A STUDY ON P-2X PURINOCEPTORS MEDIATING THE ELECTROPHYSIOLOGICAL AND CONTRACTILE EFFECTS OF PURINE NUCLEOTIDES IN RAT VAS-DEFERENS

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 P-2X 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 alpha beta methylene adenosine-5'-triphosphate (alpha beta B-meATP) all evoked inward currents over a similar concentration range (0.3-10 mu M), being approximately equipotent with similar concentrations for threshold effects (0.3 mu M). ADP (10 mu M) also evoked a rapid current of similar peak amplitude to that seen with ATP (10 mu M). 3 alpha beta-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 mu M). 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 alpha beta-meATP. ADP, AMP and adenosine were inactive at concentrations up to 1 mM. The rank order of agonist potencies observed for contraction was alpha beta-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 alpha beta-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 alpha beta-meATP>>ATP = 2-MeSATP is not in fact characteristic of smooth muscle P-2X-purinoceptors as commonly believed.