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Biochemical and Molecular Actions of Nutrients

Exogenous Nucleosides Stimulate Proliferation of Fetal Rat Hepatocytes¹

María José Sáez-Lara, Manuel Manzano^*, Antonio José Angulo^*,2, Antonio Suárez^*, María Isabel Torres, Carolina Gómez-Llorente^**, Ángel Gil and Luis Fontana³

Department of Biochemistry and Molecular Biology, School of Pharmacy, University of Granada, Spain; ^* R & D Department, Abbott Laboratories, Granada, Spain; Department of Experimental Biology, School of Sciences, University of Jaén, Spain; ^** Department of Biochemistry and Molecular Biology, School of Medicine, University of Granada, Spain

³To whom correspondence should be addressed. E-mail: fontana{at}ugr.es.

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Exogenous nucleotides (NT) have been reported to exert a reparative role in animal models of intestinal and hepatic damage. Thus, the administration of NT in the diet of rats with thioacetamide-induced liver cirrhosis normalized many of the histological and biochemical alterations produced by this hepatotoxin. We are currently studying the mechanism by which NT exert this effect using cell culture models. The aim of this work was to investigate whether exogenous nucleosides (NS) modulate the proliferation of hepatocytes. We used fetal rat hepatocytes, which, unlike adult hepatocytes, are proliferative cells. Fetal rat primary hepatocytes were incubated with mixtures of NS, and cell proliferation was studied. NS added to the medium of fetal hepatocytes were taken up in a selective fashion by the cells. Cell proliferation was enhanced, as demonstrated by the induction of c-myc and h-ras gene expression as well as by the higher percentage of cells in S phase, and exogenous NS increased the expression of -fetoprotein. These results suggest that exogenous NS may in fact stimulate proliferation of hepatic cells and help preserve the undifferentiated state of fetal rat hepatocytes.

KEY WORDS: • exogenous nucleosides • fetal hepatocytes • proliferation • rats

Exogenous nucleotides (NT)⁴ have been reported to have various biological effects. They modulate lipid metabolism, immune function, and intestinal microbiota, and have a reparative effect in certain tissues (1). The last-mentioned effect was shown in animal models of liver and intestine damage. Thus, the administration of a NT supplement in the diet of rats with liver cirrhosis induced by thioacetamide (TAA) normalized many of the histological and biochemical alterations produced by this hepatotoxic agent (2,3). Our research group recently showed that the improvement in liver histology seems to be due to a higher expression of matrix metalloproteinase-1 (MMP-1) and to a lower expression of tissue inhibitor of MMP-1 observed in the cirrhotic rats fed the NT supplement (3). However, other mechanisms responsible for the histological improvement should not be ruled out.

We are currently studying the mechanism of action of NT using cell culture models. We recently investigated the effects of the addition of nucleosides (NS) to the medium of 3 different cell culture systems: adult rat primary hepatocytes, a rat liver stellate cell line, and cocultures of both cell types (4,5). Our results suggested that exogenous NS are taken up by the cells and increase the intracellular NT pool, subsequently inducing changes in cell function and in the expression of extracellular matrix genes such as 1(I) procollagen, fibronectin, laminin, and MMP-1 (4,5). Exogenous NS did not modify cell proliferation in any of the above-mentioned culture systems. However, 2 different reports contradicted our results (6,7). Ohyanagi et al. (6) showed that a mixture of NT and NS stimulated DNA and RNA syntheses of both adult rat hepatocytes and hepatoma cells. Although increased proliferation of adult hepatocytes would enhance their growth and could be beneficial, particularly during liver regeneration, increased proliferation of hepatoma cells would enhance the proliferation of tumor cells. In the other report, Torres-López et al. (7) showed that the oral administration of NT increased the percentage of binucleated hepatocytes in both control and cirrhotic rats, suggesting that NT may in fact stimulate hepatocyte proliferation.

The aim of this work was to investigate the hypothesis that exogenous NS may modulate proliferation of hepatic cells. For this purpose, we used cultures of fetal rat hepatocytes which, unlike adult hepatocytes, are proliferative cells. Our goal was to reconcile the conflicting data regarding the effects of NS on proliferation of hepatic cells. We used fetal hepatocytes to investigate the effects of NS in fetal cells because they express various genes typical of tumor cells.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
    Isolation and culture of fetal hepatocytes. Hepatocytes from 20-d-old fetal Wistar rats were isolated by collagenase disruption following the method described by Roncero et al. (8), and plated on dishes containing nucleotide-, nucleoside-, and arginine-free medium 199 (Imperial Laboratories), supplemented with 200 µmol/L ornithine, 10% fetal calf serum (FCS), and penicillin/streptomycin. Cells were incubated in 5% CO₂ at 37°C for 4 h to allow attachment to plates. The medium was changed at that time and replaced by one of the same composition except that 10% FCS was replaced by 2% newborn bovine serum. After 18–20 h, the medium was again replaced by one of identical composition but in the absence of serum. NS were added 2 h later. All procedures were approved by the Animal Care Committee of the University of Granada and conformed to European Union Animal Care Regulations governing the use of animals for research.

    Nucleosides. Thymidine, uridine, cytidine, guanosine, and inosine were from Sigma Chemical. Two mixtures of these NS containing equimolecular concentrations of cytidine, guanosine, inosine, and thymidine or uridine (TCGI or UCGI) were added to the culture medium for various times. Unless otherwise stated, experiments were carried out at a final NS concentration of 100 µmol/L and for 24 and 48 h. This concentration is within physiologic ranges found in some biological fluids (9,10). These mixtures were chosen so as to use NS that are normally present in plasma and whose nitrogenous bases are those found in DNA and RNA, respectively. Inosine was used instead of adenosine to avoid the hormonal effects of the latter (11).

    Nucleoside uptake. For these experiments, we first determined that there was no loss of NS due to degradation or adsorption phenomena. The stability of each individual nucleoside was studied by incubating Petri dishes containing culture medium with NS but without cells for 1 wk at 37°C and 5% CO₂, and NS were found to be stable for this period of time.

The NS remaining in the culture medium after 0, 3, 8, 24, and 48 h of incubation were analyzed by gradient reverse-phased HPLC, following the method described by Leach et al. (12). The culture medium was harvested and filtered through a 0.2-µm filter. NS were separated on an LC18 Supelcosyl column (3-µm particle size, 4.6 x 150 mm, Supelco), with a linear gradient obtained from 100% buffer A (0.1 mol/L potassium acetate in 2 mmol/L hexanesulfonic acid, pH 4.5) to 100% buffer B (7% acetonitrile in buffer A) in 25 min, at a flow rate of 1 mL/min, using a Waters 2690 HPLC separation module. Peaks were registered at 245 nm with a diode array detector (Waters), and integrated automatically using Millennium 32-integration software (Waters). The amounts of NS in the culture medium were calculated on the basis of peak areas of nucleoside standards.

    Electron microscopy. After 48 h of incubation with NS, the cells were fixed in 2% glutaraldehyde in 0.1 mol/L cacodylate buffer, pH 7.3, and postfixed in 1% osmium tetroxide. The samples were then dehydrated in acetone and embedded in Epon 812 resin. Ultra-thin sections (50 nm) were double-stained with uranyl acetate and lead citrate and examined under a Zeiss 902 transmission electron microscope.

    Cell viability assay. Cell viability was assessed by the crystal-violet assay (13). After 24 h of incubation with NS, the medium was discarded and the remaining viable adherent cells were stained with a solution of 0.2% crystal violet in 2% ethanol for 30 min. Then, plates were rinsed with tap water, allowed to dry, and 1% SDS was added to elute crystal violet. The absorbance values at 540 nm were directly correlated with the number of remaining viable cells. Cell viability was also assessed by a colorimetric assay based on the cleavage of the tetrazolium salt XTT by the mitochondrial succinate dehydrogenase of the viable cells to a formazan dye (Roche Molecular Biochemicals), following the manufacturer’s directions.

    Protein content. After incubation with NS for 24 h, cell layers were dissolved in 24.9 mmol/L HEPES, 2.5 mmol/L EDTA, and 0.01% Triton X-100, and total protein was determined with a dye-binding assay using an acidic solution of Coomassie Blue G-250 (BioRad) (14).

    Cell-cycle analysis. These experiments were conducted after 24 and 48 h of incubation with NS. After trypsinization, cells were washed with PBS and resuspended in a permeabilization buffer containing 0.05% Triton X-100 and 0.1% bovine serum albumin for 15 min at room temperature. The cell suspension was then treated with Vindelov’s solution (1.2 g/L Tris-base, 0.6 g/L sodium chloride, 0.01 g/L RNAse, 0.05 g/L propidium iodide, 1 mL/L Nonidet P-40; pH 8.0) for 15 min at room temperature while protected from light. Red fluorescence was measured in a flow cytometer FACSCalibur (Becton Dickinson).

    c-myc, h-ras and -fetoprotein mRNAs semiquantitative analysis. Semiquantitative measurement of c-myc, h-ras and -fetoprotein gene expression was performed by a modification of the multiplex competitive PCR assay (3,15). Total RNA was extracted from fetal rat hepatocytes according to Chomczynski and Sacchi (16). To exclude possible amplification of contaminating genomic DNA, a DNA digestion was performed with 10 U of DNase RNase-free for 30 min at 37°C. cDNA was synthesized with 1 µg total fetal hepatocytes RNA and 0.5 µg of oligo-dT₁₆ in 20 µL of solution containing 1X first-strand buffer, 10 mmol/L dithiothreitol, 500 µmol/L each dNTP, 1 µCi of [-³²P] dCTP, and 200 U of reverse transcriptase. After 1 h at 42°C, the efficiency of cDNA synthesis was estimated by incorporation of [-³²P] dCTP. All RT reactions were performed in duplicate.

From each RT reaction, PCR reactions were conducted in duplicate with quantities of cDNA in all samples estimated by [-³²P] dCTP incorporation. The competitive amplifications were made by using specific sets of primers for c-myc, h-ras, -fetoprotein and glyceraldehyde-3-phosphate dehydrogenase (gapdh) within each PCR reaction. The sequence of the primers was as follows: forward primers, 5'-GGCGAGAACAGTTGAAACAC-3', 5'-CACTAGTACGTGAGATTCGG-3' and 5'-TGAAATGACAGAGGAGCAGC-3'; reverse primers, 5'-TGAGGCAGTTAACATTATGGC-3', 5'-GAGCAGGGACATCAAAGTG-3' and 5'-CACCAAAGCGTCGACACATT-3' for c-myc, h-ras and -fetoprotein, respectively; gapdh was used as an internal control, which was coamplified with a forward primer (5'-TAAAGGGCATCCTGGGCTAC-3') and a reverse primer (5'-TTACTCCTTGGAGGCCATG-3'). Forward specific primers were 5'-labeled with tetrachloro-6-carbixyfluorescein for c-myc and with 6-carboxyfluorescein for h-ras, -fetoprotein and gapdh. Because an inverse exponential relation between template size and amplification efficiency was observed (17), the lengths of the amplified products were 187, 200, 200, and 205 bp for h-ras, -fetoprotein, gapdh, and c-myc, respectively.

When PCR reactions were performed within the exponential phase of PCR DNA synthesis, a direct correlation between initial cDNA template amount and PCR DNA production was observed (3,15,17). In our case, the exponential phase of the PCR reaction occurred between cycles 26 and 34 (data not shown). PCR reactions were performed in the same set with an Applied Biosystems DNA thermal cycler model 9700 as follows: an initial denaturing for 10 min at 94°C; 40 cycles at 94°C for 30 s, 55°C for 30 s, and 72°C for 30 s; and a final extension at 72°C for 10 min. The fluorescently labeled PCR products of c-myc, h-ras, -fetoprotein and gapdh were separated, identified by their size and distinct 5'-fluorescent label, and quantified by capillary electrophoresis in a Genetic Analyzer model 500 (Applied Biosystems).

    Statistical analysis. All results are expressed as means ± SEM. Statistical analysis was performed after testing for variance homogeneity (Levene’s test) by either two-way ANOVA (Table 2; sources of variation: type of NS mixture and time) or one-way ANOVA (Table 1 and Figs. 3and 4; source of variation: type of NS mixture) with a Bonferroni post-test to compare means from experimental incubations with controls. Differences with P < 0.05 were considered significant. The software used for the statistical analysis was SPSS version 11.0.

View larger version (33K): [in this window] [in a new window]   FIGURE 3 Effect of the addition of nucleosides to fetal rat hepatocyte medium on the expression of c-myc (A) and h-ras (B) mRNA levels. Values are means ± SEM, n = 3. Bars without a common letter differ, P < 0.05.

View larger version (47K): [in this window] [in a new window]   FIGURE 4 Effect of the addition of nucleosides to fetal rat hepatocyte medium on the expression of -fetoprotein mRNA levels. Values are mean ± SEM, n = 3. Bars without a common letter differ, P < 0.05.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

View larger version (19K): [in this window] [in a new window]   FIGURE 1 Time course of clearance of exogenous nucleosides from fetal rat hepatocyte medium. Cells were incubated with 100 µmol/L TCGI (A) or UCGI (B) mixtures for various times, and the remaining nucleosides were analyzed. Results show concentrations of remaining nucleosides in the culture medium as means ± SEM, n = 3.

View larger version (188K): [in this window] [in a new window]   FIGURE 2 Electron micrographs of fetal rat hepatocytes incubated with the UCGI mixture. Cells incubated with UCGI grew as a monolayer containing a greater number of hepatocytes, and their appearance resembled that of liver tissue (A), with bile canaliculi (B), binucleated hepatocytes (C), and abundant lysosomes (D).

    Cell proliferation. Total protein content (Table 1), cell cycle phases (Table 2), and mRNA levels of c-myc and h-ras (Fig. 3) were examined as markers of cell proliferation. Three different concentrations of NS (10, 50, and 100 µmol/L) were used to study the effects on total protein content. Both mixtures of exogenous NS significantly increased protein content, TCGI at concentrations of 50 and 100 µmol/L, and UCGI at 50 µmol/L (Table 1).

Analysis of the cell cycle revealed that the percentage of control fetal hepatocytes in phase S increased over time, with a parallel decrease in the percentage of cells in phases G0–G1 (Table 2). The most striking finding of these experiments was a significant increase in the percentage of cells in phase S for those fetal hepatocytes that were incubated for 48 h with the UCGI mixture compared with controls for the same time period (Table 2). This result is consistent with the positive effects that this mixture of NS had on cell growth and maturation (Fig. 2).

In addition, exogenous NS, mainly the mixture that contained uridine, significantly induced the expression of two genes involved in cell proliferation (Fig. 3). The TCGI mixture had a modest but significant effect on c-myc expression (1.3-fold compared with controls) but no effect on h-ras expression. In contrast, the UCGI mixture had a more potent effect. This mixture induced expression of c-myc 2.3-fold, and that of h-ras 1.6-fold.

    Cell function. -Fetoprotein mRNA levels were determined to assess the effect of exogenous NS on fetal hepatocyte function. Both NS mixtures significantly induced expression of -fetoprotein compared with controls (Fig. 4), 2.1-fold and 1.7-fold in cells incubated with mixtures TCGI and UCGI, respectively.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
    Nucleoside clearance. NS uptake processes in mammals can be divided into two categories on the basis of the underlying transport mechanism. In equilibrative transport (facilitated diffusion), the flux of NS across the membrane is driven solely by the concentration gradient, whereas in concentrative (active) transport, the flux is coupled to that of sodium ions such that the electrochemical ion gradient can drive cellular uptake of NS against their concentration gradients (18).

In this study, we observed selective uptake of the different NS by the cells (Fig. 1). Our results show that 58 and 65% of purine NS (guanosine and inosine, respectively) were taken up by fetal hepatocytes regardless of the mixture used, TCGI or UCGI. Fetal hepatocytes took up 23% of the initial uridine concentration. These data are different from those obtained by Arnaud et al. (4) in cultures of adult rat primary hepatocytes, which completely took up these NS.

Neither thymidine nor cytidine was taken up by fetal hepatocytes. The behavior of thymidine again differed from that observed in adult hepatocytes in which it totally disappeared from the culture medium after 24 h of incubation (4). However, the cytidine clearance observed in the fetal hepatocytes coincided with that observed in adult rat primary hepatocytes by Arnaud et al. (4) and Ohyanagi et al. (6).

Valdés et al. (19) reported that NS transporter expression is modulated by the presence or absence of NT in the diet. Thus, CNT1 (which encodes N2, a pirimidine-preferring active transporter) expression increases in the liver when the diet contains NT, whereas it decreases with NT-free diets. These data suggest that the addition of NS might change the expression of their transporters in fetal hepatocytes.

    Proliferation of fetal hepatocytes by NS. The fact that exogenous NS were taken up by fetal hepatocytes prompted us to study the effect of these compounds on cell proliferation. Several authors described a marked inhibition of DNA synthesis, paralleling a reduced cell viability, caused by the addition of NS/NT mixtures at concentrations > 1 mmol/L to media of different types of cells (6,20). In the present study, the range of NS concentrations tested was below the inhibitory concentrations of NS used by the above-mentioned authors and was more physiologic (10–100 µmol/L). This concentration range was selected to match the NS concentrations in some biological fluids, such as human, sheep, dog, mouse, and rat plasma (uridine: 0.5–10 µmol/L), and human milk (cytidine: 14.2 µmol/L, uridine: 37.3 µmol/L) (9,10). On the basis of cell viability, our work demonstrated that TCGI and UCGI mixtures of NS were not toxic to hepatocytes.

In contrast, the stimulatory effect of NS/NT on cell proliferation was reported by others (21–25), suggesting that the response to NS is stimulatory or inhibitory depending on the state of cell differentiation, the phase of the cell cycle, and the time of incubation.

Our results indicate that exogenous NS enhanced proliferation of fetal rat hepatocytes, differing as follows from cells that did not receive NS: 1) the cultures that were incubated with either TCGI or UCGI mixtures had a higher total protein content (Table 1); 2) those incubated with TCGI mixture had a higher expression of c-myc (Fig. 3); and 3) those incubated with UCGI mixture had a higher percentage of cells in phase S of the cell cycle (Table 2) and higher expressions of c-myc and h-ras (Fig. 3).

It is noteworthy that the UCGI mixture exerted greater effects than did TCGI. The electron microscopy analysis appears to confirm these results (Fig. 2). A possible explanation is that cytidine could be deaminated and transformed into uridine. Consequently, UCGI would then contain a double dose of available uridine. In addition, only receptors for adenine- and uracil-derivatives were reported to date (26,27). As mentioned above, we did not use adenine to rule out its hormonal effects. Consequently, uridine would appear to be the responsible NS capable of activating receptors and triggering signal transduction pathways. NS were reported to activate transduction pathways mediated by adenylate cyclase, phospholipase C, and mitogen-activated protein kinases (27).

In summary, we showed that NS added to the medium of fetal hepatocytes were taken up in a selective fashion by the cells with the following effects after the addition of NS: 1) cell proliferation was enhanced, as demonstrated by the induction of c-myc and h-ras expression and by the higher percentage of cells in S phase; 2) exogenous NS also increased the expression of -fetoprotein gene, which suggests that these compounds may help preserve the undifferentiated state of fetal hepatocytes.

	FOOTNOTES

 
¹ Supported by a research grant from the European Union (FEDER 1FD97–0410-ALI1), by the Red Temática de Investigación Cooperativa G03/015 of the Instituto de Salud Carlos III (Fondo de Investigación Sanitaria), and by Abbott Laboratories.

² Dr. A. J. Angulo passed away August 2003. This publication is dedicated to his memory.

⁴ Abbreviations used: FCS, fetal calf serum; MMP-1, matrix metalloproteinase-1; NS, nucleosides; NT, exogenous nucleotides; TAA, thioacetamide; TCGI, fetal hepatocytes cultured in the presence of thymidine, cytidine, guanosine, and inosine; UCGI, fetal hepatocytes cultured in the presence of uridine, cytidine, guanosine, and inosine.

Manuscript received 5 December 2003. Initial review completed 21 January 2004. Revision accepted 18 February 2004.

	LITERATURE CITED

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 

1. Sánchez-Pozo, A., Rueda, R., Fontana, L. & Gil, A. (1998) Dietary nucleotides and cell growth. Pandalai, S. G. eds. Trends Comp. Biochem. Physiol. 5:99-111 Transworld Research Network, Trivandrum, India.

2. Fontana, L., Moreira, E., Torres, M. I., Fernández, I., Ríos, A., Sánchez de Medina, F. & Gil, A. (1998) Dietary nucleotides correct plasma and liver microsome fatty acid alterations in rats with liver cirrhosis induced by oral intake of thioacetamide. J. Hepatol. 28:662-669.[Medline]

3. Pérez, M. J., Suárez, A., Gómez-Capilla, J. A., Sánchez de Medina, F. & Gil, A. (2002) Dietary nucleotide supplementation reduces thioacetamide-induced liver fibrosis in rats. J. Nutr. 132:652-657.[Abstract/Free Full Text]

4. Arnaud, A., Fontana, L., Angulo, A. J., Gil, A. & López-Pedrosa, J. M. (2003) Exogenous nucleosides alter the intracellular nucleotide pool in hepatic cell cultures. Implications in cell proliferation and function. Clin. Nutr. 22:391-399.[Medline]

5. Arnaud, A., Fontana, L., Sáez-Lara, M. J., Gil, A. & López-Pedrosa, J. M. (2004) Exogenous nucleosides modulate the expression of rat liver extracellular matrix genes in single cultures of primary hepatocytes and a liver stellate cell line and in their co-culture. Clin. Nutr. 23:43-51.[Medline]

6. Ohyanagi, H., Nishimatsu, S., Kanbara, Y., Usami, M. & Saitoh, Y. (1989) Effects of nucleosides and a nucleotide on DNA and RNA syntheses by the salvage and de novo pathway in primary monolayer cultures of hepatocytes and hepatoma cells. J. Parenter. Enteral Nutr. 13:51-58.[Abstract/Free Full Text]

7. Torres-López, M. I., Fernández, I., Fontana, L., Gil, A. & Ríos, A. (1996) Influence of dietary nucleotides on liver structural recovery and hepatocyte binuclearity in cirrhosis induced by thioacetamide. Gut 38:260-264.[Abstract/Free Full Text]

8. Roncero, C., Lorenzo, M., Fabregat, I. & Benito, M. (1989) Rates of lipogenesis in fetal hepatocytes in suspension and in primary culture: hormonal effects. Biochim. Biophys. Acta 1012:320-324.[Medline]

9. Moyer, J. D., Oliver, J. T. & Handschumacher, R. E. (1981) Salvage of circulating pyrimidine nucleosides in the rat. Cancer Res. 41:3010-3017.[Abstract/Free Full Text]

10. Karle, J. M., Anderson, L. W. & Cyssyk, R. L. (1984) Effect of plasma concentrations of uridine on pyrimidine biosynthesis in cultures of L1210 cells. J. Biol. Chem. 10:67-72.

11. Jiménez, A., Pubill, D., Payás, M., Camins, A., Yadó, S., Camarasa, J. & Escubedo, E. (2000) Further characterization of an adenosine transport system in the mitochondrial fraction of rat testis. Eur. J. Pharmacol. 398:31-39.[Medline]

12. Leach, J. L., Baxter, J. H., Molitor, B. E., Ramstack, M. B. & Masor, M. L. (1995) Total potentially available nucleosides of human milk by stage of lactation. Am. J. Clin. Nutr. 61:1224-1230.[Abstract/Free Full Text]

13. Drysdale, B. E., Zacharchuk, C. N. & Shin, H. S. (1983) Mechanism of macrophage-mediated cytotoxicity: production of a soluble cytotoxic factor. J. Immunol. 131:2362-2367.[Abstract]

14. Bradford, M. M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72:248-254.[Medline]

15. Apostolakos, M. J., Schermann, W.H.T., Frampton, M. W., Utell, M. J. & Willey, J. C. (1993) Measurement of gene expression by multiplex competitive polymerase chain reaction. Anal. Biochem. 213:277-284.[Medline]

16. Chomczynski, P. & Sacchi, N. (1987) Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162:156-159.[Medline]

17. McCulloch, R. K., Choong, C. S. & Hurley, D. M. (1995) An evaluation of competitor type and size for use in the determination of mRNA by competitive PCR. PCR Methods Appl. 4:219-226.[Medline]

18. Griffith, D. A. & Jarvis, S. M. (1996) Nucleoside and nucleobase transport systems of mammalian cells. Biochim. Biophys. Acta 1286:153-181.[Medline]

19. Valdés, R., Ortega, M. A., Casado, F. J., Felipe, A., Gil, A., Sánchez-Pozo, A. & Pastor-Anglada, M. (2000) Nutritional regulation of nucleoside transporter expression in rat small intestine. Gastroenterology 119:1623-1630.[Medline]

20. Rathbone, M. P., Middlemiss, P. J., Gysbers, J. W., DeForge, S., Costello, P. & Del Maestro, R. F. (1992) Purine nucleosides and nucleotides stimulate proliferation of a wide of cell types. In Vitro Cell Dev. Biol. 28A:7-8.[Medline]

21. Schor, S. & Rozengurt, E. (1973) Enhancement by purine nucleosides and nucleotides of serum-induced DNA synthesis in quiescent 3T3 cells. J. Cell. Physiol. 81:339-346.[Medline]

22. Engstrom, W. & Zetterberg, A. (1984) The relationship between purines, pyrimidines, nucleosides and glutamine for fibroblast cell proliferation. J. Cell. Physiol. 120:233-240.[Medline]

23. Huang, N., Wang, D. J. & Heppel, L. A. (1989) Extracellular ATP is a mitogen for 3T3, 3T6, and A431 cells and acts synergistically with other growth factors. Proc. Natl. Acad. Sci. U.S.A. 86:7904-7908.[Abstract/Free Full Text]

24. Meininger, C. & Granger, J. H. (1990) Mechanisms leading to adenosine-stimulated proliferation of microvascular endothelial cells. Am. J. Physiol. 258:H198-H206.

25. Middlemiss, P. J., Rathbone, M. P. & Hoofman, E. (1990) Purigenic stimulation of astrocytes proliferation: Evidence for involvement of P₁-A₂ and P_2Y receptors. Can. Coll. Neuro. Pharm. Abstracts 13:7 (abs.).

26. Harden, T. K., Lazarowski, E. R. & Boucher, R. C. (1997) Release, metabolism and interconversion of adenine and uridine nucleotides: implications for G protein-coupled P2 receptor agonist selectivity. Trends Pharmacol. Sci. 18:43-46.[Medline]

27. Boarder, M. R. & Hourani, S. M. (1998) The regulation of vascular function by P2 receptors: multiple sites and multiple receptors. Trends Pharmacol. Sci. 19:99-107.[Medline]

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