[3H]-(R)-N6-Phenylisopropyladenosine Agonist Binding to the Solubilized A1Adenosine Receptor from Bovine Epidydimal Spermatozoa

The high-affinity agonist radioligand [3H]-(R)-N6-phenylisopropyladenosine ([3H]-R-PIA) was used to investigate agonist A1adenosine receptors interactions in soluble preparations from bovine spermatozoa. The digitonin-solubilized receptor shows a high-affinity state with aKdof 5.32 ± 1.17 nMand aBmaxof 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), [3H]-R-PIA binds to the soluble receptor with aKdof 7.97 ± 1.44 nMand aBmaxof 400.8 ± 27 fmol/mg protein. The radioligand rapidly dissociates with aK−1of 0.125 min−1although specific [3H]-R-PIA is still found in solubilized A1receptor. The A1agonistN6-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 A1receptors tightly coupled to Gi, the pertussin toxin-sensitive component of the G protein family.