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 GietDagger

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
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 alpha ,beta -methylene ATP (alpha ,beta -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
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 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, alpha ,beta -methyleneadenosine 5'-triphosphate (alpha ,beta -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 alpha ,beta -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 alpha ,beta -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, alpha ,beta -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 alpha ,beta -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
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.

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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Table I
Fragment ionic masses measured by PSD-MALDI MS


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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.

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).

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 alpha ,beta -meATP elicited a rapidly desensitizing vasoconstriction.

Fig. 5 demonstrates the action of 2-meSATP, 2-ClATP, alpha ,beta -meATP, and Ap4 on coronary vasculature. At base-line perfusion pressure the nucleotides, except for alpha ,beta -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 (black-square), 2-ClATP (), alpha ,beta -meATP (), and Ap4 (open circle ). 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, alpha ,beta -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).

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 alpha ,beta -meATP remained unaffected (Fig. 6A). In the absence of intact endothelium, vasoconstrictor responses to Ap4, 2-ClATP, 2-meSATP, and alpha ,beta -meATP disappeared after the addition of either the P2X receptor antagonist PPADS (30 µM, Fig. 6B) or the P2X receptor agonist alpha ,beta -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 alpha ,beta -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.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 alpha ,beta -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 alpha ,beta -meATP (27, 28). The P2X4 receptor cannot be inhibited by PPADS (29). The P2X1 receptor is inhibitable by PPADS, sensitive to alpha ,beta -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.

Dagger 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; alpha , beta -meATP, alpha ,beta -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
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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