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Research Communications |
a Medizinische Klinik I, Universitätsklinik Marienhospital der Ruhr-Universität Bochum,
b Institut für Analytische Chemie, J. W. Goethe-Universität, Frankfurt, Germany;
c Institut für Pharmakologie und Toxikologie, Universität Münster, Germany;
d Klinik u. Poliklinik für Thorax-, Herz-, Gefässchirurgie, Universität Münster, Germany; and
e Institut für Pharmazeutische Chemie, Universität Münster, Germany
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: diadenosine polyphosphates • cardiac-specific granules • vasoactivity • force contraction • L-type calcium current
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Purification of the diadenosine polyphosphates 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 exclusively to isolate
diadenosine polyphosphates from myocardial tissue. The coronary tissue
was cut into small pieces (~1 cm3), frozen in
liquid nitrogen, and stored at -80°C for one night. Then the tissue
was lyophilized and powdered (step 1). The powder was suspended in 200
ml 0.6 mol/l 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 4000 rpm for 10
min at 4°C, the supernatant was titrated to pH 6.5 with HCl and
centrifuged again as described above (step 2).
A reversed-phase column (Lichroprep RP-18, 310 x 25 mM, Merck, Rahway, N.J.) was used to concentrate the nucleotides (step 3). The column was equilibrated with 40 mmol/l triethylammonium acetate (TEAA) in water. The supernatant with 40 mmol/l TEAA added (final concentration) was pumped to the column, washed with 40 mmol/l TEAA and eluted with 40% acetonitrile (ACN) in water at a flow rate of 1 ml/min. The 40% ACN-eluate was collected, frozen at -80°C, and lyophilized.
Size exclusion chromatography was performed according to Schlüter and Zidek (13) (step 4). Size exclusion gel Sephacryl S-100 high resolution (1000 x 16 mM, S100 HR; Pharmacia BioTech, Piscataway, N.J.) was equilibrated with water. The dried sample from the preparative reversed-phase column resolved in 5 ml water was loaded onto the column. The eluent (water) was pumped with a flow rate of 1 ml/min and monitored with a UV detector at 254 nm.
An anion exchange column (Fractogel EMD DEAE-650, 300 x 25 mM, Merck) was equilibrated with 10 mmol/l ammonium acetate (pH 7.4) (step 5). After adding 10 mmol/l ammonium acetate (final concentration), pH 7.4, to the fraction from the size exclusion chromatography, it was pumped through the column. Nucleotides were eluted by 1 mol/l ammonium acetate (pH 7.4) at a flow rate of 3.0 ml/min. The eluate 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, Richmond, Calif.), was synthesized according to Barnes et al. (14) . The affinity resin was packed into a glass column (150 x 20 mM) and equilibrated with 1 mol/l ammonium acetate (pH 9.5). The pH of the eluate from the anion exchange column was adjusted to pH 9.5 and loaded to the affinity column. The column was washed with 1 mol/l ammonium acetate (pH 9.5) with a flow rate of 1 ml/min. Binding substances were eluted with 1 mmol/l HCl. The eluate was frozen and lyophilized. Fractions were monitored with a UV detector at 254 nm.
Fractions from affinity chromatography were desalted by reversed-phase high-performance liquid chromatography (HPLC) (Superspher 100 RP C18 end-capped, 250 x 4 mM, Merck) (step 7). The fractions dissolved in eluent A (40 mmol/l 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 absorbing fraction was collected.
The desalted fraction from affinity chromatography was fractionated by anion exchange chromatography (step 8). The anion exchange column (50 x 5 mM, Mono-Q HR 5/5; Pharmacia Biotech) was equilibrated with eluent A (10 mmol/l K2HPO4, pH 8.0). The sample dissolved in eluent A was injected to 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 concentrations of eluent B (50 mmol/l K2HPO4 + 1 mol/l NaCl, pH 8.0). The time program of the gradient was 0–10 min 0–5% B, 10–100 min 5–35% B, 100–105 min 35–40% B, and 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 x 4 mM, Merck) (step 9). The fractions dissolved in eluent A (40 mmol/l TEAA) were injected to HPLC. The following gradient was programmed: 0–4 min 0–2% B, 4–79 min 2–7% B, 79–85 min 7–60%. The flow rate was 0.5 ml/min. The wavelength of the UV detector was 254 nm; 1 ml fractions were collected.
Purification of the diadenosine polyphosphates from porcine
myocardial-specific granules
Specific granules were obtained from porcine left ventricular
tissue according to the method of De Bold and Benscome (15)
. Briefly,
immediately after death the heart was removed, washed in ice-cold 0.25
mol/l sucrose (containing 0,2% glycogen, 1 mmol/l EDTA, pH 7), and
placed in a container with ice-cold 0.25 mol/l sucrose. After removing
large vessels and fat, the tissue was washed for a second time in
ice-cold 0.25 mol/l sucrose. After collecting 108 g myocardial
tissue, it was homogenized at 3°C. The resulting pulp was washed into
a glass homogenizer with 10 volumes of 0.25 mol/l sucrose. The
suspension was homogenized by 20 strokes of a Teflon pestle. The
resulting homogenate was filtered by cheesecloth. This filtrate was
centrifuged at 1900 x g for 10 min. The supernatant
was filtered again by cheesecloth, centrifuged at 1900 x
g for 10 min, and centrifuged at 32,000 x g
for 10 min. The pellet containing specific granules was resuspended in
0.25 mol/l sucrose and centrifuged again at 32,000 x g
for 10 min. The pellet was resuspended by addition of 4 ml 1.6 mol/l
sucrose (containing 0,2% glycogen, 1 mmol/l EDTA, pH 7). The remaining
pellet was further resuspended by homogenizing in a glass homogenizer
by a Teflon pestle. The suspension was pipetted on top of 0.5 ml of 2
mol/l sucrose (containing 0,2% glycogen, 1 mmol/l EDTA, pH 7) and
topped with 1 ml of 0.25 mol/l sucrose. The resulting gradient was
centrifuged immediately at 154,000 x g for 60 min,
when five fractions were obtained. The protein concentration was
measured according to Bradford (16)
. 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 x 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 (17)
. In MALDI-MS, large fractions of the desorbed
analyte ions undergo postsource decay (PSD) during flight in the field
free drift path. Using a RETOF setup, sequence information from PSD
fragment ions of precursors produced by MALDI were obtained (18)
. For
MALDI-MS and PSD MALDI-MS, speed vacuum-dried samples were dissolved in
10 µl bidistilled water. One microliter of the 3-hydroxyl-picolinic
acid matrix solution (50 g/l) 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 106-107
W/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 by a LeCroy 9400 recorder
(19)
.
UV spectroscopy
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/Vis-Spectrophotometer, DU-600; Beckman, Fullerton, Calif.). UV
absorption was scanned from 400 nm 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 1) 20 µl 200 mmol/l Tris buffer (pH 8.9), incubated
with 5'-nucleotide hydrolase 3 mU from Crotalus durissus, EC
3.1.15.1 (Boehringer Mannheim, Mannheim, Germany), and purified
according to Sulkowski and Laskowski (20)
for 9 min at 37°C;
2) 20 µl 200 mmol/l Tris and 20 mmol/l EDTA buffer (pH
7.4) and incubated with 3'-nucleotide hydrolase (1 mU) from calf
spleen, EC 3.1.16.1 (Boehringer Mannheim) for 1 h at 37°C; and
3) 20 µl 10 mmol/l Tris, 1 mmol/l
ZnCl2, and 1 mmol/l MgCl2
buffer (pH 8) and incubated with alkaline phosphatase (1 mU; EC 3.1.3.1
from calf intestinal mucosa, Boehringer Mannheim) 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 mmol/l K2HPO4, pH 7),
was subjected to anion exchange chromatography (MiniQ PC 3.2/3,
Pharmacia). The gradient was 0–3 min: 0% B (50 mmol/l
K2HPO4, pH 7 with 1 mol/l
NaCl); 3–20 min: 0–50% B; 20–21 min: 50–100% B. The flow rate was
100 µl/min.
Identification and quantification of Ap2A and
Ap3A in myocardial-specific granules
The specific granule pellet was suspended in water. After
addition of 1.5 µg di-etheno-Ap6A as internal
standard (for preparation and purification see ref. (21)
to the
specific granule suspension, 10 ml 50% ethanol (with 10 mmol/l
K2HPO4) was added. This
mixture was sonicated three times for 20 s. The resulting
suspension was passed through a reversed-phase column (HPLC column,
4 x 250 mM, Superspher, Merck) to remove lipids. Ammonium acetate
(final concentration, 1 mol/l, pH 9.5) was added to the eluate and
chromatographed by affinity chromatography (phenyl boronic acid coupled
to an cation exchange support, as described above). After addition of
TEAA (40 mmol/l final concentration), the eluate was desalted by a HPLC
reversed-phase column (4 x 250 mM, Superspher, Merck). The
nucleotides were eluted by 30% acetonitrile in water. The dried eluate
was chromatographed by anion exchange chromatography (as described
above, MonoQ, Pharmacia). The substances eluting from the column were
fractionated (peak fractionation) and desalted as described. The dried
fractions were analyzed by MALDI-MS. The absolute amounts of the
diadenosine polyphosphates and the internal standard were calculated by
their individual calibration curves. For the internal standard, a
recovery of 32 ± 7% was calculated. The amount of the
diadenosine polyphosphates was corrected according to the recovery of
the internal standard.
Quantification of (ADP + ATP)/ApnA ratio in
myocardial-specific granules by capillary electrophoresis
The ratio of the mononucleotides ADP and ATP to
Ap2A or Ap3A was determined
by capillary electrophoresis, which, in contrast to HPLC analysis,
allows the simultaneous detection of mononucleotides and
ApnAs. The specific granule pellet was
delipidated and concentrated by reversed-phase chromatography, as
described above. The dried eluate of the reversed-phase chromatography
was dissolved in water and analyzed by a capillary electrophoresis
system (P/ACE 2100, software Gold V 7.11, Beckman) equipped with an
in-line, variable wavelength detector (254 nm). The samples were
injected hydrodynamic for 1 s at 55 psi. The separations were run
at -20 kV (65–70 µA, 20°C; inlet: cathode, outlet: anode) with an
untreated, fused silica capillary (50 µM I.D., 37 cm length, 30 cm to
the detector); 50 mmol/l citric acid (pH 4.75) was used as buffer.
In the electropherogram, ADP, ATP, Ap2A, and Ap3A were identified by spiking their peaks with the authentic nucleotides. By using the peak areas, the amount of the nucleotides was calculated by the calibration curves of the individual nucleotides.
Isolated perfused heart
Isolated perfused rat hearts were prepared according to van der
Giet et al. (22)
. Briefly, adult male Wistar Kyoto rats (250–300 g)
were killed by cervical dislocation a minimum of 15 min after heparin
(100 units/kg, i.p.) was administered. Hearts were rapidly excised; the
Langendorff technique was used to perfuse hearts with Krebs-Henseleit
solution maintained at 37 ± 0.5°C, pH 7.4, and gassed with 5%
O2/95% CO2. The
composition of Krebs-Henseleit solution was as follows (mmol/l): NaCl
120, KCl 4.7, NaH2PO4, 1.18
MgSO4, CaCl2 1.25,
NaHCO3 25 and glucose 5.5. The heart was allowed
to beat spontaneously and was perfused at a constant flow rate. The
perfusion pressure was measured by a pressure transducer attached to a
side arm of the aortic cannula. Changes in perfusion pressure were
recorded on a chart recorder. All hearts were equilibrated for 20 min
in Krebs-Henseleit solution. The flow rate was gradually adjusted to
obtain a perfusion pressure of 65–75 mmHg and kept constant. Under the
experimental conditions, vasodilation resulted in a decrease in
perfusion pressure and vasoconstriction resulted in an increase in
perfusion pressure, as described by Man et al. (23)
.
Drug preparation and administration
All test solutions were made fresh daily from stock solutions (1
mmol/l, concentrates stored frozen). All substances were applied as 100
µl bolus into a sample loop proximal to the preparation. Ten minutes
elapsed between each bolus injection. The nitric oxide synthase
inhibitor NG-nitro-L-arginine methyl ester
(L-NAME, 50 µmol/l) was added to the perfusate 30 min before
challenge with Ap2A, Ap3A,
or acetylcholine. The nitric oxide synthase inhibitor elevated
perfusion pressure by 25 ± 4 mmHg.
Force of contraction experiments
Experiments were performed as described previously (24)
. In
brief, right papillary muscles were isolated from hearts of
reserpinized (5 mg/kg, 16 h before death) male guinea pigs
(300–400 g). Reserpinization was performed to exclude effects of
endogenous catecholamines and for reasons of comparability with our
previous work (24)
.
Isolated papillary muscles were mounted in organ baths. The bathing solution contained (in mmol/l) NaCl 119.8, KCl 5.4, CaCl2 1.8, MgCl2 1.05, NaH2PO4 0.42, NaHCO3 22.6, Na2EDTA 0.05, ascorbic acid 0.28, and glucose 5.0, continuously gassed with 95% O2 and 5% CO2, and maintained at 35°C, resulting in a pH of 7.4. Isometric force of contraction was measured after stretching each muscle to optimal length, i.e., the length at which force of contraction was maximal. Papillary muscles were electrically stimulated with rectangular pulses 5 min duration at 1 Hz (24) ; the voltage was ~10–20% greater than the threshold. Preparations were allowed to contract until a stable mechanical recording was reached (at least 30 min) before 0.2 U/ml adenosine deaminase (ADA) was added for an additional 30 min. ADA was used to exclude the possibility that, after degradation of Ap2A to adenosine, the latter might be the active principle in the experiments. ADA converts adenosine to inactive inosine. In the presence of 0.2 U/ml ADA, the negative inotropic effect of 1 mmol/l adenosine was abolished (data not shown). After the ADA incubation period, muscles were stimulated with 0.01 µmol/l isoproterenol for 10 min, then 100 µmol/l Ap2A was added for 10 min.
Isolation of guinea pig ventricular myocytes
Guinea pig ventricular myocytes were isolated by a
collagenase/protease digestion of Langendorff-perfused hearts (37°C,
52 mmHg), as published (24)
. In brief, hearts were perfused for 5 min
with calcium-free solution A (in mmol/l: NaCl 135, KCl 4,
NaH2PO4 0.3,
MgCl2 1,
4-(2-hydroxyethyl)-1-piperazineethane-sulfonic acid (HEPES) 10,
dextrose 10, pH 7.4) before enzymes were added to this solution at a
flow-independent dose rate of 1.4 mg/min collagenase (type 1,
Worthington, Freehold, N.J.) and 0.6 mg/min protease (type XIV, SIGMA,
Deisenhofen, Germany) over a period of 5 min. Afterward the hearts were
perfused for 10 min with enzyme-free solution A containing 0.2 mmol/l
calcium. Cells were harvested after mincing the hearts with fine
scissors, gentle agitation of the tissue, and filtering through a nylon
mesh.
Measurement of L-type calcium current (ICaL)
Freshly isolated cardiomyocytes were plated in petri dishes,
which served as recording chambers (volume approx. 1 ml) on the stage
of an inverted microscope (Leica, Köln, Germany).
Electrophysiological experiments were performed as described previously
(24)
. In brief, solution A supplemented with 2 mmol/l calcium served as
the extracellular solution, and recording pipettes (soft glass coated
with Sylgard, 1.5–2.5 M
) were filled with (in mmol/l) K-aspartate
80, KCl 50, KH2PO4 10,
MgCl2 0.5, MgATP 3, HEPES 5, and ethylene glycol
bis(ß-aminoethyl ether) N,N',N'-tetraacetic acid (EGTA) 1, pH 7.4.
L-type calcium currents were elicited by voltage steps from a holding
potential of -40 mV to a test potential of +10 mV for 300 min, applied
every 10 s. Current was recorded by an L/M-PC-amplifier
(LIST-Electronic, Darmstadt, Germany) connected to a 486 computer
equipped with ISO2 software (version 1.2, MFK, Niedernhausen, Germany).
Currents were evaluated as the difference between peak inward current
and the current level at the end of the test pulse. Series resistance
was compensated to the maximum possible extent, using the feedback
circuitry of the amplifier.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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A. Anionic substances in the fraction containing molecules in
the molecular mass range of 300-2000 Da were concentrated with an anion
exchange column (step 5). The eluate was passed through an affinity
column to concentrate dinucleoside polyphosphates (step 6). The eluate
of the affinity chromatography, desalted by a reversed-phase
chromatography (step 7), was fractionated by anion exchange
chromatography (step 8). The chromatogram of step 8 is given in Fig. 1B
. Every fraction with a significant UV absorbance at 254
nm was fractionated by reversed-phase chromatography (step 9). Typical
chromatograms are presented in Fig. 1C, D
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Fractions from the reversed-phase chromatography (step 9) -absorbing UV
light at a wavelength of 254 nm were analyzed by MALDI-MS. In the
fraction labeled Ap2A and
Ap3A in Fig. 1C, D
, the molecular
masses of 676 Da and 756 Da were found, respectively (data not shown).
These masses correspond with those of Ap2A and
Ap3A, respectively.
UV spectroscopy of these fractions showed a curve essentially identical
to that of adenosine, confirming that an adenosine-containing
nucleotide was present in both fractions. Figure 2
and Fig. 3
summarize the identification of Ap2A and
Ap3A. Figure 2A
shows the PSD-MALDI
mass spectrum from the fraction of the reversed-phase chromatography
labeled Ap2A in Fig. 1C
. In
Table 1
, the masses obtained by PSD-MALDI mass spectrometry are assigned to the
respective fragment ions. The fragmentation pattern obtained was
identical to that from commercially available
Ap2A. To answer the question of the position of
the phospho-ester bonds, aliquots of the fraction were incubated with a
5'-nucleotide hydrolase, a 3'-nucleotide hydrolase, and an alkaline
phosphatase. The substances in the two fractions were cleaved by the
5'-nucleotide hydrolase (phosphodiesterase from C. durissus)
only, yielding AMP (Fig 2B, C
). These results demonstrate
that the phosphates in the two molecules are linked via the 5'-position
to the ribose. The 3'-nucleotide hydrolase and the alkaline phosphatase
had no effect on the substance, showing that the molecule contains no
phosphoester connected to the 3'-position of the ribose and that the
phosphoester interconnect the two adenosines. Cleavage experiments with
authentic substance yielded the same results.
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Figure 3
shows the analogous experiments with the fraction labeled
Ap3A in Fig. 1D
. PSD-MALDI mass
spectrometry revealed a fragmentation pattern identical to that of
commercially available Ap3A (Fig. 3A
).
The cleavage experiment with 5'-nucleotide hydrolase together with the
control chromatography is shown in Fig. 3B, C
. 5'-Nucleotide
hydrolase produced AMP and ADP as split products, indicating that the
phosphates in the two molecules are linked via the 5'-position to the
ribose. The 3'-nucleotide hydrolase and the alkaline phosphatase had no
effect on the substance, showing that the molecule contains no
phosphoester connected to the 3'-position of the ribose and that the
phosphoester interconnect the two adenosines. Cleavage experiments with
authentic substance yielded the same results. Taken together, the
results confirm the identity of Ap2A and
Ap3A in cardiac tissue.
To further localize Ap2A and
Ap3A within myocardial tissue, specific granules
were tested for the presence of the dinucleotides. In the specific
granule fraction isolated from 108 g tissue, 0.18 µmol
Ap2A and 0.02 µmol Ap3A
were measured by HPLC analysis (Fig. 4
A). From these amounts, the concentrations for
Ap2A and Ap3A in specific
granules can be estimated to be 9 mmol/l and 1 mmol/l, respectively.
The estimation was based on the final pellet volume after granule
isolation, neglecting the trapped fluid within the pellet. The identity
of the peaks labeled Ap2A and
Ap3A was confirmed by PSD-MALDI mass spectrometry
and retention time comparison.
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The ratio between the sum of ADP and ATP and Ap2A
or Ap3A, respectively, was calculated with
capillary electrophoresis from the peak areas of the nucleotides. The
ratio of (ADP + ATP):Ap2A in specific granules
was 17.6 ± 1.5 and the ratio of (ADP +
ATP):Ap3A was 187.5 ± 11.6 (mean ±
SD, each n=5, Fig. 4B
).
Figure 5
summarizes the results of the action of Ap2A and
Ap3A to the vasculature of the perfused rat
heart. Both Ap2A and Ap3A
caused a dose-dependent coronary vasodilation, being less potent by a
factor of 10 than acetylcholine. When nitric oxide synthase was
inhibited by L-NAME, the vasodilation by Ap2A and
Ap3A was essentially unchanged, whereas the
response to acetylcholine expectedly was completely abrogated.
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In isolated, electrically driven guinea pig papillary muscles,
Ap2A alone had no effect on force of contraction
(data not shown). Stimulation with 0.01 µmol/l isoproterenol
increased force of contraction to 187.2 ± 14% (n=5)
of control and applied Ap2A (100 µmol/l)
reduced force of contraction to 150 ± 11% (n=5) of
control, i.e., the isoproterenol-effect was reduced by 20 ± 2%.
The control values amounted to 1.0 ± 0.1 mN. A typical original
recording illustrating the effects of Ap2A on
force of contraction is depicted in Fig. 6
A.
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Application of 100 µmol/l Ap2A alone to
isolated guinea pig ventricular myocytes did not affect the amplitude
of L-type calcium current (n=4, data not shown).
Measurements were performed in four individual cells obtained from
three guinea pig hearts. Stimulation with 10 nmol/l isoproterenol
increased the calcium current to 290.8 ± 31% of control
(n=4). Additional application of 100 µmol/l
Ap2A attenuated the isoproterenol-stimulated
calcium current to 131.5 ± 15% of control (n=4).
Measurements were performed in four individual cells obtained from
three guinea pig hearts. The control values amounted to 6.2 ± 0.4
pA/pF. A typical experiment illustrating the original time course of
the amplitude of the calcium current through L-type calcium channels is
depicted in Fig. 6B
. Ap3A had nearly
identical effects on myocardial muscle preparations (data not shown).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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To prove the existence of diadenosine polyphosphates in myocardial tissue, a purification scheme was developed to concentrate these substances specifically. The purification scheme includes an affinity chromatographic step, which binds specifically substances with cis-diol groups. This purification step eliminates mononucleotides like ATP, although they have a cis-diol group. This elimination was achieved by coupling phenylboronic acid to a cation exchange resign according to the method of Barnes et al. (14) . Nucleotides that are negatively charged are repulsed by the functional groups of the cation exchange resign, which are also negatively charged. The buffer system used in this purification step, 1 mol/l ammonium acetate, partly suppresses the electrostatic interactions. As a result, negatively charged substances like diadenosine polyphosphates with two cis-diol groups bind to the stationary phase. Negatively charged substances with only one cis-diol group like mononucleotides do not bind because the repulsion overcomes the binding via the cis-diol group.
The search for diadenosine polyphosphates contained another important analytical tool, MALDI mass spectrometry. With this instrument, fractions absorbing UV light at 254 nm were systematically analyzed in order to find molecular masses identical to those of the diadenosine polyphosphates (e.g., Ap2A: 676 Da, Ap3A: 756 Da). Furthermore, PSD-MALDI-MS and enzymatic cleavage analysis were used to confirm the existence of the diadenosine polyphosphates.
To establish a physiological role of Ap2A and Ap3A, it has to be shown that these agents indeed act on myocardium and/or coronary arteries and are secreted into the extracellular space. First, in guinea pig papillary muscles, Ap2A and Ap3A attenuate the effect of sympathetic overstimulation. This is in accordance with the observation that the calcium current of guinea pig ventricular myocytes, stimulated by isoproterenol, is attenuated by Ap2A and Ap3A.
Second, it was tested whether Ap2A and Ap3A are present in myocardial-specific granules, which are known to be released into the extracellular space. Analysis revealed that Ap2A and Ap3A exist in the millimolar range in specific granules. The (ADP + ATP)/Ap2A ratio appears to be low compared with data from platelets (26) . This may be explained by taking into account that ADP and ATP are much more labile than Ap2A. The granule fraction contains acidic phosphatase, which degrades both mononucleotides, but not dinucleotides. According to the above results, relatively high local concentrations of Ap2A and Ap3A in the extracellular space can be assumed. Admittedly, Ap2A and Ap3A may have been partly destroyed during the purification procedure, too. However, given a significant degradation of ApnAs, we should underestimate rather than overestimate the tissue ApnA concentration. The concentrations reached in the extracellular space are limited by several factors like volume of distribution in the extracellular space, clearance of the diadenosine polyphosphates by degradation, diffusion, or uptake into the cells. Therefore, the concentrations within specific granules may represent a peak value that might be reached under optimum conditions and for a short time in a restricted area. The attempt to estimate the locally effective ApnA concentrations discloses several uncertainties concerning the assumptions on which the estimations are based. It should be emphasized therefore that the primary aim of the study was the qualitative, not quantitative, detection of Ap2A and Ap3A in human myocardial tissue.
The greater stability of ApnAs compared with ATP also has significant implications with respect to their extracellular actions. In the extracellular space, ATP appears to be much more rapidly degraded than the diadenosine polyphosphates by ectohydrolases and plasma nucleotidases. Therefore, despite lower tissue concentrations, Ap2A and Ap3A could be at least as effective as ATP.
The existence of the diadenosine polyphosphates opens the question
about its biosynthesis. Aminoacyl-tRNA synthetases catalyze the
formation of Ap3A and Ap4A
(aminoacyl-AMP + ADP 
Ap3A, aminoacyl-AMP +
ATP Ap4A) (27)
. Adenosine 5'-monophosphate
does not react (28)
; therefore, this type of enzymatic reaction cannot
synthesize Ap2A.
Ap4A-phosphorylases are another class of
diadenosine polyphosphate-synthesizing enzymes according to the
following reaction ADP + ATP Ap4A +
pi (27)
. Theoretically the reaction of a
diadenosine polyphosphate phosphorylase catalyzing the formation of
Ap2A should be AMP + ADP
Ap2A + pi. Nevertheless,
neither the enzyme Ap4A phosphorylase nor a
potential gene sequence has been detected in metazoan animals. The
lower eukaryotic phosphorylases are unable to synthesize
Ap2A.
We cannot yet identify the cell type containing Ap2A and Ap3A in the human heart. The samples examined stem from human left ventricle; hence, it is most likely that ventricular cardiomyocytes contain these diadenosine polyphosphates. It seems probable that Ap2A and Ap3A exist not only in specific granules of porcine myocardial tissue, but also in human myocardium. The presence of diadenosine polyphosphates is reported as the main component in myocardial-specific granules Ap2A, whereas Ap4A, Ap5A, and Ap6A were not detected.
It is interesting that one report shows Ap5A in guinea pig heart (12) . We also looked for diadenosine polyphosphates with a higher number of phosphate groups, but failed to detect any of these compounds either in human heart tissue or in specific granules prepared from porcine myocardial tissue. Although the possibility that interspecies differences account for this cannot be dismissed, those compounds with a higher number of phosphate groups may also have been degraded in our human material. These discrepancies with regard to the literature might also be explained by taking into account that we examined tissue from hearts suffering from severe ischemia, although we used only macroscopically intact tissue. Considering the role of diadenosine polyphosphates as `alarmones' in other tissues or cells, an altered synthesis of these substances under pathological conditions such as hypoxia cannot be excluded (31) .
The experiments revealed that Ap2A and Ap3A, like adenosine, act as coronary vasodilators. However, there are important differences from the action of adenosine. Half-lives of the diadenosine polyphosphates are at least 10-fold longer than that of adenosine (32) . Among the diadenosine polyphosphates, Ap2A was shown to be the most stable compound in vivo. The comparatively high stability of Ap2A may be explained by the selectivity of the hydrolases accounting for a considerable part of the diadenosine polyphosphate-degrading activity. Asymmetrically cleaving hydrolases generate AMP and Apn-1 from ApnA. These enzymes have a low affinity to Ap2A. Furthermore, the symmetrically cleaving hydrolases do not degrade Ap2A. Mainly, Ap2A is degraded by phosphodiesterases occurring in plasma and endothelial cells (33) . Compared with Ap2A and Ap3A, ATP is degraded far more rapidly. A degradation experiment with guinea pig left atrium tissue showed that ATP is degraded much faster than any of the dinucleotides. Ap3A was the least stable of the diadenosine compounds in this experiment; Ap2A was the most stable diadenosine polyphosphate. The relative order of stability was Ap2A > Ap4A = Ap5A > Ap3A >> ATP (8). In conclusion, it can be assumed that Ap2A is the most stable diadenosine polyphosphate present in circulation.
Besides their role as A1-stimulating agonists, the diadenosine polyphosphates (but not adenosine) may also act as modulators of the ATP-dependent K outward current in cardiomyocytes. Several studies demonstrated an inhibition of the IK(ATP) by Ap4A, Ap5A, and Ap6A 10, 11, 34) , but Ap2A and Ap3A have not been tested yet as to their direct effects on transmembrane K current.
The vasodilator action of Ap2A and Ap3A was not mediated via nitric oxide. Thus, vasodilation induced by Ap2A and Ap3A mediated via A2-receptors seems unlikely 35, 36) . Binding of adenosine to the A2 receptor releases nitric oxide from the coronary endothelial cells, which then induce vasodilation (35) . Therefore, the diadenosine polyphosphates obviously present a vasodilatory mechanism other than that elicited by adenosine.
Earlier studies showed that, in renal vasculature, Ap2A and Ap3A act via P2Y receptors and A2 receptors, whereas the affinity of Ap2A and Ap3A toward P2X receptors is very low. Vasoconstrictive effects of Ap2A and Ap3A in the rat isolated perfused kidney were mostly due to stimulation of A1 receptors (22) . In basal tone preparations in the isolated perfused rat, mesenteric arterial bed Ap2A and Ap3A had no vasoconstrictive effect in contrast to Ap4A, Ap5A, and Ap6A (37) . In raised tone preparations, Ap2A and Ap3A evoked vasodilation, which was blocked by the A2 receptor blocker 3,7-dimethyl-1-propargylxanthine (22) . In raised tone preparations in the isolated perfused rat mesenteric arterial bed, Ap2A elicited endothelium-dependent vasodilation partly via P1 and possibly via P2 U purinoceptors. In this model, Ap3A mediates vasodilation via P2Y purinoceptors (38) .
Together, the pertinent literature reveals that Ap2A and Ap3A exert vasodilation either via A2 or P2Y receptors. Both receptors appear to elicit nitric oxide release. Therefore, the present results from the coronary vessels suggest that, in this preparation, still another mechanism is active.
The existence of Ap2A and Ap3A in human hearts and specific granules of myocardial tissue as well as their effects on coronary artery tone and myocardial function indicate a regulatory role of these diadenosine polyphosphates in the human heart.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Abbreviations: ACN, acetonitrile; ADA, adenosine
deaminase; Ap2A,
5',5'''-P1,P2-diphosphate; Ap3A,
5',5'''-P1,P3-triphosphate; Ap4A,
5',5'''-P1,P4-tetraphosphate; HEPES,
4-(2-hydroxyethyl)-1-piperazineethane-sulfonic acid; HPLC,
high-performance liquid chromatography; L-NAME,
NG-nitro-L-arginine methyl ester; ICaL, L-type
calcium current; MALDI-MS, matrix-assisted laser desorption/ionization
mass spectrometry; PSD, postsource decay; RETOF, reflectron-type
time-of-flight; TEAA, triethylammonium acetate.
Received for publication June 16, 1998.
Revision received November 9, 1998.
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-Ap6A: dietheno-Ap6A (internal
standard). B) Electropherogram of an extract from
myocardial granules. Detector: UV photometer, 254 nm. Samples
injection: hydrodynamic (1 s at 55 psi). 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 mmol/l citric acid (pH 4.75).
