Selective antigen-stimulated release of proteolytic activity from rat mast cells

Abstract

Stimulation of rat mast cell suspension from actively sensitized rats with antigen in vitro produced a parallel release of histamine and enzyme, probably proteolytic activity, which releasesp-nitrophenol from anl-tyrosine-p-nitrophenyl ester derivative (TPNE). The histamine and enzyme release correlated with respect to their dependence on antigen concentration, reaction time and inhibition by 2,4-DNP and papaverine. In contrast, more than 50% of total histamine but nearly no enzyme was released by the ionophore A 23.187 and C 48/80 (each ≤1 μg/ml). The enzyme was apparently secreted predominantly in a particular form. It was approximately 50% inactivated by heating for 1 h at 56°C or by incubation for 3 h at 37°C with the chymotrypsin inactivator tosyl-phenylalanine chloromethylketone (TPCK; 2.5×10⁻⁴ M) or for 5 min at 37°C with benzyl sulphonyl fluoride (2.5×10⁻⁴ M), which reacts with SH groups. Heating for 3 min at 100°C destroyed it completely. On the basis of these properties we suggest that the antigen-released enzyme is the known granulabound chymase from rat mast cells.

TPNE was not only a cleavable substrate for the enzymatic activity in the 800g cell supernatant following antigen stimulation, but also a strong inhibitor of the histamine release on administration before antigen (IC₅₀∼10⁻⁶ M). It appears that the same enzyme activity acts initially intracellularly as activator of the histamine secretion and then is subsequently released along with histamine as a further mediator. Extracellularly this enzyme may act as a modulator of inflammatory reactions in type I allergy both locally and systemically.