Nucleoside and nucleobase transporters of primary human cardiac microvascular endothelial cells: characterization of a novel nucleobase transporter

• Published in: American journal of physiology. Heart and circulatory physiology 2007-12, Vol.293 (6), p.H3325-H3332
• Main Authors: Bone, Derek B. J, Hammond, James R
• Format: Article
• Language: English
• Subjects: 2-Chloroadenosine - metabolism; Adenosine; Animals; Biochemistry; Cardiovascular system; Cell Culture Techniques; Cell Line; Cells; Cells, Cultured; Child, Preschool; Coronary Vessels - drug effects; Coronary Vessels - metabolism; Dilazep - pharmacology; Dipyridamole - pharmacology; Dogs; Endothelial Cells - drug effects; Endothelial Cells - metabolism; Equilibrative Nucleoside Transporter 1 - antagonists & inhibitors; Equilibrative Nucleoside Transporter 1 - genetics; Equilibrative Nucleoside Transporter 1 - metabolism; Equilibrative-Nucleoside Transporter 2 - antagonists & inhibitors; Equilibrative-Nucleoside Transporter 2 - metabolism; Female; Heart; Humans; Hypoxanthine - metabolism; Kinetics; Microcirculation - metabolism; Nucleobase Transport Proteins - antagonists & inhibitors; Nucleobase Transport Proteins - metabolism; Organic chemicals; Piperazines - pharmacology; Protein Binding; Purines - metabolism; Purines - pharmacology; Rats; Swine; Thioinosine - analogs & derivatives; Thioinosine - metabolism; Transfection; Tritium
• ISSN: 0363-6135; 1522-1539
• DOI: 10.1152/ajpheart.01006.2007

Department of Physiology and Pharmacology, Schulich School of Medicine and Dentistry, The University of Western Ontario, London, Canada Submitted 30 August 2007 ; accepted in final form 3 October 2007 Levels of cardiovascular active metabolites, like adenosine, are regulated by nucleoside transporters of endothelial cells. We characterized the nucleoside and nucleobase transport capabilities of primary human cardiac microvascular endothelial cells (hMVECs). hMVECs accumulated 2-[ 3 H]chloroadenosine via the nitrobenzylmercaptopurine riboside-sensitive equilibrative nucleoside transporter 1 (ENT1) at a V max of 3.4 ± 1 pmol·µl –1 ·s –1 , with no contribution from the nitrobenzylmercaptopurine riboside-insensitive ENT2. Inhibition of 2-chloroadenosine uptake by ENT1 blockers produced monophasic inhibition curves, which are also compatible with minimal ENT2 expression. The nucleobase [ 3 H]hypoxanthine was accumulated within hMVECs ( K m = 96 ± 37 µM; V max = 1.6 ± 0.3 pmol·µl –1 ·s –1 ) despite the lack of a known nucleobase transport system. This novel transporter was dipyridamole-insensitive but could be inhibited by adenine ( K i = 19 ± 7 µM) and other purine nucleobases, including chemotherapeutic analogs. A variety of other cell types also expressed the nucleobase transporter, including the nucleoside transporter-deficient PK(15) cell line (PK15NTD). Further characterization of [ 3 H]hypoxanthine uptake in the PK15NTD cells showed no dependence on Na + or H + . PK15NTD cells expressing human ENT2 accumulated 4.5-fold more [ 3 H]hypoxanthine in the presence of the ENT2 inhibitor dipyridamole than did PK15NTD cells or hMVECs, suggesting trapping of ENT2-permeable metabolites. Understanding the nucleoside and nucleobase transporter profiles in the vasculature will allow for further study into their roles in pathophysiological conditions such as hypoxia or ischemia. adenosine; hypoxanthine; human; cardiovascular Address for reprint requests and other correspondence: J. R. Hammond, Dept. of Physiology and Pharmacology, MSB 266, Medical Sciences Bldg., Univ. of Western Ontario, London, ON, Canada N6A 5C1 (e-mail: james.hammond{at}schulich.uwo.ca )