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

Am J Physiol Endocrinol Metab 290:E940-E951, 2006. First published 13 December 2005; doi: 10.1152/ajpendo.00173.2005 You might find this additional info useful... This article cites 66 articles, 23 of which you can access for free at: http://ajpendo.physiology.org/content/290/5/E940.full#ref-list-1 This article has been cited by 6 other HighWire-hosted articles: http://ajpendo.physiology.org/content/290/5/E940#cited-by Updated information and services including high resolution figures, can be found at: http://ajpendo.physiology.org/content/290/5/E940.full Additional material and information about American Journal of Physiology - Endocrinology and Metabolism can be found at: http://www.the-aps.org/publications/ajpendo This information is current as of July 31, 2013.

Inosine released after hypoxia activates hepatic glucose liberation through A 3 adenosine receptors

Downloaded from http://ajpendo.physiology.org/ at Inst De Investigaciones on July 31, 2013

American Journal of Physiology - Endocrinology and Metabolism publishes results of original studies about endocrine and metabolic systems on any level of organization. It is published 12 times a year (monthly) by the American Physiological Society, 9650 Rockville Pike, Bethesda MD 20814-3991. Copyright 2006 by American Physiological Society. ISSN: 0193-1849, ESSN: 1522-1555. Visit our website at http://www.the-aps.org/.

Am J Physiol Endocrinol Metab 290: E940 E951, 2006. First published December 13, 2005; doi:10.1152/ajpendo.00173.2005.

Inosine released after hypoxia activates hepatic glucose liberation through A3 adenosine receptors
Raquel Guinzberg,1 Daniel Corte s,1 Antonio D az-Cruz,2 1 He ctor Riveros-Rosas, Rafael Villalobos-Molina,3 and Enrique Pin a1
Departamentos de Bioqu mica, Facultad de Medicina; 2Nutricio n Animal y Bioqu mica, Facultad de Medicina Veterinaria y Zootecnia; and 3Unidad de Biomedicina, Facultad de Estudios Superiores-Iztacala, Universidad Nacional Auto noma de Me xico, Mexico City, Mexico.
Submitted 20 April 2005; accepted in nal form 3 December 2005
1

Downloaded from http://ajpendo.physiology.org/ at Inst De Investigaciones on July 31, 2013

Guinzberg, Raquel, Daniel Corte s, Antonio D az-Cruz, He ctor Riveros-Rosas, Rafael Villalobos-Molina, and Enrique Pin a. Inosine released after hypoxia activates hepatic glucose liberation through A3 adenosine receptors. Am J Physiol Endocrinol Metab 290: E940E951, 2006. First published December 13, 2005; doi:10.1152/ajpendo.00173.2005.Inosine, an endogenous nucleoside, has recently been shown to exert potent effects on the immune, neural, and cardiovascular systems. This work addresses modulation of intermediary metabolism by inosine through adenosine receptors (ARs) in isolated rat hepatocytes. We conducted an in silico search in the GenBank and complete genomic sequence databases for additional adenosine/inosine receptors and for a feasible physiological role of inosine in homeostasis. Inosine stimulated glycogenolysis (40%, EC50 4.2 109 M), gluconeogenesis (40%, EC50 7.8 109 M), and ureagenesis (130%, EC50 7.0 108 M) compared with basal values; these effects were blunted by the selective A3 AR antagonist 9-chloro-2-(2-furanyl)-5-[(phenylacetyl)amino][1,2,4]-triazolo[1,5-c]quinazoline (MRS 1220) but not by selective A1, A2A, and A2B AR antagonists. In addition, MRS 1220 antagonized inosine-induced transient increase (40%) in cytosolic Ca2 and enhanced (90%) glycogen phosphorylase activity. Inosineinduced Ca2 mobilization was desensitized by adenosine; in a reciprocal manner, inosine desensitized adenosine action. Inosine decreased the cAMP pool in hepatocytes when A1, A2A, and A2B AR were blocked by a mixture of selective antagonists. Inosine-promoted metabolic changes were unrelated to cAMP decrease but were Ca2 dependent because they were absent in hepatocytes incubated in EGTA- or BAPTA-AM-supplemented Ca2-free medium. After in silico analysis, no additional cognate adenosine/inosine receptors were found in human, mouse, and rat. In both perfused rat liver and isolated hepatocytes, hypoxia/reoxygenation produced an increase in inosine, adenosine, and glucose release; these actions were quantitatively greater in perfused rat liver than in isolated cells. Moreover, all of these effects were impaired by the antagonist MRS 1220. On the basis of results obtained, known higher extracellular inosine levels under ischemic conditions, and inosines higher sensitivity for stimulating hepatic gluconeogenesis, it is suggested that, after tissular ischemia, inosine contributes to the maintainence of homeostasis by releasing glucose from the liver through stimulation of A3 ARs. ischemia; calcium; urea; phylogenetic analysis; homeostasis.
INOSINE IS A NATURALLY OCCURRING PURINE NUCLEOSIDE formed by adenosine deamination. Its normal interstitial concentrations in rat plasma and serum have been reported in the range of 0.520 M (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 rst to describe a stimulatory action of inosine on ureagenesis and gluconeogenesis in isolated hepatocytes (23, 68). However, over the last decade several reports (e.g., Refs. 19, 32, 59) appeared regarding the role of inosine in regulating the immunologic and cardiovascular systems. Although in the majority of cases inosine binds to A3 adenosine 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 afnity, and signal-transducing mechanism (for a review, see Ref. 49). All four AR subtypes are present in isolated hepatocytes, where they stimulate glycogenolysis, gluconeogenesis, and ureagenesis rates (49). Signal transduction systems for obtaining these increases were via adenylyl cyclase for A2A and A2B AR, whereas A1 and A3 AR involved changes in cytosolic Ca2 (20 22, 60, 66). The purpose of this work included the following: 1) to dene the receptor type involved in inosine responses in isolated hepatocytes; 2) to identify the signal transduction pathway mediating these inosine responses; 3) to explore the possibility of nding additional adenosine/inosine receptors; and 4) to obtain insight into the physiological meaning of these inosine actions.
MATERIALS AND METHODS

Selective AR agonists and antagonists used in this work are included in Table 1 and are listed in alphabetical order of their abbreviations. Full chemical names, the receptor-binding constant for AR agonist, reported data on the Ki for AR antagonists, and pertinent references are additionally included. All of these compounds were purchased from Sigma RBI. All animal experiments were conducted in accordance with the Federal Guidelines for the Care and Use of Animals (NOM-062ZOO-1999, Ministry of Agriculture, Mexico) and were approved by the Institutional Ethics Committee of the National Autonomous University of Mexicos Faculty of Medicine (FM-UNAM). Isolation of hepatocytes. Male Wistar rats (150 200 g) were anesthetized with ether, and cells were isolated by the method of Berry and Friend (7) as modied by Guinzberg et al. (23). Hepatocytes were used when viability was at least 95%, as assayed by the trypan blue exclusion method. Experiments were conducted by duplicate with 30 40 mg wet wt hepatocytes.

Address for reprint requests and other correspondence: E. Pin a, Departamento de Bioqu mica, Facultad de Medicina, Universidad Nacional Auto noma de Me xico, Apdo. Postal 70159, Mexico City, 04510, Mexico (e-mail: epgarza@servidor.unam.mx). E940

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. http://www.ajpendo.org

0193-1849/06 $8.00 Copyright 2006 the American Physiological Society

HOMEOSTATIC ROLE OF INOSINE IN THE LIVER

E941

Table 1. Specic agonists and antagonists for ARs used in this work
Abbreviation Chemical Name Receptor Action ReceptorBinding Value Ki Reference

ADSPX Alloxazine CCPA CGS-15943 CGS-21680 CSC DPCPX IB-MECA MRS 1220 NECA

1-allyl-3,7-dimethyl-8-p-sulfophenylxanthine Benzo[g]pteridine 2,4(1H,3H)-dione 2-chloro-N6-cyclopentyladenosine 9-chloro-2-(2-furanyl)[1,2,4]triazolo[1,5c]quinazoline-5-amine 2-P(2-carboxyethyl)phenethylamino-5-Nethylcarboxamidoadenosine 1,3,7-trimethyl-8-(3-chlorostyryl) xanthine 8-cyclopentyl-1,3-dipropylxanthine 1-deoxy-1-[6-[((3iodophenyl)methyl)amino]-9H-purin-9yl]-N-methyl--D-ribofuranuronamide 9-chloro-2-(2-furanyl)-5-((phenylacetyl) amino)-[1,2,4]triazol[1,5-c]quinazoline 5-N-ethylcarboxamidoadenosine

A2B A2B A1 A1 A2A A2A A1 A3 A3 A1, A2B

Antagonist Antagonist Agonist Antagonist Agonist Antagonist Antagonist Agonist Antagonist Agonist

0.6 nM 13 nM 0.4 nM 4 nM 15 nM 54 nM 0.69 nM 1.1 nM 14 nM A1 11 nM A2B 16 nM

(28) (40) (43) (31) (30) (29) (25) (64)

Downloaded from http://ajpendo.physiology.org/ at Inst De Investigaciones on July 31, 2013

(36) (9)

AR, adenosine receptor.

Ureagenesis. Hepatocytes from 24-h-starved rats were incubated for 1 h at 37C in an atmosphere of O2-CO2 (95%-5%) for 60 min in a gyratory water bath in Krebs-Ringer buffer (KRB) containing 10 mM glucose, 5 mM (NH4)2CO3, and 3 mM ornithine. Urea synthesis was assayed after 60 min (24). Gluconeogenesis. Hepatocytes from 24-h-starved rats were incubated for 1 h in KRB containing 10 mM lactate. Glucose synthesis was measured in the supernatant of cells by the glucose oxidase method (18). Glycogenolysis. Hepatocytes from rats fed ad libitum were incubated for 45 min in KRB without lactate or any other substrate. Glucose release was measured (18). Glycogen phosphorylase activity. This activity was assayed by measuring the incorporation of [U-14C]glucose 1-phosphate into glycogen, as described by Starke et al. (57). Hepatocytes were exposed to the 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 of phosphorylase assay medium containing 50 mM NaF, 4.8 mM caffeine, 86 mM glucose 1-phosphate, 2% glycogen, and 8.5 Ci of [U-14C]glucose 1-phosphate and incubated at 37C. The reaction was stopped after 30 min by the addition of 25 l of glacial acetic acid. A 50-l sample was spotted onto lter paper and washed twice with 66% ethanol, washed with acetone, and placed in a cocktail for liquid scintillation counting. cAMP accumulation. Hepatocytes from fed rats were incubated at 37C for 2 min in KRB. cAMP was measured using the Amersham kit TRK4312. Ca2 measurement in fura 2-AM loaded hepatocytes. This was performed as described by Llopis et al. (42). Briey, isolated hepatocytes from fed rats were diluted in KRB to a nal concentration of 40 mg wet wt/ml and incubated for 10 min at 37C in an atmosphere of O2-CO2 (95%-5%). Cells were incubated for an additional 20 min in the presence of 3 M fura 2-AM and were washed twice by centrifugation at 500 rpm for 3 min. Liver cells were divided into 200-l aliquots, immersed in ice, and used within the subsequent 5 min. Ca2 was measured in these cells as in Llopis et al. (42) using a Kd 224 nM. Hypoxia/reoxygenation in isolated hepatocytes and perfused liver. In experiments with fed rats, isolated hepatocytes were used to measure 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
AJP-Endocrinol Metab VOL

constant ow rate of 16 ml/min. Hepatic venous efuents were obtained via a cannula in the vena cava. Adenosine and inosine release quantication. Nucleosides were measured by enzymatic assay in double-beam spectrophotometer by the method described by Olsson (47). Statistical methods. Values are reported as means SE. Students t-test was applied to assess differences between groups. Statistical signicance was set at P 0.05. Identication of cognate ARs on protein databases. Initially, sequences of known ARs from the rhodopsin superfamily were retrieved from the Swiss-Prot protein database at http://au.expasy.org/sprot/ (3). The amino acid sequence from each of these known ARs was used as bait for BLASTP (1) searches at the National Center for Biotechnology Information GenBank nonredundant protein database (6). To determine the number of sequences encoding ARs in animals with complete genome sequence, we repeated the BLAST search with the tBLASTn program (1), using amino acid sequences of characterized adenosine GPCRs as queries against whole genomic DNA sequences or the high-throughput genomic sequence database from human, mouse, rat, zebra sh, Japanese puffer sh (International Fugu Genome Consortium, assembly version 3.0; http://genome.jgi-psf.org/ fugu6/fugu6.home.html), and the ascidian Ciona intestinalis (assembly version 1.0; http://genome.jgipsf.org/ciona4/ciona4.home.html). Ab initio gene predictions were performed with the GeneComber system (54), which provides increased gene recognition accuracy by combining predictions from the gene-nding Genscan (10) and HMMgene (37) programs. GeneComber-predicted exons were veried by multiple alignments with amino acid sequences from adenosine GPCRs to gather additional support for constructing gene models. Multiple sequence alignment and phylogenetic analysis. Multiple sequence alignments were performed by using ClustalX v1.81 (58) and corrected according to gapped BLASTP results (1). Phylogenetic analyses were carried out with MEGA v2.1 (38) software, using both the maximum parsimony and distance-based methods UPGMA (unweighted pair group method with arithmetic mean) and neighbor joining, along with minimum evolution with the Poisson correction distance method, and gaps were treated by pairwise deletion. Accuracy of reconstructed trees was examined by the bootstrap test with 1,000 replications. Phylogenetic trees were rooted with the bovine rhodopsin sequence. Complete names of organisms included in the phylogenetic analysis are as follows: ANOGA, Anopheles gambiae (Arthropoda, insecta); ASTMI, Asterina miniata (starsh; Echinodermata); BOVIN, Bos taurus (Chordata, vertebrata, mammalia); CAEBR, Caenorhabditis briggsae (Nematoda); CAEEL, Caenorhabditis elegans (Nematoda); CANFA, Canis familiaris (Chordata, verwww.ajpendo.org

290 MAY 2006

E942

HOMEOSTATIC ROLE OF INOSINE IN THE LIVER

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 sh; Chordata, vertebrata, teleostei); DROME, Drosophila melanogaster (Arthropoda, insecta); FUGRU, Fugu rubripes (Japanese puffer sh; Chordata, vertebrata, teleostei); HORSE, Equus caballus (Chordata, vertebrata, mammalia); HUMAN, Homo sapiens (Chordata, vertebrata, mammalia); MOUSE, Mus musculus (Chordata, vertebrata, mammalia); RABBIT, Oryctolagus cuniculus (Chordata, vertebrate, mammalia); RAT, Rattus norvegicus (Chordata, vertebrata, mammalia); SHEEP, Ovis aries (Chordata, vertebrata, mammalia), and XENLA, Xenopus laevis (Chordata, vertebrata, amphibia).
RESULTS

Table 2. EC50 values for adenosine and inosine to stimulate glycogenolysis, gluconeogenesis, and ureagenesis in isolated rat hepatocytes
EC50 Values for Adenosine EC50 Values for Inosine Ratio, EC50 Adenosine to EC50 Inosine

Pathway

Glycogenolysis Gluconeogenesis Ureagenesis

3.8109M 1.7108M 1.8107M

4.2109M 7.8109M 7.0108M

0.90 2.2 2.6

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

Downloaded from http://ajpendo.physiology.org/ at Inst De Investigaciones on July 31, 2013

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

Fig. 1. Dose-response curves of inosine (E) or adenosine (F) for glycogenolysis (A), gluconeogenesis (B), and ureagenesis (C) in hepatocytes. Basal values in the absence of nucleosides (). Plotted values are means, and vertical lines represent SE of duplicate incubation of 6 independent cell preparations, except for control sample, where 8 10 independent cell preparations were included. Statistical signicance vs. control values are indicated. *P 0.05; **P 0.001. AJP-Endocrinol Metab VOL

were calculated, along with ratios for (adenosine EC50 value)/ (inosine EC50 value) in each activated pathway (Table 2). These data indicated that gluconeogenesis and ureagenesis might be activated at lower concentrations of inosine than of adenosine. The stimulating effect of 1 M inosine on glycogenolysis, gluconeogenesis, and ureagenesis was blunted specically with the selective A3 AR antagonist 9-chloro-2-(2furanyl)-5-[((phenylacetyl)amino)-[1,2,4]triazolo[1,5-c]quinazoline (MRS 1220) but was not modied when inosine was simultaneously incubated with 9-chloro-2-(2-furanyl)[1,2,4] triazolo[1,5-c]quinazolin-5-amine (CGS-15943), 1,3,7-trimethyl-8-(3-chlorostyryl)xanthine (CSC), and 1-allyl-3,7dimethyl-8-p-sulfophenylxanthine (ADSPX), or alloxazine, selective antagonists for A1, A2A, and A2B AR, respectively (Fig. 2); i.e, inosine stimulated these three metabolic routes in isolated rat liver cells only if A3 AR was not blocked. Two selective A2B AR antagonists were used in these experiments because the required ADSPX solvent [A2B antagonists with lower receptor-binding constant (Table 1)] is dimethyl sulfoxide, which, when used at a concentration of 1 mM to quantify urea, interfered with the assay (results not shown) (24). Thus, in this case, ADSPX was substituted for a water-soluble selective A2B AR antagonist such as alloxazine. Inosine-induced Ca2 mobilization to stimulate glycogenolysis, gluconeogenesis, and ureagenesis. A common action of adenosine and an AR-specic agonist is to increase [Ca2]i in isolated hepatocytes (22). Results in Table 3 show that inosine shares in this action. It is noteworthy that stimulation with either inosine or the individual AR agonists employed resulted in a rise in Ca2 similar to the rise obtained with adenosine, which might activate all four ARs. We performed three series of experiments to investigate the role of calcium in liver metabolic pathway inosine-mediated activation. In the rst series, Ca2 was eliminated from KRB; in the second series, EGTA was included in Ca2-free KRB to chelate extracellular Ca2; and in the third series, BAPTA-AM was added to Ca2-free KRB to chelate intracellular Ca2. Inosine elicited a lesser stimulation in studied metabolic pathway rates when cells were incubated in Ca2-free KRB. In addition, these pathways were not stimulated at all by the nucleoside when either of the used chelating agents was present (Fig. 3). To identify AR involved in the transient inosine-mediated increase of free Ca2, we conducted the experiment presented in Fig. 4. Inosine alone produced a temporary increase in Ca2 (Fig. 4A) that was not modied by A1, A2A, and A2B ARselective antagonists (Fig. 4, BD) but was blunted by A3 AR antagonist (Fig. 4E).
www.ajpendo.org

290 MAY 2006

HOMEOSTATIC ROLE OF INOSINE IN THE LIVER

E943

Table 3. [Ca2]i in isolated hepatocytes treated with adenosine, inosine, or selective AR agonists
Additions AR-Stimulated [Ca2]i, nmol/l Values, %

None Adenosine Inosine CCPA CGS-21680 NECA plus DPCPX IB-MECA

All 4 ? A1 A2A A2B A3

1956.3 2847.3 2746.3 3016.9 2816.9 2987.9 2797.9

100 146 141 154 144 153 143

Numbers are means SE of duplicates from 4 independent cell preparations. [Ca2]i, cytosolic Ca2 concentration. Experimental conditions as in MATERIALS AND METHODS. To stimulate A2B AR alone, an AR agonist for A2B and A1, such as NECA (Table 1), was mixed with DPCPX, a selective antagonist for A1 AR. Nucleosides, agonists, and antagonists were used at a 1-M nal concentration. Statistical signicance, nucleoside or agonist vs. control; P 0.001 in all cases.

Downloaded from http://ajpendo.physiology.org/ at Inst De Investigaciones on July 31, 2013

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 in MATERIALS AND METHODS with 1 M inosine alone or combined with 1 M nal concentration of the following AR-selective antagonists: 9-chloro-2-(2furanyl)[1,2,4]triazolo[1,5-c]quinazolin-5-amine (CGS-15943) for A1; 1,3,7trimethyl-8-(3-chlorostyryl)xanthine (CSC) for A2A; 1-allyl-3,7-dimethyl-8-psulfophenylxanthine (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. In ureagenesis studies, alloxazine was used instead of ADSPX as an A2Bselective antagonist (see text). Values represent means SE of duplicate incubation from 4 to 6 independent cell preparations. *Statistical signicance vs. control sample without inosine, P 0.001; **statistical signicance inosine alone vs. inosine MRS 1220, P 0.01.

Desensitization experiments were conducted to test whether inosine acted through GPCR. Isolated hepatocytes were stimulated with 1 M adenosine or inosine, and Ca2-transient rises were monitored. After recovery to initial values in 2 min, cells were stimulated again. Under this protocol, adenosine failed to reinitiate cell activation independently of whether rst activation was produced by adenosine (Fig. 5A) or by inosine (Fig. 5D). Similarly, inosine failed to reinitiate cell activation independently of whether initial activation was obtained with adenosine (Fig. 5B) or inosine (Fig. 5C). Inosine- and adenosine-stimulated phosphorylase activity. Incubation of isolated hepatocytes with either inosine or adenosine resulted in a statistically signicant increase in glycogen phosphorylase activity (Fig. 6). Phosphorylase activity in inosine-stimulated cells reached a nearly twofold increase over basal 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 potent than inosine, and BAPTA-AM additionally blunted adenosine stimulation of phosphorylase (Fig. 6). A3 AR antagonist partial inhibitory action on adenosine-mediated stimulation (Fig. 6) might be explained by the effect of adenosine through activation of AR other than A3. Inosine and cAMP pool. To investigate cAMP involvement in the inosine response, hepatocytes were incubated with graded concentrations of the nucleoside. Changes in the cAMP pool were compared with those produced by adenosine and AR-selective agonists. Results show that stimulation of A2A AR with the selective agonist 2-P(2-carboxyethyl)phenethylamino-5-N-ethylcarboxamidoadenosine and A2B AR with a mixture of 5-(N-ethylcarboxamido)adenosine (an A1, A2B AR agonist) plus 8-cyclopentyl-1,3-dipropylxanthine (an A1-selective AR antagonist) produced a dose-dependent increase in cAMP content (Fig. 7). In contrast, stimulation with 2-chloroN6-cyclopentyladenosine, an A1-selective AR agonist, or 1-deoxy-1-[6-[((3-iodophenyl)methyl)amino]-9H-purin-9-yl]-Nmethyl--D-ribofuranuronamide (IB-MECA), an A3 ARselective agonist, originated a dose-related decrease in cAMP content (Fig. 7). According to all of the ndings in this paper, inosine actions resemble those of A3 AR agonists. Therefore, it would be expected that inosine might decrease cAMP in the same manner as A3 AR agonists; however, adenosine and, unexpectedly, inosine were unable to modify the cAMP pool in
www.ajpendo.org

AJP-Endocrinol Metab VOL

290 MAY 2006

E944

HOMEOSTATIC ROLE OF INOSINE IN THE LIVER

Downloaded from http://ajpendo.physiology.org/ at Inst De Investigaciones on July 31, 2013

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

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

hepatocytes (Fig. 7). Next, hepatocytes were incubated with AR antagonist-supplemented inosine. Thus selective antagonists for each of the four ARs, CGS-15943 for A1, CSC for A2A, ADSPX for A2B, and MRS 1220 for A3, were used so that different mixtures of three of these antagonists added to hepatocytes would maintain three of the four ARs blocked, leaving only one AR able to be activated, which might or might not be stimulated by inosine. Only in experiments in which A3 AR was not antagonized by the adequate mixture of AR agents did inosine decrease the cAMP cellular pool (Fig. 8), similarly to IB-MECA, an A3 AR agonist, whereas, if inosine was added to cells in which A1, A2A, or A2B AR were not antagonized by adequate AR blocker mixtures, cAMP values remained unmodied (Fig. 8). Additional experiments are required to understand why inosine alone did not modify the cAMP cellular pool (Fig. 7), whereas inosine did indeed decrease the cAMP pool if A1, A2A, and A2B AR were blocked by their selective antagonists (Fig. 8).
www.ajpendo.org

290 MAY 2006

HOMEOSTATIC ROLE OF INOSINE IN THE LIVER

E945

Fig. 5. Inosine and adenosine desensitize hepatocytes to each other. Cells were labeled with fura 2-AM and stimulated initially with 106 M adenosine (A and B) or 106 M of inosine (C and D), and free [Ca2]i was measured as a function of time. After recovery to initial values, a second stimulation with inosine (B and C) or adenosine (A and D) was performed. Experiment was repeated 4 times with identical results.

In any event, cAMP does not appear to be involved in inosineactivated metabolic routes in hepatocytes. Phylogenetic analyses ruled out the existence of an additional GPCR homologous to ARs in mammals. The results in this paper, as well as those of other authors, clearly demonstrate that GPCR, mainly through A3 AR, mediates some inosine effects. However, this does not discard the possibility that other adenosine-related GPCRs might exist, including a cognate inosine GPCR. To explore the latter possibility, we conducted an extensive search for homologous protein sequences to the adenosine/inosine receptor in whole genomic DNA sequences from human, mouse, rat, zebra sh (D. rerio),
AJP-Endocrinol Metab VOL

Japanese puffer sh (F. rubripes), and the ascidian C. intestinalis. Subsequently, we conducted a phylogenetic analysis for retrieved adenosine/inosine receptor sequences. We found no additional cognate adenosine/inosine receptors in addition to the four known adenosine GPCRs in human, mouse, and rat. Unexpectedly, however, we did nd three additional ARhomologous protein sequences in puffer sh and one in zebra sh. Recently, a similar observation was reported with 2adrenoceptors, because the zebra sh possesses ve 2-adrenoceptors instead of the three found in mammals and the puffer sh possesses eight 2-adrenoceptors (11, 52, 53). Figure 9 shows a phylogenetic tree constructed with a total of 46 full-length protein sequences identied as ARs (all belonging exclusively to animals). It can be observed that all AR protein sequences in mammals belong to one of the four known AR types. No additional AR types were found in mammals; however, in the puffer sh, two distinct A1 AR were found (designated provisionally as A1A and A1B) along with one additional A2 AR (provisionally denominated A2C). On the other hand, in the complete genome of C. intestinalis (a nonvertebrate chordate that diverged very early from other chordates, including vertebrates) we identied only three ARhomologous protein sequences, although none resulted orthologous (same gene in different species) to the four AR types known in mammals. These three C. intestinalis ARs are grouped with other AR sequences found in zebra sh, puffer sh, starsh (echinodermata), arthropoda, and nematoda; these sequences probably comprise a fth AR type. Within this group, only the AR from the starsh A. miniata has been experimentally demonstrated as an AR coupled to a Gi-linked protein (35). Adenosine, inosine, and glucose are released by the liver under hypoxia/reoxygenation conditions. Once we dened which AR was involved in inosine action in liver, the signal transduction pathway mediating inosine action, and the absence of an additional adenosine/inosine receptor participating in these responses, we focused on the physiological meaning of inosine-mediated action in liver. It is known that adenosine and inosine 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 under hypoxic conditions (34, 41). Isolated rat hepatocytes also release adenosine under hypoxic conditions (5); however, the metabolic effect of endogenous adenosine and inosine release in liver has not been tested. Thereafter, we subjected both perfused rat liver and isolated hepatocytes to hypoxia/reoxygenation conditions and measured inosine, adenosine, and glucose release. During hypoxic incubation, isolated hepatocytes accumulated inosine, adenosine, and glucose in extracellular volume (Table 4). Both nucleosides and glucose accumulation were observed additionally under conditions of hypoxia/reoxygenation. The selective antagonist for A3 AR, MRS 1220, impaired liberation of glucose from intracellular sources, but interestingly, it also impaired inosine and adenosine release from hepatocytes. We obtained similar results in perfused rat livers that were subjected to hypoxia and hypoxia/reoxygenation conditions (Fig. 10). Once experimental conditions were set, inosine, adenosine, and glucose release began after an initial lag of 2.5 min. Inosine reached a plateau after 10 min and adenosine after 5 min, but glucose increased progressively during the following 30 min (Fig. 10).
www.ajpendo.org

Downloaded from http://ajpendo.physiology.org/ at Inst De Investigaciones on July 31, 2013

290 MAY 2006

E946

HOMEOSTATIC ROLE OF INOSINE IN THE LIVER

Fig. 6. Effect of inosine and adenosine of glycogen phosphorylase activity in hepatocytes from fed rats. Isolated hepatocytes (20 3 mg of protein) were incubated in 5 ml of Krebs-Ringer bicarbonate containing 1.2 mM CaCl2. Glycogen phosphorylase activity was measured as detailed in MATERIALS AND METHODS. A: samples were incubated 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 adenosine 106 M MRS 1220 (E), or 106 M adenosine 10 M BAPTA-AM (). Values are means SE of 3 independent experiments by duplicate. Statistical signicance: P 0.001 by comparing inosine at 0 min vs. inosine at 2.5, 5, and 10 min; P 0.001 by comparing 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 comparing adenosine alone vs. adenosine MRS 1220 or adenosine BAPTAAM.

Downloaded from http://ajpendo.physiology.org/ at Inst De Investigaciones on July 31, 2013

Under hypoxia and hypoxia/reoxygenation conditions, gluconeogenesis and ureagenesis activities were assayed in rat hepatocytes that were isolated from fasted rats (16 h). Both ATPdependent glucose and urea production diminished by 50% in

isolated hepatocytes incubated under hypoxia or hypoxia/reoxygenation conditions (data not shown). These latter results can be explained because under low oxygen tension, insufcient ATP production precludes ux through anabolic pathways (8).

Fig. 7. Effect of adenosine, inosine, and AR-selective agonists on cAMP production in hepatocytes. Cells were incubated for 2 min in KRB with adenosine (), inosine (F), and the following AR selective agonists: 2-chloroN6-cyclopentyladenosine for A1 (); 2-P(2-carboxyethyl)phenethyl-amino-5N-ethylcarboxyamidoadenosine for A2A (E), and 1-deoxy-1-[6-[((3-iodophenyl)methyl)amino]-9H-purin-9-yl]-N-methyl--D-ribofuranuronamide for A3 (); to stimulate A2B AR (), a mixture of 5-(N-ethylcarboxamido)adenosine and 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) was used. Results are expressed as %basal value, which was 0.74 0.03 pmol of cAMP formed in 2 min/mg (wet wt). Each value represents means SE of 4 independent experiments, each performed in duplicate. Statistical signicance vs. basal is indicated: *P 0.05; **P 0.01; ***P 0.001. AJP-Endocrinol Metab VOL

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

290 MAY 2006

HOMEOSTATIC ROLE OF INOSINE IN THE LIVER

E947

Downloaded from http://ajpendo.physiology.org/ at Inst De Investigaciones on July 31, 2013

Fig. 9. Phylogenetic analysis of ARs. Phylogenetic tree constructed with available protein sequences belonging to the AR subfamily by using minimum evolution method. Trees were calculated using MEGA 2.1 (38). Dotted bars indicate nodes supported in 70 (open), 80 (gray), or 90% (lled) of 1,000 random bootstrap 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 group to which the protein sequence belongs and ne vertical bars the type of AR to which the protein sequence belongs. Sequence names are indicated according to a Swiss-Prot-like identier (gene organism) followed by the database accession number (GenBank, PIR, Swiss-Prot, etc.) and protein amino acid length. AR sequences 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/), the Fugu rubripes Genome Project v3.0 (2), and the Ciona intestinalis Genome Project v1.0 (16), the last 2 both at 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.

AJP-Endocrinol Metab VOL

290 MAY 2006

www.ajpendo.org

E948

HOMEOSTATIC ROLE OF INOSINE IN THE LIVER

Table 4. Release of inosine and adenosine and glycogenolysis rate in isolated rat hepatocytes mantained in Ringer-HEPES buffer and subjected to different oxygenation conditions
Inosine Experimental Conditions Adenosine Glycogenolysis mol glucose/g wet wt in 45 min

molmin1mg prot1

Oxygenation Hypoxia Hypoxia/reoxygenation

O2-CO2 O2-CO2 N2-CO2 N2-CO2 N2-CO2 N2-CO2 O2-CO2

(19:1) (19:1) 106 MRS 1220 (19:1) (19:1) 106 M-MRS 1220 for 25 min, then O2-CO2 for 20 min 106 M MRS 1220 for 25 min, then 106 M MRS 1220 for 20 min

2.220.01 1.410.06* 4.690.04* 1.480.01 4.670.11* 2.000.02

1.860.02 1.650.02 3.890.01* 1.340.02 3.030.01* 1.680.01

54.32.2 50.81.7 123.83.2* 72.52.7 125.32.3* 62.71.9

Values are means SE in 3 independent experiments with duplicate samples. Hepatocytes were incubated for 45 min in Ringer-HEPES buffer containing the 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 gassed under conditions indicated in the table. Aliquots were withdrawn for analysis at the end of the incubation period. Statistical signicance: * P 0.01 vs. control value 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. DISCUSSION

Downloaded from http://ajpendo.physiology.org/ at Inst De Investigaciones on July 31, 2013

Data from this paper conrm our preliminary nding (23, 68) and greatly extend previous information on the hepatic actions of inosine. Adenosine- or inosine-stimulated rat liver cells showed a dose-related increase in the rates of three of the main hepatic metabolic pathways, namely glycogenolysis, gluconeogenesis, and ureagenesis (Table 1 and Fig. 1). Considering the EC50 values obtained for adenosine or inosine to stimulate the three metabolic pathways and the reported physiological concentrations of adenosine and inosine in rat serum (61), both nucleosides might be effective in producing activation of these metabolic pathways. Converging evidence suggests a preeminent role of inosine over adenosine in stimulating hepatic metabolic routes through A3 AR activation, because inosine serum concentration is higher than adenosine and inosine concentration was 25-fold higher than adenosine in hepatic venous efuents of isolated perfused liver (51). In addition, EC50 values for inosine are similar to EC50 values for adenosine to stimulate glycogenolysis, but they are 2- to 2.5-fold lower in stimulating gluconeogenesis and ureagenesis. Furthermore, a very active adenosine deaminase is present in rat serum that converts adenosine into inosine (48). The previously mentioned considerations led us to explore some characteristics of inosine-mediated actions on liver cells, although additional evidence in favor of inosine as an important intercellular messenger will be included in the nal part of this discussion. The three main inosine-stimulated metabolic pathways were equally blunted by the selective A3 AR antagonist (Fig. 2); in contrast, incubation of liver cells with selective A1, A2A, or A2B AR antagonists did not modify the inosinemediated rise in glycogenolysis, gluconeogenesis, and ureagenesis rates (Fig. 2). Hence, A3 AR seems to be the initial target 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 the transduction mechanism elicited by A3 AR stimulation with adenosine or A3 AR agonists to obtain glycogenolysis, gluconeogenesis, and ureagenesis rate increases. Similar results were obtained after stimulation of isolated hepatocytes with inosine: an increase in [Ca2]i (Table 3), dependence of such an increase to activate metabolic pathway rates (Fig. 3), and blockade in the rise of [Ca2]i observed exclusively with MRS 1220, the A3 AR antagonist, but not with the use of selective A1, A2A, and A2B AR antagonists (Fig. 4).
AJP-Endocrinol Metab VOL

Main metabolic pathway stimulation in liver by inosine is absolutely dependent on an increase in free [Ca2]i (Fig. 3). Thus incubation of cells in Ca2-free KRB supplemented with the intracellular chelant BAPTA-AM impaired any inosinemediated activation in glycogenolysis, gluconeogenesis, and ureagenesis rates (Fig. 3). Nonetheless, when hepatocytes were incubated in Ca2-free KRB in the absence of chelant agents, inosine produced minor stimulation in the metabolic pathway rates that we studied compared with stimulation observed in KRB containing 1.2 mM Ca2 (Fig. 3). All these data point to a relevant role of extracellular Ca2 in inosine-mediated transduction actions in liver and to a minor contribution of intracellular Ca2 storage compartments to drive the same actions. Unpublished experiments (Guinzberg R and Pin a E) using isolated hepatocytes, incubated in KRB with 1.2 mM Ca2 and challenged with MRS 1220, an A3 AR agonist, are conrmatory. Thapsigargin, an inhibitor of Ca2 release from intracellular storage compartments, decreases stimulation of urea synthesis by nearly 40%. The following experiment presents another property of the inosine-sensitive AR. This nucleoside desensitizes AR toward adenosine (Fig. 4); a lower concentration of serum adenosine will be quantitatively less important compared with inosine to promote further metabolic responses in liver. In addition, these data support that a GPCR is involved in inosine-mediated actions in liver. Intracellular Ca2 increase has been shown to stimulate glycogenolysis (33, 63). In particular, two Ca2-mobilizing agents, namely epinephrine and ionophore A-23187, promoted hepatocyte glycogen phosphorylase activation that led to an increase in cell glucose release (62). Thereafter, a [Ca2]i rise by A3 AR stimulation in hepatocytes by any of the studied nucleosides (Fig. 4) in turn activated glycogen phosphorylase to a greater extent with inosine than with adenosine (Fig. 6). With the information recorded to this point in this work, we could anticipate a blockade in nucleoside-mediated phosphorylase activation with the use of either a selective A3 AR antagonist (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 as a signal transduction pathway for inosine-mediated metabolic actions gave negative results. Inosine alone, as well as adenosine alone, did not modify cAMP pool in liver cells (Fig. 7). In another set of experiments with isolated hepatocytes (Fig. 8),
www.ajpendo.org

290 MAY 2006

HOMEOSTATIC ROLE OF INOSINE IN THE LIVER

E949

inosine lowered the cAMP pool and behaved similarly to the selective A3 AR agonist IB-MECA (Fig. 7) but only when selective A1, A2A, and A2B AR antagonists were supplemented in the incubation mixture (Fig. 8). The signicance of these experiments remains to be evaluated but is inconsistent with any participation of cAMP in inosine-mediated activation of metabolic pathways in liver. Phylogenetic analysis results excluded the existence of additional cognate adenosine/inosine receptors in mammals, but this analysis leads us to propose that the four AR types observed in mammals, A1, A2A, A2B, and A3, arose during the evolution of early vertebrates. Their origin is related to genome duplications produced before radiation of jawed vertebrates some 500 million years ago (26, 55). Phylogenetic analysis also suggests the probable existence of a fth type of AR in invertebrates and lower vertebrates (shes). This latter nding

agrees with previous papers that claim the presence of adenosine GPCR in nonvertebrate animals such as the blowy Calliphora vicina (44), the bloodsucking bug Rhodnius prolixus (12), the mussels Mytilus californianus (13) and Mytilus edulis (4), and the spiny lobster Panulirus argus (17). Furthermore, one protein within this group (accession no. AAN33001) has been experimentally demonstrated as an AR in the starsh A. miniata (35), reinforcing the idea that this group of proteins probably corresponds to a fth type of AR. It should be mentioned that Clark et al. (15), after a great effort to identify novel human transmembrane proteins, reported an additional putative AR of 347 amino acid (AA) length (accession no. AAQ89007). However, this novel protein, predicted from isolated full-length cDNA, is a chimeric protein comprising an NH2-terminal domain identical to the rst 119 AA from the A3 AR and a COOH-terminal domain homologous to single Ig domain receptor (140 347 AA) (14). This chimeric protein results from alternative mRNA splicing, fusing the rst exon of A3 AR (ADORA3) gene and ADO26 gene located downstream of ADORA3 gene. However, on the basis of modeling studies of A3 AR (46) it can be predicted that the adenosine-binding domain in this chimeric protein is disrupted and, therefore, cannot be considered as an AR. In short, the considered exclusion of additional cognate adenosine/inosine receptors in humans and rats (Fig. 9) reinforces the previously suggested central role of hepatic A3 AR as the physiological receptor for inosine in preference to adenosine. Inosine-mediated stimulation of A3 AR in the liver, through a Ca2-dependent process (Figs. 3 and 4E) and independent from cAMP involvement (Figs. 7 and 8), activates a glycogen phosphorylase (Fig. 6) and raises glucose release from hepatic cells (Figs. 1 and 2), an oxidizable substrate that is particularly useful under ischemic conditions. The nal experiments (Table 4 and Fig. 10) link cellular ischemia with inosine/adenosine release and glucose liberation from liver. For both experimental designs, isolated hepatocytes and perfused liver, cellular ischemia produced a nearly twofold increase in inosine/adenosine release, slightly higher with inosine than adenosine. The huge increase in glucose liberation from hepatic glycogen

Downloaded from http://ajpendo.physiology.org/ at Inst De Investigaciones on July 31, 2013

Fig. 10. Release of inosine, adenosine, and glucose from perfused rat liver under different oxygenation conditions. All experiments were performed after a 30-min equilibration period in which liver was perfused with KRB solution saturated with O2-CO2 mixture (19:1). Under control conditions, the same perfusion solution (KRB) saturated with an O2-CO2 mixture (19:1) was passed through the liver for an additional 30 min (F). In hypoxia experiments, the perfusion solution was replaced with KRB saturated with an N2-CO2 mixture (19:1) and passed through the liver for additional 30 min (). In hypoxia/ reoxygenation experiments, the perfusion solution was replaced rst with KRB solution saturated with N2-CO2 mixture (19:1) followed 5 min later with KRB solution saturated with O2-CO2 mixture (19:1) for 25 min (). An additional hypoxia/reoxygenation experiment was performed in identical form, but 106 M MRS 1220 was included in the KRB solutions ({). Liver efuent samples (100 l) were withdrawn at time intervals. Values are means SE for 3 independent and duplicated experiments. Statistical signicance for inosine values: P 0.001 by comparing control vs. the other 3 experimental groups at all tested times; P 0.001 by comparing hypoxia/reoxygenation vs. hypoxia/ reoxygenation MRS 1220 at all tested times. Statistical signicance for adenosine 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 signicance for glucose values: P 0.001 by comparing control or hypoxia/reoxygenation MRS 1220 vs. hypoxia or hypoxia/reoxygenation at all times tested. AJP-Endocrinol Metab VOL
290 MAY 2006

www.ajpendo.org

E950

HOMEOSTATIC ROLE OF INOSINE IN THE LIVER pighian tubules isolated from Rhodnius prolixus. Arch Insect Biochem Physiol 43: 7277, 2000. Chen JH and Bayne CJ. Hemocyte adhesion in the California mussel (Mytilus californianus): regulation by adenosine. Biochim Biophys Acta 1268: 178 184, 1995. Chung DH, Humphrey MB, Nakamura MC, Ginzinger DG, Seaman WE, and Daws MR. CMRF-35-like molecule-1, a novel mouse myeloid receptor, can inhibit osteoclast formation. J Immunol 171: 6541 6548, 2003. Clark HF, Gurney AL, Abaya E, Baker K, Baldwin D, Brush J, Chen J, Chow B, Chui C, Crowley C, Currell B, Deuel B, Dowd P, Eaton D, Foster J, Grimaldi C, Gu Q, Hass PE, Heldens S, Huang A, Kim HS, Klimowski L, Jin Y, Johnson S, Lee J, Lewis L, Liao D, Mark M, Robbie E, Sanchez C, Schoenfeld J, Seshagiri S, Simmons L, Singh J, Smith V, Stinson J, Vagts A, Vandlen R, Watanabe C, Wieand D, Woods K, Xie MH, Yansura D, Yi S, Yu G, Yuan J, Zhang M, Zhang Z, Goddard A, Wood WI, Godowski P, and Gray A. The secreted protein discovery initiative (SPDI), a large-scale effort to identify novel human secreted and transmembrane proteins: a bioinformatics assessment. Genome Res 13: 22652270, 2003. Dehal P, Satou Y, Campbell RK, Chapman J, Degnan B, De Tomaso A, Davidson B, Di Gregorio A, Gelpke M, Goodstein DM, Harafuji N, Hastings KE, Ho I, Hotta K, Huang W, Kawashima T, Lemaire P, Martinez D, Meinertzhagen IA, Necula S, Nonaka M, Putnam N, Rash S, Saiga H, Satake M, Terry A, Yamada L, Wang HG, Awazu S, Azumi K, Boore J, Branno M, Chin-Bow S, DeSantis R, Doyle S, Francino P, Keys DN, Haga S, Hayashi H, Hino K, Imai KS, Inaba K, Kano S, Kobayashi K, Kobayashi M, Lee BI, Makabe KW, Manohar C, Matassi G, Medina M, Mochizuki Y, Mount S, Morishita T, Miura S, Nakayama A, Nishizaka S, Nomoto H, Ohta F, Oishi K, Rigoutsos I, Sano M, Sasaki A, Sasakura Y, Shoguchi E, Shin-i T, Spagnuolo A, Stainier D, Suzuki MM, Tassy O, Takatori N, Tokuoka M, Yagi K, Yoshizaki F, Wada S, Zhang C, Hyatt PD, Larimer F, Detter C, Doggett N, Glavina T, Hawkins T, Richardson P, Lucas S, Kohara Y, Levine M, Satoh N, and Rokhsar DS. The draft genome of Ciona intestinalis: insights into chordate and vertebrate origins. Science 298: 21572167, 2002. Derby CD, Ache BW, and Carr WE. Purinergic modulation in the brain of the spiny lobster. Brain Res 421: 57 64, 1987. Fales FW. Glucose (enzymatic). Stand Methods Clin Chem 4: 101112, 1963. Go mez G and Sitkovky MV. Differential requirement for A2A and A3 adenosine receptors for the protective effect of inosine in vivo. Blood 102: 4472 4478, 2003. Gonza lez-Ben tez E, Guinzberg R, D az-Cruz A, and Pin a E. Regulation of glycogen metabolism in hepatocytes through adenosine receptors. Role of Ca2 and cAMP. Eur J Pharmacol 437: 105111, 2002. Guinzberg R, D az-Cruz A, Uribe S, and Pin a E. Inhibition of adenosine mediated responses in isolated hepatocytes by depolarizing concentrations of K. Biochem Biophys Res Commun 197: 229 234, 1993. Guinzberg R, D az-Cruz A, Uribe S, and Pin a E. Ca2 dependence of the response of three adenosine type receptors in rat hepatocytes. Eur J Pharmacol 340: 243247, 1997. Guinzberg RP, Laguna I, Zentella A, Guzman R, and Pin a E. Effect of adenosine and inosine on ureagenesis in hepatocytes. Biochem J 245: 371374, 1987. Gutman I and Bergmeyer HU. Determination of urea. In: Methods of Enzymatic Analysis. New York: Academic, 1974, p. 17911794. Haleen SJ, Steffen RP, and Hamilton HW. PD 116,948, a highly selective A1 adenosine receptor antagonist. Life Sci 40: 555561, 1987. Holland PW, Garc a-Ferna ndez J, Williams NA, and Sidow A. Gene duplications and the origins of vertebrate development. Dev Suppl 125 133, 1994. Idzko M, Panther E, Bremer HC, Windisch W, Sorichter S, Herouy Y, Elsner P, Mockenhaupt T, Girolomoni G, and Norgauer J. Inosine stimulates chemostaxis, Ca2-transients and actin polymerization in immature human dendritic cells via a pertussis toxin-sensitive mechanism independent of adenosine receptors. J Cell Physiol 199: 149 156, 2004. Jacobson KA, Nikodijevic O, Pedgett WL, Gallo-Rodr guez C, Millard M, and Daly JW. 8-(3-Chlorostyryl)caffeine (CSC) is a selective A2-adenosine antagonist in vitro and in vivo. FEBS Lett 323: 141144, 1993. Jacobson KA, Shi D, Gallo-Rodr guez C, Manning M Jr, Muller C, Daly JW, Neumeyer JI, Kiriasis I, and Peiderer W. Effect of triuwww.ajpendo.org

(22.7-fold in Fig. 10) followed the release of nucleosides. Because this liberation was blunted with the A3 AR antagonist, we can conclude that glucose release was due to the presence of nucleosides. Relevance of the herein-reported studies on inosine and its physiological role in the liver, stimulating glucose release, is further supported by the documented protective role of inosine for a variety of ischemic and inammatory injuries, particularly in muscular tissues (67). Therefore, it appears plausible that release of inosine/adenosine under hypoxia conditions in tissues other than liver (34, 41, 50, 51, 56, 61) might promote liberation of glucose from hepatic cells responding to the activation of both nucleosides, preferably inosine. In conclusion, we propose that in situations of tissular ischemia, inosine liberated from different tissues has a physiological role of paramount importance, i.e., to contribute in maintaining body homeostasis by providing blood glucose from liver glycogen through A3 AR activation. In contrast, although stimulation of specic A1, A2A, and A2B hepatic ARs resulted in glycogenolysis, gluconeogenesis, and ureagenesis activation, presently, the effective function of these receptors in liver cells has not been described.
ACKNOWLEDGMENTS We are grateful to Adriana Julia n-Sa nchez (FM-UNAM) for help with phylogenetic analyses, Alejandra Palomares for secretarial contribution, and Ingrid Masher and Maggie Brunner for careful reading of the manuscript. GRANTS This work was partially supported by Grant IN2115022 from Direccion General de Asuntos del Personal Academico, UNAM, and 45003-A1 from the Mexican Council of Science and Technology. REFERENCES 1. Altschul SF and Lipman DJ. Protein database searches for multiple alignments. Proc Natl Acad Sci USA 87: 5509 5513, 1990. 2. Aparicio S, Chapman J, Stupka E, Putnam N, Chia JM, Dehal P, Christoffels A, Rash S, Hoon S, Smit A, Gelpke MD, Roach J, Oh T, Ho IY, Wong M, Detter C, Verhoef F, Predki P, Tay A, Lucas S, Richardson P, Smith SF, Clark MS, Edwards YJ, Doggett N, Zharkikh A, Tavtigian SV, Pruss D, Barnstead M, Evans C, Baden H, Powell J, Glusman G, Rowen L, Hood L, Tan YH, Elgar G, Hawkins T, Venkatesh B, Rokhsar D, and Brenner S. Whole-genome shotgun assembly and analysis of the genome of Fugu rubripes. Science 297: 13011310, 2002. 3. Bairoch A and Apweiler R. The SWISS-PROT protein sequence database and its supplement TrEMBL in 2000. Nucleic Acids Res 28: 45 48, 2000. 4. Barraco RA and Stefano GB. Pharmacological evidence for the modulation of monoamine release by adenosine in the invertebrate nervous system. J Neurochem 54: 20022006, 1990. 5. Belloni FL, Elkin PL, and Giannotto B. The mechanism of adenosine release from hypoxic rat liver cells. Br J Pharmacol 85: 441 446, 1985. 6. Benson DA, Karsch-Mizrachi I, Lipman DJ, Ostell J, Rapp BA, and Wheeler DL. GenBank. Nucleic Acids Res 28: 1518, 2000. 7. Berry MN and Friend DS. High-yield preparation of isolated rat liver parenchymal cells. J Cell Biol 43: 506 520, 1969. 8. Boon L and Meijer AJ. Oxygen tension does not affect urea synthesis in perfused rat hepatocytes. Eur J Biochem 195: 455 457, 1991. 9. Bruns RF, Lu GH, and Pugsley TA. Characterization of the A2 adenosine receptor labeled by [3H]NECA in rat striatal membranes. Mol Pharmacol 29: 331346, 1986. 10. Burge C and Karlin S. Prediction of complete gene structures in human genomic DNA. J Mol Biol 268: 78 94, 1997. 11. Bylund DB. Alpha-2 adrenoceptor subtypes: are more better? Br J Pharmacol 144: 159 160, 2005. 12. Caruso-Neves C, Monteiro SO, de Oliveira CF, Filho CC, and Lopes AG. Adenosine modulates the (Na()K())ATPase activity in malAJP-Endocrinol Metab VOL

13. 14.

15.

Downloaded from http://ajpendo.physiology.org/ at Inst De Investigaciones on July 31, 2013

16.

17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27.

28.

29.

290 MAY 2006

HOMEOSTATIC ROLE OF INOSINE IN THE LIVER oromethyl and other substituents on activity of xanthines at adenosine receptors. J Med Chem 36: 2639 2644, 1993. Jarvis MF, Schulz R, Hutchinson AJ, Do UH, Sills MA, and Williams M. [3H]CGS 21680, a selective A2 adenosine receptor agonist directly labels A2 receptors in rat brain. J Pharmacol Exp Ther 251: 888 893, 1989. Jarvis MF, Williams M, Do UH, and Sills MA. Characterization of the binding of a novel nonxanthine adenosine antagonist radioligand, [3H]CGS 15943, to multiple afnity states of the adenosine A1 receptor in the rat cortex. Mol Pharmacol 39: 49 51, 1990. Jin X, Shepherd RK, Duling BR, and Linden J. Inosine binds to A3 adenosine receptors and stimulates mast cell degranulation. J Clin Invest 100: 2849 2857, 1997. Joseph SK and Williamson JR. The origin, quantitation, and kinetics of intracellular calcium mobilization by vasopressin and phenylephrine in hepatocytes. J Biol Chem 258: 1042510432, 1983. Kekesi V, Zima E, Barat E, Huszar E, Nagy A, Losonczi L, Merkely B, Horkay F, and Juhasz-Nagy A. Pericardial concentrations of adenosine, inosine and hypoxanthine in an experimental canine model of spastic ischaemia. Clin Sci (Lond) 48: 198S201S, 2002. Kalinowski RR, Jaffe LA, Foltz KR, and Giusti AF. A receptor linked to a Gi-family G-protein functions in initiating oocyte maturation in starsh but not frogs. Dev Biol 253: 139 149, 2003. Kim YC, Ji XD, and Jacobson KA. Derivatives of the triazoloquinazoline adenosine antagonist (CGS15943) are selective for the human A3 receptor subtype. J Med Chem 39: 4142 4148, 1996. Krogh A. Two methods for improving performance of an HMM and their application for gene nding. Proc Int Conf Intell Syst Mol Biol 5: 179 186, 1997. Kumar S, Tamura K, Jakobsen IB, and Nei M. MEGA2: molecular evolutionary genetics analysis software. Bioinformatics 17: 1244 1245, 2001. Latini S and Pedate F. Adenosine in the central nervous system: release mechanisms and extracellular concentrations. J Neurochem 79: 463 484, 2001. Liang BT and Haltiwanger B. Adenosine A2a and A2b receptors in cultured fetal chick heart cells. High- and low-afnity coupling to stimulation of myocyte contractility and cAMP accumulation. Circ Res 76: 242251, 1995. Linden J. Molecular approach to adenosine receptors: receptor-mediated mechanisms of tissue protection. Annu Rev Pharmacol Toxicol 41: 775 787, 2001. Llopis J, Kass GE, Gahm A, and Orrenius S. Evidence for two pathways of receptor-mediated Ca2 entry in hepatocytes. Biochem J 384: 243247, 1992. Lohse MJ, Klotz KN, Schwabe U, CristaLLi G, Vittori S, and Grifantini M. 2-Chloro-N6-cyclopentyladenosine: a highly selective agonist at A1 adenosine receptors. Naunyn Schmiedebergs Arch Pharmacol 33: 687 689, 1988. Magazanik LG and Fedorova IM. Modulatory role of adenosine receptors in insect motor nerve terminals. Neurochem Res 28: 617 624, 2003. Mentzer RM Jr, Rubio R, and Berne RM. Release of adenosine by hypoxic canine lung tissue and its possible role in pulmonary circulation. Am J Physiol 229: 16251631, 1975. Moro S, Spalluto G, and Jacobson KA. Techniques: recent developments in computer-aided engineering of GPCR ligands using the human adenosine A3 receptor as an example. Trends Pharmacol Sci 26: 44 51, 2005. Olsson RA. Changes in content of purine nucleoside in canine myocardium during coronary occlusion. Circ Res 26: 301306, 1970. Plagemann PG, Wohlhueter RM, and Kraupp M. Adenosine uptake, transport, and metabolism in human erythrocytes. J Cell Physiol 125: 330 336, 1985. Ralevic V and Burnstock G. Receptors for purines and pyrimidines. Pharmacol Rev 50: 413 492, 1998.

E951

30.

31.

32. 33. 34.

35. 36. 37. 38. 39. 40.

41. 42. 43.

44. 45. 46.

47. 48. 49.

50. Roth S, Rosenbaum PS, Osinski J, Park SS, Toledano AY, Li B, and Moshfeghi AA. Ischemia induces signicant changes in purine nucleoside concentration in the retina-choroid in rats. Exp Eye Res 65: 771779, 1997. 51. Rubio R and Berne RM. Localization of purine and pyridine nucleoside phosphorylases in heart, kidney, and liver. Am J Physiol Heart Circ Physiol 239: H721H730, 1980. 52. Ruuskanen JO, Laurila J, Xhaard H, Rantanen VV, Vuoriluoto K, Wurster S, Marjamaki A, Vainio M, Johnson MS, and Scheinin M. Conserved structural, pharmacological and functional properties among the three human and ve zebra sh alpha2-adrenoceptors. Br J Pharmacol 144: 165177, 2005. 53. Ruuskanen JO, Xhaard H, Marjamaki A, Salaneck E, Salminen T, Yan YL, Postlethwait JH, Johnson MS, Larhammar D, and Scheinin M. Identication of duplicated fourth alpha2-adrenergic receptor subtype by cloning and mapping of ve receptor genes in zebra sh. Mol Biol Evol 21: 14 28, 2004. 54. Shah SP, McVicker GP, Mackworth AK, Rogic S, and Ouellette BF. GeneComber: combining outputs of gene prediction programs for improved results. Bioinformatics 19: 1296 1297, 2003. 55. Sidow A. Gen(om)e duplications in the evolution of early vertebrates. Curr Opin Genet Dev 6: 715722, 1996. 56. Silva PH, Dillon D, and Van Wylen DG. Adenosine deaminase inhibition augments interstitial adenosine but does not attenuate myocardial infarction. Cardiovasc Res 29: 616 623, 1995. 57. Starke PE, Hoek JB, and Farber JL. Calcium-dependent and calciumindependent mechanism or irreversible cell injury in cultured hepatocytes. J Biol Chem 261: 3006 3012, 1986. 58. Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, and Higgins DG. The CLUSTAL_X windows interface: exible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 25: 4876 4882, 1997. 59. Tilley SL, Wagoner VA, Salvatore CA, Jacobson MA, and Koller BH. Adenosine and inosine increase cutaneous vasopermeability by activating A(3) receptors on mast cells. J Clin Invest 105: 361367, 2000. 60. Tinton SA, Chow SC, Buc-Caldero n PM, and Kass GE. Adenosine stimulates calcium inux in isolated rat hepatocytes. Eur J Biochem 238: 576 581, 1996. 61. Traut TW. Physiological concentrations of purines and pyrimidines. Mol Cell Biochem 140: 122, 1994. 62. Villalobos-Molina R and Devlin TM. Effects of tri-Calciphor (trimmer of 16,16-dimethyl-15-dehydroprostaglandin B1) on glucose metabolism in liver cells. Biochem Biophys Res Commun 201: 14571463, 1994. 63. Villalobos-Molina R, Saavedra-Molina A, and Devlin TM. Effect of hypoxia and reoxygenation on metabolic pathways in rat hepatocytes. Arch Med Res 29: 219 223, 1998. 64. Von Lubitz DK, Carter MF, Deutsch SI, Lin RC, Mastropaolo J, Meshulam Y, and Jacobson KA. The effects of adenosine A3 receptor stimulation on seizures in mice. Eur J Pharmacol 275: 2329, 1995. 65. Winn HR, Rubio R, and Berne RM. Brain adenosine concentration during hypoxia in rat. Am J Physiol Heart Circ Physiol 241: H235H242, 1981. 66. Yasuda N, Inove T, Horizoe T, Nagata K, Minami H, Kawata T, Hoshino Y, Harada H, Yoshikawa S, Asano O, Nagaoka J, Murakami M, Abe S, Kobayashi S, and Tanaka I. Funtional characterization of the adenosine receptor contributing to glycogenolysis and gluconeogenesis in rat hepatocytes. Eur J Pharmacol 459: 159 166, 2003. 67. Wakai A, Winter DC, Street JT, OSullivan RG, Wang JH, and Redmond HP. Inosine attenuates tourniquet-induced skeletal muscle reperfusion injury. J Surg Res 99: 311315, 2001. 68. Zentella de Pin a M, D az-Cruz A, Guinzberg PR, and Pin a E. Hormone-like effect of adenosine and inosine gluconeogenesis from lactate in isolated hepatocytes. Life Sci 44: 2269 2274, 1989.

Downloaded from http://ajpendo.physiology.org/ at Inst De Investigaciones on July 31, 2013

AJP-Endocrinol Metab VOL

290 MAY 2006

www.ajpendo.org