Transport characteristics of HL-1 cells: a new model for the study of adenosine physiology in cardiomyocytes

Abstract

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 (Km= 51.3 ± 12.9 μM), NBTI-sensitive adenosine transport. NBTI binds at a high-affinity single site (Bmax= 520 ± 10 fmol/mg protein, Kd= 0.11 ± 0.04 nM, 1.6 × 105NBTI-binding 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.Key words: adenosine, nucleoside transport, HL-1 cells, cardiovascular, glucose transport, protein kinase C.