The extraneuronal O-methylation of ³H-(±)isoprenaline by guinea-pig tracheal rings in vitro

Summary

Tracheal rings isolated from male guinea-pigs and incubated in Krebs' solution at 37° C O-methylated ³H-(±)isoprenaline by a saturable, high affinity mechanism.

• 1. With ³H-isoprenaline at 1 μmol·l⁻¹, O-methyl ³H-(±)isoprenaline (³H-OMI) appeared in the tissue with a half-time for approach to steady state of approximately 10 min and was measured in the incubation medium after about 5 min, its concentration increasing linearly thereafter. With ³H-isoprenaline concentrations ranging from 1 to 200 μmol·l⁻¹, the total formation of ³H-OMI (estimated from that contained in the tissue and the medium) was maintained at steady state rates for up to 60 min, after initial lag times of between 1 and 3 min. O-methylation obeyed Michaelis-Menten saturation kinetics; K _m=6.14±0.13 μmol·l⁻¹, V _max=0.31±0.01 nmol·g⁻¹·min⁻¹.

• 2. The catechol-O-methyl transferase (COMT) inhibitor U-O521 and the extraneuronal uptake inhibitor corticosterone both reduced O-methylation of ³H-isoprenaline (0.1 μmol·l⁻¹) by tracheal rings. However, U-O521 was fully inhibitory (IC₅₀=2.6 μmol·l⁻¹), but corticosterone inhibited by only 46% at concentrations up to 1 mmol·l⁻¹.

• 3. The O-methylating activity of the “smooth muscle-rich” component of the trachea was approximately three-times greater than for complete tracheal rings. However, considerable activity was also associated with “cartilage-rich”, “smooth muscle-poor” sections. This activity did not seem to be associated with endothelial cells.

• 4. Histamine strongly inhibited O-methylation (IC₅₀=30 μmol·l⁻¹), but two other contractile agonists, 5-hydroxytryptamine and bethanechol, were weakly active and inactive, respectively.