The metabolism of 3Hnoradrenaline released by electrical stimulation from the isolated nictitating membrane of the cat and from the vas deferens of the rat

The noradrenaline (NA) stores of the isolated medial muscle of the cat's nictitating membrane were labelled with (3)HNA and the tissue was set up in an isolated organ bath for field stimulation. The tritiated NA and its metabolites released spontaneously and by field stimulation were determined by scintillation counting following chromatographic separation.2. NA represented 11.8 +/- 1.0% of the total radioactivity of the spontaneously released tritiated compounds. The rest was accounted for by NA metabolites: (a) normetanephrine (NMN), 35%; (b) 4-hydroxy-3-methoxymandelic acid (VMA), 20%; (c) 3,4-dihydroxyphenylglycol (DOPEG), 10%; (d) 3,4-dihydroxymandelic acid (DOMA), 10%; (e) 4-hydroxy-3-methoxyphenylglycol (MOPEG), 14%.3. Field stimulation at 25 shocks/sec with supramaximal stimuli of 1 msec duration increased the outflow of NA six to eightfold and that of NMN, DOPEG and VMA two to threefold. The increase in outflow of DOMA and MOPEG was small. NA represented 35.8 +/- 4.1% of the total increase in radioactivity.4. After pargyline pretreatment field stimulation increased the outflow of NA and NMN. Stimulation in the presence of pyrogallol raised the release of NA, DOMA and DOPEG. Neither pargyline nor pyrogallol affected the total release induced by stimulation.5. Cocaine 0.3 mug/ml. increased the release due to stimulation at 4 shocks/sec but not at 25 shocks/sec. Cocaine did not affect the metabolism of the released transmitter.6. Phenoxybenzamine (10 mug/ml.) increased release by stimulation at 4 and 25 shocks/sec. Metabolism of the released transmitter was prevented in the presence of phenoxybenzamine.7. Phentolamine (3 mug/ml.), like phenoxybenzamine, blocked responses to field stimulation, but failed to modify release and subsequent metabolism of NA liberated by field stimulation.8. The main NA metabolites in the rat vas deferens were DOPEG, DOMA and MOPEG. Stimulation at 4 shocks/sec resulted in an increased outflow of NA and of DOPEG. Fifty per cent of the total increase of radioactive compounds was accounted for by NA metabolites.9. These experiments show that for the calculation of the actual output of transmitter it is important to include the metabolites and not to rely on the determination of (3)HNA alone.