COMPETITION OF ADENINE NUCLEOTIDES FOR A l,3‐[3H]‐DIPROPYL‐8‐CYCLOPENTYLXANTHINE BINDING SITE IN RAT VAS DEFERENS

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Abstract
SUMMARY: 1. The binding of l,3‐[3H]‐dipropyl‐8‐cyclopentylxanthine ([3H]‐DPCPX), a specific adenosine Ai receptor antagonist, was examined in rat vas deferens membrane preparations using radioligand binding techniques.2. l,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‐phenyliso‐propyladenosine (R‐PIA) 2‐chloroadenosine (2‐CI ADO) cyclopentyladenosine (CPA) N‐ethylcarboxamidoadenosine (NECA)>s‐phenylisopropyladenosine (s‐PIA). Kj values were in the low nmol/L range. The rank order of nucleoside potency at the lower affinity site corresponded to R‐PIA CPA NECA>=2‐ClADO> S‐PIA. Ki values were in the low μmol/L range.4. Nucleotides competed for [3H]‐DPCPX binding at a single site only. The rank order of potency at this site corresponded to a,β‐methylene ATP βγ‐methylene ATP = ATP. Ki values were in the high |xmol/L range. This 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 Ai 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.

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