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doi: 10.1152/ajpendo.00173.2005290:E940-E951, 2006. First published 13 December 2005;Am J Physiol Endocrinol Metab

Villalobos-Molina and Enrique PiaRaquel Guinzberg, Daniel Corts, Antonio Daz-Cruz, Hctor Riveros-Rosas, Rafael

adenosine receptors3liberation through AInosine released after hypoxia activates hepatic glucose

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Inosine released after hypoxia activates hepatic glucose liberation

through A3 adenosine receptors

Raquel Guinzberg,1 Daniel Cortes,1 Antonio Daz-Cruz,2

Hector Riveros-Rosas,1 Rafael Villalobos-Molina,3 and Enrique Pina1

1

Departamentos de Bioqumica, Facultad de Medicina; 2

Nutricion Animal y Bioqumica,Facultad de Medicina Veterinaria y Zootecnia; and 3Unidad de Biomedicina, Facultad

de Estudios Superiores-Iztacala, Universidad Nacional Autonoma de Mexico, Mexico City, Mexico.

Submitted 20 April 2005; accepted in final form 3 December 2005

Guinzberg, Raquel, Daniel Cortes, Antonio Daz-Cruz, Hector Ri-veros-Rosas, Rafael Villalobos-Molina, and Enrique Pina. Inosine re-leased after hypoxia activates hepatic glucose liberation through A3adeno-sine receptors.Am J Physiol Endocrinol Metab 290: E940E951, 2006. Firstpublished December 13, 2005; doi:10.1152/ajpendo.00173.2005.Inosine,an endogenous nucleoside, has recently been shown to exert potenteffects on the immune, neural, and cardiovascular systems. This workaddresses modulation of intermediary metabolism by inosine throughadenosine receptors (ARs) in isolated rat hepatocytes. We conducted

an in silico search in the GenBank and complete genomic sequencedatabases for additional adenosine/inosine receptors and for a feasiblephysiological role of inosine in homeostasis. Inosine stimulated gly-cogenolysis (40%, EC504.2 10

9 M), gluconeogenesis (40%,EC507.8 10

9 M), and ureagenesis (130%, EC507.0 108 M)

compared with basal values; these effects were blunted by the selec-tive A3 AR antagonist 9-chloro-2-(2-furanyl)-5-[(phenylac-etyl)amino][1,2,4]-triazolo[1,5-c]quinazoline (MRS 1220) but not byselective A1, A2A, and A2B AR antagonists. In addition, MRS 1220antagonized inosine-induced transient increase (40%) in cytosolicCa2 and enhanced (90%) glycogen phosphorylase activity. Inosine-induced Ca2 mobilization was desensitized by adenosine; in areciprocal manner, inosine desensitized adenosine action. Inosinedecreased the cAMP pool in hepatocytes when A1, A2A, and A2BARwere blocked by a mixture of selective antagonists. Inosine-promotedmetabolic changes were unrelated to cAMP decrease but were Ca2

dependent because they were absent in hepatocytes incubated inEGTA- or BAPTA-AM-supplemented Ca2-free medium. After insilico analysis, no additional cognate adenosine/inosine receptorswere found in human, mouse, and rat. In both perfused rat liver andisolated hepatocytes, hypoxia/reoxygenation produced an increase ininosine, adenosine, and glucose release; these actions were quantita-tively greater in perfused rat liver than in isolated cells. Moreover, allof these effects were impaired by the antagonist MRS 1220. On thebasis of results obtained, known higher extracellular inosine levelsunder ischemic conditions, and inosines higher sensitivity for stim-ulating hepatic gluconeogenesis, it is suggested that, after tissularischemia, inosine contributes to the maintainence of homeostasis byreleasing glucose from the liver through stimulation of A3 ARs.

ischemia; calcium; urea; phylogenetic analysis; homeostasis.

INOSINE IS A NATURALLY OCCURRING PURINE NUCLEOSIDEformed byadenosine deamination. Its normal interstitial concentrations inrat plasma and serum have been reported in the range of 0.520M (51, 61), and inosine accumulates to even higher levels(100 M) than adenosine does in ischemic tissues (34, 41,

50, 51, 56). Our laboratory was the first to describe a stimu-latory action of inosine on ureagenesis and gluconeogenesis inisolated hepatocytes (23, 68). However, over the last decadeseveral reports (e.g., Refs. 19, 32, 59) appeared regarding therole of inosine in regulating the immunologic and cardiovas-cular systems. Although in the majority of cases inosine bindsto A3adenosine receptors (ARs) to promote its effects (19, 32,59), there are reports in which A2A AR (19) or even an

AR-independent G protein-coupled receptor (GPCR) pathway(27) were involved.

To date, four AR subtypes have been cloned (A1, A2A, A2B,and A3), each with unique tissue distributions, ligand affinity,and signal-transducing mechanism (for a review, see Ref. 49).All four AR subtypes are present in isolated hepatocytes,where they stimulate glycogenolysis, gluconeogenesis, andureagenesis rates (49). Signal transduction systems for obtain-ing these increases were via adenylyl cyclase for A2Aand A2BAR, whereas A1 and A3 AR involved changes in cytosolicCa2 (2022, 60, 66). The purpose of this work included thefollowing: 1) to define the receptor type involved in inosineresponses in isolated hepatocytes; 2) to identify the signaltransduction pathway mediating these inosine responses; 3) toexplore the possibility of finding additional adenosine/inosinereceptors; and4) to obtain insight into the physiological mean-ing of these inosine actions.

MATERIALS AND METHODS

Selective AR agonists and antagonists used in this work areincluded in Table 1 and are listed in alphabetical order of theirabbreviations. Full chemical names, the receptor-binding constant forAR agonist, reported data on the Kifor AR antagonists, and pertinentreferences are additionally included. All of these compounds werepurchased from Sigma RBI.

All animal experiments were conducted in accordance with theFederal Guidelines for the Care and Use of Animals (NOM-062-

ZOO-1999, Ministry of Agriculture, Mexico) and were approved bythe Institutional Ethics Committee of the National Autonomous Uni-versity of Mexicos Faculty of Medicine (FM-UNAM).

Isolation of hepatocytes. Male Wistar rats (150 200 g) wereanesthetized with ether, and cells were isolated by the method ofBerry and Friend (7) as modified by Guinzberg et al. (23). Hepato-cytes were used when viability was at least 95%, as assayed by thetrypan blue exclusion method. Experiments were conducted by dupli-cate with 3040 mg wet wt hepatocytes.

Address for reprint requests and other correspondence: E. Pina, Departa-mento de Bioqumica, Facultad de Medicina, Universidad Nacional Autonomade Mexico, Apdo. Postal 70159, Mexico City, 04510, Mexico (e-mail:[email protected]).

The costs of publication of this article were defrayed in part by the paymentof page charges. The article must therefore be hereby marked advertisementin accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Am J Physiol Endocrinol Metab290: E940E951, 2006.First published December 13, 2005; doi:10.1152/ajpendo.00173.2005.

0193-1849/06 $8.00 Copyright 2006 the American Physiological Society http://www.ajpendo.orgE940


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Ureagenesis.Hepatocytes from 24-h-starved rats were incubatedfor 1 h at 37C in an atmosphere of O2-CO2(95%-5%) for 60 min ina gyratory water bath in Krebs-Ringer buffer (KRB) containing 10mM glucose, 5 mM (NH4)2CO3, and 3 mM ornithine. Urea synthesiswas assayed after 60 min (24).

Gluconeogenesis.Hepatocytes from 24-h-starved rats were incu-bated for 1 h in KRB containing 10 mM lactate. Glucose synthesiswas measured in the supernatant of cells by the glucose oxidasemethod (18).

Glycogenolysis.Hepatocytes from rats fed ad libitum were incu-bated for 45 min in KRB without lactate or any other substrate.Glucose release was measured (18).

Glycogen phosphorylase activity. This activity was assayed bymeasuring the incorporation of [U-14C]glucose 1-phosphate into gly-cogen, as described by Starke et al. (57). Hepatocytes were exposed tothe agents, and aliquots were withdrawn at time intervals and placed

in 0.2 ml of ice-cold medium containing 10 mM MES, 20 mM NaF,25 mM glycerophosphate, 10 mM EDTA, and 0.8 mM digitonin.Hepatocyte extracts (25 l) were mixed with an equal volume ofphosphorylase assay medium containing 50 mM NaF, 4.8 mM caf-feine, 86 mM glucose 1-phosphate, 2% glycogen, and 8.5 Ci of[U-14C]glucose 1-phosphate and incubated at 37C. The reaction wasstopped after 30 min by the addition of 25 l of glacial acetic acid. A50-l sample was spotted onto filter paper and washed twice with66% ethanol, washed with acetone, and placed in a cocktail for liquidscintillation counting.

cAMP accumulation. Hepatocytes from fed rats were incubated at37C for 2 min in KRB. cAMP was measured using the Amersham kitTRK4312.

Ca2 measurement in fura 2-AM loaded hepatocytes. This wasperformed as described by Llopis et al. (42). Briefly, isolated hepa-

tocytes from fed rats were diluted in KRB to a final concentration of40 mg wet wt/ml and incubated for 10 min at 37C in an atmosphereof O2-CO2(95%-5%). Cells were incubated for an additional 20 minin the presence of 3 M fura 2-AM and were washed twice bycentrifugation at 500 rpm for 3 min. Liver cells were divided into200-l aliquots, immersed in ice, and used within the subsequent 5min. Ca2 was measured in these cells as in Llopis et al. (42) usinga Kd 224 nM.

Hypoxia/reoxygenation in isolated hepatocytes and perfused liver.In experiments with fed rats, isolated hepatocytes were used tomeasure inosine, adenosine, and glycogenolysis release. Fasted rats(16 h) were used to measure gluconeogenesis and ureagenesis rates.Rat livers were perfused in situ by placing a cannula in the portal vein,and KRB was equilibrated with an O2-CO2 mixture (19:1) at a

constant flow rate of 16 ml/min. Hepatic venous effluents wereobtained via a cannula in the vena cava.

Adenosine and inosine release quantification. Nucleosides weremeasured by enzymatic assay in double-beam spectrophotometer bythe method described by Olsson (47).

Statistical methods.Values are reported as means SE. Studentst-test was applied to assess differences between groups. Statisticalsignificance was set at P 0.05.

Identification of cognate ARs on protein databases. Initially, se-quences of known ARs from the rhodopsin superfamily were retrievedfrom the Swiss-Prot protein database at http://au.expasy.org/sprot/ (3).The amino acid sequence from each of these known ARs was used asbait for BLASTP (1) searches at the National Center for Biotechnol-ogy Information GenBank nonredundant protein database (6). Todetermine the number of sequences encoding ARs in animals withcomplete genome sequence, we repeated the BLAST search with the

tBLASTn program (1), using amino acid sequences of characterizedadenosine GPCRs as queries against whole genomic DNA sequencesor the high-throughput genomic sequence database from human,mouse, rat, zebra fish, Japanese puffer fish (International Fugu Ge-nome Consortium, assembly version 3.0; http://genome.jgi-psf.org/fugu6/fugu6.home.html), and the ascidian Ciona intestinalis (assem-bly version 1.0; http://genome.jgipsf.org/ciona4/ciona4.home.html).Ab initio gene predictions were performed with the GeneCombersystem (54), which provides increased gene recognition accuracy bycombining predictions from the gene-finding Genscan (10) andHMMgene (37) programs. GeneComber-predicted exons were veri-fied by multiple alignments with amino acid sequences from adeno-sine GPCRs to gather additional support for constructing gene models.

Multiple sequence alignment and phylogenetic analysis.Multiplesequence alignments were performed by using ClustalX v1.81 (58)

and corrected according to gapped BLASTP results (1). Phylogeneticanalyses were carried out with MEGA v2.1 (38) software, using boththe maximum parsimony and distance-based methods UPGMA (un-weighted pair group method with arithmetic mean) and neighbor

joining, along with minimum evolution with the Poisson correctiondistance method, and gaps were treated by pairwise deletion. Accu-racy of reconstructed trees was examined by the bootstrap test with1,000 replications. Phylogenetic trees were rooted with the bovinerhodopsin sequence. Complete names of organisms included in thephylogenetic analysis are as follows: ANOGA, Anopheles gambiae(Arthropoda, insecta); ASTMI, Asterina miniata (starfish; Echinoder-mata); BOVIN, Bos taurus (Chordata, vertebrata, mammalia);CAEBR,Caenorhabditis briggsae(Nematoda); CAEEL, Caenorhab-ditis elegans (Nematoda); CANFA, Canis familiaris (Chordata, ver-

Table 1. Specific agonists and antagonists for ARs used in this work

Abbreviation Chemical Name Receptor ActionReceptor-

Binding Value Ki Reference

ADSPX 1-allyl-3,7-dimethyl-8-p-sulfophenylxanthine A2B Antagonist 0.6 nM (28)Alloxazine Benzo[g]pteridine 2,4(1H,3H)-dione A2B Antagonist 13 nM (40)CCPA 2-chloro-N6-cyclopentyladenosine A1 Agonist 0.4 nM (43)CGS-15943 9-chloro-2-(2-furanyl)[1,2,4]triazolo[1,5-

c]quinazoline-5-amine

A1 Antagonist 4 nM (31)

CGS-21680 2-P(2-carboxyethyl)phenethylamino-5-N-ethylcarboxamidoadenosine

A2A Agonist 15 nM (30)

CSC 1,3,7-trimethyl-8-(3-chlorostyryl) xanthine A2A Antagonist 54 nM (29)DPCPX 8-cyclopentyl-1,3-dipropylxanthine A1 Antagonist 0.69 nM (25)IB-MECA 1-deoxy-1-[6-[((3-

iodophenyl)methyl)amino]-9H-purin-9-yl]-N-methyl--D-ribofuranuronamide

A3 Agonist 1.1 nM (64)

MRS 1220 9-chloro-2-(2-furanyl)-5-((phenylacetyl)amino)-[1,2,4]triazol[1,5-c]quinazoline

A3 Antagonist 14 nM (36)

NECA 5-N-ethylcarboxamidoadenosine A1, A2B Agonist A1 11 nM (9)A2B 16 nM

AR, adenosine receptor.

E941HOMEOSTATIC ROLE OF INOSINE IN THE LIVER

AJP-Endocrinol Metab VOL 290 MAY 2006 www.ajpendo.org


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tebrata, mammalia); CAVPO, Cavia porcellus (domestic guinea pig;Chordata, vertebrata, mammalia); CHICK, Gallus gallus (Chordata,vertebrata, aves); CIOIN, Ciona intestinalis (Chordate, urochordata,ascidiacea); DANRE, Danio rerio (zebra fish; Chordata, vertebrata,teleostei); DROME, Drosophila melanogaster(Arthropoda, insecta);FUGRU, Fugu rubripes (Japanese puffer fish; Chordata, vertebrata,teleostei); HORSE,Equus caballus(Chordata, vertebrata, mammalia);HUMAN,Homo sapiens(Chordata, vertebrata, mammalia); MOUSE,

Mus musculus (Chordata, vertebrata, mammalia); RABBIT, Orycto-lagus cuniculus(Chordata, vertebrate, mammalia); RAT, Rattus nor-vegicus(Chordata, vertebrata, mammalia); SHEEP, Ovis aries(Chor-data, vertebrata, mammalia), and XENLA, Xenopus laevis(Chordata,vertebrata, amphibia).

RESULTS

Inosine stimulates glycogenolysis, gluconeogenesis, andureagenesis in hepatocytes via A3 AR. Adenosine and inosineconcentration-response curves to stimulate glycogenolysis,gluconeogenesis, and ureagenesis rates are presented in Fig. 1.Effective concentration (EC50) values of adenosine and inosine

were calculated, along with ratios for (adenosine EC50value)/(inosine EC50 value) in each activated pathway (Table 2).These data indicated that gluconeogenesis and ureagenesismight be activated at lower concentrations of inosine than ofadenosine. The stimulating effect of 1 M inosine on glyco-genolysis, gluconeogenesis, and ureagenesis was blunted spe-cifically with the selective A3 AR antagonist 9-chloro-2-(2-furanyl)-5-[((phenylacetyl)amino)-[1,2,4]triazolo[1,5-c]quina-zoline (MRS 1220) but was not modified when inosine wassimultaneously incubated with 9-chloro-2-(2-furanyl)[1,2,4]triazolo[1,5-c]quinazolin-5-amine (CGS-15943), 1,3,7-tri-methyl-8-(3-chlorostyryl)xanthine (CSC), and 1-allyl-3,7-dimethyl-8-p-sulfophenylxanthine (ADSPX), or alloxazine, se-lective antagonists for A1, A2A, and A2BAR, respectively (Fig.2); i.e, inosine stimulated these three metabolic routes inisolated rat liver cells only if A3 AR was not blocked. Twoselective A2BAR antagonists were used in these experimentsbecause the required ADSPX solvent [A2B antagonists withlower receptor-binding constant (Table 1)] is dimethyl sulfox-ide, which, when used at a concentration of 1 mM to quantifyurea, interfered with the assay (results not shown) (24). Thus,

in this case, ADSPX was substituted for a water-solubleselective A2BAR antagonist such as alloxazine.

Inosine-induced Ca2 mobilization to stimulate glycogenol-

ysis, gluconeogenesis, and ureagenesis. A common action ofadenosine and an AR-specific agonist is to increase [Ca2]iinisolated hepatocytes (22). Results in Table 3 show that inosineshares in this action. It is noteworthy that stimulation witheither inosine or the individual AR agonists employed resultedin a rise in Ca2 similar to the rise obtained with adenosine,which might activate all four ARs. We performed three seriesof experiments to investigate the role of calcium in livermetabolic pathway inosine-mediated activation. In the firstseries, Ca2 was eliminated from KRB; in the second series,

EGTA was included in Ca2

-free KRB to chelate extracellularCa2; and in the third series, BAPTA-AM was added toCa2-free KRB to chelate intracellular Ca2. Inosine elicited alesser stimulation in studied metabolic pathway rates whencells were incubated in Ca2-free KRB. In addition, thesepathways were not stimulated at all by the nucleoside wheneither of the used chelating agents was present (Fig. 3).

To identify AR involved in the transient inosine-mediatedincrease of free Ca2, we conducted the experiment presentedin Fig. 4. Inosine alone produced a temporary increase in Ca 2

(Fig. 4A) that was not modified by A1, A2A, and A2B AR-selective antagonists (Fig. 4, BD) but was blunted by A3ARantagonist (Fig. 4E).

Fig. 1. Dose-response curves of inosine (E) or adenosine (F) for glycogenol-ysis (A), gluconeogenesis (B), and ureagenesis (C) in hepatocytes. Basal valuesin the absence of nucleosides (). Plotted values are means, and vertical linesrepresent SE of duplicate incubation of 6 independent cell preparations, exceptfor control sample, where 810 independent cell preparations were included.Statistical significance vs. control values are indicated. *P 0.05; **P 0.001.

Table 2. EC50values for adenosine and inosine to stimulateglycogenolysis, gluconeogenesis, and ureagenesisin isolated rat hepatocytes

PathwayEC50 Values for

AdenosineEC50 Values for

Inosine

Ratio, EC50Adenosine toEC50Inosine

Glycogenolysis 3.810

9

M 4.210

9

M 0.90Gluconeogenesis 1.7108M 7.8109M 2.2Ureagenesis 1.8107M 7.0108M 2.6

Data obtained from experiments in Fig. 1. EC50, effective concentration.

E942 HOMEOSTATIC ROLE OF INOSINE IN THE LIVER



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Desensitization experiments were conducted to test whetherinosine acted through GPCR. Isolated hepatocytes were stim-ulated with 1 M adenosine or inosine, and Ca2-transientrises were monitored. After recovery to initial values in 2min, cells were stimulated again. Under this protocol, adeno-sine failed to reinitiate cell activation independently of whetherfirst activation was produced by adenosine (Fig. 5A) or byinosine (Fig. 5D). Similarly, inosine failed to reinitiate cellactivation independently of whether initial activation was ob-tained with adenosine (Fig. 5B) or inosine (Fig. 5C).

Inosine- and adenosine-stimulated phosphorylase activity.Incubation of isolated hepatocytes with either inosine or adeno-sine resulted in a statistically significant increase in glycogenphosphorylase activity (Fig. 6). Phosphorylase activity in in-osine-stimulated cells reached a nearly twofold increase overbasal levels, and this stimulation was blunted with the A3 AR

antagonist or the intracellular Ca2

chelant agent BAPTA-AM(Fig. 6). At equimolecular doses, adenosine was less potentthan inosine, and BAPTA-AM additionally blunted adenosinestimulation of phosphorylase (Fig. 6). A3AR antagonist partialinhibitory action on adenosine-mediated stimulation (Fig. 6)might be explained by the effect of adenosine through activa-tion of AR other than A3.

Inosine and cAMP pool. To investigate cAMP involvementin the inosine response, hepatocytes were incubated withgraded concentrations of the nucleoside. Changes in the cAMPpool were compared with those produced by adenosine andAR-selective agonists. Results show that stimulation of A2AAR with the selective agonist 2-P(2-carboxyethyl)phenethyl-amino-5-N-ethylcarboxamidoadenosine and A2B AR with a

mixture of 5-(N-ethylcarboxamido)adenosine (an A1, A2BARagonist) plus 8-cyclopentyl-1,3-dipropylxanthine (an A1-selec-tive AR antagonist) produced a dose-dependent increase incAMP content (Fig. 7). In contrast, stimulation with 2-chloro-N6-cyclopentyladenosine, an A1-selective AR agonist, or 1-de-oxy-1-[6-[((3-iodophenyl)methyl)amino]-9H-purin-9-yl]-N-methyl--D-ribofuranuronamide (IB-MECA), an A3 AR-selective agonist, originated a dose-related decrease in cAMPcontent (Fig. 7). According to all of the findings in this paper,inosine actions resemble those of A3AR agonists. Therefore, itwould be expected that inosine might decrease cAMP in thesame manner as A3 AR agonists; however, adenosine and,unexpectedly, inosine were unable to modify the cAMP pool in

Fig. 2. Effect of inosine in the absence or presence of adenosine receptor

(AR)-selective antagonists on the rate of glycogenolysis (A), gluconeogenesis(B), and ureagenesis (C) in hepatocytes. Cells were incubated as detailed inMATERIALS AND METHODS with 1 M inosine alone or combined with 1 Mfinal concentration of the following AR-selective antagonists: 9-chloro-2-(2-furanyl)[1,2,4]triazolo[1,5-c]quinazolin-5-amine (CGS-15943) for A1; 1,3,7-trimethyl-8-(3-chlorostyryl)xanthine (CSC) for A2A; 1-allyl-3,7-dimethyl-8-p-sulfophenylxanthine (ADSPX) for for A2B; and 9-chloro-2-(2-furanyl)-5-((phenylacetyl)amino)-[1,2,4]triazolo[1,5-c]quinazoline (MRS 1220) for A3.Control samples were incubated without added inosine or antagonist. Inureagenesis studies, alloxazine was used instead of ADSPX as an A2B-selective antagonist (see text). Values represent means SE of duplicateincubation from 4 to 6 independent cell preparations. *Statistical significancevs. control sample without inosine, P 0.001; **statistical significanceinosine alone vs. inosine MRS 1220, P 0.01.

Table 3. [Ca2]i in isolated hepatocytes treated withadenosine, inosine, or selective AR agonists

Additions AR-Stimulated [Ca2]i, nmol/l Values, %

None 1956.3 100Adenosine All 4 2847.3 146Inosine ? 2746.3 141

CCPA A1

3016.9 154CGS-21680 A2A 2816.9 144NECA plus DPCPX A2B 2987.9 153IB-MECA A3 2797.9 143

Numbers are means SE of duplicates from 4 independent cell prepara-tions. [Ca2]i, cytosolic Ca2 concentration. Experimental conditions as inMATERIALS AND METHODS. To stimulate A2BAR alone, an AR agonist for A2Band A1, such as NECA (Table 1), was mixed with DPCPX, a selectiveantagonist for A1 AR. Nucleosides, agonists, and antagonists were used at a1-M final concentration. Statistical significance, nucleoside or agonist vs.control; P 0.001 in all cases.




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hepatocytes (Fig. 7). Next, hepatocytes were incubated withAR antagonist-supplemented inosine. Thus selective antago-nists for each of the four ARs, CGS-15943 for A1, CSC forA2A, ADSPX for A2B, and MRS 1220 for A3, were used so that

different mixtures of three of these antagonists added to hepa-tocytes would maintain three of the four ARs blocked, leavingonly one AR able to be activated, which might or might not bestimulated by inosine. Only in experiments in which A3 ARwas not antagonized by the adequate mixture of AR agents didinosine decrease the cAMP cellular pool (Fig. 8), similarly toIB-MECA, an A3AR agonist, whereas, if inosine was added tocells in which A1, A2A, or A2B AR were not antagonized byadequate AR blocker mixtures, cAMP values remained unmodi-fied (Fig. 8). Additional experiments are required to understandwhy inosine alone did not modify the cAMP cellular pool (Fig. 7),whereas inosine did indeed decrease the cAMP pool if A1, A2A,and A2BAR were blocked by their selective antagonists (Fig. 8).

Fig. 3. Calcium participation in inosine-mediated stimulation of glycogenolysis (A),gluconeogenesis (B), and ureagenesis (C) in hepatocytes. Cells were placed under 4different conditions: 1) complete Krebs-Ringer buffer (KRB) containing 1.2 mMCa2; 2) Ca2-free KRB; 3) cells were preincubated for 15 min in Ca2-freeKRB supplemented with 1.2 mM EGTA; and 4) cells were preincubated for 20 minin Ca2-free KRB supplemented with 10 M BAPTA-AM. Control cells at left(filled bars) of each experimental condition and hepatocytes were supplemented with1 106 M inosine at the right(open bars) of each experimental condition. Eachdatum in the figure corresponds to mean SE of duplicate incubations from 4 to 6independent cell preparations. *Statistical significance for cells incubated with 1) KRBwith Ca2 inosine vs. 2) Ca2-free KRB inosine, P 0.01; **3) Ca2-freeKRB with EGTA inosine and 4) Ca2-free-KRB with BAPTA-AM inosine,bothP 0.001.

Fig. 4. Effect of inosine in the absence or presence of AR-selective antagonistson cytosolic Ca2 concentration ([Ca2]i) in hepatocytes. Cells were labeledwith fura 2-AM and stimulated with 106 M inosine alone or supplementedwith 106 M for each AR-selective antagonist indicated. Experiment wasrepeated 3 times with identical results.




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In any event, cAMP does not appear to be involved in inosine-activated metabolic routes in hepatocytes.

Phylogenetic analyses ruled out the existence of an addi-tional GPCR homologous to ARs in mammals. The results inthis paper, as well as those of other authors, clearly demon-strate that GPCR, mainly through A3 AR, mediates someinosine effects. However, this does not discard the possibilitythat other adenosine-related GPCRs might exist, including acognate inosine GPCR. To explore the latter possibility, weconducted an extensive search for homologous protein se-quences to the adenosine/inosine receptor in whole genomicDNA sequences from human, mouse, rat, zebra fish (D. rerio),

Japanese puffer fish (F. rubripes), and the ascidian C. intesti-nalis. Subsequently, we conducted a phylogenetic analysis forretrieved adenosine/inosine receptor sequences. We found noadditional cognate adenosine/inosine receptors in addition tothe four known adenosine GPCRs in human, mouse, and rat.Unexpectedly, however, we did find three additional AR-homologous protein sequences in puffer fish and one in zebra

fish. Recently, a similar observation was reported with 2-adrenoceptors, because the zebra fish possesses five 2-adre-noceptors instead of the three found in mammals and the pufferfish possesses eight 2-adrenoceptors (11, 52, 53). Figure 9shows a phylogenetic tree constructed with a total of 46full-length protein sequences identified as ARs (all belongingexclusively to animals). It can be observed that all AR proteinsequences in mammals belong to one of the four known ARtypes. No additional AR types were found in mammals; how-ever, in the puffer fish, two distinct A1 AR were found(designated provisionally as A1A and A1B) along with oneadditional A2 AR (provisionally denominated A2C). On theother hand, in the complete genome of C. intestinalis (anonvertebrate chordate that diverged very early from otherchordates, including vertebrates) we identified only three AR-homologous protein sequences, although none resulted or-thologous (same gene in different species) to the four AR typesknown in mammals. These three C. intestinalis ARs aregrouped with other AR sequences found in zebra fish, pufferfish, starfish (echinodermata), arthropoda, and nematoda; thesesequences probably comprise a fifth AR type. Within thisgroup, only the AR from the starfish A. miniata has beenexperimentally demonstrated as an AR coupled to a Gi-linkedprotein (35).

Adenosine, inosine, and glucose are released by the liverunder hypoxia/reoxygenation conditions. Once we definedwhich AR was involved in inosine action in liver, the signal

transduction pathway mediating inosine action, and the ab-sence of an additional adenosine/inosine receptor participatingin these responses, we focused on the physiological meaning ofinosine-mediated action in liver. It is known that adenosine andinosine can be released by different organs, e.g., brain (39, 65),heart (34, 41, 56), eye (50), lung (45), kidney, and liver (51).Furthermore, release of these nucleosides is induced underhypoxic conditions (34, 41). Isolated rat hepatocytes alsorelease adenosine under hypoxic conditions (5); however, themetabolic effect of endogenous adenosine and inosine releasein liver has not been tested. Thereafter, we subjected bothperfused rat liver and isolated hepatocytes to hypoxia/reoxygen-ation conditions and measured inosine, adenosine, and glucoserelease. During hypoxic incubation, isolated hepatocytes accumu-

lated inosine, adenosine, and glucose in extracellular volume(Table 4). Both nucleosides and glucose accumulation were ob-served additionally under conditions of hypoxia/reoxygenation.The selective antagonist for A3AR, MRS 1220, impaired libera-tion of glucose from intracellular sources, but interestingly, it alsoimpaired inosine and adenosine release from hepatocytes.

We obtained similar results in perfused rat livers that weresubjected to hypoxia and hypoxia/reoxygenation conditions(Fig. 10). Once experimental conditions were set, inosine,adenosine, and glucose release began after an initial lag of 2.5min. Inosine reached a plateau after 10 min and adenosine after5 min, but glucose increased progressively during the follow-ing 30 min (Fig. 10).

Fig. 5. Inosine and adenosine desensitize hepatocytes to each other. Cells werelabeled with fura 2-AM and stimulated initially with 106 M adenosine (Aand

B) or 106 M of inosine (C and D), and free [Ca2]i was measured as afunction of time. After recovery to initial values, a second stimulation withinosine (B and C) or adenosine (A and D) was performed. Experiment wasrepeated 4 times with identical results.




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Under hypoxia and hypoxia/reoxygenation conditions, glu-coneogenesis and ureagenesis activities were assayed in rat hepa-tocytes that were isolated from fasted rats (16 h). Both ATP-dependent glucose and urea production diminished by 50% in

isolated hepatocytes incubated under hypoxia or hypoxia/reoxy-genation conditions (data not shown). These latter results can beexplained because under low oxygen tension, insufficient ATPproduction precludes flux through anabolic pathways (8).

Fig. 6. Effect of inosine and adenosine of glycogen phosphorylaseactivity in hepatocytes from fed rats. Isolated hepatocytes (20 3 mgof protein) were incubated in 5 ml of Krebs-Ringer bicarbonatecontaining 1.2 mM CaCl2. Glycogen phosphorylase activity wasmeasured as detailed in MATERIALS AND METHODS. A: samples wereincubated with 106 M inosine alone (F), 106 M inosine 106 M

MRS 1220 (E), or 106 M inosine 10 M BAPTA-AM (). B:samples were incubated with 106 M adenosine (F), 106 M adeno-sine 106 M MRS 1220 (E), or 106 M adenosine 10 MBAPTA-AM (). Values are means SE of 3 independent experi-ments by duplicate. Statistical significance: P 0.001 by comparinginosine at 0 min vs. inosine at 2.5, 5, and 10 min; P 0.001 bycomparing inosine alone vs. inosine MRS 1220 or inosine BAPTA-AM; P 0.01 (at least) by comparing adenosine at 0 min vs.adenosine at 2.5, 5, and 10 min; P 0.01 (at least) by comparingadenosine alone vs. adenosine MRS 1220 or adenosine BAPTA-AM.

Fig. 7. Effect of adenosine, inosine, and AR-selective agonists on cAMPproduction in hepatocytes. Cells were incubated for 2 min in KRB withadenosine (), inosine (F), and the following AR selective agonists: 2-chloro-

N6-cyclopentyladenosine for A1(); 2-P(2-carboxyethyl)phenethyl-amino-5-N-ethylcarboxyamidoadenosine for A2A (E), and 1-deoxy-1-[6-[((3-iodophe-nyl)methyl)amino]-9H-purin-9-yl]-N-methyl--D-ribofuranuronamide for A3(); to stimulate A2BAR (), a mixture of 5-(N-ethylcarboxamido)adenosineand 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) was used. Results are ex-pressed as %basal value, which was 0.74 0.03 pmol of cAMP formed in 2min/mg (wet wt). Each value represents means SE of 4 independentexperiments, each performed in duplicate. Statistical significance vs. basal isindicated: *P 0.05; **P 0.01; ***P 0.001.

Fig. 8. Effect of inosine on cAMP values of hepatocytes, to which 3 of 4 ARswere inhibited by mixtures of selective AR antagonists as detailed in the text.Inosine (1 M, final concentration) was added to each tube in which the ARnoninhibited remnant AR was contained: A1 () when mixing 1 M (finalconcentration) CSC 1 M ADSPX 1 M MRS 1220; A2A(E) when mixing1 M DPCPX 1M ADSPX 1M MRS 1220; A2B() when mixing 1 MDPCPX 1 M CSC 1 M MRS 1220; and A3 () when mixing 1 MDPCPX 1 M CSC 1 M ADSPX. Cells were incubated in KRB with theindicated additions. Results are expressed as %basal value, which was 0.74 0.03pmol of cAMP formed in 2 min/mg (wet wt). Each value represents means SEof 4 independent experiments, each performed in duplicate. Statistical significancevs. basal is indicated: *P 0.001.




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Fig. 9. Phylogenetic analysis of ARs. Phylogenetic tree constructed with available protein sequences belonging to the AR subfamily by using minimum evolutionmethod. Trees were calculated using MEGA 2.1 (38). Dotted bars indicate nodes supported in 70 (open), 80 (gray), or 90% (filled) of 1,000 randombootstrap replicates of all UPGMA (unweighted pair group method with arithmetic mean), neighbor-joining, minimum-evolution, and maximum-parsimony trees.Scale bar represents 0.2 amino acid substitutions per site. Obtained trees were rooted by use of bovine rhodopsin. Thick vertical bars indicate the taxonomic groupto which the protein sequence belongs and fine vertical bars the type of AR to which the protein sequence belongs. Sequence names are indicated according toa Swiss-Prot-like identifier (gene organism) followed by the database accession number (GenBank, PIR, Swiss-Prot, etc.) and protein amino acid length. ARsequences deduced from genomic sequences were obtained from the following sources: the Danio rerio Sequencing Group at the Sanger Institute(http://www.sanger.ac.uk/Projects/D_rerio/), theFugu rubripes Genome Project v3.0 (2), and the Ciona intestinalis Genome Project v1.0 (16), the last 2 bothat the US Department of Energy Joint Genome Institute (http://genome.jgi-psf.org/fugu6/fugu6.home.html and http://genome.jgi-psf.org/ciona4/ciona4.home.html). Experimentally characterized ARs are underlined. A full list of organism names included in the tree is provided in MATERIALS AND METHODS.




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DISCUSSION

Data from this paper confirm our preliminary finding (23,68) and greatly extend previous information on the hepaticactions of inosine. Adenosine- or inosine-stimulated rat livercells showed a dose-related increase in the rates of three of themain hepatic metabolic pathways, namely glycogenolysis, glu-coneogenesis, and ureagenesis (Table 1 and Fig. 1). Consider-ing the EC50 values obtained for adenosine or inosine tostimulate the three metabolic pathways and the reported phys-iological concentrations of adenosine and inosine in rat serum(61), both nucleosides might be effective in producing activa-tion of these metabolic pathways. Converging evidence sug-gests a preeminent role of inosine over adenosine in stimulat-ing hepatic metabolic routes through A3 AR activation, be-cause inosine serum concentration is higher than adenosine andinosine concentration was 25-fold higher than adenosine inhepatic venous effluents of isolated perfused liver (51). In

addition, EC50values for inosine are similar to EC50values foradenosine to stimulate glycogenolysis, but they are 2- to2.5-fold lower in stimulating gluconeogenesis and ureagenesis.Furthermore, a very active adenosine deaminase is present inrat serum that converts adenosine into inosine (48). The pre-viously mentioned considerations led us to explore some char-acteristics of inosine-mediated actions on liver cells, althoughadditional evidence in favor of inosine as an important inter-cellular messenger will be included in the final part of thisdiscussion. The three main inosine-stimulated metabolic path-ways were equally blunted by the selective A3 AR antagonist(Fig. 2); in contrast, incubation of liver cells with selective A1,A2A, o r A2B AR antagonists did not modify the inosine-mediated rise in glycogenolysis, gluconeogenesis, and ure-

agenesis rates (Fig. 2). Hence, A3 AR seems to be the initialtarget of inosine for stimulating metabolic actions in liver cells.

In previous work with isolated hepatocytes (20, 22, 60, 66),it was established that the change in the [Ca2]i pool was thetransduction mechanism elicited by A3 AR stimulation withadenosine or A3 AR agonists to obtain glycogenolysis, glu-coneogenesis, and ureagenesis rate increases. Similar resultswere obtained after stimulation of isolated hepatocytes withinosine: an increase in [Ca2]i (Table 3), dependence of suchan increase to activate metabolic pathway rates (Fig. 3), andblockade in the rise of [Ca2]iobserved exclusively with MRS1220, the A3 AR antagonist, but not with the use of selectiveA1, A2A, and A2BAR antagonists (Fig. 4).

Main metabolic pathway stimulation in liver by inosine isabsolutely dependent on an increase in free [Ca2]i (Fig. 3).Thus incubation of cells in Ca2-free KRB supplemented withthe intracellular chelant BAPTA-AM impaired any inosine-

mediated activation in glycogenolysis, gluconeogenesis, andureagenesis rates (Fig. 3). Nonetheless, when hepatocytes wereincubated in Ca2-free KRB in the absence of chelant agents,inosine produced minor stimulation in the metabolic pathwayrates that we studied compared with stimulation observed inKRB containing 1.2 mM Ca2 (Fig. 3). All these data point toa relevant role of extracellular Ca2 in inosine-mediated trans-duction actions in liver and to a minor contribution of intra-cellular Ca2 storage compartments to drive the same actions.Unpublished experiments (Guinzberg R and Pina E) usingisolated hepatocytes, incubated in KRB with 1.2 mM Ca2 andchallenged with MRS 1220, an A3 AR agonist, are confirma-tory. Thapsigargin, an inhibitor of Ca2 release from intracel-lular storage compartments, decreases stimulation of urea syn-thesis by nearly 40%.

The following experiment presents another property of theinosine-sensitive AR. This nucleoside desensitizes AR towardadenosine (Fig. 4); a lower concentration of serum adenosinewill be quantitatively less important compared with inosine topromote further metabolic responses in liver. In addition, thesedata support that a GPCR is involved in inosine-mediatedactions in liver.

Intracellular Ca2 increase has been shown to stimulateglycogenolysis (33, 63). In particular, two Ca2-mobilizingagents, namely epinephrine and ionophore A-23187, promotedhepatocyte glycogen phosphorylase activation that led to anincrease in cell glucose release (62). Thereafter, a [Ca2]irise

by A3 AR stimulation in hepatocytes by any of the studiednucleosides (Fig. 4) in turn activated glycogen phosphorylaseto a greater extent with inosine than with adenosine (Fig. 6).With the information recorded to this point in this work, wecould anticipate a blockade in nucleoside-mediated phosphor-ylase activation with the use of either a selective A3 ARantagonist (Fig. 4) or an intracellular chelating agent (Fig. 3).In fact, both inhibitory actions were recorded (Fig. 6).

Two additional experiments analyzing the role of cAMP asa signal transduction pathway for inosine-mediated metabolicactions gave negative results. Inosine alone, as well as adeno-sine alone, did not modify cAMP pool in liver cells (Fig. 7). Inanother set of experiments with isolated hepatocytes (Fig. 8),

Table 4. Release of inosine and adenosine and glycogenolysis rate in isolated rat hepatocytes mantained in Ringer-HEPESbuffer and subjected to different oxygenation conditions

Experimental Conditions

Inosine Adenosine Glycogenolysismol glucose/g wet

wt in 45 minmolmin1mg prot1

Oxygenation O2-CO2(19:1) 2.220.01 1.860.02 54.32.2O2-CO2 (19:1) 106 MRS 1220 1.410.06* 1.650.02 50.81.7

Hypoxia N2-CO2(19:1) 4.690.04* 3.890.01* 123.83.2*N2-CO2(19:1) 106 M-MRS 1220 1.480.01 1.340.02 72.52.7

Hypoxia/reoxygenation N2-CO2for 25 min, then O2-CO2 for 20 min 4.670.11* 3.030.01* 125.32.3*N2-CO2 106 M MRS 1220 for 25 min, thenO2-CO2 106 M MRS 1220 for 20 min

2.000.02 1.680.01 62.71.9

Values are means SE in 3 independent experiments with duplicate samples. Hepatocytes were incubated for 45 min in Ringer-HEPES buffer containingthe following: 120 mM NaCl, 1.2 mM CaCl2, 1.2 mM MgSO4, 1.2 mM KH2PO4, and 20 mM HEPES (pH7.4). Cell aliquots were incubated at 37C and gassedunder conditions indicated in the table. Aliquots were withdrawn for analysis at the end of the incubation period. Statistical significance: * P 0.01 vs. controlvalue with O2-CO2 (19:1); P 0.001 vs. value with N2-CO2 (19:1); P 0.001 vs. value with N2-CO2 for 25 min, then O2-CO2 for 20 min.




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inosine lowered the cAMP pool and behaved similarly to theselective A3 AR agonist IB-MECA (Fig. 7) but only whenselective A1, A2A, and A2BAR antagonists were supplementedin the incubation mixture (Fig. 8). The significance of theseexperiments remains to be evaluated but is inconsistent withany participation of cAMP in inosine-mediated activation ofmetabolic pathways in liver.

Phylogenetic analysis results excluded the existence of ad-ditional cognate adenosine/inosine receptors in mammals, butthis analysis leads us to propose that the four AR typesobserved in mammals, A1, A2A, A2B, and A3, arose during theevolution of early vertebrates. Their origin is related to genomeduplications produced before radiation of jawed vertebratessome 500 million years ago (26, 55). Phylogenetic analysisalso suggests the probable existence of a fifth type of AR ininvertebrates and lower vertebrates (fishes). This latter finding

agrees with previous papers that claim the presence of adeno-sine GPCR in nonvertebrate animals such as the blowflyCalliphora vicina (44), the bloodsucking bug Rhodnius pro-lixus (12), the mussels Mytilus californianus (13) and Mytilusedulis(4), and the spiny lobster Panulirus argus(17). Further-more, one protein within this group (accession no. AAN33001)has been experimentally demonstrated as an AR in the starfish

A. miniata(35), reinforcing the idea that this group of proteinsprobably corresponds to a fifth type of AR. It should bementioned that Clark et al. (15), after a great effort to identifynovel human transmembrane proteins, reported an additionalputative AR of 347 amino acid (AA) length (accession no.AAQ89007). However, this novel protein, predicted from iso-lated full-length cDNA, is a chimeric protein comprising anNH2-terminal domain identical to the first 119 AA from the A3AR and a COOH-terminal domain homologous to single Igdomain receptor (140347 AA) (14). This chimeric proteinresults from alternative mRNA splicing, fusing the first exon ofA3AR (ADORA3) gene and ADO26 gene located downstreamof ADORA3 gene. However, on the basis of modeling studiesof A3 AR (46) it can be predicted that the adenosine-bindingdomain in this chimeric protein is disrupted and, therefore,cannot be considered as an AR. In short, the consideredexclusion of additional cognate adenosine/inosine receptors inhumans and rats (Fig. 9) reinforces the previously suggestedcentral role of hepatic A3AR as the physiological receptor forinosine in preference to adenosine.

Inosine-mediated stimulation of A3AR in the liver, througha Ca2-dependent process (Figs. 3 and 4E) and independentfrom cAMP involvement (Figs. 7 and 8), activates a glycogenphosphorylase (Fig. 6) and raises glucose release from hepaticcells (Figs. 1 and 2), an oxidizable substrate that is particularlyuseful under ischemic conditions. The final experiments (Table4 and Fig. 10) link cellular ischemia with inosine/adenosine

release and glucose liberation from liver. For both experimen-tal designs, isolated hepatocytes and perfused liver, cellularischemia produced a nearly twofold increase in inosine/aden-osine release, slightly higher with inosine than adenosine. Thehuge increase in glucose liberation from hepatic glycogen

Fig. 10. Release of inosine, adenosine, and glucose from perfused rat liverunder different oxygenation conditions. All experiments were performed aftera 30-min equilibration period in which liver was perfused with KRB solutionsaturated with O2-CO2 mixture (19:1). Under control conditions, the sameperfusion solution (KRB) saturated with an O2-CO2mixture (19:1) was passedthrough the liver for an additional 30 min (F). In hypoxia experiments, theperfusion solution was replaced with KRB saturated with an N2-CO2mixture(19:1) and passed through the liver for additional 30 min (). In hypoxia/

reoxygenation experiments, the perfusion solution was replaced first with KRBsolution saturated with N2-CO2mixture (19:1) followed 5 min later with KRBsolution saturated with O2-CO2 mixture (19:1) for 25 min (). An additionalhypoxia/reoxygenation experiment was performed in identical form, but 106

M MRS 1220 was included in the KRB solutions ({). Liver effluent samples(100 l) were withdrawn at time intervals. Values are means SE for 3independent and duplicated experiments. Statistical significance for inosinevalues:P 0.001 by comparing control vs. the other 3 experimental groups atall tested times; P 0.001 by comparing hypoxia/reoxygenation vs. hypoxia/reoxygenation MRS 1220 at all tested times. Statistical significance foradenosine values: P 0.01 by comparing control vs. hypoxia or hypoxia/reoxygenation;P 0.05 by comparing control vs. hypoxia/reoxygenation MRS 1220;P 0.001 by comparing hypoxia/reoxygenation MRS 1220 vs.hypoxia or hypoxia/reoxygenation. Statistical significance for glucose values:P 0.001 by comparing control or hypoxia/reoxygenation MRS 1220 vs.hypoxia or hypoxia/reoxygenation at all times tested.




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(22.7-fold in Fig. 10) followed the release of nucleosides.Because this liberation was blunted with the A3AR antagonist,we can conclude that glucose release was due to the presenceof nucleosides. Relevance of the herein-reported studies oninosine and its physiological role in the liver, stimulatingglucose release, is further supported by the documented pro-tective role of inosine for a variety of ischemic and inflamma-

tory injuries, particularly in muscular tissues (67). Therefore, itappears plausible that release of inosine/adenosine under hyp-oxia conditions in tissues other than liver (34, 41, 50, 51, 56,61) might promote liberation of glucose from hepatic cellsresponding to the activation of both nucleosides, preferablyinosine. In conclusion, we propose that in situations of tissularischemia, inosine liberated from different tissues has a physi-ological role of paramount importance, i.e., to contribute inmaintaining body homeostasis by providing blood glucosefrom liver glycogen through A3 AR activation. In contrast,although stimulation of specific A1, A2A, and A2Bhepatic ARsresulted in glycogenolysis, gluconeogenesis, and ureagenesisactivation, presently, the effective function of these receptorsin liver cells has not been described.

ACKNOWLEDGMENTS

We are grateful to Adriana Julian-Sanchez (FM-UNAM) for help withphylogenetic analyses, Alejandra Palomares for secretarial contribution, andIngrid Masher and Maggie Brunner for careful reading of the manuscript.

GRANTS

This work was partially supported by Grant IN2115022 from DireccionGeneral de Asuntos del Personal Academico, UNAM, and 45003-A1 from theMexican Council of Science and Technology.

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