Characterization of [3H]-Adenosine Binding to Media Membranes of Hog Carotid Arteries

Abstract

Binding of [3H]-adenosine to a microsomal fraction derived from media strips of hog carotid arteries was studied by a centrifugation technique. Specific binding was very rapid, readily and completely reversible and temperature-dependent. Under the chosen conditions of incubation, at 4.degree. C and in the presence of deoxycoformycin, .apprx. 95% of the bound radioactivity was chromatographically identified as adenosine. Scatchard analysis revealed a small number (1.7 pmol/mg protein) of high affinity (Kd = 0.23 .mu.mol/l) and a large number (21.6 pmol/mg protein) of low affinity bindings sites (Kd = 4.3 .mu.mol/l). 5''-Phosphorylated adenosine compounds, 5''-deoxyadenosine, 2-chloroadenosine and adenosine-5''-N-ethylcarboxamide, all derivatives of adenosine with pronounced vasodilatory properties, were most potent in displacing [3H]-adenosine from its binding sites with IC50 [mean inhibitory concentration dosage] values ranging from 1.8 to 14.3 .mu.mol/l. 2-Chloroadenosine and (-)N6-phenylisopropyladenosine were weaker competitors of binding than adenosine, which disagrees with the pharmacological findings. The IC50 of aminophylline was much higher than the concentration required for half-maximal inhibition of the biological actions of adenosine. This partial lack of specificity may be due to nonreceptor binding of [3H]-adenosine at the low affinity sites.