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Iain L. O. Buxton, Robert A. Kaiser, Brian C. Oxhorn and Dennis J.

Cheek
Am J Physiol Heart Circ Physiol 281:1657-1666, 2001.

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on the following topics:
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Cell Biology .. Endothelial Cells
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Am J Physiol Heart Circ Physiol
281: H1657–H1666, 2001.

Evidence supporting the Nucleotide Axis Hypothesis:


ATP release and metabolism by coronary endothelium

IAIN L. O. BUXTON, ROBERT A. KAISER, BRIAN C. OXHORN, AND DENNIS J. CHEEK


Department of Pharmacology, University of Nevada School of Medicine, Reno, Nevada 89557
Received 12 March 2001; accepted in final form 13 June 2001

Buxton, Iain L. O., Robert A. Kaiser, Brian C. Ox- states (8). We (37) and others (4) have shown that
horn, and Dennis J. Cheek. Evidence supporting the Nu- endothelial cells release ATP in response to numerous
cleotide Axis Hypothesis: ATP release and metabolism by stimuli, such as shear stress and vasoactive agonists,
coronary endothelium. Am J Physiol Heart Circ Physiol 281: including ATP itself. Furthermore, ATP acting at the
H1657–H1666, 2001.—The Nucleotide Axis Hypothesis, de-
endothelial cell P2y1 receptor, stimulates the release of
fined and supported herein, proposes that ATP stimulates
the release of vasoactive mediators from endothelium, in- the endothelium-dependent vasodilators nitric oxide
cluding ATP itself. Here, we show rapid endothelium-depen- (NO) and prostacyclin I2 (PGI2) (26). We have hypoth-

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dent, agonist-stimulated ATP elaboration in coronary vessels esized that the release of ATP by coronary endothelium
of guinea pigs. Measurement of extracellular ADP metabo- and its subsequent actions at ATP receptors on nearby
lism in intact vessels results in the time- and substrate- endothelial cells and downstream from the site of re-
dependent formation of ATP in the coronary perfusate in lease where it acts as the ATP metabolites (ADP and
amounts greater than can be accounted for by release from adenosine) constitute fundamental elements of an in-
endothelium alone. ATP formation by endothelial cells is travascular axis that serves to regulate vascular tone
saturable (KM ⫽ 38.5 ␮mol/l, where KM is substrate concen- (37).
tration at which rate is half-maximal.) and trypsin-sensitive,
The Nucleotide Axis Hypothesis proposes that in
membranes from [␥-32P]ATP-labeled cells support ADP-de-
pendent transphosphorylation by a 20-kDa protein, Western response to discrete stimuli, such as the action of
blots reveal the presence of a nucleoside diphosphate kinase vasoactive agonists on endothelium or acute changes in
(NDPK) of ⬃20 kDa in endothelial membranes, and analysis shear stress, ATP is released from endothelium and
of NDPK antibody binding by flow cytometry is consistent acts as an autocrine and paracrine hormone able to do
with the presence of an ecto-NDPK on cardiac endothelial the following. First, it may cause receptor-mediated
cells. Sequencing of the endothelial cell ecto-NDPK reveals a release of vasoactive factors such as NO, PGI2, and
predicted amino acid sequence with 85% identity to human ATP from endothelium. Second, it may act in the blood
Nm23-H1 and consistent with a protein whose properties vessel lumen at P2y1 and P2y2 receptors on nearby
may confer membrane association as well as sites of regula- endothelium downstream from the site of release to
tion of activity. Our data underscore the potential impor- amplify the ATP and vasoactive factor release re-
tance of a nucleotide axis in cardiac blood vessels.
sponse. Third, it may act on platelets in the blood-
release of vasoactive factors; ectoenzyme; ecto-Nm23 stream adjacent to endothelium as an antagonist at the
thrombocyte P2t receptor to block platelet aggregation.
Fourth, once dephosphorylated to ADP by ecto-ADPase
THE NOTION THAT ATP is, despite its ubiquitous role in of endothelium, it may act again at P2Y1 receptors and
fueling cellular processes, strictly an intracellular pe- be (in part) converted back to ATP by the endothelial
troleuse is invalid. It is now well established that both ectonucleoside diphosphate kinase (NDPK) to act
nucleosides and nucleotides, released as local hor- again, thus propagating the actions of ATP in time and
mones and neurotransmitters (35), act as extracellular space within the blood vessel. Fifth, it may eventually
signaling molecules in many parts of the body (16, 26). break down to adenosine downstream from the site of
Extracellularly directed receptors for ATP exist as both release/conversion in the coronary artery, resulting in
ion channels (P2x) and G protein-coupled receptors continued inhibition of platelet aggregation, vasodila-
(P2Y) and are ubiquitously expressed in all mamma- tion of resistance vessels, and growth maintenance/
lian tissues examined to date (26). Indeed, the extra- promotion of endothelium.
cellular role of nucleotides in the regulation of multi- Whereas the documented release of ATP by endothe-
cellular communication is evident as early in the lial cells in tissue culture is suggestive of a physiolog-
passage of evolution as the Protista (29). In the ische- ical role for ATP release in vivo, it is possible that such
mic heart, adenosine released from myocytes aug- release is the result of the alteration or activation of
ments blood flow (3), and ATP may play a role in the endothelium during cell isolation or is a result of the
regulation of cardiac blood flow in nonpathological
The costs of publication of this article were defrayed in part by the
Address for reprint requests and other correspondence: I. L. O. payment of page charges. The article must therefore be hereby
Buxton, Univ. of Nevada School of Medicine, Howard Research Bldg., marked ‘‘advertisement’’ in accordance with 18 U.S.C. Section 1734
Laboratory Suite 216, Reno, NV 89557. solely to indicate this fact.

http://www.ajpheart.org 0363-6135/01 $5.00 Copyright © 2001 the American Physiological Society H1657
H1658 NUCLEOTIDE AXIS IN CARDIAC BLOOD VESSELS

conditions of cell culture. Thus we have employed both without trypsin. The employment of trypsin to remove cells
primary cultures of endothelial cells as well as a mod- markedly reduced the density of bands that stained with
ified perfused heart model to determine whether ATP antibodies for NDPK in immunoblots. Cell pellets were ho-
release occurs in intact coronary arteries of guinea pigs mogenized on ice in Kontes 1-ml glass-glass homogenizers in
PBS containing leupeptin (1 ␮mol/l). Membrane and cytosolic
and to investigate the likely fate of purine nucleotides
fractions were prepared by a slow-speed initial centrifugation
in the coronary circulation. Here we demonstrate a role of the cell homogenate (⫻1 K, 10 min), followed by centrifu-
for nucleotides and the peptide bradykinin in the mo- gation of the resulting supernatant at ⫻88 K for 45 min.
ment-to-moment regulation of ATP release by endothe- Samples for PAGE were boiled in SDS using standard pro-
lium and offer evidence for the extracellular production tocols. Proteins separated by SDS-PAGE (15%) were blotted
of ATP from ADP by endothelium in a heretofore un- to nitrocellulose and probed with a rabbit polyclonal antibody
appreciated manner. (19) directed against rat liver NDPK or a monoclonal anti-
body directed against Nm23-H1.
METHODS Endothelial NDPK-phosphotransferase activity. Endothe-
lial cell membranes were incubated with 100 ␮mol/l ATP
Coronary artery perfusion. Hearts, removed from CO2- ([␥-32P]ATP, 100 ␮Ci) ⫾ the NDPK substrate ADP (30
asphyxiated female guinea pigs (300–350 g) under an insti- ␮mol/l) added as a phosphate acceptor. Reactions carried out
tutionally approved protocol, were cannulated via the aorta in endothelial cell membranes at 21°C for 30 min in the
and perfused with 37°C heart perfusion buffer (HPM) at pH absence of a phosphoryl donor were separated on 15% SDS
7.2 (37). In Ca2⫹-free HPM buffer, the right and left epicar- PAGE gels exposed to Kodak X-OMAT AR film in cassettes
dial coronary vessels could be cut 10 mm from their origin with intensifying screens held at 4° for 48–72 h. Film was

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and cannulated with polyethelene tubing for collection of the developed (Konica QX-70 film processor) and quantified by
perfusate. The preparation was then perfused at 4 ml/min desitometry.
with buffer as described above containing CaCl2 at 1.8 NDPK-A surface labeling by flow cytometry. Endothelial
mmol/l. In some experiments, the right coronary artery was cells were incubated with mouse monoclonal antibody
perfused with distilled H2O for 3 min to remove a functional (IgG2a) directed against human Nm23-H1 antigen (Santa
endothelium (9) and was verified microscopically (17). Exper- Cruz Biotechnology; Santa Cruz, CA). A secondary rabbit
iments performed in this way were unaffected by treatments antibody-FITC conjugate directed against mouse IgG2a was
altering flow because the perfusion was controlled at a con- employed to detect binding of the primary antibody. Controls
stant rate and only the epicardial portion of the vessel was were employed for nonimmune mouse IgG2a/secondary anti-
employed. body binding. Cells (10,000) were analyzed by flow cytometry
Preparation of endothelial cells. Guinea pig hearts (6 fe- using a Beckman-Coulter EPICS Elite ESP instrument, and
males/prep) were perfused with Ca2⫹-free HPM containing 1 flow experiments were repeated five times with endothelial
mg/ml collagenase (Worthington, type II) and purified as cells from different cell isolations.
previously described (37). Endothelial cells liberated in this RNA isolation and RT-PCR. Total RNA was isolated from
manner were grown to confluence on polymer (F-5147, Sig- 50 mg of freshly dissected guinea pig ventricle or 1 ⫻ 106
ma)-coated plastic for 5 to 7 days. Cells were subcultured and plated cardiac endothelial cells using the TRIzol method
employed as first (P-1) through third passage. In flow cytom- (GIBCO-BRL, Life Technologies; Grand Island, NY). Isolated
etry studies, freshly isolated cells were first labeled with a RNA was DNAse I-treated before cDNA synthesis and re-
mouse anti-human monoclonal antibody (IgG1) directed verse transcribed using Superscript II. Quality of cDNA was
against the endothelial marker CD31 (clone EN4) and posi- tested for genomic contamination by performing ␤-actin con-
tive cells retrieved by fluorescence-activated cell sorting (10). trol RT-PCRs and checking for the presence of the genomic
CD31⫹ cells were grown as above and employed for detection (700 bp) or cDNA (500 bp) product. RT-PCR was performed
of Nm23-H1 as described. In studies not described here, for 40 cycles at 94°C 25 s, 58°C 25 s, and 72°C 60 s in an
RT-PCR studies of RNA from cells as early as the fifth Eppendorf Mastercylcer Gradient (Eppendorf Scientific;
passage were found to loose expression of P2 receptors. Westbury, NY) with primers of our own design obtained from
Measurement of ATP by chemiluminescence. The presence Integrated DNA Technologies (Coralville, IA). Oligonucleo-
of ATP in the coronary perfusate or cell culture incubation tide primers for NDPK were designed for PCR based on
media was determined using luciferin-luciferase chemilumi- conserved sequences of the human (GenBank X73066),
nescence (37). Effects of all treatments on the assay were mouse (M65037), and rat (D13374) NDPK genes as follows:
always carefully controlled, such as the use of ADP at con- forward GAGCGTACCTTCATTGC(G/C)ATC, reverse GCA-
centrations employed in our experiments, which produces a CTCTCCAC(A/T)GAATCACTGC. Products were analyzed on
small signal in the assay. 1.0% agarose gels, stained with 2 ug/ml ethidium bromide
Measurement of purines by HPLC. Metabolism of [2,8- and imaged. RACE-ready cDNA was synthesized according
3
H]ADP sodium salt, and [8,5⬘-3H] GDP sodium salt by cells to the SMART-RACE protocol (Clontech; Palo Alto, CA) using
and coronary vessels was determined by injecting filtered 3 ␮g of ␤-actin verified total RNA. The 3⬘ reaction was
control or experimental buffer solutions (400 ␮l) over a performed using the RACE primer and the forward primer
100-mm Whatman Partisil SAX (5 ␮m) HPLC column. Pu- given above. The 5⬘ reaction was performed using the RACE
rines were separated by a linear gradient of NH4PO4 (pH 3.8) primer and the reverse primer GATGTTCCTGCCGACTTG.
from 0 to 2 M. Radioactivity was measured continuously RACE PCRs were performed using the following variation of
using an INUS ␤-RAM pumping flow scintillation cocktail at touchdown PCR: 5 cycles 94°C 30 s/72°C 3 min, 5 cycles 94°C
a ratio of 3:1 resulting in an efficiency for tritium of 36%. The 30 s/70°C 30 s/72°C 3 min, and 28 cycles 94°C 30 s/68°C 30
identity of peaks for adenine and guanine nucleoside and s/72°C 3 min. Potential bands were isolated from a 1.0%
nucleotides were determined by separation of bona fide stan- agarose gel (QIAgen Gel extraction method, Valencia, CA)
dards. and sequenced using an ABI/prism sequencer.
Western blots. Endothelial cells were removed from the Materials. All drugs and chemicals were obtained from
growth surface with PBS (pH 7.2) containing 1 mmol/l EGTA Sigma (St. Louis, MO) unless otherwise noted. Collagenase
AJP-Heart Circ Physiol • VOL 281 • OCTOBER 2001 • www.ajpheart.org
NUCLEOTIDE AXIS IN CARDIAC BLOOD VESSELS H1659

type-II was obtained from Worthington Biochemical (Lake-


wood, NJ). Radioactive nucleotides were obtained from NEN
Life Science Products, now Perkin-Elmer Life Sciences (Bos-
ton, MA). Polyclonal antibody to rat liver NDPK was the
generous gift of N. Kimura, Tokyo Metropolitan Institute of
Gerontology (Tokyo, Japan). Monoclonal antibody to
Nm23-H1 employed in immunoblots was obtained from
Seikagaku America (Rockville, MD). DNAse I was obtained
from Promega, Madison, WI and Superscript II from Gibco
(Rockville, MD).

RESULTS

In perfused coronary artery of the guinea pig, intro-


duction of BK results in the immediate release of ATP
that can be measured in the perfusate (Fig. 1A). The
time course of release to BK (1 nM) in the continued
presence of the agonist (flow, 4 ml/min) peaks within
the first minute; desensitizes, thereafter, consistent
with action of BK at the BK2 receptor on endothelium;

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and is blocked by the addition of the BK receptor
antagonist HOE-140. Release of ATP by coronary ar-
tery was endothelium-dependent, because removal of
endothelium in the right, but not the left, coronary
artery by prior perfusion with distilled water elimi-
nated the response to BK (Fig. 1B). Addition of 2-meth-
ylthio-ATP or ADP, both agonists at the endothelial
P2Y1 receptor (1), led to the immediate appearance of
ATP in the perfusate (Fig. 1A). However, unlike the
action of BK, stimulation of the endothelial P2Y1 re-
ceptor led to the sustained appearance of ATP in the
perfusate despite the fact that ATP receptor responses
are known to desensitize rapidly (36). Because ATP is
known to stimulate its own release from endothelial
cells in culture (37), we reasoned that such a phenom-
enon could explain the continued presence of ATP in
the coronary perfusate. However, studies in cultured
cells suggested an additional possibility.
When ADP was added in increasing concentrations
to cultured guinea pig endothelial cells, ATP elabora-
tion measured at concentrations above 10 ␮mol/l was
significantly greater than that seen with BK at any
dose (Fig. 2A, inset) and represented as much as 10% Fig. 1. Agonist-mediated ATP release from guinea pig coronary
(at 100 ␮M ADP) of total cellular ATP (Fig. 2B). Such arteries in situ. Female guinea pig hearts were cannulated for
a large amount of ATP from cells could not be ex- collection of the perfusate. ATP, expressed as picomoles of ATP per
milliliter of perfusate, was measured using the luciferin-luciferase
plained by release alone. Indeed, all of the ATP in the method. A: agonists were added continuously at the concentrations
cell, defined by treatment with perchloric acid, totaled shown, and the perfusate was collected at the indicated times (rate ⫽
18.8 ⫾ 2.45 nmol/million cells, whereas total “releas- 4 ml/min). A lag seen is due to the start time being marked just
able” ATP defined by treatment with ionomycin (1 ␮M) before switching flow to a buffer containing drug. B: hearts were
prepared as in A but perfusate from right and left vessels were
for 2 min (Fig. 2B), measured 12.34 ⫾ 2.23 nmol/ segregated. Agonists were added at the indicated times and flow
million cells. To test our hypothesis that ATP could be collected over 2 min. Removal of endothelium in the right coronary
generated extracellularly by endothelium, we intro- only, by perfusing with distilled water for 3 min at min 90–93,
duced [3H]ADP (1 ␮Ci, 100 ␮M) into the coronary prevented the response to bradykinin (BK), which persisted in the
artery perfusate in the presence of a phosphoryl donor left coronary artery and again could be prevented by the addition of
antagonist. 2-Me-S-ATP, 2-methylthio-ATP. Bsl, basal; H, HOE-140.
(GTP, 1 mM). The perfusate was collected and sub- Means ⫾ SE, n ⫽ 8 experiments (A), n ⫽ 4 experiments (B).
jected to HPLC anion-exchange separation with radio-
active flow detection. In the presence of GTP, there was
a rapid formation of [3H]ATP at the expense of thelium is not prevented, it is clear that within this
[3H]ADP along with the expected formation of 1-min exposure to endothelium, the disappearance of
[3H]AMP and [3H]adenosine (Fig. 3A). Although it is [3H]ADP can be approximately accounted for by the
not strictly possible to account for all adenyl purine in [3H] products of metabolism seen on the chromato-
these experiments where adenosine uptake into endo- gram. Control perfusions through endothelium-de-
AJP-Heart Circ Physiol • VOL 281 • OCTOBER 2001 • www.ajpheart.org
H1660 NUCLEOTIDE AXIS IN CARDIAC BLOOD VESSELS

(1 mM) yielded the rapid appearance ATP in the buffer


bathing the cells in a fashion (15) suggesting the pres-
ence of an ectoenzyme (Fig. 4A). The substrate concen-
tration at which the maximal rate of reaction (Vmax) is
half-maximal (Km) obtained in these experiments
(Km ⫽ 38.5 ␮mol/l) was consistent with the action of an
NDPK as was blockade by addition of UDP at high (5
mM) concentration, the absence of an effect of the
adenylate kinase inhibitor diadenosine pentaphos-
phate (Fig. 3D), and the ability to see the same activity
(albeit with a much decreased Vmax) at 4°C (data not
shown). Consistent with an NDPK is its promiscuity
with respect to nucleotide substrate and phosphoryl
donor (23). Endothelial cells were able to convert
[3H]GDP to [3H]GTP in the presence of ATP (Fig. 3C)
and could also utilize IDP or CDP as substrate (data
not shown). The ability of the nonneutralizing anti-
NDPK antibody we employed to activate the enzyme
(Fig. 3D) was also seen when the antibody was em-

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ployed with the purified rat liver NDPK (Sigma) used
as a control.
We employed mild trypsin treatment of the cultures
(Fig. 4B) and obtained results demonstrating an 85%
reduction in ATP generated within 1 min. The effect of
trypsin was reversible after a 45-min recovery period in
growth medium. The presence of an ecto-NDPK was
confirmed in Western blots, employing polyclonal anti-
bodies directed against rat liver NDPK (19), which
showed the presence of a 20.5-kDa membrane-associated
NDPK (Fig. 5A). This same nonneutralizing antibody,
when added to activity assays, stimulated the enzyme
twofold (Fig. 3D). A monoclonal antibody directed against
Fig. 2. Measurement of ADP-stimulated ATP elaboration by pri- the human Nm23-H1 gene product, an NDPK isoform
mary cultures of cardiac endothelial cells suggests a process other expressed by numerous transformed cells (14, 31, 32) and
than release. A: guinea pig cardiac endothelial cells, isolated and
cultured as previously described (37), were exposed to increasing proposed as a metastasis associated gene (27), further
concentrations of ADP or BK (Inset) in Krebs buffer for 2 min, and identified the endothelial NDPK as the guinea pig homo-
ATP was measured using the chemiluminescent luciferin-luciferase logue of human Nm23-H1 (Fig. 5B).
assay. B: confluent monolayers of endothelial cells were washed, and Partially purified endothelial cell membranes were in-
total cellular ATP was measured after perchloric acid extraction
(PCA, 2%) using chemiluminescence. Treatment of cultures with
cubated in the presence of [␥-32P]ATP, washed, incubated
ionomycin (IONO, 1 ␮M, 2 min) defined all ATP that could possibly with the NDPK acceptor (ADP), and separated by SDS
be made available for release under any physiological circumstance. PAGE followed by autoradiography. Autoradiograms
Data are means ⫾ SE, n ⫽ 5 experiments. demonstrated decreasing concentrations of [␥-32P]phos-
phate associated with NDPK over time (Fig. 6) dependent
on the presence of the acceptor, ADP.
nuded vessels demonstrated that this [3H]ATP forma- While the expression of NDPK as an ectoenzyme is
tion is a consequence of an intact endothelium (Fig. strongly suggested by the ability to measure enzyme
3B). In particular, the fact that one observes ATP activity in a coronary vessel or in a cell monolayers,
formation comparable to that of AMP formation in additional evidence for the surface expression was ob-
intact vessels, suggests the presence of an ecto-ADP tained using flow cytometry of intact cells. Incubation
kinase present on endothelium. The ability of endothe- of cells with a fluorescent antibody directed at human
lium to convert ADP to ATP in the presence of GTP was NDPK (Nm23-H1 specific) and analysis of fluorescence
not exclusive. When [3H]UDP was employed as sub- by flow cytometry revealed that the entire population
strate and ITP employed as a phosphoryl donor, of cells acquired a fluorescence signal consistent with
[3H]UTP was formed (data not shown). expression of an ecto-NDPK (Fig. 7).
The existence of an endothelial ectoenzyme was also RT-PCR was employed to sequence a guinea pig
suggested in experiments employing primary cultures NDPK based on the notion that the activity we mea-
of cardiac endothelial cells. The candidate enzyme for sure is homologous to Nm23-H1. The following open
ATP production from ADP in the presence of a phos- reading frame was obtained from the sequences of the
phoryl donor is a NDPK that catalyzes the reaction [N1 NDPK RACE products (Genbank accession number
DP ⫹ N2TP 7 N1TP ⫹ N2DP] (23). Intact cell culture AY017306): ATGGCCAGCAGTGAGCGGACCTTCA-
monolayers exposed to ADP in the presence of GTP TTGCCATCAAGCCGGATGGTGTCCAGCGGGGAC-
AJP-Heart Circ Physiol • VOL 281 • OCTOBER 2001 • www.ajpheart.org
NUCLEOTIDE AXIS IN CARDIAC BLOOD VESSELS H1661

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Fig. 3. Guinea pig coronary endothelium expresses nucleoside diphosphate kinase (NDPK) activity. A: represen-
tative HPLC tracings of [3H]ADP metabolism by coronary artery. Radioactive ADP ([3H]ADP, 1 ␮Ci) in the
presence of nonradioactive ADP (30 ␮mol/l) was introduced into the perfusion (4 ml/min) as a 400-␮l bolus and the
perfusate collected at 20 (A), 40 (B), and 60 s (C). Retention time for ATP was determined using bona fide standard.
B: results from several experiments demonstrate that guinea pig coronary arteries generate significant amounts
of ATP from ADP in an endothelium-dependent fashion (nd ⫽ none detected). Endothelium was removed as
detailed in Fig. 1. Total recovery of radioactivity was 3 ⫾ 0.5% less in the endothelium intact vessels. Data are
mean ⫾ SE, n ⫽ 5 experiments. C: endothelial cell monolayers, convert [3H]GDP to [3H]GTP in the presence of ATP
added as a phosphoryl donor. Representative HPLC tracings performed as in A are (trace A) HPLC separation of
the radioactive label before addition to cells (trace B), HPLC tracing of sample incubated with cells for 2 min.
Retention time for GTP was determined using bona fide standard. D: determination of activity converting [3H]GDP
to [3H]GTP in the absence and presence of the adenylate kinase inhibitor Ap5A (100 ␮mol/l), the putative NDPK
inhibitor UDP (5 mmol/l) and a nonneutralizing polyclonal antibody to NDPK. cpm, Counts per minute. Data are
means ⫾ SE, n ⫽ 4–6 experiments.

TTGTGGGAGACATCATCAAACGCTTTGAGCAGAA- (phenylalanine-59), and catalysis of the phosphate trans-


GGGATTCCGCCTTGTGGCCCTGAAACTTATACAG- fer (histidine-118) (20). Serines-123 and -126 correspond
GCCTCGGAGGACCTGCTCAAGGAGCACTACGTCG- to -122 and -125 in all other published NDPKs that lack
ACCTGAAGGACCGCCCGTTCTTCCCTGGCCTGGT- glycine-121. The guinea pig NDPK contains one of the
GCAGTACATGCACTCGGGACCTGTGGTTGCCATG- three amino acids associated with DNA binding (argi-
GTCTGGGAGGGGCTGAACGTGGTGAAGACGGGC- nine-34); however, it lacks the other two (asparagine-69
CGTGTGATGCTGGGAGAGACCAACCCTGCAGACT- and lysine-135) of the human Nm23-H2 (25).
CCAAGCCGGGCACCATCCGAGGGGACTTCTGCAT-
CCAAGTCGGCAGGAACATCATCCACGGCAGCGGC- DISCUSSION
GACTCGGTGGAGAGCGCCGAGAAGGAGATCGCG-
CTGTGGTTCCAGCCGGAGGAGCTGGTGGACTACA- Our results demonstrate that critical elements of our
GGAGCTGTGCTCAGGACTGGATCTATGAGTGA. proposed nucleotide axis hypothesis can be established
This sequence is up to 86% identical to the published in coronary arteries in situ and support the notion that
mouse, human, and rat sequences and translates into a the elaboration of ATP through release and extracellu-
154 amino acid protein that is up to 91% identical and lar formation by endothelium could serve to regulate
over 94% homologous to these other NDPK sequences the production of endothelium-dependent vasodilators
(Fig. 8). This sequence contains one more codon than and prevent thrombosis. Indeed, when agonist is deliv-
the comparable sequences corresponding to a glycine at ered to the perfused coronary, the effluent is seen to
position 121. This sequence contains key conserved contain significant amounts of ATP as soon as a mea-
residues believed to be associated with phosphorylation surement can be taken. This result is consistent with
(serines-44, -120, -123, -126), protein:protein interaction the notion that the regulated release of ATP could be
AJP-Heart Circ Physiol • VOL 281 • OCTOBER 2001 • www.ajpheart.org
H1662 NUCLEOTIDE AXIS IN CARDIAC BLOOD VESSELS

Fig. 5. Western blot demonstration of NDPK protein in cardiac


endothelial cell membranes. Samples for PAGE were boiled in SDS
using standard protocols. A: proteins separated by SDS PAGE (15%)
were blotted to nitrocellulose and probed with a polyclonal antibody
directed to rat liver to NDPK [gift of N. Kimura (19)]. The blot was
developed using a goat anti-rabbit antibody conjugated to alkaline
phosphatase and revealed a 20.5-kDa membrane associated NDPK.
Lane 1, molecular weight marker (lysozyme); lane 2, bovine NDPK
(Sigma N-2635, 2 ␮g); lane 3, 4 ␮g of endothelial cell homogenate;

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lane 4, 2 ␮g of endothelial cell cytoplasm; lane 5, 3 ␮g of endothelial
cell membrane fraction; lane 6, 4 ␮g of K562 leukemia cell homoge-
nate (positive control). B: a monoclonal antibody (Seikagaku Amer-
ica) directed against human Nm23-H1 protein was employed to
detect cardiac endothelial cell NDPK after incubation with rabbit
anti-mouse antibody conjugated to horseradish peroxidase and de-
tected using chemiluminescence. Lane 1, 1 ␮g of bovine NDPK; lane
2, 4 ␮g of endothelial cell homogenate; lane 3, 4 ␮g of endothelial cell
homogenate from cells pretreated with trypsin (0.5% w/v ⫻ 5 min);
lane 4, 4 ␮g of endothelial cell cytoplasm; lane 5, 4 ␮g of endothelial
cell membrane; lane 6, 4 ␮g of K562 leukemia cell homogenate
control. Blots in (A) and (B) were scanned to text and are represen-
tative of 7–10 experiments.

Fig. 4. Endothelial cells express an ecto-NDPK. A: intact endothelial


cell monolayers were exposed to increasing concentrations of ADP in
the presence of GTP (1 mmol/l) and ATP in the Krebs incubation
buffer was determined at 2 min using the luciferin-luciferase assay.
Data were normalized to cell number and computer fit to obtain KM
and Vmax using the equation: v/Vmax ⫽ [S]/Ks ⫹ [S] where v, rate
observed; Vmax, maximal rate of reaction; [S], substrate concentra-
tion; Ks, affinity of enzyme for substrate; Km, substrate concentration
at which rate is one-half maximal. Data are the means ⫾ SE, n ⫽ 10.
B: NDPK activity was assessed in similar cultures in the absence of
ADP and GTP (basal) or in the presence of both substrate and donor
(ADP 10 ␮mol/l ⫹ GTP 1 mmol/l) for 1 min. Treatment of the same
cultures with trypsin (0.3% wt/vol in Ca2⫹-free Krebs buffer) for 90 s
followed by washing (posttrypsin) reduced the ability of the cells to
convert ADP to ATP (623 ⫾ 35 7 132 ⫾ 16 pmol/106 cells). Cells
reacquired the ability to generate ATP from added ADP after cells
were returned to growth conditions for 45 min, washed, and the
experiment repeated (recovery). Data are means ⫾ SE, n ⫽ 6.

involved in acute alterations in blood flow and preven-


tion of platelet aggregation and in agreement with the Fig. 6. Autoradiographic analysis of ADP-ATP phosphotransferase
measurement of significant amounts of ATP seen in activity in cardiac endothelial cell membranes. A: endothelial cell
arterial blood (5). The fact that such release can be membranes were incubated with [␥-32P]ATP (100 ␮Ci; 100 ␮M) in
the presence (lanes 3, 5, and 7) and absence (lanes 2, 4, and 6) of the
blocked and is not seen, if endothelium is intentionally NDPK substrate ADP (30 ␮M) as a phosphate acceptor. Enzymatic
damaged, supports the notion that ATP release is a reactions carried out at 21°C for 10 min (lanes 2, 3), 20 min (lanes 4,
receptor-mediated endothelial cell phenomenon in a 5), or 30 min (lanes 6, 7) were separated on 15% SDS gels and
real blood vessel. developed using a radiographic imager. Lanes 1 and 8 are purified
bovine NDPK under identical conditions in the absence of acceptor
Studying ATP release from endothelium with ago- for 1 min (lane 1) to 34 min (lane 8) as an assay control. Radiogram
nists such as BK does not suffer ambiguity as was the is representative of six experiments yielding similar results. B:
case with the P2y1 receptor agonist ADP. Our efforts to densitometric analysis. Means ⫾ SE, n ⫽ 4 experiments.

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NUCLEOTIDE AXIS IN CARDIAC BLOOD VESSELS H1663

be generated by addition of ADP to endothelial cell


monolayers was enormous in contrast to the amounts
of ATP that could be measured by maximal stimulation
with nonnucleotide agonists and was more than could
be reasonably explained by release alone (2,800 pmoles
vs. 100 pmol/106 cells; data of Fig. 2). If ATP elaborated
after the addition of 100 ␮M ADP (Fig. 2A) were the
result of P2y1 receptor stimulation alone, it would
constitute ⬃20% of the ATP available after membrane
compartments were accessed by ionomycin treatment
(⬃10% total cellular ATP). Such a possibility was not
intellectually pleasing and, furthermore, does not fit
with the fact that coronary endothelium remains intact
after ADP perfusions as evidenced by the tissue’s abil-
ity to release ATP to subsequent introduction of BK
(data not shown). The alternate possibility that ADP
Fig. 7. Flow cytometric analysis of Nm23-H1 binding to intact
added in this fashion enters the cell, is phosphorylated,
guinea pig cardiac endothelial cells. CD31-sorted endothelial cells and returns to the extracellular space without obvious
were grown to confluence in tissue culture flasks, removed without damage to the cell is also remote in the extreme. In

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the aid of trypsin, and immunostained as described in METHODS. Cells addition, the ability to measure NDPK activity in cell
were analyzed using a Beckman Coulter EPICS Elite ESP FACS and
side scatter from blue laser (488 nm) excitation of the cells quantified
cultures at 4°C suggests that under these conditions,
from PMT2 as fluorescence isothiocyanate fluorescence for each cell well below the phase transition temperature of a mam-
event under isotype control and Nm23-H1-positive conditions. Con- malian cell membrane, nucleotides could not possibly
trol cells received only the secondary antibody (IgG1) conjugated to enter and exit cells freely. We hypothesized, therefore,
FITC as an isotype control. Data are from a single experiment
repeated 5 times with qualitatively similar results.
that ATP formation after ADP perfusion of the coro-
nary vessel was due not to regulated release alone, but
included in large measure, extracellular enzymatic for-
measure the effect of ADP stimulation led to the un- mation of ATP.
mistakable conclusion that ADP was not only acting at Although substituted nucleotides such as 8-chloro-
a purinergic receptor to stimulate ATP release from cAMP have been employed as NDPK inhibitors (2),
cells but was, in addition, supporting ADP phosphory- there is no known potent and selective nonnucleotide
lation extracellularly. The amounts of ATP that could inhibitor of NDPK described to date that offers utility

Fig. 8. Sequence alignment of endo-


thelial NDPK-A. Alignment of guinea
pig NDPK-A (AY017306) with human
Nm23-H1 (X73066) and Nm23-H2
(X58965), mouse Nm23 (M65037), and
rat NDPK (D13374). A consensus se-
quence is presented below the align-
ment. A phylogenetic tree is also given
that demonstrates the guinea pig
NDPK is most closely related to the
human NM23-H1 protein. Alignment
and tree were prepared in Vector NTI
Suite 6.0 (Informax; Bethesda, MD).

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H1664 NUCLEOTIDE AXIS IN CARDIAC BLOOD VESSELS

in our studies. The enzyme’s ability to phosphorylate serve to decrease the chances of platelet activation (5).
either adenyl or guanyl purine dinucleotides as well as Such possibilities need not be rejected on the basis of
pyrimidine dinucleotide, together with its inhibition by perceived dilution of the nucleotide. ATP, released
high levels (in mM) of pyrimidine dinucleotide sub- from or generated by endothelium, might reasonably
strate or EGTA, are well known (14, 30). Whereas its be expected to act as ATP (or a metabolite) immedi-
presence as an endothelial ectoenzyme was not known, ately adjacent to the site of release in vivo (34). On the
such a possibility was apparent in perfused hearts (21), basis of our data where ATP is measured in the bulk
not without precedence in transformed cells (18, 19), perfusate, ATP reaches concentrations as high as 0.1
and could be directly tested in cultured cardiac endo- ␮M in the coronary vessel. Whereas the subsequent
thelial cells. actions of released ATP may be diffusion limited, this
The effect of limited proteolysis to remove the ability limitation is a special case for endothelium where the
of cells to support ADP to ATP conversion, together laminar flow fluid layer above endothelial cells is in
with the return of the activity in the same cells after motion continuously and not in immediate equilibrium
trypsin removal and a period of control incubation, with the majority of liquid flowing in the vessel (11).
supports the notion that the NDPK activity we mea- Although there is no objective method to determine the
sure is assembled as an ectoenzyme and that the cells concentration of released ATP at the microenviron-
are capable of rapid recovery of activity under these ment next to the endothelial surface, if restricted there
conditions. for a time, the resulting nucleotide concentration (10 to
The identity of the NDPK activity of endothelium as 100 times or more that determined for the bulk phase)

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Nm23-G1, the guinea pig homologue of Nm23-H1, is would be vasoactive in guinea pigs (7) and may lead to
evident from immunoblots and is consistent with the formation of ADP that would achieve a local concen-
ability of purified endothelial cell membranes to dis- tration sufficient to support the activity of NDPK.
play a phosphoprotein that operates as a phosphoryl Nucleotide axis hypothesis may offer a context in
donor capable of transferring phosphate to ADP. The which to consider the elegant work of those who have
fact that this ping-pong transphosphorylase is an ec- established our understanding of the critical impor-
toenzyme is supported by our finding that intact cells tance of NO in the regulation of vascular tone. It
can be immunostained with Nm23-H1-specific anti- appears that the list of those agonists and perturba-
body, analyzed by flow cytometry, and found to express tions that stimulate endothelial cells to liberate NO (6,
the antigen on the surface of the cell. 12, 13, 28, 33, 37) also stimulates ATP release. Our
The proposed sequence of guinea pig ecto-NDPK con- hypothesis suggests that ATP plays a central role in
tains three consensus protein kinase C sites at serine-4, the signaling and responsiveness of endothelium in
threonine-86, and threonine-103. These sites are con- vivo and serves to amplify and carry forward in time
served in all of the NDPKs aligned (Fig. 8). Sequence and space the effects of other vasoactive agonists or
analysis reveals that there are no transmembrane re- shear stress beyond their initial sites of action in the
gions in NDPK, yet we find NDPK to be associated with blood vessel. ATP release by endothelium stimulates
the membrane. There are no typical glycosylation sites, its own release, thus amplifying local vasodilation.
but there are two conserved N-myristoylation sites Once metabolized to ADP, the nucleotide can act again
shared by all of the NDPKs at glycines-15 and -102, as at an endothelial cell P2y1 receptor to again amplify
well as an additional myristoylation site in Nm23-G1 the response (downstream) and, to the extent that ADP
created at glycine-119 due to the unique presence of is rephosphorylated to ATP, can act yet again as ATP
glycine-121. This is absent in the human, mouse, and rat and then ADP, etc., furthering the axis cascade. The
proteins. This site is near the catalytic histidine-118 and movement of the nucleotide in the bloodstream, to-
interrupts the potentially phosphorylated serines at -120, gether with the estimated fraction of ADP that can be
-123, and -126. The role of this extra glycine is unclear, rephosphorylated to ATP in the vessel (12%; data of
and particularly interesting due to its location in such an Fig. 4B), mitigates this seemingly positive feedback
active region of the enzyme. The GDP binding pocket and system. Moreover, the presence of ATP elaboration by
active site are conserved with histidine-118, leucine-48, endothelium will serve to limit platelet activation both
and serine-123 (24, 25). because ATP receptor activation leads to NO release
Current dogma does not predict ADP-kinase activity and because ATP [and its ultimate vascular end prod-
of endothelium but rather predicts the rapid metabo- uct adenosine (22)], like NO, is directly antithrombo-
lism of adenyl purine nucleotides to adenosine (15). genic. Once metabolized to adenosine, the purine acts
The notion that endothelium could generate ATP ex- again to dilate terminal arterioles. This concept offers
tracellularly was intriguing because it would be con- the pleasing feature of biological economy by employ-
sistent with maintaining the actions of ATP in time ing a single purine molecule acting multiple times at
and space within the blood vessel and would allow the disparate purinergic receptor sites in different loca-
purine nucleotide signal, originally generated by acute tions within the vessel to achieve the local regulation of
changes in shear or the actions of rapidly degraded blood flow. Finally, the avid uptake of adenosine by
peptide agonists such as BK to be carried forward well capillary endothelium, where it can be reutilized, offers
beyond the site of initial endothelial response. Further- the ultimate in biological economy.
more, the removal of the aggregatory agonist ADP by In studies presented herein, we have employed an
rephosphorylation to platelet antagonist (ATP) would extracellular phosphoryl donor in the form of either
AJP-Heart Circ Physiol • VOL 281 • OCTOBER 2001 • www.ajpheart.org
NUCLEOTIDE AXIS IN CARDIAC BLOOD VESSELS H1665

GTP or ATP to favor the formation of the correspond- 2. Anciaux K, Van Dommelen K, Willems R, Roymans D, and
ing purine triphosphate by the action of guinea pig Slegers H. Inhibition of nucleoside diphosphate kinase (NDPK/
Nm23) by cAMP analogues. FEBS Lett 400: 75–79, 1997.
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the NDPK assay in this manner permits an unmistak- regulation of blood flow. In: Methods in Pharmacology, edited by
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1996.
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Expression of a cloned P2Y purinergic receptor that couples to
The framework we offer for considering the nucleo- phospholipase C. Mol Pharmacol 46: 8–14, 1994.
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our understanding of the role of purines in protecting and Kimura N. Recombinant rat nucleoside diphosphate kinase
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tion to immunological detection of native isoforms in rat tissues.
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purines also regulate blood flow. To the extent that extracellular adenine nucleotides by cultured endothelial cells
endothelial cells release ATP and that ATP is eventu- from pig aorta. Feed-forward inhibition of adenosine production
ally hydrolyzed to adenosine, the nucleotide axis hy- at the cell surface. J Biol Chem 261: 15496–15507, 1986.
16. Hoyle CHV and Burnstock G. ATP receptors and their phys-
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We are grateful to David Schooley for critical review of the manu- Physiol Heart Circ Physiol 257: H1096–H1103, 1989.
script and to Qaio Zhong for flow cytometry assistance. 18. Kimura N and Johnson GS. Increased membrane-associated
This work was supported by grants from the American Heart nucleoside diphosphate kinase activity as a possible basis for
Association (National Center), National Heart, Lung, and Blood enhanced guanine nucleotide-dependent adenylate cyclase activ-
Institute Grant HL-56422 and a grant from the Foundation for ity induced by picolinic acid treatment of simian virus 40-trans-
Research (to I. L. O. Buxton), a postdoctoral fellowship from the formed normal rat kidney cells. J Biol Chem 258: 12609–12617,
American Heart Association (Nevada Affiliate) (to D. J. Cheek), a 1983.
National Institutes of Health predoctoral fellowship award (NR- 19. Kimura N and Shimada N. Membrane-associated nucleoside
07379) (to B. C. Oxhorn), and a predoctoral fellowship from the diphosphate kinase from rat liver purification, characterization,
American Heart Association (Western States Affiliate) (to R. A. and comparison with cytosolic enzyme. J Biol Chem 263: 4647–
Kaiser). 4653, 1988.
Current address of D. J. Cheek: University of North Carolina at 20. La Rosa AD, Williams RL, and Steeg PS. Nm23/nucleoside
Chapel Hill, Carrington Hall, CB 7460, Chapel Hill, NC 27599-7460 diphosphate kinase: toward a structural and biochemical under-
(E-mail: djcheek@email.unc.edu). standing of its biological functions. Bioessays 17: 53–62, 1995.
21. Moir TW and Downs TD. Myocardial reactive hyperemia:
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