期刊
JOURNAL OF CEREBRAL BLOOD FLOW AND METABOLISM
卷 21, 期 8, 页码 929-936出版社
LIPPINCOTT WILLIAMS & WILKINS
DOI: 10.1097/00004647-200108000-00005
关键词
adenosine; biologic transport; blood-brain barrier; cotransporters; sodium
资金
- NIMH NIH HHS [R01-MH-61138] Funding Source: Medline
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 [H-3]-adenosine uptake. After dilutional cloning, a full-length, 2905-nucleotide adenosine transporter cDNA, designated clone A-11, was isolated. The A-11 clone yielded [H-3]-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 K-m and V-max of adenosine transport in the cRNA-injected oocytes were 23.1 +/- 3.7 mu mol/L and 10.8 +/- 0.9 pmol/oocyte . min. The K-0.5 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.
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