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E. Evans, L. Chiang, W. Zhang, C. Okamoto, S. Hamm–Alvarez; Polymeric IgA Receptor Trafficking and Secretory Component Secretion in Rabbit Lacrimal Gland Acinar Cells . Invest. Ophthalmol. Vis. Sci. 2006;47(13):5433.
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© ARVO (1962-2015); The Authors (2016-present)
Tear proteins secreted by the lacrimal gland maintain the health of the ocular surface. One abundant tear protein is secretory IgA (sIgA), which is abundant in tear fluid. According to studies in transfected MDCK cells, binding of dimeric IgA to the polymeric IgA receptor (pIgR) at the basolateral membrane promotes secretory trafficking of the complex and release of the extracellular fragment, as cleaved sIgA, at the apical membrane. Unoccupied pIgR can also be cleaved at the apical membrane with the terminal domain released as free secretory component (SC), which is also abundant in tear fluid. Here we investigate the pathways involved in pIgR trafficking and SC secretion in lacrimal gland acinar cells (LGAC).
Rabbit LGAC cultured for 3 days were stimulated with carbachol (100 µM) and media was collected at time points up to 30 min. Some collected media was concentrated and analyzed by Western blotting for SC or the transduced secretory marker, syncollin–GFP. ß–hexosaminidase and bulk protein release were also assayed. Values for all secreted proteins were normalized to cell pellet protein and secretion studies are summarized from n=6 assays. Rab3D, rab11, and pIgR localization were monitored by confocal fluorescence microscopy using appropriate primary and secondary fluorescent antibodies.
Upon carbachol–stimulation, syncollin–GFP and ß–hexosaminidase showed an initial rapid burst of release (0–10 min), a second flat phase of release (10–15 min), and a third rapid burst of release (15–30 min), similar to the first phase. Surprisingly, carbachol–stimulated release of SC showed an initial rapid burst of release similar to that of syncollin–GFP and ß–hexosaminidase (0–10 min), followed by a slower sustained rate of SC release (10–30 min). Bulk protein exhibited a sustained release profile from 0–30 min, showing no initial burst. Confocal fluorescence microscopy revealed that the immunofluorescence associated with subapical pIgR was significantly colocalized with rab11 immunofluorescence associated with apical endosomes, and also with rab3D immunofluorescence associated with secretory vesicles. This is consistent with previous findings that recombinant Rab3D interacts with the cytoplasmic tail of LGAC pIgR.
Besides being trafficked through the transcytotic pathway, the release of SC from unoccupied pIgR in LGAC also appears to be released from pIgR stored in secretory vesicles which is mobilized by carbachol stimulation.
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