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.