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L Qian, J Xie, T Nakamura, AK Mircheff; Chronic Carbachol Stimulation Down-regulates Cholinergic Response and Alters Endomembrane Traffic in Rabbit Acinar Cells . Invest. Ophthalmol. Vis. Sci. 2002;43(13):3119.
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Purpose: To determine whether chronic cholinergic stimulation, which might occur in the presence of receptor-activating autoantibodies, alters the spectra of secreted and surface-expressed autoantigens by perturbing traffic between the basal-lateral membrane (blm) and endomembrane compartments. Methods: Isolated lacrimal acinar cell were cultured in serum-free media overnight, then divided into parallel control samples and samples treated with 10 µM carbachol. Both control and treated cells were incubated for further 20 hr, harvested, lysed, and analyzed by isopycnic centrifugation on sorbitol density gradients. Results: Chronic stimulation increased the total content of membrane-associated protein, and it appeared to increase the contents of ß-hexosaminidase and VAMPs in the Golgi complex and in domains of the trans-Golgi network (tgn-svr and tgn-lr) involved in traffic to the secretory vesicles (sv) and pre-lysosomes (preLys), and it decreased the content of ß-hexosaminidase in the lysosomes (Lys). It increased the content of immature cathepsin B in the blm and fractions containing an endocytic compartment with blm-like composition (e-blml) and tgn domains related to the blm (tgn-blmr), decreased the content of an intermediate form of cathepsin B in fractions containing the tgn-svr, tgn-lr, preLys, and Lys, and, similarly, decreased the content of the mature form of cathepsin B in the preLys and Lys. Chronic stimulation decreased levels of M3 receptors, G protein α-subunits Gq, G11, and Gi3, and PKCα in the endomembrane compartments where they were primarily localized. Chronic stimulation also significantly decreased secretory responses to acute stimulation with 100 µM carbachol and 1 µM phorbol-dibutyrate, but it had no effect on secretion induced by 1 µM ionomycin. Conclusion: Chronic stimulation with carbachol: (1) down-regulates intracellular signaling and certain effectors elicited by muscarinic receptor activation; (2) decreases traffic into the lysosomal pathway, apparently by blocking traffic from tgn-lr to prelys; and (3) leads to increased intracellular accumulation, blm expression, and, possibly, basal-lateral secretion of secretory proteins and immature lysosomal proteins. Thus, chronic stimulation may alter the acinar cell’s autoantigenic spectra by increasing blm-expression and basal-lateral secretion of intracellular proteins and by changing the nature of the compartments in which autoantigens are proteolytically processed.
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