A study on P2x purinoceptors mediating the electrophysiological and contractile effects of purine nucleotides in rat vas deferens

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Abstract
1 We have studied both the electrophysiological and contractile effects of the purine nucleotide, adenosine-5′-triphosphate (ATP), as well as a number of its structural analogues as agonists at P2X purinoceptors in the rat vas deferens in vitro. 2 Electrophysiological effects were investigated by a whole cell voltage clamp technique (holding potential − 70 mV) with fast flow concentration-clamp applications of agonists in single isolated smooth muscle cells. ATP, 2-methylthio adenosine-5′-triphosphate (2-MeSATP) and α, β methylene adenosine-5′-triphosphate (αβ-meATP) all evoked inward currents over a similar concentration range (0.3–10 μM), being approximately equipotent with similar concentrations for threshold effects (0.3 μM). ADP (10 μM) also evoked a rapid current of similar peak amplitude to that seen with ATP (10 μM). 3 α,β-meATP was the most potent agonist in producing contractions of the rat vas deferens whole tissue preparation, with a threshold concentration equal to that in the electrophysiological studies (0.3 UM). However, ATP and 2-MeSATP were at least ten times less potent in studies measuring contraction than in the electrophysiological studies. Furthermore, their concentration-effect curves were shallow with smaller maximal responses than could be achieved with α,β-meATP. ADP, AMP and adenosine were inactive at concentrations up to 1 mM. The rank order of agonist potencies observed for contraction was α,β-meATP≫ATP = 2-MeSATP. 4 Measurement of inorganic phosphate (iP), as a marker of purine nucleotide metabolism in the vas deferens whole tissue preparation, indicated that ATP and 2-MeSATP were rapidly metabolized, whereas α,β-meATP was stable for up to 2 h. Removal of divalent cations prevented breakdown of ATP and 2-MeSATP, suggesting that metabolism involved a Ca2+/Mg2+-dependent enzyme. 5 It appears that in isolated preparations of rat vas deferens, the low potency of ATP and 2-MeSATP can be explained by rapid agonist breakdown by ectonucleotidases. However, this is not the case in the single cell studies where the use of rapid concentration-clamp applications revealed the true potency of the agonists. Under such conditions the three agonists were all equal in potency indicating that the rank order of agonist potencies of α,β-meATP≫ATP = 2-MeSATP is not in fact characteristic of smooth muscle P2X-purinoceptors as commonly believed.