Am J Physiol Cell Physiol 296: C828–C839, 2009.
First published February 4, 2009; doi:10.1152/ajpcell.00619.2008.
Autocrine ATP release coupled to extracellular pyrophosphate accumulation
in vascular smooth muscle cells
Domenick A. Prosdocimo,1 Dezmond C. Douglas,1 Andrea M. Romani,1 W. Charles O’Neill,2
and George R. Dubyak1
1
Department of Physiology and Biophysics, School of Medicine, Case Western Reserve University, Cleveland, Ohio;
and 2Department of Medicine, Renal Division, Emory University School of Medicine, Atlanta, Georgia
Submitted 2 December 2008; accepted in final form 29 January 2009
adenosine 5⬘-triphosphate release; vascular calcification; ectonucleotide pyrophosphatase; ectoadenosine 5⬘-triphosphatase
EXTRACELLULAR ATP ACTS AS an agonist for the P2X (44) and
P2Y (8, 29, 61) families of nucleotide receptors. To prevent
activation of these receptors in resting conditions, concentrations of ATP in extracellular compartments are maintained
within a narrow nanomolar range through the coordinated
coupling of ATP release mechanisms and metabolism of released ATP via various ectonucleotidases (28, 29). Recent
studies indicate that extracellular ATP metabolism also contributes to the regulation of cardiovascular calcification in
blood vessels and valves through generation of inorganic
pyrophosphate (PPi), a potent inhibitor of calcification (22, 51).
Address for reprint requests and other correspondence: G. R. Dubyak, Dept.
of Physiology and Biophysics, Case Western Reserve Univ. School of Medicine, 2109 Adelbert Rd., Cleveland, OH 44106 (e-mail: george.dubyak
@case.edu).
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At micromolar levels, extracellular PPi inhibits the ability of
Ca2⫹ and phosphate (Pi) to form insoluble hydroxyapatite
crystals in both in vitro solutions (60) and intact tissues such as
aortic rings cultured ex vivo (37). The importance of extracellular PPi homeostasis in human cardiovascular function is
highlighted by the often fatal syndrome of idiopathic infantile
arterial calcification (51, 52) that has been linked to inactivating mutations in the ENPP1 gene, which encodes an ectonucleotide pyrophosphatase/phosphodiesterase (eNPP1) also known
as PC-1 (57). Calcification of large-capacitance arteries also
markedly disrupts normal cardiovascular hemodynamics in
more common human diseases such as renal failure and Type
II diabetes (10, 45, 56).
The suppressive effect of PPi on pathological calcification in
soft tissues, such as blood vessels, requires its regulated accumulation within local extracellular compartments, and three
distinct plasma membrane proteins regulate this local accumulation. These include eNPP1 (59), which catabolizes extracellular ATP to produce PPi, and tissue nonselective alkaline
phosphatase (TNAP), which hydrolyzes PPi into phosphate and
thereby opposes the action of eNPP1 (40). The progressive
ankylosis disease susceptibility gene product (ANK) is a
plasma membrane protein that either directly mediates efflux of
cytosolic PPi or regulates the activity of another as-yet-undefined PPi transporter (17). Thus, sustained activities of NPP1
and/or ANK are required for the active, steady-state suppression of pathological calcification in soft tissues. TNAP is a
marker protein for osteoblasts and, via its ability to clear
locally generated PPi, facilitates normal mineralization of bone
(15). Similarly, aberrant accumulation of TNAP-expressing
cells within vascular lesions can promote arterial calcification
(10, 45, 56).
Calcification of large arteries in rodents can be induced by
experimental manipulations that induce renal failure or conditions, such as hyperphosphatemia and hyperparathyroidism,
which accompany chronic kidney disease (38, 42). The capacity of PPi-regulatory proteins to counteract vascular calcification is also contingent on local extracellular matrix proteins
and the ambient levels of Ca2⫹ and Pi (13, 33). While deletion
of eNPP1 or inactivation of ANK predisposes mice to aortic
calcification (22), the rate and severity of calcification is
greatly potentiated when enpp1⫺/⫺ or ank mice are fed a
high-phosphate diet (42).
That loss of either eNPP1 or ANK activity potentiates
vascular calcification suggests that these proteins act in nonThe 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.
0363-6143/09 $8.00 Copyright © 2009 the American Physiological Society
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Prosdocimo DA, Douglas DC, Romani AM, O’Neill WC,
Dubyak GR. Autocrine ATP release coupled to extracellular pyrophosphate accumulation in vascular smooth muscle cells. Am J
Physiol Cell Physiol 296: C828 –C839, 2009. First published February
4, 2009; doi:10.1152/ajpcell.00619.2008.—Extracellular inorganic
pyrophosphate (PPi) is a potent suppressor of physiological calcification in bone and pathological calcification in blood vessels. Ectonucleotide pyrophosphatase/phosphodiesterases (eNPPs) generate PPi via
the hydrolysis of ATP released into extracellular compartments by
poorly understood mechanisms. Here we report that cultured vascular
smooth muscle cells (VSMC) from rat aorta generate extracellular PPi
via an autocrine mechanism that involves ATP release tightly coupled
to eNPP activity. The nucleotide analog ␤,␥-methylene ATP (MeATP
or AMPPCP) was used to selectively suppress ATP metabolism by
eNPPs but not the CD39-type ecto-ATPases. In the absence of
MeATP, VSMC generated extracellular PPi to accumulate ⱖ600 nM
within 2 h while steadily maintaining extracellular ATP at 1 nM.
Conversely, the presence of MeATP completely suppressed PPi accumulation while increasing ATP accumulation. Probenecid, which
inhibits PPi efflux dependent on ANK, a putative PPi transporter or
transport regulator, reduced extracellular PPi accumulation by approximately twofold. This indicates that autocrine ATP release coupled to
eNPP activity comprises ⱖ50% of the extracellular PPi-generating
capacity of VSMC. The accumulation of extracellular PPi and ATP
was markedly attenuated by reduced temperature but was insensitive
to brefeldin A, which suppresses constitutive exocytosis of Golgiderived secretory vesicles. The magnitude of extracellular PPi accumulation in VSMC cultures increased with time postplating, suggesting that ATP release coupled to PPi generation is upregulated as
cultured VSMC undergo contact-inhibition of proliferation or deposit
extracellular matrix.
EXTRACELLULAR ATP AND PPi ACCUMULATION IN VASCULAR SMOOTH MUSCLE
EXPERIMENTAL PROCEDURES
Materials. Low-glucose (1 g/l) Dulbecco’s modified Eagle’s medium (LG-DMEM), firefly luciferase assay mix (FL-AAM), ATP
standards (FL-AAS), sodium pyrophosphate, adenosine 5⬘-phosphosulfate (APS), adenosine-5⬘-triphosphate sulfurylase (ATP-sulfurylase), ␤,␥-methylene ATP (MeATP or AMPPCP), probenecid (PB),
brefeldin A (BFA), levamisole, methylene diphosphonate (PCP), and
TRIzol were from Sigma-Aldrich (St. Louis, MO); 1,N6-ethenoadenosine 5⬘-triphosphate (ε-ATP) was from Invitrogen/Molecular
Probes (Eugene, OR). Fetal bovine serum, newborn calf serum (CS),
and penicillin-streptomycin (P/S) were from Hyclone (Logan, UT).
Oligo(dT)15 primer and RNase inhibitor were purchased from Promega (Madison, WI). Avian myeloblastosis virus (AMV) reverse
transcriptase, Taq polymerase, and PCR nucleotide mix were from
Roche (Indianapolis, IN) while the dNTP mix was from Stratagene.
PCR primers were obtained from Operon Biotechnologies (Huntsville, AL). Anti-human eNPP1 (L-20), anti-green fluorescent protein
(GFP) (FL), and anti-actin (C-11) primary antibodies and secondary
antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA). A
plasmid expression vector encoding human eNPP1 was provided by
Dr. Robert Terkeltaub (University of California, San Diego).
VSMC isolation and cell culture. Sprague-Dawley rats were maintained and handled in accordance with the Guide for the Care and Use
of Laboratory Animals (Institute of Laboratory Animal Resources,
Commission on Life Science, National Research Council; published
by National Institutes of Health, Publication No. 85-23, Revised
1996), as approved by the Institutional Animal Use and Care Committee of Case Western Reserve University. Rats (250 g, male) were
anesthetized by intraperitoneal injection of a saturated pentobarbital
sodium solution (40 mg/kg body wt). Following achievement of deep
anesthesia, the chest was opened and the thoracic aorta was rapidly
removed under aseptic surgical conditions. Rat aortic VSMC were
isolated using an explanted aortic ring model previously described
(11). All subsequent steps were performed using a dissection microscope in a cell culture hood under sterile airflow. The adventitial and
endothelial layers were gently removed, and the aorta was sectioned
into 2- to 3-mm rings and placed onto the prescratched (by a scalpel
blade) and washed surfaces of wells within a six-well culture plate.
Three rings were placed per well together with 2 ml growth media
(LG-DMEM supplemented with 10% fetal bovine serum, 1% P/S, and
AJP-Cell Physiol • VOL
0.11 g/l sodium-pyruvate) and maintained at 37°C in a 10% CO2
atmosphere. After initial culture for 2 days, the rings were transferred
to the wells of a fresh six-well plate and cultured for an additional 5
days during which significant outgrowth of VSMC was observed.
These primary VSMC were then passed weekly by trypsinization and
1:4 splits for subculture. Unless otherwise noted, experiments were
performed using VSMC at passages 2-4. The cells were ⱖ95%
positive for smooth muscle ␣-actin as measured by immunofluorescence (data not shown).
HEK-293 cells were stably transfected with expression plasmids
encoding either GFP or human eNPP1. The transfected HEK-293
lines were selected and maintained in the presence of G418 (400
␮g/ml) in DMEM supplemented with 10% CS and antibiotics. Mouse
L-cells differentiated by culture in the presence of 1 mM 8-chlorophenylthio-cyclic AMP (CPT-cAMP) for 2 days were used as positive
control cells that express TNAP at high levels (5).
RT-PCR analysis of ectonucleotidase subtypes. Semiquantitative
RT-PCR was used to compare the expression profiles of ectonucleotidases and PPi homeostatic proteins in the cultured VSMC. Total
RNA was isolated using TRIzol following the manufacturer’s protocol. First-strand cDNA synthesis was accomplished using 1–2 ␮g total
RNA, primed with 1 ␮g oligo(dT) primer, and incubated with 2 units
AMV reverse transcriptase, 8 mM dNTPs, and 1 unit RNase inhibitor
at 42°C for 1 h. The following cDNA transcripts were analyzed:
eNPTDase1/CD39, eNTPDase2, eNTPDase3, eNPP1/PC1, eNPP3/
B10/gp130, CD73, TNAP, and ANK. The PCR primer pairs and
amplicon sizes for the various ectonucleotidases have been previously
reported (25). The primer pair (5⬘-GCCGCTGCTCATCCCCATCC,
antisense, 5⬘-GGCCGAGGTGACCGTGTTGTTC) used to analyze
ANK cDNA transcripts was designed using DNAStar v.5 (DNASTAR)
to produce a 400-bp ANK amplicon. Glyceraldehyde phosphate dehydrogenase was used as a high-copy housekeeping gene product. PCR
reactions were performed in 20-␮l reaction volumes containing 10-fold
dilutions of RT reaction product (1:10 to 1:10,000) with 10 mM Tris䡠HCl
(pH 8.3), 1.5 mM MgCl2, 50 mM KCl, 250 ␮M dNTPs, 1 ␮M primer
mix, and 2.5 U/␮l Taq DNA polymerase. PCR running conditions
were as follows: 94°C for 2 min, followed by 35 cycles of 94°C for
40 s, 58°C for 40 s, and 74°C for 3 min with a final extension step for
5 min at 37°C. PCR products were electrophoretically separated on
1.5% agarose gels containing ethidium bromide (1 ␮g/ml), and the
fluorescent bands were visualized and recorded with a Bio-Rad
GelDoc 1000 detector.
Ectonucleotidase assays. Ecto-ATPase activities of VSMC (as
adherent monolayers) or HEK-293 cells (as trypsin-detached suspensions) were monitored by luciferase-based methods. HEK-293 cells
(106) were resuspended in 200 ␮l basal salt solution (BSS) containing
(in mM) 130 NaCl, 5 KCl, 1.5 CaCl2, 1 MgCl2, 25 Na-HEPES
(adjusted to pH 7.5 at room temperature, 22–24°C), 5 glucose, and
0.1% BSA, supplemented with 8 ␮l from a concentrated luciferase/
luciferin stock solution (FL-AAM). The cell suspensions were then
pulsed with 100 –500 nM exogenous ATP; decreases in ATP-dependent luminescence over the next 20 –30 min were continuously monitored using a Turner Designs (TD 20/20) luminometer. Where indicated, the cell suspensions were supplemented with 300 ␮M MeATP
immediately before addition of the ATP pulse. Ecto-ATPase activities
of adherent VSMC were performed using cell monolayers (0.3– 0.6 ⫻
106 cells/well of 6-well plates) bathed in 1 ml BSS. These experiments
were performed at 37°C in the absence or presence of 300 ␮M
MeATP. The cells were preincubated for 20 –30 min to clear endogenously released ATP before the addition of exogenous ATP. Extracellular samples (100 ␮l) taken at various times after ATP addition
were boiled for 5 min (to inactivate any shed ectonucleotidase activity) and then stored on ice before analysis of ATP and PPi levels as
described below.
Enzyme assays for quantitation of ATP and PPi levels in extracellular medium samples. To quantitate extracellular PPi we adapted an
enzyme-linked bioluminescence assay originally developed to detect
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redundant, parallel pathways for the maintenance of appropriate extracellular PPi homeostasis. Johnson et al. (22) observed
that steady-state levels of PPi in the conditioned media of
cultured aortic vascular smooth muscle cells (VSMC) from
either enpp1⫺/⫺ or ank mice were similarly reduced by ⬃50%,
relative to wild-type cultures, during induction of in vitro
calcification by elevated phosphate. However, increased phosphate also alters expression of both eNPP1 and ANK (18).
Thus, the relative contribution of the eNPP1 versus ANK
pathway to extracellular PPi generation by VSMC in the basal
state is unclear.
ENPP1 is part of a broader purinergic signaling network in
blood vessels that regulates not only calcification but also
vascular tone, inflammation, and remodeling (8). Notably, the
source and mechanism for delivery of the extracellular ATP
that drives eNPP1-mediated generation of PPi in vascular
compartments remains undefined. Here we characterize an
autocrine pathway of ATP release that is tightly coupled to the
production of extracellular PPi in a rat aortic VSMC model.
Our data indicate that eNPP1-mediated hydrolysis of endogenous ATP released from VSMC operates in parallel with
another pathway involving direct efflux of intracellular PPi to
maintain extracellular PPi levels in the micromolar range.
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EXTRACELLULAR ATP AND PPi ACCUMULATION IN VASCULAR SMOOTH MUSCLE
AJP-Cell Physiol • VOL
Western blot analysis. Adherent cultures of HEK-293 cells or
VSMC were extracted and analyzed by SDS-PAGE and Western
blotting using previously described methods (3).
Statistical analysis. All experiments with VSMC were repeated at
least three times using different preparations of cultured VSMC. All
data, unless otherwise stated, represent means ⫾ SE, and statistical
significance was defined as P ⬍ 0.05 using two- or three-way
ANOVA with the Bonferroni post hoc test.
RESULTS
Clearance of extracellular ATP is coupled to PPi accumulation in rat aortic VSMC. We used rat aortic VSMC at low
passage number (2– 4) as a tissue culture model for quantitatively assaying extracellular ATP release and metabolism and
coupled PPi accumulation. These cultures expressed smooth
muscle-specific ␣-actin in ⬎95% of the cells for at least six
passages, as well as mRNA for multiple VSMC gene markers,
including smooth muscle myosin-1 heavy chain, SM22-␣,
caldesmon, and calponin (data not shown). Analysis of mRNA
transcripts for different ectonucleotidase subtypes indicated
significant expression of eNPP1, NTPDase3, and CD73, while
TNAP, NTPDase1 (CD39), and eNPP3 were present at lower
levels (Fig. 1A); mRNA for the ANK PPi transporter/regulator
was also abundant. The intact VSMC expressed robust ectoATPase activity as indicated their ability to completely hydrolyze an exogenously added pulse of 100 nM ATP within 10
min (Fig. 1B). Notably, the rate at which the VSMC hydrolyzed extracellular ATP was reduced twofold in the presence of
MeATP (also known as AMPPCP). We previously reported
that MeATP selectively suppresses the catabolism of extracellular ATP by eNPP-family, but not CD39-family, ectoATPases in several rat and human cell types (3, 24, 25). EctoATPase measurements in HEK-293 cells stably transfected
with human eNPP1 verified this selectivity of MeATP (supplemental Fig. 1; supplemental data and online article are
available at the American Journal of Physiology-Cell Physiology website). To demonstrate that eNPP1 in VSMC functions
as a PPi-generating ecto-ATPase, we pulsed monolayers of
adherent cells (⬃5 ⫻ 105 cells䡠ml⫺1 䡠35-mm dish⫺1) with 10
␮M ATP in the absence or presence of MeATP (300 ␮M) and
measured extracellular levels of both ATP and PPi over a
60-min test period. The 10 ␮M pulse of exogenous ATP was
almost completely cleared by the VSMC monolayers within 60
min (Fig. 1C) and correlated with the progressive accumulation
of extracellular PPi (Fig. 1D). However, the extracellular PPi
concentration ([PPi]) at 60 min was only 3 ␮M, indicating that
only 30% of the hydrolyzed ATP was coupled to PPi generation or that PPi itself was further metabolized. MeATP modestly retarded clearance of the 10 ␮M ATP pulse (Fig. 1C) but
completely suppressed the accumulation of PPi (Fig. 1D). This
indicates that rat aortic VSMC express MeATP-sensitive
eNPP-family ectonucleotidases that generate extracellular PPi
as an end-product and also MeATP-insensitive NTDPasefamily ecto-ATPases that produce Pi rather than PPi. Direct
analysis of Pi production with 500 ␮M ATP as the substrate
indicated an ecto-ATPase rate of 3.23 ⫾ 0.12 nmol䡠h⫺1 䡠␮g
protein⫺1 in the absence of MeATP versus an ecto-ATPase of
3.38 ⫾ 0.26 nmol䡠h⫺1 䡠␮g protein⫺1 in the presence of 300
␮M MeATP.
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liberated PPi from RNA reverse transcription reactions (27, 50). In
this assay system, PPi reacts with APS in the presence of ATPsulfurylase to generate ATP which is measured by a coupled luciferin/
luciferase reaction. Our adaptation of this assay facilitated quantitative
measurements of both the ATP and PPi content within the same
sample of extracellular medium. Briefly, 75 ␮l of heat-inactivated
(100°C, 5 min) extracellular sample was mixed with 20 ␮l of 25 ␮M
APS, and 4 ␮l concentrated FL-AAM to yield a final reaction volume
of 99 ␮l (with 5 ␮M APS). The initial steady-state bioluminescence,
measured at room temperature with the TD 20/20 luminometer, was
an indicator of the ATP content in the extracellular medium sample.
When this initial ATP-dependent bioluminescence reached a steady
value, ATP-sulfurylase (0.01 units in 1 ␮l) was added to drive
conversion of PPi to ATP, and bioluminescence was continuously
measured until a new steady state was reached. This value was
recorded and subtracted from the original ATP-dependent bioluminescence value to yield PPi-dependent bioluminescence. Calibration
curves were generated for each experiment using identical coupled
enzymes reactions with BSS containing ATP and PPi standards (1 nM
to 1 ␮M) in place of the cell-conditioned media samples. For extracellular samples containing pharmacological inhibitors of ATP release
pathways or ectonucleotidases, additional calibration curves were
generated in the presence of the indicated inhibitor to assess possible
inhibitory effects on the ATP-sulfurylase or luciferase reactions.
This assay system was also used to measure extracellular ATP and
PPi concentrations produced endogenously by rat aortic VSMC. For
these experiments, unless otherwise stated, adherent monolayers of rat
aortic VSMC (0.3– 0.6 ⫻ 106 cells/well) were used between passages
2 and 4 and 3–9 days postplating on six-well plates at 37°C. For these
experiments, the cell monolayers were rapidly washed twice with PBS
(after aspiration of tissue culture medium) and then transferred to 1.2
ml fresh BSS to initiate the test incubations at 37°C in the absence or
presence of the indicated inhibitor(s). An initial 100-␮l aliquot of
extracellular media was immediately taken (⬃1- to 3-min time point),
and additional 100-␮l samples were taken at various intervals (5– 60
min as indicated in specific experiments) for up to 2 h after transfer of
the cells to the BSS test medium. Each extracellular medium sample
was heated (100°C, 5 min) and centrifuged before quantitative analysis of ATP and PPi content.
Alkaline phosphatase activity in lysates of VSMC or CPT-cAMPdifferentiated L-cells was assayed in the absence or presence of
levamisole by previously described methods (5, 38).
Ectonucleotidase assays by high-performance liquid chromatography. Reverse-phase high-performance liquid chromatography (HPLC)
was used to measure the extracellular metabolism of fluorescent,
etheno-derivitized, adenine nucleotide analogs ε-ATP (Molecular
Probes) and ε-MeATP (25, 31). Cell monolayers were grown in
six-well tissue culture plates, washed twice with 1 ml PBS pH 7.4, and
equilibrated for 30 min in 1 ml BSS at room temperature. Cells
(HEK-293 or VSMC) were then pulsed with indicated concentrations
of either ε-ATP or ε-MeATP. At indicated time points, 100 ␮l of
extracellular medium were removed and boiled for 5 min. Adenine
nucleotide derivatives were resolved using an Alltech C18 Ad-sorbosphere column eluted at 1.3 ml/min. A methanol gradient was formed
by mixing buffer A (0.1 M KH2PO4, pH 6, 5% methanol) with buffer
B (0.1 M KH2PO4, pH 6, 15% methanol) using the following protocol:
0 –2 min (100% buffer A); 2–3 min (ramp to 75% buffer A/25% buffer
B); 3–26 min (ramp to 35% buffer A/65% buffer B); 26 –30 min (ramp
to 100% buffer A). Fluorescent adenine analogs were detected with a
Linear LC305 fluorescence detector using 270 nm excitation and 410
nm emission wavelengths. Alternatively, isocratic reverse-phase
HPLC was used to assay the extracellular metabolism of 300 ␮M
MeATP by VSMC monolayers grown in six-well dishes. Extracellular
samples were processed as described before injection onto the Alltech
C18 column. Nucleotides were resolved by isocratic elution (1.3
ml/min) with buffer A and detected by absorbance at 254 nm.
EXTRACELLULAR ATP AND PPi ACCUMULATION IN VASCULAR SMOOTH MUSCLE
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ATP released from VSMC is efficiently coupled to extracellular PPi accumulation. Most mammalian cell types constitutively release ATP at low but significant rates thereby conditioning extracellular compartments to contain nanomolar concentrations of ATP and ATP metabolites at steady state (24,
29). Perturbation of the steady-state relationship between the
ATP release rate and the ATP clearance rate, by either increasing the rate of ATP release (e.g., due to mechanical stimulation) or repressing activity of the opposing ecto-ATPases, leads
to higher levels of extracellular ATP. We assessed the ability
of adherent VSMC to condition their extracellular medium
with endogenous ATP and PPi during 2-h test incubations in
serum-free BSS. It is important to note that transfer of the
VSMC monolayers from the regular tissue culture media to this
experimental test media (including two intermediate washes
with PBS) constitutes a transient mechanical stimulus that
triggers rapid release of endogenous ATP and other lowmolecular-mass metabolites into the extracellular compartment. Thus, Fig. 2, B (log scale) and C (linear scale), shows
that 10 –20 nM extracellular ATP was present at the earliest
time point (3 min) at which the assay medium was sampled.
Notably, ⬃150 nM extracellular PPi was also rapidly accumulated at this early time point (Fig. 1A) followed by a sustained
increase in extracellular [PPi] to ⬎600 nM as the VSMC
progressively conditioned the assay medium over the 2-h
AJP-Cell Physiol • VOL
experimental time course (Fig. 2, A and D). In contrast, the
initial burst of accumulated extracellular ATP progressively
decreased at the later time points to a steady-state level of 1 nM
ATP within 30 min. Notably, when VSMC were transferred to
MeATP-containing assay medium, the accumulation of both
extracellular ATP (Fig. 2, B and C) and PPi (Fig. 2A) was
markedly altered. Although the initial burst of PPi accumulation was not affected, the VSMC did not accumulate additional
extracellular PPi but slowly cleared the initially accumulated
PPi. Conversely, inclusion of MeATP reversed the clearance of
the initially released ATP and facilitated accumulation of
extracellular [ATP] in the 20 – 60 nM range during the 2 h
of subsequent incubation (Fig. 2, B and C). These data indicate
that VSMC continue to release ATP after the initial mechanical
stimulus at a rate that is matched by an opposing MeATPsensitive ecto-ATPase (and MeATP-insensitive ecto-ATPases)
to yield a steady-state level of 1 nM extracellular ATP in the
absence of MeATP. The sustained increase in extracellular PPi
levels during maintenance of this low steady-state ATP level
indicates that ATP release from the VSMC is very efficiently
coupled to PPi generation via an eNPP-type ecto-ATPase.
Suppression of this eNPP-mediated ATP metabolism in the
presence of MeATP coordinately blocks accumulation of extracellular PPi and facilitates increased levels of ATP.
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Fig. 1. Expression and activities of ectonucleotidases and inorganic pyrophosphate (PPi) homeostatic proteins in rat aortic vascular smooth muscle
cells (VSMC). A: RT-PCR analysis of mRNAs for
ectonucleotidases and PPi homeostatic proteins in
VSMC. NPP, nucleotide pyrophosphatase/phosphodiesterase; ANK, progressive ankylosis gene; TNAP,
tissue nonselective alkaline phosphatase; NTPD, nucleoside 5⬘-triphosphate diphosphohydrolase. B: suspensions of intact VSMC [106 cells in 200 ␮l basal
salt solution (BSS)] at 23°C were pulsed with 100
nM exogenous ATP in the absence (■) or presence
(䊐) of 300 ␮M ␤,␥-methylene ATP (MeATP); F,
cell-free control. ATP was assayed by luciferasebased luminescence. Data points represent the average ⫾ range of duplicates from a representative
experiment repeated three times using different
VSMC preparations. C and D: metabolism of exogenous (10 ␮M) ATP coupled to extracellular PPi
generation by adherent VSMC monolayers in the
absence (■) or presence (䊐) of 300 ␮M MeATP.
Data represent the average ⫾ range of duplicates
from a representative experiment repeated three
times using adherent VSMC monolayers bathed in 1
ml BSS at 37°C.
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EXTRACELLULAR ATP AND PPi ACCUMULATION IN VASCULAR SMOOTH MUSCLE
Tissue nonselective alkaline phosphatase (TNAP) acts as a
potent PPi hydrolyzing ectoenzyme (40, 42, 43). We used 5
mM levamisole, a well-characterized TNAP inhibitor, to test
whether basally expressed TNAP significantly limited the magnitude of extracellular PPi accumulation by the cultured VSMC
(43). If TNAP activity is high, then the presence of levamisole
should potentiate PPi accumulation. However, this was not
observed. In the presence of levamisole, extracellular PPi
accumulation (measured at 2 h as described in Fig. 2D) by
intact VSMC was 71 ⫾ 29% (n ⫽ 3 experiments) of the
normalized values measured in the absence of levamisole. This
concentration of levamisole strongly inhibited alkaline phosphatase activity (supplemental Fig. 2), as measured by standard
p-nitrophenyl phosphate hydrolysis assays, in lysates of VSMC
or lysates of cAMP-differentiated L-cells that express high levels
of TNAP (5). Notably, the levamisole-sensitive alkaline phosphatase-specific activity in VSMC lysates was ⬃16-fold lower
than in L-cell lysates. Thus, basally expressed TNAP is not a
major regulator of extracellular PPi homeostasis in cultured
VSMC. Direct measurements indicated that exogenous PPi was
only slowly cleared (20% of a 1 ␮M pulse was metabolized
within 2 h) by these cultured VSMC (data not shown).
Attenuation of extracellular PPi accumulation by methylene
diphosphonate, a product of eNPP1-mediated metabolism of
MeATP. We previously reported that MeATP inhibits ATP
hydrolysis by eNPP-type ecto-ATPases by acting as a competing substrate (25) and have verified this rapid catabolism of
extracellular MeATP in both HEK-293 cells stably transfected
with human eNPP1 (Fig. 3A) and in rat VSMC (Fig. 3B). Thus,
eNPP1 will catalyze the reaction (MeATP 3 PCP ⫹ AMP) to
generate PCP, a bisphosphonate analog, as a by-product. In
turn, the PCP that accumulates during incubation of VSMC
AJP-Cell Physiol • VOL
with MeATP may affect the autocrine mechanisms that drive
accumulation of extracellular PPi. This was supported by our
observation that PPi accumulation was reduced in a dosedependent manner by exogenously added PCP, with 300 ␮M
producing an eightfold decrease (Fig. 3C). Notably, we verified
that these concentrations of PCP had no inhibitory actions on
either the luciferase or ATP-sulfurylase reactions used to
quantitate the PPi levels in conditioned medium. PCP (300
␮M) by itself did not affect the transient accumulation of
extracellular ATP induced by the medium exchange stimulus.
Rather, the presence of both PCP and MeATP in the exchange
medium further potentiated the marked ATP accumulation
facilitated by MeATP alone (Fig. 3D). This suggests that PCP
may act as a product inhibitor of eNPP1 and thereby attenuate
both PPi accumulation and ATP clearance.
PCP is similar to the non-nitrogen-containing bisphosphonates used in antiosteoporosis therapy. The therapeutic action
of such bisphosphonates involves their accumulation within
osteoclasts and intracellular incorporation into cytotoxic ATP
analogs via a back reaction with AMP-aminoacyl tRNA synthetase conjugates (48). We tested whether the observed ability
of PCP to suppress accumulation of extracellular PPi by VSMC
might involve toxic effects that result in decreased intracellular
ATP content. However, exposure of VSMC to 300 ␮M PCP
for up to 2 h (the usual duration of the test incubations for
measuring PPi accumulation rates) did not result in significant
reduction in intracellular ATP levels (Fig. 3E).
Attenuation of extracellular PPi generation by probenecid.
Given the high expression of ANK mRNA in VSMC (Fig. 1A),
we tested the possible contribution of ANK-regulated PPi
efflux by comparing PPi accumulation and ATP release in the
absence or presence of PB, MeATP, or combined PB plus
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Fig. 2. Autocrine ATP release and coupled extracellular PPi accumulation in rat aortic VSMC.
A–C: extracellular levels of PPi (A, linear scale)
and ATP (B, log scale; C, linear scale) following
transfer of cultured VSMC to basal saline assay
medium in the absence [■,control (Con)] or presence (䊐) of 300 ␮M MeATP. Data points in A–C
represent means ⫾ SE from 11–16 separate experiments (each performed in duplicate) with different VSMC preparations at passages 3-4; for
some data points, the error bars are smaller than
the symbols. D: statistical analysis at the 120-min
time points. *P ⬍ 0.05, two-way ANOVA with
the Bonferroni post hoc test.
EXTRACELLULAR ATP AND PPi ACCUMULATION IN VASCULAR SMOOTH MUSCLE
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MeATP. PB acts as an inhibitor of multiple anion transporters
and has been shown to reduce PPi efflux mediated or regulated
by ANK (17). Fig. 4, A and C, shows that extracellular PPi
accumulation was reduced twofold in the presence of 2.5 mM
PB, which contrasted with the near-complete suppression of
PPi generation by MeATP. These experiments indicated that a
PB-sensitive mechanism accounts for ⬃50% of the PPi that
accumulates in extracellular medium acutely conditioned by
VSMC. PB alone had no obvious effect on the initial mechanically induced ATP release or the clearance of this released
ATP (Fig. 4B). However, exposure of VSMC to both PB and
MeATP resulted in lower (albeit not statistically significant)
accumulation of extracellular ATP (at the later times following
medium transfer) compared with cultures incubated with only
MeATP (Fig. 4, B and C). This suggested that the inhibitory
effects of PB on PPi levels might additionally involve reduction in the ATP release that is correlated with the MeATPsensitive PPi accumulation.
Accumulation of extracellular PPi and ATP by VSMC is
attenuated by reduced temperature but not brefeldin A. Basal
nucleotide release can reflect export of nucleotides contained
AJP-Cell Physiol • VOL
within the Golgi-derived secretory vesicles that continuously
bring new protein and lipid cargo to the plasma membrane
(26). Because secretion of Golgi-derived vesicles is a highly
temperature-sensitive process, we tested the effect of reduced
incubation temperature (23°C relative to the control 37°C) on
PPi accumulation (Fig. 5, A and C) and ATP release (Fig. 5, B
and C) by VSMC. These experiments showed that the sustained rate, but not the initial burst, of extracellular PPi accumulation was greatly reduced at the lower assay temperature.
This could reflect reduced rates of 1) eNPP1-catalyzed conversion of released ATP to PPi, 2) ANK-dependent PPi efflux, or
3) an ATP release process per se. The latter possibility was
supported by reduced ATP accumulation observed at 23°C in
the presence of MeATP. Reduced temperature did not decrease
the initial burst of ATP released after medium transfer, but no
additional accumulation of ATP was observed in the presence
of MeATP at 23°C. BFA inhibits the GTP/GDP exchange
cycle of ARF-family small GTPases and thereby attenuates
anterograde membrane traffic both from the endoplasmic reticulum to the Golgi and from the Golgi to the plasma membrane to suppress constitutive secretion (4, 41) in most cells,
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Fig. 3. MeATP is rapidly metabolized by eNPP1
in VSMC to generate methylene diphosphonate
(PCP) that acts as an inhibitor of autocrine PPi
accumulation. A: HPLC chromatograms of extracellular media from intact HEK-green fluorescent
protein (GFP) cells (left) or HEK-NPP1 cells
(right) pulsed with 20 ␮M exogenous etheno-␤,␥methylene ATP (ε-MeATP) and incubated for the
indicated times. B: HPLC chromatograms of extracellular media from rat VSMC pulsed with 300 ␮M
exogenous MeATP and incubated for the indicated
times. Inset: time course of MeATP (300 ␮M)
hydrolysis based on calibrated absorbance from the
HPLC chromatogram in B. C: extracellular PPi
generation by VSMC incubated in the absence (■)
or presence of 30 ␮M (䊐), 100 ␮M (F), or 300 ␮M
(E) PCP. Data points represent the average ⫾ range
of duplicates from representative experiments
(each repeated three times with similar results)
using passage 4 VSMC. D: extracellular ATP release by VSMC incubated in the absence (■) or
presence of 300 ␮M MeATP (䊐), 300 ␮M PCP (F),
or 300 ␮M MeATP ⫹ 300 ␮M PCP (E). PCP data
points indicated by solid circles overlap with the
control data points indicated by solid squares.
E: total intracellular ATP was measured in control
or PCP-treated VSMC by rapid permeabilization of
the cell monolayers with digitonin (50 ␮g/ml) to
release ATP for quantification by the luciferasebased assay. VSMC were incubated in the absence
or presence of 300 ␮M PCP for the indicated times
before digitonin permeabilization. Data points in D
and E represent means ⫾ SE from 3 separate
experiments (each performed in triplicate) with
VSMC preparations at passages 2-4 (in D, error
bars are smaller than symbols for some data
points).
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EXTRACELLULAR ATP AND PPi ACCUMULATION IN VASCULAR SMOOTH MUSCLE
including VSMC (39). Given the marked temperature sensitivity of the PPi and ATP accumulation, we tested whether the
effects of reduced temperature would be mimicked by BFA.
VSMC were preincubated with 3 ␮g/ml BFA for 2 h before the
transfer to serum-free test medium and were also incubated
with this concentration of BFA throughout the media conditioning period. The BFA-treated cells were characterized by
extracellular PPi accumulation (measured at 2 h as described in
Fig. 5C) that was 86 ⫾ 12% (n ⫽ 4 experiments) of the
normalized values measured in the absence of BFA. Likewise,
BFA treatment did not affect the release or metabolism of
extracellular ATP.
PPi accumulation in rat aortic VSMC cultures is modulated
by time postplating. The ATP release and PPi accumulation
experiments illustrated in Figs. 2–5 used rat aortic VSMC
cultures at passages 3-4, and most were performed at ⬃4 days
postplating of 4 ⫻ 105 cells per well of the six-well assay
dishes. In vitro calcification experiments routinely involve
more prolonged in vitro culture (7–14 days postplating) in the
presence of various promineralizing stimuli (e.g., elevated
phosphate, osteogenic cytokines) (22); the controls for such
experiments use VSMC cultured for similarly prolonged times
but in the absence of promineralizing factors (13). We observed increases in the rate and extent of PPi accumulation
when control VSMC from the same aortic isolate and passage
were assayed at 9 days postplating (Fig. 6, B and D) versus 3
days postplating (Fig. 6, A and D). Control experiments with
cells plated at increasing densities indicated that this effect of
time postplating could not be ascribed simply to increased cell
numbers per well (data not shown). Differences in PPi accumulation between short-term (3– 4 days postplating) versus
longer-term (7–9 days) cultures were observed at all passages
AJP-Cell Physiol • VOL
(Fig. 6D). This may reflect differences in 1) ATP release,
2) hydrolysis of ATP to PPi by eNPP1, 3) competing hydrolysis of released ATP by ecto-ATPases other than eENPP1, or
4) efflux of intracellular PPi via anion transporters. Figure 6C
shows that reduced expression or activity of eNPP1 per se is an
unlikely explanation for the low rates of PPi generation observed in shorter-term VSMC cultures because these cells
readily generated extracellular PPi via a MeATP-sensitive
mechanism when challenged with a 500 nM pulse of exogenous ATP to supplement the ATP released from endogenous
stores.
Steady-state PPi accumulation by rat aortic VSMC in tissue
culture. The fetal bovine serum used for tissue culture of
VSMC contains substantial alkaline phosphatase activity,
which efficiently hydrolyzes PPi to Pi. In the experiments
shown thus far, VSMC were transferred to serum-free assay
medium to analyze extracellular PPi and ATP accumulation
during short-term (ⱕ2 h) incubations under defined in vitro
conditions. We also tested how VSMC accumulate extracellular PPi when maintained in standard serum-supplemented
DMEM-type tissue culture medium (Fig. 7). Our stocks of
DMEM, in the absence of serum or conditioning by cells,
contained ⬃2 ␮M PPi as presumed contaminant from added
antibiotics or other cofactors. However, the addition of serum,
which contains alkaline phosphatase, rapidly reduces this to
100 nM PPi. Rat aortic VSMC maintained in serum-supplemented DMEM steadily conditioned this medium to produce
an extracellular level of 10 ␮M PPi within 4 days. The
VSMC-conditioned DMEM contained very low (⬍1 nM) extracellular ATP due to the combined ecto-ATPase activities of
both cells and the serum (data not shown). Thus, VSMC
constitutively generate extracellular PPi at a rate sufficient to
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Fig. 4. Attenuation of extracellular PPi accumulation by probenecid (PB). A and B: extracellular levels of PPi (A, linear
scale) and ATP (B, log scale) following transfer of cultured
VSMC to basal saline assay medium in the absence (■,
control) or presence of inhibitors; Œ, 300 ␮M MeATP; 䊐, 2.5
mM PB; ‚, MeATP ⫹ PB. Data points in A and B represent
means ⫾ SE from 3 separate experiments (each performed in
duplicate) with different VSMC preparations at passages 3-4;
for some data points, the error bars are smaller than the
symbols. C: statistical analysis at the 120-min time points.
*P ⬍ 0.05, two-way ANOVA with the Bonferroni post hoc
test.
EXTRACELLULAR ATP AND PPi ACCUMULATION IN VASCULAR SMOOTH MUSCLE
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counteract serum-associated pyrophosphatase activity and
thereby elevate PPi to the supramicromolar levels required for
suppression of vascular calcification.
DISCUSSION
This study provides the first direct characterization of an
autocrine pathway wherein the release of endogenous ATP
stores is tightly coupled to extracellular ATP hydrolysis and
the sustained accumulation of extracellular pyrophosphate in
cultured VSMC. Our experiments indicate that this coupled
pathway of ATP release and extracellular hydrolysis operates
in synchrony with another autocrine mechanism wherein intracellular PPi, generated as a by-product of multiple intracellular nucleotide-consuming metabolic reactions (60), is directly released into the extracellular compartment via a probenecid-sensitive transport process. This probenecid-sensitive
PPi accumulation may be mediated or regulated by ANK,
which is highly expressed in these VSMC. Other results
suggest that the expression and/or activities of the signaling
proteins that underlie these autocrine pathways of PPi accumulation may be modulated as the growth state of VSMC and
their secretion of extracellular matrix elements changes during
sustained tissue culture. Taken together, these observations
support significant roles for local release of both ATP and PPi
in the steady-state suppression of pathological calcification in
blood vessels as in the model illustrated in Fig. 8. Perturbation
of either autocrine pathway may contribute to the complex
etiology of cardiovascular calcification that is associated with
dysregulation of multiple endocrine, paracrine, and metabolic
factors (23, 56).
AJP-Cell Physiol • VOL
These data extend previous findings using cultured aortic
VSMC isolated from enpp1⫺/⫺ and ank/ank murine models,
which indicated that eNPP1- and ANK-dependent mechanisms
provided equal and additive contributions to the accumulation
of extracellular PPi (22). That study used VSMC cultured
under promineralizing conditions (elevated extracellular phosphate and reduced serum) that favor upregulation of PPidegrading TNAP, whereas the present experiments used standard synthetic phenotype VSMC that express much lower
levels of TNAP. Thus, our finding that a PB-sensitive pathway
comprised ⬃50% of the acute PPi-generating capacity in these
latter VSMC further suggests that the autocrine ANK and
eNPP1 pathways are coordinately utilized for PPi accumulation
under both basal conditions and during induction of proosteogenic gene expression. This reinforces in vivo observations
that absence of either eNPP1 or ANK predisposes mice to
spontaneous aortic calcification and markedly accelerates the
aortic mineralization induced by hyperphosphatemia (42). Although our experiments focused on VSMC, the coordinate
roles of eNPP1- and ANK-based pathways in the suppression
of mineralization have been most extensively characterized in
osteoblasts and chrondrocytes (15, 16). These latter cells share
a mesenchymal stem cell lineage with VSMC, fibroblasts, and
adipocytes (21). ANK expression in murine fibroblasts is
increased during growth stimulation in response to multiple
mitogens (14), while downregulation of eNPP1 is required for
optimal adipogenic differentiation of murine fibroblasts (34).
Whether similar dynamic regulation of coordinate ATP release, eNPP1, or ANK activity occurs as VSMC switch be-
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Fig. 5. Accumulation of extracellular PPi and ATP by VSMC is
attenuated by reduced temperature. A and B: extracellular levels
of PPi (A, linear scale) and ATP (B, log scale) following transfer
of cultured VSMC to basal saline assay medium in the absence
(■, 䊐: control) or presence (Œ, ‚) of 300 ␮M MeATP at 37°C
(filled symbols) and 23°C (open symbols). Data points in A and
B represent means ⫾ SE from 7 separate experiments (each
performed in duplicate) with different VSMC preparations at
passages 2-4; for some data points, the error bars are smaller
than the symbols. C: statistical analysis at the 120-min time
points. *P ⬍ 0.05, three-way ANOVA with the Bonferroni post
hoc test.
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EXTRACELLULAR ATP AND PPi ACCUMULATION IN VASCULAR SMOOTH MUSCLE
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Fig. 6. Extracellular PPi accumulation in VSMC cultures is
modulated by time postplating. A: extracellular levels of PPi
(top, linear scale) and ATP (bottom, log scale) following
transfer of cultured VSMC to basal saline assay medium in
the absence (■, control) and presence (䊐) of 300 ␮M MeATP.
Data points represent means ⫾ SE from the triplicates of a
representative experiment repeated three times using passage
2 VSMC at 3 days postplating (dpp). B: VSMC from the
same preparation as in A, but cultured 6 additional days
before the analysis of PPi and ATP accumulation. Data points
represent means ⫾ SE from the triplicates of a representative
experiment repeated three times. C: VSMC from the same
preparation as in A and assayed for PPi and ATP levels
following transfer of basal saline assay medium alone (■,
control; ⫺ATP) or assay medium supplemented with 500 nM
exogenous ATP (䊐) or with 500 nM ATP plus 300 ␮M
MeATP (F). Data represent means ⫾ SE from the triplicates
of a representative experiment repeated three times. D: statistical analysis of net PPi generation (⌬PPi) by VSMC as a
function of passage number and days postplating. Data points
represent means ⫾ SE from 3 separate experiments (each
performed in triplicate) with different VSMC preparations.
*P ⬍ 0.05, two-way ANOVA with the Bonferroni post hoc test.
⌬PPi ⫽ [PPi]120 min ⫺ [PPi]3 min.
tween contractile and synthetic phenotypes is an important
question for future studies.
Our use of MeATP as an experimental tool presented both
advantages and limitations. Ecto-ATPases encompass two
families of structurally and functionally distinct cell surface
proteins: 1) the ectonucleoside 5⬘-triphosphate diphosphohydrolases (NTPDases), which include CD39 (NTPDase1) and
four related members (47); and 2) the eNPPs, which include
eNPP2 and eNPP3 in addition to eNPP1 (57). Although both
NTPDases and eNPPs can use ATP as a preferred substrate,
their catalytic properties are distinct in that NTPDases serially
hydrolyze ATP to produce AMP plus two phosphates (Pi)
while eNPPs act as pyrophosphatases hydrolyzing ATP to
generate AMP plus PPi. RT-PCR analysis and direct measurement of ecto-ATPase activity confirmed that the cultured
VSMC express multiple ecto-ATPases (NTPDase1, NTPDase3,
AJP-Cell Physiol • VOL
and eNPP3) in addition to eNPP1 (Fig. 1). At present, there are no
selective inhibitors of either ecto-ATPase family (32). We previously described (and confirmed in this study) pharmacological
experiments indicating that MeATP competitively inhibits
ATP utilization by eNPPs but not NTPDases (25). Significantly, recent analysis of the crystal structure of NTPDase2
showed that MeATP/AMPPCP, in contrast to AMPPNP (5⬘adenylyl-␤,␥-imidodiphosphate), cannot fit into the ATP-binding site of that ecto-ATPase (63). This explains our findings
that even submillimolar MeATP does not affect the hydrolysis
of micromolar ATP by cells expressing only NTDPases and no
eNPPs (25) (supplemental Fig. 1). This extraordinary selectivity also underlies our observation that MeATP completely
suppresses the generation of extracellular PPi by VSMC challenged with a 10 ␮M pulse of exogenous ATP while only
modestly slowing clearance of this added ATP by the com-
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EXTRACELLULAR ATP AND PPi ACCUMULATION IN VASCULAR SMOOTH MUSCLE
bined ecto-ATPase activities of eNPP1, eNPP3, NTPDase1/
CD39, and NTPDase3. It is important to note that MeATP also
acts as a substrate for eNPP1-mediated hydrolysis in VSMC
(Fig. 3B) with its by-products being extracellular AMP and
PCP. Notably, we observed that PCP, which is a nonhydrolyzable PPi analog, potently inhibited the autocrine accumulation
of extracellular PPi by VSMC (Fig. 3C). Under our experimental conditions, PCP did not suppress ATP accumulation (Fig.
3D) nor did it induce a decrease in intracellular ATP content
(Fig. 3E). These findings indicate that PCP may suppress
accumulation of extracellular PPi in two ways: 1) by blocking
PPi efflux via ANK; and 2) by acting as a product-inhibitor of
eNPP1-mediated hydrolysis of released ATP into PPi and AMP
(Fig. 8). Although short-term PCP exposure did not induce
obvious cytotoxic or proapoptotic responses, we have observed
that more prolonged incubation (⬎6 h) in the presence of PCP
induces a significant rounding up and detachment of cultured
VSMC. This suggests that PCP may eventually induce proapoptotic effects similar to those triggered by non-nitrogencontaining bisphosphonates.
Extracellular PPi homeostasis and mineralization reactions
are further regulated by the expression and functional activity
of TNAP, a glycosylphosphatidylinositol-anchored ectoenzyme that degrades locally accumulated extracellular PPi into
Pi (40). However, we observed that the cultured rat aortic
VSMC express only low levels of TNAP mRNA (Fig. 1A) and
levamisole-sensitive alkaline phosphatase activity (supplemental Fig. 2). Thus, TNAP does not significantly limit the rate or
extent of PPi accumulation during acute medium conditioning
in this cultured VSMC model. However, TNAP is markedly
upregulated in several VSMC models during culture in the
presence of promineralizing stimuli, such as elevated phosphate and various proosteogenic cytokines (33, 43, 46, 58). A
robust levamisole-sensitive pyrophosphatase activity is also
AJP-Cell Physiol • VOL
observed in freshly isolated rat aortic rings, and this activity is
increased in aortic segments derived from uremic rats (38, 43).
Thus, the relative expression of TNAP appears to vary with the
well-known phenotype plasticity of VSMC (55): modest expression in the differentiated, contractile phenotype cells that
predominate in intact blood vessels or aortic rings, low expression in the proliferating, synthetic phenotype cells that predominate in tissue culture, and high expression in the calcifying phenotype cells that accumulate in response to osteogenic
stimuli. However, it remains possible that levamisole-insensitive phosphatase(s) also described in explanted rat aortic rings
may act as predominant pyrophosphatases in the synthetic
phenotype VSMC (43).
A major unresolved question is the mechanism(s) by which
ATP is released from VSMC to drive the eNPP1-mediated
accumulation of extracellular PPi. A significant limitation
in addressing this issue is the very efficient coupling between
release of ATP into the cell surface microenvironment and its
rapid metabolism by eNPP1 and other ecto-ATPases (28). We
have previously characterized the functional colocalization of
ATP release pathways and eNPP activity during receptormediated stimulation of ATP release from human 1321N1
astrocytes (3, 24). Significant accumulation of ATP in the bulk
extracellular compartment was observed only when eNPP
activity was inhibited by MeATP. In contrast, accumulation of
submicromolar ATP in the cell surface microenvironment was
measured in the absence of eNPP inhibition using a surfacelocalized luciferase sensor that effectively competed with
eNPP for the released ATP. In the present study, we observed
that extracellular ATP in the conditioned bulk medium compartment of VSMC was steadily maintained in the 1–2 nM
range in the absence of ecto-ATPase inhibitors but at higher
levels, ranging from 10 – 80 nM, in the presence of MeATP.
These results suggest that eNPP1 is in close proximity to ATP
release sites in VSMC. This model of continuous ATP release
and eNPP1-dependent hydrolysis is further supported by the
sustained and MeATP-sensitive increase in extracellular PPi by
VSMC.
The ability of reduced temperature to attenuate this continuous ATP release from nominally unstimulated VSMC (Fig. 6)
initially suggested that constitutive exocytosis of Golgi-derived secretory vesicles might provide the pathway for ATP
export. Recent studies by Lazarowski and colleagues have
Fig. 8. Model of extracellular ATP metabolism and PPi generation in VSMC.
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Fig. 7. Extracellular PPi accumulation by VSMC maintained under tissue
culture conditions. VSMC were cultured in serum-supplemented medium as
described in EXPERIMENTAL PROCEDURES. Media samples were assayed for PPi
levels (F) at the indicated days following transfer of the VSMC to fresh culture
medium. Data points represent means ⫾ SE of triplicates from a representative
experiment repeated 3 times using passage 2 VSMC. Day 0 represents addition
of fresh growth media to VSMC that were seeded 3 days prior. ■, PPi levels
in serum-containing tissue culture media; 䊐, PPi levels in serum-free tissue
culture media.
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EXTRACELLULAR ATP AND PPi ACCUMULATION IN VASCULAR SMOOTH MUSCLE
AJP-Cell Physiol • VOL
both the ATP/eNPP1 and the ANK pathways used by VSMC
for the autocrine regulation of extracellular PPi levels.
ACKNOWLEDGMENTS
We thank Dr. Robert Terkeltaub (University of California, San Diego,
School of Medicine, La Jolla, CA) for providing the human eNPP1 plasmid;
Dr. Steve Fisher and lab members for technical assistance in the isolation of rat
aortic VSMC; Gregg DiNuoscio for technical assistance; and Andrew E. Blum
for useful discussions.
GRANTS
This work was supported in part by National Institutes of Health (NIH)
Grants P01-HL-18708 (to G. R. Dubyak and A. M. Romani), RO1-GM-36387
(to G. R. Dubyak), and RO1-DK-69681 (to W. C. O’Neill) and by a grant from
the Genzyme Renal Innovations Program (to W. C. O’Neill). D. A. Prosdocimo was supported by NIH Grant T32-HL-07887.
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demonstrated that a BFA-sensitive pathway underlies the constitutive release of UDP-glucose from several cell types (extracellular UDP-glucose functions as an agonist for Gi-coupled
P2Y14 receptors) (26, 30). However, using similar BFA treatment protocols, we failed to observe any significant attenuation
of ATP and PPi accumulation in the conditioned medium of
VSMC. In addition to exocytosis, cells can release ATP by
mechanisms that involve facilitated efflux of the cytosolic ATP
pool via channels or transporters (see review in Ref. 29). Given
the very favorable electrochemical driving force for ATP
efflux, even low-frequency opening of such channels can
deliver significant amounts of ATP to extracellular compartments.
Multiple transport proteins or conductances have been suggested as “ATP channels,” including some ATP-binding cassette-family transporters, volume-regulated anion channels,
maxi-anion channels, and plasma membrane variants of the
mitochondrial voltage-dependent anion channel porins (29).
However, there is growing support for the involvement of
so-called hemichannels, composed of protein subunits from the
well-characterized connexin (Cx) family or the recently described pannexin (Px) family (1, 9, 19, 20, 35, 36, 54).
Although Cx-based channels are generally associated with the
transcellular movement of molecules through gap junction
channels, Cx (and Px) hemichannels at nonjunctional membrane sites can also be gated to the open state to act as possible
conduits for ATP and other small organic metabolites. VSMC
express multiple connexins, including Cx43. In preliminary
studies, we have observed that extracellular accumulation of
ATP and PPi is significantly attenuated in VSMC treated with
100 ␮M carbenoxolone, a nonselective inhibitor of connexin
and pannexin channels. This suggests that low-frequency opening of such hemichannels may comprise a major ATP release
pathway in these cells. Previous studies have indicated that the
gating of connexin gap junction channels is sensitive to temperature (6, 7) and that this might underlie the attenuated
accumulation of ATP and PPi observed at 23°C versus 37°C.
Alternatively, the lower temperature may act via indirect effects on the homeostasis of ions (e.g., H⫹ or Ca2⫹) that
allosterically modulate hemichannel gating.
Many recent studies have shown increased activity of Cx- or
Px-based hemichannels in response to various types of mechanical stimulation, such as fluid shear, cell swelling, or direct
non-lytic deformation of the plasma membrane (2, 12, 49, 53,
62). In this regard, unavoidable mechanical stimulation accompanies routine manipulations used in our experimental assays
including 1) the initial transfer of VSMC from growth medium
to assay medium and 2) periodic removal of extracellular
medium samples for analysis of ATP and PPi content. These
seemingly innocuous manipulations may generate mechanical
stimuli sufficient to gate nonjunctional hemichannels or other
mechanosensitive channels with appreciable permeability to
ATP. The possible role of mechanical stimulation as a regulator of the ATP/eNPP1-mediated pathway for PPi accumulation
is mechanistically appealing given the basal physiological state
of VSMC within the aorta and other calcification-prone elastic
arteries. These cells undergo constant cyclic mechanical perturbation due to the repetitive systolic stretch and diastolic
recoil entrained by the cardiac cycle. Thus, future experiments
should test the effects of graded cyclic mechanical strain on
EXTRACELLULAR ATP AND PPi ACCUMULATION IN VASCULAR SMOOTH MUSCLE
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