Povidone iodine treatment is deleterious to human ocular surface conjunctival cells in culture.

OBJECTIVE: To determine the effect of povidone iodine (PI), an antiseptic commonly used prior to ocular surgery, on viability of mixed populations of conjunctival stratified squamous and goblet cells, purified conjunctival goblet cells and purified conjunctival stromal fibroblasts in primary culture. METHODS AND ANALYSIS: Mixed population of epithelial cells (stratified squamous and goblet cells), goblet cells and fibroblasts were grown in culture from pieces of human conjunctiva using either supplemented DMEM/F12 or RPMI. Cell type was evaluated by immunofluorescence microscopy. Cells were treated for 5 min with phosphate-buffered saline (PBS); 0.25%, 2.5%, 5% or 10% PI in PBS; or a positive control of 30% H2O2. Cell viability was determined using Alamar Blue fluorescence and a live/dead kit using calcein/AM and ethidium homodimer-1 (EH-1). RESULTS: Mixed populations of epithelial cells, goblet cells and fibroblasts were characterised by immunofluorescence microscopy. As determined with Alamar Blue fluorescence, all concentrations of PI significantly decreased the number of cells from all three preparation types compared with PBS. As determined by calcein/EH-1 viability test, mixed populations of cells and fibroblasts were less sensitive to PI treatment than goblet cells. All concentrations of PI, except for 0.25% used with goblet cells, substantially increased the number of dead cells for all cell populations. The H2O2 control also significantly decreased the number and viability of all three types of cells in both tests. CONCLUSION: We conclude that PI, which is commonly used prior to ocular surgeries, is detrimental to human conjunctival stratified squamous cells, goblet cells and fibroblasts in culture.

Resolvin D2 elevates cAMP to increase intracellular [Ca2+] and stimulate secretion from conjunctival goblet cells.

Under physiologic conditions, conjunctival goblet cells (CGCs) secrete mucins into the tear film to preserve ocular surface homeostasis. Specialized proresolving mediators (SPMs), like resolvins (Rvs), regulate secretion from CGCs and actively terminate inflammation. The purpose of this study was to determine if RvD2 stimulated mucin secretion and to investigate the cellular signaling components. Goblet cells were cultured from rat conjunctiva. Secretion was measured by an enzyme-linked lectin assay, change in intracellular [Ca2+] ([Ca2+]i) using Fura-2, and cellular cAMP levels by ELISA. RvD2 (10-11-10-8 M) stimulated secretion, increased cellular cAMP levels and the [Ca2+]i. RvD2-stimulated increase in [Ca2+]i and secretion was blocked by Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis and the PKA inhibitor N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide dihydrochloride but not by the cAMP exchange protein inhibitor α-[2-(3-chlorophenyl)hydrazinylidene]-5-(1,1-dimethylethyl)-b-oxo-3-isoxazolepropanenitrile. Forskolin, 3-isobutyl-1-methylxanthine, and 8-bromo-cAMP (8-Br-cAMP) increased [Ca2+]i. Increasing cAMP with 8-Br-cAMP inhibited the increase in [Ca2+]i stimulated by the cAMP-independent agonist cholinergic agonist carbachol. In conclusion, RvD2 uses both cellular cAMP and [Ca2+]i to stimulate glycoconjugate secretion from CGCs, but the interaction of cAMP and [Ca2+]i is context dependent. Thus RvD2 likely assists in the maintenance of the mucous layer of the tear film to sustain ocular surface homeostasis and has potential as a novel treatment for dry eye disease.-Botten, N., Hodges, R. R., Li, D., Bair, J. A., Shatos, M. A., Utheim, T. P., Serhan, C. N., Dartt, D. A. Resolvin D2 elevates cAMP to increase intracellular [Ca2+] and stimulate secretion from conjunctival goblet cells.

Lipoxin A4 Counter-regulates Histamine-stimulated Glycoconjugate Secretion in Conjunctival Goblet Cells.

Conjunctival goblet cells synthesize and secrete mucins which play an important role in protecting the ocular surface. Pro-resolution mediators, such as lipoxin A4 (LXA4), are produced during inflammation returning the tissue to homeostasis and are also produced in non-inflamed tissues. The purpose of this study was to determine the actions of LXA4 on cultured human conjunctival goblet cell mucin secretion and increase in intracellular [Ca2+] ([Ca2+]i) and on histamine-stimulated responses. LXA4 increased mucin secretion and [Ca2+]i, and activated ERK1/2 in human goblet cells. Addition of LXA4 before resolvin D1 (RvD1) decreased RvD1 responses though RvD1 did not block LXA4 responses. LXA4 inhibited histamine-stimulated increases in mucin secretion, [Ca2+]i, and ERK1/2 activation through activation of ß-adrenergic receptor kinase 1. We conclude that conjunctival goblet cells respond to LXA4 through the ALX/FPR2 receptor to maintain homeostasis of the ocular surface and regulate histamine responses and could provide a new therapeutic approach for allergic conjunctivitis and dry eye diseases.

Alteration in cellular turnover and progenitor cell population in lacrimal glands from thrombospondin 1-/- mice, a model of dry eye.

The purpose of this study was to investigate the changes that occur in the lacrimal glands (LGs) in female thrombospondin 1 knockout (TSP1-/-) mice, a mouse model of the autoimmune disease Sjogren's syndrome. The LGs of 4, 12, and 24 week-old female TSP1-/- and C57BL/6J (wild type, WT) mice were used. qPCR was performed to measure cytokine expression. To study the architecture, LG sections were stained with hematoxylin and eosin. Cell proliferation was measured using bromo-deoxyuridine and immunohistochemistry. Amount of CD47 and stem cell markers was analyzed by western blot analysis and location by immunofluorescence microscopy. Expression of stem cell transcription factors was performed using Mouse Stem Cell Transcription Factors RT2 Profiler PCR Array. Cytokine levels significantly increased in LGs of 24 week-old TSP1-/- mice while morphological changes were detected at 12 weeks. Proliferation was decreased in 12 week-old TSP1-/- mice. Three transcription factors were overexpressed and eleven underexpressed in TSP1-/- compared to WT LGs. The amount of CD47, Musashi1, and Sox2 was decreased while the amount of ABCG2 was increased in 12 week-old TSP1-/- mice. We conclude that TSP1 is necessary for maintaining normal LG homeostasis. Absence of TSP1 alters cytokine levels and stem cell transcription factors, LG cellular architecture, decreases cell proliferation, and alters amount of stem cell markers.

Goblet cell response after photorefractive keratectomy and laser in situ keratomileusis.

PURPOSE: To determine whether patients without dry eye preoperatively have an altered conjunctival goblet cell density and mucin secretion postoperatively and to explore what factors affect changes in goblet cell density and mucin secretion. SETTING: The former Walter Reed Army Medical Center, Washington, DC, USA. DESIGN: Prospective nonrandomized clinical study. METHODS: Impression cytology was used to determine conjunctival goblet cell density before and 1 week, 1 month, and 3 months after photorefractive keratectomy (PRK) or laser in situ keratomileusis (LASIK). The McMonnies questionnaire, Schirmer test, tear breakup time, corneal sensitivity, rose bengal staining, and computerized videokeratoscopy were also performed to assess tear-film and ocular-surface health. RESULTS: The ratio of goblet cell to total cells changed postoperatively from baseline in both groups (P < .001). The most significant change was a median 29% decrease 1 month postoperatively. However, there were no significant differences between groups over time (P = .772). The ratio of filled goblet cell to total goblet cell did not change significantly over the same time period (P = .128), and there were no significant differences between the PRK group and the LASIK group over time (P = .282). CONCLUSIONS: Patients without apparent dry eye had an altered conjunctival goblet cell population after PRK or LASIK. The conjunctival goblet cell population tended to decrease in the early postoperative period after either surgery and was most affected by preoperative goblet cell density. The changes in the tear film and ocular surface did not seem to affect goblet cell mucin secretion after either procedure. FINANCIAL DISCLOSURE: None of the authors has a financial or proprietary interest in any material or method mentioned.

Role of PKCα activation of Src, PI-3K/AKT, and ERK in EGF-stimulated proliferation of rat and human conjunctival goblet cells.

PURPOSE: To determine the order and components of the signaling pathway utilized by epidermal growth factor (EGF) to stimulate conjunctival goblet cell proliferation. METHODS: Goblet cells from rat bulbar and forniceal conjunctiva and human bulbar conjunctiva were grown in organ culture. Goblet cells (GCs) were serum starved for 24 hours and preincubated with inhibitors for 30 minutes or small interfering RNA (siRNA) for 48 hours prior to addition of EGF. Proliferation was then measured or Western blot analysis was performed using antibodies against phosphorylated protein kinase B (AKT), extracellular signal-regulated kinase 1/2 (ERK1/2), or the non-receptor tyrosine kinase Src. Rat GCs were also incubated with adenoviruses expressing dominant negative protein kinase Cα (DNPKCα) or constitutively activated protein kinase Cα (myrPKCα), and activation of AKT and ERK1/2 was determined by Western blot analysis. RESULTS: Inhibitors of phosphoinositol-3 kinase (PI-3K)/AKT pathway blocked EGF-stimulated ERK1/2 activation and GC proliferation. Inhibitors of EGF-stimulated ERK1/2 activity did not inhibit AKT activation but blocked proliferation. DNPKCα blocked EGF-stimulated activation of AKT and ERK1/2 while myrPKCα increased activation of these kinases. Inhibitors of PI-3K, ERK1/2, and protein kinase C (PKC) blocked myrPKCα-stimulated GC proliferation. EGF and myrPKCα increased phosphorylation of Src, and inhibition of Src with the chemical inhibitor PP1 or siRNA inhibited EGF-stimulated GC proliferation. CONCLUSIONS: We found that EGF activates a major pathway to stimulate goblet cell proliferation. This pathway consists of induction of phospholipase C (PLC)γ to activate PKCα. Active PKCα phosphorylates Src to induce PI-3K to phosphorylate AKT that subsequently activates the ERK1/2 cascade to stimulate goblet cell proliferation.

Effect of VIP on intracellular [Ca2+], extracellular regulated kinase 1/2, and secretion in cultured rat conjunctival goblet cells.

PURPOSE: To determine the intracellular signaling pathways that vasoactive intestinal peptide (VIP) uses to stimulate high molecular weight glycoconjugate secretion from cultured rat conjunctival goblet cells. METHODS: Goblet cells from rat bulbar and forniceal conjunctiva were grown in organ culture. Presence and localization of VIP receptors (VPAC1 and 2) were determined by RT-PCR, immunofluorescence microscopy and Western blot analysis. Intracellular [Ca(2+)] ([Ca(2+)]i) was measured using fura-2. Extracellular signal-regulated kinase (ERK)-1/2 activity was determined by Western blot analysis. High molecular weight glycoconjugate secretion was measured with an enzyme-linked lectin assay on cultured goblet cells that were serum-starved for 2 hours before stimulation with VIP, VPAC1-, or VPAC2-specific agonists. Inhibitors were added 30 minutes prior to VIP. Activation of epidermal growth factor receptor (EGFR) was measured by immunoprecipitation using an antibody against pTyr followed by Western blot analysis with an antibody against EGFR. RESULTS: Both VIP receptors were present in rat conjunctiva and cultured goblet cells. VIP- and VPAC-specific agonists increased [Ca(2+)]i and secretion in a concentration-dependent manner. VIP also increased ERK1/2 activity, VIP-stimulated increase in [Ca(2+)]i. Secretion, but not ERK1/2 activity, was inhibited by the protein kinase A inhibitor, H89. VIP-stimulated secretion was inhibited by siRNA for ERK2 but not by siRNA for EGFR. VIP did not increase the phosphorylation of the EGFR. CONCLUSIONS: In conclusion, in cultured rat conjunctival goblet cells, VPAC1 and 2 receptors are functional. VIP stimulates a cAMP-dependent increase in [Ca(2+)]i and glycoconjugate secretion, but not ERK1/2 activation. VIP does not activate with EGFR.

Effect of histamine on Ca(2+)-dependent signaling pathways in rat conjunctival goblet cells.

PURPOSE: The purpose of this study was to determine the Ca(2+)-dependent cellular signaling pathways used by histamine to stimulate conjunctival goblet cell secretion. METHODS: Cultured rat goblet cells were grown in RPMI 1640. Goblet cell secretion of high molecular weight glycoconjugates was measured by an enzyme-linked lectin assay. Intracellular [Ca(2+)] ([Ca(2+)](i)) was measured by loading cultured cells with the Ca(2+) sensitive dye fura-2. The level of [Ca(2+)](i) was measured using fluorescence microscopy. Extracellular regulated kinase (ERK) 2 was depleted using small interfering RNA (siRNA). RESULTS: Histamine-stimulated conjunctival goblet cell secretion of high molecular weight glycoproteins was blocked by removal of extracellular Ca(2+) and depletion of ERK2 by siRNA. Histamine increase in [Ca(2+)](i) was desensitized by repeated addition of agonist and blocked by a phospholipase C antagonist. Histamine at higher doses increased [Ca(2+)](i) by stimulating influx of extracellular Ca(2+), but at a lower dose released Ca(2+) from intracellular stores. Activation of each histamine receptor subtype (H(1)-H(4)) increased [Ca(2+)](i) and histamine stimulation was blocked by antagonists of each receptor subtype. The H(2) receptor subtype increase in [Ca(2+)](i) was cAMP dependent. CONCLUSIONS: We conclude that histamine activates phospholipase C to release intracellular Ca(2+) that induces the influx of extracellular Ca(2+) and activates ERK1/2 to stimulate conjunctival goblet cell mucous secretion, and that activation of all four histamine receptor subtypes can increase [Ca(2+)](i).

Signaling pathways used by EGF to stimulate conjunctival goblet cell secretion.

The purpose of this study was to identify the signaling pathways that epidermal growth factor (EGF) uses to stimulate mucin secretion from cultured rat conjunctival goblet cells and to compare the pathways used by EGF with those used by the known secretagogue muscarinic, cholinergic agonists. To this end, goblet cells from rat conjunctiva were grown in culture using RPMI media. For immunofluorescence experiments, antibodies against EGF receptor (EGFR) and ERK 2 as well as muscarinic receptors (M(1)AchR, M(2)AchR, and M(3)AchR) were used, and the cells viewed by fluorescence microscopy. Intracellular [Ca(2+)] ([Ca(2+)](i)) was measured using fura 2/AM. Glycoconjugate secretion was determined after cultured goblet cells were preincubated with inhibitors, and then stimulated with EGF or the cholinergic agonist carbachol (Cch). Goblet cell secretion was measured using an enzyme-linked lectin assay with UEA-I or ELISA for MUC5AC. In cultured goblet cells EGF stimulated an increase in [Ca(2+)](i) in a concentration-dependent manner. EGF-stimulated increase in [Ca(2+)](i) was blocked by inhibitors of the EGF receptor and removal of extracellular Ca(2+). Inhibitors against the EGFR and ERK 1/2 blocked EGF-stimulated mucin secretion. In addition, cultured goblet cells expressed M(1)AchR, M(2)AchR, and M(3)AchRs. Cch-stimulated increase in [Ca(2+)](i) was blocked by inhibitors for the M(1)AchRs, matrix metalloproteinases, and EGF receptors. Inhibitors against the EGF receptor and ERK 1/2 also blocked Cch-stimulated mucin secretion. We conclude that in conjunctival goblet cells, EGF itself increases [Ca(2+)](i) and activates ERK 1/2 to stimulate mucin secretion. EGF-stimulated secretion is dependent on extracellular Ca(2+). This mechanism of action is similar to cholinergic agonists that use muscarinic receptors to transactivate the EGF receptor, increase [Ca(2+)](i), and activate ERK 1/2 leading to an increase in mucin secretion.

Effect of biopsy location and size on proliferative capacity of ex vivo expanded conjunctival tissue.

PURPOSE: To evaluate the effect of location and size of biopsy on phenotype and proliferative capacity of cultured rat conjunctival epithelial cells. METHODS: Pieces of conjunctiva were used from six areas: superior and inferior areas of bulbus, fornix, and tarsus of male Sprague-Dawley rats (n = 6). Explants were grown in RPMI 1640 with 10% fetal bovine serum on coverslips for 8 days or assayed for colony-forming efficiency (n = 9). Analysis included immunofluorescence microscopy and outgrowth measurements with ImageJ software. The Mann-Whitney test and Spearman's rank-order correlation test were used. RESULTS: Superior (23.9 ± 2.9-fold growth) and inferior (22.4 ± 1.2-fold growth) forniceal tissues yielded significantly more outgrowth with respect to explant size than superior bulbar (13.4 ± 1.9-fold growth; P < 0.05 and P < 0.01, respectively), inferior bulbar (13.6 ± 1.6-fold growth; P = 0.01 and P < 0.01, respectively), and inferior tarsal tissues (14.0 ± 1.3-fold growth; P = 0.01). Outgrowth size correlated positively with explant size (r(s) = 0.54; P < 0.001), whereas explant size correlated negatively with fold growth (r(s) = 0.36; P < 0.001). Superior forniceal cells displayed higher colony-forming efficiency (3.6% ± 0.9%) than superior bulbar (1.1% ± 0.3%; P < 0.05) and inferior bulbar cells (1.6% ± 0.8%; P < 0.05). Percentage of p63+ and PCNA+ cells correlated positively with explant and outgrowth size. CONCLUSIONS: Small forniceal conjunctival explants grow the most effectively; however, for transplantation purposes, the loss of p63+ and PCNA+ cells with small explants must be considered.

Isolation and characterization of progenitor cells in uninjured, adult rat lacrimal gland.

PURPOSE: The purpose of this study was to investigate the presence of progenitor cells in the uninjured, adult rat lacrimal gland (LG). METHODS: The presence of progenitor cells was examined in LG sections from male rats using antibodies against selected stem cell markers and α-smooth muscle actin (SMA), which marks myoepithelial cells (MECs), by immunofluorescence microscopy (IF). Small, immature cells were isolated after digestion of LG with collagenase and culture in RPMI 1640 for 2 weeks. Immature cells were examined for expression of stem cell markers by IF. Immature cell were grown in neuronal, epithelial, and myoepithelial cell media, and examined by light morphology and IF using antibodies to markers of different cell lineages. RESULTS: In the intact LGs, MECs expressed the stem cell markers nestin, Musashi 1, ABCG2, Pax6, Chx 10, ΔN p63, and Sox 2. All markers colocalized with SMA. Isolated immature cells contained Ki-67, nestin, Musashi 1, Pax 6, and CHX 10. In neuronal media, immature cells differentiated and assumed a neuronal cell morphology expressing neurofilament 200. In media for human corneal endothelial cells, immature cells differentiated, assumed cobblestone morphology, and labeled with the epithelial marker AE1/AE3. In RPMI media immature cells differentiated into cells with MEC-like morphology, and expressed the MEC markers SMA, α-actinin, adenylate cyclase II, and vimentin. CONCLUSIONS: We conclude that uninjured, adult LG contains progenitor cells that may be MECs, which can be isolated and differentiated into multiple lineages.

Application of human persistent fetal vasculature neural progenitors for transplantation in the inner retina.

Persistent fetal vasculature (PFV) is a potentially serious developmental anomaly in human eyes, which results from a failure of the primary vitreous and the hyaloid vascular systems to regress during development. Recent findings from our laboratory indicate that fibrovascular membranes harvested from subjects with PFV contain neural progenitor cells (herein called NPPFV cells). Our studies on successful isolation, culture, and characterization of NPPFV cells have shown that they highly express neuronal progenitor markers (nestin, Pax6, and Ki67) as well as retinal neuronal markers (ß-III-tubulin and Brn3a). In the presence of retinoic acid and neurotrophins, these cells acquire a neural morphological appearance in vitro, including a round soma and extensive neurites, and express mature neuronal markers (ß-III-tubulin and NF200). Further experiments, including real-time qRT-PCR to quantify characteristic gene expression profiles of these cells and Ca(2+) imaging to evaluate the response to stimulation with different neurotransmitters, indicate that NPPFV cells may resemble a more advanced stage of retinal development and show more differentiation toward inner retinal neurons rather than photoreceptors. To explore the potential of inner retinal transplantation, NPPFV cells were transplanted intravitreally into the eyes of adult C57BL/6 mice. Engrafted NPPFV cells survived well in the intraocular environment in presence of rapamycin and some cells migrated into the inner nuclear layer of the retina 1 week posttransplantation. Three weeks after transplantation, NPPFV cells were observed to migrate and integrate in the inner retina. In response to daily intraperitoneal injections of retinoic acid, a portion of transplanted NPPFV cells exhibited retinal ganglion cell-like morphology and expressed mature neuronal markers (ß-III-tubulin and synaptophysin). These findings indicate that fibrovascular membranes from human PFV harbor a population of neuronal progenitors that may be potential candidates for cell-based therapy for degenerative diseases of the inner retina.

Increase of intracellular Ca2+ by purinergic receptors in cultured rat lacrimal gland myoepithelial cells.

PURPOSE: To isolate and characterize cultured myoepithelial cells (MECs) from rat lacrimal gland and determine which purinergic receptor subtypes are present and functional in MECs. METHODS: Rat lacrimal glands were subjected to collagenase digestion, and MECs were grown. RT-PCR was performed for the purinergic receptors P2X(7), P2Y(1), P2Y(11), and P2Y(13) on RNA isolated from the MECs. Immunofluorescence experiments were performed with antibodies against MEC markers and P2X(7), P2Y(1), P2Y(11), and P2Y(13) purinergic receptors. Proteins from MECs were separated using Western blot analysis techniques. In addition, cells were incubated with Fura 2 tetra acetoxymethyl ester, and intracellular [Ca(2+)] ([Ca(2+)](i)) was determined in response to P2 purinergic agonists. RESULTS: MECs expressed the MEC proteins α-smooth muscle actin, vimentin, α-actinin, and adenylyl cyclase II. RT-PCR, Western blot, and immunofluorescence techniques demonstrated the presence of the purinergic receptors P2X(7), P2Y(1), P2Y(11), and P2Y(13). The purinergic agonists ATP, benzoylbenzoyl ATP (BzATP), α,ß methylene ATP, UTP, 2-methylthioATP (MeSATP), and ATPγS increased [Ca(2+)](i). As BzATP binds to the P2X(7) receptor, specific characteristics of this receptor were investigated. Neither inhibitors of P2X(7) receptors nor removal of extracellular Mg(2+) or Ca(2+) had an effect on the BzATP-stimulated increase in [Ca(2+)](i). Repeated applications of BzATP desensitized this response. Inhibitors for P2Y(1), P2Y(11), and P2Y(13) each decreased the BzATP-stimulated increase in [Ca(2+)](i) with the P2Y(1) inhibitor most effective. CONCLUSIONS: MECs can be isolated from rat lacrimal glands, and they express P2X(7), P2Y(1), P2Y(11), and P2Y(13) purinergic receptors. Surprisingly, BzATP binds the P2Y(1) receptor, which is primarily responsible for the BzATP-stimulated increase in [Ca(2+)](i).

Conjunctival goblet cell secretion stimulated by leukotrienes is reduced by resolvins D1 and E1 to promote resolution of inflammation.

The conjunctiva is a mucous membrane that covers the sclera and lines the inside of the eyelids. Throughout the conjunctiva are goblet cells that secrete mucins to protect the eye. Chronic inflammatory diseases such as allergic conjunctivitis and early dry eye lead to increased goblet cell mucin secretion into tears and ocular surface disease. The purpose of this study was to determine the actions of the inflammatory mediators, the leukotrienes and the proresolution resolvins, on secretion from cultured rat and human conjunctival goblet cells. We found that both cysteinyl leukotriene (CysLT) receptors, CysLT(1) and CysLT(2,) were present in rat conjunctiva and in rat and human cultured conjunctival goblet cells. All leukotrienes LTB(4), LTC(4), LTD(4), and LTE(4), as well as PGD(2), stimulated goblet cell secretion in rat goblet cells. LTD(4) and LTE(4) increased the intracellular Ca(2+) concentration ([Ca(2+)](i)), and LTD(4) activated ERK1/2. The CysLT(1) receptor antagonist MK571 significantly decreased LTD(4)-stimulated rat goblet cell secretion and the increase in [Ca(2+)](i). Resolvins D1 (RvD1) and E1 (RvE1) completely reduced LTD(4)-stimulated goblet cell secretion in cultured rat goblet cells. LTD(4)-induced secretion from human goblet cells was blocked by RvD1. RvD1 and RvE1 prevented LTD(4)- and LTE(4)-stimulated increases in [Ca(2+)](i), as well as LTD(4) activation of ERK1/2. We conclude that cysteinyl leukotrienes stimulate conjunctival goblet cell mucous secretion with LTD(4) using the CysLT(1) receptor. Stimulated secretion is terminated by preventing the increase in [Ca(2+)](i) and activation of ERK1/2 by RvD1 and RvE1.

Phospholipase D1, but not D2, regulates protein secretion via Rho/ROCK in a Ras/Raf-independent, MEK-dependent manner in rat lacrimal gland.

PURPOSE: A prior study showed that cholinergic agonists activate phospholipase D (PLD). The purpose of this study was to determine whether cholinergic agonists use the PLD pathway to alter protein secretion and to identify the molecular signaling components of this pathway in rat lacrimal gland acini. METHODS: Rat lacrimal gland acini were isolated by collagenase digestion. Presence and localization of PLD1 and -2 were determined by immunofluorescence and Western blot experiments. Acini were incubated with adenoviruses overnight or the inhibitors 1-butanol, Y-27632, or C3 exotoxin before stimulation with the cholinergic agonist carbachol (Cch, 10(-4) M) for 5 minutes. Western blot analysis was performed for 20 minutes, and protein secretion was measured spectrophotometrically. Activation of ERK, MEK, Pyk2, Ras, and Raf was determined by Western blot analysis. RESULTS: 1-Butanol increased Cch-stimulated protein secretion and decreased ERK activity. Incubation with catalytically inactive PLD1, but not catalytically inactive mutant PLD2 adenovirus, also increased Cch-stimulated protein secretion and decreased ERK activity. Inhibition of Rho with C3 exotoxin and a dominant negative Rho adenovirus and inhibition of ROCK with Y-27632 inhibited Cch-stimulated PLD1 activity, increased protein secretion, and decreased ERK activity. The association of PLD1 and ROCK increased with Cch stimulation, as determined by immunoprecipitation. PMA-stimulated ERK activity was also inhibited by 1-butanol. 1-Butanol had no effect on Cch-stimulated Pyk2, Ras, and Raf activity, but decreased MEK activity. CONCLUSIONS: Cholinergic agonists activate PLD1 through Rho and ROCK, which in turn activate MEK and ERK, which attenuate protein secretion in freshly isolated epithelial cells.

Characterization of P2X7 purinergic receptors and their function in rat lacrimal gland.

PURPOSE: To characterize the effects of P2X(7) purinergic receptors on lacrimal gland function. METHODS: P2X(7) purinergic receptors were identified by RT-PCR, Western blot analysis, and immunofluorescence techniques. Rat lacrimal gland acini were isolated by collagenase digestion. Acini were incubated with the fluorescent indicator molecule fura 2, and [Ca(2+)](i) was measured by a fluorescence imaging system. Protein secretion was measured with a fluorescence assay system. Activation of ERK 1/2 was determined by Western blot analysis with an antibody against phosphorylated (active) ERK 1/2. RESULTS: P2X(7) receptors were present in the lacrimal gland by RT-PCR and Western blot analysis. These receptors were located in the membranes of acinar and ductal cells and the cytoplasm of acinar cells. Activation of P2X(7) receptors with (benzoylbenzoyl)adenosine 5'-triphosphate increased [Ca(2+)](i), peroxidase secretion, and ERK 1/2 activation, each of which was inhibited by the P2X(7) receptor inhibitors Brilliant Blue G or A 438079. CONCLUSIONS: P2X(7) purinergic receptors are present in rat lacrimal gland and when stimulated increase [Ca(2+)](i), protein secretion, and ERK 1/2 activation.

Role of cPKCalpha and nPKCepsilon in EGF-stimulated goblet cell proliferation.

PURPOSE: The authors determined the role of the protein kinase C (PKC) isoforms cPKCalpha and nPKCepsilon in EGF-stimulated proliferation of cultured rat and human conjunctival goblet cells. METHODS: Rat and human conjunctivas were minced, and goblet cells were allowed to grow. Passage 1 cells were serum starved for 24 to 48 hours and were incubated with the PKC inhibitors calphostin C and Gö 6983 (10(-10)-10(-7) M) for 20 minutes before stimulation with EGF (10(-7) M) for 24 hours. The presence and localization of PKC isoforms in cultured rat goblet cells were determined by Western blot analysis and immunofluorescence microscopy, respectively. Cultured rat goblet cells were serum starved and incubated with adenoviruses containing genes for dominant-negative cPKCalpha (Ad DNPKCalpha, 10(4) pfu), dominant-negative nPKCepsilon (Ad DNPKCepsilon, 10(4) pfu), and wild-type cPKCalpha (Ad WTPKCalpha, 10(7) pfu), and proliferation was measured. RESULTS: In rat goblet cells, EGF-stimulated proliferation was completely inhibited by calphostin C, whereas Gö 6983 inhibited proliferation by 53%+/-15%. In human goblet cells, EGF-stimulated proliferation was completely inhibited by calphostin C. PKCalpha, -betaI, -betaII, -delta, -epsilon, -iota/lambda, -theta, -gamma, and -zeta were found in cultured rat goblet cells. Ad DNPKCalpha and Ad DNPKCepsilon inhibited EGF-stimulated proliferation in rat goblet cells by 78%+/-6% and 92%+/-8%, respectively. Incubation with Ad WTPKCalpha alone significantly increased proliferation. CONCLUSIONS: cPKCalpha and nPKCepsilon play key roles in conjunctival goblet cell proliferation.

Stimulatory role of PKCalpha in extracellular regulated kinase 1/2 pathway in conjunctival goblet cell proliferation.

PURPOSE: To determine whether a constitutively active protein kinase C (PKC)-alpha stimulates rat and human conjunctival goblet cell proliferation through activation of ERK 1/2. METHODS: Conjunctivas from rat and human were minced and goblet cells were allowed to grow. Goblet cells were serum starved and incubated with an adenovirus containing a constitutively active form of PKCalpha (Ad-myr-PKCalpha, 1 x 10(7) pfu), EGF (10(-7) M), or both. The location of myrPKCalpha was determined by immunofluorescence microscopy. Cultured goblet cells were preincubated with the PKC inhibitor calphostin C (10(-10)-10(-7) M) or the ERK 1/2 inhibitor U0126 (10(-9)-10(-6) M) before incubation with Ad-myr-PKCalpha. Cell proliferation was measured. RESULTS: Transduction of rat goblet cells with Ad-myr-PKCalpha did not change PKC location compared with nontransduced cells. Incubation with Ad-myr-PKCalpha caused an increase in cell proliferation by 2.5+/-0.3-fold, whereas EGF increased proliferation by 2.1+/-0.2-fold. Simultaneous addition of Ad-myr-PKCalpha and EGF did not further increase proliferation. U0126 inhibited Ad-myr-PKCalpha-stimulated proliferation a maximum of 70%. In human goblet cells, incubation with Ad-myr-PKCalpha caused an increase in cell proliferation by 2.3+/-0.3-fold, whereas EGF increased proliferation by 3.1+/-0.4-fold. Simultaneous addition of Ad-myr-PKCalpha and EGF decreased proliferation compared with either compound alone. Ad-myr-PKCalpha caused ERK 1/2 to translocate to the nucleus in rat and human cells, but the translocation was blocked by U0126. CONCLUSIONS: Activation of PKCalpha alone by inducing phosphorylation of ERK 1/2 and translocating it to the nucleus is necessary and sufficient to cause conjunctival cell proliferation in rat, and probably human, goblet cells.

ERK/p44p42 mitogen-activated protein kinase mediates EGF-stimulated proliferation of conjunctival goblet cells in culture.

PURPOSE: To determine whether activation of the ERK pathway by EGF leads to rat and human goblet cell proliferation. METHODS: The conjunctiva was removed from male Sprague-Dawley rats. Human conjunctiva was removed during ocular surgery. The tissue was minced and goblet cells were grown. The cells were stimulated with EGF (10(-7) M) for 1 and 5 minutes and Western blot analysis was performed with an antibody against phosphorylated EGFR, to measure the activation of the EGF receptor (EGFR). The cells were incubated with EGF (10(-7) M) for 24 hours, and cell proliferation was measured by WST-8. Inhibitors were added either 20 minutes before EGF or 2 hours after. The cells were stimulated with EGF (10(-7) M) for 1 minute to 24 hours. The number of cells expressing phosphorylated ERK (pERK) in the nucleus and Ki-67 was determined by immunofluorescence. RESULTS: EGF increased the activation of EGFR in rat conjunctival goblet cells. EGF-stimulated proliferation was inhibited by the EGFR inhibitor AG1478 and the MEK inhibitor U0126 in rat and human cultured goblet cells. EGF caused the translocation of pERK to the nucleus in a biphasic manner. Inhibition of the second peak with U0126 prevented proliferation. EGF-stimulated goblet cells progressed through the cell cycle expressing pERK in the nucleus. CONCLUSIONS: EGF stimulated human and rat conjunctival goblet cell proliferation by activating the EGFR. EGFR stimulated ERK causing its biphasic translocation to the nucleus. The second peak response is responsible for cell proliferation, but the role of the first peak is not known.

Effect of OPC-12759 on EGF receptor activation, p44/p42 MAPK activity, and secretion in conjunctival goblet cells.

The purpose of the study was to determine if OPC-12759 stimulates secretion from conjunctival goblet cells in culture and if it activates the EGF receptor (EGFR) and p44/p42 mitogen-activated protein kinase (MAPK) to cause mucin secretion. Conjunctival goblet cells were cultured from pieces of male rat conjunctiva. OPC-12759 was added at increasing concentrations and for varying times to the cultured cells. The cholinergic agonist carbachol was used as a positive control. In selected experiments an inhibitor of the EGFR, AG1478, or an inhibitor of the kinase that activates MAPK, U0126, were added before OPC-12759. Goblet cell secretion of high molecular weight glycoconjugates was measured by an enzyme-linked lectin assay using the lectin UEA-1. Activation of the EGFR and MAPK were determined with Western blotting analysis using antibodies specific to the phosphorylated and the total amounts of these proteins. We found that OPC-12759 induced goblet cell secretion in a time- and concentration-dependent manner. Inhibition of the EGFR with AG1478 blocked secretion stimulated by OPC-12759. Inhibition of MAPK with U0126 also blocked secretion stimulated by OPC-12759. OPC-12759 increased the phosphorylation of the EGFR and MAPK in a time-dependent manner. We concluded that OPC-12759 stimulates secretion from cultured conjunctival goblet cells by activating the EGFR, which then induces MAPK activity.