The difference of aquaporin 5 distribution in acinar and ductal cells in lacrimal and parotid glands

PURPOSE: This study was designed to clarify the physiological function and tissue distribution of aquaporin 5 (AQP5) in the lacrimal and parotid glands. METHODS: Saliva and tear volumes were compared in AQP5 knockout (AQP5-/-) mice and wild-type mice. Immunohistochemistry and immunoblot analysis were performed in wild-type and AQP5-/- mice. RESULTS: Immunofluorescence of AQP5 staining showed that AQP5 was localized mainly in the ductal cells rather than in the acinar cells of the lacrimal gland. In contrast, in the parotid gland, AQP5 was observed abundantly in acinar cells with undetectable staining in ductal cells. Tear secretion was not changed in AQP5-/- mouse, although saliva secretion was significantly reduced. CONCLUSIONS: AQP5 distribution in acinar cells and ductal cells was completely opposite in the lacrimal and parotid glands. The physiological role of AQP5 might be dependent on the characteristic tissue distribution of the protein in the lacrimal and parotid glands.     

New insight into lacrimal gland function: Role of the duct epithelium in tear secretion

Tear secretion is a complex process with the involvement of the main and accessory lacrimal glands, corneal and conjunctival epithelial cells and the Meibomian glands. The lacrimal gland is the main source of fluid, electrolytes and proteins in tear fluid. Deficient ion and water secretion results in aqueous deficient dry eye with serious consequences on the integrity of the ocular surface. Functions of acinar cells are widely studied, whereas less information is available about the duct system of the lacrimal gland. Secretory mechanisms of duct epithelium may play an important role in tear production, but only limited studies have tried to elucidate the role of the duct system in tear secretion. Significant progress has been made in the past few years, resulting in new insight into lacrimal gland duct function. New experimental techniques were introduced, which contributed to the exploration of the role of lacrimal gland ducts in more detail. Therefore, the aim of this review is to summarize our present knowledge about the role of ducts in lacrimal gland function and tear secretion, which appears to be the first review with a focus on this topic. Short outline of pancreatic and salivary gland duct functions is also given for the purposes of comparison.     

Leakage of aquaporin 5 in the tear of dacryoadenitis mice

PURPOSE: The objective of this study was to investigate whether leakage of aquaporin 5 (AQP5) in tear is associated with damage of lacrimal glands (LGs) in dacryoadenitis models. METHODS: Female MRL/lpr (24-week-old), male NOD/Shi Jci (5-, 8-, and 10-week-old), female NFS/s-TX (10-week-old), and lipopolysaccharide (LPS)-induced dacryoadenitis model mice were used. Tear fluid was collected by a cotton thread. Tear proteins in the thread were dissolved in sodium dodecyl sulfate buffer, and AQP5 proteins were analyzed by the Western blot technique using anti-AQP5 antibody. LGs were prepared for hematoxylin and eosin staining or immunostaining of AQP5. RESULTS: In MRL/lpr, NFS/s-TX, 8- and 10-week-old NOD/Shi Jci mice, AQP5 protein was detected in the tear by Western blot analysis. Inflammatory lymphocyte infiltrations were observed in LGs of these dacryoadenitis model mice. In contrast, AQP5 leakage and damage of LG were not observed in normal mice. In 5-week-old NOD/Shi Jci mice, infiltration was not seen in LG, and AQP5 leakage was not detected in the tear. In LPS-induced dacryoadenitis model mice, either tissue destruction with inflammation in LG or AQP5 leakage in the tear was observed. AQP5 in the tear and tissue inflammation in LGs was not found in control mice. These results indicate that AQP5 is leaked in tears when LGs are damaged by dacryoadenitis. CONCLUSIONS: Leakage of AQP5 in the tear was found to be related to LG damage. This finding suggests that detection of AQP5 in tear is useful for specific diagnosis of LG disorders with tissue destruction.     

Absence of 4 1BB gene function exacerbates lacrimal gland inflammation in autoimmune-prone MRL-Faslpr mice

PURPOSE: To define the role of endogenous 4-1BB (an important T-cell costimulatory molecule) in the regulation of ocular disease, MRL-Fas(lpr) mice deficient in 4-1BB were generated, and their lacrimal gland function was studied. METHODS: 4-1BB(-/-)MRL/MpJ-Tnfrs(lpr)/Tnfrs(lpr) (lpr/4-1BB(-/-)) mice were generated and used at the ninth backcross. Mice were killed at various times, and lacrimal gland cellularity was analyzed by flow cytometry. Tear and tissue samples were analyzed by Western blotting for the presence of aquaporin 5 (AQP5) and 120-kDa fragments of alpha-fodrin. Cytokine expression of lacrimal glands was assessed by flow cytometry and RT-PCR analysis. RESULTS: Absence of the 4-1BB gene function in lpr mice resulted in early and increased infiltration of mononuclear cells into lacrimal glands compared with 4-1BB intact lpr mice. The severity of lesions in lpr/4-1BB(-/-) mice was closely associated with enhanced accumulation of primarily CD4(+) T cells within the lacrimal glands and with increased expression of IL-4. Elevated levels of AQP5 and cleaved 120-kDa fragments of alpha-fodrin were found in tears and lacrimal gland lysates, respectively, of lpr/4-1BB(-/-) but not lpr/4-1BB(+/+) mice. CONCLUSIONS: Deletion of 4-1BB in lpr mice accelerates lacrimal gland lesions through increased CD4(+) T-cell infiltration and their production of immune modulators.     

Role of aquaporin water channels in eye function

The aquaporins (AQPs) are a family of more than 10 homologous water transporting proteins expressed in many mammalian epithelia and endothelia. At least five AQPs are expressed in the eye: AQP0 (MIP) in lens fiber, AQP1 in cornea endothelium, ciliary and lens epithelia and trabecular meshwork, AQP3 in conjunctiva, AQP4 in ciliary epithelium and retinal Müller cells, and AQP5 in corneal and lacrimal gland epithelia. This cell-specific expression pattern suggests involvement of AQPs in corneal and lens transparency, intraocular pressure (IOP) regulation, retinal signal transduction, and tear secretion. Indeed, humans with mutant AQP0 develop cataracts. Mice lacking AQP1 have reduced IOP and impaired corneal transparency after swelling, and mice lacking AQP4 have reduced light-evoked potentials by electroretinography. There is evidence for impaired cellular processing of AQP5 in lacrimal glands of humans with Sjogren's syndrome. AQPs facilitate fluid secretion and absorption in the eye, and hence are involved in the regulation of pressure, volume and tissue hydration. Pharmacological alteration of AQP function may provide a new approach for therapy of glaucoma, corneal edema, and other diseases of the eye associated with abnormalities in IOP or tissue hydration.     

A single injection of interleukin-1 induces reversible aqueous-tear deficiency, lacrimal gland inflammation, and acinar and ductal cell proliferation

Emerging studies from our laboratory demonstrate that interleukin-1 (IL-1) family members play a major role in impairing lacrimal gland functions. Here we have extended our investigations to observe the effects of IL-1 on aqueous tear production, lacrimal gland secretion, lacrimal gland histology, and acinar and ductal cell proliferation. We demonstrate that a single injection of IL-1 into the lacrimal glands inhibited neurally- as well as agonist-induced protein secretion resulting in decreased tear output. Meanwhile, IL-1 injection induced a severe, but reversible (7-13 days), inflammatory response that led to destruction of lacrimal gland acinar epithelial cells. Finally, we demonstrate that as the inflammatory response subsided and lacrimal gland secretion and tear production returned to normal levels, there was increased proliferation of acinar and ductal epithelial cells. Our work uncovers novel effects of IL-1 on lacrimal gland functions and the potential regenerative capacity of the mouse lacrimal gland.     

Fluid transport across cultured layers of corneal endothelium from aquaporin-1 null mice

We explored the role of AQP1, the only known aquaporin in corneal endothelium, on active fluid transport and passive osmotic water movements across corneal endothelial layers cultured from AQP1 null mice and wildtype mice. AQP1 null mice had grossly transparent corneas, just as wildtype mice. Endothelial cell layers grown on permeable supports transported fluid at rates of (in microl h(-1) cm(-2), n = 9 mean+/-s.e.): 4.3+/-0.6, wildtype mice (MCE); 3.5+/-0.6, AQP1 null mice (KMCE; difference not significant). The osmotic water flow (also in microl h(-1) cm(-2)) induced by a 100 mOsm sucrose gradient across MCE cell layers (8.7+/-0.6, n = 8) was significantly greater than that across KMCE (5.7+/-0.7, n = 6, p = 0.007). When plated on glass coverslips, plasma membrane osmotic water permeability determined by light scattering was significantly higher for cells from wildtype vs. AQP1 null mice (in microm sec(-1): 74+/-4, n = 19 vs. 44+/-4 microm sec(-1), n = 11, p < 0.001). Unexpectedly, after 10% hypo-osmotic challenge, the extent of the regulatory volume recovery was significantly reduced for AQP1 null mice cells (in%: MCE controls, 99+/-1, n = 19 vs. KMCE: 64+/-5, n = 11, p < 0.001). Thus, as in other 'low rate' fluid transporting epithelia, deletion of AQP1 in mice corneal endothelium reduces osmotic water permeability but not active transendothelial fluid transport. However, that deletion impaired the extent of regulatory volume decrease after a hypo-osmotic challenge, suggesting a novel role for AQP1 in corneal endothelium.     

Characterization of beta-hexosaminidase secretion in rabbit lacrimal gland

The present study was aimed at validating the use of the lysosomal enzyme beta-hexosaminidase as a marker of secretory function in cultured rabbit lacrimal gland acinar cells. The secretory response and morphological characteristics of isolated acinar cells cultured in a serum-free medium supplemented with an extracellular matrix extract were monitored over time as part of optimization of our culturing protocol. Secreted beta-hexosaminidase activity was analyzed and compared with that of another lysosomal enzyme, cathepsin B, as well as protein secreted into the media, w or w/o the presence of secretagogues or protein kinase C activators and inhibitors. Lacrimal gland fluid was obtained from pilocarpine stimulated rabbits, and the activities of beta-hexosaminidase and cathepsin B were measured. A membrane fraction and a soluble fraction were obtained from isolated acinar cells and used for kinetic studies of beta-hexosaminidase in comparison with that released from cultured cells, in the lacrimal gland fluid and in serum. Optimal secretory response was obtained when the cells had been in culture for 2-3 days, coinciding with the formation of acinus-like structures. Stimulation of the cultured cells by carbachol or phorbol esters resulted in a more than 3-fold increase of beta-hexosaminidase release over basal, whereas no effect on cathepsin B release could be detected. Treatment with the protein kinase C inhibitor, chelerythrine chloride, significantly decreased the carbachol and phorbol ester-stimulated secretion. Cathepsin B could not be detected in rabbit lacrimal fluid, but beta-hexosaminidase was easily measured in quantities corresponding to as low as 0.4 microl of tear fluid. Using 4-methylumbelliferyl N-acetyl-beta-D-glucosaminide as a substrate for beta-hexosaminidase, the K(m) in lacrimal gland fluid (1.22+/-0.15 mM) was not significantly different from that of the membrane-associated fraction, the soluble fraction, rabbit serum or activity secreted from cultured cells. Beta-hexosaminidase is secreted by rabbit lacrimal gland, in vivo, and by acinar cells in primary culture, whereas cathepsin B is not secreted under the conditions described. Beta-hexosaminidase therefore provides a versatile marker for secretion in studies of tear production utilizing the rabbit as a model. Our results also indicate that PKC is an important regulator of rabbit lacrimal gland secretion.     

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