Profound secretory dysfunction can be associated with relatively modest lymphocytic infiltration of the lacrimal and salivary glands of Sjögren's syndrome (SjS) patients. SjS patients' sera contain autoantibodies to M3 muscarinic acetylcholine receptors (MAChR) that have variously been reported to have agonistic and antagonistic effects. We sought to identify consequences of chronic agonist stimulation by maintaining acinar cells from rabbit lacrimal glands for 20 h in the presence or absence of 10 mM carbachol (CCh). Exposure to CCh diminished the cells' ability to elevate cytosolic Ca 2þ and secrete b-hexosaminidase in response to acute stimulation with 100 mM CCh, but it enhanced their secretory responses to phenylephrine and ionomycin. Secretory vesicles appeared normal by electron microscopy, but confocal fluorescence microscopy revealed depletion of the secretory vesicle membrane marker, rab3D, and decreased ability to recruit secretory transport vesicles in response to acute 100 mM CCh. Additionally, the apical cortical actin cytoskeleton was disrupted and diminished compared to the basal-lateral cortical network. Subcellular fractionation analyses revealed that total membrane phase protein content was increased. The contents of b-hexosaminidase and MAChR relative to total protein were not significantly altered, and MAChR abundance in the plasma membrane fraction was increased as the result of redistribution from endomembrane pools. However, relative cellular contents of the heterotrimeric guanosine triphosphate (GTP)-binding proteins, G q and G 11 , were decreased. Additional biochemical changes included decreased contents of 47 kDa G s and G i3 , protein kinase Ca and rab3D and polymeric immunoglobulin (Ig) receptors; internalization of Na,K-ATPase from the plasma membranes to endomembrane compartments and decreased content of b-hexosaminidase in the lysosomes. The observations demonstrate that chronic exposure to a MAChR agonist induces refractoriness to optimal stimulation, without causing receptor downregulation, by downregulating postreceptor-signalling mediators and effectors. The cells' secretory mechanisms for IgA and electrolytes also appear to be impaired, as does their ability to properly sort proteins to the lysosomes.

Chronic muscarinic stimulation induces functional quiescence (Scand J Immunol 2003;58:550-65) 125 I]-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.