THE PHARMACOLOGY AND KINETICS OF ECTO-NUCLEOTIDASES IN THE PERILYMPHATIC COMPARTMENT OF THE GUINEA-PIG COCHLEA

This study investigated the characteristics of ecto-nucleotidases in t issues lining the perilymphatic cavity of the cochlea. The perilymphat ic space of the isolated guinea-pig cochlea was maintained with oxygen ated artificial perilymph (AP) perfused at a rate of 100 mu l/min. Fol lowing AP perfusion, either adenosine triphosphate (ATP), adenosine di phosphate (ADP) or adenosine monophosphate (AMP) was introduced into s cala tympani, and perfusion arrested for 2 min for substrate incubatio n with cochlear tissues. Effluent collected from the cochlea was assay ed for adenine nucleotide metabolites by reverse-phase high-performanc e liquid chromatography (RP-HPLC). Extracellular ATP and ADP were rapi dly and sequentially hydrolysed to adenosine by Ca2+/Mg2+-dependent an d Ca2+/Mg2+-independent enzymatic mechanisms. The degradation of extra cellular 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 intracellula r ATPases (oligomycin, ouabain, N-ethylmaleimide, 100 mu M NaN3) and n on-specific alkaline phosphatase (beta-glycerophosphate). The hydrolys is of ATP was inhibited by 5 mM NaN3, suramin, ATP gamma S, La3+ and C TP, the hydrolysis of ADP by beta,gamma-imidoATP, and AMP degradation by alpha,beta-methyleneADP. Ecto-ATPase, ecto-ADPase and ecto-5'-nucle otidase followed Michaelis-Menten hyperbolic kinetics, with estimated K-m values of 2282 mu M, 6619 mu M and 881 mu M, respectively. Our res ults indicate the presence of considerable ecto-nucleotidase activity within scala tympani of the cochlea, and support its role as the termi nating mechanism for P2 receptor signalling known to occur in the coch lea. A competition plot is consistent with ATP and ADP degradation med iated by the same enzyme (ecto-ADP diphosphohydrolase) with two differ ent catalytic sites. (C) 1998 Elsevier Science B.V.