Evidence of A1 Adenosine Receptor on Epidydimal Bovine Spermatozoa

Abstract

3H-R-phenylisopropyladenosine (PIA) was used to characterize adenosine receptors on bovine epidydimal spermatozoa membranes. Dypiridamole, an adenosine uptake inhibitor, did not effect the radioligand binding, indicating an external site for the interaction of adenosine with spermatozoa. Steady-state binding was achieved after 45 min at 25°C and lasted for at least 3 h. Scatchard plots were linear with a K d of 6.98 ± 1.02 nM and B max of 34 ± 8 fmol/mg protein. N-6-cyclopentyladenosine (CPA), with a K i of approx, 0.196 nM, was the most potent inhibitor of binding and the agonist order potency series was CPA > R-PIA = N-6-cyclohexyladenosine > N-6-phenyladenosine > 5′- N-ethylcarboxamidoadenosine > 2-chloroadenosine > 2-( p-2-carboxyethyl)phenylamine)-5′- N-ethylcarboxy-amidoadenosine. 1-3-Dipropyl-8-cyclopentylxanthine (DPCPX), an A1 receptor selective antagonist, produced the strongest inhibition with a K i of 0.46 ± 0.1 nM. Antagonist order potency series DPCPX > xanthine amine congener > cyclopentyltheophilline = theophylline > caffeine > 1-3-dipropylxanthine > 8-phenyltheophilline was consistent with A1 adenosine receptor (A1AR). Guanylyl-5′-imidodiphosphate did not decrease bound 3-H-R-PIA nor accelerate its dissociation, a behavior consistent with inhibitory receptors only. The incubation of isolated membranes with N-ethylmaleimid followed by a reduction of 57% of the ligand binding further supports the existence of A1AR on bovine epidydimal spermatozoa.