Characterization of the ATPase released during sympathetic nerve stimulation of the guinea-pig isolated vas deferens.

The release of ATPase activity evoked by electrical field stimulation (EFS) (8 Hz, 25 s) was investigated in several tissues in which adenosine 5'-triphosphate (ATP) acts as a neurotransmitter. Superfusate collected during EFS of sympathetic nerves of the guinea-pig, rat and mouse isolated vas deferens and parasympathetic nerves of the guinea-pig isolated urinary bladder contained ATPase activity. ATP breakdown was fastest in superfusate collected from the guinea-pig isolated vas deferens. However, EFS of the enteric nerves of the guinea-pig isolated taenia coli did not release any detectable ATPase. The ATPase released from the guinea-pig isolated vas deferens metabolized ATP at similar rates at incubation temperatures of 37 degrees C and 20 degrees C. Lineweaver-Burke analysis of the initial rates of ATP hydrolysis gave a K(M) of 39 microM and a V(max) of 1039 pmol ATP metabolized min(-1) ml(-1) superfusate. 6-N,N-diethyl-D-beta,gamma-dibromomethyleneATP (ARL 67156), pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS) and pyridoxal-5'-phosphate (P-5-P) all inhibited the ATPase activity in a concentration-dependent manner with a potency order of ARL 67156 = PPADS>P-5-P. In conclusion, EFS of several tissues in which ATP is a neurotransmitter causes the release of an ATPase and activity is greatest in the guinea-pig vas deferens. The enzyme has pharmacological and kinetic characteristics that are similar to ectonucleoside triphosphate diphosphohydrolases.

Release of a soluble ATPase from the rabbit isolated vas deferens during nerve stimulation.

The properties of the ATPase released during electrical field stimulation (EFS) (8 Hz, 25 s) of the sympathetic nerves of the superfused rabbit isolated vas deferens were investigated. Superfusate collected during EFS rapidly metabolised exogenous ATP (100 microM) and 50% was broken down in 5.67+/-0.65 min. The main metabolite was ADP, virtually no AMP was produced and adenosine was absent. No enzyme activity was seen in samples collected in the absence of EFS. Lineweaver-Burke analysis of the initial rates of ATP hydrolysis gave a K(M) of 40 microM and V(max) of 20.3 nmol ATP metabolized min(-1) ml(-1) superfusate. ATPase activity was unaffected by storage at room temperature for 24 h, but was abolished at pH4 or by heating at 80 degrees C for 10 min. ARL 67156 inhibited ATP breakdown in a concentration-dependent manner (IC(50)=25 microM (95% confidence limits=22-27 microM), Hill slope=-1.06+/-0.04). When EFS was applied three times at 30 min intervals, ATP metabolism was 20-30% less in superfusate collected during the second and third stimulation periods compared with the first. ATPase activity was released in a frequency-dependent manner, with significantly greater activity seen after stimulation at 4 and 8 Hz than at 2 Hz. In conclusion, EFS of the sympathetic nerves in the rabbit vas deferens causes release of substantial ATPase, but little ADPase activity into the extracellular space. This contrasts with the guinea-pig vas deferens, which releases enzymes that degrade ATP to adenosine. Thus, the complement of enzymes released by nerve stimulation is species-dependent.