- Email: [email protected]
No effect of aluminium upon the hydrolysis of ATP in the coronary circulation of the isolated working rat heart
www.elsevier.nl/locate/jinorgbio Journal of Inorganic Biochemistry 76 (1999) 121–126
No effect of aluminium upon the hydrolysis of ATP in the coronary circulation of the isolated working rat heart Olga Korchazhkina a,*, Gordon Wright b, Christopher Exley a a
Birchall Centre for Inorganic Chemistry and Materials Science, Department of Chemistry, Keele University, Keele, Staffordshire, UK b Centre for Science and Technology in Medicine, Keele University, Keele, Staffordshire, UK
Abstract Adenosine 59-triphosphate (ATP) is now recognised as an important extracellular signalling molecule. Its action at a number of specific receptors is mediated by the activity of ectonucleotidases. We have optimised a high performance liquid chromatography (HPLC) method to allow the simultaneous determination of ATP, and the products of its hydrolysis, in the coronary effluent of an isolated working rat heart. The method is extremely sensitive allowing picomolar quantities of product to be determined. We have used this method to investigate the influence of aluminium on the hydrolysis of ATP by an ecto-ATPase located in the luminal surface of the coronary endothelium of the rat heart. Aluminium did not influence the hydrolysis of ATP by this enzyme. q 1999 Elsevier Science Inc. All rights reserved. Keywords: Aluminium; Extracellular adenosine 59-triphosphate; Ectonucleotidase; Isolated working rat heart; High performance liquid chromatography
1. Introduction Adenosine 59-triphosphate (ATP) is a major extracellular signalling molecule throughout the body [1]. It is actively released by cells into extracellular fluids where it acts upon both metabotropic (P2Y) and ionotropic (P2X) receptors. The extracellular concentration, and therefore activity as a signalling molecule, of ATP is tightly controlled by ectonucleotidases located upon luminal surfaces of plasma membranes of cells expressing ATP receptors [2]. In the brain, soluble ectonucleotidases are released from intracellular stores into the synaptic cleft where they mediate the action of ATP in neurotransmission [3]. The ectonucleotidase responsible for the hydrolysis of ATP to ADP in the coronary endothelium is activated by both Ca2q and Mg2q and is described as a Ca2q/Mg2q ecto-ATPase [4,5]. Since both of these divalent cations are present in the majority of extracellular fluids at mM concentrations and will be bound by ATP at physiological pH to leave only nM concentrations of free ATP, it is likely that Ca–ATP and Mg– ATP are the preferred substrates for the enzyme. Ecto-ATPase activities are inhibited by metal ions, for example, La, Zn and Cu, though only at concentrations approaching mM [6]. Al, though not an essential metal, is present in extracellular fluids in the concentration range 1–10 mM total Al (AlT). * Corresponding author. Tel. q44-1782-584080; fax: q44-1782-715944; e-mail: [email protected]
The higher concentrations are usually only found in individuals using Al-based medications, often in association with kidney dialysis [7]. Al is a known inhibitor of a number of phosphoryl-transferring enzymes [8,9], for example, inhibiting the phosphorylation of glucose by hexokinase at mM AlT [10]. Although Al is bound by ATP with a significantly higher affinity than either Ca or Mg [11] it is unlikely that significant ()nM) concentrations of Al–ATP would form in plasma at pH 7.4 (personal communications with R.J.P. ¨ Williams, W.R. Harris, T. Kiss and S. Sjoberg). A plasma [ATP] of 40 mM would be required before the concentration of Al–ATP complexes exceeded 0.1 mm. Whilst this [ATP] is unlikely to occur in bulk solution it has been measured at the extracellular surface of a number of ATP-secreting cell types [12–14]. In the coronary endothelium of the isolated working rat heart ATP is a potent vasodilator. The vasodilation induced by ATP is actually the product of the activities of adenosine and ADP acting at P1 and P2Y1 receptors, respectively, and is entirely dependent upon the efficient hydrolysis of ATP by ecto-ATPases [15]. Any interference with the hydrolysis of ATP, for example, by Al, could therefore have implications for extracellular signalling via ATP [16]. We have perfused isolated rat hearts with a plasma-like solution containing concentrations of ATP and Al which, being typical of those found in the bulk plasma, would not be predicted to form significant concentrations of Al–ATP, and we have determined the influ-
0162-0134/99/$ - see front matter q 1999 Elsevier Science Inc. All rights reserved. PII S 0 1 6 2 - 0 1 3 4 ( 9 9 ) 0 0 1 2 3 - 3
Friday Oct 22 11:59 AM
StyleTag -- Journal: JIB (Journal of Inorganic Biochemistry)
Article: 6221
122
O. Korchazhkina et al. / Journal of Inorganic Biochemistry 76 (1999) 121–126
ence of Al on ecto-ATPase activity in rat coronary endothelium. The latter was achieved using a development of a high performance liquid chromatography (HPLC) technique to measure nucleotide and nucleoside concentrations before and after single passage of the perfusate through the coronary circulation. HPLC has already been used in biological applications to separate and determine the products of nucleotide metabolism [17–19]. Since nucleotides (e.g. ATP, ADP, AMP) usually carry a negative charge, ion exchange and ion pairing techniques have traditionally been used in their separation whilst reversed phase techniques have been applied to the separation of nucleosides (e.g. adenosine, inosine) and bases (e.g. xanthine, hypoxanthine). A number of procedures for the simultaneous separation of each of these classes of compounds have now been described. In this paper we describe a new reversed phase method for the simultaneous separation and determination of ATP, ADP, AMP, uric acid, xanthine, hypoxanthine, inosine and adenosine in the coronary effluent of a perfused rat heart. We have used this technique to demonstrate that, for the conditions of this study, the hydrolysis of ATP in the coronary endothelium of the isolated rat heart was unaffected by Al.
2. Materials and methods 2.1. Analyses using HPLC 2.1.1. Equipment and conditions A Waters HPLC system (Waters Corp., MA, USA) incorporating a 2690 separations module and a 996 photodiode array detector (DAD) was used in this study. Separation of the nucleotides, nucleosides and bases was achieved using a Waters Nova-Pak C18 column (3.9=300 mm). A guard column, Waters Nova-Pak C18 (3.9=20 mm), was placed in front of the main column to eliminate contaminating moieties. Potassium dihydrogen phosphate buffer (30 mM, adjusted to pH 6.0 with 5 M KOH, at 308C) was freshly prepared on the day of the experiment, degassed and filtered under reduced pressure through a 0.45 mm Nylon membrane filter (Whatman, UK). The buffer was sparged with helium to prevent the dissolution of air gases causing pH shifts and the formation of micro-bubbles during in-line mixing with acetonitrile. Nucleotides, nucleosides and bases were separated on the column as peaks eluted at discrete retention times. To achieve high reproducibility of retention times the column temperature was maintained at 308C. The flow rate of the mobile phase was set at 0.5 ml miny1. A reverse-phase two-step gradient was employed. Step one of the elution comprised 30 mM KH2PO4 buffer at pH 6.0 for 7 min, during which time uric acid, ATP and ADP were separated. In step two hypoxanthine, xanthine, AMP, inosine and adenosine were eluted using a linear gradient of acetonitrile (0–15%) over a further 18 min period. After each complete run the column was equilibrated by pumping 5 column volumes of 100% 30 mM
Friday Oct 22 11:59 AM
KH2PO4. The range of wavelengths scanned was 190–300 nm. Resolution, or the degree of disengagement of two adjacent peaks, was measured according to the formula: R ss1.18(t 2yt1)/(W1qW2) where t2 and t1 are the retention times of two adjacent peaks, and W1 and W2 are their widths at half-height [20]. 2.1.2. Column care At the end of each experiment both the analytical and guard columns were removed and the fluidic path of the HPLC system was washed with ultrapure water (3 ml miny1 for 30 min) to prevent the precipitation of salts. Columns were flushed with 20–30 volumes of ultrapure water followed by 30–40 volumes of acetonitrile. In between use, columns were stored in acetonitrile. Prior to re-use the analytical column was flushed with 5 column volumes of acetonitrile. Periodic flushing of the analytical column with a 1/1 mixture of isopropanol and chloroform helped to minimise baseline noise, ensure good and stable resolution and prolong the column life. Guard columns were replaced as frequently as required. 2.1.3. Preparation of standards and samples for analysis Standards (1.0–3.0 mM) of ATP, ADP, AMP, inosine, adenosine, uric acid, hypoxanthine, and xanthine were prepared in ultrapure water. Dissolution of xanthine and uric acid required sonication and alkalinisation using 5 M NaOH. Samples of coronary effluent were collected, filtered through 0.20 mm Nylon membrane filters (‘Pall’, UK) and maintained at 48C in the autosampler compartment. The injection volume for both samples and standards was 25 ml. Standards were injected after every five samples to ensure the stabilities of both the resolution and the retention times. 2.2. Perfusion of the isolated working rat heart The investigation was performed in accordance with the Home Office Guidance on the Operation of the Animals (Scientific Procedures) Act 1986, published by HMSO, London. We have used an adaptation of an isolated working heart preparation first described by Westerhof et al. [21]. Briefly, male Wistar rats, 230–280 g in weight, were anaesthetised, and the heart excised and perfused as described previously [7,8]. The perfusion buffer contained 25.0 mM HEPES, 118.5 mM NaCl, 1.2 mM MgSO4, 1.4 mM CaCl2 and 11.0 mM glucose, and was maintained at pH 7.42, 378C and pO2)85 kPa. The experimental protocol consisted of a 15 min control perfusion with this buffer, a 5 min perfusion with either (i) bufferqATP (0.2, 1.0 or 5.0 mM prepared from a 10 mM acidic stock solution) or (ii) buffer (including a background of 5 mM AlT)qAl–ATP (0.2, 1.0 or 5.0 mM prepared from a 8 mM acidic stock solution), followed by a wash-out with buffer for a further 15 min period. Samples of coronary effluent for HPLC were collected at the end of the control period, after 4 min of the treatment period and at the end of the period of wash-out. Samples of bufferqATP/Al–
StyleTag -- Journal: JIB (Journal of Inorganic Biochemistry)
Article: 6221
O. Korchazhkina et al. / Journal of Inorganic Biochemistry 76 (1999) 121–126
ATP were also taken to confirm the concentration of ATP entering the coronary circulation. 2.3. Reagents AnalaR grade NaCl, KCl, MgSO4, CaCl2 and glucose, AristaR grade KOH, NaOH and HCl, HiperSolv HPLC-grade acetonitrile, chloroform and isopropanol were all purchased from BDH (Poole, UK). HEPES and inosine were obtained from Sigma (Poole, UK). Adenosine, ATP, ADP and AMP were supplied by Boehringer Mannheim (Lewes, UK). HPLC-grade KH2PO4 was obtained from Fisher Scientific Ltd (UK). Al was added from a certified standard solution (37 mM in 2% HNO3; Perkin-Elmer, Beaconsfield, UK). All solutions, including buffers, were made up in ultrapure water (conductivity below 0.05 mS cmy1).
3. Results
123
use of the column (Table 1). Both retention times and peak resolution were dependent upon the procedure of stabilisation and conditioning of the column. For example, the time for the removal of acetonitrile with ultrapure water prior to the introduction of 100% KH2PO4 was required to be minimised in order to maintain peak resolution. A change in the number of column volumes of ultrapure water used to replace the acetonitrile from 2 to 6 volumes reduced the resolution of ATP and ADP by 10% (Table 1). To counter column effects we developed the following standard protocol. At the beginning of each experimental run 10 volumes of acetonitrile followed by 2 volumes of ultrapure water were pumped through the column. The column was then conditioned by running through three separation gradients (see Section 2.1) without sample injection. This was followed by three injections of standard to ensure that resolution was reproducible. These procedures prepared the column for analyses and ensured good and reproducible performance. 3.2. Method sensitivity
3.1. Standard calibration curves A typical chromatogram taken at 260 nm and showing each standard is presented in Fig. 1. Peak resolution was excellent with an average value of 1.32"0.06 for the separation of ATP and ADP. Typical calibration curves for ATP, ADP, AMP, adenosine, inosine, hypoxanthine, xanthine and uric acid are shown in Fig. 2. To increase sensitivity, peak areas were not all recorded at 260 nm but at their wavelength of maximum absorption (Table 1). Retention times for standards were constant over many hours ()72 h) of continuous
The signal-to-noise ratios (S/N) for each of the measured metabolites for injections of 1.25 and 2.50 pmol are shown in Table 2. For S/N)3 the sensitivity of the method was 1.25 pmol for ATP and ADP; 2.50 pmol for uric acid, hypoxanthine, xanthine and AMP; 0.70 pmol for inosine; and 0.50 pmol for adenosine. 3.3. Hydrolysis of intravascular ATP and Al–ATP ATP was broken down during a single passage through the coronary vessels. Some of the metabolites of ATP breakdown
Fig. 1. A typical chromatogram showing the simultaneous determination by RP-HPLC of ATP and related metabolites.
Friday Oct 22 11:59 AM
StyleTag -- Journal: JIB (Journal of Inorganic Biochemistry)
Article: 6221
124
O. Korchazhkina et al. / Journal of Inorganic Biochemistry 76 (1999) 121–126
measured in coronary effluents are given in Table 3. The proportions of ATP hydrolysed were approximately 100, 99 and 90% for initial ATP concentrations of 0.20, 1.00 and 5.00 mM, respectively. The presence of Al did not influence ATP hydrolysis. The amount of ATP hydrolysed was not significantly different in the presence of Al (Table 4). The hydrolysis of ADP to AMP was at least as efficient as ATP to ADP (Table 3) and, like the hydrolysis of ATP, was unaffected by the presence of Al. AMP and adenosine accumulated in the coronary effluent to a greater extent than either ATP or ADP (Table 3). Al had no significant influence upon either the breakdown of AMP to adenosine or the deamination of adenosine.
4. Discussion We have optimised a method of reverse-phase high performance liquid chromatography (RP-HPLC) for the simultaneous determination of nucleotides, nucleosides and bases produced during the passage of ATP through the coronary vessels of the isolated working rat heart (Fig. 1). The method proved to be extremely sensitive, allowing measurement of sub-picomolar quantities. The success of the method was critically dependent upon the stability of the analytical column. The judicious application of appropriate standard solutions avoided problems associated with any variability in retention times. We have used RP-HPLC to demonstrate the breakdown of ATP during a single passage through the coronary vessels of the isolated working rat heart. The hydrolysis of ATP to ADP was extremely efficient. When the concentration of ATP which entered the coronary vessels was less than 1.00 mM the concentration of ATP which remained in the coronary
Fig. 2. (a,b) Typical calibration curves used in the measurement of ATP and related metabolites in coronary effluents.
Table 1 Detection wavelengths and typical retention times of standards a
l (nm) RT2vol (min) RT6vol (min)
UA
ATP
ADP
Hypo
Xan
AMP
Ino
Ado
290 5.745"0.003
260 6.372"0.003
260 7.682"0.003
250 10.842"0.006
267 12.148"0.006
260 13.992"0.005
247 19.1"0.006
260 22.521"0.008
5.446"0.003
6.064"0.007
7.186"0.011
9.988"0.013
11.151"0.016
12.709" 0.026
18.585"0.012
21.978"0.016
a
Mean and s.e.m. are given, ns5. The values represented under RT2vol were obtained when the standard column stabilisation procedure was used (Section 3.1). The values represented under RT6vol were obtained when the volume of water pumped through the column was increased three times. All of the other steps of column stabilisation were the same as in the standard procedure (Section 3.1).
Table 2 The signal-to-noise ratio of the standards a
1.25 pmol 2.50 pmol a
UA
ATP
ADP
Hypo
Xan
AMP
Ino
Ado
2.13"0.072 3.81"0.135
3.70"0.084 7.76"0.118
3.01"0.095 6.21"0.179
1.93"0.051 3.92"0.091
2.27"0.088 4.18"0.111
2.26"0.23 4.72"0.067
5.36"0.089 10.63"0.072
7.89"0.048 16.18"0.318
Mean and s.e.m. are given, ns5.
Friday Oct 22 11:59 AM
StyleTag -- Journal: JIB (Journal of Inorganic Biochemistry)
Article: 6221
O. Korchazhkina et al. / Journal of Inorganic Biochemistry 76 (1999) 121–126
125
Table 3 The products of the hydrolysis of ATP in the coronary effluent of the isolated working rat heart a [Al]nominal (mM)
0 0 5.2 0 6.0 0 10.0
Metabolites in the coronary effluent (nmol miny1 gy1)
ATP delivery [ATP]nominal (mM)
(nmol miny1 gy1)
ATP
ADP
AMP
Ado
Others
0 0.2 0.2 1.0 1.0 5.0 5.0
0 4.59"0.41 5.42"0.54 29.62"3.03 32.81"2.99 150.74"6.87 133.50"10.58
0 0 0 0.14"0.10 0.14"0.10 15.70"5.72 7.08"1.80
0 0 0 0 0 3.40"0.77 2.01"0.48
0 0 0 4.38"0.32 6.31"1.53 38.90"6.05 32.95"3.60
0.51"0.106 1.65"0.21 2.37"0.28 8.80"0.91 8.85"1.69 44.57"5.49 52.15"3.43
13.59"0.62 13.58"0.88 14.57"1.48 29.81"1.14 29.81"1.39 55.10"6.84 62.27"5.84
a
The concentrations of metabolites measured are presented as nmol miny1 gy1 to account for differences in coronary flow and weight of individual hearts. Mean and s.e.m. are given, ns5. Table 4 The influence of Al on the hydrolysis of two different concentrations of ATP a
[ATP]nominal (mM)
1.0 5.0
possible secretion of Al–ATP, as opposed to Mg–ATP or free ATP, by cells remains to be tested [26].
ATP hydrolysed (nmol miny1 gy1)
5. Abbreviations yAl
qAl
29.5"3.14 135.1"8.84
31.7"2.80 129.6"10.04
Ps0.622 Ps0.543
a
The concentrations of metabolites measured are presented as nmol miny1 g to account for differences in coronary flow and weight of individual hearts. Mean and s.e.m. are given, ns5. y1
effluent was below detection limits. ATP was shown to be hydrolysed at all concentrations of ATP tested, including concentrations as low as 50 nM [15]. The hydrolysis of ATP was unaffected by the presence of an excess of Al (Table 4). The further breakdown of ADP to AMP and AMP to adenosine was also extremely efficient and similarly unaffected by the presence of Al. The hydrolysis of ATP in the coronary vessels is achieved through the action of an ecto-ATPase located in the luminal surface of the coronary endothelium [22–24]. The preference of this enzyme towards using either Ca–ATP or Mg–ATP as substrates [4,5] suggested that its activity would be influenced by Al [8,9,11]. The lack of effect demonstrated in this study does not preclude this possibility for either higher concentrations of AlT or for a perfusate in which the concentration of Al–ATP complexes was sufficiently high to be competitive with Ca/Mg–ATP in binding the enzyme. The concentrations of AlT and ATP used in this study were chosen primarily for their physiological significance. The [ATP] values were characteristic of bulk plasma but they may be underestimates of local concentrations of the nucleotide secreted at cell surfaces [12–14]. For this reason the question of whether Al–ATP could form in plasma and influence ectoATPase activity in vivo is unresolved. Al–ATP complexes have been identified in cell cytosol [25]. The significance for the role of ATP in extracellular signal transduction of the
Friday Oct 22 11:59 AM
UA ATP ADP Hypo Xan AMP Ino Ado
uric acid adenosine 59-triphosphate adenosine 59-diphosphate hypoxanthine xanthine adenosine 59-monophosphate inosine adenosine
Acknowledgements This research was supported by the Wellcome Trust, the Royal Society, the W.E. Dunn Trust and the North Staffordshire Heart Committee. The authors acknowledge critical discussions with R.J.P. Williams, N.C. Price, W.R. Harris, ¨ T. Kiss and S. Sjoberg.
References [1] M.P. Abbracchio, G. Burnstock, Jpn. J. Pharmacol. 78 (1998) 113. [2] L. Plesner, Int. Rev. Cytol. 158 (1995) 141. [3] L.D. Todorov, S.M. Todorova, T.D. Westfall, P. Sneddon, C. Kennedy, R.A. Bjur, D.P. Westfall, Nature 387 (1997) 76. [4] N.S. Dhalla, D. Zhao, Prog. Biophys. Mol. Biol. 52 (1988) 1. [5] P. Meghji, G. Burnstock, Life Sci. 57 (1995) 763. [6] A.U. Ziganshin, L.E. Ziganshina, C.H.V. Hoyle, G. Burnstock, Br. J. Pharmacol. 114 (1995) 632. [7] D.N.S. Kerr, M.K. Ward, H.A. Ellis, W. Simpson, I.S. Parkinson, Ciba Found. Symp. 169 (1992) 123. [8] N.C. Furumo, R.E. Viola, Inorg. Chem. 28 (1989) 820. [9] G.A. Trapp, Kidney Int. 29 (1986) S-12. [10] C. Exley, N.C. Price, J.D. Birchall, J. Inorg. Biochem. 54 (1994) 297.
StyleTag -- Journal: JIB (Journal of Inorganic Biochemistry)
Article: 6221
126
O. Korchazhkina et al. / Journal of Inorganic Biochemistry 76 (1999) 121–126
[11] R.B. Martin, Clin. Chem. 32 (1986) 1797. [12] R. Beigi, E. Kobatake, M. Aizawa, G. Dubyak, Am. J. Physiol. 276 (1998) C267. [13] A. Hazama, S. Hayashi, Y. Okada, Pflugers Arch. Eur. J. Physiol. 437 (1998) 31. [14] A.L. Taylor, B.A. Kudlow, K.L. Marrs, D.C. Gruenert, W.B. Guggino, E.M. Schwiebert, Am. J. Physiol. 275 (1998) C1391. [15] O. Korchazhkina, G. Wright, C. Exley, Br. J. Pharmacol. 127 (1999) 701. [16] O. Korchazhkina, G. Wright, C. Exley, J. Inorg. Biochem. 69 (1998) 153. [17] R.A. Hartwick, S.P. Assenza, P.R. Brown, J. Chromatogr. 186 (1979) 647.
Friday Oct 22 11:59 AM
[18] V. Stocchi, L. Cucchiarini, M. Magnani, L. Chiarantini, P. Palma, G. Crescentini, Anal. Biochem. 146 (1985) 118. [19] D.A. Mei, G.J. Gross, K. Nithipatikom, Anal. Biochem. 238 (1996) 34. [20] L.R. Snyder, J.L. Glajch, J.J. Kirkland, Practical HPLC Method Development, Wiley, New York, 1988. [21] N. Westerhof, G. Elzinga, P. Sipkema, J. Appl. Physiol. 31 (1971) 776. [22] G.P. Frick, J.M. Lowenstein, J. Biol. Chem. 253 (1978) 1240. [23] J.D. Pearson, J.S. Carleton, J.L. Gordon, Biochem. J. 190 (1980) 421. [24] N.J. Cusack, J.D. Pearson, J.L. Gordon, Biochem. J. 214 (1983) 975. [25] K. Panchalingam, S. Sachedina, J.W. Pettegrew, T. Glonek, Int. J. Biochem. 23 (1991) 1453. [26] C. Exley, J. Inorg. Biochem. 76 (1999) 133.
StyleTag -- Journal: JIB (Journal of Inorganic Biochemistry)
Article: 6221
No effect of aluminium upon the hydrolysis of ATP in the coronary circulation of the isolated working rat heart Olga Korchazhkina a,*, Gordon Wright b, Christopher Exley a a
Birchall Centre for Inorganic Chemistry and Materials Science, Department of Chemistry, Keele University, Keele, Staffordshire, UK b Centre for Science and Technology in Medicine, Keele University, Keele, Staffordshire, UK
Abstract Adenosine 59-triphosphate (ATP) is now recognised as an important extracellular signalling molecule. Its action at a number of specific receptors is mediated by the activity of ectonucleotidases. We have optimised a high performance liquid chromatography (HPLC) method to allow the simultaneous determination of ATP, and the products of its hydrolysis, in the coronary effluent of an isolated working rat heart. The method is extremely sensitive allowing picomolar quantities of product to be determined. We have used this method to investigate the influence of aluminium on the hydrolysis of ATP by an ecto-ATPase located in the luminal surface of the coronary endothelium of the rat heart. Aluminium did not influence the hydrolysis of ATP by this enzyme. q 1999 Elsevier Science Inc. All rights reserved. Keywords: Aluminium; Extracellular adenosine 59-triphosphate; Ectonucleotidase; Isolated working rat heart; High performance liquid chromatography
1. Introduction Adenosine 59-triphosphate (ATP) is a major extracellular signalling molecule throughout the body [1]. It is actively released by cells into extracellular fluids where it acts upon both metabotropic (P2Y) and ionotropic (P2X) receptors. The extracellular concentration, and therefore activity as a signalling molecule, of ATP is tightly controlled by ectonucleotidases located upon luminal surfaces of plasma membranes of cells expressing ATP receptors [2]. In the brain, soluble ectonucleotidases are released from intracellular stores into the synaptic cleft where they mediate the action of ATP in neurotransmission [3]. The ectonucleotidase responsible for the hydrolysis of ATP to ADP in the coronary endothelium is activated by both Ca2q and Mg2q and is described as a Ca2q/Mg2q ecto-ATPase [4,5]. Since both of these divalent cations are present in the majority of extracellular fluids at mM concentrations and will be bound by ATP at physiological pH to leave only nM concentrations of free ATP, it is likely that Ca–ATP and Mg– ATP are the preferred substrates for the enzyme. Ecto-ATPase activities are inhibited by metal ions, for example, La, Zn and Cu, though only at concentrations approaching mM [6]. Al, though not an essential metal, is present in extracellular fluids in the concentration range 1–10 mM total Al (AlT). * Corresponding author. Tel. q44-1782-584080; fax: q44-1782-715944; e-mail: [email protected]
The higher concentrations are usually only found in individuals using Al-based medications, often in association with kidney dialysis [7]. Al is a known inhibitor of a number of phosphoryl-transferring enzymes [8,9], for example, inhibiting the phosphorylation of glucose by hexokinase at mM AlT [10]. Although Al is bound by ATP with a significantly higher affinity than either Ca or Mg [11] it is unlikely that significant ()nM) concentrations of Al–ATP would form in plasma at pH 7.4 (personal communications with R.J.P. ¨ Williams, W.R. Harris, T. Kiss and S. Sjoberg). A plasma [ATP] of 40 mM would be required before the concentration of Al–ATP complexes exceeded 0.1 mm. Whilst this [ATP] is unlikely to occur in bulk solution it has been measured at the extracellular surface of a number of ATP-secreting cell types [12–14]. In the coronary endothelium of the isolated working rat heart ATP is a potent vasodilator. The vasodilation induced by ATP is actually the product of the activities of adenosine and ADP acting at P1 and P2Y1 receptors, respectively, and is entirely dependent upon the efficient hydrolysis of ATP by ecto-ATPases [15]. Any interference with the hydrolysis of ATP, for example, by Al, could therefore have implications for extracellular signalling via ATP [16]. We have perfused isolated rat hearts with a plasma-like solution containing concentrations of ATP and Al which, being typical of those found in the bulk plasma, would not be predicted to form significant concentrations of Al–ATP, and we have determined the influ-
0162-0134/99/$ - see front matter q 1999 Elsevier Science Inc. All rights reserved. PII S 0 1 6 2 - 0 1 3 4 ( 9 9 ) 0 0 1 2 3 - 3
Friday Oct 22 11:59 AM
StyleTag -- Journal: JIB (Journal of Inorganic Biochemistry)
Article: 6221
122
O. Korchazhkina et al. / Journal of Inorganic Biochemistry 76 (1999) 121–126
ence of Al on ecto-ATPase activity in rat coronary endothelium. The latter was achieved using a development of a high performance liquid chromatography (HPLC) technique to measure nucleotide and nucleoside concentrations before and after single passage of the perfusate through the coronary circulation. HPLC has already been used in biological applications to separate and determine the products of nucleotide metabolism [17–19]. Since nucleotides (e.g. ATP, ADP, AMP) usually carry a negative charge, ion exchange and ion pairing techniques have traditionally been used in their separation whilst reversed phase techniques have been applied to the separation of nucleosides (e.g. adenosine, inosine) and bases (e.g. xanthine, hypoxanthine). A number of procedures for the simultaneous separation of each of these classes of compounds have now been described. In this paper we describe a new reversed phase method for the simultaneous separation and determination of ATP, ADP, AMP, uric acid, xanthine, hypoxanthine, inosine and adenosine in the coronary effluent of a perfused rat heart. We have used this technique to demonstrate that, for the conditions of this study, the hydrolysis of ATP in the coronary endothelium of the isolated rat heart was unaffected by Al.
2. Materials and methods 2.1. Analyses using HPLC 2.1.1. Equipment and conditions A Waters HPLC system (Waters Corp., MA, USA) incorporating a 2690 separations module and a 996 photodiode array detector (DAD) was used in this study. Separation of the nucleotides, nucleosides and bases was achieved using a Waters Nova-Pak C18 column (3.9=300 mm). A guard column, Waters Nova-Pak C18 (3.9=20 mm), was placed in front of the main column to eliminate contaminating moieties. Potassium dihydrogen phosphate buffer (30 mM, adjusted to pH 6.0 with 5 M KOH, at 308C) was freshly prepared on the day of the experiment, degassed and filtered under reduced pressure through a 0.45 mm Nylon membrane filter (Whatman, UK). The buffer was sparged with helium to prevent the dissolution of air gases causing pH shifts and the formation of micro-bubbles during in-line mixing with acetonitrile. Nucleotides, nucleosides and bases were separated on the column as peaks eluted at discrete retention times. To achieve high reproducibility of retention times the column temperature was maintained at 308C. The flow rate of the mobile phase was set at 0.5 ml miny1. A reverse-phase two-step gradient was employed. Step one of the elution comprised 30 mM KH2PO4 buffer at pH 6.0 for 7 min, during which time uric acid, ATP and ADP were separated. In step two hypoxanthine, xanthine, AMP, inosine and adenosine were eluted using a linear gradient of acetonitrile (0–15%) over a further 18 min period. After each complete run the column was equilibrated by pumping 5 column volumes of 100% 30 mM
Friday Oct 22 11:59 AM
KH2PO4. The range of wavelengths scanned was 190–300 nm. Resolution, or the degree of disengagement of two adjacent peaks, was measured according to the formula: R ss1.18(t 2yt1)/(W1qW2) where t2 and t1 are the retention times of two adjacent peaks, and W1 and W2 are their widths at half-height [20]. 2.1.2. Column care At the end of each experiment both the analytical and guard columns were removed and the fluidic path of the HPLC system was washed with ultrapure water (3 ml miny1 for 30 min) to prevent the precipitation of salts. Columns were flushed with 20–30 volumes of ultrapure water followed by 30–40 volumes of acetonitrile. In between use, columns were stored in acetonitrile. Prior to re-use the analytical column was flushed with 5 column volumes of acetonitrile. Periodic flushing of the analytical column with a 1/1 mixture of isopropanol and chloroform helped to minimise baseline noise, ensure good and stable resolution and prolong the column life. Guard columns were replaced as frequently as required. 2.1.3. Preparation of standards and samples for analysis Standards (1.0–3.0 mM) of ATP, ADP, AMP, inosine, adenosine, uric acid, hypoxanthine, and xanthine were prepared in ultrapure water. Dissolution of xanthine and uric acid required sonication and alkalinisation using 5 M NaOH. Samples of coronary effluent were collected, filtered through 0.20 mm Nylon membrane filters (‘Pall’, UK) and maintained at 48C in the autosampler compartment. The injection volume for both samples and standards was 25 ml. Standards were injected after every five samples to ensure the stabilities of both the resolution and the retention times. 2.2. Perfusion of the isolated working rat heart The investigation was performed in accordance with the Home Office Guidance on the Operation of the Animals (Scientific Procedures) Act 1986, published by HMSO, London. We have used an adaptation of an isolated working heart preparation first described by Westerhof et al. [21]. Briefly, male Wistar rats, 230–280 g in weight, were anaesthetised, and the heart excised and perfused as described previously [7,8]. The perfusion buffer contained 25.0 mM HEPES, 118.5 mM NaCl, 1.2 mM MgSO4, 1.4 mM CaCl2 and 11.0 mM glucose, and was maintained at pH 7.42, 378C and pO2)85 kPa. The experimental protocol consisted of a 15 min control perfusion with this buffer, a 5 min perfusion with either (i) bufferqATP (0.2, 1.0 or 5.0 mM prepared from a 10 mM acidic stock solution) or (ii) buffer (including a background of 5 mM AlT)qAl–ATP (0.2, 1.0 or 5.0 mM prepared from a 8 mM acidic stock solution), followed by a wash-out with buffer for a further 15 min period. Samples of coronary effluent for HPLC were collected at the end of the control period, after 4 min of the treatment period and at the end of the period of wash-out. Samples of bufferqATP/Al–
StyleTag -- Journal: JIB (Journal of Inorganic Biochemistry)
Article: 6221
O. Korchazhkina et al. / Journal of Inorganic Biochemistry 76 (1999) 121–126
ATP were also taken to confirm the concentration of ATP entering the coronary circulation. 2.3. Reagents AnalaR grade NaCl, KCl, MgSO4, CaCl2 and glucose, AristaR grade KOH, NaOH and HCl, HiperSolv HPLC-grade acetonitrile, chloroform and isopropanol were all purchased from BDH (Poole, UK). HEPES and inosine were obtained from Sigma (Poole, UK). Adenosine, ATP, ADP and AMP were supplied by Boehringer Mannheim (Lewes, UK). HPLC-grade KH2PO4 was obtained from Fisher Scientific Ltd (UK). Al was added from a certified standard solution (37 mM in 2% HNO3; Perkin-Elmer, Beaconsfield, UK). All solutions, including buffers, were made up in ultrapure water (conductivity below 0.05 mS cmy1).
3. Results
123
use of the column (Table 1). Both retention times and peak resolution were dependent upon the procedure of stabilisation and conditioning of the column. For example, the time for the removal of acetonitrile with ultrapure water prior to the introduction of 100% KH2PO4 was required to be minimised in order to maintain peak resolution. A change in the number of column volumes of ultrapure water used to replace the acetonitrile from 2 to 6 volumes reduced the resolution of ATP and ADP by 10% (Table 1). To counter column effects we developed the following standard protocol. At the beginning of each experimental run 10 volumes of acetonitrile followed by 2 volumes of ultrapure water were pumped through the column. The column was then conditioned by running through three separation gradients (see Section 2.1) without sample injection. This was followed by three injections of standard to ensure that resolution was reproducible. These procedures prepared the column for analyses and ensured good and reproducible performance. 3.2. Method sensitivity
3.1. Standard calibration curves A typical chromatogram taken at 260 nm and showing each standard is presented in Fig. 1. Peak resolution was excellent with an average value of 1.32"0.06 for the separation of ATP and ADP. Typical calibration curves for ATP, ADP, AMP, adenosine, inosine, hypoxanthine, xanthine and uric acid are shown in Fig. 2. To increase sensitivity, peak areas were not all recorded at 260 nm but at their wavelength of maximum absorption (Table 1). Retention times for standards were constant over many hours ()72 h) of continuous
The signal-to-noise ratios (S/N) for each of the measured metabolites for injections of 1.25 and 2.50 pmol are shown in Table 2. For S/N)3 the sensitivity of the method was 1.25 pmol for ATP and ADP; 2.50 pmol for uric acid, hypoxanthine, xanthine and AMP; 0.70 pmol for inosine; and 0.50 pmol for adenosine. 3.3. Hydrolysis of intravascular ATP and Al–ATP ATP was broken down during a single passage through the coronary vessels. Some of the metabolites of ATP breakdown
Fig. 1. A typical chromatogram showing the simultaneous determination by RP-HPLC of ATP and related metabolites.
Friday Oct 22 11:59 AM
StyleTag -- Journal: JIB (Journal of Inorganic Biochemistry)
Article: 6221
124
O. Korchazhkina et al. / Journal of Inorganic Biochemistry 76 (1999) 121–126
measured in coronary effluents are given in Table 3. The proportions of ATP hydrolysed were approximately 100, 99 and 90% for initial ATP concentrations of 0.20, 1.00 and 5.00 mM, respectively. The presence of Al did not influence ATP hydrolysis. The amount of ATP hydrolysed was not significantly different in the presence of Al (Table 4). The hydrolysis of ADP to AMP was at least as efficient as ATP to ADP (Table 3) and, like the hydrolysis of ATP, was unaffected by the presence of Al. AMP and adenosine accumulated in the coronary effluent to a greater extent than either ATP or ADP (Table 3). Al had no significant influence upon either the breakdown of AMP to adenosine or the deamination of adenosine.
4. Discussion We have optimised a method of reverse-phase high performance liquid chromatography (RP-HPLC) for the simultaneous determination of nucleotides, nucleosides and bases produced during the passage of ATP through the coronary vessels of the isolated working rat heart (Fig. 1). The method proved to be extremely sensitive, allowing measurement of sub-picomolar quantities. The success of the method was critically dependent upon the stability of the analytical column. The judicious application of appropriate standard solutions avoided problems associated with any variability in retention times. We have used RP-HPLC to demonstrate the breakdown of ATP during a single passage through the coronary vessels of the isolated working rat heart. The hydrolysis of ATP to ADP was extremely efficient. When the concentration of ATP which entered the coronary vessels was less than 1.00 mM the concentration of ATP which remained in the coronary
Fig. 2. (a,b) Typical calibration curves used in the measurement of ATP and related metabolites in coronary effluents.
Table 1 Detection wavelengths and typical retention times of standards a
l (nm) RT2vol (min) RT6vol (min)
UA
ATP
ADP
Hypo
Xan
AMP
Ino
Ado
290 5.745"0.003
260 6.372"0.003
260 7.682"0.003
250 10.842"0.006
267 12.148"0.006
260 13.992"0.005
247 19.1"0.006
260 22.521"0.008
5.446"0.003
6.064"0.007
7.186"0.011
9.988"0.013
11.151"0.016
12.709" 0.026
18.585"0.012
21.978"0.016
a
Mean and s.e.m. are given, ns5. The values represented under RT2vol were obtained when the standard column stabilisation procedure was used (Section 3.1). The values represented under RT6vol were obtained when the volume of water pumped through the column was increased three times. All of the other steps of column stabilisation were the same as in the standard procedure (Section 3.1).
Table 2 The signal-to-noise ratio of the standards a
1.25 pmol 2.50 pmol a
UA
ATP
ADP
Hypo
Xan
AMP
Ino
Ado
2.13"0.072 3.81"0.135
3.70"0.084 7.76"0.118
3.01"0.095 6.21"0.179
1.93"0.051 3.92"0.091
2.27"0.088 4.18"0.111
2.26"0.23 4.72"0.067
5.36"0.089 10.63"0.072
7.89"0.048 16.18"0.318
Mean and s.e.m. are given, ns5.
Friday Oct 22 11:59 AM
StyleTag -- Journal: JIB (Journal of Inorganic Biochemistry)
Article: 6221
O. Korchazhkina et al. / Journal of Inorganic Biochemistry 76 (1999) 121–126
125
Table 3 The products of the hydrolysis of ATP in the coronary effluent of the isolated working rat heart a [Al]nominal (mM)
0 0 5.2 0 6.0 0 10.0
Metabolites in the coronary effluent (nmol miny1 gy1)
ATP delivery [ATP]nominal (mM)
(nmol miny1 gy1)
ATP
ADP
AMP
Ado
Others
0 0.2 0.2 1.0 1.0 5.0 5.0
0 4.59"0.41 5.42"0.54 29.62"3.03 32.81"2.99 150.74"6.87 133.50"10.58
0 0 0 0.14"0.10 0.14"0.10 15.70"5.72 7.08"1.80
0 0 0 0 0 3.40"0.77 2.01"0.48
0 0 0 4.38"0.32 6.31"1.53 38.90"6.05 32.95"3.60
0.51"0.106 1.65"0.21 2.37"0.28 8.80"0.91 8.85"1.69 44.57"5.49 52.15"3.43
13.59"0.62 13.58"0.88 14.57"1.48 29.81"1.14 29.81"1.39 55.10"6.84 62.27"5.84
a
The concentrations of metabolites measured are presented as nmol miny1 gy1 to account for differences in coronary flow and weight of individual hearts. Mean and s.e.m. are given, ns5. Table 4 The influence of Al on the hydrolysis of two different concentrations of ATP a
[ATP]nominal (mM)
1.0 5.0
possible secretion of Al–ATP, as opposed to Mg–ATP or free ATP, by cells remains to be tested [26].
ATP hydrolysed (nmol miny1 gy1)
5. Abbreviations yAl
qAl
29.5"3.14 135.1"8.84
31.7"2.80 129.6"10.04
Ps0.622 Ps0.543
a
The concentrations of metabolites measured are presented as nmol miny1 g to account for differences in coronary flow and weight of individual hearts. Mean and s.e.m. are given, ns5. y1
effluent was below detection limits. ATP was shown to be hydrolysed at all concentrations of ATP tested, including concentrations as low as 50 nM [15]. The hydrolysis of ATP was unaffected by the presence of an excess of Al (Table 4). The further breakdown of ADP to AMP and AMP to adenosine was also extremely efficient and similarly unaffected by the presence of Al. The hydrolysis of ATP in the coronary vessels is achieved through the action of an ecto-ATPase located in the luminal surface of the coronary endothelium [22–24]. The preference of this enzyme towards using either Ca–ATP or Mg–ATP as substrates [4,5] suggested that its activity would be influenced by Al [8,9,11]. The lack of effect demonstrated in this study does not preclude this possibility for either higher concentrations of AlT or for a perfusate in which the concentration of Al–ATP complexes was sufficiently high to be competitive with Ca/Mg–ATP in binding the enzyme. The concentrations of AlT and ATP used in this study were chosen primarily for their physiological significance. The [ATP] values were characteristic of bulk plasma but they may be underestimates of local concentrations of the nucleotide secreted at cell surfaces [12–14]. For this reason the question of whether Al–ATP could form in plasma and influence ectoATPase activity in vivo is unresolved. Al–ATP complexes have been identified in cell cytosol [25]. The significance for the role of ATP in extracellular signal transduction of the
Friday Oct 22 11:59 AM
UA ATP ADP Hypo Xan AMP Ino Ado
uric acid adenosine 59-triphosphate adenosine 59-diphosphate hypoxanthine xanthine adenosine 59-monophosphate inosine adenosine
Acknowledgements This research was supported by the Wellcome Trust, the Royal Society, the W.E. Dunn Trust and the North Staffordshire Heart Committee. The authors acknowledge critical discussions with R.J.P. Williams, N.C. Price, W.R. Harris, ¨ T. Kiss and S. Sjoberg.
References [1] M.P. Abbracchio, G. Burnstock, Jpn. J. Pharmacol. 78 (1998) 113. [2] L. Plesner, Int. Rev. Cytol. 158 (1995) 141. [3] L.D. Todorov, S.M. Todorova, T.D. Westfall, P. Sneddon, C. Kennedy, R.A. Bjur, D.P. Westfall, Nature 387 (1997) 76. [4] N.S. Dhalla, D. Zhao, Prog. Biophys. Mol. Biol. 52 (1988) 1. [5] P. Meghji, G. Burnstock, Life Sci. 57 (1995) 763. [6] A.U. Ziganshin, L.E. Ziganshina, C.H.V. Hoyle, G. Burnstock, Br. J. Pharmacol. 114 (1995) 632. [7] D.N.S. Kerr, M.K. Ward, H.A. Ellis, W. Simpson, I.S. Parkinson, Ciba Found. Symp. 169 (1992) 123. [8] N.C. Furumo, R.E. Viola, Inorg. Chem. 28 (1989) 820. [9] G.A. Trapp, Kidney Int. 29 (1986) S-12. [10] C. Exley, N.C. Price, J.D. Birchall, J. Inorg. Biochem. 54 (1994) 297.
StyleTag -- Journal: JIB (Journal of Inorganic Biochemistry)
Article: 6221
126
O. Korchazhkina et al. / Journal of Inorganic Biochemistry 76 (1999) 121–126
[11] R.B. Martin, Clin. Chem. 32 (1986) 1797. [12] R. Beigi, E. Kobatake, M. Aizawa, G. Dubyak, Am. J. Physiol. 276 (1998) C267. [13] A. Hazama, S. Hayashi, Y. Okada, Pflugers Arch. Eur. J. Physiol. 437 (1998) 31. [14] A.L. Taylor, B.A. Kudlow, K.L. Marrs, D.C. Gruenert, W.B. Guggino, E.M. Schwiebert, Am. J. Physiol. 275 (1998) C1391. [15] O. Korchazhkina, G. Wright, C. Exley, Br. J. Pharmacol. 127 (1999) 701. [16] O. Korchazhkina, G. Wright, C. Exley, J. Inorg. Biochem. 69 (1998) 153. [17] R.A. Hartwick, S.P. Assenza, P.R. Brown, J. Chromatogr. 186 (1979) 647.
Friday Oct 22 11:59 AM
[18] V. Stocchi, L. Cucchiarini, M. Magnani, L. Chiarantini, P. Palma, G. Crescentini, Anal. Biochem. 146 (1985) 118. [19] D.A. Mei, G.J. Gross, K. Nithipatikom, Anal. Biochem. 238 (1996) 34. [20] L.R. Snyder, J.L. Glajch, J.J. Kirkland, Practical HPLC Method Development, Wiley, New York, 1988. [21] N. Westerhof, G. Elzinga, P. Sipkema, J. Appl. Physiol. 31 (1971) 776. [22] G.P. Frick, J.M. Lowenstein, J. Biol. Chem. 253 (1978) 1240. [23] J.D. Pearson, J.S. Carleton, J.L. Gordon, Biochem. J. 190 (1980) 421. [24] N.J. Cusack, J.D. Pearson, J.L. Gordon, Biochem. J. 214 (1983) 975. [25] K. Panchalingam, S. Sachedina, J.W. Pettegrew, T. Glonek, Int. J. Biochem. 23 (1991) 1453. [26] C. Exley, J. Inorg. Biochem. 76 (1999) 133.
StyleTag -- Journal: JIB (Journal of Inorganic Biochemistry)
Article: 6221