EPR spectroscopy. Both direct and spin trapping
EPR techniques have confirmed that there is a burst of oxygen free
radical generation in the heart after postischemic
reperfusion(5, 6, 7, 8) . Although
several mechanisms have been proposed to be involved in the generation
of oxygen free radicals, xanthine oxidase has been shown to be a
central mechanism in a variety of postischemic cells and
tissues(9, 10, 11, 12, 13, 14) .
In reperfused tissues, xanthine oxidase in the presence of its
substrate hypoxanthine or xanthine reduces molecular oxygen to Over the last
decade the therapeutic use of inhibitors of xanthine oxidase has been
proposed as an approach to prevent reperfusion injury (15) .
This was based on evidence that xanthine oxidase is a major source of
reperfusion-associated free radical generation; therefore, blocking the
enzyme would be expected to inhibit radical generation. The importance
of this approach was further supported by the hypothesis that in
ischemic tissues xanthine dehydrogenase, which reduces NAD to NADH, may
be converted via proteolytic cleavage to xanthine oxidase with this
increase in xanthine oxidase activity triggering postischemic free
radical generation(9, 14, 16) . Indeed,
xanthine oxidase blockers have been reported to decrease radical
generation and prevent postischemic heart
injury(12, 17, 18, 19, 20) .
But there was also evidence of possible adverse effects of these drugs,
and a number of reports failed to show in vivo efficacy in
preventing cell death. It has been reported that both the xanthine
oxidase blockers allopurinol and oxypurinol failed to decrease
infarction size in in vivo regional models of myocardial
necrosis(21, 22, 23) . There is also evidence
that these drugs may either exert direct dose-dependent toxicity or
toxicities due to increased xanthine or hypoxanthine
concentrations(12) . Therefore, there has been a need to look
for other alternative ways to prevent free radical-mediated injury. Recently, the kinetics of free radical generation and the
relationship of substrate and enzyme control in the mechanism of free
radical generation from xanthine oxidase during ischemia and
reperfusion have been elucidated in isolated rat hearts(24) .
It was demonstrated that the burst of xanthine oxidase-mediated free
radical generation in the reperfused heart is triggered and its time
course controlled by a large increase in substrate formation, which
occurs secondary to the degradation of ATP during ischemia. Since
the availability of xanthine oxidase substrates was shown to be the
primary factor that triggers and controls the burst of oxygen free
radical generation in the postischemic heart, blocking the formation of
these enzyme substrates should inhibit radical generation and prevent
radical-mediated myocardial dysfunction. Therefore, studies were
performed to determine the effect of adenosine deaminase inhibition on
the formation of xanthine oxidase substrates as well as upon free
radical generation and free radical-mediated contractile dysfunction.
These studies demonstrate that inhibition of xanthine oxidase substrate
formation by blockade of adenosine deaminase can greatly decrease free
radical generation and contractile dysfunction in the postischemic
heart.
Reversed-phase HPLC was
performed using a procedure similar to that of Hull-Ryde et al.(26) using a Waters µBondapak C18 column and a Waters
HPLC system (Waters Associates, Milford, MA) with a model 484 UV
detector, two model 510 reciprocating pumps, and Maxima software, as
described previously(27) .
EPR spectra were recorded in flat
cells at room temperature with a Bruker-IBM ER 300 spectrometer
operating at X-band with a TM 110 cavity using a modulation frequency
of 100 kHz, modulation amplitude of 0.5 G, microwave power of 20
milliwatts, microwave frequency of 9.77 GHz, and acquisition of 10
1-min scans. Quantitation of the free radical signals was performed by
comparing the double integral of the observed signal with that of a
known concentration of the 2,2,6,6-tetramethylpiperidinoxy free radical
in aqueous solution as described previously(6, 11) .
Figure 1:
Schematic diagram illustrating the
reaction pathways by which adenine nucleotides are degraded to form
substrates for the oxygen free radical-generating enzyme xanthine
oxidase. The deamination of adenosine is the rate-determining factor
for the subsequent generation of xanthine oxidase substrates. Adenosine
deaminase inhibition with EHNA blocks the formation of these substrates
and thus may decrease free radical
generation.
Figure 2:
Representative chromatograms of
reversed-phase HPLC nucleotide separation. A 10-µm µBondapak
C18 column (3.9
Figure 3:
Effect of EHNA preischemic administration
on the adenine nucleotide pool in hearts subjected to 30-min ischemia.
As shown, preischemic infusion with 250 µM EHNA
significantly blocked the formation of hypoxanthine and xanthine during
ischemia. Each bar represents the mean ± S.E. values
obtained from measurements of four hearts.
Only trace amounts of inosine and
adenosine could be detected in preischemic heart. At the end of 30 min
of ischemia, the concentration of inosine and adenosine increased to
9.9 ± 0.8 and 8.6 ± 2.6 nmol/mg protein, respectively. In
the presence of EHNA, a marked further increase in myocardial adenosine
level was observed to a concentration of 17.1 ± 0.8 nmol/mg
protein (p < 0.01 versus control hearts). However,
the large rise of inosine that occurred in untreated ischemic hearts
was dramatically decreased to 2.6 ± 0.2 nmol/mg protein (p < 0.001, versus control) ( Fig. 2and Fig. 3B). These data confirmed that adenosine deaminase
had been inhibited by the preischemic infusion of EHNA. Inosine can
be further degraded by inosine phosphorylase to form the xanthine
oxidase substrates hypoxanthine and xanthine. While no hypoxanthine or
xanthine was detected in preischemic hearts, after 30 min ischemia
significant hypoxanthine and xanthine formation and accumulation
occurred with average concentrations of 2.2 ± 0.3 and 2.4
± 0.2 nmol/mg protein. In EHNA-treated hearts, hypoxanthine and
xanthine concentrations were much lower than in untreated hearts with
mean concentrations of 0.6 ± 0.3 and 0.5 ± 0.4 nmol/mg
protein (p < 0.01). Thus, EHNA infusion was effective at
blocking adenosine deaminase within the heart, and it prevented the
formation and accumulation of xanthine oxidase substrates, hypoxanthine
and xanthine, in postischemic myocardium.
Figure 4:
EPR spectra measuring free radical
generation in hearts perfused with 50 mM DMPO prior to
ischemia (A) and after 40-60 s of reperfusion (B) in untreated or EHNA-treated hearts. The predominant
radical signal was the 1:2:2:1 quartet of DMPO-OH, though small trace
amounts of the six peaked DMPO-R adducts were also present. As shown,
the radical signals were markedly attenuated by the preischemic
infusion of 250 µM EHNA.
Figure 5:
Graph of the effect of EHNA on the time
course of free radical generation measured by EPR spin trapping, with
50 mM DMPO. In untreated hearts (circles) a burst of
radical generation during the first 2 min of reperfusion was observed
and gradually declined over the next 5 min. In EHNA-treated hearts (squares) radical concentrations were significantly
decreased.
To evaluate
if EHNA in the concentration used could have any direct effects in
scavenging free radicals, studies were performed evaluating the effect
of 250 µM EHNA on both superoxide and hydroxyl free
radicals generated in vitro from xanthine oxidase using the
spin trap DMPO, 50 mM. In the presence of xanthine, xanthine
oxidase generates superoxide with a prominent DMPO-OOH signal, and EHNA
had no effect on the magnitude of this radical generation (Fig. 6, A and B). Similarly in the presence
of the iron chelate Fe(III)-nitrilotriacetate, xanthine oxidase and
xanthine generate the hydroxyl radical with a prominent DMPO-OH signal,
and this radical generation was not altered by EHNA (Fig. 6, C and D). Thus, EHNA had no measurable direct effect
as a free radical scavenger, which confirms that the observed decrease
in radical generation is due to its effect on decreasing the formation
of xanthine oxidase substrates.
Figure 6:
EPR spectra showing the effects of EHNA on
superoxide or hydroxyl radical generation from xanthine and xanthine
oxidase. Spin trapping measurements were performed using 50 mM DMPO in the absence (A and C) or the presence (B and D) of 250 µM EHNA. The reaction
mixture for superoxide generation (A and B) contained
0.05 units/ml xanthine oxidase, 0.5 mM xanthine, and 1 mM deferoxamine in 50 mM phosphate buffer (pH 7.4). For
hydroxyl radical generation (C and D), deferoxamine
was excluded, and 50 µM Fe
Figure 7:
Recovery of left ventricular developed
pressure, LVDP, in hearts reperfused after 30 min of global ischemia,
expressed as a percentage of preischemic values. Square,
hearts infused with vehicle; circle, hearts infused with 250
µM EHNA prior to ischemia; triangle, hearts
infused with EHNA and 500 µM hypoxanthine and xanthine.
Preischemic infusion of EHNA significantly improved the recovery of
LVDP over the entire duration of reflow (p < 0.01), and
this was totally reversed by hypoxanthine and xanthine. Each point
corresponds to the mean ± S.E. values obtained from the
measurements of seven hearts.
Figure 8:
Recovery of rate pressure product, RPP, in
hearts subjected to 30 min of ischemia followed by reperfusion. Data
are shown as described in the legend to Fig. 7. EHNA
significantly improved the recovery of RPP over the entire duration of
reflow (p < 0.01), and this was totally reversed by
hypoxanthine and xanthine.
Figure 9:
Recovery of coronary flow, expressed as a
percentage of preischemic values. Data are shown as described in the
legend to Fig. 7. EHNA treatment did not alter the observed
coronary flow after reperfusion.
While EHNA
pretreatment resulted in significant beneficial effects on the recovery
of contractile function, with inhibition of the formation of the
xanthine oxidase substrates hypoxanthine and xanthine, and decreased
free radical generation, it also increased the concentrations of
adenosine, and this also might exert protective effects. To further
confirm the importance of the decreased formation of radical-generating
substrates in the protective action of EHNA, additional experiments
were performed to determine whether supplementation of hypoxanthine and
xanthine could reverse this protection. As reported previously, the
concentrations of hypoxanthine and xanthine within the heart are
approximately 500 µM after 30 min of global ischemia (24) . In a series of seven hearts, hypoxanthine (500
µM) and xanthine (500 µM) were infused into
hearts together with EHNA for 10 min followed by subsequent ischemia
and reperfusion. This restoration of hypoxanthine and xanthine totally
reversed the increased recovery of contractile function in EHNA-treated
hearts ( Fig. 7and Fig. 8). In the presence of
hypoxanthine and xanthine, the recovery of LVDP and RPP in EHNA-treated
hearts after 45-min reperfusion were similar to levels observed in
untreated hearts with values of 25.2 ± 5.2% and 18.0 ±
4.7% of preischemic levels. This suggests that the protective action of
EHNA was mainly due to the decreased formation of hypoxanthine and
xanthine, not the elevation of adenosine. Oxidative reperfusion injury is thought to be a central
mechanism of the cellular damage affecting all organs and tissues after
ischemia. There is considerable direct and indirect evidence that
oxygen free radical generation occurs in the early minutes after
reperfusion and results in cellular
injury(1, 2, 3, 4) . It has been
demonstrated that reactive oxygen species including In order to
develop a rational and effective approach to prevent free radical
generation and radical-mediated injury in the postischemic heart, it is
necessary to understand the biochemical process that triggers and
controls this radical generation. It has been demonstrated that
xanthine oxidase is present and is a major source of radical generation
in the isolated rat heart(12) . While this was known for some
time, questions remained regarding the magnitude and relative
importance of enzyme formation versus substrate formation in
triggering the burst of radical generation seen upon reperfusion. It
was originally proposed that in ischemic tissues xanthine dehydrogenase
undergoes proteolytic cleavage to form the oxidase and that a large
proportion of enzyme conversion would trigger the oxidant burst. It was
also recognized that substrate formation would concomitantly occur,
further supporting this process of radical generation(9) .
Recently, studies performed in the isolated rat heart demonstrated that
xanthine oxidase-mediated radical generation is triggered and
controlled primarily by the formation of large concentrations of the
substrates xanthine and hypoxanthine due to the breakdown of ATP during
myocardial ischemia(24) . While no substrate was detectable
prior to ischemia, during ischemia both xanthine and hypoxanthine
concentrations increased by over 1000-fold. Prior to ischemia, however,
xanthine oxidase was present, but in the absence of its substrates no
radical generation was observed. After ischemia only modest 30%
increases were seen. Thus, it was demonstrated that the burst of
radical generation in the ischemic heart was triggered and controlled
primarily by the formation of xanthine and hypoxanthine during
ischemia. In principle, either blocking xanthine oxidase or
inhibiting the formation of enzyme substrates could have identical
effects on radical generation, but the latter strategy would be
expected to quench the initiator that triggered the free radical burst.
A large number of experiments have been performed to evaluate the
effect of xanthine oxidase blockers on reperfusion damage, and variable
efficacy has been
reported(17, 18, 19, 20, 21, 22, 23) .
In contrast, relatively little research has been reported to assess the
potential of inhibiting enzyme substrate formation in attenuating
oxidative reperfusion injury. Abd-Elfattah and colleagues (35, 36) have noted with in vivo surgical
canine models of global ischemia and reflow that pharmacological
interventions aimed at decreasing substrate concentrations can decrease
the severity of injury. However, the exact mechanism by which this
protection occurred was not elucidated. In the present study, we
systematically evaluated how blocking substrate formation affected the
burst of free radical generation and radical-mediated heart
dysfunction. While from prior studies it was clear that radical
generation was triggered by xanthine oxidase substrate formation, it
was not known if blocking this substrate formation could be effective
at preventing free radical generation and free radical-mediated
reperfusion injury. To confirm the functional importance of this
substrate formation in the pathogenesis of postischemic injury and in
an effort to elucidate effective pharmacological approaches to prevent
this tissue injury, the present studies were performed evaluating the
effects of adenosine deaminase inhibition on xanthine oxidase substrate
formation, free radical generation, and functional injury in the
isolated rat heart. It was observed that the adenosine deaminase
inhibitor EHNA was effective at blocking the formation of the xanthine
oxidase substrates xanthine and hypoxanthine. More than 70% block of
this substrate formation was seen in the presence of 250 µM EHNA, with no adverse functional effects. EPR spin trapping
measurements demonstrated that this inhibition of xanthine oxidase
substrate formation was sufficient to decrease radical generation by
more than 80%. While radical generation was inhibited in the heart,
EHNA had no in vitro efficacy as a scavenger of superoxide or
hydroxyl radicals and no measurable effect on xanthine oxidase-mediated
radical generation in the presence of added xanthine. Studies of heart
contractile function demonstrated that this blockade of xanthine
oxidase substrate formation and radical generation resulted in more
than a 2-fold increase in the recovery of contractile function upon
reperfusion. Therefore, these findings suggest that decreasing the
formation of xanthine oxidase substrates by blocking adenosine
deaminase can inhibit oxygen free radical generation and prevent
myocardial reperfusion injury. Inhibition of adenosine deaminase not
only decreased the concentration of hypoxanthine and xanthine, but it
also increased the levels of adenosine within the ischemic heart. This
elevation of adenosine could have also potentially exerted a protective
effect on the heart beyond that which occurs due to prevention of
xanthine oxidase-mediated radical generation. It is well known that
adenosine is a potent coronary vasodilator, and adenosine-mediated
increases in coronary flow could potentially result in enhanced
recovery of cardiac function(37) . Indeed, it has been
suggested that adenosine may have an important role in the process of
myocardial preconditioning, by which short periods of ischemia protect
the heart from subsequent longer periods of ischemia (38) . In
untreated hearts, it was observed that the adenosine concentration
after 30 min of ischemia was increased by about 20-fold, while in the
presence of EHNA a 40-fold increase was seen. Even though the greater
increase in adenosine concentrations in the EHNA-treated hearts could
have potentially resulted in increased coronary flow and subsequent
protection, no such increase occurred, and the recovery of coronary
flow was identical in EHNA and untreated hearts. This suggested that
the further increases in adenosine seen with EHNA did not exert
significant functional effects on myocardial circulation and function.
It is probable that the large increase of adenosine in untreated hearts
is more than sufficient to saturate adenosine receptors within the
heart. In the EHNA-treated hearts a higher recovery of ATP was also
observed, with values of 22% of preischemic values in the untreated
hearts (corresponding to an intracellular concentration of 2.0
mM), while values of 36% were measured in the EHNA-treated
hearts (intracellular concentration of 3.2 mM). Since it has
previously been shown that ATP concentrations of greater than 1 mM are more than sufficient to saturate the myocardial
ATPases(39, 40) , this change in ATP concentration in
itself would not be expected to explain the enhanced recovery of
contractile function that was observed with EHNA. In order to
further demonstrate that the beneficial effects of adenosine deaminase
inhibition were due to the decreased formation of xanthine oxidase
substrates, experiments were performed to determine if the EHNA-induced
protection could be reversed by hypoxanthine and xanthine. We observed
that an exogenous supply of hypoxanthine and xanthine at concentrations
equal to those that occur in the absence of EHNA completely reversed
the EHNA-induced protection of the postischemic heart. These results
confirm that the EHNA-induced protective effects on postischemic heart
function were mainly due to decreased formation of xanthine oxidase
substrates. Adenosine deaminase blockade is only one of several ways
to inhibit the formation and accumulation of hypoxanthine and xanthine
in ischemic tissues. As shown in the ATP degradation diagram,
interrupting any link in the chain of reactions prior to hypoxanthine
formation would limit the production of hypoxanthine and xanthine (Fig. 1). In fact, these enzymatic steps provide multiple
possible sites of pharmacological intervention. For instance, it has
been reported that blocking adenine nucleotide transport protein with p-nitrobenzylthioinosine significantly reduced the formation
of hypoxanthine as well as xanthine and improved postischemic heart
function(35, 36) . Several studies have demonstrated
that inhibition of 5`-nucleotidase exerted protective effects on the
postischemic heart and suggested that this protection was related to
enhanced ATP resynthesis during reperfusion(41, 42) .
Our observations could provide an alternative explanation for these
beneficial effects on postischemic function, since inhibition of
5`-nucleotidase would also be expected to decrease the formation of
hypoxanthine and xanthine, which would decrease free radical
generation. In conclusion, we have demonstrated that the formation
and accumulation of xanthine oxidase substrates in ischemic myocardium
could be largely inhibited by blocking the degradation of adenine
nucleotides using the adenosine deaminase blocker EHNA. Limiting
substrate formation greatly decreased free radical generation, which in
turn resulted in decreased reperfusion injury with increased recovery
of contractile function. Thus, inhibition of adenine nucleotide
breakdown was found to be highly effective at preventing xanthine
oxidase-mediated free radical generation and subsequent contractile
dysfunction in the postischemic heart.
Volume 271,
Number 17,
Issue of April 26, 1996 pp. 10096-10102
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
O
and
HO, which can further react to form the more
reactive OH(12, 13) . The OH and O radicals produced by the enzyme can then in turn oxidize cellular
proteins and membranes resulting in cellular injury.
Isolated Heart Perfusion
Female Sprague-Dawley
rats (250-350 g) were heparinized and anesthetized with
intraperitoneal pentobarbital. The hearts were excised, the aorta was
cannulated, and retrograde perfusion was initiated. Hearts were
perfused at a constant pressure of 80 mm Hg using Krebs bicarbonate
buffer consisting of 120 mM NaCl, 17 mM glucose, 25
mM NaHCO
, 5.9 mM KCl, 1.2 mM MgCl, 1.25 mM CaCl, 0.5 mM EDTA and bubbled with 95% O and 5% CO gas
at 37 °C as described previously(24) . A sidearm in the
perfusion line allowed infusion of different agents according to the
experimental purpose. To monitor cardiac contractile function, a latex
balloon was inserted through an opening in the left atrium across the
mitral valve into the left ventricular cavity and connected to a
pressure transducer as described previously(12) . The balloon
was initially inflated with a volume of distilled water sufficient to
produce an end diastolic pressure in the range of 8-14 mm Hg.
Subsequent measurements of developed pressures were calculated as the
difference between the peak systolic and end diastolic pressure. Left
ventricular pressure was recorded with a Gould RS3400 four-channel
recorder. Coronary flow and heart rates were measured periodically
every 5 min prior to ischemia and after 30 min of global ischemia for
45 min of reperfusion.High Performance Liquid Chromatography (HPLC) (
After 15 min of perfusion with
equilibration of contractile function, hearts were frozen in liquid
nitrogen as a preischemic control. For ischemic and reperfusion
measurements, hearts were subjected to further 30-min periods of global
ischemia or 30 min of ischemia followed by variable duration of reflow.
The hearts were immediately frozen in liquid nitrogen at the desired
time point and then ground under liquid nitrogen. Then the frozen
tissue was transferred to a glass homogenizer with a Teflon pastel and
allowed to thaw with homogenization in 10 ml of 0.5 M perchloric acid at 4 °C. A small amount of homogenate was
removed for assay of total protein content by Lowry assay(25) .
Acid extraction continued on ice for 15 min, at which time cellular
debris was pelleted by centrifugation at 14,000 
)Measurement
g for
1 min. The acid extract was neutralized by mixing with 20 ml of
Freon/trioctylamine (4:1) for 30 s. The mixture was centrifuged at
14,000 g for 1 min, and the upper aqueous layer was
recovered and passed through a 0.45-µm Millipore filter and then
stored at -80 °C for analysis.EPR Spectroscopy and Spin Trapping
Studies were
performed using the spin trap 5,5-dimethyl-1-pyrroline-N-oxide
(DMPO). The DMPO (>97% pure) was purchased from Aldrich and further
purified by double distillation. For the in vitro studies
assessing the effects of EHNA as a radical scavenger, 50 mM DMPO was used. Superoxide was generated from 0.05 units/ml of
xanthine oxidase and 0.5 mM xanthine in 50 mM phosphate buffer containing 1 mM deferoxamine at pH 7.4,
as described previously(13) . To generate the hydroxyl radical,
deferoxamine was excluded and 50 µM Fe(III)-nitrilotriacetate (1:3) was added to facilitate rapid
formation of the hydroxyl radical via the iron-mediated Fenton
reaction(6, 13) . For studies measuring free radical
generation in the isolated heart, DMPO was infused through a sidearm
located just proximal to the heart perfusion cannula with a final
concentration of 50 mM. Spin trap containing effluent was
collected in 20-s aliquots prior to ischemia and at different times of
reflow(12, 14) .Statistical Analysis
Data are expressed as mean
± S.E. Differences in the recovery of various physiology
parameters among the groups were tested using a repeated measures
two-way analysis of variance. Individual comparisons were analyzed by
Student's t test for independent means. The values of p < 0.05 were accepted as statistically significant.
Effect of EHNA on the Adenine Nucleotide Pool in the
Postischemic Heart
It is well established that ATP depletion
during myocardial ischemia leads to accumulation of the diffusible
adenine nucleotides, adenosine and inosine, as well as the
nucleopurines, hypoxanthine and xanthine (Fig. 1). In these
degradation reactions, a limiting step is conversion of adenosine to
inosine catalyzed by adenosine deaminase(28, 29) .
EHNA has been demonstrated to be an adenosine deaminase blocker with K
= 1
nM(30, 31) . Studies were performed to
confirm that EHNA administration prior to ischemia effectively blocked
adenosine deaminase with decreased formation of hypoxanthine and
xanthine. Either untreated or EHNA-pretreated (250 µM infusion for 10 min prior to ischemia) hearts were studied, with 4
hearts in each group. The hearts were subjected to 30-min global
ischemia, and reversed-phase HPLC of heart extracts was performed to
measure the intracellular adenosine nucleotide pool. As shown in Fig. 2a and reported previously(27) , each of
the cellular nucleotides and their metabolites have unique retention
times, allowing measurement and quantitation. Myocardial adenine
nucleotide levels in EHNA-treated and vehicle control groups before and
after ischemia are shown in Fig. 2and Fig. 3. The mean
preischemic levels of adenosine nucleotides in EHNA and control groups
were similar, with no significant differences observed. At the end of
30-min ischemia, ATP and ADP levels in control hearts markedly dropped
to 7.2 ± 0.4 and 4.9 ± 1.1 nmol/mg protein from the
preischemic values of 33.4 ± 0.5 and 16.3 ± 0.2 nmol/mg
protein (p < 0.01, versus preischemia). While
preischemic administration of EHNA resulted in higher mean ATP and ADP
levels after ischemia with values of 12.6 ± 2.7 and 9.6 ±
2.4 nmol/mg protein, respectively, due to interexperiment variation
these differences did not reach statistical significance (p = 0.17 and p = 0.09, respectively, for EHNA versus untreated hearts). Myocardial AMP and NAD levels were
also not significantly affected by the inhibitor (Fig. 3A).
150 mm) was used with a 20-µl injection.
The peaks are labeled as follows: A, uric acid; B,
ATP; C, ADP; D, hypoxanthine; E, xanthine; F, AMP; G, inosine; H, NAD; I,
adenosine. Panel a shows a chromatogram of a mixture of
standards containing a 100 µM concentration of each
compound. Chromatograms from extracts of an untreated heart (b) or an EHNA-treated heart (c) (preischemic
infusion of 250 µM EHNA) after 30-min ischemia are shown.
In the untreated hearts, significant inosine, hypoxanthine, and
xanthine peaks were seen (b). In EHNA-treated hearts, there
was a marked decrease in the inosine peak and a large increase in the
adenosine peak (c). No significant hypoxanthine or xanthine
peaks were observed in the EHNA-treated
hearts.
Effect of EHNA on Free Radical Generation in the
Postischemic Heart
Preischemic administration of EHNA
significantly reduces the formation and accumulation of
radical-generating substrates hypoxanthine and xanthine. In order to
determine whether this results in inhibition of free radical
generation, experiments were performed comparing radical generation in
untreated and EHNA-treated hearts, with three hearts in each group. The
coronary effluent containing the spin trap DMPO was collected in 20-s
aliquots during both preischemic and postischemic infusion, and EPR
measurements were performed. No significant signal was observed prior
to ischemia in either untreated control or EHNA-treated hearts. In
control hearts, a prominent spectrum was seen immediately after the
onset of reflow. As reported previously, this spectrum consisted of a
large 1:2:2:1 quartet signal, a
= a
= 14.9 G, indicative of DMPO-OH, and a small 1:1:1:1:1:1
sextet signal, a = 16.0 and a =
22.0 G, indicative of DMPO-R (Fig. 4). Maximum signal intensity
was observed within the first minute of reflow followed by a gradual
decline over the next 5 min. In the presence of EHNA, the amplitude of
the observed free radical generation was decreased by more than 80%,
compared with that in untreated hearts (Fig. 5).
-nitrilotriacetate was added. As shown, EHNA
did not scavenge either superoxide or hydroxyl radicals generated from
xanthine oxidase.
Effect of EHNA on Postischemic Injury
To determine
if adenosine deaminase inhibition prevented postischemic injury and
enhanced the recovery of contractile function after reperfusion, a
series of 14 hearts were subjected to 30 min of global ischemia
followed by 45 min of reperfusion, and hemodynamic indexes were
continuously recorded. 250 µM EHNA or vehicle were infused
into the heart prior to ischemia. Preischemic administration of EHNA
had no effect on contractile function or coronary flow. However,
EHNA-treated hearts had significantly improved contractile performance
with higher recovery of left ventricular developed pressure (LVDP) and
rate-pressure product (RPP). EHNA-treated hearts exhibited more than
2-fold increased recovery of LVDP and RPP over the entire course of
reflow ( Fig. 7and Fig. 8). Average final recovery of
LVDP and RPP after 45 min of reperfusion was 48.2 ± 8.4% and
44.6 ± 8.9% in the EHNA group, compared with only 25.0 ±
2.5% and 23.3 ± 3.7% in the untreated controls (p <
0.01). EHNA pretreatment did not significantly alter the recovery of
coronary flow upon reperfusion (Fig. 9).
O,
HO, and OH are produced at the
time of postischemic reperfusion(5, 6, 7) .
These oxidants can be cytotoxic to cells by initiating lipid
peroxidation of cell membranes and reacting with proteins causing amino
acid oxidation and polypeptide chain scission. While several mechanisms
have been described for the production of oxygen free radicals in
postischemic tissues, xanthine oxidase has been widely accepted as a
major primary source of this radical
generation(9, 10, 11, 12, 13, 14) .
This theory was initially supported by many studies that demonstrated
that administration of xanthine oxidase inhibitors reduced the
incidence of reperfusion arrhythmias and improved functional
recovery(17) . More recently, further direct evidence for the
presence of xanthine oxidase-mediated free radical generation in
postischemic cells and tissues has been provided by EPR studies that
demonstrate that xanthine oxidase inhibition markedly decreases radical
generation in both reoxygenated endothelial cells and in the isolated
rat heart(12, 13) . While it had been questioned
whether this mechanism of radical generation occurs in humans tissues (32, 33) , recently it has been demonstrated that
xanthine oxidase is present in both human arterial and venous
endothelial cells and that under conditions of anoxia and reoxygenation
the enzyme gives rise to free radical generation, which can result in
cellular injury and death (27, 34) .
)
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
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