Cloned Blood–Brain Barrier Adenosine Transporter is Identical to the Rat Concentrative Na⁺ Nucleoside Cotransporter CNT2

Abstract

Adenosine transport into brain is regulated by the activity of the adenosine transporter located at the brain capillary endothelial wall, which forms the blood-brain barrier (BBB) in vivo. To facilitate cloning of BBB adenosine transporters, poly A+ RNA was purified from isolated rat brain capillaries for production of a rat BBB cDNA library in the pSPORT vector. The cloned RNA (cRNA) generated from in vitro transcription of this library was injected into frog oocytes followed by measurement of [3H]-adenosine uptake. After dilutional cloning, a full-length, 2905-nucleotide adenosine transporter cDNA, designated clone A-11, was isolated. The A-11 clone yielded [3H]-adenosine flux ratios of 400 to 500 after injection of cRNA in oocytes. The adenosine uptake was sodium-dependent and insensitive to inhibition by S-(4-nitrobenzyl)-6-thioinosine (NBTI). The Km and Vmax of adenosine transport in the cRNA-injected oocytes were 23.1 +/- 3.7 micromol/L and 10.8 +/- 0.9 pmol/oocyte. min. The K0.5 for sodium was 2.4 +/- 0.1 mmol/L, with a Hill coefficient (n) of 1.06 +/- 0.07. DNA sequence analysis indicated the rat BBB A-11 adenosine cDNA was identical to rat concentrative nucleoside transporter type 2 (CNT2). The adenosine transporter activity of the rat BBB A-11 CNT2 clone is 50-fold more active than previously reported rat CNT2 clones. In summary, these studies describe the expression cloning of CNT2 from a rat BBB library and show that the pattern of sodium dependency and NBTI insensitivity of the cloned CNT2 are identical to patterns of adenosine transport across the BBB in vivo. These results suggest that BBB adenosine transport in vivo is mediated by CNT2, which would make CNT2 one of the few known sodium-dependent cotransporters that mediate substrate transport across the BBB in the blood to brain direction.

Dietary supplementation with uridine-5′-monophosphate (UMP), a membrane phosphatide precursor, increases acetylcholine level and release in striatum of aged rat

“…Consumption of UMP (2000 mg) by humans raises plasma uridine levels by 3 fold (from 6.0 to 21.9 μM), for 5-6 hours (Cansev, 2006). Circulating uridine crosses the blood-brain barrier via an adenosine transporter (the concentrative nucleoside transporter type 2; CNT2) (Li et al, 2001), which is unsaturated at physiologic plasma uridine concentrations (Cansev et al, 2005). Thus, dietary UMP can increase brain levels of uridine and subsequently, as shown in gerbils, brain levels of UTP, CTP and CDP-choline (Cansev et al, 2005).…”

“…Brain PtdCho synthesis can be enhanced by its three circulating precursors (Kennedy and Weiss, 1956): choline, a pyrimidine nucleoside like uridine or cytidine, and a polyunsaturated fatty acid (PUFA) like docosahexaenoic acid (DHA) (Araki and Wurtman, 1998;Rapoport, 2001;Marszalek and Lodish, 2005); all three readily cross the blood-brain barrier (Li et al, 2001;Spector, 2001). Brain uridine can be phosphorylated by uridine kinase (Suzuki et al, 2004) to form uridine-5′-triphosphate (UTP), which can be aminated (by CTP synthetase) to cytidine-5′-triphosphate (CTP) (Genchev and Mandel, 1974).…”

The blood-brain barrier: Bottleneck in brain drug development

“…However, the BBB choline transporter is sodium independent 45 and is likely a member of a different SLC gene family. The BBB adenosine carrier transports adenosine, guanosine, and certain pyrimidine nucleosides such as uridine, 46 and is derived from the CNT2 gene, 47 where CNT ϭ concentrative nucleoside transporter. Purine nucleosides are also transported by sodium independent or equilibrative nucleoside transporters (ENT), which are members of the SLC29 gene family (Table 2).…”

CONSTITUTIVE EXPRESSION OF VARIOUS XENOBIOTIC AND ENDOBIOTIC TRANSPORTER mRNAs IN THE CHOROID PLEXUS OF RATS

“…Studies using rodent and human tissues have reported the existence of specific proteins that transport a variety of xenobiotics and endobiotics, such as drugs, flavonoids, metals, nucleotides and nucleosides, di-and tripeptides, and bile acids (Saito et al, 1995;Lomri et al, 1996;Miyamoto et al, 1996;Yao et al, 1997;Suzuki and Sugiyama, 1998;Cousins and McMahon, 2000;Ferguson et al, 2001;Li et al, 2001). The total number of transporters in rodents reported in the literature independent of the discoveries through the genome sequencing projects is about 50, and many more seem to await characterization.…”