Competition of adenine nucleotides for a 1,3-[3H]-dipropyl-8-cyclopentylxanthine binding site in rat vas deferens.
1. The binding of 1,3-[3H]-dipropyl-8-cyclopentylxanthine ([3H]-DPCPX), a specific adenosine A1 receptor antagonist, was examined in rat vas deferens membrane preparations using radioligand binding techniques. 2. 1,3-[3H]-Dipropyl-8-cyclopentylxanthine bound to these preparations with a KD of 1.07 +/- 0.14 nmol/L (n = 6). The density of [3H]-DPCPX binding sites was 133.38 +/- 5.57 fmol/mg protein. 3. Computer analysis indicated that nucleosides competed for [3H]-DPCPX binding at two distinct sites. The rank order of potency at the higher affinity site corresponded to R-phenylisopropyladenosine (R-PIA) > or = 2-chloroadenosine (2-CIADO) > or = cyclopentyladenosine (CPA) > or = N-ethylcarboxamidoadenosine (NECA) > s-phenylisopropyladenosine (s-PIA). Ki values were in the low nmol/L range. The rank order of nucleoside potency at the lower affinity site corresponded to R-PIA > or = CPA > or = NECA > or = 2-CIADO > S-PIA. Ki values were in the low mumol/L range. 4. Nucleotides competed for [3H]-DPCPX binding at a single site only. The rank order of potency at this site corresponded to alpha, beta-methylene ATP > or = beta, gamma-methylene ATP > or = ATP. Ki values were in the high mumol/L range. The site seemed to correspond with one of the two binding sites predicted by nucleoside competition binding. 5. The ATP-regenerating compound myokinase did not significantly change the competition curve for ATP, indicating that the competition for [3H]-DPCPX binding observed in the presence of ATP was due to an effect of ATP per se and not to an action of a degradation product. 6. The results demonstrate that in rat vasa deferentia there exist two distinct binding sites for [3H]-DPCPX. One of these sites binds only nucleosides and may represent an adenosine A1 receptor, as usually defined. The other site binds both nucleosides and nucleotides and may represent an atypical adenosine A1 receptor, an atypical P2 or a P3 purinoceptor.
Smith
A D
AD
Department of Pharmacology, School of Medicine, University of Nevada, Reno 89557, USA.
Cheek
D J
DJ
Buxton
I L
IL
Westfall
D P
DP
D015762:2-Chloroadenosine / Q000378:metabolism / Q000494:pharmacology; D000227:Adenine Nucleotides / Q000378:metabolism* / Q000494:pharmacology; D000241:Adenosine / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000378:metabolism / Q000494:pharmacology; D019830:Adenosine-5'-(N-ethylcarboxamide); D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive / Q000187:drug effects; D066298:In Vitro Techniques; D007700:Kinetics; D008297:Male; D058906:Purinergic P1 Receptor Agonists; D058915:Purinergic P1 Receptor Antagonists; D011869:Radioligand Assay; D051381:Rats; D017207:Rats, Sprague-Dawley; D018047:Receptors, Purinergic P1 / Q000378:metabolism; D014649:Vas Deferens / Q000378:metabolism*; D014970:Xanthines / Q000378:metabolism*
D000227:Adenine Nucleotides; D058906:Purinergic P1 Receptor Agonists; D058915:Purinergic P1 Receptor Antagonists; D018047:Receptors, Purinergic P1; D014970:Xanthines; D015762:2-Chloroadenosine; C047235:N-(1-methyl-2-phenylethyl)adenosine; D019830:Adenosine-5'-(N-ethylcarboxamide); C048599:N(6)-cyclopentyladenosine; D000255:Adenosine Triphosphate; C051360:1,3-dipropyl-8-cyclopentylxanthine; D000241:Adenosine