Identification and Characterization of Adenosine
5'-Tetraphosphate in Human Myocardial Tissue*
Timm
Westhoff,
Joachim
Jankowski,
Sven
Schmidt,
Jiankai
Luo,
Günter
Giebing,
Hartmut
Schlüter,
Martin
Tepel,
Walter
Zidek, and
Markus
van der Giet
From the Freie Universität Berlin, Universitätsklinikum
Benjamin Franklin, Medizinische Klinik IV, Hindenburgdamm 30, 12200 Berlin, Germany
Received for publication, January 10, 2003, and in revised form, February 4, 2003
 |
ABSTRACT |
Endocrine functions of the human heart have been
studied extensively. Only recently, nucleotidergic mechanisms have been
studied in detail. Therefore, an isolation strategy was developed to
isolate novel nucleotide compounds from human myocardium. The human
myocardial tissue was fractionated by several chromatographic
studies. A substance purified to homogeneity was identified as
adenosine 5'-tetraphosphate (Ap4) by matrix-assisted
laser desorption/ionization mass spectrometry (MALDI MS), post-source
decay MALDI MS, and enzymatic cleavage analysis. Furthermore,
Ap4 was also identified in ventricular specific granules.
In the isolated perfused rat heart, Ap4 elicited
dose-dependent vasodilations. Vasodilator responses were
abolished in the presence of the P2Y1 receptor antagonist
MRS 2179 (1 µM) or the NO synthase inhibitor
NG-nitro-L-arginine methyl ester
(50 µM). After removal of the endothelium by Triton
X-100, Ap4 induced dose-dependent
vasoconstrictions. Inhibition of P2X receptors by
pyridoxal phosphate-6-azophenyl-2',4'-disulfonic acid (30 µM) or desensitization of P2X receptors by
,
-methylene ATP (
,
-meATP, 1 µM) diminished
these vasoconstrictor responses completely. In the present study
Ap4 has been isolated from human tissue. Ap4
was shown to exist in human myocardial tissue and was identified in
ventricular specific granules. In coronary vasculature the
nucleotide exerted vasodilation via endothelial P2Y1
receptors and vasoconstriction via P2X receptors on
vascular smooth muscle cells. Ap4 acts as an endogenous
extracellular mediator and might contribute to the regulation of
coronary perfusion.
 |
INTRODUCTION |
The endocrine functions of myocardial tissue have been
investigated widely in the past. Well known myocardium-derived hormonal factors include the atrial natriuretic peptide (1) and angiotensin II (2). Except for the long known nucleotide, ATP, new
myocardium-derived nucleotides such as diadenosine polyphosphates have
only recently been isolated (3).
As in myocardial tissue a great variety of nucleotidergic receptor
subtypes (P1 and P2 receptors) are expressed (4, 5), the secretion of
nucleotides from myocardial cells represents one of the autocrine
functions of the heart (6, 7). In many studies it has been demonstrated
that nucleotides exert numerous effects on cardiac functions (4, 5).
One of the important mechanisms of nucleotidergic signaling in the
heart is the regulation of coronary perfusion. A2A and
P2X1 receptors, as well as P2Y1 receptors,
contribute to the modulation of coronary vascular tone (6-11).
The present study was aimed at extending our knowledge of autocrine
loops in myocardial tissue. To this purpose, an isolation procedure was
constructed that was suitable to detect further novel nucleotides
secreted by human myocardium. The experiments revealed adenosine
5'-tetraphosphate
(Ap4)1 in human
myocardial tissue. Moreover, secretory granules contained this
nucleotide, and studies in an animal model of coronary circulation revealed potent effects of Ap4 on coronary vascular tone.
 |
EXPERIMENTAL PROCEDURES |
Materials--
All reagents were purchased from Sigma
unless otherwise specified. Human myocardial tissue was obtained from
two graft recipients after heart transplantation. The study was
approved by the local ethical committee, and written consent was
obtained from the transplant patients. In the hearts used for isolation
of nucleotides, coronary heart disease was the primary disorder leading
to transplantation. For isolation of adenosine 5'-tetraphosphate only
macroscopically intact tissue from the left ventricle was used.
Purification of Adenosine 5'-Tetraphosphate from Human Myocardial
Tissue--
After excision from the heart transplant recipient, human
myocardial tissue (10 g) was immediately placed in ice-cooled
physiological saline solution and processed within 30 min. The
following isolation procedure was designed to isolate adenosine
4'-tetraphosphate exclusively from myocardial tissue. The coronary
tissue was cut into small pieces (about 1 cm3), frozen in
liquid nitrogen, and stored at
80 °C for 1 night. Then the tissue
was lyophilized and powdered (step 1). The powder was suspended in 200 ml of 0.6 M ice-cold perchloric acid and homogenized at
25,000 rpm three times for 1 min. The homogenate was ultracentrifuged
at 30,000 rpm for 60 min at 4 °C. The supernatant was adjusted with
KOH to pH 8.5 and stored at 4 °C for 30 min to precipitate
KClO4. After centrifugation at 4,000 rpm for 10 min at
4 °C, the supernatant was titrated to pH 6.5 with HCl and centrifuged again as above (step 2).
A reversed-phase column (Lichroprep RP-18, 310 × 25 mm, Merck)
was used to concentrate the nucleotide (step 3). The column was
equilibrated with 40 mM triethylammonium acetate (TEAA) in water. The supernatant with 40 mM TEAA added (final
concentration) was pumped to the column, washed with 40 mM
TEAA, and eluted with 40% acetonitrile in water at a flow rate of 1 ml/min. The 40% acetonitrile eluate was collected, frozen at
80 °C, and lyophilized.
Size exclusion chromatography was performed according to
Hillenkamp and Karas (12) (step 4). Size exclusion gel Sephacryl S-100
high resolution (1000 × 16 mm, S100 HR, Amersham Biosciences) was
equilibrated with water. The dried sample from the preparative reversed-phase column resolved in 5 ml of water was loaded onto the
column. The eluent (water) was pumped at a flow rate of 1 ml/min. The effluent was monitored with a UV detector at 254 nm.
An anion-exchange column (Fractogel EMD DEAE-650, 300 × 25 mm,
Merck) was equilibrated with 10 mM ammonium acetate (pH
7.4) (step 5). After the addition of 10 mM (final
concentration) ammonium acetate, pH 7.4, the fraction from the size
exclusion chromatography was pumped through the column. Nucleotides
were eluted by 1 M ammonium acetate (pH 7.4) at a flow rate
of 3.0 ml/min. The effluent was detected with a UV detector at 254 nm.
The eluate from the anion-exchange column (step 6) was purified further
with affinity chromatography. The affinity chromatography gel, phenyl
boronic acid coupled to a cation-exchange resin (BioRex 70, Bio-Rad),
was synthesized according to Barnes et al. (13). The
affinity resin was packed into a glass column (150 × 20 mm) and
equilibrated with 1 M ammonium acetate (pH 9.5). The pH of the eluate from the anion-exchange column was adjusted to 9.5 and
loaded to the affinity column. The column was washed with 1 M ammonium acetate (pH 9.5) with a flow rate of 1 ml/min.
Binding substances were eluted with 1 mM HCl. The eluate
was frozen and lyophilized. Fractions were monitored with a UV detector
at 254 nm.
The fractions from the affinity chromatography were desalted by
reversed-phase high-performance liquid chromatography (HPLC) (Superspher 100 RP C18 end-capped, 250 × 4 mm, Merck) (step 7). The fractions dissolved in eluent A (40 mM TEAA) were
injected to the HPLC. After a washing period of 10 min with eluent A,
the nucleotide-containing fraction was eluted with 30% acetonitrile in
water. The UV-absorbing fraction was collected.
The desalted fraction from the affinity chromatography was fractionated
by an anion-exchange chromatography (step 8). The anion-exchange column
(50 × 5 mm, Mono-Q HR 5/5, Amersham Biosciences) was equilibrated
with eluent A (10 mM K2HPO4, pH
8.0). The sample dissolved in eluent A was injected into the column at
a flow rate of the mobile phase of 0.5 ml/min. Binding substances were
eluted using a linear gradient with increasing concentration of eluent B (50 mM K2HPO4 + 1 M
NaCl, pH 8.0). The time course of the gradient was: 0-10 min, 0-5%
B; 10-100 min, 5-35% B; 100-105 min, 35-40% B; 105-110 min,
40-100% B. The wavelength of the UV detector was fixed to 254 nm.
Fractions were collected every 2 min.
The fractions from anion-exchange chromatography were further separated
by reversed-phase HPLC (Superspher 100 RP C18 end-capped, 250 × 4 mm, Merck) (step 9). The fractions dissolved in eluent A (40 mM TEAA) were injected into the HPLC. The following
gradient was programmed: 0-4 min, 0-2% B; 4-79 min, 2-7% B;
79-85 min, 7-60% B. The flow rate was 0.5 ml/min. The
wavelength of the UV detector was 254 nm. 1-ml fractions were collected.
Purification of Adenosine 5'-tetraphosphate from Porcine
Myocardium-specific Granules--
Specific granules were obtained from
porcine left ventricular tissue according to De Bold and Bencosme (14).
Briefly, immediately after sacrifice the heart was removed, washed in
ice-cold 0.25 M sucrose (containing 0.2% glycogen, 1 mM EDTA, pH 7), and placed in a container with ice-cold
0.25 M sucrose. After large vessels and fat were removed,
the tissue was washed for a second time in ice-cold 0.25 M
sucrose. After 10 g of myocardial tissue was collected, it was
homogenized at 4 °C. The resulting pulp was washed into a glass
homogenizer with 10 volumes of 0.25 M sucrose. The
suspension was homogenized by 20 strokes of a Teflon pestle. The
resulting homogenate was filtered using cheesecloth. This filtrate was
centrifuged at 1,900 × g for 10 min. The supernatant was filtered again using cheesecloth at 1,900 × g for
10 min and centrifuged at 32,000 × g for 10 min. The
pellet containing specific granules was resuspended in 0.25 M sucrose and centrifuged again at 32,000 × g for 10 min. The pellet was resuspended by the addition of
4 ml of 1.6 M sucrose (containing 0.2% glycogen, 1 mM EDTA, pH 7). The remaining pellet was further
resuspended by homogenizing it in a glass homogenizer by a
Teflon pestle. The suspension was pipetted on top of 0.5 ml of 2 M sucrose (containing 0.2% glycogen, 1 mM
EDTA, pH 7) and topped with 1 ml of 0.25 M sucrose. The
resulting gradient was centrifuged immediately at 154,000 × g for 60 min. After centrifugation, 5 fractions were
obtained. The protein concentration was measured according to Bradford
(15). The fraction with less than 10% of the total protein amount
contained the specific granules. This fraction was collected, diluted
to a 10-fold volume with water, and centrifuged at 32,000 × g for 10 min.
MALDI MS and PSD-MALDI MS--
The molecular masses of the
molecules in the fractions from reversed-phase HPLC (step 9) were
determined by matrix-assisted laser desorption/ionization mass
spectrometry (MALDI MS). A reflectron-type time-of-flight (RETOF) mass
spectrometer, equipped with a nitrogen laser (337 nm; pulse length, 3 ns) was used for ion generation and mass analysis (12). In MALDI MS
large fractions of the desorbed analyte ions undergo post-source decay
(PSD) during flight in the field-free drift path. Using a RETOF
set-up, sequence information from PSD fragment ions of precursors
produced by MALDI were obtained (16). For MALDI MS and PSD MALDI MS,
speed vacuum-dried samples were dissolved in 10 µl of bidistilled
water. 1.0 µl of the 3-hydroxyl-picolinic acid matrix solution (50 g/liter) in water was mixed with 0.5 µl of the sample on a flat
metallic support and dried in a stream of cold air. Desorption of
analyte ions was performed by laser shots of irradiances in the range
of 106 to 107 watts/cm2
focused to spot sizes of typically 50 to 100 µm in diameter. The ions
generated were accelerated with an energy of 12 KeV for detection. The
spectra were recorded using a LeCroy 9400 recorder (17).
UV Spectroscopy--
The substances in the fractions of the
reversed-phase HPLC (step 9), dissolved in 100 µl (pH 6.5), were
analyzed by a UV spectrometer (UV-visible spectrophotometer, DU-600,
Beckman). UV absorption was scanned from 400 to 190 nm with a scan
speed of 400 nm/min.
Enzymatic Cleavage Experiments--
Aliquots of the fractions
from the reversed-phase column (step 9) were incubated with enzymes as
follows. The samples were dissolved in: (a) 20 µl of 200 mM Tris buffer (pH 8.9) and incubated with 3 milliunits of 5'-nucleotide hydrolase from Crotalus durissus (EC 3.1.15.1, Roche Applied Science), purified according to Sulkowski
and Laskowski (18), for 9 min at 37 °C; (b) 20 µl of
200 mM Tris and 20 mM EDTA buffer (pH 7.4) and
incubated with 3'-nucleotide hydrolase (1 milliunit; from calf spleen,
EC 3.1.16.1, from Roche Applied Science) for 1 h at 37 °C; and
(c) 20 µl of 10 mM Tris, 1 mM
ZnCl2, and 1 mM MgCl2 buffer (pH 8)
and incubated with alkaline phosphatase (1 milliunit; from calf
intestinal mucosa, EC 3.1.3.1, from Roche Molecular
Biochemicals) 1 h at 37 °C. The reaction was terminated by an
ultrafiltration with a centrifuge filter (exclusion limit 10 kDa,
Millipore). After filtration of the enzymatic cleavage products, the
filtrate, dissolved in 980 µl eluent A (10 mM
K2HPO4, pH 7), was subjected to an
anion-exchange chromatography (MiniQ PC 3.2/3, Amersham
Biosciences). The gradient was: 0-3 min, 0% B (50 mM
K2HPO4, pH 7 with 1 M NaCl); 3-20
min, 0-50% B; 20-21 min, 50-100% B. The flow rate was 100 µl/min.
Isolated Perfused Heart--
Isolated perfused rat hearts were
taken from male Wistar-Kyoto rats, aged 3 months, weighing 250-300 g,
and were prepared according to van der Giet et al. (11).
Responses of preparations to Ap4,
,
-methyleneadenosine 5'-triphosphate (
,
-meATP),
2-methylthioadenosine 5'-triphosphate (2-meSATP), and 2-chloroadenosine
5'-triphosphate (2-ClATP) were assessed at basal tone. Dose-response
curves were constructed for each substance, with 5 min allowed to
elapse between consecutive doses. This procedure allowed for
dose-response curves for several agonists to be constructed for the
same preparation. Desensitization was not detected when 5 min
was allowed to elapse between consecutive doses. Single doses of sodium
nitroprusside (10 nmol) and acetylcholine (10 nmol) were used as
controls for endothelium-independent or endothelium-dependent vasodilations mediated via NO.
Further experiments were done after endothelium was removed by Triton
X-100, to investigate the endothelium-independent effects of all of the
agonists used. The endothelium was removed by perfusion of the isolated
heart for 5 s with 0.1% Triton X-100. Successful removal was
verified by the acetylcholine response. Unaffected K+-induced contractions (130 mM bolus)
indicated intact vascular smooth muscle cell function. In some
experiments with removed endothelia, P2X receptors were
desensitized by permanent perfusion with
,
-meATP (1 µM) to block P2X-mediated vasoconstriction.
The specific P2Y1 receptor antagonist,
2'-deoxy-N6-methyl-adenosine
3',5'-diphosphate diammonium (MRS2179, 1 µM), the
P2X receptor antagonist pyridoxal
phosphate-6-azophenyl-2',4'-disulfonic acid (PPADS, 10 µM), and the nitric-oxide synthase inhibitor
NG-nitro-L-arginine methyl ester
(L-NAME, 10 µM) were added to the perfusate
30 min before challenge with mono- or dinucleotides. MRS2179 and PPADS
did not significantly influence vascular tone, whereas
L-NAME significantly (p < 0.05) increased
perfusion pressure. Permanent perfusion with
,
-meATP showed a
rapid and completely desensitizing vasoconstriction.
Each of the monoadenosinephosphates, sodium nitroprusside, and
acetylcholine was applied as a 100-µl bolus into a valve
proximal to the preparation. Drug dilutions were performed daily from
stock solutions of 10 mM (concentrates were stored frozen
in bidistilled water) with Krebs-Henseleit buffer unless indicated
otherwise. Heparin (sodium salt), PPADS, MRS2179,
,
-meATP,
2-meSATP, and 2ClATP were from Research Biochemicals Inc. (Deisenhofen, Germany).
Responses were measured as changes in perfusion pressure (mm Hg), and
results were presented as the means ± S.E. Two-way
repeated measures of variance followed by Bonferroni's multiple
comparison test (for changes in coronary perfusion pressure) were used
to identify where statistically significant differences had occurred. The effects of L-NAME, MRS2179, PPADS, and
,
-meATP on
coronary perfusion pressure were compared with drug-free control
conditions using the Mann-Whitney test. All p values
presented are two-tailed, and p values < 0.05 were
considered significant.
 |
RESULTS |
Human ventricular myocardial tissue from transplant recipients was
lyophilized and powdered (step 1). Homogenized powder was deproteinated
by perchloric acid (step 2). Nucleotides were concentrated from the
supernatant by reversed-phase chromatography (step 3). The concentrate
was fractionated with size exclusion chromatography (step 4). The
resulting chromatogram is presented in Fig.
1A. Anionic substances with a
molecular mass between 300 and 2000 Da (indicated by a solid
line in Fig. 1A) were concentrated by an anion-exchange
HPLC (step 5). The resulting chromatogram is shown in Fig.
1B. The fraction indicated by a solid line in
Fig. 1B was further fractionated by reversed-phase HPLC
(steps 6 and 7) and by anion-exchange chromatography (step 8). The
corresponding chromatogram is shown in Fig. 1C. The
arrow in Fig. 1C indicates the fraction that was
further screened for nucleotides. This fraction was passed through an
affinity column to concentrate mononucleoside polyphosphates. The
resulting eluate was desalted by a reversed-phase column and
fractionated for a final time by anion-exchange
chromatography.

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Fig. 1.
Purification of AP4
from ventricular heart tissue. A, size exclusion
chromatography (purification step 4) of human heart tissue extract:
column, S100 HF (1000 × 16 mM, Amersham Biosciences);
eluent, water; flow rate, 1 ml/min; detector, UV photometer, 254 nm,
0.5 AUFS. Black bar, fraction collected for further
purification. B, anion-exchange HPLC (purification
step 8) of the eluent from affinity chromatography: column, Mono-Q HR
5/5 (50 × 5 mM, Amersham Biosciences).
Eluent A, 10 mM K2HPO4, pH 8.0;
eluent B, 50 mM K2HPO4 + 1 M NaCl, pH 8.0. Gradient, 0-10 min 0-5% B, 10-100 min
5-35% B, 100-105 min 35-40%, 105-110 min 40-100% B. Flow rate,
0.5 ml/min. Detector, UV photometer, 254 nm, 0.5 AUFS. The fraction,
which was further purified by reversed-phase HPLC, is indicated by a
solid line (at 10 min retention time). The fraction
in panel A containing anionic low molecular mass substances
is indicated by a solid line. This fraction was
chromatographed by anion-exchange chromatography. The fraction, which
was further purified by reversed-phase HPLC, is indicated by a
solid line (at 70 min retention time). C,
reversed-phase HPLC (purification step 9) of the desalted fraction 1 from anion-exchange chromatography: column, Superspher 100 RP C18
end-capped (250 × 4 mM, Merck); eluent A, 40 mM TEAA in water; eluent B, 100% CAN; gradient, 0-4 min
0-2% B, 4-79 min 2-7% B, 79-85 min 7-60%; flow rate, 0.5 ml/min; detector, UV photometer at 154 nm and 0.5 AUFS.
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The fraction indicated by an arrow in Fig. 1C was
analyzed by MALDI MS. The UV-absorbing fraction revealed a molecular
mass of 588.2 Da [M+H]+. The UV spectrum of this fraction
at pH 7.0 showed maximum and minimum absorbances at 259 nm and 230 nm,
respectively, indicating the presence of an adenine moiety. The
fraction labeled in Fig. 1C was analyzed by PSD-MALDI MS to
obtain fragment ion masses (Fig. 2). The
fragment ions showed masses identical to those of phosphate, adenosine,
AMP, and ADP. The PSD-MALDI MS fragment spectrum of authentic
AP4 showed an identical fragment ion pattern (Table
I). The measured fragment ions of the
fraction labeled in Fig. 1C and of authentic AP4
are listed in Table I together with an interpretation of the
fragments. The above measurements therefore revealed a molecule
containing adenosine together with four phosphate moieties. In a
further series of experiments, the connection of the adenosine to the
phosphate moieties was studied. To this end, enzymatic cleavage
experiments were done. 3'-Exonuclease had no effect on the purified
nucleotide, whereas alkaline phosphatase yielded AMP, ADP, and ATP.
After incubation of the fraction with 5'-exonuclease, AMP was detected
(Fig. 3). The results suggested that in
the molecule a chain of four phosphate groups is bound to adenosine via
a 5'-phosphodiester bond.

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Fig. 2.
Identification of Ap4
by PSD-MALDI mass spectrometry. PSD-MALDI mass spectrum of
the fraction labeled Ap4 from reversed-phase HPLC (Fig.
1C).
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Fig. 3.
MALDI mass spectrum of the reaction products
after incubation of the fraction, labeled by an arrow
in Fig. 1C, with 3'-exonuclease
(A), alkaline phosphatase (B),
5'-exonuclease (C), respectively.
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To determine the localization of Ap4 in ventricular
myocardium, specific granules were examined for the presence of
Ap4. As shown in Fig. 4,
Ap4 was found in ventricular specific granules. The amount
of Ap4 was estimated to be 4.3 µmol/g wet tissue
according to the UV absorption signal in the chromatogram.

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Fig. 4.
Identification and quantification of
Ap4 in myocardial granules. A,
anion-exchange HPLC chromatogram of an extract from myocardial
granules: column, Mono-Q HR 5/2 (50 × 2 mM, Amersham
Biosciences); eluent A, 10 mM
K2HPO4, pH 8.0; eluent B, 50 mM
K2HPO4 + 1 M NaCl, pH 8.0. Gradient: 0-2 min, 0-5% B; 2-22 min, 5-40% B; 22-22.5 min,
40-100% B; 22.5-23.5 min, 100% B. Flow rate, 0.1 ml/min; detector,
UV photometer, 254 nm, 0.5 AUFS. B, electropherogram of an
extract from myocardial granules: detector, UV photometer, 254 nm.
Samples of injection: hydrodynamic pressure (1 s at 55 p.s.i.); capillary, untreated fused silica (50 µM I.D.,
37-cm length, 30 cm to the detector). 20 kV (65-70 µA, 20 °C;
inlet, cathode; outlet, anode). Buffer: 50 mM citric acid
(pH 4.75).
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In isolated perfused rat heart experiments, the mean coronary flow rate
as measured in a total of 51 experiments amounted to 8.3 ± 1.8 ml/min to achieve a constant coronary perfusion pressure of 60 ± 1 mm Hg. MRS2179 and PPADS did not significantly change vascular tone,
whereas L-NAME significantly (p < 0.05)
increased perfusion pressure. Permanent perfusion with
,
-meATP
elicited a rapidly desensitizing vasoconstriction.
Fig. 5 demonstrates the action of
2-meSATP, 2-ClATP,
,
-meATP, and Ap4 on coronary
vasculature. At base-line perfusion pressure the nucleotides, except
for
,
-meATP, caused dose-dependent vasodilation. The
dose-response curves were not parallel, and the maximal contractions induced varied considerably, which makes calculation of potency ratios
difficult; but based on an estimation of concentrations equi-effective
to 1 nmol of 2-ClATP, the rank order of potency was
Ap4
2-ClATP
2-meSATP.

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Fig. 5.
Changes in perfusion pressure in the isolated
perfused rat heart induced by 2-meSATP ( ), 2-ClATP ( ),
, -meATP ( ), and
Ap4 ( ). Each point is the mean of at
least eight determinations; error bars show S.E. Where error
bars do not occur, they are within the symbol size. Significant
differences from base-line perfusion pressure: 2-ClATP 10 11 mol, 2-meSATP and Ap4 10 10.5 mol, , -meATP 10 9.5
mol. ED50 (in-log mol) was 10.9 ± 0.4 for 2-ClATP,
10.1 ± 0.2 for 2-meSATP, and 9.7 ± 0.2 for Ap4
(each, n = 7).
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After removal of the endothelium by Triton X-100, vasodilator responses
to a 10-nmol bolus of Ap4, 2-ClATP, and 2-meSATP were abolished, and vasoconstrictor responses increased, as shown in Fig. 6B. Vasodilations induced
by sodium nitroprusside did not change after removal of the
endothelium. With intact endothelium, both inhibition of
P2Y1 receptors by MRS2179 (1 µM) and
inhibition of NO generation by L-NAME (50 µM)
abolished vasodilator responses to Ap4, 2-ClATP, and
2-meSATP (Fig. 6A); vasodilations induced by sodium
nitroprusside and vasoconstrictions induced by
,
-meATP remained
unaffected (Fig. 6A). In the absence of intact endothelium, vasoconstrictor responses to Ap4, 2-ClATP, 2-meSATP, and
,
-meATP disappeared after the addition of either the
P2X receptor antagonist PPADS (30 µM, Fig.
6B) or the P2X receptor agonist
,
-meATP (1 µM, Fig. 6B). In this experimental setting,
Ap4, 2-ClATP and 2-meSATP did not affect basal tone
significantly.

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Fig. 6.
Changes in perfusion pressure in the isolated
perfused rat heart induced by bolus injection of 1 nmol of each agonist
before (A) and after endothelium removal
(B). A, open bars, in the
absence of any antagonist (control); filled bars,
with MRS2179 (1 µM); hatched bars, with
L-NAME (50 µM). B, open
bars, in the absence of any antagonist (control);
filled bars, with , -methylene ATP (1 µM); hatched bars, with PPADS (10 µM) in the perfusate. Each column is the mean of at least
nine determinations; error bars show S.E. *,
p < 0.05, antagonist versus control.
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 |
DISCUSSION |
In the present study the presence of Ap4 in human
myocardial tissue has been demonstrated. Furthermore, Ap4
has also been identified in ventricular specific granules.
Ap4 exerts P2Y1 receptor-mediated vasodilation
and P2X receptor-mediated vasoconstriction on coronary vasculature.
Although to the best of our knowledge Ap4 has not yet been
demonstrated in human metabolism, its presence in mammalian tissues has
been documented in a number of thorough experiments, most of which were
performed several decades ago (19-23).
Like most nucleotides Ap4 may have either intracellular or
extracellular functions. On the one hand, Ap4 may be
involved in intracellular metabolic pathways, serving either for
cellular energy supply or for intracellular signal transduction. On the other hand, Ap4 may indeed serve as an extracellular
messenger, as suggested by its vascular effects. An extracellular
action cannot be inferred from the mere existence of Ap4 in
myocardial tissue. However, the isolation of this compound from
myocardial secretory granules strongly suggests that Ap4 is
produced in cardiomyocytes to act as an extracellular messenger.
The characterization of the vasoactive properties of
Ap4 revealed several interesting findings. 1) Although
the structure of Ap4 is very similar to that of ATP,
Ap4 has a longer biological half-life than ATP. ATP is
degraded to more than 95% during a single coronary passage. The levels
of ATP in the coronary circulation increase during conditions of
hypoxia or ischemia, suggesting a possible role of nucleotides as
extracellular mediators of pathological processes in atherosclerotic
vessels. The ATP content in human myocardial tissue is about 40 µmol/g wet tissue (24). Compared with ATP, the concentration of
Ap4 is about 10 times lower, but Ap4 has a
longer half-life in the circulation, as it is more resistant to
extracellular nucleotidases than ATP (25). Therefore the findings
suggest that Ap4 may exert a least equivalent actions to
those of ATP from a quantitative point of view. 2) The findings reveal
that the activity of Ap4 is profoundly dependent on
endothelial integrity. Ap4 is a potent vasodilator in
intact coronary vasculature. In the absence of endothelial
P2Y1 receptors, Ap4 is a vasoconstrictor. This
endothelium-dependent action may be relevant with respect to coronary pathology. It may be speculated that in severely
atherosclerotic coronary vessels, Ap4 may be a mediator of
vasospastic processes. 3) The actions of Ap4 are mediated
by several of the known purinoceptor subtypes.
Ap4-induced vasoconstriction depends on the activation of
an
,
-meATP-sensitive, completely desensitizable P2X
receptor subtype. Only mRNAs for P2X1,
P2X2, and P2X4 have been detected in coronary
vasculature thus far (26). Additionally the P2X5 receptor
was cloned in rat heart (27). In accordance with pertinent literature,
the P2X2 receptor and the P2X5 receptor are not
activated by
,
-meATP (27, 28). The P2X4 receptor
cannot be inhibited by PPADS (29). The P2X1 receptor is
inhibitable by PPADS, sensitive to
,
-meATP, and completely
desensitizable (28). Therefore it can be speculated that the
P2X1 receptor subtype underlies Ap4-induced
vasoconstriction. P2X receptor subtypes are known to form
hetero-oligomeric channels with new pharmacological properties. Among
P2X1, P2X2, P2X4, and
P2X5, only P2X1 and P2X5 are
reportedly able to assemble (30). It cannot finally be excluded that a hetero-oligmeric form of P2X1 and P2X5, in
addition to the P2X1 receptor, contributes to
Ap4-induced vasoconstriction. On the other hand,
Ap4-induced vasodilation is mediated by the
P2Y1 receptor subtype, as evidenced by specific inhibition
of P2Y1 receptor with MRS2179. From the literature, it is
known that P2Y1 receptors is expressed abundantly on
endothelial cell surfaces and therefore is responsible for the
vasodilator action of Ap4 (31).
Beyond the isolation and characterization of Ap4,
our experiments suggest that endogenous myocardium-derived
nucleotides may be an important part of the spectrum of myocardial
endocrine functions. Using the isolation procedure established in the
present study, it will be possible to screen extracts from myocardial
tissue for further functionally relevant, novel nucleotides. Because several other vasoactive nucleotides have been described recently in
human myocardial tissue (3), it may be speculated that the myocardium-derived nucleotide hormones comprise a larger and more complex family of extracellular messengers than known in the last few decades.
 |
FOOTNOTES |
*
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed: Medizinische Klinik
IV, Universitätsklinikum Benjamin-Franklin-Freie
Universität Berlin, Hindenburgdamm 30, 12200 Berlin, Germany.
Tel.: 49-30-8445-3021; Fax: +49-30-8445-4235; E-mail:
vdgiet@zedat.fu-berlin.de.
Published, JBC Papers in Press, February 27, 2003, DOI 10.1074/jbc.M300288200
 |
ABBREVIATIONS |
The abbreviations used are:
Ap4, adenosine 5'-tetraphosphate;
TEAA, triethylammonium acetate;
HPLC, high-performance liquid chromatography;
MALDI, matrix-assisted laser desorption/ionization;
MS, mass
spectrometry;
PSD, post-source decay;
,
-meATP,
,
-methylene ATP;
2-meSATP, 2-methylthioadenosine
5'-triphosphate;
2-ClATP, 2-chloroadenosine 5'-triphosphate;
MRS2179, 2'-deoxy-N6-methyl-adenosine
3',5'-diphosphate diammonium;
PPADS, pyridoxal
phosphate-6-azophenyl-2',4'-disulfonic acid;
L-NAME, NG-nitro-L-arginine methyl
ester.
 |
REFERENCES |
1.
|
Currie, M. G.,
Geller, D. M.,
Cole, B. R.,
Siegel, N. R.,
Fok, K. F.,
Adams, S. P.,
Eubanks, S. R.,
Galluppi, G. R.,
and Needleman, P.
(1984)
Science
223,
67-69[Medline]
[Order article via Infotrieve]
|
2.
|
Zisman, L. S.,
Abraham, W. T.,
Meixell, G. E.,
Vamvakias, B. N.,
Quaife, R. A.,
Lowes, B. D.,
Roden, R. L.,
Peacock, S. J.,
Groves, B. M.,
and Raynolds, M. V.
(1995)
J. Clin. Invest.
96,
1490-1498[Medline]
[Order article via Infotrieve]
|
3.
|
Luo, J.,
Jankowski, J.,
Knobloch, M.,
Van der Giet, M.,
Gardanis, K.,
Russ, T.,
Vahlensieck, U.,
Neumann, J.,
Schmitz, W.,
Tepel, M.,
Deng, M. C.,
Zidek, W.,
and Schluter, H.
(1999)
FASEB J.
13,
695-705[Abstract/Free Full Text]
|
4.
|
Broad, R.,
and Linden, J.
(2001)
in
Purinergic and Pyrimidinergic Signalling II: Cardiovascular, Respiratory, Immune, Metabolic and Gastrointestinal Tract Function
(Abbracchio, M.
, and Williams, M., eds), Vol. 151/II
, pp. 3-23, Springer, Berlin
|
5.
|
Pelleg, A.,
and Vassort, G.
(2001)
in
Purinergic and Pyrimidinergic Signalling II
(Abbracchio, M.
, and Williams, M., eds), Vol. 151/II
, pp. 73-100, Springer, Berlin
|
6.
|
Vassort, G.
(2001)
Physiol. Rev.
81,
767-806[Abstract/Free Full Text]
|
7.
|
Flores, N. A.,
Stavrou, B. M.,
and Sheridan, D. J.
(1999)
Cardiovasc. Res.
42,
15-26[CrossRef][Medline]
[Order article via Infotrieve]
|
8.
|
Ralevic, V.,
and Burnstock, G.
(1998)
Pharmacol. Rev.
50,
413-492[Abstract/Free Full Text]
|
9.
|
Phillis, J. W.,
Song, D.,
and O'Regan, M. H.
(1998)
Eur. J. Pharmacol.
356,
199-206[CrossRef][Medline]
[Order article via Infotrieve]
|
10.
|
Giannella, E.,
Mochmann, H. C.,
and Levi, R.
(1997)
Circ. Res.
81,
415-422[Abstract/Free Full Text]
|
11.
|
van der Giet, M.,
Schmidt, S.,
Tolle, M.,
Jankowski, J.,
Schluter, H.,
Zidek, W.,
and Tepel, M.
(2002)
Eur. J. Pharmacol.
448,
207-213[CrossRef][Medline]
[Order article via Infotrieve]
|
12.
|
Hillenkamp, F.,
and Karas, M.
(1990)
Methods Enzymol.
193,
280-295[Medline]
[Order article via Infotrieve]
|
13.
|
Barnes, L. D.,
Robinson, A. K.,
Mumford, C. H.,
and Garrison, P. N.
(1985)
Anal. Biochem.
144,
296-304[Medline]
[Order article via Infotrieve]
|
14.
|
De Bold, A. J.,
and Bencosme, S. A.
(1973)
Cardiovasc. Res.
7,
364-369[Medline]
[Order article via Infotrieve]
|
15.
|
Bradford, M. M.
(1976)
Anal. Biochem.
72,
248-254[CrossRef][Medline]
[Order article via Infotrieve]
|
16.
|
Kaufmann, R.,
Kirsch, D.,
and Spengler, B.
(1994)
Methods Enzymol.
193,
263-279
|
17.
|
Nordhoff, E.,
Ingendoh, A.,
Cramer, R.,
Overberg, A.,
Stahl, B.,
Karas, M.,
Hillenkamp, F.,
and Crain, P. F.
(1992)
Rapid Commun. Mass Spectrom.
6,
771-776[Medline]
[Order article via Infotrieve]
|
18.
|
Sulkowski, E.,
and Laskowski, S.
(1971)
Biochim. Biophys. Acta
240,
443-447
|
19.
|
Marrian, D.
(1953)
Biochim. Biophys. Acta
12,
492
|
20.
|
Lieberman, I.
(1955)
J. Am. Chem. Soc.
77,
3373-3375
|
21.
|
Small, G.,
and Cooper, C.
(1966)
Biochemistry
5,
26-33[Medline]
[Order article via Infotrieve]
|
22.
|
Van Dyke, K.,
Robinson, R.,
Urquilla, P.,
Smith, D.,
Taylor, M.,
Trush, M.,
and Wilson, M.
(1977)
Pharmacology
15,
377-391[Medline]
[Order article via Infotrieve]
|
23.
|
Gualix, J.,
Abal, M.,
Pintor, J.,
and Miras-Portugal, M. T.
(1996)
FEBS Lett.
391,
195-198[CrossRef][Medline]
[Order article via Infotrieve]
|
24.
|
Starling, R. C.,
Hammer, D. F.,
and Altschuld, R. A.
(1998)
Mol Cell Biochem.
180,
171-177[CrossRef][Medline]
[Order article via Infotrieve]
|
25.
|
Lee, J. W.,
Kong, I. D.,
and Park, K. S.
(1995)
Korean J. Physiol.
29,
217-223
|
26.
|
Nori, S.,
Fumagalli, L.,
Bo, X.,
Bogdanov, Y.,
and Burnstock, G.
(1998)
J. Vasc. Res.
35,
179-185[CrossRef][Medline]
[Order article via Infotrieve]
|
27.
|
Garcia-Guzman, M.,
Soto, F.,
Laube, B.,
and Stuhmer, W.
(1996)
FEBS Lett.
388,
123-127[CrossRef][Medline]
[Order article via Infotrieve]
|
28.
|
Evans, R. J.,
Lewis, C.,
Buell, G.,
Valera, S.,
North, R. A.,
and Surprenant, A.
(1995)
Mol. Pharmacol.
48,
178-183[Abstract]
|
29.
|
Bo, X.,
Zhang, Y.,
Nassar, M.,
Burnstock, G.,
and Schoepfer, R.
(1995)
FEBS Lett.
375,
129-133[CrossRef][Medline]
[Order article via Infotrieve]
|
30.
|
Torres, G. E.,
Egan, T. M.,
and Voigt, M. M.
(1999)
J. Biol. Chem.
274,
6653-6659[Abstract/Free Full Text]
|
31.
|
Hopwood, A. M.,
and Burnstock, G.
(1987)
Eur. J. Pharmacol.
136,
49-54[CrossRef][Medline]
[Order article via Infotrieve]
|
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