Chymotryptic activity in perfusates of isolated rat trachea: correlation with mucosal and connective tissue mast cell secretion.

Serine proteinases participate in many inflammatory events in the airway. We therefore screened perfusates of isolated rat tracheas for tryptic, elastolytic, and chymotryptic serine proteinases. Only chymotryptic activity, indicated by hydrolysis of the synthetic substrate N-succinylalanylalanylprolylphenylalanyl p-nitroaniline (AAPF), was consistently detected in these perfusates. Basal levels of chymotryptic activity were not increased significantly by electrical field stimulation (EFS) (mean change +/- SEM: -0.05 +/- 0.05 m o.d. units, n = 4) or by 10(-7) M substance P (SP) (+0.04 +/- 0.02 m o.d. units, n = 14). However, the mean change after the stimuli were jointly administered (0.17 +/- 0.06 m o.d. units, n = 12) was significantly greater than control or after EFS (P = 0.01, one-way ANOVA). The SP + EFS-induced chymotryptic activity was inhibited by PMSF, soybean trypsin inhibitor, and chymostatin and was associated with an increase in histamine concentration and immunoreactivity to rat mast cell proteases (RMCP), indicating that the activity is due to mast cell degranulation. However, the activity was not significantly decreased by pretreating rats with systemic compound 48/80. SP + EFS-induced chymotryptic activity peaked rapidly and was associated with modest histamine release and an immediate peak in immunoreactivity to RMCP II, a marker of mucosal mast cells. Immunoreactivity to RMCP I, a marker of connective tissue mast cells, also increased after SP + EFS, but this immunoreactivity was either delayed or more sustained and did not coincide with the peak of chymotryptic activity. We conclude that in the isolated rat trachea, chymotryptic activity can be used as a measure of mast cell degranulation, and that SP and EFS synergistically degranulate both mucosal and connective tissue populations of tracheal mast cells. These findings emphasize the likely importance of both types of mast cells and their proteinases in neurogenic responses in the trachea.