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.
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 microM, 21.0 +/- 2.3 microM for ADP, and 10.0 +/- 1.1 microM 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 microM, but had no effect on the breakdown of AMP. The 5'-nucleotidase inhibitor alpha,beta-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-beta,gamma-dibromomethylene-D-ATP (ARL67156), an ecto-ATPase inhibitor, suppressed ATPase and AMPase activities, whereas NaN(3) (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.
Mihaylova-Todorova
Svetlana T
ST
Department of Pharmacology, University of Nevada School of Medicine, Howard Medical Sciences Building, Room 222, ms 318, Reno, NV 39557-0046, USA. mihay_s@med.unr.edu
Todorov
Latchezar D
LD
Westfall
David P
DP
D015720:5'-Nucleotidase / Q000009:adverse effects / Q000378:metabolism*; D000227:Adenine Nucleotides / Q000378:metabolism; D000241:Adenosine / Q000378:metabolism; D000244:Adenosine Diphosphate / Q000031:analogs & derivatives* / Q000494:pharmacology; D000251:Adenosine Triphosphatases / Q000009:adverse effects / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000494:pharmacology; D000818:Animals; D002118:Calcium / Q000502:physiology; D002851:Chromatography, High Pressure Liquid; D004558:Electric Stimulation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006168:Guinea Pigs; D066298:In Vitro Techniques; D007700:Kinetics; D008274:Magnesium / Q000502:physiology; D008297:Male; D009708:Nucleotidases / Q000009:adverse effects / Q000378:metabolism*; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism; D010744:Phosphoric Monoester Hydrolases / Q000378:metabolism; D010940:Plant Proteins / Q000494:pharmacology; D011755:Pyrophosphatases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D013498:Suramin / Q000494:pharmacology; D013564:Sympathetic Nervous System / Q000201:enzymology; D014649:Vas Deferens / Q000187:drug effects / Q000201:enzymology* / Q000294:innervation
C092431:6-N,N-diethyl-beta,gamma-dibromomethylene-D-ATP; D000227:Adenine Nucleotides; D004791:Enzyme Inhibitors; D010940:Plant Proteins; C523965:alpha,beta-methyleneadenosine 5'-diphosphate; C007110:concanavalin B protein, Canavalia ensiformis; D013498:Suramin; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D009708:Nucleotidases; C018775:adenosine monophosphatase; D010744:Phosphoric Monoester Hydrolases; D015720:5'-Nucleotidase; D010727:Phosphoric Diester Hydrolases; D000251:Adenosine Triphosphatases; D011755:Pyrophosphatases; C439652:nucleoside-triphosphate diphosphohydrolase 3; D008274:Magnesium; D000241:Adenosine; D002118:Calcium