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 contractions and electrically evoked contractions, overflow of ATP and overflow of tritium (after labelling with [3H]-noradrenaline) was studied in rat vas deferens. Pieces of vas deferens degraded 83 to 85% of added ATP, ADP and 2-methyl-thio ATP (all 100 microM) over 30 min. Evans blue (100 microM) reduced this degradation to 22 to 26%. Nucleotides elicited contraction with potency declining in the order alpha,beta-methylene ATP > 2-methylthio ATP > ATP > ADP. Evans blue (100 microM) shifted the concentration-response curve of alpha, beta-methylene ATP to the right and increased the maximum. Concentration-response curves of ATP, ADP and 2-methylthio ATP, in contrast, were shifted to the left and responses were much potentiated. In the presence of Evans blue, the rank order of potency was ATP > 2-methylthio ATP > alpha, beta-methylene ATP > ADP. Electrical field stimulation (100 pulses at 10 Hz) elicited contraction and an overflow of tritium and ATP. Evans blue (100 microM) did not alter the contraction and the evoked overflow of tritium but increased 24-fold the evoked overflow of ATP. The results indicate that Evans blue may serve as an-albeit impure-ecto-nucleotidase inhibitor in functional experiments. Such experiments demonstrate that the low potency of ATP (and also ADP and 2-methylthio ATP) in eliciting contraction, and the small size of the overflow of ATP upon sympathetic nerve stimulation, are due to rapid breakdown.