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Am J Physiol Heart Circ Physiol 281:1657-1666, 2001.
This article cites 31 articles, 12 of which you can access free at:
http://ajpheart.physiology.org/cgi/content/full/281/4/H1657#BIBL
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Am J Physiol Heart Circ Physiol
281: H1657–H1666, 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-
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
RESULTS
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)
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/
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