Role of cysteine proteases and protease inhibitors in gastric mucosal damage induced by ethanol or ammonia in the rat.

Since recent studies suggest an imbalance betweencathepsin B and its tissue protease inhibitors (PI) in the pathogenesis of acute and chronic diseases, we tested the hypothesis that release of activated cysteine proteases (P) such as cathepsins B, H, and L might play a role in the pathogenesis of gastric hemorrhagic mucosal lesions (HML) induced by ethanol (E) or ammonia (A).Anesthetized rats received 1 ml of 50% E or 1% A solution intragastrically for 1 min during in situ gastric luminal perfusion.Rapid activation and release of cathepsins B, L, and H into the luminal perfusate preceded the formation of HML quantified by planimetry.Mucosal presence and activity of cysteine PI and cathepsin B have also been investigated in the pathogenesis of chemically induced HML.We extracted and partially isolated acid and thermostable inhibitors of cathepsin B in the gastric mucosa, and found rapid inactivation of PI and activation of cathepsin B in the early phase of E-or A-induced HML.Negative correlations were found between P and PI activities by E or A solutions.Both the activation of cathepsins B, L, and H and the development of E-induced HML were prevented by pretreatment with the sulfhydryl alkylator N -ethylmaleimide.These results suggest that cysteine P may be activated in the rat stomach after E or A exposure, and cysteine P may have a role in the pathogenesis of E-or A-induced gastric HML.Endogenous PI may also participate in the mechanisms of gastric mucosal lesions and gastroprotection.( J. Clin.Invest.1996.98:1047-1054.)Key words: thiol proteases and inhibitors • gastric mucosal injury • Helicobacter pylori • gastroprotection Methods Materials.Cathepsin B (EC 3.4.22.1)(from bovine spleen, 22 U/mg protein, 8.substrates, and 4-methoxy-2 naphthylamide-HCl (HCl-MNA) were purchased from Enzyme Systems Products (Livermore, CA).BSA, l -cysteine free base, polyoxyethylene 23 lauryl ether (Brij 35), NEM, EDTA disodium, DMSO, perchloric acid, sodium chloroacetate, sodium phosphate dibasic, potassium phosphate monobasic, and 10% neutral buffered formalin, were obtained from Sigma Chemical Co.(St.Louis, MO).Glacial acetic acid, sodium acetate and ammonium hydroxide were from Fisher Scientific Co. (Springfield, NJ).Pentobarbital sodium (Nembutal R ) was from Abbot Laboratories (North Chicago, IL).Absolute ethanol from Florida Distillers (Lake Alfred,