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Diadenosine Polyphosphates’ Action on Calcium and Vessel Contraction
AJH
1997;10:1404 –1410
Diadenosine Polyphosphates’ Action on Calcium and Vessel Contraction Martin Tepel, Joachim Jankowski, Hartmut Schlu¨ter, Ju¨rgen Bachmann, Marcus van der Giet, Christian Ruess, Jo¨rg Terliesner, and Walter Zidek
The effects of the endogenous, platelet-derived vasoactive compounds, diadenosine tetraphosphate (AP4A), diadenosine pentaphosphate (AP5A), and diadenosine hexaphosphate (AP6A) on the vasoconstriction of isolated rat renal resistance vessels and rat aortic strips were measured using a vessel myograph. In addition, the effects of AP4A, AP5A, and AP6A on the cytosolic free calcium concentration ([Ca21]i) were evaluated in cultured rat vascular smooth muscle cells (VSMC) using the fluorescent dye technique. Diadenosine polyphosphates dose-dependently increased the force of renal resistance vessels and isolated aortic strips. The administration of 10 mmol/L AP4A, AP5A, or AP6A significantly increased the force of isolated renal resistance vessels by 3.48 6 0.43 mN (n 5 8), 2.14 6 0.40 mN (n 5 12), or 2.70 6 0.31 mN (n 5 11, each P < .01 compared with resting tension), respectively. The administration of 10 mmol/L AP4A, AP5A, or AP6A significantly increased the force of isolated aortic strips by
2.45 6 0.97 mNewton (n 5 10), 2.70 6 0.30 mN (n 5 6), or 1.48 6 0.20 mN (each P < .01 compared with resting tension), respectively. The administration of 10 mmol/L AP4A, AP5A, or AP6A significantly increased [Ca21]i in VSMC to a peak concentration of 314 6 60 nmol/L (n 5 6), 247 6 25 nmol/L (n 5 15), or 332 6 100 nmol/L (n 5 5), respectively (each P < .01 compared with resting value). Both the diadenosine polyphosphateinduced vasoconstriction and [Ca21]i increase was significantly reduced in the absence of extracellular calcium or after administration of a specific inhibitor of P2 purinoceptors. It is concluded that diadenosine polyphosphates increase [Ca21]i and hence cause vessel constriction. Am J Hypertens 1997;10:1404 –1410 © 1997 American Journal of Hypertension, Ltd.
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in vivo.1,2 Both AP5A and AP6A were shown to constrict renal vasculature,2 to stimulate growth, and to increase cytosolic free calcium concentration ([Ca21]i) in renal mesangial cells.3–5 To further assess the role of AP5A and AP6A in blood pressure regulation, the action of both agents on the tone of a large artery and of resistance arteries was compared in the present study. Furthermore, it is recognized that cytosolic free calcium concentration ([Ca21]i) is a major component in signal transduction and a major regulator of vascular smooth muscle force generation.6 – 8 Therefore the effects of these diadenosine polyphosphates on [Ca21]i were related to the vasoconstrictive effects, and were
ecently, endogenous platelet-derived vasoconstrictors, diadenosine pentaphosphate (AP5A) and diadenosine hexaphosphate (AP6A), have been identified.1,2 Platelet-derived diadenosine polyphosphates have been related to the local and systemic regulation of blood pressure
Received February 14, 1997. Accepted June 24, 1997. From the Universita¨tsklinik Marienhospital, Medizinische Klinik I, Ruhr-Universita¨t-Bochum, Herne, Germany. Address correspondence and reprint requests to Dr. M. Tepel, Medizinische Klinik I, Universita¨tsklinik Marienhospital, Ruhr-Universita¨t-Bochum, Ho¨lkeskampring 40, D-44625 Herne, Germany.
© 1997 by the American Journal of Hypertension, Ltd. Published by Elsevier Science, Inc.
KEY WORDS:
Diadenosine polyphosphates, cytosolic free Ca21, vascular smooth muscle cells, vasoconstriction.
0895-7061/97/$17.00 PII S0895-7061(97)00305-1
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compared with that of diadenosine tetraphosphate (AP4A), a known coronary and mesenterial vasodilator.9,10 The experiments showed that the vasoconstrictive effects of the diadenosine polyphosphates were even more pronounced in resistance vessels.
tetraacetic acid was added. The P2 purinoceptor blocker pyridoxal-phosphate-6-azophenyl-29,49-disulphonic acid (PPADS, Research Biochemical International, Cologne, Germany) was used at a concentration of 10 mmol/L.
METHODS
Cell Culture of Vascular Smooth Muscle Cells Vascular smooth muscle cells (VSMC) were obtained from thoracic aortas of Wistar-Kyoto rats and cultured by the tissue explant method according to published procedures.15–17 Briefly, cells were incubated in Dulbecco’s modified Eagle’s medium (Gibco, Eggenstein, Germany), containing 10% (vol/vol) fetal calf serum (Boehringer, Mannheim, Germany), 100 U/mL penicillin G, and 100 mg/mL streptomycin. Cultures were incubated at 37°C in a humidified atmosphere of 95% air and 5% CO2. The medium was changed initially after 24 h and then every 2 to 3 days. After the first subculture, cells were subcultured every week at a seeding density of about 1 3 104 cells/cm2 and reached confluence in 8 to 10 days. Thereafter they were harvested by adding 0.05% trypsin, and the culture was continued up to 8 passages as previously described.18 Separate experiments confirmed recent reports that resting [Ca21]i in cultured VSMC was not significantly different in these passages.7 To ascertain that cultured cells were VSMC, immunocytochemical localization of smooth muscle-specific a-actin was carried out using monoclonal antibodies ASM-1 (Progen, Heidelberg, Germany) raised against smooth muscle a-actin and labeled with a fluorescence marker.18 Staining of cultured VSMC with that antibody revealed that all cells in the preparation were labeled, and actin stress fibers were seen throughout the cytosol. It was confirmed that cultured VSMC were free from contamination with endothelial cells or fibroblasts by immunocytochemical staining of cells with antibodies against von Willebrand factor coupled to a fluorescence marker. A viability of VSMC higher than 95% was observed by trypan blue exclusion. Cells were made quiescent by incubation in serum free medium containing 0.1% bovine serum albumin, 100 U/mL penicillin and 100 mg/mL streptomycin for 48 h prior to [Ca21]i measurements.
Preparation of Aortic Strips and Renal Resistance Vessels Six-month-old male Wistar-Kyoto rats from the Mu¨nster strain, as described previously,11 were fed a standard pellet diet and water ad libitum. The vasoconstrictions of aortic strips or renal resistance vessels from male Wistar-Kyoto rats were measured using a vessel myograph introduced by Mulvany and Nyborg.12 The abdominal aorta was excised and transferred into a medium containing (mmol/L) NaCl 115; KCl 4.6; MgSO4 1.2; NaH2PO4 1.2; NaHCO3 22; CaCl2 1; d-glucose 5.5; equilibrated with 95% O2/5% CO2; pH 7.4 at 4°C. The vessels were freed of connective tissue under a dissecting microscope. 10 3 1.5 mm strips were suspended in an organ bath containing 5 mL of the medium kept at 37°C. To obtain renal resistance vessels the kidney was removed, incised from cortex to medulla, washed, and kept unfolded at a temperature of 4°C in the solution mentioned above. By microdissection, renal proximal resistance vessels with internal diameters between 200 mm and 250 mm were prepared from the medulla under a dissecting microscope. The active force and wall tension of the vasculature was measured under isometric conditions using the methodology recently described.13,14 Briefly, the vessel was mounted by wires between the two vessel supports of the vessel myograph that were facing each other. The right vessel support was movably connected to a force transducer (Swema, Stockholm, Sweden, maximum amplitude 6 25 mN). The left vessel support was linked to a linear slide that was attached to a micrometer to adjust the resting wall tension of the vessel. The aortic strips were equilibrated for 20 min. By stretching the aortic strips the resting tension was set to 10 mN. Stimulation of the aortic strips with 130 mmol/L potassium was repeated every 15 min until a reproducible contractile response was obtained. An isolated renal resistance vessel segment was threaded onto two parallel 40 mm steel wires that were attached to the two vessel supports of the vessel myograph. The vasoconstriction was analyzed after normalization of the data with respect to control responses to 130 mmol/L potassium, because even if the strips had the same width, there still remains the possibility of a different cross-sectional area. The vasoconstriction was also measured in the presence of 10 mmol/L nifedipine or in the absence of extracellular calcium. For calcium free medium, calcium was omitted and 50 mmol/L ethyleneglycol-bis-amino-ethylether-N,N9-
Spectrophotometric Measurements of Cytosolic Free Calcium Concentration ([Ca21]i) in VSMC Measurements of [Ca21]i were performed using the calcium-sensitive dye fura2 using VSMC grown on round coverslips with a diameter of 13 mm according to previously published methodology.19 –21 Briefly, VSMC on cover slips (2.5 z 104 cells/cover slip) were washed twice in physiological salt solution (PSS, containing in mmol/L: NaCl 135, KCl 5, CaCl2 1, MgCl2 1, d-glucose 5.5, N-2-hydroxyethyl-piperazine-N9-2-ethanesulfonic acid (HEPES) 10, pH 7.4) and then incubated with 2 mmol/L cell-permeant fura2-acetoxymethylester (Calbiochem, Bad Soden, Germany) and
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0.02% (wt/vol) nonionic surfactant pluronic F-127 (Molecular Probes, Eugene, OR) for 60 min at 37°C. Additional experiments showed that the administration of pluronic at the concentrations used to facilitate the loading of fura2 into VSMC did not change cellular [Ca21]i. After loading of the cells with fura2 the experiments were continued only when a viability higher than 95% was observed by trypan blue exclusion. At the end of the loading period, the coverslips were washed twice in physiological saline solution (PSS) and inserted into quartz glass cuvettes with 2 mL PSS. The fluorescence intensity of fura2-loaded VSMC was measured at 37°C using a spectrofluorophotometer RF-5001 PC (Shimadzu, Tokyo, Japan) equipped with a thermostatically controlled cuvette holder, and with intracellular calcium measurement software (Shimadzu). The light source was a 150-W xenon lamp with ozone self-dissociation function. The wavelength drive motors and slit control motors were operated by the computer. The wavelength accuracy was better than 65 nm. Output signals from the monitor detector and fluorescence detector (photomultiplier) were processed via the analog-to-digital converter. The complete intracellular hydrolysis of fura2acetoxymethylester to fura2 was judged by changes in the excitation and emission spectra. The fluorescence of fura2 was measured using a data sampling interval of 1 s with alternate excitation wavelengths of 340 nm and 380 nm (bandwidth, 5 nm), and emission was collected at 510 nm (bandwidth, 5 nm). Autofluorescence was measured in similar cells that had not been loaded with fura2-acetoxymethylester and was less than 5% of the total fluorescence of fura2 loaded VSMC. After the subtraction of autofluorescence for each wavelength, the ratio (R) of the measured fluorescence values at 340 nm and 380 nm excitation was calculated.19,22 The F340nm/F380nm excitation ratio of resting VSMC remained constant during the whole experiment, indicating a stable resting [Ca21]i in VSMC. Calibration of the fluorescence signal in terms of [Ca21]i was performed with digitonin and ethylene glycol-bis-(b-aminoethyl ether)-N,N,N9N9-tetraacetic acid (EGTA) at each cover slip. 1 mmol/L digitonin and 5 mmol/L EGTA were sequentially added to determine the maximum (Rmax) and the minimum (Rmin) of the F340nm/F380nm excitation ratio, respectively. Control experiments confirmed that further increase of the digitonin or EGTA concentration had no effect on Rmax or Rmin, respectively. [Ca21]i was calculated following the equation of Grynkiewicz et al:19 [Ca21]i 5 K 3 (R-Rmin)/(Rmax-R); K stands for KD 3 Fmin380/ Fmax380, the latter representing the ratio of the fluorescence at F380nm excitation measured in EGTA plus digitonin to that measured in 1 mmol/L external calcium plus digitonin, and KD represents the dissociation constant of fura2 for calcium, which was set to be
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224 nmol/L according to Grynkiewicz et al.19 Leakage during the measurement was less than 4% of the total fluorescence as observed by quenching external fluorescence with 100 mmol/L MnCl2 as described elsewhere.23 Purification of the Diadenosine Polyphosphates The diadenosine polyphosphates, AP4A, AP5A, and AP6A, were used after purification of the preparations obtained from Sigma Chemical Co. (Deisenhofen, Germany) according to the previously described methods.3 The high pressure liquid chromatography (HPLC) equipment consisted of a L-6200 gradient pump (Merck, Darmstadt, Germany), coupled to a Rheodyne injector (Latek, Heidelberg, Germany) an UV-HPLC detector (Lambda-Max 481, Waters), a twochannel compensation recorder (Pharmacia Biosystems, Freiburg, Germany), and a RediFrac fraction collector (Pharmacia Biosystems, Freiburg, Germany). A Mono Q HR 5/5 (Cl2-form) anion-exchange column from Pharmacia (Freiburg, Germany) and a Lichrospher RP select B reversed phase column (4 mm 3 250 mm) from Merck (Darmstadt, Germany) were used. HPLC-grade water and HPLC-grade acetonitrile were from Baker (Groß-Gerau, Germany). Diadenosine polyphosphates, 10 mg, dissolved in 1 mL of eluent A, were purified with an anion exchange column. Eluent A was made up of 10 mmol/L K2HPO4 (pH 8) in water and eluent B of 10 mmol/L K2HPO4 (pH 8) with 1 mol/L NaCl. The following gradient was run: 0 to 11 min: 100% A, 11 to 21 min 0 to 15% B, 21 to 71 min: 15 to 40% B. The main UV absorbing peak (measured at 254 nm) was collected and concentrated to dryness in a Speed-Vac concentrator. Next the sample was dissolved in 1 mL eluent C (40 mmol/L triethylammonium acetate in water) and chromatographed on a reversed phase column. Elution was performed from 0 to 20 min with 100% eluent C, with a linear gradient in 4 min from 0 to 2% eluent D (80% acetonitrile), in 46 min from 2% to 7% eluent D, in 6 min from 7% to 60% eluent D and in 1 min from 60% to 100% eluent D with a flow rate of 0.5 mL/min. The main UV-absorbing peak was collected and the amount of the purified dinucleotide was calculated after UV-spectroscopic measurement of the absorbance of the sample. The fraction was divided into fractions containing 1 mg of the dinucleotide and then concentrated to dryness in a Speed-Vac concentrator. The purified samples were analyzed using matrix-assisted laser desorption/ionisation mass spectrometry (MALDI-MS).24 A reflectortype time-of-flight mass spectrometer, equipped with a nitrogen laser (337 nm, pulselength, 4 ns) was used for ion generation and mass analysis. After purification, MALDI-MS revealed a single mass peak of 837, 917, and 997 Da, which could be attributed to AP4A, AP5A, and AP6A, respectively. AP4A, AP5A, AP6A, adenosine triphosphate (ATP), adenosine diphosphate
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(ADP), the P2x purinoceptor agonist, a,b-methyleneATP, and all other substances were obtained from Sigma Chemical Co. (Deisenhofen, Germany) if not indicated otherwise. Statistics All data are presented as mean 6 standard error. For statistical evaluation of the data Student’s t test was used. Two-tailed P values ,.05 were considered to be significant. Original tracings shown in the figures are representative for at least 5 separate experiments. Original tracings were superimposed on the figures using the computer software GraphPad Prism 2.0 (GraphPad Software Inc., San Diego, CA). RESULTS Effect of Diadenosine Polyphosphates on Vessel Tension Figure 1A shows typical tracings of the vasoconstriction of isolated renal resistance vessels after administration of AP4A, AP5A, or AP6A. Diadenosine polyphosphates increased the tension of isolated renal resistance vessels at concentrations above 0.1 mmol/L. The administration of 10 mmol/L AP4A, AP5A, or AP6A significantly increased the force of isolated renal resistance vessels by 3.48 6 0.43 mN (n 5 8), 2.14 6 0.40 mN (n 5 12), or 2.70 6 0.31, mN (n 5 11, each P , .01 compared with resting tension), respectively. The concentration–response curves of AP4A, AP5A, or AP6A induced vasoconstriction of renal resistance vessels are shown in Figure 1B. In addition the concentration–response curves of AP4A, AP5A, or AP6A induced vasoconstriction of isolated aortic strips are shown. The administration of 10 mmol/L AP4A, AP5A, or AP6A significantly increased the force of isolated aortic strips by 2.45 6 0.97 mN (n 5 10), 2.70 6 0.30 mN (n 5 6), or 1.48 6 0.20 mN (each P , .01 compared with resting tension). Using renal resistance vessels the apparent median effective concentrations (EC50) values were 0.80 mmol/L, 0.80 mmol/L, and 0.56 mmol/L for AP4A, AP5A, and AP6A, respectively. Using isolated aortic strips the apparent EC50 values were 1.02 mmol/L, 5.46 mmol/L, and 6.69 mmol/L for AP4A, AP5A, and AP6A, respectively. The vasoconstriction was also analyzed after normalization of the data with respect to control responses to 130 mmol/L potassium, because even if the strips had the same width, there still remains the possibility of a different cross-sectional area. After normalization of the data with respect to control responses to 130 mmol/L potassium it appeared that the response to diadenosine polyphosphates is more pronounced in renal resistance vessels compared to isolated aortic strips (Figure 2A). The vasoconstriction was significantly higher in renal resistance vessels compared to isolated aortic strips for AP4A (70 6 8%, n 5 8, v 26 6 6%, n 5 10, P , .01), AP5A (43% 6 3%, n 5 12, v 27% 6 4%, n 5 6; P , .05), or AP6A (54% 6 6%, n 5 11, v 17% 6 4%, n 5 11, P , .01), respectively.
FIGURE 1. Diadenosine polyphosphate (APxA) induced vasoconstriction in renal resistance vessels and aorta. A. The contractile response of isolated renal resistance vessels from Wistar-Kyoto rats was measured under isometric conditions using a vessel myograph. The tracings are representative of 6 to 12 similar experiments. B. Concentration–response curves. The contractile responses of isolated aortic strips (upper panel) or renal resistance vessels (lower panel) are shown. Data were plotted as percentage of the maximal response to each diadenosine polyphosphate. Ctrl, control; AP4A, diadenosine tetraphosphate, filled squares; AP5A, diadenosine pentaphosphate, filled circles; AP6A, diadenosine hexaphosphate, open circles.
As shown in Figure 2B the vasoconstriction induced by AP4A was dependent on the presence of extracellular calcium. In the absence of extracellular calcium the AP4A induced vasoconstriction of renal resistance vessels was significantly reduced from 70% 6 8%, n 5
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FIGURE 2. Bar graph shows summary data of the maximum effects of diadenosine polyphosphates on the tension of isolated aortic strips and renal resistance vessels. A. The contractile responses of isolated aortic strips (open bars) or renal resistance vessels (filled bars) from Wistar-Kyoto rats were measured under isometric conditions using a vessel myograph. AP4A, diadenosine tetraphosphate; AP5A, diadenosine pentaphosphate AP6A, diadenosine hexaphosphate. *P , .05, **P , .01 compared with aortic strips. B. The AP4A induced vasoconstriction was measured under control conditions, in the absence of extracellular calcium (Calcium-free), in the presence of 10 mmol/L nifedipine, or in the presence of the P2 purinoceptor blocker pyridoxal-phosphate-6azophenyl-29,49-disulphonic acid (PPADS, 10 mmol/L). Data represent mean 6 SEM from 6 to 12 similar experiments. **P , .01 compared to control conditions.
8, to 28% 6 7%, n 5 8, P , .01). The AP4A induced vasoconstriction was also significantly reduced in the presence of 10 mmol/L of the calcium channel blocker nifedipine (9% 6 1%, n 5 5; P , .01). The administration of the P2 purinoceptor blocker pyridoxal-phosphate-6-azophenyl-29,49-disulphonic acid (PPADS) significantly reduced the AP4A induced vasoconstriction to 1.4 6 0.5%, n 5 6, P , .01). These data indicate that diadenosine polyphosphate produced vasoconstriction after interaction with P2 purinoceptors via activation of calcium influx through calcium channels. For further characterization of the changes of cytosolic calcium after the administration of diadenosine polyphosphates [Ca21]i was measured in vascular smooth muscle cells.
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Effect of Diadenosine Polyphosphates on [Ca21]i in VSMC The cytosolic free calcium concentration ([Ca21]i) in resting cultured VSMC was 80 6 4 nmol/L (n 5 71). The administration of AP4A, AP5A, or AP6A significantly increased [Ca21]i in VSMC (Figure 3). The administration of 10 mmol/L AP4A, AP5A, or AP6A significantly increased [Ca21]i in VSMC to a peak concentration of 314 6 60 nmol/L (n 5 6), 247 6 25 nmol/L (n 5 15), or 332 6 100 nmol/L (n 5 5), respectively (each P , .01 compared with resting value; Figure 3B). The sustained [Ca21]i increase after administration of 10 mmol/L AP4A, AP5A, or AP6A was 183 6 41 nmol/L (n 5 6), 149 6 15 nmol/L (n 5 15), or 146 6 44 nmol/L (n 5 5), respectively. 10 mmol/L adenosine triphosphate (ATP) increased [Ca21]i in VSMC to a peak concentration of 301 6 59 nmol/L (n 5 7, P , .01), whereas the agonist of P2x purinoceptors a,b-methylene adenosine triphosphate (a,b-methylene ATP) had no significant effect on [Ca21]i in VSMC (87 6 10 nmol/L; n 5 5). 10 mmol/L adenosine diphosphate (ADP) increased [Ca21]i in VSMC to a peak concentration of 286 6 25 nmol/L (n 5 5; P , .05). The sustained [Ca21]i increase after administration of ATP or ADP was 171 6 39 nmol/L (n 5 7, P , .05), or 155 6 21 nmol/L (n 5 5, P , .05), respectively. As indicated in Figure 3C the AP4A induced [Ca21]i increase in VSMC was significantly reduced in the absence of extracellular calcium from 314 6 60 nmol/L (n 5 6) to 93 6 10 nmol/L (n 5 5; P , .01). In addition, the administration of the P2 purinoceptor PPADS significantly reduced the AP4A induced [Ca21]i increase to 96 6 6 nmol/L (n 5 5; P , .01). The calcium channel blocker nifedipine could not be used in that setting due to its autofluorescence interfering with the spectrophotometric measurements. DISCUSSION The results show that the diadenosine polyphosphates, AP4A, AP5A, and AP6A induce vasoconstriction in both large arteries and renal resistance arteries. The findings further support the concept that diadenosine polyphosphates may contribute to blood pressure regulation. Moreover, the results show that resistance vessels also were contracted by diadenosine polyphosphates at concentrations that are liberated during the platelet release reaction.1,2 The release of diadenosine polyphosphates during platelet aggregation may produce sufficient extracellular concentrations of diadenosine polyphosphates to elicit vasoconstriction. Although up to now no plasma levels of diadenosine polyphosphates have been reported, platelet diadenosine polyphosphates concentrations may be in the micromolar range,25 and by thrombin about 80% of the diadenosine polyphosphates are liberated during the platelet release reaction.1,2 The present study shows that diadenosine poly-
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FIGURE 3. Effect of diadenosine polyphosphates and related compounds on cytosolic free calcium concentration ([Ca21]i) in cultured rat vascular smooth muscle cells (VSMC). [Ca21]i was measured spectrophotometrically using fura2. A. Original tracings. AP4A, diadenosine tetraphosphate; AP5A, diadenosine pentaphosphate AP6A, diadenosine hexaphosphate. The figure shows representative tracings of 5 to 11 similar experiments. B. Summary data of the maximum effects of AP4A, AP5A, AP6A, adenosine triphosphate (ATP), a,b-methylene-ATP, or adenosine diphosphate (ADP) on [Ca21]i. Control indicates the [Ca21]i in resting VSMC. C. The AP4A induced [Ca21]i increase was measured under control conditions, in the absence of extracellular calcium (Calcium-free), or in the presence of the P2 purinoceptor blocker pyridoxal-phosphate-6-azophenyl-29,49-disulphonic acid (PPADS, 10 mmol/L). Data represent mean 6 SEM. **P , .01 compared with control conditions.
phosphates act via activation of transplasmamembrane calcium influx. Both the diadenosine polyphosphates induced vasoconstriction and changes of [Ca21]i were significantly reduced in the absence of extracellular calcium. The experiments show that the increase in [Ca21]i induced by diadenosine polyphos-
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phates is almost exclusively due to a transplasmamembrane Ca21 influx, whereas Ca21 release from cellular stores does not appear to be involved in diadenosine polyphosphate-induced changes of [Ca21]i in VSMC from rats. Similar findings have been reported in human fibroblast cells. In other cell types, diadenosine polyphosphates also elicit a release of intracellular stored calcium.26 Which receptors are activated by the diadenosine polyphosphates? There are several reports suggesting that the vasoconstrictive response may be mediated by vascular P2 purinoceptors. The finding that both the diadenosine polyphosphates-induced vasoconstriction and [Ca21]i increase was significantly reduced in the presence of a P2 purinergic receptor blocker, PPADS, is in accordance with that view. The experiments using a,b-methylene-adenosine triphosphate (a,b-methylene-ATP), which was formerly thought to be a specific agonist of P2x purinoceptors, might be surprising at first sight. However, recent data show that the P2x(2) and P2x(5) purinoceptors that have been identified in myenteric neurons and PC12 cells are insensitive to a,b-methylene-ATP, are blocked by PPADS, and are related to calcium influx.27,28 Therefore it might be concluded that diadenosine polyphosphates act on vessels after activation of P2x(2) or P2x(5) receptors. Furthermore, when comparing the vasoconstrictive effects of diadenosine polyphosphates in large arteries and renal resistance arteries, marked differences are found. After normalization of the data with respect to control responses to 130 mmol/L potassium it appeared that the responses to diadenosine polyphosphates were significantly more pronounced in renal resistance vessels compared to isolated aortic strips. As indicated by the apparent EC50 values, the renal resistance arteries showed an increased sensitivity to diadenosine polyphosphates. One explanation of these differences may be the different distribution or density of purinoceptors on different vessels. It is known that diadenosine polyphosphates activate different receptors depending on the number of phosphate groups. Diadenosine polyphosphates simultaneously activate several different vascular purinoceptors. Recently, AP4A has been shown to be a coronary and mesenterial dilator,29 whereas the present study indicates that it is an aortic and renal vasoconstrictor. Activation of P2x receptors produces vasoconstriction, whereas the vasodilatory effect of AP4A may be mediated by recently, not completely characterized receptors. Differences of receptor density and receptor distribution in different organs may be responsible for the different action of AP4A in different vascular beds. The use of receptor binding analysis could be useful to assess the contribution of the relative receptor density. A detailed discussion on
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the involved mechanisms and receptors have recently been published.30 In summary, the present study emphasizes that endogenous platelet-derived diadenosine polyphosphates produce vasoconstriction in large arteries and renal resistance vessels by activation of purinoceptors leading to an increase of [Ca21]i. REFERENCES 1.
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effect of recombinant human erythropoietin on renal resistance vessels. Kidney Int 1991;39:259 –265. Franks DJ, Plamondon J, Hamet P: An increase in adenylate cyclase activity precedes DNA synthesis in cultured vascular smooth muscle cells. J Cell Physiol 1984; 119:41– 45. Ross R: The smooth muscle cell. II. Growth of smooth muscle in culture and formation of elastic fibers. Cell Biol 1971;50:172–186. Zhu Z, Tepel M, Neusser M, et al: Effect of captopril on vasoconstriction and Ca21 fluxes in aortic smooth muscle. Hypertension 1993;22:806 – 811. Bendhack LM, Sharma RV, Bhalla RC: Altered signal transduction in vascular smooth muscle cells of spontaneously hypertensive rats. Hypertension 1992;19: II42–II48. Grynkiewicz G, Poenie M, Tsien RY: A new generation of Ca21 indicators with greatly improved fluorescence properties. J Biol Chem 1985;260:3440 –3450. Zhu Z, Tepel M, Neusser M, Zidek W: The role of Na1-Ca21 exchange on agonist-induced changes of cytosolic Ca21 in vascular smooth muscle cells. Am J Physiol 1994;266:C794 –C799. Tepel M, Heidenreich S, Zhu Z, et al: Captopril inhibits the agonist-induced increase of cytosolic free Ca21 in glomerular mesangial cells. Kidney Int 1994;46:696 – 702. Williams DA, Fogarty KE, Tsien RY, Fay FS: Calcium gradients in single smooth muscle cells revealed by the digital imaging microscope using fura-2. Nature 1985; 318:558 –561. Simonson MS, Dunn MJ: Ca21 signaling by distinct endothelin peptides in glomerular mesangial cells. Exp Cell Res 1991;192:148 –156. Nordhoff E, Ingendoh A, Cramer R, Karas M, et al: Matrix-assisted laser desorption/ionization mass spectrometry of nucleic acids with wavelengths in the ultraviolet and infrared. Rapid Comm Mass Spectrom. 1992;6:771–776. McLennan AG: Ap4A and Other Dinucleoside Polyphosphates. CRC Press, Ann Arbor, 1992. Green AK, Dixon CJ, McLennan AG, et al: Adenine dinucleotide-mediated cytosolic free Ca21 oscillations in single hepatocytes. FEBS Lett 1993;322:197–200. Zhou XP, Galligan JJ: P2x purinoceptors in cultured myenteric neurons of guinea pig small intestine. J Physiol (Lond) 1996;486:719 –729. Michel AD, Grahames CBA, Humphrey PPA: Functional characterisation of P2 purinoceptors in PC12 cells by measurement of radiolabelled calcium influx. Naunyn-Schmiedebergs Arch Pharmacol 1996;354:562– 571. Ralevic V, Burnstock G: Effects of purines and pyrimidines on the rat mesenteric arterial bed. Circ Res 1991; 69:1583–1590. van der Giet M, Khattab M, Bo¨rgel J, et al: Differential effects of diadenosine phosphates on purinoceptors in the rat isolated perfused kidney. Br J Pharmacol 1997; 120:1453–1460.
1997;10:1404 –1410
Diadenosine Polyphosphates’ Action on Calcium and Vessel Contraction Martin Tepel, Joachim Jankowski, Hartmut Schlu¨ter, Ju¨rgen Bachmann, Marcus van der Giet, Christian Ruess, Jo¨rg Terliesner, and Walter Zidek
The effects of the endogenous, platelet-derived vasoactive compounds, diadenosine tetraphosphate (AP4A), diadenosine pentaphosphate (AP5A), and diadenosine hexaphosphate (AP6A) on the vasoconstriction of isolated rat renal resistance vessels and rat aortic strips were measured using a vessel myograph. In addition, the effects of AP4A, AP5A, and AP6A on the cytosolic free calcium concentration ([Ca21]i) were evaluated in cultured rat vascular smooth muscle cells (VSMC) using the fluorescent dye technique. Diadenosine polyphosphates dose-dependently increased the force of renal resistance vessels and isolated aortic strips. The administration of 10 mmol/L AP4A, AP5A, or AP6A significantly increased the force of isolated renal resistance vessels by 3.48 6 0.43 mN (n 5 8), 2.14 6 0.40 mN (n 5 12), or 2.70 6 0.31 mN (n 5 11, each P < .01 compared with resting tension), respectively. The administration of 10 mmol/L AP4A, AP5A, or AP6A significantly increased the force of isolated aortic strips by
2.45 6 0.97 mNewton (n 5 10), 2.70 6 0.30 mN (n 5 6), or 1.48 6 0.20 mN (each P < .01 compared with resting tension), respectively. The administration of 10 mmol/L AP4A, AP5A, or AP6A significantly increased [Ca21]i in VSMC to a peak concentration of 314 6 60 nmol/L (n 5 6), 247 6 25 nmol/L (n 5 15), or 332 6 100 nmol/L (n 5 5), respectively (each P < .01 compared with resting value). Both the diadenosine polyphosphateinduced vasoconstriction and [Ca21]i increase was significantly reduced in the absence of extracellular calcium or after administration of a specific inhibitor of P2 purinoceptors. It is concluded that diadenosine polyphosphates increase [Ca21]i and hence cause vessel constriction. Am J Hypertens 1997;10:1404 –1410 © 1997 American Journal of Hypertension, Ltd.
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in vivo.1,2 Both AP5A and AP6A were shown to constrict renal vasculature,2 to stimulate growth, and to increase cytosolic free calcium concentration ([Ca21]i) in renal mesangial cells.3–5 To further assess the role of AP5A and AP6A in blood pressure regulation, the action of both agents on the tone of a large artery and of resistance arteries was compared in the present study. Furthermore, it is recognized that cytosolic free calcium concentration ([Ca21]i) is a major component in signal transduction and a major regulator of vascular smooth muscle force generation.6 – 8 Therefore the effects of these diadenosine polyphosphates on [Ca21]i were related to the vasoconstrictive effects, and were
ecently, endogenous platelet-derived vasoconstrictors, diadenosine pentaphosphate (AP5A) and diadenosine hexaphosphate (AP6A), have been identified.1,2 Platelet-derived diadenosine polyphosphates have been related to the local and systemic regulation of blood pressure
Received February 14, 1997. Accepted June 24, 1997. From the Universita¨tsklinik Marienhospital, Medizinische Klinik I, Ruhr-Universita¨t-Bochum, Herne, Germany. Address correspondence and reprint requests to Dr. M. Tepel, Medizinische Klinik I, Universita¨tsklinik Marienhospital, Ruhr-Universita¨t-Bochum, Ho¨lkeskampring 40, D-44625 Herne, Germany.
© 1997 by the American Journal of Hypertension, Ltd. Published by Elsevier Science, Inc.
KEY WORDS:
Diadenosine polyphosphates, cytosolic free Ca21, vascular smooth muscle cells, vasoconstriction.
0895-7061/97/$17.00 PII S0895-7061(97)00305-1
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compared with that of diadenosine tetraphosphate (AP4A), a known coronary and mesenterial vasodilator.9,10 The experiments showed that the vasoconstrictive effects of the diadenosine polyphosphates were even more pronounced in resistance vessels.
tetraacetic acid was added. The P2 purinoceptor blocker pyridoxal-phosphate-6-azophenyl-29,49-disulphonic acid (PPADS, Research Biochemical International, Cologne, Germany) was used at a concentration of 10 mmol/L.
METHODS
Cell Culture of Vascular Smooth Muscle Cells Vascular smooth muscle cells (VSMC) were obtained from thoracic aortas of Wistar-Kyoto rats and cultured by the tissue explant method according to published procedures.15–17 Briefly, cells were incubated in Dulbecco’s modified Eagle’s medium (Gibco, Eggenstein, Germany), containing 10% (vol/vol) fetal calf serum (Boehringer, Mannheim, Germany), 100 U/mL penicillin G, and 100 mg/mL streptomycin. Cultures were incubated at 37°C in a humidified atmosphere of 95% air and 5% CO2. The medium was changed initially after 24 h and then every 2 to 3 days. After the first subculture, cells were subcultured every week at a seeding density of about 1 3 104 cells/cm2 and reached confluence in 8 to 10 days. Thereafter they were harvested by adding 0.05% trypsin, and the culture was continued up to 8 passages as previously described.18 Separate experiments confirmed recent reports that resting [Ca21]i in cultured VSMC was not significantly different in these passages.7 To ascertain that cultured cells were VSMC, immunocytochemical localization of smooth muscle-specific a-actin was carried out using monoclonal antibodies ASM-1 (Progen, Heidelberg, Germany) raised against smooth muscle a-actin and labeled with a fluorescence marker.18 Staining of cultured VSMC with that antibody revealed that all cells in the preparation were labeled, and actin stress fibers were seen throughout the cytosol. It was confirmed that cultured VSMC were free from contamination with endothelial cells or fibroblasts by immunocytochemical staining of cells with antibodies against von Willebrand factor coupled to a fluorescence marker. A viability of VSMC higher than 95% was observed by trypan blue exclusion. Cells were made quiescent by incubation in serum free medium containing 0.1% bovine serum albumin, 100 U/mL penicillin and 100 mg/mL streptomycin for 48 h prior to [Ca21]i measurements.
Preparation of Aortic Strips and Renal Resistance Vessels Six-month-old male Wistar-Kyoto rats from the Mu¨nster strain, as described previously,11 were fed a standard pellet diet and water ad libitum. The vasoconstrictions of aortic strips or renal resistance vessels from male Wistar-Kyoto rats were measured using a vessel myograph introduced by Mulvany and Nyborg.12 The abdominal aorta was excised and transferred into a medium containing (mmol/L) NaCl 115; KCl 4.6; MgSO4 1.2; NaH2PO4 1.2; NaHCO3 22; CaCl2 1; d-glucose 5.5; equilibrated with 95% O2/5% CO2; pH 7.4 at 4°C. The vessels were freed of connective tissue under a dissecting microscope. 10 3 1.5 mm strips were suspended in an organ bath containing 5 mL of the medium kept at 37°C. To obtain renal resistance vessels the kidney was removed, incised from cortex to medulla, washed, and kept unfolded at a temperature of 4°C in the solution mentioned above. By microdissection, renal proximal resistance vessels with internal diameters between 200 mm and 250 mm were prepared from the medulla under a dissecting microscope. The active force and wall tension of the vasculature was measured under isometric conditions using the methodology recently described.13,14 Briefly, the vessel was mounted by wires between the two vessel supports of the vessel myograph that were facing each other. The right vessel support was movably connected to a force transducer (Swema, Stockholm, Sweden, maximum amplitude 6 25 mN). The left vessel support was linked to a linear slide that was attached to a micrometer to adjust the resting wall tension of the vessel. The aortic strips were equilibrated for 20 min. By stretching the aortic strips the resting tension was set to 10 mN. Stimulation of the aortic strips with 130 mmol/L potassium was repeated every 15 min until a reproducible contractile response was obtained. An isolated renal resistance vessel segment was threaded onto two parallel 40 mm steel wires that were attached to the two vessel supports of the vessel myograph. The vasoconstriction was analyzed after normalization of the data with respect to control responses to 130 mmol/L potassium, because even if the strips had the same width, there still remains the possibility of a different cross-sectional area. The vasoconstriction was also measured in the presence of 10 mmol/L nifedipine or in the absence of extracellular calcium. For calcium free medium, calcium was omitted and 50 mmol/L ethyleneglycol-bis-amino-ethylether-N,N9-
Spectrophotometric Measurements of Cytosolic Free Calcium Concentration ([Ca21]i) in VSMC Measurements of [Ca21]i were performed using the calcium-sensitive dye fura2 using VSMC grown on round coverslips with a diameter of 13 mm according to previously published methodology.19 –21 Briefly, VSMC on cover slips (2.5 z 104 cells/cover slip) were washed twice in physiological salt solution (PSS, containing in mmol/L: NaCl 135, KCl 5, CaCl2 1, MgCl2 1, d-glucose 5.5, N-2-hydroxyethyl-piperazine-N9-2-ethanesulfonic acid (HEPES) 10, pH 7.4) and then incubated with 2 mmol/L cell-permeant fura2-acetoxymethylester (Calbiochem, Bad Soden, Germany) and
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0.02% (wt/vol) nonionic surfactant pluronic F-127 (Molecular Probes, Eugene, OR) for 60 min at 37°C. Additional experiments showed that the administration of pluronic at the concentrations used to facilitate the loading of fura2 into VSMC did not change cellular [Ca21]i. After loading of the cells with fura2 the experiments were continued only when a viability higher than 95% was observed by trypan blue exclusion. At the end of the loading period, the coverslips were washed twice in physiological saline solution (PSS) and inserted into quartz glass cuvettes with 2 mL PSS. The fluorescence intensity of fura2-loaded VSMC was measured at 37°C using a spectrofluorophotometer RF-5001 PC (Shimadzu, Tokyo, Japan) equipped with a thermostatically controlled cuvette holder, and with intracellular calcium measurement software (Shimadzu). The light source was a 150-W xenon lamp with ozone self-dissociation function. The wavelength drive motors and slit control motors were operated by the computer. The wavelength accuracy was better than 65 nm. Output signals from the monitor detector and fluorescence detector (photomultiplier) were processed via the analog-to-digital converter. The complete intracellular hydrolysis of fura2acetoxymethylester to fura2 was judged by changes in the excitation and emission spectra. The fluorescence of fura2 was measured using a data sampling interval of 1 s with alternate excitation wavelengths of 340 nm and 380 nm (bandwidth, 5 nm), and emission was collected at 510 nm (bandwidth, 5 nm). Autofluorescence was measured in similar cells that had not been loaded with fura2-acetoxymethylester and was less than 5% of the total fluorescence of fura2 loaded VSMC. After the subtraction of autofluorescence for each wavelength, the ratio (R) of the measured fluorescence values at 340 nm and 380 nm excitation was calculated.19,22 The F340nm/F380nm excitation ratio of resting VSMC remained constant during the whole experiment, indicating a stable resting [Ca21]i in VSMC. Calibration of the fluorescence signal in terms of [Ca21]i was performed with digitonin and ethylene glycol-bis-(b-aminoethyl ether)-N,N,N9N9-tetraacetic acid (EGTA) at each cover slip. 1 mmol/L digitonin and 5 mmol/L EGTA were sequentially added to determine the maximum (Rmax) and the minimum (Rmin) of the F340nm/F380nm excitation ratio, respectively. Control experiments confirmed that further increase of the digitonin or EGTA concentration had no effect on Rmax or Rmin, respectively. [Ca21]i was calculated following the equation of Grynkiewicz et al:19 [Ca21]i 5 K 3 (R-Rmin)/(Rmax-R); K stands for KD 3 Fmin380/ Fmax380, the latter representing the ratio of the fluorescence at F380nm excitation measured in EGTA plus digitonin to that measured in 1 mmol/L external calcium plus digitonin, and KD represents the dissociation constant of fura2 for calcium, which was set to be
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224 nmol/L according to Grynkiewicz et al.19 Leakage during the measurement was less than 4% of the total fluorescence as observed by quenching external fluorescence with 100 mmol/L MnCl2 as described elsewhere.23 Purification of the Diadenosine Polyphosphates The diadenosine polyphosphates, AP4A, AP5A, and AP6A, were used after purification of the preparations obtained from Sigma Chemical Co. (Deisenhofen, Germany) according to the previously described methods.3 The high pressure liquid chromatography (HPLC) equipment consisted of a L-6200 gradient pump (Merck, Darmstadt, Germany), coupled to a Rheodyne injector (Latek, Heidelberg, Germany) an UV-HPLC detector (Lambda-Max 481, Waters), a twochannel compensation recorder (Pharmacia Biosystems, Freiburg, Germany), and a RediFrac fraction collector (Pharmacia Biosystems, Freiburg, Germany). A Mono Q HR 5/5 (Cl2-form) anion-exchange column from Pharmacia (Freiburg, Germany) and a Lichrospher RP select B reversed phase column (4 mm 3 250 mm) from Merck (Darmstadt, Germany) were used. HPLC-grade water and HPLC-grade acetonitrile were from Baker (Groß-Gerau, Germany). Diadenosine polyphosphates, 10 mg, dissolved in 1 mL of eluent A, were purified with an anion exchange column. Eluent A was made up of 10 mmol/L K2HPO4 (pH 8) in water and eluent B of 10 mmol/L K2HPO4 (pH 8) with 1 mol/L NaCl. The following gradient was run: 0 to 11 min: 100% A, 11 to 21 min 0 to 15% B, 21 to 71 min: 15 to 40% B. The main UV absorbing peak (measured at 254 nm) was collected and concentrated to dryness in a Speed-Vac concentrator. Next the sample was dissolved in 1 mL eluent C (40 mmol/L triethylammonium acetate in water) and chromatographed on a reversed phase column. Elution was performed from 0 to 20 min with 100% eluent C, with a linear gradient in 4 min from 0 to 2% eluent D (80% acetonitrile), in 46 min from 2% to 7% eluent D, in 6 min from 7% to 60% eluent D and in 1 min from 60% to 100% eluent D with a flow rate of 0.5 mL/min. The main UV-absorbing peak was collected and the amount of the purified dinucleotide was calculated after UV-spectroscopic measurement of the absorbance of the sample. The fraction was divided into fractions containing 1 mg of the dinucleotide and then concentrated to dryness in a Speed-Vac concentrator. The purified samples were analyzed using matrix-assisted laser desorption/ionisation mass spectrometry (MALDI-MS).24 A reflectortype time-of-flight mass spectrometer, equipped with a nitrogen laser (337 nm, pulselength, 4 ns) was used for ion generation and mass analysis. After purification, MALDI-MS revealed a single mass peak of 837, 917, and 997 Da, which could be attributed to AP4A, AP5A, and AP6A, respectively. AP4A, AP5A, AP6A, adenosine triphosphate (ATP), adenosine diphosphate
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(ADP), the P2x purinoceptor agonist, a,b-methyleneATP, and all other substances were obtained from Sigma Chemical Co. (Deisenhofen, Germany) if not indicated otherwise. Statistics All data are presented as mean 6 standard error. For statistical evaluation of the data Student’s t test was used. Two-tailed P values ,.05 were considered to be significant. Original tracings shown in the figures are representative for at least 5 separate experiments. Original tracings were superimposed on the figures using the computer software GraphPad Prism 2.0 (GraphPad Software Inc., San Diego, CA). RESULTS Effect of Diadenosine Polyphosphates on Vessel Tension Figure 1A shows typical tracings of the vasoconstriction of isolated renal resistance vessels after administration of AP4A, AP5A, or AP6A. Diadenosine polyphosphates increased the tension of isolated renal resistance vessels at concentrations above 0.1 mmol/L. The administration of 10 mmol/L AP4A, AP5A, or AP6A significantly increased the force of isolated renal resistance vessels by 3.48 6 0.43 mN (n 5 8), 2.14 6 0.40 mN (n 5 12), or 2.70 6 0.31, mN (n 5 11, each P , .01 compared with resting tension), respectively. The concentration–response curves of AP4A, AP5A, or AP6A induced vasoconstriction of renal resistance vessels are shown in Figure 1B. In addition the concentration–response curves of AP4A, AP5A, or AP6A induced vasoconstriction of isolated aortic strips are shown. The administration of 10 mmol/L AP4A, AP5A, or AP6A significantly increased the force of isolated aortic strips by 2.45 6 0.97 mN (n 5 10), 2.70 6 0.30 mN (n 5 6), or 1.48 6 0.20 mN (each P , .01 compared with resting tension). Using renal resistance vessels the apparent median effective concentrations (EC50) values were 0.80 mmol/L, 0.80 mmol/L, and 0.56 mmol/L for AP4A, AP5A, and AP6A, respectively. Using isolated aortic strips the apparent EC50 values were 1.02 mmol/L, 5.46 mmol/L, and 6.69 mmol/L for AP4A, AP5A, and AP6A, respectively. The vasoconstriction was also analyzed after normalization of the data with respect to control responses to 130 mmol/L potassium, because even if the strips had the same width, there still remains the possibility of a different cross-sectional area. After normalization of the data with respect to control responses to 130 mmol/L potassium it appeared that the response to diadenosine polyphosphates is more pronounced in renal resistance vessels compared to isolated aortic strips (Figure 2A). The vasoconstriction was significantly higher in renal resistance vessels compared to isolated aortic strips for AP4A (70 6 8%, n 5 8, v 26 6 6%, n 5 10, P , .01), AP5A (43% 6 3%, n 5 12, v 27% 6 4%, n 5 6; P , .05), or AP6A (54% 6 6%, n 5 11, v 17% 6 4%, n 5 11, P , .01), respectively.
FIGURE 1. Diadenosine polyphosphate (APxA) induced vasoconstriction in renal resistance vessels and aorta. A. The contractile response of isolated renal resistance vessels from Wistar-Kyoto rats was measured under isometric conditions using a vessel myograph. The tracings are representative of 6 to 12 similar experiments. B. Concentration–response curves. The contractile responses of isolated aortic strips (upper panel) or renal resistance vessels (lower panel) are shown. Data were plotted as percentage of the maximal response to each diadenosine polyphosphate. Ctrl, control; AP4A, diadenosine tetraphosphate, filled squares; AP5A, diadenosine pentaphosphate, filled circles; AP6A, diadenosine hexaphosphate, open circles.
As shown in Figure 2B the vasoconstriction induced by AP4A was dependent on the presence of extracellular calcium. In the absence of extracellular calcium the AP4A induced vasoconstriction of renal resistance vessels was significantly reduced from 70% 6 8%, n 5
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FIGURE 2. Bar graph shows summary data of the maximum effects of diadenosine polyphosphates on the tension of isolated aortic strips and renal resistance vessels. A. The contractile responses of isolated aortic strips (open bars) or renal resistance vessels (filled bars) from Wistar-Kyoto rats were measured under isometric conditions using a vessel myograph. AP4A, diadenosine tetraphosphate; AP5A, diadenosine pentaphosphate AP6A, diadenosine hexaphosphate. *P , .05, **P , .01 compared with aortic strips. B. The AP4A induced vasoconstriction was measured under control conditions, in the absence of extracellular calcium (Calcium-free), in the presence of 10 mmol/L nifedipine, or in the presence of the P2 purinoceptor blocker pyridoxal-phosphate-6azophenyl-29,49-disulphonic acid (PPADS, 10 mmol/L). Data represent mean 6 SEM from 6 to 12 similar experiments. **P , .01 compared to control conditions.
8, to 28% 6 7%, n 5 8, P , .01). The AP4A induced vasoconstriction was also significantly reduced in the presence of 10 mmol/L of the calcium channel blocker nifedipine (9% 6 1%, n 5 5; P , .01). The administration of the P2 purinoceptor blocker pyridoxal-phosphate-6-azophenyl-29,49-disulphonic acid (PPADS) significantly reduced the AP4A induced vasoconstriction to 1.4 6 0.5%, n 5 6, P , .01). These data indicate that diadenosine polyphosphate produced vasoconstriction after interaction with P2 purinoceptors via activation of calcium influx through calcium channels. For further characterization of the changes of cytosolic calcium after the administration of diadenosine polyphosphates [Ca21]i was measured in vascular smooth muscle cells.
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Effect of Diadenosine Polyphosphates on [Ca21]i in VSMC The cytosolic free calcium concentration ([Ca21]i) in resting cultured VSMC was 80 6 4 nmol/L (n 5 71). The administration of AP4A, AP5A, or AP6A significantly increased [Ca21]i in VSMC (Figure 3). The administration of 10 mmol/L AP4A, AP5A, or AP6A significantly increased [Ca21]i in VSMC to a peak concentration of 314 6 60 nmol/L (n 5 6), 247 6 25 nmol/L (n 5 15), or 332 6 100 nmol/L (n 5 5), respectively (each P , .01 compared with resting value; Figure 3B). The sustained [Ca21]i increase after administration of 10 mmol/L AP4A, AP5A, or AP6A was 183 6 41 nmol/L (n 5 6), 149 6 15 nmol/L (n 5 15), or 146 6 44 nmol/L (n 5 5), respectively. 10 mmol/L adenosine triphosphate (ATP) increased [Ca21]i in VSMC to a peak concentration of 301 6 59 nmol/L (n 5 7, P , .01), whereas the agonist of P2x purinoceptors a,b-methylene adenosine triphosphate (a,b-methylene ATP) had no significant effect on [Ca21]i in VSMC (87 6 10 nmol/L; n 5 5). 10 mmol/L adenosine diphosphate (ADP) increased [Ca21]i in VSMC to a peak concentration of 286 6 25 nmol/L (n 5 5; P , .05). The sustained [Ca21]i increase after administration of ATP or ADP was 171 6 39 nmol/L (n 5 7, P , .05), or 155 6 21 nmol/L (n 5 5, P , .05), respectively. As indicated in Figure 3C the AP4A induced [Ca21]i increase in VSMC was significantly reduced in the absence of extracellular calcium from 314 6 60 nmol/L (n 5 6) to 93 6 10 nmol/L (n 5 5; P , .01). In addition, the administration of the P2 purinoceptor PPADS significantly reduced the AP4A induced [Ca21]i increase to 96 6 6 nmol/L (n 5 5; P , .01). The calcium channel blocker nifedipine could not be used in that setting due to its autofluorescence interfering with the spectrophotometric measurements. DISCUSSION The results show that the diadenosine polyphosphates, AP4A, AP5A, and AP6A induce vasoconstriction in both large arteries and renal resistance arteries. The findings further support the concept that diadenosine polyphosphates may contribute to blood pressure regulation. Moreover, the results show that resistance vessels also were contracted by diadenosine polyphosphates at concentrations that are liberated during the platelet release reaction.1,2 The release of diadenosine polyphosphates during platelet aggregation may produce sufficient extracellular concentrations of diadenosine polyphosphates to elicit vasoconstriction. Although up to now no plasma levels of diadenosine polyphosphates have been reported, platelet diadenosine polyphosphates concentrations may be in the micromolar range,25 and by thrombin about 80% of the diadenosine polyphosphates are liberated during the platelet release reaction.1,2 The present study shows that diadenosine poly-
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FIGURE 3. Effect of diadenosine polyphosphates and related compounds on cytosolic free calcium concentration ([Ca21]i) in cultured rat vascular smooth muscle cells (VSMC). [Ca21]i was measured spectrophotometrically using fura2. A. Original tracings. AP4A, diadenosine tetraphosphate; AP5A, diadenosine pentaphosphate AP6A, diadenosine hexaphosphate. The figure shows representative tracings of 5 to 11 similar experiments. B. Summary data of the maximum effects of AP4A, AP5A, AP6A, adenosine triphosphate (ATP), a,b-methylene-ATP, or adenosine diphosphate (ADP) on [Ca21]i. Control indicates the [Ca21]i in resting VSMC. C. The AP4A induced [Ca21]i increase was measured under control conditions, in the absence of extracellular calcium (Calcium-free), or in the presence of the P2 purinoceptor blocker pyridoxal-phosphate-6-azophenyl-29,49-disulphonic acid (PPADS, 10 mmol/L). Data represent mean 6 SEM. **P , .01 compared with control conditions.
phosphates act via activation of transplasmamembrane calcium influx. Both the diadenosine polyphosphates induced vasoconstriction and changes of [Ca21]i were significantly reduced in the absence of extracellular calcium. The experiments show that the increase in [Ca21]i induced by diadenosine polyphos-
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phates is almost exclusively due to a transplasmamembrane Ca21 influx, whereas Ca21 release from cellular stores does not appear to be involved in diadenosine polyphosphate-induced changes of [Ca21]i in VSMC from rats. Similar findings have been reported in human fibroblast cells. In other cell types, diadenosine polyphosphates also elicit a release of intracellular stored calcium.26 Which receptors are activated by the diadenosine polyphosphates? There are several reports suggesting that the vasoconstrictive response may be mediated by vascular P2 purinoceptors. The finding that both the diadenosine polyphosphates-induced vasoconstriction and [Ca21]i increase was significantly reduced in the presence of a P2 purinergic receptor blocker, PPADS, is in accordance with that view. The experiments using a,b-methylene-adenosine triphosphate (a,b-methylene-ATP), which was formerly thought to be a specific agonist of P2x purinoceptors, might be surprising at first sight. However, recent data show that the P2x(2) and P2x(5) purinoceptors that have been identified in myenteric neurons and PC12 cells are insensitive to a,b-methylene-ATP, are blocked by PPADS, and are related to calcium influx.27,28 Therefore it might be concluded that diadenosine polyphosphates act on vessels after activation of P2x(2) or P2x(5) receptors. Furthermore, when comparing the vasoconstrictive effects of diadenosine polyphosphates in large arteries and renal resistance arteries, marked differences are found. After normalization of the data with respect to control responses to 130 mmol/L potassium it appeared that the responses to diadenosine polyphosphates were significantly more pronounced in renal resistance vessels compared to isolated aortic strips. As indicated by the apparent EC50 values, the renal resistance arteries showed an increased sensitivity to diadenosine polyphosphates. One explanation of these differences may be the different distribution or density of purinoceptors on different vessels. It is known that diadenosine polyphosphates activate different receptors depending on the number of phosphate groups. Diadenosine polyphosphates simultaneously activate several different vascular purinoceptors. Recently, AP4A has been shown to be a coronary and mesenterial dilator,29 whereas the present study indicates that it is an aortic and renal vasoconstrictor. Activation of P2x receptors produces vasoconstriction, whereas the vasodilatory effect of AP4A may be mediated by recently, not completely characterized receptors. Differences of receptor density and receptor distribution in different organs may be responsible for the different action of AP4A in different vascular beds. The use of receptor binding analysis could be useful to assess the contribution of the relative receptor density. A detailed discussion on
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the involved mechanisms and receptors have recently been published.30 In summary, the present study emphasizes that endogenous platelet-derived diadenosine polyphosphates produce vasoconstriction in large arteries and renal resistance vessels by activation of purinoceptors leading to an increase of [Ca21]i. REFERENCES 1.
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