Mechanism of Histamine Release by Alpha-Chymotrypsin from Isolated Rat Mast Cells

Abstract

Abstract-Alpha-chymotrypsin (CT) was modified chemically and physically by the treatments with diisopropyl fluorophosphatc, L-(1-tosylamide-2-phenyl) ethylchloro methylketone, hydrogen peroxide and heat. After these treatments, CT lost or de creased both the enzymic activity and ability of releasing histamine from rat mast cells. Ca++ was essential for histamine release by CT, while it enhanced only slightly the enzymic activity. Process of histamine release by CT could be separated into two stages: CT-dependent but not Ca--dependent, and Ca--dependcrit but not CT-dependent.The activated state of mast cells produced by CT decayed rapidly at 37'C in the absence of Ca++, but these cells responded to Ca++ by adding CT once again, suggesting reconstitution of cell membrane structure affected by CT. Iso proterenol, epinephrine, prostaglandin E,, and dibutyryl-cyclic AMP (0.01-0.1 mM) did not inhibit release of histamine induced by CT. Neither theophylline (0.01 0.1 mM) alone nor the combinations of these cyclic AMP-active agents with theo phylline inhibited the release of histamine. But, in the presence of papaverine (0.01 0.1 mM) a marked, dose-dependent inhibition was observed. These data suggest that 1) release of histamine by CT from rat mast cells is causally related to its hydrolytic activity, 2) this activity causes a reversible change on mast cell membrane which pro bably facilitates Ca++-influx through the cell membrane, and 3) there are subtle differences among CT, compound 48/80 and antigens concerning the effect of cyclic AMP-active agents in histamine-releasing mechanisms in mast cells. Alpha-chymotrypsin(CT)2 has been known to degranulate or release histamine from isolated (1, 2) or mesentery mast cells (2, 3) of rats. This action of CT has been reported to be influenced by the same factors which have controlled the similar action of compound 48/80, sinomenine and antigens, e.g. Ca", pH, temperature, metabolic inhibitors, oxygen, glucose (2, 3). It therefore seems reasonable to assume that the degranulation or histamine release process evoked by CT is essentially the same as that initiated by these histamine releasing substances. But a question as to whether or not these actions of CT causally relate to its enzymic activity still remains to be solved, despite the fact that much I This work was supported in part by the Ministry of Education of Japan, the Scientific Research Fund (No. 757030). 2 Abbreviations used in this paper are: CT, (r-chymotrypsin; cyclic AMP, adenosine 3',5' cyclic monophosphate; DB-cyclic AMP, N'-2'-O-dibutyryl adenosine 3%5'-cyclic monophos phate; PGE,, prostaglandin E,; DFP, diisopropyl fluorophosphate; TPCK, L-(l-tosylamido 2-phenyl) ethylchloromethylketone; TCA, trichloroacetic acid; ATEE, N-acetyl L-tyrosine ethylester; AT, N-acetyl L-tyrosine: BSA, bovine serum albumin (Fraction V); PS, physiolo gical buffer solution containing NaCI 154 mM, KCI 2.7 mM, CaCI2 0.9 mM and SOrensen phosphate buffer 6.7 mM (pH 7.2).