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

1 The properties of the ATPase released during electrical field stimulation (EFS) (8 Hz, 25 s) of the sympathetic nerves of the superfused rabbit isol ated vas deferens were investigated. 2 Superfusate collected during EFS rapidly metabolised exogenous ATP (100 m uM) and 50% was broken down in 5.67 +/- 0.65 min. The main metabolite was A DP, virtually no AMP was produced and adenosine was absent. No enzyme activ ity was seen in samples collected in the absence of EFS. 3 Lineweaver-Burke analysis of the initial rates of ATP hydrolysis gave a K -M of 40 muM and V-max of 20.3 nmol ATP metabolized min(-1) ml(-1) superfus ate. ATPase activity was unaffected by storage at room temperature for 24 h , but was abolished at pH4 or by heating at 80 degreesC for 10 min. 4 ARL 67156 inhibited ATP breakdown in a concentration-dependent manner (IC 50 = 25 muM (95% confidence limits = 22 - 27 muM), Hill slope = - 1.06 +/- 0.04). 5 When EFS was applied three times at 30 min intervals, ATP metabolism was 20-30% less in superfusate collected during the second and third stimulatio n periods compared with the first. ATPase activity was released in a freque ncy-dependent manner, with significantly greater activity seen after stimul ation at 4 and 8 Hz than at 2 Hz. 6 In conclusion, EFS of the sympathetic nerves in the rabbit vas deferens c auses release of substantial ATPase, but little ADPase activity into the ex tracellular space. This contrasts with the guinea-pig vas deferens, which r eleases enzymes that degrade ATP to adenosine. Thus, the complement of enzy mes released by nerve stimulation is species-dependent.