Accumulation of Catalytically Active Proteases in Lacrimal Gland Acinar Cell Endosomes During Chronic Ex Vivo Muscarinic Receptor Stimulation

Chronic muscarinic stimulation induces functional quiescence (Scand J Immunol 2003;58:550–65) and alters the traffic of immature cathepsin B (Exp Eye Res 2004;79:665–75) in lacrimal acinar cells. To test whether active proteases aberrantly accumulate in the endosomes, cell samples were cultured 20 h with and without 10‐µm carbachol (CCh), incubated with [125I]‐bovine serum albumin and then lysed and analysed by subcellular fractionation. CCh decreased total cysteine protease and cathepsin S activities in the isolated lysosome, redistributing them to early endocytic and biosynthetic compartments. CCh decreased [125I] accumulation in all compartments of cells loaded in the absence of protease inhibitors; the cysteine protease inhibitor, leupeptin, prevented the endosomal decrease but not the lysosomal decrease. Sodium dodecyl sulphate‐polyacrylamide gel electrophoresis and autoradiography demonstrated [125I]‐labelled proteolytic products in endomembrane compartments of both control and CCh‐stimulated cells, even in the presence of leupeptin, but analysis indicated that CCh increased the amount in endosomes. Two‐dimensional fractionation analyses suggest that the CCh‐induced redistributions result from blocks in traffic to the late endosome from both the early endosome and the trans‐Golgi network. Therefore, we conjecture that chronic muscarinic acetylcholine receptor stimulation leads to aberrant proteolytic processing of autoantigens in endosomes, from whence previously cryptic epitopes may be secreted to the underlying interstitial space.