Characterization of 5′- N ethylcarboxamido[ 3H]adenosine binding to pig aorta smooth muscle membranes

Abstract

Binding of 5 prime:; N-ethylcarboxamido[ 3H]adenosine ([ 3H]NECA) to pig aorta smooth muscle membranes was rapid, reversible and dependent on protein concentration and temperature. Due to a rapid rate of dissociation binding was highest at 0°. Binding was saturable and Scatchard analysis revealed two different binding sites for [ 3]NECA with K D values of 0.29 and 4.64 μM and B max values of 9.3 and 35.5pmol mg −1. GTP, Mg 2+, Mn 2+ and Ca 2+ did not affect the binding. (−)[N 6]-[ 3 H] phenylisopropyladenosine ([ 3H]PIA) bound to pig aorta smooth muscle membranes with very low affinity and non-specific binding was high (50%), in contrast to that for [ 3H]NECA (<10%). In competition studies, NECA and 5′- N-methylcarboxamidoadenosine were the most potent displacers of [ 3H]Neca followed by adenosine, 2-chloroadenosine and 2′,5′-dideoxyadenosine. (−)PIA and N 6-cyclohexyladenosine, potent A 1 receptor agonists, did not compete for [ 3H]NECA binding sites. The xanthines, 3-isobutyl- l-methylxanthine and theophylline, inhibited [ 3H]NECA binding, but, in contrast, 8-phenyltheophylline, a potent adenosine antagonist in other systems, did not compete for binding sites. No effect of NECA nor (−)PIA on adenylate cyclase activity could be demonstrated, whereas forskolin increased activity 17-fold. Similarly, the same adenosine analogues incubated with intact slices of rat aorta smooth muscle failed to elevate tissue cAMP levels, although forskolin elicited a 37-fold increase. These results demonstrate low affinity [ 3H]NECA binding sites in pig aorta smooth muscle with properties similar to those described in lung and platelet membranes but which differ from characteristic A 2-receptors in certain features.