Characterization of an NBTI-sensitive Equilibrative Nucleoside Transporter in Vascular Smooth Muscle

Abstract

Adenosine plays a significant role in various physiological and regulatory processes including coronary vasodilatation. In the current study, a high-affinity adenosine transporter in freshly dissociated porcine coronary smooth muscle (PCSM) cells and cultured human coronary smooth muscle (HCSM) cells was characterized. Kinetic analysis of the transport process revealed a Vmaxof 82±17 pm/mg protein/min and a Kmof 4.3±2.1μ m for PCSM cells, whereas a Kmof 4.8 μ m and Vmaxof 254 pm/mg/min was observed for cultured HCSM. Concentration-dependent inhibition of adenosine uptake by S -(4-nitrobenzyl)-6-thioinosine (NBTI) was observed in both PCSM (IC50, 0.08 μ m) and HCSM (0.1 μ m) cells. Both cell types also demonstrate a high-affinity, single binding site for NBTI (PCSM,Bmax 144.8±23 fmol/mg protein and Kd1.1±0.35 n m; HCSM,Bmax 672±62 fmol/mg protein and Kd0.45±0.14 n m). Adenosine uptake in these cells was not affected by extracellular sodium concentration. RT-PCR analysis of mRNA from individually selected PCSM and HCSM cells demonstrated expression of an NBTI-sensitive equilibrative transporter. Smooth muscle cells isolated from porcine brachial and femoral arteries also transported adenosine at levels similar to that of coronaries. These data demonstrate that vascular coronary smooth muscle possess an NBTI-sensitive equilibrative transporter for adenosine which could function in regulation of vasodilation.