Nucleoside Transporter Expression and Function in Cultured Mouse Astrocytes
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GLIA 52:25–35 (2005) Nucleoside Transporter Expression and Function in Cultured Mouse Astrocytes LIANG PENG,1 RONG HUANG,2 ALBERT C.H. YU,1 KING Y. FUNG,1 MICHEL P. RATHBONE,3 AND LEIF HERTZ1,2* 1Hong Kong DNA Chips, Ltd., Kowloon, Hong Kong, China 2Department of Pharmacology, University of Saskatchewan, Saskatoon, Saskatchewan, Canada 3Department of Medicine, McMaster University, Hamilton, Ontario, Canada KEY WORDS and play important roles in energy homeostasis and sig- adenosine; CNT2; ENT1; ENT2; guanosine; thymidine naling; (3) astrocytes synthesize guanine nucleotides (Pelled et al., 1999); (4) ATP is released from astrocytes during the propagation of Ca21 waves (Guthrie et al., ABSTRACT 1999) and degraded extracellularly; and (5) adenosine Uptake of purine and pyrimidine nucleosides in astrocytes is and guanosine function as neuromodulators, acting on P1 important for several reasons: (1) uptake of nucleosides con- tributes to nucleic acid synthesis; (2) astrocytes synthesize purinergic receptors of different subtypes in neurons and/ AMP, ADP, and ATP from adenosine and GTP from guano- or astrocytes (van Calker and Hamprecht, 1979; Kim sine; and (3) adenosine and guanosine function as neuromo- et al., 1991; Rathbone et al., 1999; Ciccarelli et al., 2001; dulators, whose effects are partly terminated by cellular Chen et al., 2001; Di Iorio et al., 2004; Wittendorp et al., uptake. It has previously been shown that adenosine is 2004), and cellular uptake contributes to the termination rapidly accumulated by active uptake in astrocytes (Hertz of their neuromodulator effects. However, except for ade- and Matz, Neurochem Res 14:755–760, 1989), but the ratio nosine (Hertz, 1978; Thampy and Barnes, 1983; Bender between active uptake and metabolism-driven uptake of ade- and Hertz, 1986; Hosli€ and Hosli,€ 1988; Matz and Hertz, nosine is unknown, as are uptake characteristics for guano- 1990; Bender et al., 1994; Othman et al., 2002), only little sine. The present study therefore aims at providing detailed information is available about the mechanisms by which information of nucleoside transport and transporters in pri- mary cultures of mouse astrocytes. Reverse transcription- purine and pyrimidine nucleosides are accumulated into polymerase chain reaction identified the two equilibrative astrocytes, and even in the case of adenosine the relative nucleoside transporters, ENT1 and ENT2, together with the importance of different uptake mechanisms is unknown. 1 concentrative nucleoside transporter CNT2, whereas CNT3 Three major Na -dependent nucleoside transport sys- was absent, and CNT1 expression could not be investigated. tems are expressed in nontransformed cells: cif (N1) Uptake studies of tritiated thymidine, formycin B, guano- accepts purine nucleosides, including the inosine deriva- sine, and adenosine (3-s uptakes at 1–4°C to study diffu- tive formycin B, and uridine as permeants; cit (N2 and ° sional uptake and 1–60-min uptakes at 37 C to study con- N4) is pyrimidine selective, but it also transports adeno- centrative uptake) demonstrated a fast diffusional uptake of 1 sine and, in the case of N4, guanosine; and cib (N3) all four nucleosides, a small, Na -independent and probably metabolism-driven uptake of thymidine (consistent with accepts both purine and pyrimidine nucleosides (Cass DNA synthesis), larger metabolism-driven uptakes of guano- et al., 1999; Ritzel et al., 2001a). Additional transporters have been described in transformed cells, including a sine (consistent with synthesis of DNA, RNA, and GTP) and 1 especially of adenosine (consistent with rapid nucleotide guanosine-specific Na -dependent transporter, csg (Fla- 1 synthesis), and Na1-dependent uptakes of adenosine (consis- nagan and Mecklin-Gill, 1997). The Na gradient pro- tent with its concentrative uptake) and guanosine, render- vides the driving force for the uptake, and cif and cit ing neuromodulator uptake independent of nucleoside meta- transport the nucleoside and Na1 in a 1:1 ratio, whereas bolism. Astrocytes are accordingly well suited for both cib requires uptake of 2 Na1 for each molecule nucleo- intense nucleoside metabolism and metabolism-independent side accumulated. Both cif and cit are widespread in uptake to terminate neuromodulator effects of adenosine brain, whereas cib is expressed to a minor extent in and guanosine. VC 2005 Wiley-Liss, Inc. brain and only in selected regions (Ritzel et al., 1998, 2001b). In addition, one or both of the two equilibrative, 1 INTRODUCTION non-Na -dependent nucleoside transporters es and ei are expressed rather ubiquitously and are also present Uptake of purine and pyrimidine nucleosides in astro- cytes is important for several reasons: (1) even in the adult brain astrocytes are capable of cell division and may Grant sponsor: Canadian Spinal Research Organization. partly utilize cellular uptake to acquire nucleosides for *Correspondence to: Leif Hertz, RR 2, Box 245, Gilmour, Ontario, K0L 1W0, Canada. E-mail: [email protected] synthesis of not only RNA but also DNA (Fontana et al., Received 8 December 2004; Accepted 22 February 2005 1980; Huff et al., 1990; Korr et al., 1994; Kornack and DOI 10.1002/glia.20216 Rakic, 2001); (2) AMP, ADP, and ATP are avidly synthe- Published online 12 May 2005 in Wiley InterScience (www.interscience. sized from adenosine in astrocytes (Matz and Hertz, 1989) wiley.com). VC 2005 Wiley-Liss, Inc. 26 PENG ET AL. in brain (Jennings et al., 2001) and in C-6 cells, an astrocytic model system (Sinclair et al., 2000), although little es is found in the latter (Sinclair et al., 2001). The difference between es and ei is that es, but not ei,is inhibited by low nanomolar concentrations of nitroben- zyl-thio-inosine (NBTI). With the exception of cgs, none of the Na1-dependent transporters is NBTI-sensitive. The facilitated diffusion mediated by the equilibrative transporters es and ei is only capable of bringing the intracellular nucleoside concentration up to the same level as the extracellular concentration (or vice versa during nucleoside efflux). Nevertheless, they can facili- tate maintained uptake, if the accumulated nucleoside is metabolized, thereby reducing the concentration of free nucleoside and allowing continuous facilitated diffusion Fig. 1. Primer sequences used for RT-PCR of mRNA for ENT1 (A), ENT2 (B), CNT2 (C), CNT3 (D)andb-actin (E). The primers were con- along the resulting concentration gradient (e.g., Hertz, structed based on the published nucleobase sequences for ENT1, ENT2, 1991a,b). Such a ‘‘metabolism-driven’’ uptake is capable CNT2 and CNT3 and the publications by Leung et al. (2001) and Ritzel of supplying nucleosides for synthesis of RNA, DNA, et al. (2001b), and the primer sequence for b-actin was from Hayashi et al. (1999). and adenosine nucleotides at the rates at which these are formed, but it cannot be used for efficient uptake of 1 presence and absence of Na ) and a kinetic analysis of neuromodulator adenosine or guanosine independently the nucleoside transport systems operating in these cul- of subsequent intracellular metabolism. It may therefore 1 tures. Kinetics and Na dependence of the uptakes were be consistent with the neuromodulator activity of adeno- determined for the purine nucleosides guanosine and sine that accumulation of extracellular adenosine into adenosine, the poorly metabolizable inosine analogue adjacent astrocytes and neurons (Hertz, 1978; Hosli€ and formycin B, and the pyrimidine nucleoside thymidine, Hosli,€ 1988; Bender et al., 1994; Othman et al., 2002) which has no known neuromodulator action and is a can occur as a metabolism-independent, concentrative precursor of DNA but not of RNA. Uptakes were deter- uptake (Hertz and Matz, 1989; Matz and Hertz, 1990). mined as total accumulation of radioactivity in the pre- However, it has not been established whether a similar sence of tritiated adenosine, guanosine, formycin B, or uptake mechanism exists for guanosine. 1 thymidine during either a 3-s incubation period in ice- The existence of different Na -dependent nucleoside cold medium or long-term (1–60 min) incubation at transport systems is reflected by the demonstration of 37°C. The former approach provides information about three concentrative nucleoside transporters (CNTs) in diffusional uptake (simple [passive] or facilitated diffu- nontransformed cells: CNT1, which mediates uptake by sion, distinguished in the present study by saturability system cit and N2 (but not N4); CNT2, which is the or delay at high concentrations and in the literature by transporter responsible for the cif system, and CNT3, sensitivity to specific inhibitors) in the more or less com- corresponding to cib. The two equilibrative, nonconcen- plete absence of metabolism of the accumulated nucleo- trative adenosine transporters ENT1 and ENT2 mediate side (on account of the short uptake period and the low the uptake by facilitated diffusion (systems es and ei), temperature), the second about total uptake (diffusional with ENT1 sensitive, and ENT2 resistant, to low nano- 1 uptake plus Na -independent metabolism-driven uptake molar concentrations of NBTI. The complete nucleotide or Na1-dependent, concentrative uptake). base sequence has been determined for mouse ENT1 and ENT2 as well as for mouse CNT2 and CNT3 (Choi et al., 2000; Kiss et al., 2000; Patel et al., 2000; Handa MATERIALS AND METHODS et al., 2001; Ritzel et al., 2001b; Strausberg et al., 2002), Materials whereas the complete sequence for CNT1 has not been determined in the mouse, and the transporter