FUNCTIONAL CONSEQUENCES OF INHIBITION OF NUCLEOTIDE BREAKDOWN IN RAT VAS-DEFERENS - A STUDY WITH EVANS BLUE

The effect of Evans blue on nucleotide breakdown, nucleotide-evoked co ntractions and electrically evoked contractions, overflow of ATP and o verflow of tritium (after labelling with [H-3]-noradrenaline) was stud ied in rat vas deferens. Pieces of vas deferens degraded 83 to 85% of added ATP, ADP and 2-methylthio ATP (all 100 mu M) over 30 min. Evans blue (100 mu M) reduced this degradation to 22 to 26%. Nucleotides eli cited contraction with potency declining in the order alpha,beta-methy lene ATP > 2-methylthio ATP > ATP > ADP. Evans blue (100 mu M) shifted the concentration-response curve of alpha,beta-methylene ATP to the r ight and increased the maximum. Concentration-response curves of ATP, ADP and 2-methylthio ATP, in contrast, were shifted to the left and re sponses were much potentiated. In the presence of Evans blue, the rank order of potency was ATP > 2-methylthio ATP > alpha,beta-methylene AT P > ADP. Electrical field stimulation (100 pulses at 10 Hz) elicited c ontraction and an overflow of tritium and ATP. Evans blue (100 mu M) d id not alter the contraction and the evoked overflow of tritium but in creased 24-fold the evoked overflow of ATP. The results indicate that Evans blue may serve as an - albeit impure - ecto-nucleotidase inhibit or in functional experiments. Such experiments demonstrate that the lo w potency of ATP (and also ADP and 2-methylthio ATP) in eliciting cont raction, and the small size of the overflow of ATP upon sympathetic ne rve stimulation, are due to rapid breakdown.