Vol. 302, Issue 3, 992-1001, September 2002

Enzyme Kinetics and Pharmacological Characterization of Nucleotidases Released from the Guinea Pig Isolated Vas Deferens during Nerve Stimulation: Evidence for a Soluble Ecto-Nucleoside Triphosphate Diphosphohydrolase-Like ATPase and a Soluble Ecto-5'-Nucleotidase-Like AMPase

Svetlana T. Mihaylova-Todorova, Latchezar D. Todorov and David P. Westfall

Department of Pharmacology, University of Nevada School of Medicine, Reno, Nevada

Previously, we have demonstrated that stimulation of the sympathetic nerves of the guinea pig vas deferens evokes release not only of the cotransmitters ATP and norepinephrine but also of soluble nucleotidases that break down extracellular ATP, ADP, and AMP into adenosine. In this study we show that the apparent K_m values of the releasable enzyme activity vary depending on which of these adenine nucleotides is used as initial substrate. The K_m value for ATP was 33.6 ± 2.3 µM, 21.0 ± 2.3 µM for ADP, and 10.0 ± 1.1 µM for AMP. The ratios of the V_max values for each enzyme reaction were 4:2:3. We have also found a different sensitivity of the metabolism of ATP and AMP by releasable nucleotidases to known nucleotidase inhibitors. Suramin inhibited the breakdown of ATP by releasable nucleotidases in a noncompetitive manner and with a K_i value of 53 µM, but had no effect on the breakdown of AMP. The 5'-nucleotidase inhibitor ,-methylene ADP inhibited the breakdown of AMP but not that of ATP. Concanavalin A inhibited the breakdown of AMP but had neither inhibitory nor facilitatory effects on the breakdown of ATP. 6-N,N-Diethyl-,-dibromomethylene-D-ATP (ARL67156), an ecto-ATPase inhibitor, suppressed ATPase and AMPase activities, whereas NaN₃ (10 mM) affected neither reaction, but inhibited the ADP metabolism. Phosphatase- and phosphodiesterase inhibitors did not affect the activity of the releasable nucleotidases. This evidence suggests that the soluble nucleotidases released during neurogenic stimulation of the guinea pig vas deferens combine an ecto-5'-nucleotidase-like and an ecto-nucleoside triphosphate diphosphohydrolase-like activity.