[H-3]-(R)-N-6-phenylisopropyladenosine agonist binding to the solubilized A(1) adenosine receptor from bovine epidydimal spermatozoa

The high-affinity agonist radioligand [H-3]-(R)-N-6-phenylisopropyladenosine ([H-3]-R-PIA) was used to investigate agonist A(1) adenosine receptors interactions in soluble preparations from bovine spermatozoa. The digitonin-solubilized receptor shows a high-affinity state with a K-d of 5.32 +/- 1.17 nM and a B-max of 460 +/- 33 fmol/mg protein. The binding capacity, higher than that of the membrane-bound form, indicates that the soluble preparation is likely enriched with binding sites. In the presence of guanylyl-5'-imidodiphosphate (Gpp(NH)p), [H-3]-R-PIA binds to the soluble receptor with a K-d of 7.97 +/- 1.44 nM and a B-max of 400.8 +/- 27 fmol/ mg protein. The radioligand rapidly dissociates with a K--1 of 0.125 min(-1) although specific [H-3]-R-PIA is still found in solubilized A(1) receptor. The A(1) agonist N-6-cyclopentyladenosine differentiates two affinity states, whereas Gpp(NH)p shifts the agonist curve to the right and all the receptors are in the low-affinity state. In the presence of NaCl, the agonist still recognizes two affinity states with a lower affinity than that observed in the absence of NaCl. Analyses of sedimentation profiles show the existence of a population of A(1) receptors tightly coupled to G(i), the pertussin toxin-sensitive component of the G protein family. (C) 1997 Academic Press, Inc.