Published online before print October 2, 2008, doi: 10.1161/CIRCRESAHA.108.177865
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
© 2008 American Heart Association, Inc.
Integrative Physiology |
Adenosine 5'-Tetraphosphate Is a Highly Potent Purinergic Endothelium-Derived Vasoconstrictor
From the Medizinische Klinik IV–Nephrology (M.T., V.J., M.S., A.W., T.H., F.H., W.Z., J. Jankowski, M.v.d.G.) and Insitut für Pathologie (C.L.), Charite–Campus Benjamin Franklin, Berlin, Germany; Department of Biophysics (J.K., J. Jemielity), Institute of Experimental Physics, Warsaw University, Poland; and Department of Biochemistry and Biotechnology (A.G.), University of Life in Sciences Pozna
, Poland.
Correspondence to Prof Dr Markus van der Giet, Charité–Campus Benjamin Franklin, Med. Klinik IV–Nephrology, Hindenburgdamm 30, 12203 Berlin, Germany. E-mail markus.vandergiet{at}charite.de
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Besides serving as a mechanical barrier, the endothelium has important regulatory functions. The discovery of nitric oxide revolutionized our understanding of vasoregulation. In contrast, the identity of endothelium-derived vasoconstrictive factors still remains uncertain. The supernatant from mechanically stimulated human microvascular endothelial cells elicited a potent vasoconstrictive response in the isolated perfused rat kidney. Whereas a nonselective purinoceptor blocker blocked this vasoactivity most potently, the inhibition of the endothelin receptor by BQ123 weakly affected that vasoconstrictive response. As a compound responsible for that vasoconstrictive effect, we have isolated from HMECs and identified the mononucleotide adenosine 5'-tetraphosphate (AP4). This nucleotide proved to be the most potent vasoactive purinergic mediator identified to date, exerting the vasoconstriction predominantly through activation of the P2X1 receptor. The intraarterial application of AP4 in a Wistar–Kyoto rat induced a strong increase of the mean arterial pressure. The plasma concentration of AP4 is in the nanomolar range, which, in vivo, induces a significant change in the mean arterial pressure. To our knowledge, AP4, which exerts vasoactive effects, is the most potent endogenous mononucleotide identified to date in mammals. The effects of AP4, the plasma concentration of AP4, and its release suggest that this compound functions as an important vasoregulator.
Key Words: purinoceptors • P2X • adenosine 5'-tetraphosphate • endothelium-derived contracting factor
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
For more than 2 decades, it has been known that the endothelium plays an essential role in vascular function. In 1980, Furchgott and Zawadzki reported that an essential role of the endothelium on the vascular tone is the induction of vasorelaxation1 resulting from the production and release of nitric oxide (NO).2 After these ground-breaking findings, other endothelium-derived vasorelaxing factors (EDRFs) were isolated, like prostacyclin3 or the endothelium-derived hyperpolarizing factor (EDHF).4 In the last few years, the perception that the endothelium mainly releases vasorelaxing factors to control vascular tone has changed. There is increasing evidence that the endothelial layer also releases potent vasoconstrictive mediators, which are called endothelium-derived vasoconstricting factors (EDCFs). Several EDCFs, like endothelin-1,5 prostaglandins,6 or reactive oxygen species,7 have been identified to date. It is generally accepted that the endothelium serves as a unique mechanoreceptor, sensing and transducing physical stimuli (eg, shear forces, pressure) into changes in vascular tone by the release of EDRFs or EDCFs.8 Under physiological conditions, a precise balance exists within the endothelial excretion of EDCFs and EDRFs. Alterations in this balance result in a local and generalized increase in vascular tone, leading to local vasospasm and hypertension. Furthermore, this facilitates thrombus formation and the progression of inflammatory vascular disease. A disequilibrium within this tightly regulated system is most often characterized by a low EDRF concentration and a high EDCF concentration, which is a key feature of the endothelial dysfunction.8 Therefore, isolation and characterization of yet unknown EDCFs is helpful to understand their relevance in the progression of vascular disease. This might give the opportunity to establish new pharmacological strategies for the prevention of endothelial dysfunction and consecutively cardiovascular disease.
Recently, our group analyzed EDCFs released from stimulated human microvascular endothelial cells (HMECs). It has been shown that more than 70% of vasoconstrictive activity produced by these mechanically stimulated endothelial cells was caused by mono- or dinucleoside polyphosphates.9 Following a specialized isolation procedure, we identified a pyrimidine-containing dinucleoside polyphosphate, uridylyl(5')tetraphospho(5')adenosine (UP4A),9 which has been proven to be a potent EDCF.9,10 The strategy for the isolation of this dinucleoside polyphosphate from supernatants of HMECs eliminated chromatographically mononucleoside polyphosphates from the fractions of interest. Therefore, our previous work focused on the isolation of dinucleoside polyphosphates. Interestingly, the mononucleoside polyphosphate–containing fraction showed even more pronounced vasoactive properties than the dinucleoside polyphosphate–containing one. Therefore, we speculated that HMECs might also secrete potent vasoactive mononucleoside polyphosphates. From previous experiments, we knew that mononucleoside phosphates, such as ATP, ADP, GTP, GDP, UTP, or UDP, show potent vasoactive properties only in millimolar concentrations.9,11 Thus, the existence of other potent vasoconstrictive mononucleoside polyphosphates in the supernatant of HMECs had to be assumed.
In this report, we describe an isolation strategy that allowed us to obtain from the HMEC supernatant a highly potent vasoactive purinergic compound that proved to be adenosine 5'-tetraphosphate (AP4). We also show that AP4 is present in human plasma in nanomolar concentrations, exhibits potent local and systemic blood pressure–increasing effects, and is the most potent endogenous vasoconstrictive purinergic compound currently known.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
An expanded Materials and Methods section is available in the online data supplement at http://circres.ahajournals.org.
Culture and Stimulation of HMECs
Cultivation and stimulation of HMECs were performed as previously published.9
Chromatographic Fractionation of the Supernatants From Endothelial Cells
Triethylammonium acetate (40 mmol/L final concentration) was added to the supernatants and titrated to pH 6.5. Next, 2 C18 reversed-phase columns interconnected in series were used to concentrate the supernatant of endothelial cells. The eluate fractions were frozen at –80°C and lyophilized. In addition, these eluated fractions of the first reversed-phase chromatographic step were purified further by affinity chromatography. The pH of the eluate from the first reversed-phase chromatographic step was adjusted to pH 9.5 and loaded to the affinity column. Nonbinding substances were eluted with the ammonium acetate solution with a flow rate of 1 mL/min. The solution was monitored at 254 nm. Then the sample was thawed, adjusted with 1 mol/L triethylammonium acetate to 40 mmol/L concentration, and injected into a reversed-phase high-performance liquid column (Chromolith Performance RP-18e 100–4.6, Merck) for fractionation and desalting as described before.12
Matrix-Assisted Laser Desorption/Ionization Mass Analysis
The high-performance liquid chromatography (HPLC) fraction with a significant absorption at 254 nm was analyzed by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS).
Extraction of Human Plasma
Peripheral blood (40 mL) was obtained by catheterization of the cubital vein and was collected in tubes containing K2-EDTA (7.2 mg). The blood samples were centrifuged at 2100g for 10 minutes to isolate plasma after a standardized interval of 10 minutes of postsampling. The resulting plasma was deproteinized with 0.6 mol/L (final concentration) perchloric acid and centrifuged (2100g, 4°C, 5 minutes). After adjusting the pH to 9.0 with 5 mol/L KOH, the precipitated proteins and KClO4 were removed by centrifugation (2100g, 4°C, 5 minutes).
Isolation and Identification of AP4 From Human Plasma
Isolation and identification of AP4 was performed using various HPLC purification steps, as described in detail in the online data supplement.
Isolated Perfused Rat Kidney and Arterial Tension Studies
The following procedures were performed in accordance with the Guiding Principles for the Care and Use of Animals in the Field of Physiological Science recommended by the German Physiological Society. The rat kidney was prepared according to van der Giet et al.11,13,14 Arterial tension studies using renal arteries from rats were performed as previously published.15
Measurement of Mean Arterial Blood Pressure
Measurement of mean arterial blood pressure (MAP) was done as previously published.15 Norepinephrine (NE), UP4A, AP4, ATP, and 
,β-meATP were applied intraaortically via a polyethylene catheter inserted into the right carotid artery.
Statistics
Responses were measured as changes in perfusion pressure (mm Hg), and results are presented as means±SEM. Statistical analysis was performed with the Mann–Whitney test. The probability values obtained with this test were corrected for multiple comparisons with Bonferroni correction, where appropriate. All probability values presented are 2-tailed. Probability values <0.05 were considered significant.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Isolation of AP4 From Supernatants From Stimulated HMECs
The supernatant obtained from HMECs stimulated by mechanical stress elicited a vasoconstriction response when injected into an isolated perfused rat kidney model (Figure 1A). Endothelin is a potent vasoactive peptide secreted by endothelial cells5 after stimulation. To verify the contribution of endothelin to the endothelium-derived vasoconstriction induced by supernatant of mechanically stimulated HMECs, we first blocked the endothelin receptor A (ET-A) with the highly specific antagonist BQ123 (1 µmol/L). The contribution of endothelin on the total vasoconstrictive response was only weak (11.4±9.2% of the maximal perfusion pressure increase induced by supernatant from HMECs, n=12) (Figure 1A), which is comparable to our previously published results.9 Next, we determined the contribution of vasoactive mononucleoside polyphosphates in the supernatant of mechanically stimulated HMECs. Therefore, we added alkaline phosphatase to the supernatant to degrade all mononucleoside polyphosphates. Matrix-assisted laser desorption/ionization mass spectrometry (MALDI) mass spectra of the supernatant confirmed that ATP and UTP were efficiently degraded (data not shown). This procedure diminished the vasopressor activity of the supernatant by 42.1±4.3% (Figure 1A). It is well known that activation of P2X receptors induces a strong vasoactive response to mononucleoside polyphosphates, eg, ATP, or dinucleoside polyphosphates, eg, UP4A. To analyze the vasoactive effect of the supernatant by activating P2X receptors, we performed additional experiments using the nonselective P2 receptor antagonist suramin (50 µmol/L) or the more specific P2 receptor antagonist pyridoxalphosphate-6-azophenyl-2-disulfonic acid (PPADS 10 µmol/L). The inhibition of P2X and P2Y receptors by suramin and PPADS diminished the vasoactivity by 64.8±6.1% (suramin) or 61.1±7.1% (PPADS). The combination of suramin and AP led to a slightly more pronounced inhibitory effect of 71.5±6.2%. Therefore, most of the supernatant-induced vasoactive effects could be explained by activation of P2X or P2Y receptors by mononucleoside polyphosphates, which are the most effective agonists. In the next step, we analyzed the vasoactive effect of 10nmol doses of the common nucleotides ATP, ADP, AMP, GTP, GDP, GMP, UTP, UDP, UMP, and the synthetic ATP derivative
,β-methylene ATP (,β-meATP), which has been known to be the most potent P2X receptor agonist to date. As anticipated, 10 nmol of ,β-meATP induced a very strong increase of the perfusion pressure (108.2±8.1 mm Hg). Of all the nucleotides tested, only ATP (11.35±5.21 mm Hg) and UTP (7.23±1.24 mm Hg) increased perfusion pressure of more than 5 mm Hg (Figure 1B). Because of these findings, we screened the supernatant for a new potent mononucleoside polyphosphate compound, which might explain the strong vasoactive effect of the supernatant.
|
We first performed reversed-phase HPLC experiments for the isolation of a new compound. The endothelial supernatant was deproteinized with perchloric acid and concentrated by preparative ion-pair reversed-phase chromatography using a stepwise gradient (Figure 2). The peak indicated by the arrow in Figure 2A (nucleotide-containing fraction) represents the UV absorption of the fraction containing nucleoside polyphosphates, as identified by retention time obtained for authentic nucleoside polyphosphates. Because the endothelial supernatant contains mononucleoside polyphosphates, as well as dinucleoside polyphosphates, we used an affinity chromatography next, which offered the possibility to separate mononucleoside and dinucleoside polyphosphates, as described earlier.9 Phenyl boronic acid resin retains nucleotides containing two or more 1,2-cis-diol groups. On the other hand, mononucleoside polyphosphates like ATP or AP4 with 1 cis-diol group do not bind to the boronate gel in the presence of 1 mmol/L ammonium acetate because of charge repulsions between the negative phosphate groups and the carboxyl groups of the cation-exchange gel. Only the boryl ester formation of the 2 cis-diol groups of dinucleoside polyphosphates is sufficient to overcome charge repulsion.16 The fraction containing nucleotides with 1 cis-diol group is labeled by an arrow in Figure 2B. Next, the fraction was desalted and fractionated by an analytic ion-pair reversed-phase chromatography. Each fraction of this reversed-phase chromatography with a significant UV absorption at 254 nm (Figure 2C) was analyzed by MALDI-TOF/TOF mass spectrometry. Hereby, we were able to identify 1 fraction (labeled by an arrow in Figure 2C) containing the new endothelial mononucleotide compound. In this fraction, a mononucleoside polyphosphate with the molecular weight of m/z of 588.4 Da (M+H+) was detected (Figure 2D). This substance was fragmented to obtain structural information that could be used for its identification by searching in a reference in-house database. The fragmentation pattern of the isolated substance was identical to the fragmentation pattern of AP4 (Table 1, column 2). This result was confirmed by comparison to a fragment MALDI-TOF/TOF spectrum of synthetic AP4 (Table 1, column 4). There were no signals for other potential vasoactive mononucleotides, eg, adenosine 5'-pentaphosphate or guanosine 5'-tetraphosphate.
|
|
Vasoactive Properties of AP4
Next, we tested the vasoactive properties of AP4 in the isolated perfused rat kidney. In Figure 3A, an original tracing is presented showing the strong and dose-dependent vasoactivity of AP4 (ED50[log mol]=–10.3± 0.1; 
Pmax=114.9±3.8 mm Hg; n=8; Figure 3B). We compared the vasoactive response of AP4 with these of other known vasoactive substances such as ,β-meATP, NE, UP4A, and ATP (Figure 3B). AP4 was the most potent vasoactive substance in the isolated perfused rat kidney compared to all other tested substances (NE: ED50[log mol]=–9.5±0.1; Pmax=109.6±3.8 mm Hg; n=8; UP4A: ED50=–8.3±0.1; Pmax=105.1±5.3 mm Hg; n=8; ATP: ED50=–8.9±0.1; Pmax=27.1±2.1 mm Hg; n=8). In particular, in comparison with the most potent synthetic nucleoside polyphosphate, ,β-meATP (ED50=–9.8±0.1; Pmax=119.1±4.2 mm Hg; n=8), AP4 is significantly (P<0.05) more potent. The rank order of potency is: AP4>,β-meATP>NE>>UP4A>>ATP (Table 2). Next, we investigated by which receptor AP4 exerts its highly potent vasoconstriction in the isolated perfused rat kidney. Therefore, we desensitized the P2X1/3 receptor using ,β-meATP (1 µmol/L). After desensitization of the P2X1 and P2X3 receptor, AP4 did not show any change of the perfusion pressure (Figure 4A). To differentiate vasoactive actions of AP4 on P2X1 or P2X3 receptors, we performed additional experiments using IP5I, which inhibits P2X1 receptors by a factor of 1000 more potently than P2X3 receptors. With concentrations of IP5I inhibitory for P2X1 receptors and not P2X3 receptors, the AP4-induced vasoconstriction was abolished (Figure 4A). In the next step, we investigated possible vasodilatory effects of AP4. Therefore, the perfusion pressure was raised by the continuous perfusion of angiotension (Ang) II (100 nmol/L). After reaching a stable precontraction, ,β-meATP (10 µmol/L) was added to the perfusion medium to desensitize the P2X1 and P2X3 receptor. Acetylcholine was used to test the endothelial integrity. The application of AP4 induced a very slight dose-dependent decrease of the perfusion pressure (Figure 4B). The application of ,β-meATP (10nmol) did not induce any change of perfusion pressure excluding an unspecific effect. To investigate a more simple arterial model, we performed arterial tension studies using the renal artery in a wire myograph. In this model, AP4 and ,β-meATP induced a dose-dependent vasoconstriction (EC50[–log mol/L]: AP4=7.4±0.1 and ,β-meATP= 7.1±0.1), whereas AP4 was significantly more potent than ,β-meATP (P<0.05, n=5). UP4A did not reach maximum possible vasoconstriction, whereas ATP showed only weak vasoconstriction at high concentrations (Figure 4C). After endothelium denudation of the renal artery, vasoconstriction of AP4 and ,β-meATP was not significantly affected with respect to potency and maximal induced vasoconstriction, but vasoconstriction of UP4A and ATP increased significantly (Figure I in the online data supplement) but still had lower potency in comparison to AP4 or ,β-meATP. After desensitization of P2X1/3 receptors using ,β-meATP or in the presence of the IP5I, using concentrations inhibiting P2X1 receptors, AP4 did not show a significant vasoconstriction, revealing that the P2X1 receptor is the main vasoconstrictive receptor activated by AP4 in the present model (supplemental Figure I).
|
|
|
Using immunostaining of the kidney and the renal artery, we could find a strong positivity of P2X1 receptor expression in the vascular smooth muscle layer of various arterial vessels of the kidney and the renal artery (Figure 4D). There was only very weak (P2X3, P2Y1) or no expression of P2Y2, P2Y4, or P2Y6 receptors in vascular smooth muscle cells of renal arteries (supplemental Figure II).
To exclude any vasoactive effects of metabolites arising from degradation of AP4, we investigated the effect of methylene derivatives of AP4, ,β-meAP4, β,
-meAP4, and ,
-meAP4,17,18 which are less susceptible to hydrolysis than AP4. All these derivatives induced a strong and dose-dependent increase of the perfusion pressure in the isolated perfused rat kidney. The potency slightly declined from ,β-meAP4 to ,-meAP4 (,β-meAP4: ED50=–10.4± 0.1; Pmax=114.1±6.8 mm Hg; β,-meAP4: ED50=–10.2± 0.1; Pmax=112.4±5.4 mm Hg; ,-meAP4: ED50=–9.8±0.1; Pmax=116.0±7.3 mm Hg; n=8) (Figure 4E). These experiments exclude that the vasoconstrictive response is primarily attributable to degradation products of AP4. Both the bolus application of AP4 and of each stable AP4 analog showed no vasoactive response during continuous perfusion with ,β-meATP (Figure 4F), revealing that the AP4 derivatives also activate P2X1 or P2X3 receptors. The AP4 analogs did not show any vasodilatory properties in Ang II–increased tone preparations of the isolated perfused rat kidney in the presence of ,β-meATP (Figure 4B).
In Vivo Effects of AP4
In the next step, we investigated the in vivo effects of AP4 on the systemic MAP of a Wistar–Kyoto rat. The maximum change in the MAP induced by vasoactive compounds was analyzed. The arterial bolus application of AP4 induced a strong increase in MAP in a dose-dependent manner (Figure 5A and 5B), with a short terminal MAP decrease. Interestingly, the same dose of ATP induced a strong and short decrease of the MAP also in a dose-dependent manner (Figure 5A and 5B). ,β-meAP4 induced only vasoconstriction and did not show any decrease in MAP, suggesting that degradation products of AP4 induce the terminal decrease in MAP. The stabilized analogs of AP4 are as potent as NE. Rank order of potency: ,β-meAP4=NE
,β-meATPAP4>>ATP.
|
Quantification of AP4 in Human Plasma
At last, the question arose whether AP4 is also present in human plasma. The purification procedure was comparable to that described for isolation of AP4 from supernatants of stimulated endothelial cells. To purify AP4, we used a preparative reversed-phase (Figure 6A), an affinity (Figure 6B), and an analytic reversed-phase chromatography (Figure 6C) from plasma of healthy subjects (n=6 probands; healthy donors with no intercurrent illness; age: 28±5 years; 3 males and 3 females). AP4 was identified by MALDI-TOF/TOF mass spectrometry (Figure 6D) and fragment MALDI-TOF/TOF mass spectrometry (Table 1, column 3). AP4 was quantified by integration of the UV absorption and by using a calibration curve of synthetic AP4. We detected a AP4 plasma concentration of 255.6±82.4 nmol/L.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
In the present study, we identified AP4 as a highly potent EDCF activating mainly P2X1 receptors. First, our experiments revealed that AP4 is the most potent endogenous vasoactive purinergic compound currently known. Firstly, the synthetic substance
,β-meATP19,20 has been regarded as the most potent vasoconstrictive purinergic agonist. In the present experiments, AP4 exceeded the vasoconstrictive potency of ,β-meATP in vitro significantly. In vivo, however, AP4 was not so potent because there is a enzymatic degradation of the substance which is not present with ,β-meATP. The more stable variant of AP4, ,β-meAP4 is again equally to more potent than ,β-meATP in vivo. Secondly, the experiments revealed that AP4 is an endothelium-derived vasoconstrictor. It is known that endothelial cells are also capable of secreting other nucleotides. In particular, ATP has been repeatedly demonstrated to be released from these cells.21–23 ATP, in contrast to AP4, is a well-known unspecific purinergic agonist activating numerous P2 purinoceptors, which exert constrictive, dilatory, or no effects on vascular tone. On the other hand, AP4 appears to be a highly potent and highly specific vasoconstrictor, possibly the natural ligand for P2X1 receptors with the highest affinity.
The results of our experiments, using the isolated perfused rat kidney and isolated renal artery, show that AP4 potently activates the P2X1/3 receptor subtype. The selective P2X1/3 desensitizer ,β-meATP24 markedly inhibited AP4 induced vasoconstriction. In vascular smooth muscle cells, P2X1 is the dominant P2X receptor subtype,25–27 although P2X2, P2X3, P2X4, and P2X5 have also been found.28–30 The dominant effect of ,β-meATP renders major contributions from P2X receptors other than P2X1 or P2X3 unlikely. As far as we know, P2X3 has not been detected in rat renal tissue or renal vessels.30 We also detected a strong positive expression of P2X1 receptor in the vascular smooth muscle layer of arterial vessels of the kidney and the renal artery. In contrast to the observations by Turner et al,30 we observed a weak expression of P2X3 in the renal artery. Currently, no ideal inhibitor is known that discriminates between P2X1 and P2X3 receptors.31 Therefore, we applied IP5I, which shows a P2X1/P2X3 receptor affinity ratio of 1000.32 IP5I applied in a concentration inhibitory for P2X1, but far below the IC50 reported for the P2X3 receptor, completely inhibited the vasoconstrictive AP4 response. This result is in accordance with findings obtained in P2X1 receptor knockout mice showing that vasoconstrictive responses to ATP are abolished in arteries lacking this P2X receptor subtype.33
After preconstriction of renal vessels using Ang II and desensitization of P2X1/3 receptors using ,β-meATP, there was a small vasodilation induced by high doses of AP4. It has been shown previously that AP4 can activate endothelial P2Y1 receptors in coronary arteries to induce vasodilation,34 but in renal endothelium, P2Y1 receptors are not present.30 Therefore, we speculate that the vasodilation is rather attributable to degradation of AP4 to ATP and its derivatives. These substances are also potent vasodilators in the rat kidney.19 We used methylene AP4 analogs ,β-meAP4, β,-meAP4, and ,-meAP4,17,18 which are less susceptible to enzymatic hydrolysis than AP4. In the Ang II preconstricted vessels and after ,β-meATP desensitization, there was no constriction by the stable AP4 analogs and no vasodilation, indicating that the vasodilatory AP4 effects are indeed mediated by degradation products.
It has been demonstrated that AP4 is much more stable than ATP.35 The rate of hydrolysis of AP4 is 1.89% in 2 minutes, whereas that of ATP is 24.64% in the same time. The fact that AP4 is more resistant to degradation further favors potent and prolonged vasoconstrictive effects, besides higher affinity of AP4 to P2X receptors.
The vasoconstrictive in vitro effects of AP4 are paralleled by hypertensive effects in vivo. In the present study, AP4 also showed in vivo a dose dependent increase in blood pressure after intraaortic injection in anesthetized rats. The vasoconstriction induced by the stabilized AP4 analog ,β-meAP4 was as equally potent to that of NE and equally to more potent than ,β-meATP. Therefore, it may be speculated that AP4 is a vasoconstrictor secreted by human endothelial cells, which also play a role in the regulation of systemic blood pressure under physiological and pathological conditions. This hypothesis is supported by the finding that AP4 plasma concentrations are in a range effecting vasoconstriction. Moreover, the plasma levels of AP4 are about 2 times higher than ATP levels. Plasma ATP levels are in the range of 138 nmol/L, which has been confirmed by other groups previously.36
In conclusion, these findings may be of interest for several reasons. First, AP4 appears to be a new, highly potent nonpeptidic endothelium-derived vasoconstrictor that is even more potent in vitro than NE. Second, AP4 is the most potent endogenous purinergic vasoconstrictor currently known. Because AP4 is secreted by human endothelial cells and is present in effective concentrations in human plasma, a vasoregulatory role of AP4 seems to be likely.
|
Acknowledgments |
|---|
We thank Simone Spiekermann for technical support.
Sources of Funding
This work was supported by a grant from the Deutsche Nierenstiftung (M.T.), Deutsche Forschungsgemeinschaft grant JA972/11-1 (to M.v.d.G. and J. Jankowski), a Rahel-Hirsch scholarship from the Charité (to V.J.), the Sonnenfeld-Stiftung (to M.v.d.G. and M.T.), Bundesministerium für Bildung und Forschung grant 0313920D, the Deutsche Hochdruckliga (M.T.), Polish Government grant PBZ-MNiSW-07/I/2007 (to J.K., J. Jemielity, and A.G.), and the Dr Werner Jackstädt-Stiftung (M.v.d.G. and M.T.).
Disclosures
None.
|
Footnotes |
|---|
Original received April 21, 2008; revision received August 14, 2008; accepted September 18, 2008.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
1. Furchgott RF, Zawadzki JV. The obligatory role of endothelial cells in the relaxation of arterial smooth muscle by acetylcholine. Nature. 1980; 288: 373–376.[CrossRef][Medline] [Order article via Infotrieve]
2. Ignarro LJ, Buga GM, Wood KS, Byrns RE, Chaudhuri G. Endothelium-derived relaxing factor produced and released from artery and vein is nitric oxide. Proc Natl Acad Sci U S A. 1987; 84: 9265–9269.
3. Moncada S, Vane JR. Pharmacology and endogenous roles of prostaglandin endoperoxides, thromboxane A2, and prostacyclin. Pharmacol Rev. 1978; 30: 293–331.[Medline] [Order article via Infotrieve]
4. Feletou M, Vanhoutte PM. Endothelium-derived hyperpolarizing factor. Clin Exp Pharmacol Physiol. 1996; 23: 1082–1090.[Medline] [Order article via Infotrieve]
5. Yanagisawa M, Kurihara H, Kimura S, Tomobe Y, Kobayashi M, Mitsui Y, Yazaki Y, Goto K, Masaki T. A novel potent vasoconstrictor peptide produced by vascular endothelial cells. Nature. 1988; 332: 411–415.[CrossRef][Medline] [Order article via Infotrieve]
6. Coleman RA, Smith WL, Narumiya S. International Union of Pharmacology classification of prostanoid receptors: properties, distribution, and structure of the receptors and their subtypes. Pharmacol Rev. 1994; 46: 205–229.[Medline] [Order article via Infotrieve]
7. Rubanyi GM, Vanhoutte PM. Superoxide anions and hyperoxia inactivate endothelium-derived relaxing factor. Am J Physiol. 1986; 250: H822–H827.[Medline] [Order article via Infotrieve]
8. Feletou M, Vanhoutte PM. Endothelial dysfunction: a multifaceted disorder (The Wiggers Award Lecture). Am J Physiol Heart Circ Physiol. 2006; 291: H985–H1002.
9. Jankowski V, Tolle M, Vanholder R, Schonfelder G, van der Giet M, Henning L, Schluter H, Paul M, Zidek W, Jankowski J. Uridine adenosine tetraphosphate: a novel endothelium- derived vasoconstrictive factor. Nat Med. 2005; 11: 223–227.[CrossRef][Medline] [Order article via Infotrieve]
10. Gui Y, Walsh MP, Jankowski V, Jankowski J, Zheng XL. Up4A stimulates endothelium-independent contraction of isolated rat pulmonary artery. Am J Physiol Lung Cell Mol Physiol. 2008; 294: L733–L738.
11. van der Giet M, Westhoff T, Cinkilic O, Jankowski J, Schluter H, Zidek W, Tepel M. The critical role of adenosine and guanosine in the affinity of dinucleoside polyphosphates to P(2X)-receptors in the isolated perfused rat kidney. Br J Pharmacol. 2001; 132: 467–474.[CrossRef][Medline] [Order article via Infotrieve]
12. Jankowski V, Vanholder R, Henning L, Karadogan S, Zidek W, Schluter H, Jankowski J. Isolation and quantification of dinucleoside polyphosphates by using monolithic reversed phase chromatography columns. J Chromatogr B Analyt Technol Biomed Life Sci. 2005; 819: 131–139.[CrossRef][Medline] [Order article via Infotrieve]
13. van der Giet M, Jankowski J, Schluter H, Zidek W, Tepel M. Mediation of the vasoactive properties of diadenosine tetraphosphate via various purinoceptors. J Hypertens. 1998; 16: 1939–1943.[CrossRef][Medline] [Order article via Infotrieve]
14. van der Giet M, Khattab M, Borgel J, Schluter H, Zidek W. Differential effects of diadenosine phosphates on purinoceptors in the rat isolated perfused kidney. Br J Pharmacol. 1997; 120: 1453–1460.[CrossRef][Medline] [Order article via Infotrieve]
15. Nofer JR, van der Giet M, Tolle M, Wolinska I, von Wnuck Lipinski K, Baba HA, Tietge UJ, Godecke A, Ishii I, Kleuser B, Schafers M, Fobker M, Zidek W, Assmann G, Chun J, Levkau B. HDL induces NO-dependent vasorelaxation via the lysophospholipid receptor S1P3. J Clin Invest. 2004; 113: 569–581.[CrossRef][Medline] [Order article via Infotrieve]
16. Barnes LD, Robinson AK, Mumford CH, Garrison PN. Assay of diadenosine tetraphosphate hydrolytic enzymes by boronate chromatography. Anal Biochem. 1985; 144: 296–304.[CrossRef][Medline] [Order article via Infotrieve]
17. Jemielity J, Pietrowska-Borek M, Starzynska E, Kowalska J, Stolarski R, Guranowski A, Darzynkiewicz E. Synthesis and enzymatic characterization of methylene analogs of adenosine 5'-tetraphosphate (P4A). Nucleosides Nucleotides Nucleic Acids. 2005; 24: 589–593.[CrossRef][Medline] [Order article via Infotrieve]
18. Guranowski A, Starzynska E, Pietrowska-Borek M, Jemielity J, Kowalska J, Darzynkiewicz E, Thompson MJ, Blackburn GM. Methylene analogues of adenosine 5'-tetraphosphate. Their chemical synthesis and recognition by human and plant mononucleoside tetraphosphatases and dinucleoside tetraphosphatases. FEBS J. 2006; 273: 829–838.[CrossRef][Medline] [Order article via Infotrieve]
19. Eltze M, Ullrich B. Characterization of vascular P2 purinoceptors in the rat isolated perfused kidney. Eur J Pharmacol. 1996; 306: 139–152.[CrossRef][Medline] [Order article via Infotrieve]
20. Ralevic V, Hoyle CH, Burnstock G. Pivotal role of phosphate chain length in vasoconstrictor versus vasodilator actions of adenine dinucleotides in rat mesenteric arteries. J Physiol (Lond). 1995; 483: 703–713.
21. Bodin P, Burnstock G. ATP-stimulated release of ATP by human endothelial cells. J Cardiovasc Pharmacol. 1996; 27: 872–875.[CrossRef][Medline] [Order article via Infotrieve]
22. Ralevic V, Milner P, Kirkpatrick KA, Burnstock G. Flow-induced release of adenosine 5'-triphosphate from endothelial cells of the rat mesenteric arterial bed. Experientia. 1992; 48: 31–34.[CrossRef][Medline] [Order article via Infotrieve]
23. Yang S, Cheek DJ, Westfall DP, Buxton IL. Purinergic axis in cardiac blood vessels. Agonist-mediated release of ATP from cardiac endothelial cells. Circ Res. 1994; 74: 401–407.
24. Kasakov L, Burnstock G. The use of the slowly degradable analog, alpha, beta-methylene ATP, to produce desensitisation of the P2-purinoceptor: effect on non-adrenergic, non-cholinergic responses of the guinea-pig urinary bladder. Eur J Pharmacol. 1982; 86: 291–294.[CrossRef][Medline] [Order article via Infotrieve]
25. Bo X, Sexton A, Xiang Z, Nori SL, Burnstock G. Pharmacological and histochemical evidence for P2X receptors in human umbilical vessels. Eur J Pharmacol. 1998; 353: 59–65.[CrossRef][Medline] [Order article via Infotrieve]
26. Kunapuli SP, Daniel JL. P2 receptor subtypes in the cardiovascular system. Biochem J. 1998; 336: 513–523.[Medline] [Order article via Infotrieve]
27. Wang L, Karlsson L, Moses S, Hultgardh-Nilsson A, Andersson M, Borna C, Gudbjartsson T, Jern S, Erlinge D. P2 receptor expression profiles in human vascular smooth muscle and endothelial cells. J Cardiovasc Pharmacol. 2002; 40: 841–853.[CrossRef][Medline] [Order article via Infotrieve]
28. Pulvirenti TJ, Yin JL, Chaufour X, McLachlan C, Hambly BD, Bennett MR, Barden JA. P2X (purinergic) receptor redistribution in rabbit aorta following injury to endothelial cells and cholesterol feeding. J Neurocytol. 2000; 29: 623–631.[CrossRef][Medline] [Order article via Infotrieve]
29. Nori S, Fumagalli L, Bo X, Bogdanov Y, Burnstock G. Coexpression of mRNAs for P2X1, P2X2 and P2X4 receptors in rat vascular smooth muscle: an in situ hybridization and RT-PCR study. J Vasc Res. 1998; 35: 179–185.[CrossRef][Medline] [Order article via Infotrieve]
30. Turner CM, Vonend O, Chan C, Burnstock G, Unwin RJ. The pattern of distribution of selected ATP-sensitive P2 receptor subtypes in normal rat kidney: an immunohistological study. Cells Tissues Organs. 2003; 175: 105–117.[CrossRef][Medline] [Order article via Infotrieve]
31. North RA. Molecular physiology of P2X receptors. Physiol Rev. 2002; 82: 1013–1067.
32. King BF, Liu M, Pintor J, Gualix J, Miras-Portugal MT, Burnstock G. Diinosine pentaphosphate (IP5I) is a potent antagonist at recombinant rat P2X1 receptors. Br J Pharmacol. 1999; 128: 981–988.[CrossRef][Medline] [Order article via Infotrieve]
33. Vial C, Evans RJ. P2X(1) receptor-deficient mice establish the native P2X receptor and a P2Y6-like receptor in arteries. Mol Pharmacol. 2002; 62: 1438–1445.
34. Westhoff T, Jankowski J, Schmidt S, Luo J, Giebing G, Schluter H, Tepel M, Zidek W, van der Giet M. Identification and characterization of adenosine 5'-tetraphosphate in human myocardial tissue. J Biol Chem. 2003; 278: 17735–17740.
35. Gomez-Villafuertes R, Gualix J, Miras-Portugal MT, Pintor J. Adenosine 5'-tetraphosphate (Ap(4)), a new agonist on rat midbrain synaptic terminal P2 receptors. Neuropharmacology. 2000; 39: 2381–2390.[CrossRef][Medline] [Order article via Infotrieve]
36. Gorman MW, Marble DR, Ogimoto K, Feigl EO. Measurement of adenine nucleotides in plasma. Luminescence. 2003; 18: 173–181.[CrossRef][Medline] [Order article via Infotrieve]
Related Article:
Circ. Res. 2008 103: 1056-1057.
This article has been cited by other articles:
 |
|
 |
|
  G. Burnstock Endothelium-Derived Vasoconstriction by Purines and Pyrimidines Circ. Res., November 7, 2008; 103(10): 1056 - 1057. [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||