Alpha-Adrenergic Agonists Stimulate Fluid Secretion in Lacrimal Gland Ducts.

Purpose: The role of adrenergic innervation in the regulation of lacrimal gland (LG) ductal fluid secretion is unknown. The Aim of the present study was to investigate the effect of adrenergic stimulation on fluid secretion in isolated LG duct segments and to study the underlying intracellular mechanisms. Methods: Fluid secretion of isolated mouse LG ducts was measured using video-microscopy. Effect of various adrenergic agonists (norepinephrine, phenylephrine, and isoproterenol) on fluid secretion as well as inhibitory effects of specific antagonists on adrenergic agonist-stimulated secretory response were analyzed. Changes in intracellular Ca2+ level [Ca2+i] were investigated with microfluorometry. Results: Both norepinephrine and phenylephrine initiated a rapid and robust fluid secretory response, whereas isoproterenol did not cause any secretion. Phenylephrine-induced secretion was completely blocked by α1D-adrenergic receptor blocker BMY-7378. The endothelial nitric oxide synthase (eNOS) inhibitor L-NAME or guanylyl cyclase inhibitor ODQ reduced but not completely abolished the phenylephrine-induced fluid secretion, whereas co-administration of Ca2+-chelator BAPTA-AM resulted in a complete blockade. Phenylephrine stimulation induced a small, but statistically significant elevation in [\(Ca_i^{2 + }\)]. Conclusions: Our results prove the direct role of α1-adrenergic stimulation on LG ductal fluid secretion. Lack of isoproterenol-induced fluid secretory response suggests the absence of ß-receptor mediated pathway in mouse LG ducts. Complete blockade of phenylephrine-induced fluid secretion by BMY-7378 and predominant inhibition of the secretory response either by L-NAME or ODQ suggest that α-adrenergic agonists use the NO/cGMP pathway through α1D receptor. Ca2+ signaling independent from NO/cGMP pathway may also play an at least partial role in α-adrenergic induced ductal fluid secretion.

The regulatory role of vasoactive intestinal peptide in lacrimal gland ductal fluid secretion: A new piece of the puzzle in tear production.

Purpose: Vasoactive intestinal peptide (VIP) is an important regulator of lacrimal gland (LG) function although the effect of VIP on ductal fluid secretion is unknown. Therefore, the aim of the present study was to investigate the role of VIP in the regulation of fluid secretion of isolated LG ducts and to analyze the underlying intracellular mechanisms. Methods: LGs from wild-type (WT) and cystic fibrosis transmembrane conductance regulator (CFTR) knockout (KO) mice were used. Immunofluorescence was applied to confirm the presence of VIP receptors termed VPAC1 and VPAC2 in LG duct cells. Ductal fluid secretion evoked by VIP (100 nM) was measured in isolated ducts using videomicroscopy. Intracellular Ca2+ signaling underlying VIP stimulation was investigated with microfluorometry. Results: VIP stimulation resulted in a robust and continuous fluid secretory response in isolated duct segments originated from WT mice. In contrast, CFTR KO ducts exhibited only a weak pulse-like secretion. A small but statistically significant increase was detected in the intracellular Ca2+ level [Ca2+]i during VIP stimulation in the WT and in CFTR KO ducts. VIP-evoked changes in [Ca2+]i did not differ considerably between the WT and CFTR KO ducts. Conclusions: These results suggest the importance of VIP in the regulation of ductal fluid secretion and the determining role of the adenylyl cyclase-cAMP-CFTR route in this process.

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.

Molecular Evidence for Precursors of Sjögren's Foci in Histologically Normal Lacrimal Glands.

Understanding the formation of Sjogren's lymphocytic infiltrates could permit earlier diagnosis and better outcomes. We submitted gene transcript abundances in histologically normal rabbit lacrimal glands to principal component analysis. The analysis identified a cluster of transcripts associated with Sjögren's foci, including messenger RNAs (mRNAs) for C⁻X⁻C motif chemokine ligand 13 (CXCL13) and B-cell activating factor (BAFF), which dominated the major principal component. We interpreted the transcript cluster as the signature of a cluster of integrally functioning cells. Pregnancy and dryness increased the likelihood that the cluster would develop to high levels, but responses were subject to high levels of stochasticity. Analyzing microdissected samples from high- and low-cluster-level glands, we found that certain transcripts, including mRNAs for C⁻C motif chemokine ligand 21 (CCL21), CXCL13, cluster of differentiation 4 (CD4), CD28, CD25, BAFF, and interleukin 18 (IL-18) were significantly more abundant in immune cell clusters (ICs) from the high-cluster-level gland; mRNAs for CCL2, CD25, and IL-1RA were significantly more abundant in acinus-duct axis samples; mRNAs for CCL4, BAFF, IL-6, and IL-10 were more abundant in some acinus-duct samples; cells with high prolactin immunoreactivity were more frequent in interacinar spaces. In conclusion, integrated functional networks comprising Sjögren's infiltrates, such as ICs, acinar cells, ductal cells, and interacinar cells, can form in histologically normal glands, and it is feasible to detect their molecular signatures.

Novel Insight Into the Role of CFTR in Lacrimal Gland Duct Function in Mice.

Purpose: The role of cystic fibrosis transmembrane conductance regulator (CFTR) in lacrimal gland (LG) function has only recently received some attention, mainly from our group. In the present study, we investigated the potential changes of LG pathology, tear secretion, ocular surface integrity, and fluid secretion in isolated LG ducts from CFTR knockout (KO) mice. Methods: Tear production and ocular surface integrity were investigated in anesthetized wild-type (WT) and KO mice using cotton threads and fluorescein staining, respectively. Immunofluorescence was used to localize CFTR protein in the LGs. Ductal fluid secretions evoked by forskolin (10 µM); cell-permeable cAMP analogue (8-bromo cAMP, 100 µM); or carbachol (100 µM) were measured in isolated LG ducts using video-microscopy. Intracellular Ca2+ homeostasis underlying carbachol stimulation was investigated with microfluorometry. Results: Significant decrease in tear secretion and impaired ocular surface integrity were observed in KO mice. Immunofluorescence demonstrated the predominant presence of CFTR protein in the apical membranes of the duct cells from WT mice. Continuous fluid secretion was evoked by forskolin and 8-bromo cAMP in LG ducts from WT mice, while no secretory response was observed in ducts from KO mice. Carbachol caused similar secretory responses in ducts from WT and KO animals without significant differences in cytosolic Ca2+ signaling. Conclusions: Our results suggest the important role of CFTR in LG ductal secretion and in the maintenance of ocular surface integrity, suggesting that CFTR may be a promising target of novel therapeutic approaches in the treatment of dry eye.

Pregnancy probabilistically augments potential precursors to chronic, immune-mediated or autoimmune lacrimal gland infiltrates.

PURPOSE: This study asked whether pregnancy, a risk factor for dry eye disease associated with both chronic, immune-mediated- and autoimmune etiologies, augments development of clusters of coordinately functioning cells (CCFC) that may be precursors to pathological lacrimal gland infiltrates. METHODS: Lacrimal glands were from six virgin- and six term-pregnant rabbits of the same age and environmental exposure history. Seventy-two immune response-related gene transcripts were assayed by real time RT-PCR. Principal component (PC) analysis identified transcript signatures of CCFC contributing negative (⊖) or positive (⊕) PC loadings and determined gland PC projections, which reflect levels of CCFC development. RESULTS: Three CCFC were of interest as potential precursors to pathological infiltrates. CCFC 1⊖ was suggestive of an ectopic lymphoid structure with resting T cells and B cells. CCFC 1⊕ was suggestive of an immune-mediated infiltrate with TH1 cells and mature, cytotoxic B cells. CCFC 2⊖ was suggestive of an ectopic lymphoid structure with activated T cells, mature B cells, germinal center, and plasmacytes. CCFC 4⊖ and CCFC 5⊖ also included plasmacytes. Pregnancy augmented CCFC 1⊖ in some glands; augmented CCFC 1⊕ in others; and augmented CCFC 2⊖, CCFC 4⊖, and CCFC 5⊖ different combinations. CONCLUSIONS: Potential precursors of pathological infiltrates form in the lacrimal glands by the time of sexual maturity. Pregnancy augments lacrimal gland plasmacyte populations, and it can augment development of potential precursors to either chronic, immune-mediated infiltrates or autoimmune infiltrates of various phenotypes. Systemic and strictly local, probabilistic phenomena interact with pregnancy to determine which combinatorial phenotypes are favored.

Rapamycin Eye Drops Suppress Lacrimal Gland Inflammation In a Murine Model of Sjögren's Syndrome.

Purpose: To evaluate the efficacy of topical rapamycin in treating autoimmune dacryoadenitis in a mouse model of Sjögren's syndrome. Methods: We developed rapamycin in a poly(ethylene glycol)-distearoyl phosphatidylethanolamine (PEG-DSPE) micelle formulation to maintain solubility. Rapamycin or PEG-DSPE eye drops (vehicle) were administered in a well-established Sjögren's syndrome disease model, the male nonobese diabetic (NOD) mice, twice daily for 12 weeks starting at 8 weeks of age. Mouse tear fluid was collected and tear Cathepsin S, a putative tear biomarker for Sjögren's syndrome, was measured. Lacrimal glands were retrieved for histological evaluation, and quantitative real-time PCR of genes associated with Sjögren's syndrome pathogenesis. Tear secretion was measured using phenol red threads, and corneal fluorescein staining was used to assess corneal integrity. Results: Lymphocytic infiltration of lacrimal glands from rapamycin-treated mice was significantly (P = 0.0001) reduced by 3.8-fold relative to vehicle-treated mice after 12 weeks of treatment. Rapamycin, but not vehicle, treatment increased tear secretion and decreased corneal fluorescein staining after 12 weeks. In rapamycin-treated mice, Cathepsin S activity was significantly reduced by 3.75-fold in tears (P < 0.0001) and 1.68-fold in lacrimal gland lysates (P = 0.003) relative to vehicle-treated mice. Rapamycin significantly altered the expression of several genes linked to Sjögren's syndrome pathogenesis, including major histocompatibility complex II, TNF-α, IFN-γ, and IL-12a, as well as Akt3, an effector of autophagy. Conclusions: Our findings suggest that topical rapamycin reduces autoimmune-mediated lacrimal gland inflammation while improving ocular surface integrity and tear secretion, and thus has potential for treating Sjögren's syndrome-associated dry eye.

Characterization of Na+-K+-2Cl- Cotransporter Activity in Rabbit Lacrimal Gland Duct Cells.

PURPOSE: We recently reported that isolated duct segments from rabbit lacrimal gland (LG) were able to secrete fluid in response to secretagogues, which were blocked completely by bumetanide. This suggests the functional involvement of Na+-K+-2Cl- cotransporter (NKCC1) in ductal fluid secretion. Therefore, the aim of this study was to investigate the activity profile of NKCC1 in isolated rabbit LG duct segments. METHODS: Interlobular ducts were isolated from fresh rabbit LG tissue. Microfluorometry with the ammonium (NH4+)-pulse technique was used to elicit pH changes in duct cells, and the rate of bumetanide-sensitive cytosolic acidification after addition of NH4+ was used to quantify the activity of NKCC1. RESULTS: While basal activity of NKCC1 was undetectable, low cytosolic chloride (Cl-) level and hyperosmotic challenge (390 mOsm) were able to increase the activity of NKCC1. Carbachol (100 µM) had no significant effect on NKCC1 activity. Elevation of cytosolic calcium (Ca2+) level with Ca2+-ionophore (A 23187, 1 µM) did not cause any alteration in the activity of the cotransporter while direct activation of protein kinase C (phorbol myristate acetate, 100 nM) increased its activity slightly but in a significant manner. Addition of either forskolin (10 µM), cell-permeable cAMP analogue (8-bromo cAMP, 100 µM) or vasoactive intestinal peptide (200 nM) resulted in a significant increase in the activity of NKCC1. CONCLUSIONS: These results highlight the functional involvement of NKCC1 in LG duct secretion. These findings may facilitate our understanding of LG function and may contribute to the development of targeted pharmacologic interventions in case of dry eye disease.

ENaC in the Rabbit Lacrimal Gland and its Changes During Sjögren Syndrome and Pregnancy.

PURPOSE: Epithelial sodium channel (ENaC) plays a critical role in the control of Na(+) balance and the development and progression of exocrine gland pathologic condition. The aim of the present study was to investigate the presence of ENaC in the rabbit lacrimal gland (LG) and its potential changes during induced autoimmune dacryoadenitis (IAD) and pregnancy. METHODS: Total messenger RNA (mRNA) of α, ß, and γ subunits was extracted from whole LG, acinar cells, and ductal cells by laser capture microdissection (LCM) for real-time reverse-transcriptase polymerase chain reaction. Lacrimal glands were processed for Western blot and immunofluorescence. RESULTS: Messenger RNA for both α and γ was expressed in whole LG lysates, whereas ß was undetectable. In rabbits with IAD, the levels of mRNA for α and γ were 20.9% and 58.9% lower (P<0.05), whereas no significant changes were observed in term-pregnant rabbits (P=0.152). However, we were unable to detect mRNA of any subunit in LCM specimens of ductal cells because of their low levels. Western blot demonstrated bands for both α (90 kDa) and γ (85 kDa) but ß was undetectable. In rabbits with IAD, densitometry analysis showed that expression of α decreased 22%, whereas γ decreased 26% (P<0.05). In pregnant rabbits, however, α expression was 31% lower, whereas γ expression was 34% lower (P<0.05). From immunofluorescence studies, all subunits were present in ductal cells, whereas virtually no immunoreactivity was detected in acini. No noticeable changes of their distribution pattern and intensity were found in rabbits with IAD or during pregnancy. CONCLUSIONS: The present study demonstrated the presence of ENaC in the rabbit LG and its alterations in IAD and pregnancy, suggesting that ENaC may contribute to the pathogenesis of altered LG secretion and ocular surface symptoms in these animals.

Potentially pathogenic immune cells and networks in apparently healthy lacrimal glands.

Lacrimal glands of people over 40 years old frequently contain lymphocytic infiltrates. Relationships between histopathological presentation and physiological dysfunction are not straightforward. Data from rabbit studies have suggested that at least two immune cell networks form in healthy lacrimal glands, one responding to environmental dryness, the other to high temperatures. New findings indicate that mRNAs for several chemokines and cytokines are expressed primarily in epithelial cells; certain others are expressed in both epithelial cells and immune cells. Transcript abundances vary substantially across glands from animals that have experienced the same conditions, allowing for correlation analyses, which detect clusters that map to various cell types and to networks of coordinately functioning cells. A core network--expressing mRNAs including IL-1α, IL-6, IL-17A, and IL-10--expands adaptively with exposure to dryness, suppressing IFN-γ, but potentially causing physiological dysfunction. High temperature elicits concurrent increases of mRNAs for prolactin (PRL), CCL21, and IL-18. PRL is associated with crosstalk to IFN-γ, BAFF, and IL-4. The core network reacts to the resulting PRL-BAFF-IL-4 network, creating a profile reminiscent of Sjögren's disease. In a warmer, moderately dry setting, PRL-associated increases of IFN-γ are associated with suppression of IL-10 and augmentations of IL-1α and IL-17, creating a profile reminiscent of severe chronic inflammation.

Proteomic analysis revealed the altered tear protein profile in a rabbit model of Sjögren's syndrome-associated dry eye.

Sjögren's syndrome (SS) is an autoimmune disease that results in pathological dryness of mouth and eye. The diagnosis of SS depends on both clinical evaluation and specific antibodies. The goal of this study was to use quantitative proteomics to investigate changes in tear proteins in a rabbit model of SS-associated dry eye, induced autoimmune dacryoadenitis (IAD). Proteomic analysis was performed by iTRAQ and nano LC-MS/MS on tears collected from the ocular surface, and specific proteins were verified by high resolution MRM. It was found that in the tears of IAD rabbits at 2 and 4 weeks after induction, S100 A6, S100 A9, and serum albumin were upregulated, whereas serotransferrin (TF), prolactin-inducible protein (PIP), polymeric immunoglobulin receptor (pIgR), and Ig gamma chain C region were downregulated. High resolution MRM with mTRAQ labeling verified the changes in S100 A6, TF, PIP, and pIgR. Our results indicated significant changes of tear proteins in IAD rabbits, suggesting these proteins could potentially be used as biomarkers for the diagnosis and prognosis of dry eye. Several of these proteins were also found in the tears of non-SS dry eye patients indicating a common basis of ocular surface pathology, however, pIgR appears to be unique to SS.

Na(+)/K(+)-ATPase expression changes in the rabbit lacrimal glands during pregnancy.

PURPOSE: To investigate the expressional changes of Na(+)/K(+)-ATPase subunits in the lacrimal glands (LG) of term pregnant rabbits. METHODS: LG were obtained from term pregnant rabbits and age-matched female control rabbits for laser capture microdissection (LCM), real time RT-PCR, western blot, and immunofluorescence. The mRNA and proteins of α1, α2, ß1, ß2, and ß3 subunits of Na(+)/K(+)-ATPase were detected and quantified. RESULTS: Although only the mRNA for ß3 from whole LG of pregnant rabbits was significantly different from that of normal controls, many mRNA levels for α1, α2, ß1, ß2, and ß3 from acini and epithelial cells from various duct segments that were collected by LCM were significantly different from those of normal control rabbits. Western blots demonstrated that the expressions of all three ß subunits were significantly higher in pregnant rabbits, while both α subunits remained unchanged during pregnancy. Interestingly, immunofluorescence results showed that the distribution patterns of all Na(+)/K(+)-ATPase subunits during pregnancy were similar to those of the control rabbits. CONCLUSIONS: Changes were found in mRNA and protein expressions of Na(+)/K(+)-ATPase subunits in LG from term pregnant rabbits and these changes suggest a role in the pregnancy-related LG secretion changes and dry eye symptoms observed in these animals.

CFTR-mediated Cl(-) transport in the acinar and duct cells of rabbit lacrimal gland.

PURPOSE: We investigated the role that the cystic fibrosis transmembrane conductance regulator (CFTR) may play in Cl(-) transport in the acinar and ductal epithelial cells of rabbit lacrimal gland (LG). METHODS: Primary cultured LG acinar cells were processed for whole-cell patch-clamp electrophysiological recording of Cl(-) currents by using perfusion media with high and low [Cl(-)], 10 µM forskolin and 100 µM 3-isobutyl-1-methylxanthine (IBMX), the non-specific Cl(-) channel blocker 4,4'-disothiocyanostilbene-2, 2' sulphonic acid (DIDS; 100 µM) and CFTRinh-172 (10 µM), a specific blocker for CFTR. Ex vivo live cell imaging of [Cl(-)] changes in duct cells was performed on freshly dissected LG duct with a multiphoton confocal laser scanning microscope using a Cl(-) sensitive fluorescence dye, N-[ethoxycarbonylmethyl]-6-methoxy-quinolinium bromide. RESULTS: Whole-cell patch-clamp studies demonstrated the presence of Cl(-) current in isolated acinar cells and revealed that this Cl(-) current was mediated by CFTR channel. Live cell imaging also showed the presence of CFTR-mediated Cl(-) transport across the plasma membrane of duct cells. CONCLUSIONS: Our previous data showed the presence of CFTR in all acinar and duct cells within the rabbit LG, with expression most prominent in the apical membranes of duct cells. The present study demonstrates that CFTR is actively involved in Cl(-) transport in both acinar cells and epithelial cells from duct segments, suggesting that CFTR may play a significant role in LG secretion.

Changes of chloride channels in the lacrimal glands of a rabbit model of Sjögren syndrome.

PURPOSE: To test the hypothesis that expressions of Na-K-2Cl cotransporter-1 (NKCC1), cystic fibrosis transmembrane conductance regulator (CFTR), and chloride channel 2 γ subunit (ClC2γ) in the lacrimal glands (LGs) of rabbits with induced autoimmune dacryoadenitis (IAD) are changed. METHODS: LGs were obtained from adult female rabbits with IAD and age-matched female control rabbits. LGs were processed for laser capture microdissection, real-time reverse transcription-polymerase chain reaction, Western blot, and immunofluorescence. RESULTS: In rabbits with IAD, messenger RNA (mRNA) abundance and protein expressions of NKCC1 and CFTR from whole LGs were significantly lower than those in controls. mRNA abundance of NKCC1, CFTR, and ClC2γ from rabbits with IAD was significantly different from that in acinar and ductal cells from controls. NKCC1 was localized to the basolateral membranes of all acinar and ductal cells, with weaker staining intensity in ductal cells, and the staining pattern from rabbits with IAD appeared similar to that from controls. CFTR was found as punctate aggregates in the apical cytoplasm of all acinar and ductal cells, with the intensity in ductal cells much stronger and no significant difference between controls and rabbits with IAD. ClC2γ was also localized to the apical cytoplasm as punctate aggregates of all acinar cells but not in ductal cells, and a similar staining pattern was observed in rabbits with IAD compared with control rabbits. CONCLUSIONS: Our data demonstrated significant changes of mRNA and protein expressions of NKCC1, CFTR, and ClC2γ in rabbits with IAD, suggesting that these changes may contribute to the altered lacrimal secretion, particularly Cl transport, in rabbits with IAD.

Changes of the ocular surface and aquaporins in the lacrimal glands of rabbits during pregnancy.

PURPOSE: To test the hypotheses that pregnancy represents a physiologic condition that is associated with dry eye symptoms, and the expression of aquaporin 4 (AQP4) and AQP5 are altered in the lacrimal gland (LG) from term pregnant rabbits. METHODS: Schirmer's test, tear break-up time (BUT), and Rose Bengal staining were used to evaluate ocular surface health. LG were obtained from term pregnant rabbits and age-matched female control rabbits and then processed for laser capture microdissection (LCM), real time RT-PCR, western blot, and immunofluorescence for the detection and quantification of mRNA and proteins of AQP4 and AQP5. RESULTS: Pregnant rabbits demonstrated typical clinical symptoms of dry eye, including decreased Schirmer score and BUT as well as increased Rose Bengal staining of cornea. In term pregnant rabbits, mRNA for AQP5 from whole LG was significantly lower than that of control rabbits, while mRNA for AQP4 was not. Levels of mRNA for AQP4 and AQP5 underwent significant changes in acini and epithelial cells from specific duct segments during pregnancy. Western blot from whole LG lysates demonstrated that expression of AQP4 was 24% more abundant in term pregnant rabbits while AQP5 was 22% less when compared to control rabbits respectively. At term pregnancy, AQP4 immunoreactivity (AQP4-IR) was increased in acini while its intensity remained the same in ducts. AQP5-IR was present in both apical and basolateral membranes of acinar cells in normal control and pregnant rabbits, while ductal cells in pregnant rabbits also showed significant amount of AQP5-IR. CONCLUSIONS: The data presented here demonstrated significant dry eye symptoms in pregnant rabbits. Our data also showed altered expressions of AQP4 and AQP5 during pregnancy and suggested that these changes may contribute to the altered LG secretion and dry eye symptoms during pregnancy.

Not all lacrimal epithelial cells are created equal-heterogeneity of the rabbit lacrimal gland and differential secretion.

AIMS: To test the hypotheses that some epithelial cells in the rabbit lacrimal gland (LG) are mucin-secreting cells that are also particularly rich in aquaporin 5 (AQP5) and sodium potassium ATPase ß(1) subunit (NKAß(1)), LG-secreted mucins contribute to the total mucin pool in tear film, and that the rabbit LG is a heterogenic gland where proteins secreted in response to different agonists are varied. MATERIALS AND METHODS: LGs were obtained from adult female rabbits and processed for paraffin sections for hematoxylin and eosin (HE) staining, periodic acid-Schiff (PAS), mucicarmine, and Alcian blue (pH 0.4, 1.0, and 2.5) for the detection of mucins. Serial sections were used for immunohistochemistry (IHC) and PAS. LG lysates and fluids were assayed by dot blot for detection of mucins, and by SDS-PAGE to detect differences in protein profiles of LG fluids stimulated by different agonists. RESULTS: HE staining demonstrated that the LG is a heterogeneous gland where most epithelial cells are serous, while all duct cells are mucous cells. Some acini and individual acinar cells within serous acini are also mucous or seromucous cells and these cells are particularly rich in AQP5 and NKAß(1). Dot blot assay showed the presence of mucins in the LG fluids. The protein profiles of LG fluids from pilocarpine, phenylephrine, and isoproterenol varied significantly, particularly in the mid range. CONCLUSIONS: Our data indicated that the rabbit LG is a heterogeneous gland that is composed of both serous and mucin-secreting cells, and mucins produced by the LG contribute to the mucin pool in the tear film. The heterogeneity of the rabbit LG supports the notion of differential secretion, i.e. the volume and composition of the LG fluids vary depending on various circumstances in the ocular surface and the body's needs.

Na(+)/K(+)-ATPase in the lacrimal glands of rabbits and its changes during induced autoimmune dacryoadenitis.

PURPOSE: To test the hypothesis that expression of Na(+)/K(+)-ATPase subunits in the lacrimal glands (LGs) of rabbits with induced autoimmune dacryoadenitis (IAD) changes. METHODS: LGs were obtained from adult female rabbits with IAD and age-matched female control rabbits. The LGs were processed for laser capture microdissection (LCM), real time RT-PCR, western blot, and immunofluorescence for the detection of mRNA and proteins of the α1, α2, ß1, ß2, and ß3 subunits of Na(+)/K(+)-ATPase. RESULTS: In the rabbits with IAD, mRNA levels of α1, ß1, and ß3 from whole LGs were significantly lower. In samples of acini and epithelial cells from various duct segments, collected by LCM, mRNA levels of α1, ß1, ß2, and ß3 were significantly lower in the rabbits with IAD, although mRNA for α2 could not be detected. However, western blots demonstrated that all five subunits were significantly higher in the rabbits with IAD, although their distribution patterns were similar to those of the control rabbits, as demonstrated by immunofluorescence. CONCLUSIONS: The data presented herein demonstrated significant changes in mRNA and protein expressions of Na(+)/K(+)-ATPase subunits in rabbits with IAD, suggesting that these changes may play a role in the pathogenesis of Sjögren's syndrome and altered LG secretion, as observed in these animals.

Changes of aquaporins in the lacrimal glands of a rabbit model of Sjögren's syndrome.

AIMS: To test the hypothesis that the expression of aquaporins (AQPs) 4 and 5 is altered in the lacrimal glands (LG) of rabbits with induced autoimmune dacryoadenitis (IAD). MATERIALS AND METHODS: LGs were obtained from adult female rabbits with IAD, and age-matched female control rabbits. LGs were processed for laser capture microdissection (LCM), real time RT-PCR, Western blot, and immunofluorescence for the detection and quantification of protein and mRNAs of AQP4 and AQP5 in whole LGs, and purified acinar cells and duct cells from specific duct segments. RESULTS: In rabbits with IAD, abundances of mRNAs for AQP4 and AQP5 from whole LGs were significantly lower than controls. Levels of mRNA for AQP4 were lower in most duct segments from rabbits with IAD. However, the mRNA abundance for AQP5 was significantly lower in acini from rabbits with IAD, while its abundance was higher in each duct segment. Western blot showed that the expression of AQP4 in LGs from rabbits with IAD was 36% more abundant than normal controls, whereas AQP5 was 72% less abundant. Immunofluorescence indicated that AQP4 immunoreactivity (AQP4-IR) was present on the basolateral membranes of acinar and ductal cells in control and diseased LGs, with ductal cells showing stronger AQP4-IR than acinar cells. AQP5-IR was found on apical and basolateral membranes of acinar cells, and showed a "mosaic" pattern, i.e., with some acini and/or acinar cells showing stronger AQP5-IR than others. Minimal AQP5-IR was detected in ductal cells from control animals, while its intensity was significantly increased in rabbits with IAD. CONCLUSIONS: These data strongly support our hypothesis that expressions of AQPs are altered in rabbits with IAD, and that specific ductal segment play important roles in lacrimal secretion.

Duct system of the rabbit lacrimal gland: structural characteristics and role in lacrimal secretion.

PURPOSE: To develop a nomenclature for the lacrimal duct system in the rabbit, based on the anatomic and structural characteristics of each duct segment, and to provide RT-PCR and immunofluorescence data to support the notion that the duct system plays important roles in lacrimal function. METHODS: Paraffin-embedded lacrimal glands (LGs) were stained with hematoxylin and eosin (H&E) and evaluated with a stereomicroscope. Cryosections of LG were stained with cresyl violet, and acinar cells and ductal epithelial cells were isolated from each duct segment by laser capture microdissection (LCM). mRNA levels from these cells were analyzed by real-time RT-PCR. Standard protocol was followed for immunofluorescence detection of ionic transporters. RESULTS: The lacrimal duct system was divided into six segments on the basis of morphologic characteristics: the intercalated, intralobular, interlobular, intralobar, interlobar, and main excretory ducts. Although the morphologic features change incrementally along the entire duct system, the gene expression of ionic transporters and aquaporins, including AE3, AQP4, AQP5, CFTR, ClC2gamma, KCC1, NHE1, NKAalpha1, NKAbeta1, NKAbeta2, NKAbeta3, and NKCC1 varied greatly among duct segments. Immunofluorescence results were generally in accordance with the abundance of mRNAs along the acinus-duct axis. CONCLUSIONS: Most LG research has focused on the acinar cells, with relatively little attention being paid to the lacrimal ducts. The lack of knowledge regarding the lacrimal ducts was so profound that a precise nomenclature had not been established for the duct system. The present data establish a nomenclature for each segment of the lacrimal duct system and provide evidence that ducts play critical roles in lacrimal secretion.