COMPARISON OF THE EFFECTS OF TROPOLONE, BETA-THUJAPLICIN AND U-0521 ON THE EXTRANEURONAL ACCUMULATION OF ISOPRENALINE IN GUINEA-PIG TRACHEALIS SMOOTH-MUSCLE CELLS - QUANTITATIVE FLUORESCENCE HISTOCHEMICAL-STUDY

1. Tracheal segments from guinea-pigs, pretreated with 6-hydroxydopamine, were incubated in isoprenaline for 30 min at 37°C, in the presence or absence of a catechol-O-methyl transferase (COMT) inhibitor, and then washed at 0-2°C. Tissues were prepared for fluorescence histochemistry and accumulated isoprenaline in trachealis smooth muscle cells measured as fluorescence by quantitative microphotometry. 2. Inhibition of COMT with tropolone, β-thujaplicin or U-0521 increased isoprenaline fluorescence in the cells. U-0521 (20 μM-500 μM) increased background fluorescence as did 2000 μM tropolone. Therefore all isoprenaline readings were corrected using readings from control tissues. 3. The concentrations which gave maximum enhancement of fluorescence were 200 μM β-thujaplicin (higher concentrations not studied), 200 μM tropolone and 100 μM U-0521. U-0521 at 500 μM caused less enhancement than at 100 μM, probably due to its weak extraneuronal uptake inhibitory activity. 4. Fluorescence could be measured in the cells after incubation of tissues in 5 μM isoprenaline. 5. U-0521 caused marginally greater enhancement of fluorescence than did tropolone or β-thujaplicin. Analysis of the data for U-0521 indicated a) that the intracellular O-methylation of isoprenaline had a relatively greater influence on fluorescence (accumulated isoprenaline) when tissues were incubated in 5 μM isoprenaline than in higher concentrations, and b) that COMT in the guinea-pig trachealis smooth muscle cells might be fully saturated by 50 μM isoprenaline, but only partially saturated by 5 μM isoprenaline. 6. It is concluded that extraneuronal uptake of isoprenaline in the guinea-pig trachealis smooth muscle cells is followed by subsequent metabolism by COMT and that this is particularly important when tissues are incubated in low concentrations of isoprenaline (5μM). The COMT inhibitor U-0521 at a concentration of 100μM gave optimum enhancement of fluorescence (and therefore presumably of accumulation of isoprenaline) in these cells. © 1979 Springer-Verlag.