Biochemical Changes Contributing to Functional Quiescence in Lacrimal Gland Acinar Cells after Chronic Ex Vivo Exposure to a Muscarinic Agonist

Abstract

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.

Diverse Mediators Modulate the Chloride Ion Fluxes That Drive Lacrimal Fluid Production

“…I SC then returned to its baseline value. Thus, chronic exposure to 10 lM CCh, which almost completely prevents an optimal dose of CCh from elevating Ca 2þ or eliciting protein secretion, 23 also appears to prevent the optimal dose from eliciting Cl À secretion.…”

“…Prolonged exposure to 10 lM CCh does not disrupt the integrity of the epithelial monolayers, but it causes cytopathologic changes 23,24 ; it alters membrane vesicle-mediated intracellular traffic in a way that might alter the proteolytic processing of potential autoantigens 53 ; and it significantly decreases cytosolic Ca 2þ elevation, protein secretion, 23,24 and Cl À secretion (Fig. 3) in response to an optimal dose of CCh.…”

“…21,22 Exposure to mediators that are not cytokines but, rather, G protein-coupled receptor agonists, can also interfere with cholinergically induced Ca 2þ elevation and exocrine b-hexosaminidase secretion. These include the muscarinic cholinergic agonist, carbachol (CCh), at a submaximal dose, 23 and the biogenic amines, histamine, and serotonin (5-HT). 24 The mechanisms of fluid secretion are largely distinct from the mechanism of merocrine protein secretion.…”

Elevated prolactin redirects secretory vesicle traffic in rabbit lacrimal acinar cells

“…Secretion assays were performed as described previously (29). Cells in Matrigel rafts were washed and resuspended in fresh medium.…”

Sensory Denervation Modulates eIF-2 Alpha Kinase Expression in the Rabbit Lacrimal Gland

“…26,27 Failure to release secretory granules may interfere with the control of protein synthesis, nascent granule formation, vesicular trafficking, and targeting. [28][29][30][31] Using differential mRNA display, we identified a gene, HRI eIF-2α kinase, the expression of which was significantly reduced after trigeminal ablation as shown by Northern and Western blot analysis.…”