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• W2052809940 abstract "ATP is known to act as an extracellular signal in many organs. In the heart, extracellular ATP modulates ionic processes and contractile function. This study describes a novel, metabolic effect of exogenous ATP in isolated rat cardiomyocytes. In these quiescent (i.e. noncontracting) cells, micromolar concentrations of ATP depressed the rate of basal, catecholamine-stimulated, or insulin-stimulated glucose transport by up to 60% (IC50 for inhibition of insulin-dependent glucose transport, 4 μm). ATP decreased the amount of glucose transporters (GLUT1 and GLUT4) in the plasma membrane, with a concomitant increase in intracellular microsomal membranes. A similar glucose transport inhibition was produced by P2 purinergic agonists with the following rank of potencies: ATP ≈ ATPγS ≈ 2-methylthio-ATP (P2Y-selective) > ADP > α,βmeATP (P2X-selective), whereas the P1 purinoceptor agonist adenosine was ineffective. The effect of ATP was suppressed by the poorly subtype-selective P2 antagonist pyridoxal-phosphate-6-azophenyl-2′,4′-disulfonic acid but, surprisingly, not by the nonselective antagonist suramin nor by the P2Y-specific Reactive Blue 2. Glucose transport inhibition by ATP was not affected by a drastic reduction of the extracellular concentrations of calcium (down to 10−9m) or sodium (down to 0 mm), and it was not mimicked by a potassium-induced depolarization, indicating that purinoceptors of the P2X family (which are nonselective cation channels whose activation leads to a depolarizing sodium and calcium influx) are not involved. Inhibition was specific for the transmembrane transport of glucose because ATP did not inhibit (i) the rate of glycolysis under conditions where the transport step is no longer rate-limiting nor (ii) the rate of [1-14C]pyruvate decarboxylation. In conclusion, extracellular ATP markedly inhibits glucose transport in rat cardiomyocytes by promoting a redistribution of glucose transporters from the cell surface to an intracellular compartment. This effect of ATP is mediated by P2 purinoceptors, possibly by a yet unknown subtype of the P2Y purinoceptor family. ATP is known to act as an extracellular signal in many organs. In the heart, extracellular ATP modulates ionic processes and contractile function. This study describes a novel, metabolic effect of exogenous ATP in isolated rat cardiomyocytes. In these quiescent (i.e. noncontracting) cells, micromolar concentrations of ATP depressed the rate of basal, catecholamine-stimulated, or insulin-stimulated glucose transport by up to 60% (IC50 for inhibition of insulin-dependent glucose transport, 4 μm). ATP decreased the amount of glucose transporters (GLUT1 and GLUT4) in the plasma membrane, with a concomitant increase in intracellular microsomal membranes. A similar glucose transport inhibition was produced by P2 purinergic agonists with the following rank of potencies: ATP ≈ ATPγS ≈ 2-methylthio-ATP (P2Y-selective) > ADP > α,βmeATP (P2X-selective), whereas the P1 purinoceptor agonist adenosine was ineffective. The effect of ATP was suppressed by the poorly subtype-selective P2 antagonist pyridoxal-phosphate-6-azophenyl-2′,4′-disulfonic acid but, surprisingly, not by the nonselective antagonist suramin nor by the P2Y-specific Reactive Blue 2. Glucose transport inhibition by ATP was not affected by a drastic reduction of the extracellular concentrations of calcium (down to 10−9m) or sodium (down to 0 mm), and it was not mimicked by a potassium-induced depolarization, indicating that purinoceptors of the P2X family (which are nonselective cation channels whose activation leads to a depolarizing sodium and calcium influx) are not involved. Inhibition was specific for the transmembrane transport of glucose because ATP did not inhibit (i) the rate of glycolysis under conditions where the transport step is no longer rate-limiting nor (ii) the rate of [1-14C]pyruvate decarboxylation. In conclusion, extracellular ATP markedly inhibits glucose transport in rat cardiomyocytes by promoting a redistribution of glucose transporters from the cell surface to an intracellular compartment. This effect of ATP is mediated by P2 purinoceptors, possibly by a yet unknown subtype of the P2Y purinoceptor family. It is well established that ATP acts as an extracellular signal in many tissues and is involved in a variety of regulatory processes including the control of vascular tone, muscle contraction, pain, or neuronal communication (for review see Ref. 1Burnstock G. Neuropharmacology. 1997; 36: 1127-1139Crossref PubMed Scopus (502) Google Scholar). In particular, ATP serves as a neurotransmitter or co-transmitter in central, as well as in peripheral neurons (1Burnstock G. Neuropharmacology. 1997; 36: 1127-1139Crossref PubMed Scopus (502) Google Scholar, 2Bean B.P. Trends Pharmacol. Sci. 1992; 13: 87-90Abstract Full Text PDF PubMed Scopus (301) Google Scholar). For instance, ATP is co-released with noradrenaline or acetylcholine from sympathetic or parasympathetic nerve endings, respectively, and even from neuromuscular synapses (1Burnstock G. Neuropharmacology. 1997; 36: 1127-1139Crossref PubMed Scopus (502) Google Scholar). Another source of extracellular ATP is that released from parenchymal cells under hypoxic or ischemic conditions (3Skobel E. Kammermeier H. Biochim. Biophys. Acta. 1997; 1362: 128-134Crossref PubMed Scopus (16) Google Scholar). Many biological responses to ATP are mediated via P2purinoceptors, which belong to either of two structurally and functionally distinct families of receptors: ligand-gated, nonselective cation channels (P2X-type), or G-protein-coupled receptors linked to the phospholipase C signaling cascade (P2Y-type) (1Burnstock G. Neuropharmacology. 1997; 36: 1127-1139Crossref PubMed Scopus (502) Google Scholar, 4Burnstock G. CIBA Found. Symp. 1996; 198: 1-28PubMed Google Scholar). Activation of P2X receptors leads to sodium and calcium influx and to cell depolarization and is therefore stimulatory in nature (e.g. it causes smooth muscle cell contraction). Although P2Y purinoceptor activation also results in an increased cytosolic calcium concentration (at least in part via inositoltrisphosphate-mediated calcium release), it induces both stimulatory and inhibitory effects (the latter through the opening of potassium channels or indirectly through nitric oxide production). In the heart, extracellular ATP and ATP analogues were found to exert pronounced although relatively complex effects on coronary tone and mechanical activity, depending on the type of preparation studied and on the purine concentrations used (5Burnstock G. Meghji P. Br. J. Pharmacol. 1983; 79: 211-218Crossref PubMed Scopus (51) Google Scholar, 6Legssyer A. Poggioli J. Renard D. Vassort G. J. Physiol. (Lond.). 1988; 401: 185-199Crossref Scopus (87) Google Scholar, 7Fleetwood G. Gordon J.L. Br. J. Pharmacol. 1987; 90: 219-227Crossref PubMed Scopus (41) Google Scholar, 8Scamps F. Legssyer E. Mayoux E. Vassort G. Circ. Res. 1990; 67: 1007-1016Crossref PubMed Scopus (64) Google Scholar). More recent studies using isolated cardiomyocytes have shown that micromolar levels of extracellular ATP increase (i) plasma membrane conductances for cations (9Puceat M. Clement O. Scamps F. Vassort G. Biochem. J. 1991; 274: 55-62Crossref PubMed Scopus (60) Google Scholar) and also for chloride (10Puceat M. Clement O. Vassort G. J. Physiol. (Lond.). 1991; 444: 241-256Crossref Scopus (48) Google Scholar, 11Kaneda M. Fukui K. Doi K. Br. J. Pharmacol. 1994; 111: 1355-1360Crossref PubMed Scopus (30) Google Scholar), (ii) the cytosolic calcium concentration (9Puceat M. Clement O. Scamps F. Vassort G. Biochem. J. 1991; 274: 55-62Crossref PubMed Scopus (60) Google Scholar, 12Danziger R.S. Raffaeli S. Moreno-Sanchez R. Sakai M. Capogrossi M.C. Spurgeon H.A. Hansford R.G. Lakatta E.G. Cell Calcium. 1988; 9: 193-199Crossref PubMed Scopus (65) Google Scholar, 13Christie A. Sharma V.K. Sheu S.S. J. Physiol. (Lond.). 1992; 445: 369-388Crossref Scopus (76) Google Scholar, 14Podrasky E. Xu D. Liang B.T. Am. J. Physiol. 1997; 273: H2380-H2387Crossref PubMed Google Scholar, 15Zhang B.X. Ma X. McConnell B.K. Damron D.S. Bond M. Circ. Res. 1996; 79: 94-102Crossref PubMed Scopus (16) Google Scholar), (iii) the rate of phosphoinositide hydrolysis (14Podrasky E. Xu D. Liang B.T. Am. J. Physiol. 1997; 273: H2380-H2387Crossref PubMed Google Scholar, 16Yamada M. Hamamori Y. Akita H. Yokoyama M. Circ. Res. 1992; 70: 477-485Crossref PubMed Scopus (54) Google Scholar, 17Puceat M. Vassort G. Biochem. J. 1996; 318: 723-728Crossref PubMed Scopus (51) Google Scholar), and (iv) the contraction amplitude (12Danziger R.S. Raffaeli S. Moreno-Sanchez R. Sakai M. Capogrossi M.C. Spurgeon H.A. Hansford R.G. Lakatta E.G. Cell Calcium. 1988; 9: 193-199Crossref PubMed Scopus (65) Google Scholar, 14Podrasky E. Xu D. Liang B.T. Am. J. Physiol. 1997; 273: H2380-H2387Crossref PubMed Google Scholar, 15Zhang B.X. Ma X. McConnell B.K. Damron D.S. Bond M. Circ. Res. 1996; 79: 94-102Crossref PubMed Scopus (16) Google Scholar). These results, along with pharmacological and immunohistochemical data (10Puceat M. Clement O. Vassort G. J. Physiol. (Lond.). 1991; 444: 241-256Crossref Scopus (48) Google Scholar, 11Kaneda M. Fukui K. Doi K. Br. J. Pharmacol. 1994; 111: 1355-1360Crossref PubMed Scopus (30) Google Scholar, 13Christie A. Sharma V.K. Sheu S.S. J. Physiol. (Lond.). 1992; 445: 369-388Crossref Scopus (76) Google Scholar, 14Podrasky E. Xu D. Liang B.T. Am. J. Physiol. 1997; 273: H2380-H2387Crossref PubMed Google Scholar, 18Babenko A.P. Vassort G. Br. J. Pharmacol. 1997; 120: 631-638Crossref PubMed Scopus (12) Google Scholar, 19Scamps F. Vassort G. Br. J. Pharmacol. 1994; 113: 982-986Crossref PubMed Scopus (35) Google Scholar, 20Vulchanova L. Arvidsson U. Riedl M. Wang J. Buell G. Surprenant A. North R.A. Elde R. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 8063-8067Crossref PubMed Scopus (368) Google Scholar), suggest that members of both families of P2 receptors are expressed in and linked to the electrical and contractile function of cardiac muscle cells. By contrast, it is not known whether ATP may also impinge on cardiomyocyte metabolism, whose regulation often closely matches the changes in energy demand and contractile status. The aim of the present investigation was therefore to investigate possible effects of ATP and other purinergic agents on the trans-sarcolemmal transport and utilization of glucose that, besides fatty acids and lactate, represents an essential substrate in cardiac myocytes. In these studies, we observed a pronounced inhibitory action of purinergic agonists on the rate of glucose transport and explored the underlying mechanisms. All chemicals were of the highest purity grade available. Chemicals for media used for cell isolation and the glucose transport assay were purchased from Merck (Darmstadt, Germany), except for bovine serum albumin (fraction V, fatty acid-free), which was from Boehringer Mannheim. All nucleotides (except 2MeSATP), 1The abbreviations used are: 2MeSATP, 2-methylthio-ATP; dGlc, 2-deoxy-d-glucose; ATPγS, adenosine 5′-O-(3-thiotriphosphate); α, βmeATP, α,β-methylene-ATP; PPADS, pyridoxal-phosphate-6-azophenyl-2′,4′-disulfonic acid; LDM, low density microsomes; DIDS, 4,4′-diisothiocyanostilbene. adenosine, andl-phenylephrine were from Sigma (Deisenhofen, Germany); dichloroacetic acid was from Aldrich (Steinheim, Germany); 2-methylthioadenosine triphosphate, pyridoxal-phosphate-6-azophenyl-2′,4′-disulfonic acid, suramin, and Reactive Blue 2 were from RBI (Biotrend, Cologne, Germany); purified bovine insulin was a kind gift from Prof. Axel Wollmer (Aachen, Germany). 2-Deoxy-d-[3H]glucose (glucose transport assay), [3-3H]-d-glucose (glycolytic measurements), [1-14C]-pyruvic acid (pyruvate oxidation assay), and ECL (Western blots) were from Amersham Pharmacia Biotech (Braunschweig, Germany). The antiserum used to immunodetect GLUT4 (OSCRX) was a kind gift from Prof. A. Zorzano (Barcelona, Spain), and that against GLUT1 was purchased from Biogenesis Inc. (Quartett, Berlin, Germany). Cardiomyocytes from adult, female Sprague-Dawley rats (220–250 g, fed ad libitum) were obtained as described previously (21Fischer Y. Rose H. Kammermeier H. Life Sci. 1991; 49: 1679-1688Crossref PubMed Scopus (63) Google Scholar). Unless otherwise indicated, treatment of cardiomyocytes (∼1.5 mg protein/sample) was performed in medium A (6 mm KCl, 1 mm Na2HPO4, 0.2 mm NaH2PO4, 1.4 mmMgSO4, 128 mm NaCl, 10 mm HEPES, 1 mm CaCl2, and 2% fatty acid-free bovine serum albumin, pH 7.4) at 37 °C, equilibrated with 100% oxygen. The rates of carrier-mediated 2-deoxy-d-glucose (dGlc) uptake were determined over a period of 30 min as described (21Fischer Y. Rose H. Kammermeier H. Life Sci. 1991; 49: 1679-1688Crossref PubMed Scopus (63) Google Scholar). In the experiments examining the relevance of ionic gradients (see Fig. 3), cardiomyocytes were incubated in media whose composition was identical to medium A, except that the concentration of the ion to be tested was varied as indicated in the figure. All the media used were isotonic; thus sodium was replaced by an equimolar amount of choline (see Fig. 3 A), potassium was replaced by sodium (see Fig. 3 C), or chloride was replaced by HEPES (see Fig. 3 D). The calcium concentration was adjusted according to the method of Fabiato and Fabiato (22Fabiato A. Fabiato A. J. Physiol. (Paris). 1979; 75: 463-505PubMed Google Scholar) (see Fig.3 B). The experiments in which the sodium concentration was lowered (see Fig. 3 A) were performed at a calcium concentration of 10−7m to avoid a calcium overload of the cells due to impairment of the sodium-dependent calcium extrusion via the sarcolemmal Na+/Ca2+ exchanger. The flux through the 6-phosphofructo-1-kinase reaction was estimated by measuring the rate of 3H2O formation from [3-3H]glucose with a protocol adapted from Ref. 23Hue L. Hers H.G. Biochem. Biophys. Res. Commun. 1974; 58: 532-539Crossref PubMed Scopus (58) Google Scholar. In brief, cardiomyocytes were treated (with or without ATP and insulin) as described in the legend of Fig. 5 before [3-3H]glucose (0.75 μCi/sample) was added for 30 min at 37 °C. The incubation was then stopped with perchloric acid (0.3m). The extracts were neutralized with KOH/KHCO3, and the tritiated water glycolytically formed was separated on Dowex-borate columns (Bio-Rad, Munich, Germany) as described (23Hue L. Hers H.G. Biochem. Biophys. Res. Commun. 1974; 58: 532-539Crossref PubMed Scopus (58) Google Scholar). The amount of nonspecific 3H2O (i.e. that which is not related to the glycolytic activity of the myocytes) was determined by adding perchloric acid to a parallel set of cell samples prior to the incubation with [3-3H]glucose. Flux through the pyruvate dehydrogenase complex was determined by monitoring the production of14CO2 from [1-14C]pyruvate as described (24Fischer Y. Böttcher U. Eblenkamp M. Thomas J. Jüngling E. Rösen P. Kammermeier H. Biochem. J. 1997; 321: 629-638Crossref PubMed Scopus (21) Google Scholar). In brief, cardiomyocytes were exposed to the experimental conditions to be investigated (ATP and/or insulin treatment) at 37 °C in 20-ml vials sealed with rubber stoppers (under the same conditions as for the glucose transport assays,i.e. in a total volume of 1.5 ml and with ∼1.5 mg cell protein/sample). The oxidation reaction was then started by injecting 0.1 μCi of [1-14C]pyruvate (final concentration, 0.1 mm) into the cell suspension. After another 10 min at 37 °C, the incubation was terminated by injecting 300 μl of 0.3n perchloric acid. The released14CO2 was trapped (over 18 h at 4 °C) in 0.3 ml ethanolamine/ethylene glycol (1:1, v/v) contained in a polypropylene center well (placed in the vials before the incubations). Nonspecific values (determined by injecting perchloric acid in parallel cell samples before adding [1-14C]pyruvate) were subtracted from all samples. The quenching caused by the ethanolamine/ethylene glycol mixture was determined in each experiment and taken into account for the calculation of pyruvate oxidation rates. Cardiomyocytes were treated as detailed in the legend of Fig. 4 and then rapidly washed once with TES buffer (20 mm Tris, 1 mm EDTA, 250 mmsucrose, 0.1 mm (phenylmethylsulfonyl fluoride, pH 7.4) and immediately frozen in liquid nitrogen in a ratio of 107cells/2.7 ml of TES. Preparation of purified plasma and intracellular membrane fractions (25Fischer Y. Thomas J. Rösen P. Kammermeier H. Endocrinology. 1995; 136: 412-420Crossref PubMed Google Scholar) and semi-quantitative immunodetection and quantitation of GLUT4 and GLUT1 (26Fischer Y. Thomas J. Sevilla L. Muñoz P. Becker C. Holman G.D. Kozka I.J. Palacin M. Testar X. Kammermeier H. Zorzano A. J. Biol. Chem. 1997; 272: 7085-7092Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar) were performed as described. Cell protein was measured by the biuret method. The curves shown in Fig. 1, as well as the IC50values of glucose transport inhibition, were obtained from the dGlc transport data using a computer program (Prism) from GraphPad Software, Inc. (San Diego, CA), according to the following equation. Y=[Ymin+(Ymax−Ymin)]/[1+10((logIC50−X)·S)]Equation 1 where X is the logarithm of agonist concentration,Y min and Y max are the minimum and maximum dGlc transport rates, IC50 is the agonist concentration producing a half-maximal inhibition, andS the Hill slope of the curves. For the statistical tests used to assess the difference of data sets, see the figure legends. Glucose transport is the first step and under many physiological conditions the rate-limiting step of myocardial glucose utilization and is known to be the target of a host of regulatory factors in heart muscle cells, such as insulin, catecholamines, contraction, anoxia, ischemia, or alternative substrates (24Fischer Y. Böttcher U. Eblenkamp M. Thomas J. Jüngling E. Rösen P. Kammermeier H. Biochem. J. 1997; 321: 629-638Crossref PubMed Scopus (21) Google Scholar, 27Kolter T. Uphues I. Wichelhaus A. Reinauer H. Eckel J. Biochem. Biophys. Res. Commun. 1992; 189: 1207-1214Crossref PubMed Scopus (48) Google Scholar, 28Fischer Y. Thomas J. Holman G.D. Rose H. Kammermeier H. Am. J. Physiol. 1996; 270: C1204-C1210Crossref PubMed Google Scholar, 29Eblenkamp M. Böttcher U. Thomas J. Löken C. Ionescu I. Rose H. Kammermeier H. Fischer Y. Life Sci. 1996; 59: 141-151Crossref PubMed Scopus (9) Google Scholar, 30Sun D. Nguyen N. DeGrado T.R. Schwaiger M. Brosius 3rd., F.C. Circulation. 1994; 89: 793-798Crossref PubMed Scopus (217) Google Scholar). In an initial series of experiments, we therefore investigated the action of purinergic agonists on cardiomyocyte glucose transport. As documented in Table I, ATP produced a substantial inhibition of glucose transport in basal (nonstimulated) myocytes, as well as in cells stimulated with insulin or with the α1-adrenergic agent phenylephrine. The effect of ATP was rapid; thus the extent of inhibition of insulin-stimulated glucose transport was the same, regardless of whether ATP was added to the cells 30, 20, 10, or 0 min before or even 2 min after insulin addition (not shown).Table IEffects of adenine nucleotides on dGlc uptake in cardiomyocytesAdditionsRate of dGlc uptakePercentagepBasal (nonstimulated)15.5 ± 1.0 (32)100+ Adenosine (100 μm)17.2 ± 1.6 (3)104>0.05+ ATP (30 μm)5.8 ± 0.9 (6)37<0.001Insulin203.1 ± 10.8 (31)100+ Adenosine (100 μm)200.0 ± 2.0 (3)98>0.05+ ATP (30 μm)102.0 ± 10.0 (13)51<0.01+ ADP (100 μm)149.3 ± 5.9 (4)74<0.01+ ATPγS (30 μm)114.5 ± 16.3 (3)58<0.01+ α,βmeATP (100 μm)152.8 ± 20.8 (4)75<0.01+ 2MeSATP (100 μm)123.5 ± 19.9 (5)58<0.01+ NADH (100 μm)161.1 ± 6.4 (4)79<0.01+ NAD+ (100 μm)163.4 ± 13.7 (4)81<0.05+ NADPH (100 μm)162.3 ± 11.3 (3)86>0.05+ NADP+ (100 μm)159.9 ± 7.6 (3)84>0.05+ UTP (100 μm)214.2 (2)106Phenylephrine90.6 ± 6.6 (5)100+ ATP (30 μm)33.4 ± 3.4 (5)37<0.001Cardiomyocytes were incubated for 10 min (at 37 °C in medium A) with or without nucleotides at the indicated concentrations before insulin (12 nm) or phenylephrine (120 μm) were added for another 30 min. In another series of samples, cells were treated for 60 min with oligomycin B (0.6 μm) in the presence or absence of ATP. At the end of these incubations, the rate of dGlc uptake was determined as described under “Experimental Procedures.” Data are the mean values expressed in pmol/h/mg protein ± S.E. The numbers of independent experiments are indicated in parentheses. The percentage values given in the third column in relation to the control values in the corresponding experiments. The pvalues given are in relation to the corresponding uptake rates measured in the absence of nucleotide (analysis of variance, except for the comparison done with basal and phenylephrine-stimulated values that were paired in each experiment and were therefore analyzed with a paired Student's t test). Open table in a new tab Cardiomyocytes were incubated for 10 min (at 37 °C in medium A) with or without nucleotides at the indicated concentrations before insulin (12 nm) or phenylephrine (120 μm) were added for another 30 min. In another series of samples, cells were treated for 60 min with oligomycin B (0.6 μm) in the presence or absence of ATP. At the end of these incubations, the rate of dGlc uptake was determined as described under “Experimental Procedures.” Data are the mean values expressed in pmol/h/mg protein ± S.E. The numbers of independent experiments are indicated in parentheses. The percentage values given in the third column in relation to the control values in the corresponding experiments. The pvalues given are in relation to the corresponding uptake rates measured in the absence of nucleotide (analysis of variance, except for the comparison done with basal and phenylephrine-stimulated values that were paired in each experiment and were therefore analyzed with a paired Student's t test). Similarly, other purine nucleotides known to mainly interact with P2 purinoceptors (2Bean B.P. Trends Pharmacol. Sci. 1992; 13: 87-90Abstract Full Text PDF PubMed Scopus (301) Google Scholar, 31Abbracchio M.P. Burnstock G. Pharmacol. Ther. 1994; 64: 445-475Crossref PubMed Scopus (993) Google Scholar) efficiently depressed glucose transport; thus addition of ATP and nonhydrolyzable ATP analogues (ATPγS1, 2MeSATP, and α,βmeATP) at micromolar concentrations resulted in a decrease in insulin-dependent glucose transport by up to ∼50%. Inhibitory effects were also observed with other purine nucleotides such as ADP, NADH, and NAD+ (Table I); NADPH and NADP+ also tended to inhibit glucose transport, although these effects did not quite reach statistical significance (Table I). By contrast, adenosine (100 μm), which preferentially activates members of the P1 purinoceptor family, had no effect on the basal or on the insulin-stimulated rate of glucose transport (Table I). In an attempt to discriminate the potential P2 receptor type involved, we examined the concentration dependence of the effects of some of the above agonists. As illustrated in Fig.1, the P2-subtype nonselective agonists ATP, ATPγS, and ADP were effective in the low micromolar range (IC50 = 4.0 ± 0.7, 5.4 ± 1.5, and 7.8 ± 3.4 μm, respectively). The P2Y-selective analogue 2MeSATP (31Abbracchio M.P. Burnstock G. Pharmacol. Ther. 1994; 64: 445-475Crossref PubMed Scopus (993) Google Scholar) also produced a significant inhibition (p < 0.001) at 1–10 μm, but higher concentrations than with ATP or ATPγS were required to reach a maximal degree of inhibition (Fig. 1); in this case the dose-response relationship could not be fitted to a simple sigmoidal function. The selective P2X purinoceptor agonist α,βmeATP (31Abbracchio M.P. Burnstock G. Pharmacol. Ther. 1994; 64: 445-475Crossref PubMed Scopus (993) Google Scholar) was clearly less potent (with an apparent IC50 of ∼100 μm) (Fig. 1). UTP, which specifically activates so-called P2U purinoceptors (which belong to the P2Y family and are also expressed in the myocardium; Ref. 32Webb T.E. Boluyt M.O. Barnard E.A. J. Auton. Pharmacol. 1996; 16: 303-307Crossref PubMed Scopus (65) Google Scholar), was without effect at a concentration as high as 100 μm (Table I). A second approach aimed to address the question of the P2-type mediating the inhibiting effect of ATP was to use purinoceptor antagonists. Surprisingly, neither did the P2Y-specific antagonist Reactive Blue (31Abbracchio M.P. Burnstock G. Pharmacol. Ther. 1994; 64: 445-475Crossref PubMed Scopus (993) Google Scholar) nor suramin, which is known to antagonize the binding of agonists to and the activation of both P2X and P2Y purinoceptors (31Abbracchio M.P. Burnstock G. Pharmacol. Ther. 1994; 64: 445-475Crossref PubMed Scopus (993) Google Scholar), affect the decrease in glucose transport brought about by ATP (Fig. 2). By contrast, the poorly subtype-selective antagonist pyridoxal-phosphate-6-azophenyl-2′,4′-disulfonic acid (PPADS, 100 μm; Ref. 33Lambrecht G. J. Auton. Pharmacol. 1996; 16: 341-344Crossref PubMed Scopus (57) Google Scholar) suppressed the ATP-dependent inhibition of glucose transport (Fig. 2). It should also be mentioned that the effect of ATP (30 μm) was not influenced by the P1 receptor antagonists 1,3-diethyl-8-phenylxanthine, or 3,7-dimethyl-1-propargylxanthine (100 μm each) (not shown). Because the pharmacological data presented above were not sufficient to clearly define or exclude either P2X or P2Y receptors as the mediator of the effect of ATP on glucose transport, we considered the possible involvement of the former purinoceptor type by using a functional approach. P2X purinoceptors are nonselective cation channels whose activation produces a depolarizing inward sodium and calcium current (1Burnstock G. Neuropharmacology. 1997; 36: 1127-1139Crossref PubMed Scopus (502) Google Scholar, 2Bean B.P. Trends Pharmacol. Sci. 1992; 13: 87-90Abstract Full Text PDF PubMed Scopus (301) Google Scholar, 4Burnstock G. CIBA Found. Symp. 1996; 198: 1-28PubMed Google Scholar). We therefore examined the influence of trans-sarcolemmal ionic gradients on the inhibition of glucose transport by ATP. To this end, we modified the composition of the incubation medium with respect to sodium, calcium, and potassium over a wide range of concentrations (while keeping the osmolarity constant). The results of this series of experiments are summarized in Fig.3. Even a reduction of the extracellular sodium concentration to nominally 0 mm (Fig. 3 A) or of the free calcium concentration down to 10−9m (Fig. 3 B) did not suppress the ATP-induced glucose transport inhibition. It is noteworthy that the basal rate of glucose transport (in the absence of ATP) significantly rose when the calcium level was lowered from the physiological value of 10−3m (Fig. 3); conversely, we had previously found that an increase in calcium from 1 to 5 mm depresses the rate of glucose transport in these cells (34Fischer Y. Kamp J. Thomas J. Pöpping S. Rose H. Carpéné C. Kammermeier H. Am. J. Physiol. 1996; 270: C1211-C1220Crossref PubMed Google Scholar). Hence it appears that calcium exerts an inhibiting influence of the glucose transport system of cardiomyocytes. However, our present data (Fig. 3,A and B) show that the action of ATP on glucose transport is not dependent on extracellular calcium and sodium, thus ruling out that a depolarizing influx of these ions could mediate the action of the nucleotide. In addition, depolarization with 30 mm potassium chloride (in the absence of ATP) did not produce an inhibition of glucose transport (Fig. 3 C,open bars). Since purinoceptor activation was also shown to activate cardiac potassium channels (18Babenko A.P. Vassort G. Br. J. Pharmacol. 1997; 120: 631-638Crossref PubMed Scopus (12) Google Scholar, 35Friel D.D. Bean B.P. Pflügers Arch. Eur. J. Physiol. 1990; 415: 651-657Crossref PubMed Scopus (33) Google Scholar), we also examined the possible influence of the potassium gradient across the plasma membrane. As shown in Fig.3 C (closed bars), ATP significantly reduced the rate of glucose transport at all tested potassium concentrations, although the percentage of inhibition tended to be smaller at 0.6 mm potassium. However, such a low potassium concentration may limit the function of the Na+/K+-ATPase,i.e. of Na+ extrusion, thus impairing Ca2+ efflux or even promoting Ca2+ influx via the sarcolemmal Na+/Ca2+ exchanger, which would eventually result in an increased cytosolic Ca2+concentration. We therefore also studied the effect of ATP at a very low extracellular Ca2+ concentration (10−7m) (at which the Na+/Ca2+ exchanger is not required to maintain a low cytosolic calcium concentration). We found that ATP inhibited glucose transport by 57 ± 7% even in the complete absence of extracellular potassium (two independent experiments; not shown). Moreover, the effect of ATP (as tested at 6 mm potassium and 1 mm Ca2+) was not affected by the potassium channel blockers Tedisamil, Apamin, or Glibenclamide (3–10 μm): thus, in the presence of these inhibitors, the percentage of inhibition by ATP was 48 ± 1%, 37 ± 16%, and 49 ± 2%, respectively (n = 2–3). In conclusion, we found no indication that the trans-sarcolemmal potassium gradient or potassium channels may be relevant to the action of ATP on glucose uptake. Finally, it was reported that in cardiomyocytes ATP activates trans-sarcolemmal bicarbonate/chloride exchange (10Puceat M. Clement O. Vassort G. J. Physiol. (Lond.). 1991; 444: 241-256Crossref Scopus (48) Google Scholar, 11Kaneda M. Fukui K. Doi K. Br. J. Pharmacol. 1994; 111: 1355-1360Crossref PubMed Scopus (30) Google Scholar). We therefore also investigated the influence of the extracellular chloride concentration on the effect of the nucleotide on glucose transport. As illustrated in Fig. 3 D, this parameter" @default.
• W2052809940 created "2016-06-24" @default.
• W2052809940 creator A5008057406 @default.
• W2052809940 creator A5023056990 @default.
• W2052809940 creator A5051629956 @default.
• W2052809940 date "1999-01-01" @default.
• W2052809940 modified "2023-09-29" @default.
• W2052809940 title "Purinergic Inhibition of Glucose Transport in Cardiomyocytes" @default.
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