© 2004 by European Society of Cardiology
Copyright © 2004, European Society of Cardiology
Hypoxia regulates the adenosine transporter, mENT1, in the murine cardiomyocyte cell line, HL-1
Department of Biology, York University, 4700 Keele St., Toronto, ON, Canada M3J 1P3
* Corresponding author. Tel.: +1-416-736-2100x30825; fax: +1-416-736-5698. coe{at}yorku.ca
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% O2) for 0–20 h. Cell viability, lactate dehydrogenase (LDH) release, glucose uptake, GLUT1 and GLUT4 protein, adenosine uptake, PKC activity, translocation profiles of PKC
and 
, nitrobenzylthioinosine (NBTI) binding and mENT1 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 PKC construct, pSVK3PKC1-401. 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 PKC but not PKC in HL-1 cells. Abrogation of PKC activity using chronic phorbol ester or a dominant negative PKC mimicked the effect of hypoxia on adenosine uptake suggesting that PKC is involved in regulation of mENT1. Hypoxia (4 h) decreases mENT1 mRNA, which returns to basal levels by 20 h. Conclusions: Chronic hypoxia down-regulates mENT1 activity possibly via PKC. Hypoxia and PKC also regulate mENT1 RNA levels. Cardiomyocytes may regulate mENT1 (via PKC) to modulate release and/or uptake of adenosine. However, the relationship between mENT1 mRNA levels, protein levels and functional transport is complex.
KEYWORDS Adenosine transporter; mENT1; Regulation; PKC; HL-1; Cardiomyocytes
Time for primary review 23 days
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