3H]5′‐N‐Ethylcarboxamidoadenosine selectively labels the low affinity adenosine binding protein, adenotin, on intact chinese hamster ovary cells

Abstract

AbstractThe ability of [3H]5′‐N‐ethylcarboxamidoadenosine (NECA) to specifically bind recognition sites on intact Chinese hamster ovary (CHO) cells was examined in the present study. Saturation experiments indicated that [3H]NECA bound with moderate affinity (Kd = 400 nM) and large capacity (apparent Bmax = 3.2 pmol/105 cells) to intact CHO cells. No specific binding to these cells was observed with the A1‐selective agonist 20 nM [3H]cyclohexyladenosine or with the A2‐selective agonist 20 nM [3H]CGS 21680. Competition studies revealed that close structural analogs of NECA and the xanthine phosphodiesterase inhibitor 3‐isobutyl‐1‐methylxanthine (IBMX) inhibited 20 nM [3H]NECA binding with moderate affinity (IC50s 0.5–15 μM). Adenosine also showed weak activity (IC50 = 100 μM) for inhibiting [3H]NECA binding. However, a wide variety of prototypic adenosine receptor agonists and antagonists did not significantly interact with these [3H]NECA recognition sites on CHO cells. [3H]NECA binding to CHO cell membranes was not sensitive to guanine nucleotides and NECA did not stimulate cAMP formation. These results are consistent with the previously demonstrated ability of [3H]NECA to bind low affinity adenosine binding proteins (adenotin proteins), as well as, adenosine receptors in a variety of mammalian tissues. The present results further indicate that [3H]NECA selectively labels in adenotin‐like recognition site on intact CHO cells in the absence of detectable binding to high affinity adenosine receptors. © 1993 Wiley‐Liss, Inc.