Chronic Muscarinic Receptor Stimulation Impairs Constitutive-, Regulated-, and Recruitable Secretory Pathways and Alters Actin Microfilament Organization in Lacrimal Acinar Cells

Abstract

Abstract: : Purpose: In Sjögren's syndrome (SjS), lacrimal glands can be nonfunctional even though retaining a large volume of acinar epithelium. SjS sera contain activating autoantibodies to the M3 acetylcholine receptor (M3AChR). However, contrary expectations for chronic stimulation, up-regulation of labial salivary gland M3AChR has been reported in SjS. To test the hypothesis that chronic stimulation down-regulates downstream mediators and effectors of M3AChR signaling, we maintained rabbit lacrimal gland acinar cells overnight in the presence of a half-maximally stimulating concentration of a cholinergic agonist. Methods: After a 24 h - 48 h equilibration period of under conditions favoring formation of reconstituted acini, acinar cells were cultured an additional 20 h with and without 10 µM carbachol (CCh), then analyzed by subcellular fractionation or confocal immunofluorescence microscopy. MAChR were detected with the high affinity ligand, [³H]-QNB. Results: As reported previously (Qian et al., ARVO 2002), overnight treatment with CCh increased basal rates of ß-hexosaminidase secretion modestly but made cells almost completely refractory to stimulation with CCh at the normally optimal concentration of 100 µM or with phorbol dibutyrate. Confocal microscopy revealed that treated cells failed to recruit VAMP2- and dynein-enriched secretory transport vesicles from the trans-Golgi network in response to 100 µM CCh. Rab3D, normally highly enriched in mature secretory vesicles, was largely absent from the apical cytoplasm. Apical actin staining was depleted, but basal-lateral actin staining was enhanced. Analytical fractionation revealed that total cellular MAChR content was not altered, but MAChR were recruited to the basal-lateral membranes (blm) from their major endomembrane pool. While total Na,K-ATPase content was not altered, the pumps were redistributed from the blm to late endocytic compartments. Total content of polymeric immunoglobin receptors was decreased by 50% and Rab3D by 80%. As reported previously, total contents of G_q, G₁₁, G_i3, G_s, and protein kinase Cα were decreased by 20% - 50%. Conclusions: Chronic MAChR activation, as might be caused by activating autoantibodies, causes a pervasive functional quiescence affecting signaling mediators as well as effectors of regulated and recruitable protein secretion, transcytotic IgA secretion, and transepithelial Na⁺ and Cl^- secretion.

Keywords: autoimmune disease • cytoskeleton • receptors: pharmacology/physiology