Hypoxia regulates the adenosine transporter, mENT1, in the murine cardiomyocyte cell line, HL-1

Objective: Adenosine is an important paracrine hormone in the cardiovascular system. Adenosine flux across cardiomyocyte membranes occurs mainly via equilibrative nucleoside transporters (ENTs). The role of the ENTs in adenosine physiology is poorly understood, particularly in response to metabolic stress such as hypoxia. Therefore, we investigated the effects of chronic hypoxia on ENT1 the predominant ENT isoform in cardiomyocytes. Methods: HL-1 cells (immortalized murine cardiomyocytes) were exposed to hypoxia (2% O-2) for 0-20 h. Cell viability, lactate dehydrogenase (LDH) release, glucose uptake, GLUT1 and GLUT4 protein, adenosine uptake, PKC activity, translocation profiles of PKCdelta and epsilon, nitrobenzylthiomosine (NBTI) binding and mENTl mRNA levels were measured. The role of PKC in regulating mENT1 was further investigated using phorbol ester (100 nM, 18 h) and a dominant negative PKCepsilon construct, pSVK3PKCepsilon1-401. Results: HL-1 cells have typical cardiomyocyte responses to hypoxia based on cell viability, LDH release, glucose uptake and GLUT protein levels. Hypoxia (8-20 h) down-regulates mENT1-dependent adenosine uptake, NBTI-binding and PKCepsilon but not PKC6 in HL-1 cells. Abrogation of PKCepsilon activity using chronic phorbol ester or a dominant negative PKCe mimicked the effect of hypoxia on adenosine uptake suggesting that PKCepsilon is involved in regulation of mENTl Hypoxia (4 h) decreases mENTl mRNA, which returns to basal levels by 20 h. Conclusions: Chronic hypoxia down-regulates mENT1 activity possibly via PKCe. Hypoxia and PKC also regulate mENT1 RNA levels. Cardiomyocytes may regulate mENT1 (via PKCepsilon) to modulate release and/or uptake of adenosine. However, the relationship between mENT1 mRNA levels, protein levels and functional transport is complex. (C) 2003 European Society of Cardiology. Published by Elsevier B.V All rights reserved.