title: The pharmacology and kinetics of ecto-nucleotidases in the perilymphatic compartment of the guinea-pig cochlea.
abstract: This study investigated the characteristics of ecto-nucleotidases in tissues lining the perilymphatic cavity of the cochlea. The perilymphatic space of the isolated guinea-pig cochlea was maintained with oxygenated artificial perilymph (AP) perfused at a rate of 100 microl/min. Following AP perfusion, either adenosine triphosphate (ATP), adenosine diphosphate (ADP) or adenosine monophosphate (AMP) was introduced into scala tympani, and perfusion arrested for 2 min for substrate incubation with cochlear tissues. Effluent collected from the cochlea was assayed for adenine nucleotide metabolites by reverse-phase high-performance liquid chromatography (RP-HPLC). Extracellular ATP and ADP were rapidly and sequentially hydrolysed to adenosine by Ca2+/Mg2+-dependent and Ca2+/Mg2+-independent enzymatic mechanisms. The degradation of extracellular ATP, ADP and AMP occurred in the presence of intact tissues, as demonstrated by the limited lactate dehydrogenase (LDH) activity (0-2.2%). ATPase activity was not affected by inhibitors of intracellular ATPases (oligomycin, ouabain, N-ethylmaleimide, 100 microM NaN3) and non-specific alkaline phosphatase (beta-glycerophosphate). The hydrolysis of ATP was inhibited by 5 mM NaN3, suramin, ATPgammaS, La3+ and CTP, the hydrolysis of ADP by beta,gamma-imidoATP, and AMP degradation by alpha,beta-methyleneADP. Ecto-ATPase, ecto-ADPase and ecto-5'-nucleotidase followed Michaelis-Menten hyperbolic kinetics, with estimated Km values of 2282 microM, 6619 microM and 881 microM, respectively. Our results indicate the presence of considerable ecto-nucleotidase activity within scala tympani of the cochlea, and support its role as the terminating mechanism for P2 receptor signalling known to occur in the cochlea. A competition plot is consistent with ATP and ADP degradation mediated by the same enzyme (ecto-ADP diphosphohydrolase) with two different catalytic sites.
authors.affiliation: Department of Physiology, The University of Auckland, New Zealand.
authors.forename: S M
authors.initials: SM
authors.lastname: Vlajkovic
authors.forename: P R
authors.initials: PR
authors.lastname: Thorne
authors.forename: G D
authors.initials: GD
authors.lastname: Housley
authors.forename: D J
authors.initials: DJ
authors.lastname: Muñoz
authors.forename: I S
authors.initials: IS
authors.lastname: Kendrick
mesh_terms: D015720:5'-Nucleotidase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000244:Adenosine Diphosphate / Q000378:metabolism / Q000494:pharmacology; D000249:Adenosine Monophosphate / Q000378:metabolism / Q000494:pharmacology; D000251:Adenosine Triphosphatases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism / Q000494:pharmacology; D000818:Animals; D015703:Antigens, CD; D000963:Antimetabolites / Q000494:pharmacology; D001081:Apyrase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D017301:Ca(2+) Mg(2+)-ATPase / Q000378:metabolism; D002470:Cell Survival / Q000187:drug effects; D002851:Chromatography, High Pressure Liquid; D003051:Cochlea / Q000166:cytology / Q000187:drug effects / Q000201:enzymology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005260:Female; D006168:Guinea Pigs; D006868:Hydrolysis; D066298:In Vitro Techniques; D007700:Kinetics; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D008297:Male; D010498:Perilymph / Q000187:drug effects / Q000201:enzymology*
chemical_list: D015703:Antigens, CD; D000963:Antimetabolites; D004791:Enzyme Inhibitors; D000249:Adenosine Monophosphate; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D007770:L-Lactate Dehydrogenase; D015720:5'-Nucleotidase; D000251:Adenosine Triphosphatases; D017301:Ca(2+) Mg(2+)-ATPase; C027201:ectoATPase; D001081:Apyrase; C067869:CD39 antigen