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Bristol Heart Institute, Bristol Royal Infirmary, University of Bristol, Bristol BS2 8HW, United Kingdom
Submitted 21 January 2003 ; accepted in final form 8 May 2003
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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,
-methylene-ADP, but was affected by a nucleoside transporter,
dipyridamole. The positive chronotropic effect of isoproterenol (130 ±3
vs. 100 ±4 beats/min) was inhibited (107 ±3 vs. 94 ±3
beats/min) in cells overexpressing cN-IA, and this was reversed by the
addition of the adenosine receptor antagonist
8-(p-sulfophenyl)theophilline (120 ± 3 vs. 90 ± 4
beats/min). Our results demonstrate that overexpressed cN-IA can be
sufficiently active in cardiomyocytes to generate physiologically effective
concentrations of adenosine at its receptors.
chronotropic effect; catecholamines; ATP metabolism
Ecto-5' -nucleotidase (e-N) can catalyze the terminal dephosphorylation of AMP to adenosine. Biochemical experiments with inhibitors and cloning have demonstrated conclusively that this enzyme is only capable of metabolizing extracellular nucleotides, in a cascade with other ectoenzymes (39). Consistent with this, adenosine derived from AMP perfused through the heart requires e-N (25). However, a link between intracellular energy utilization, nucleotide release, and e-N activity is at best controversial. In neonatal myocytes, adenosine formation induced by chemical hypoxia occurs exclusively intracellularly (21), although the extracellular pathway may also contribute in adult myocytes (4). The situation in the whole heart is more complicated due to the contribution made by the vasculature and nerve terminals. Experimental evidence has been presented in favor (13, 15) and against (3, 33) the involvement of the ecto pathway under normoxic and hypoxic conditions. Deussen and colleagues (7) used trapping of adenosine through S-adenosylhomocysteine hydrolase and mathematical modelling to estimate the contribution of cytosolic and extracellular pathways to adenosine production in normoxic guinea pig hearts. They concluded that 8–11% of adenosine production is derived from extracellular metabolism. However, increased adenosine formation in hearts stimulated by catecholamines (8, 9) apparently does not involve e-N (3, 19).
Alternatively, adenosine could be formed from cytosolic AMP, which rises
during ATP breakdown owing to the myokinase equilibrium, by the action of a
cytosolic 5'-nucleotidase (cN). Adenosine might then be released from
cells via equilibrative nucleoside transporters
(17). An IMP-preferring
enzyme, cN-II, was cloned and overexpressed and shown to selectively catalyze
conversion of IMP to inosine rather than AMP to adenosine
(28). An AMP-preferring enzyme
was originally cloned from the pigeon
(30) and then from the human
and mouse (14,
29). When overexpressed, this
enzyme selectively catalyzed conversion of AMP to adenosine. Furthermore,
selective inhibitors of cN-I inhibit adenosine formation from rat adult
myocytes under chemical hypoxia
(11). Interestingly, two cN-I
genes were identified; cN-IA, the form originally purified and cloned from the
pigeon, is abundantly expressed in the pigeon and human heart
(14,
30). cN-IB is abundant in the
testis, where it was also cloned as human autoimmune infertility-related
antigen (GenBank Accession No. AF356185
[GenBank]
). Doubts remain regarding the role of
cN-I in the myocardial actions of adenosine, in particular because its
Michaelis-Menten constant (Km) for AMP measured in the
test tube is 
5 mM, much higher than predicted cytosolic AMP
concentrations. In addition, most previous studies of cN-I activity have used
inhibitors of adenosine kinase and adenosine deaminase to prevent further
metabolism. While these studies provide absolute rates of adenosine formation,
they leave unanswered the question of whether sufficient adenosine can escape
metabolism so as to carry out physiological effects at extracellular
receptors. In the present study, we overexpressed pigeon cN-IA in rat
cardiomyocytes using adenovirus-mediated gene transfer. We then studied
adenosine formation under simulated hypoxia with and, importantly, without
inhibitors of adenosine metabolism. Finally, we investigated the influence of
cN-IA overexpression on a physiological response, namely, the beating rate in
cardiomyocytes stimulated with isoproterenol.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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Construction of a recombinant adenoviral vector. The coding sequence of pigeon cN-IA was excised with BamHI and KpnI and subcloned into the shuttle vector pDC 515 downstream from the mouse cytomegalovirus promoter (Microbix Biosystems Canada). Replication-deficient adenovirus was generated by site-specific FLP-mediated recombination of the cotransfected shuttle and genomic plasmids in 293 cells. Viral stocks were plaque purified, amplified, CsCl banded, and titrated according to the manufacturer's instructions.
Culture and infection of rat neonatal cardiomyocytes. Myocytes
were isolated from 2- to 3-day-old rats by four cycles of digestion in 0.1%
trypsin containing 0.02% EDTA in PBS. All the animal procedures used in these
studies were performed in accordance with the guidelines and regulations of
the University of Bristol and the United Kingdom Home Office. Digestion was
stopped by the addition of fetal calf serum (FCS) to 20%. The dispersed cells
were resuspended in MEM supplemented with 10% FCS, 100 µg/ml streptomycin,
and 100 U/ml penicillin and preplated for 1 h to allow fibroblasts to adhere.
The suspended myocytes were seeded on plates previously coated with 1% gelatin
in PBS for 1 h; 106 myocytes were seeded in 35-mm dishes for
beating rate measurements or 5 x 105 myocytes on 12-well
plates for metabolite assays. After 40 h in culture, >80% of the cells were
beating spontaneously. The culture medium was then changed to 4:1 DMEM-M199,
containing antibiotics and 1% FCS, and adenoviral infection was performed. A
previously described adenovirus expressing -galactosidase
(37) was used to control for
the nonspecific effects of viral infection.
Metabolic studies. Cells in 12-well dishes were washed in saline,
transferred to Krebs-Ringer-HEPES (KRH) buffer [containing (in mM) 120 NaCl,
4.8 KCl, 1.2 KH2PO4, 1.2 MgSO4, 1.3
CaCl2, and 25 HEPES; pH 7.3], and preincubated for 10 min at
37°C. Metabolic inhibition was triggered with 10 mM 2-deoxyglucose (DOG),
10 mM DOG plus 1 mM sodium dithionite (S2O4), or 10 mM
DOG plus 0.5 mM sodium cyanide. Control cells were incubated in control buffer
(KRH buffer containing 5.5 mM glucose and 1 mM sodium pyruvate). Adenosine
formation caused by -adrenergic stimulation with 20 µM isoproterenol
was measured in control buffer. To inhibit adenosine metabolism, 10 µM
erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA), an inhibitor of adenosine
deaminase (EC 3.5.4.4
[EC]
) (23),
and 50 µM 5'-amino-5'-deoxyadenosine (5'-NH2),
to inhibit adenosine kinase (EC 2.7.1.20
[EC]
)
(23), were included in some
experiments. The nucleoside transport inhibitor dipyridamole was added at a
final concentration of 10 µM from a 10 mM solution in DMSO. Cell
incubations were terminated by the addition of 0.75 volumes of 1.6 M
HClO4, and total purines and protein concentrations were determined
by HPLC as previously described
(30). Separate experiments
were conducted to measure adenosine in the medium by HPLC. To stop
incubations, 300 µl of the supernatant buffer were removed and frozen
immediately. After the rest of the buffer was carefully removed, 400 µl of
1.2 M HClO4 were added to assay purines and protein in the cell
pellet as described above. Cell viability was assessed by measurement of LDH
in cell supernatants at the end of the incubations and expressed as a
percentage of the total LDH present in parallel wells extracted in KRH buffer
containing 0.1% Triton X-100.
Activities of purine metabolizing enzymes. Cell extracts were
prepared in buffer containing 20 mM Na dimethylglutarate, 20 mM Na
-glycerophosphate, 100 mM KCl, 0.1 mM DTT, 0.1% Triton X-100, and a
Sigma protease inhibitory cocktail (P8340); pH 6.9. Assays for cN-I were
performed in extraction buffer with near-saturating substrate concentrations
(10 mM AMP) in the presence of the activator ADP (1 mM), the selective e-N
inhibitor, ,-methylene-ADP (50 µM), the inhibitor of adenosine
deaminase pentostatin (2 µM), and 5'-NH2 (10 µM)
(30). e-N was assayed in
buffer containing 50 mM Tris · HCl, 10 mM Na -glycerophosphate, 5
mM MgCl2, and 0.1% Triton X-100 (pH 8.0) containing 0.2 mM AMP as
the substrate in the presence of the inhibitor of adenosine deaminase
pentostatin (2 µM) and the adenosine kinase inhibitor
5'-NH2 (10 µM). AMP deaminase was assayed in buffer
containing (in mM) 20 Na dimethylglutarate, 1 EDTA, 0.1 DTT, and 150 KCl (pH
7.0) containing 15 mM AMP as the substrate and 1.5 mM ATP as the activator.
Adenosine deaminase was measured in 50 mM potassium phosphate buffer (pH 7.4)
containing 150 µM adenosine. Aliquots of extracts containing 10–20
µg protein were incubated for between 0 and 10 min at 37°C; the
reactions were stopped by the addition of HClO4, followed by
analysis of the products by HPLC as already described
(30). Adenosine kinase was
measured at 37°C in aliquots containing 1.5–3 µg protein using
[14C]adenosine as the substrate
(5).
Effects of cN-IA overexpression on beating rates. Cells in 35-mm dishes were transferred to HEPES-buffered RPMI 1640 (0.8 ml). Beating rates were determined by counting the beats of four groups of cells for 1 min using a heated microscope stage maintained at 37°C. Determinations were made after equilibration for 10 min under all conditions used.
Western blots and immunocytochemistry. The generation of the rabbit polyclonal antibody against peptides derived from the sequence of pigeon cN-IA and the methods used for Western blotting have been described previously (30).
Statistical analysis. Results are expressed as means ± SE. Statistical significance was assessed with one-way ANOVA, followed by paired Student's t-test if appropriate or unpaired Student's t-test with Bonferroni correction for repeated measures. A value of P < 0.05 was considered significant.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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-galactosidase
expression transduced >90% of cells
(Fig. 1A). Infection
with a similar virus engineered to express pigeon cN-IA led to overexpression
of a protein of the correct size by Western blotting using a previously
described antibody against a peptide epitope in pigeon cN-IA
(Fig. 1B). This was
accompanied by an 18-fold increase in 5'-nucleotidase activity from 45
± 6 to 824 ± 36 nmol · min–1
· mg protein–1 (n = 7). By
immunocytochemistry, using the same antibody as for Western blotting, >90%
of cardiomyocytes in cN-IA-adenovirus-infected cultures stained for cN-IA
(Fig. 1D). The
activity of other purine-metabolizing enzymes was similar in
-galactosidase- and cN-IA-transduced cells, as follows: e-N, 134
± 16 vs. 142 ± 4 nmol · min–1
· mg protein–1; adenosine kinase, 3.9
± 0.9 vs. 3.8 ± 1.0 nmol
·min–1 · mg
protein–1; adenosine deaminase, 12.5 ± 1.7
vs. 12.1 ± 1.4 nmol · min–1 ·
mg protein–1; and AMP deaminase, 125 ± 29
vs. 119 ± 26 nmol · min–1 ·
mg protein–1.
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Effect of cN-IA overexpression on nucleotide metabolism under simulated
hypoxia. Purine concentrations were measured in cardiomyocytes plus
medium after incubation under increasingly severe conditions of metabolic
poisoning to simulate the effects of hypoxia either in the presence or absence
of inhibitors of adenosine kinase (5'-NH2) and adenosine
deaminase (EHNA) (23). As
shown in Table 1, DOG, a
glycolytic inhibitor, alone decreased ATP concentrations by 40%. This was
increased to 55% by adding S2O4, which consumes
molecular oxygen, and to 70% by adding cyanide, an inhibitor of
cytochrome oxidase (Table 1).
Transduction with -galactosidase had no effect on baseline ATP
concentration or ATP catabolism compared with untransduced cells (results not
shown). The baseline ATP concentration was decreased significantly by 8% in
cN-IA-transduced cardiomyocytes (43 ± 3 nmol/mg protein, n =
7) compared with -galactosidase-transduced cells (47 ± 2 nmol/mg
protein, n = 7, P < 0.02). Despite this, there was no
difference in the percentage of ATP broken down between -galactosidase-
and cN-IA-transduced cells under any condition of chemical hypoxia
(Table 1). Including inhibitors
of adenosine metabolism did not affect either the baseline ATP concentration
in -galactosidase- or cN-IA-transduced cells [48 ± 3 and 44
± 3 nmol ATP/mg protein, respectively, n = 3, P = not
significant (NS)] or the percentage of ATP breakdown during chemical hypoxia
(Table 1). Cardiomyocyte
viability measured by LDH release was >98% in both -galactosidase-
and cN-IA-transduced cells. Only in cN-IA-transduced cells treated with DOG
and cyanide was LDH marginally elevated
(Table 1).
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Transduction with cN-IA profoundly altered the pattern of ATP metabolites
produced in either the presence or absence of inhibitors of adenosine
metabolism (Fig. 2, A and
B). Figure
2 summarizes the pattern of nucleotide metabolism in
representative experiments. Overexpression of cN-IA increased adenosine
production (Fig. 2, A and
B), even in the absence of inhibitors of adenosine
metabolism (Fig. 2A).
cN-IA overexpression increased adenosine production at the expense of IMP,
inosine, and hypoxanthine, as most clearly seen in the presence of inhibitors
of adenosine metabolism (Fig.
2B). This demonstrates competition between cN-IA and AMP
deaminase for the available cytosolic AMP. Only with DOG and cyanide, when ATP
is most depleted, did AMP accumulate in -galactosidase-transduced cells
(Fig. 2, A and
B). This is probably due to the inhibition of AMP
deaminase known to occur under such conditions
(20). With DOG and cyanide,
cN-IA overexpression reduced the final AMP concentration. Averaged data from
several experiments (Fig. 3, A and
B) demonstrate that even under baseline conditions, cN-IA
overexpression significantly increased the concentration of adenosine in the
presence or absence of inhibitors of adenosine metabolism
(Fig. 3, A and
B). Moreover, cN-IA overexpression dramatically
potentiated the stimulatory effects of simulated hypoxia on adenosine
concentration (Fig. 3, A and
B). The patterns of changes after simulated hypoxia were
similar in the absence of inhibitors of adenosine metabolism, although the
absolute levels of adenosine achieved were approximately halved
(Fig. 3, A and
B). As expected, there was correspondingly more inosine
and hypoxanthine in the absence of inhibitors of adenosine metabolism than in
their presence (Fig. 2, A and
B). In agreement with our previous work in rat neonatal
cardiomyocytes (21), inclusion
of the selective e-N inhibitor ,-methylene-adenosine diphosphate
(AOPCP; 50 µM) did not affect adenosine formation under any condition of
simulated hypoxia (data not shown and see below).
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We conducted a separate set of experiments in which medium and cells were analyzed separately. Values for adenosine concentration in the medium (Fig. 4) were similar to those measured in cells plus medium (Fig. 3B), which shows that most of the adenosine was present in the medium, as we (21) previously reported. Medium adenosine concentrations were not affected by inclusion of AOPCP either under baseline or after treatment with DOG and cyanide. However, the nucleoside transport inhibitor dipyridamole dramatically reduced medium adenosine concentrations. Adenosine concentrations in the cells were below the limits of accurate measurement except in the case of DOG plus cyanide plus dipyridamole, when they were fourfold the extracellular concentration. Conversely, ATP, ADP, or AMP concentrations were not measurable by HPLC in the medium under any condition (data not shown). These data confirm our previous study (21), which showed that the overwhelming majority of adenosine is formed intracellularly in neonatal rat cardiomyocytes, independently of e-N, and then transported out. Not surprisingly, overexpression of cN-IA increased intracellular adenosine formation.
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Effect of cN-IA overexpression on nucleotide metabolism and beating
frequency after stimulation with isoproterenol. The increase in total
(cells plus medium) adenosine concentration resulting from overexpression of
cN-IA persisted in the presence of 20 µM isoproterenol whether or not
inhibitors of adenosine metabolism were also present
(Fig. 5A). In a
separate series of experiments, we measured adenosine concentration in the
medium alone. Medium adenosine concentrations were also significantly greater
in cN-IA-than -galactosidase-transduced cells, and this difference again
persisted in the presence of isoproterenol
(Fig. 5B). The
difference between cN-IA- and -galactosidase-transduced cells was
preserved in the presence of AOPCP but lost in the presence of dipyridamole
(Fig. 5B). These data
confirm that cN-IA overexpression led to an increase in adenosine formation
and release, which was present both before and after isoproterenol
treatment.
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Isoproterenol significantly and reversibly increased the beating frequency
of cardiomyocytes transduced with -galactosidase
(Fig. 6A). The effect
of isoproterenol was not reversed by including inhibitors of adenosine
metabolism but was by adding 50 µM adenosine, which had no effect on
beating frequency in the absence of isoproterenol
(Fig. 4A and data not
shown). In contrast, isoproterenol did not increase beating frequency in
cardiomyocytes overexpressing cN-IA (Fig.
6A). To confirm that these effects were mediated through
adenosine receptors, the experiments were repeated in the presence of a
nonselective adenosine receptor antagonist,
8-(p-sulfophenyl)theophylline (8-SPT). 8-SPT had no effect on the
ability of isoproterenol to stimulate beating frequency but partially
antagonized the inhibition of this by added adenosine
(Fig. 6B). More
importantly, however, 8-SPT completely abolished the inhibitory effect of
cN-IA overexpression on isoproterenol-induced beating frequency
(Fig. 6B).
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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0.2 mM) over AMP
(Km 5 mM). It is activated in vitro by ATP or ADP and
inhibited by inorganic phosphate, which implies a biphasic response during ATP
breakdown that has been confirmed in whole cell experiments
(38). So-called cN-I was later
cloned from the pigeon, human, and mouse
(14,
29,
30). Although cN-I and cN-II
may share similar catalytic residues
(1), they are genetically
unrelated. The purified enzyme is a tetramer of 40-kDa subunits that has a
preference for AMP (Km 5 mM) compared with IMP
(Km 20 mM). It is insensitive to inorganic phosphate
and requires a nucleoside diphosphate (e.g., ADP) for maximum activity
(nucleoside triphosphates cannot act as substitutes). These kinetic
characteristics suggest that the enzyme is active during ATP catabolism when
AMP, ADP, and inorganic phosphate concentrations rise. Inhibitors selective
for cN-I over cN-II are dideoxynucleosides (particularly ddC and ddU), and
these have been shown to decrease adenosine production from rat myocytes
(10). This was the first
direct evidence that cN-I could contribute to adenosine formation in cells.
The role of cN-I was confirmed by plasmid-based overexpression studies, which
demonstrated the ability of cloned cN-IA to produce adenosine in established
cell lines in the presence of inhibitors of adenosine metabolism
(28,
30). Sequencing studies recently distinguished two isoforms of cN-I encoded by genes on human chromosomes 1 and 2, respectively. The form purified and kinetically characterized from the pigeon heart and later used for protein sequencing and cloning was designated cN-IA (29). The kinetic properties of cN-IB have yet to be fully elucidated, although its basic characteristics in terms of Km, activation by AMP, insensitivity to inorganic phosphate, and inhibition by dideoxynucleosides appear similar to cN-IA. Moreover, both isoforms can generate adenosine during ATP catabolism when overexpressed in COS-7 cells (29). Because cN-IA is abundantly expressed in the heart, whereas cN-IB is poorly expressed in the heart and predominates in the testis, we chose to focus on cN-IA in these experiments. Furthermore, because the kinetic properties of purified and cloned cN-IA are highly conserved across species from the pigeon to human (35, 36), we chose to overexpress the pigeon enzyme, for which we had suitable full-length clones.
The high Km for AMP of cN-I (5 mM) has cast doubts
on its physiological function in myocardial adenosine formation because the
predicted concentrations of free cytosolic AMP in cardiomyocytes are in the
micromolar range. On the other hand, AMP deaminase also has a relative high
Km for AMP, 0.6 mM, which increases to 2.4 mM in
the absence of ATP (20). Some
cellular fractionation and histological evidence has implied that both cN-I
and AMP deaminase may be associated with contractile fibers and therefore in a
micro-environment exposed to higher AMP concentrations
(27,
30). A further concern has
been that any adenosine produced in the cytoplasm would have to escape
metabolism by adenosine kinase and adenosine deaminase and be transported out
through the equilibrative nucleoside transporter before it could activate
extracellular adenosine receptors. There remain doubts, therefore, as to
whether cN-I could sufficiently elevate adenosine concentrations in primary
cardiomyocytes in the face of adenosine metabolism to act at adenosine
receptors. To investigate this question, we first had to develop an
adenovirus, which we showed capable of achieving high-level expression of
cN-IA in primary cardiomyocytes. Our second goal was to measure adenosine
formation and release during simulated hypoxia in the presence of inhibitors
of adenosine metabolism. Although these experiments were not designed to
measure the initial rates of adenosine formation, we can calculate that 20
nmol of adenosine were produced over 10 min in cN-IA-transduced cells, which
compares with a Vmax of nearly 900 nmol ·
min–1 · mg
protein–1. Clearly the enzyme operates under far
from saturating conditions, consistent with the low free cytosolic AMP
concentrations. Furthermore, by suppressing IMP, inosine, and hypoxanthine
concentrations, we showed that cN-IA effectively competed for substrate with
AMP deaminase. Most importantly, we showed, for the first time, that the
enzyme was sufficiently active to elevate adenosine concentrations even in the
absence of inhibitors of adenosine metabolism.
All previous studies demonstrating adenosine formation from recombinant
cN-I overexpression have used extreme conditions of ATP depletion. We showed
here, however, that overexpression of cN-I provoked adenosine formation and
elevated extracellular adenosine concentrations even under baseline
conditions, when ATP levels were only 8% less than in
-galactosidase-transduced cells.
Finally, we sought to investigate whether cN-IA over expression could
influence a response to adenosine mediated by extracellular adenosine
receptors. Because neonatal myocytes are anchored to the substratum, cell
shortening could not be quantified. Hence, the most convenient parameter to
measure was beating frequency. As previously shown
(12), isoproterenol exerts a
positive chronotropic effect on neonatal cardiomyocytes. This does not appear
to be under tonic control by adenosine because 8-SPT, a nonselective
competitive inhibitor of adenosine receptors, had no stimulatory effect on the
beating rate. However, added adenosine reversed the effect of isoproterenol,
and this appeared to be receptor mediated. The partial effect of 8-SPT can be
explained by the relatively high concentration of added adenosine used.
Overexpression of cN-I produced a similar effect to added adenosine, which was
in this case fully reversed by 8-SPT. The increase in adenosine formation and
release by cN-IA overexpression persisted in the presence of isoproterenol
and, from inhibitor experiments, was again independent of e-N but depended on
nucleoside transport. The concentration of adenosine found in
-galactosidase- and cN-IA-transduced cardiomyocytes in the presence of
isoproterenol ranged from 0.1 to 0.6 µM, which is in the range known to be
effective at adenosine receptors
(34). These results show,
again for the first time, that cN-IA can generate sufficient concentrations of
adenosine to exert physiological effects on cell surface receptors even in the
face of adenosine metabolism.
Limitations. While our overexpression studies demonstrate the potential of cN-IA to produce adenosine in cardiomyocytes, they clearly do not directly address the role of cN-IA in regulating myocardial adenosine concentrations and physiological actions in the heart. This will ultimately require transgenic and knockout experiments in mice for which our present experiments provide necessary justification. The extracellular compartment in the intact myocardium is much smaller in proportion to that in isolated cardiomyocyte cultures, and this would tend to potentiate the extracellular concentration of any adenosine formed in the cytoplasm and released from the nucleoside transporter. In this sense, our experiments in isolated cardiomyocytes represent rather stringent conditions to test the role of cN-IA, which we compensated for by overexpressing the enzyme. A further limitation of our study is that we focused on cardiomyocytes. Because endothelial cells also contribute to adenosine formation in the heart (6), future studies addressing the role of cN-I in those cells would also be valuable.
In conclusion, our studies demonstrate, for the first time, the activity of cloned cN-IA in primary rat cardiomyocytes. They demonstrate that cN-IA is sufficiently active to significantly increase adenosine concentration in the absence of inhibitors of adenosine metabolism and mediate physiological responses to adenosine at extracellular receptors.
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DISCLOSURES |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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ACKNOWLEDGMENTS |
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Present address of M. A. Curto: Instituto de Investigaciones en Ingenieria Genetica y Biologia Molecular, University of Buenos Aires, Vuelta de Obligado 2490, Buenos Aires 1428, Argentina.
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement"" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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P < 0.05 vs. KRH buffer.
