Journal
BIOCHEMISTRY AND CELL BIOLOGY-BIOCHIMIE ET BIOLOGIE CELLULAIRE
Volume 80, Issue 5, Pages 655-665Publisher
NATL RESEARCH COUNCIL CANADA
DOI: 10.1139/O02-143
Keywords
adenosine; nucleoside transport; HL-1 cells; cardiovascular; glucose transport; protein kinase C
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Adenosine is a physiologically important nucleoside in the cardiovascular system where it can act as a cardioprotectant and modulator of energy usage. Adenosine transporters (ATs) modulate cellular adenosine levels, which, in turn, can affect a number of processes such as receptor activation and glucose uptake, but their role in cardiac physiology is poorly understood. Therefore, we have developed a new cell model by determining various adenosine-related characteristics of HL-1, an immortalized atrial cardiomyocyte murine cell line. Adenosine uptake in HL-1 cells is sodium independent, saturable, and inhibitable by nucleoside transport inhibitors (nitrobenzylthioinosine (NBTI), dipyridamole, dilazep). Reverse transcription - polymerase chain reaction analysis confirmed that HL-1 cells possess mouse equilibrative nucleoside transporters 1 and 2 (mENT1, mENT2) and kinetic analyses indicate moderate-affinity (K-m = 51.3 +/- 12.9 muM), NBTI-sensitive adenosine transport. NBTI binds at a high-affinity single site (B-max = 520 +/- 10 finol/mg protein, K-d = 0.11 +/- 0.04 nM, 1.6 x 10(5) NBTI-bitiding sites/cell). HL-1 cells possess adenosine receptor, metabolic enzyme, protein kinase C isoform, and insulin-stimulated glucose transport profiles that match normal mouse heart. Therefore, HL-1 is an excellent model to study ATs within cardiomyocytes and the first model for evaluating in detail the role of the ATs in modulating effects of adenosine.
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