title: A study on P2X purinoceptors mediating the electrophysiological and contractile effects of purine nucleotides in rat vas deferens.
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 alpha,beta methylene adenosine-5'-triphosphate (alpha,beta-meATP) all evoked inward currents over a similar concentration range (0.3-10 microM), being approximately equipotent with similar concentrations for threshold effects (0.3 microM). ADP (10 microM) also evoked a rapid current of similar peak amplitude to that seen with ATP (10 microM). 3. alpha,beta-meATP was the most potent agonist in producing concentrations of the rat vas deferens whole tissue preparation, with a threshold concentration equal to that in the electrophysiological studies (0.3 microM). 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 vasdeferens 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 P2x-purinoceptors as commonly believed.
authors.affiliation: Glaxo Institute for Applied Pharmacology, Department of Pharmacology, University of Cambridge.
authors.forename: B S
authors.initials: BS
authors.lastname: Khakh
authors.forename: A
authors.initials: A
authors.lastname: Surprenant
authors.forename: P P
authors.initials: PP
authors.lastname: Humphrey
mesh_terms: D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000494:pharmacology*; D000818:Animals; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D004553:Electric Conductivity; D004594:Electrophysiology; D066298:In Vitro Techniques; D008297:Male; D009119:Muscle Contraction / Q000187:drug effects / Q000502:physiology; D009130:Muscle, Smooth / Q000187:drug effects / Q000502:physiology*; D010710:Phosphates / Q000378:metabolism; D058910:Purinergic P2 Receptor Agonists; D051381:Rats; D017207:Rats, Sprague-Dawley; D018048:Receptors, Purinergic P2 / Q000378:metabolism*; D013498:Suramin / Q000494:pharmacology; D014649:Vas Deferens / Q000187:drug effects / Q000502:physiology*
chemical_list: D010710:Phosphates; D058910:Purinergic P2 Receptor Agonists; D018048:Receptors, Purinergic P2; D013498:Suramin; D000255:Adenosine Triphosphate