6533b7d6fe1ef96bd1266618

RESEARCH PRODUCT

Stimulation with carbachol alters endomembrane distribution and plasma membrane expression of intracellular proteins in lacrimal acinar cells.

subject

description

The events that lead to Sjogren's autoimmune processes in the lacrimal gland remain poorly understood. The acinar cell's responses to acute cholinergic stimulation include release of secretory products across the apical plasma membrane (apm) and a number of processes related to traffic between endomembrane compartments and the basal-lateral plasma membranes (blm), such as recruitment of Na, K-ATPase, accelerated recycling, and accelerated transcytosis of secretory IgA. We tested the hypothesis that stimulation-induced acceleration of endomembrane traffic is accompanied by changes in compartmentation and increased blm expression of proteins that are normally sequestered in endomembrane compartments. Isolated rabbit lacrimal gland acinar cells were cultured in serum-free media for 2 days. After harvesting, cells were incubated with or without 10 microm carbachol at 37 degrees C for 20 min. Cells were lysed, and lysates were analysed by isopycnic centrifugation on sorbitol gradients. Galactosyltransferase catalytic activity was determined biochemically. Different forms of cathepsin B were detected by Western blotting. Carbachol stimulation decreased the contents of beta-hexosaminidase, alpha-glucosidase, and protein in secretory vesicles and increased them in specific compartments of the trans-Golgi network (ld-tgns). Stimulation also caused levels of galactosyltransferase, preprocathepsin B, and procathepsin B to increase two- to three-fold in the blm as well as increasing in the ld-tgns. Other changes caused by sustained stimulation included: (a) increased levels of protein and procathepsin B in compartments of the lysosomal pathway; (b) changes in the distributions of Rab5 within the endomembrane system; (c) changes in the distribution of Rab6 within the Golgi complex and tgn; (d) decreased expression of acid phosphatase and MHC class II molecules in the blm; and (e) decreased total content of Na,K-ATPase, which appeared to have been selectively depleted from the tgn and blmre. We propose that the normal compartmentation of certain proteins may allow them to remain cryptic, such that they are not subject to central tolerance. Stimulation-induced increases in the levels expressed at the blm or secreted to the interstitium may, therefore, contribute to initiation of local autoimmune responses.