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Darlene A Dartt

Publications and source records attributed to Darlene A Dartt.

At least 19 recordsLinked to original sources
PubMed2007

Lymphocytic infiltration and goblet cell marker alteration in the conjunctiva of the MRL/MpJ-Fas(lpr) mouse model of Sjögren's syndrome.

Sjögren's syndrome is an autoimmune disorder characterized by a progressive, immune-mediated destruction of mucosal tissues such as the lacrimal and salivary glands, leading to ocular and oral dryness. The MRL/MpJ-Fas(lpr) mouse is one of the animal models used to study this disease. However, little is known about the potential alterations in the conjunctiva in this murine model. The purpose of this study was to determine: (1) whether the conjunctiva is infiltrated by T lymphocytes, (2) characterize the type, amount and temporal sequence of the inflammatory infiltrates, and (3) investigate whether the amount of conjunctival goblet cells is altered in this murine model of Sjögren's syndrome. Female 4-, 9-, 13-, 16-, and 18-/20-wk-old MRL/MpJ-Fas(lpr) (lpr, diseased) and congenic MRL/MpJ (+/+, control) mice were used. Right eyes were either fixed, frozen, cryosectioned, and studied by immunofluorescence microscopy or the conjunctiva was removed, homogenized and analyzed by electrophoresis and Western blot analysis. The following antibodies were used: anti-CD3 (specific T lymphocyte marker), anti-cytokeratin 7 (CK-7), anti-PKD (formerly known as PKCmu, both markers of goblet cell bodies), anti-PGP 9.5 (pan-neuronal marker), anti-VIP and TH (markers for parasympathetic and sympathetic nerves, respectively), anti-adrenergic (alpha(1) and beta(1-3)) and muscarinic (M(1)-M(3)) receptor subtypes (markers for neurotransmitter receptors of the sympathetic and parasympathetic pathways, respectively). Left eyes were fixed, embedded, sectioned, and stained. Hematoxylin/eosin, Giemsa, or alcian blue/periodic acid Schiff's reagent were used to study lymphocyte infiltration; to determine the presence of eosinophils, neutrophils, and mast cells; and to count the number of goblet cells, respectively. By immunofluorescence microscopy, lymphocytes were detected in the conjunctiva of 9-wk-old lpr, but not +/+, mice. The lymphocytic infiltration became more extensive as the animals aged, with 16- and 18-/20-wk lpr mice appearing to have a greater lymphocytic infiltration than +/+ mice at the same age. By Western blot analysis, the amount of CD3 was enhanced in lpr compared to +/+ mice by the 16th wk, but not by the 9th wk. No major differences in the presence of eosinophils, neutrophils and degranulated mast cells between lpr and +/+ mice were observed. By light microscopy, a significant increase in goblet cell number was found in lpr mice compared to +/+ mice at 16 wks on. By Western blotting, the amount of CK-7 was significantly increased at 9 wks on and the amount of PKD was significantly increased at 16 wks. By immunofluorescence microscopy, there were no major differences in distribution of sympathetic and parasympathetic nerves present in the lpr conjunctiva compared to that of +/+ mice at any ages, although slight differences were observed with increased age. Muscarinic receptor expression was decreased, as less M(3) receptor subtype-associated immunofluorescence was detected in older lpr mice compared to +/+ mice and confirmed by Western blot analysis. No differences in the localization or the amount of alpha(1)- or beta(1-3)-adrenergic receptor immunodetection were observed between lpr and +/+ mice. We conclude that the conjunctiva is a target tissue in Sjögren's syndrome-related inflammation in this murine model.

Animals↗
PubMed2006

Effects of alpha1D-adrenergic receptors on shedding of biologically active EGF in freshly isolated lacrimal gland epithelial cells.

Transactivation of EGF receptors by G protein-coupled receptors is a well-known phenomenon. This process involves the ectodomain shedding of growth factors in the EGF family by matrix metalloproteinases. However, many of these studies employ transformed and/or cultured cells that overexpress labeled growth factors. In addition, few studies have shown that EGF itself is the growth factor that is shed and is responsible for transactivation of the EGF receptor. In this study, we show that freshly isolated, nontransformed lacrimal gland acini express two of the three known alpha(1)-adrenergic receptors (ARs), namely, alpha(1B)- and alpha(1D)-ARs. Alpha(1D)-ARs mediate phenylephrine (an alpha(1)-adrenergic agonist)-induced protein secretion and activation of p42/p44 MAPK, because the alpha(1D)-AR inhibitor BMY-7378, but not the alpha(1A)-AR inhibitor 5-methylurapidil, inhibits these processes. Activation of p42/p44 MAPK occurs through transactivation of the EGF receptor, which is inhibited by the matrix metalloproteinase ADAM17 inhibitor TAPI-1. In addition, phenylephrine caused the shedding of EGF from freshly isolated acini into the buffer. Incubation of freshly isolated cells with conditioned buffer from cells treated with phenylephrine resulted in activation of the EGF receptor and p42/p44 MAPK. The EGF receptor inhibitor AG1478 and an EGF-neutralizing antibody blocked this activation of p42/p44 MAPK. We conclude that in freshly isolated lacrimal gland acini, alpha(1)-adrenergic agonists activate the alpha(1D)-AR to stimulate protein secretion and the ectodomain shedding of EGF to transactivate the EGF receptor, potentially via ADAM17, which activates p42/p44 MAPK to negatively modulate protein secretion.

Adrenergic alpha-1 Receptor Agonists↗
PubMed2006

OPC-12759 increases proliferation of cultured rat conjunctival goblet cells.

PURPOSE: To determine if the gastroprotective drug OPC-12759 increased proliferation of rat conjunctival goblet cells in culture. METHODS: Cultured goblet cells were incubated with 10(-12) to 10(-8) M OPC-12759 for 1 to 7 days. Fetal bovine serum (FBS) was used as a positive control. Cell proliferation was determined by a MTT [3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide] colorimetric assay and by immunohistochemical staining with anti-Ki-67, a marker of cell division. Goblet cells were identified by double-labeling with anti-Ki-67, a marker of cell division, and Ulex europaeus agglutinin I lectin, anti-MUC5AC and anticytokeratin 7. Stratified squamous cells were identified by using Griffonia (Bandeiraea) simplicifolia lectin and anticytokeratin 4 antibody. RESULTS: As determined by MTT conversion to formazan, OPC-12579 at 10(-11) M induced an almost 2-fold increase in goblet cell proliferation on Days 1 and 3 of incubation but not on Days 5 and 7. The FBS at 10% increased cell proliferation by 2- to 3-fold at each time point. Daily replenishment of OPC-12579 for 3 consecutive days induced cell proliferation at all concentrations. Proliferation as determined by the number of Ki-67 positive cells increased by 4- and 3-fold at Days 1 and 3, respectively with addition of 10(-11) M OPC-12579. The FBS at 10% induced a 10-fold increase in goblet cell proliferation on Days 1, 3, and 5. Colocalization of Ulex europaeus agglutinin I, MUC5AC and anticytokeratin 7 with Ki-67 indicated that proliferating cells were goblet cells. Proliferating cells were negative for the nongoblet cell markers Bandeiraea lectin and anticytokeratin 4. CONCLUSIONS: The OPC-12759 stimulates proliferation of conjunctival goblet cells in primary culture.

Alanine↗
PubMed2006

Roles of protein kinase C, Ca2+, Pyk2, and c-Src in agonist activation of rat lacrimal gland p42/p44 MAPK.

PURPOSE: Although p42/p44 mitogen-activated protein kinase (MAPK) negatively modulates protein secretion stimulated by cholinergic and alpha(1D)-adrenergic agonists, it does not play a role in epidermal growth factor (EGF)-stimulated protein secretion. Therefore, this study was conducted to determine the roles that protein kinase C (PKC), intracellular Ca(2+) ([Ca(2+)](i)), and nonreceptor tyrosine kinases Pyk2 and Src play in the activation of agonist- and EGF-stimulated MAPK activation. METHODS: Lacrimal gland acini were isolated by collagenase digestion and incubated with phorbol 12-myristate 13-acetate (PMA) to activate PKC or ionomycin, a Ca(2+) ionophore. Acini were preincubated with the PKC inhibitors calphostin C or Ro-31-8220, EGTA to chelate Ca(2+), or the c-Src inhibitor PP1 before stimulation with the cholinergic agonist carbachol, the alpha(1D)-adrenergic agonist phenylephrine, or EGF. Activated MAPK, Pyk2, and c-Src amounts were measured by Western blot analysis. RESULTS: PMA and ionomycin significantly increased the activation of MAPK in a time- and concentration-dependent manner. Inhibition of PKC partially inhibited carbachol-stimulated MAPK activation while completely inhibiting phenylephrine- and EGF-stimulated MAPK activation. Chelation of Ca(2+) also partially inhibited carbachol-stimulated MAPK with no effect on phenylephrine- and EGF-stimulated MAPK activation. Carbachol increased the phosphorylation of Pyk2 on tyrosine 402 and c-src on tyrosine 416 in a time-dependent manner. The c-src inhibitor PP1 inhibited carbachol-stimulated phosphorylation of Pyk2. CONCLUSIONS: It was concluded that cholinergic agonists use Ca(2+) and PKC to phosphorylate Pyk2 and c-Src, which subsequently stimulate MAPK activity. In contrast, alpha(1D)-adrenergic agonists and EGF do not use Pyk2 and Src but do use PKC to activate MAPK.

Adrenergic alpha-Agonists↗
PubMed2005

Dominant-negative PKC-epsilon impairs apical actin remodeling in parallel with inhibition of carbachol-stimulated secretion in rabbit lacrimal acini.

We investigated the involvement of PKC-epsilon in apical actin remodeling in carbachol-stimulated exocytosis in reconstituted rabbit lacrimal acinar cells. Lacrimal acinar PKC-epsilon cosedimented with actin filaments in an actin filament binding assay. Stimulation of acini with carbachol (100 microM, 2-15 min) significantly (P < or = 0.05) increased PKC-epsilon recovery with actin filaments in two distinct biochemical assays, and confocal fluorescence microscopy showed a significant increase in PKC-epsilon association with apical actin in stimulated acini as evidenced by quantitative colocalization analysis. Overexpression of dominant-negative (DN) PKC-epsilon in lacrimal acini with replication-defective adenovirus (Ad) resulted in profound alterations in apical and basolateral actin filaments while significantly inhibiting carbachol-stimulated secretion of bulk protein and beta-hexosaminidase. The chemical inhibitor GF-109203X (10 microM, 3 h), which inhibits PKC-alpha, -beta, -delta, and -epsilon, also elicited more potent inhibition of carbachol-stimulated secretion relative to Gö-6976 (10 microM, 3 h), which inhibits only PKC-alpha and -beta. Transduction of lacrimal acini with Ad encoding syncollin-green fluorescent protein (GFP) resulted in labeling of secretory vesicles that were discharged in response to carbachol stimulation, whereas cotransduction of acini with Ad-DN-PKC-epsilon significantly inhibited carbachol-stimulated release of syncollin-GFP. Carbachol also increased the recovery of secretory component in culture medium, whereas Ad-DN-PKC-epsilon transduction suppressed its carbachol-stimulated release. We propose that DN-PKC-epsilon alters lacrimal acinar apical actin remodeling, leading to inhibition of stimulated exocytosis and transcytosis.

Actins↗
PubMed2005

Age-dependent alterations in mouse exorbital lacrimal gland structure, innervation and secretory response.

Several studies investigated the effect of aging on rat and human lacrimal gland physiology. However, in most of these studies, only two age groups were investigated. Furthermore, those studies did not correlate the age-related histological changes that occur in the lacrimal gland to the functional changes (nerve activity and protein secretion) that might occur with aging. Thus, the purpose of the present study was to investigate the effect of aging on lacrimal gland structure, innervation and function using BALB/c mice at different ages. Exorbital lacrimal glands were removed from 3, 8, 12, 24, and 32-month-old, male BALB/c mice, fixed, embedded and processed for histology and immunohistochemistry. Sections were stained with hematoxylin and eosin to determine morphological changes and lymphocytic infiltration; giemsa to identify mast cells; and Kinyoun's carbol fucsin solution to indicate lipofuscin-like inclusions. Parasympathetic and sympathetic nerves were identified by immunofluorescence techniques. To measure acetylcholine release and protein secretion, lacrimal gland pieces were incubated in Krebs Ringer buffer containing 5 mM KCl (control), 75 mM KCl (depolarizing buffer which activates nerves), carbachol (a cholinergic agonist, 10(-4) M), or phenylephrine (an alpha1-adrenergic agonist, 10(-4) M) for 20 min. The media were collected and analysed for acetylcholine and peroxidase using a spectrofluorometric assay. KCl-, carbachol- and phenylephrine-stimulated peroxidase secretion decreased in lacrimal glands from 8, 12, and 24-month-old mice when compared to 3-month-old animals. Both the density and distribution of parasympathetic and sympathetic nerves surrounding the acini decreased with increasing age. Acetylcholine release from lacrimal gland nerves decreased in 24-month-old mice compared to 3- and 12-month-old animals. Similarly, progressive morphological changes, including increased numbers of lipofuscin-like inclusions, mast cells and lymphocytic infiltration occurred in an age-dependent manner. We conclude that structural alterations of mouse lacrimal gland, including increased accumulation of lipofuscin-like inclusions, chronic inflammation and functional alterations including decreased acetylcholine release and protein secretion occurred with aging.

Acetylcholine↗
PubMed2005

Nitric oxide and cGMP mediate alpha1D-adrenergic receptor-Stimulated protein secretion and p42/p44 MAPK activation in rat lacrimal gland.

PURPOSE: To determine whether alpha(1)-adrenergic receptors use the nitric oxide (NO)/cGMP pathway to stimulate protein secretion by rat lacrimal gland. METHODS: Identification and cellular location of endothelial nitric oxide synthase (eNOS) and neuronal nitric oxide synthase (nNOS) were determined by Western blot and immunofluorescence techniques, respectively. Rat lacrimal gland acini were isolated by collagenase digestion, and protein secretion stimulated by phenylephrine, an alpha(1)-adrenergic agonist, was measured with a fluorescence assay system. Acini were preincubated with inhibitors for 20 minutes before addition of phenylephrine (10(-4) M). NO and cGMP were measured in response to phenylephrine stimulation. Activation of p42/p44 MAPK was determined by Western blot analysis with an antibody against phosphorylated (active) p42/p44 MAPK. RESULTS: eNOS and nNOS were both present in lacrimal gland. eNOS appeared to be localized with caveolae, whereas nNOS was present in the nerves surrounding the acini. Inhibition of eNOS with N(G)-nitro-l-arginine methyl ester (l-NAME; 10(-6) M) completely inhibited phenylephrine-stimulated protein secretion, whereas the inactive isomer d-NAME and inhibition of nNOS with S-methyl-l-thiocitrulline did not. Phenylephrine increased NO production in a time- and concentration-dependent manner, but the increase was abolished by the alpha(1D)-adrenergic receptor inhibitor BMY-7378. Inhibition of guanylate cyclase with oxadiazoloquinoxalin (ODQ) also inhibited phenylephrine-induced protein secretion, whereas phenylephrine caused a 2.2-fold increase in cGMP. In addition, preincubation with l-NAME and ODQ inhibited phenylephrine-stimulated p42/p44 MAPK activation. CONCLUSIONS: alpha(1D)-Adrenergic agonists stimulate eNOS to produce NO, leading to production of cGMP by guanylate cyclase, to transduce the extracellular signal through the cell and stimulate protein secretion in rat lacrimal gland.

Adrenergic alpha-1 Receptor Antagonists↗
PubMed2004

Control of mucin production by ocular surface epithelial cells.

Multiple species of mucins are synthesized and secreted by corneal and conjunctival epithelial cells. These mucins are vital components of the tear film protecting the ocular surface from the external environment by providing a physical and chemical barrier. The release of mucins must be tightly regulated as both mucin overproduction and underproduction cause ocular surface disorders. Mucin production can be regulated by controlling mucin synthesis, mucin release, or proliferation of the cells that produce the mucin. This review will focus on the evidence demonstrating the control of the mechanisms responsible for production of mucins, their secretion, and corneal and conjunctival epithelia cell proliferation. By understanding these mechanisms under normal conditions, treatments can be designed for diseases of the mucous production of the ocular surface.

Animals↗
PubMed2004

Interaction of EGF family growth factors and neurotransmitters in regulating lacrimal gland secretion.

The lacrimal gland is the primary source for the aqueous portion of the tear film. This portion contains water, electrolytes and proteins, which are necessary for the health and maintenance of the cells of the ocular surface. Afferent sensory nerves in the cornea and conjunctiva stimulate efferent parasympathetic and sympathetic nerves in the lacrimal gland. Cholinergic agonists, released from parasympathetic nevres, and norepinephrine, released from sympathetic nerves, are major stimuli of lacrimal gland secretion. These neurotransmitters activate distinct, but overlapping signal transduction pathways leading to lacrimal gland secretion. Other stimuli of lacrimal gland secretion are the EGF family of growth factors. In addition to stimulation of secretion, these growth factors can interact with the cells of the lacrimal gland themselves or with the cells of the ocular surface depending upon the location from which these growth factors are released. This review will focus on the effects of the EGF family of growth factors on the lacrimal gland and their interactions with the pathways stimulated by the neurotransmitters released from nerves.

Epidermal Growth Factor↗
PubMed2004

Dysfunctional neural regulation of lacrimal gland secretion and its role in the pathogenesis of dry eye syndromes.

Tears are a complex fluid consisting of three layers, each of which is secreted by a different set of tissues or glands. The aqueous portion of the tear film is produced predominantly by the lacrimal gland. Dry eye syndromes are diseases in which the amount and composition of tears are altered, which can lead to ocular surface damage. There are many causes for dry eye syndromes. One such cause is the alteration in the functions of nerves innervating the lacrimal gland and the ocular surface. The autoimmune disease Sjogren syndrome can deleteriously affect the innervation of the lacrimal gland. Damage to the sensory nerves in the ocular surface, specifically the cornea, as a result of refractive surgery and normal aging, prevents the normal reflex arc to the lacrimal gland. Both defects can result in decreased tear secretion and dry eye syndromes. This review will discuss the current information regarding neurally-stimulated protein, water, and electrolyte secretion from the lacrimal gland and delineate how nerve dysfunction resulting from a variety of causes decreases secretion from this gland.

Journal Article↗
PubMed2004

Effect of overexpression of constitutively active PKCalpha on rat lacrimal gland protein secretion.

PURPOSE: The lacrimal gland secretes water, electrolytes, and protein into the tear film. Decreased secretion from the lacrimal gland can lead to dry eye syndromes with deleterious effects on vision. Protein kinase C (PKC)-alpha plays a major role in cholinergic- and alpha1-adrenergic-induced protein secretion from the lacrimal gland. This study was undertaken to determine whether activation of PKCalpha alone would induce lacrimal gland protein secretion by examining the effects of overexpression of constitutively active PKCalpha. METHODS: Rat lacrimal gland acini were transduced with an adenovirus containing a gene for constitutively active PKCalpha. Protein secretion was measured in response to cholinergic and alpha1-adrenergic agonist stimulation. RESULTS: More than 84% of acinar cells were transduced, and PKCalpha expression was increased 176-fold. Western blot analysis using an antibody to phosphorylated (activated) PKCalpha indicated that the overexpressed PKCalpha was active, and basal secretion was increased. Cholinergic agonist-stimulated protein secretion was not stimulated above basal secretion, whereas alpha1-adrenergic-agonist-stimulated protein secretion was increased in transduced acini. CONCLUSIONS: Basal lacrimal gland protein secretion can be stimulated by bypassing the release of neurotransmitters and activating PKCalpha, possibly leading to the development of new treatments for dry eye syndromes.

Adenoviridae↗
PubMed2003

Ca2+-dependent inhibition of NHE3 requires PKC alpha which binds to E3KARP to decrease surface NHE3 containing plasma membrane complexes.

The intestinal brush border (BB) Na+/H+ exchanger isoform 3 (NHE3) is acutely inhibited by elevation in the concentration of free intracellular Ca2+ ([Ca2+]i) by the cholinergic agonist carbachol and Ca2+ ionophores in a protein kinase C (PKC)-dependent manner. We previously showed that elevating [Ca2+]i with ionomycin rapidly inhibited NHE3 activity and decreased the amount of NHE3 on the plasma membrane in a manner that depended on the presence of the PDZ domain-containing protein E3KARP (NHE3 kinase A regulatory protein, also called NHERF2). The current studies were performed in PS120 fibroblasts (NHE-null cell line) stably transfected with NHE3 and E3KARP to probe the mechanism of PKC involvement in Ca2+ regulation of NHE3. Pretreatment with the general PKC inhibitor, GF109203X prevented ionomycin inhibition of NHE3 without altering basal NHE3 activity. Similarly, the Ca2+-mediated inhibition of NHE3 activity was blocked after pretreatment with the conventional PKC inhibitor Gö-6976 and a specific PKCalpha pseudosubstrate-derived inhibitor peptide. [Ca2+]i elevation caused translocation of PKCalpha from cytosol to membrane. PKCalpha bound to the PDZ1 domain of GST-E3KARP in vitro in a Ca2+-dependent manner. PKCalpha and E3KARP coimmunoprecipitated from cell lysates; this occurred to a lesser extent at basal [Ca2+]i and was increased with ionomycin exposure. Biotinylation studies demonstrated that [Ca2+]i elevation induced oligomerization of NHE3 in total lysates and decreased the amount of plasma membrane NHE3. Treatment with PKC inhibitors did not affect the oligomerization of NHE3 but did prevent the decrease in surface amount of NHE3. These results suggest that PKCalpha is not necessary for the Ca2+-dependent formation of the NHE3 plasma membrane complex, although it is necessary for decreasing the membrane amounts of NHE3, probably by stimulating NHE3 endocytosis.

Animals↗
PubMed2003

Regulatory pathways in lacrimal gland epithelium.

Tears are a complex fluid that continuously cover the exposed surface of the eye, namely the cornea and conjunctiva. Tears are secreted in response to the multitude of environmental stresses that can harm the ocular surface such as cold, mechanical stimulation, physical injury, noxious chemicals, as well as infections from various organisms. Tears also provide nutrients and remove waste from cells of the ocular surface. Because of the varied function of tears, tears are complex and are secreted by several different tissues. Tear secretion is under tight neural control allowing tears to respond rapidly to changing environmental conditions. The lacrimal gland is the main contributor to the aqueous portion of the tear film and the regulation of secretion from this gland has been well studied. Despite multiple redundencies in pathways to stimulate secretion from the lacrimal gland, defects can occur resulting in dry eye syndromes. These diseases can have deleterious effects on vision. In this review, we summarize the latest information regarding the regulatory pathways, which control secretion from the lacrimal gland, and their roles in the pathogenesis of dry eye syndromes.

Animals↗
PubMed2003

VEGF-dependent conjunctivalization of the corneal surface.

PURPOSE: To investigate the mechanisms governing corneal neovascularization and the appearance of goblet cells in a murine model of limbal insufficiency. METHODS: The spatial and time-dependent relationship between corneal neovascularization and goblet cell density was analyzed in corneal flatmounts. Immunohistochemical detection of the vascular endothelial growth factor (VEGF) receptor Flt-1 (VEGFR1) was performed in paraffin-embedded sections. A transgenic mouse that expresses the reporter gene lacZ targeted to the Flt-1 locus through homologous recombination was used to analyze corneal expression of Flt-1. The presence of soluble and membranous goblet cell Flt-1 mRNA and protein content was assessed with Northern and Western blot analyses, respectively. Finally, systemic adenoviral expression of a soluble Flt-1/Fc construct was used to study the effect of inhibition of VEGF bioactivity on the appearance of goblet cells and neovascularization. RESULTS: Corneal neovascularization preceded the appearance of goblet cells, although both processes overlapped temporally. Flt-1 was abundant in the conjunctiva-like epithelium covering the cornea, as well as in the goblet cells, invading leukocytes, and vasculature. A similar expression pattern was observed in the transgenic mice expressing the lacZ gene downstream from the Flt-1 promoter. Isolated human and rat goblet cells in culture expressed Flt-1 mRNA and protein, as did freshly isolated human conjunctiva. The systemic inhibition of VEGF bioactivity potently suppressed both corneal neovascularization (8.3% +/- 8.1% vs. 41.1% +/- 15.3% corneal area; P < 0.001) and corneal goblet cell density (1.6% +/- 2.5% vs. 12.2% +/- 2.4% corneal area; P < 0.001). CONCLUSIONS: Two important features of corneal conjunctivalization, the appearance of goblet cells and neovascularization, are regulated by VEGF. Both processes are probably mediated, in part, through the Flt-1 receptor. Taken together, these data indicate that an anti-VEGF therapeutic approach may limit the visual loss associated with conjunctivalization of the corneal surface.

Animals↗
PubMed2003

Isolation and characterization of cultured human conjunctival goblet cells.

PURPOSE: To isolate and characterize goblet cells from normal human conjunctival tissue to determine whether epidermal growth factor (EGF) receptors are present and whether EGF can influence goblet cell proliferation. METHODS: Goblet cells were isolated from explant cultures established from normal conjunctival tissue harvested from patients during periocular surgery. The cells were grown in RPMI culture medium supplemented with 10% fetal bovine serum and characterized using morphology, histochemistry, indirect immunofluorescence microscopy, molecular biology, and biochemistry. Proliferation was determined with a MTT proliferation assay after exposing goblet cells, which had been serum deprived for 48 hours, to increasing concentrations of epidermal growth factor (EGF; 0-80 ng/mL) for 24 hours. RESULTS: Goblet cells were isolated from conjunctival explants by scraping nongoblet cells from the culture dish. Human goblet cells exhibited positive reactivity with alcian blue-periodic acid Schiff (PAS) reagent, goblet cell-specific cytokeratin-7, HPA lectin, and MUC5AC, but negative reactivity to the stratified squamous epithelial cell marker, cytokeratin-4. The mRNA for MUC5AC was detected using RT-PCR. The presence of the EGF receptors EGFR, ErbB2, and ErbB3 was confirmed through Western blot analysis of cell lysates. EGF elicited a concentration-dependent increase in goblet cell proliferation of 160% +/- 0.5%, 188% +/- 0.45%, 293% +/- 1.3%, and 220% +/- 0.5% of control values with 10, 20, 40, and 80 ng/mL EGF, respectively. CONCLUSIONS: Human goblet cells that retain characteristics of goblet cells in vivo can be cultured. EGF receptors are present in human goblet cells, and EGF stimulates their proliferation.

Adult↗
PubMed2003

Signal transduction pathways used by EGF to stimulate protein secretion in rat lacrimal gland.

PURPOSE: To determine the effects of epidermal growth factor (EGF) on lacrimal gland secretion of proteins and characterize its signal-transducing components. METHODS: Both exorbital lacrimal glands were removed from male Sprague-Dawley rats. Dispersed acini were isolated by collagenase digestion in Krebs-Ringer bicarbonate (KRB) buffer at 37 degrees C. Acini were incubated with EGF (10(-7) M), the cholinergic agonist carbachol (10(-4) M), or the alpha(1)-adrenergic agonist phenylephrine (10(-4) M), and peroxidase secretion was measured by a fluorescence assay. To measure intracellular calcium ([Ca(2+)](i)), acini were incubated in fura-2 tetra-acetoxymethyl ester for 60 minutes at 22 degrees C, and fluorescence was measured at 340 and 380 nm with an emission wavelength of 505 nm. Extracellular Ca(2+) was chelated with KRB-BSA without CaCl(2) and with 2 mM EGTA before measurement of peroxidase secretion. Protein kinase C (PKC) was downregulated by incubating acini overnight, with or without the phorbol ester, phorbol 12-myristate 13-acetate (PMA; 10(-6) M), and peroxidase secretion was measured. RESULTS: EGF-stimulated peroxidase secretion in a concentration-dependent manner with a significant increase at 10(-7) M. EGF-stimulated secretion was inhibited by the EGF receptor (EGFR) inhibitor AG1478, but not by the phosphoinositide-3 kinase inhibitor LY292004 or the mitogen-activated kinase kinase (MEK) inhibitor U0126. EGF increased [Ca(2+)](i), whereas chelation of extracellular Ca(2+) inhibited EGF-induced peroxidase secretion by 90%. Downregulation of PKC also inhibited EGF-stimulated peroxidase secretion. CONCLUSIONS: EGF stimulates lacrimal gland secretion of protein by activating the EGFR to increase [Ca(2+)](i) and activate PKC.

Animals↗
PubMed2003

Activation of mitogen-activated protein kinase by cholinergic agonists and EGF in human compared with rat cultured conjunctival goblet cells.

PURPOSE: To compare activation of the p42/p44 mitogen-activated protein kinase (MAPK) by cholinergic agonists and epidermal growth factor (EGF) in cultured human and rat goblet cells. METHOD: . Conjunctiva was removed from either humans during ocular surgery or male Sprague-Dawley rats and cultured in RPMI medium. These cells were incubated with the cholinergic agonist carbachol (10(-4) M) or EGF (10(-8) M) for various times. Before stimulation, cells were incubated with the EGF receptor (EGFR) inhibitor, AG1478 (10(-7) M) or the muscarinic M(3) receptor inhibitor, 4-diphenylacetoxy-N-(2-chloroethyl)-piperidine hydrochloride (4-DAMP; 10(-5) M) for 10 minutes. Proteins were analyzed by Western blot analysis, using antibodies specific to phosphorylated (activated) p42/44-MAPK or total p42-MAPK. Immunoreactive bands were quantified, and data were expressed as percentage of increase over basal. RESULTS: Carbachol (10(-4) M) increased MAPK activity in human and rat cultured goblet cells in a time-dependent manner, increasing pMAPK with a maximum at 10 minutes. EGF (10(-8) M) activated MAPK in human and rat goblet cells in a time-dependent manner with a maximum at 5 minutes. Carbachol- and EGF-induced activation of pMAPK was completely inhibited by AG1478 in cultured conjunctival goblet cells from both species. Carbachol-induced MAPK activity was also completely inhibited by 4-DAMP in both species. CONCLUSIONS: In human and rat cultured conjunctival goblet cells, cholinergic agonists and EGF activate MAPK with a similar time dependency, this activation is receptor mediated, and cholinergic agonists transactivate the EGF receptor. Thus, rat cultured conjunctival goblet cells can be used as a model to study human conjunctival goblet cells.

Adult↗
PubMed2003

Presence and localization of neurotrophins and neurotrophin receptors in rat lacrimal gland.

PURPOSE: To determine which of the neurotrophins (NTs)-nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT3), and neurotrophin-4/5 (NT4)-and their receptors (NTrs), TrkA, TrkB, TrkC, and p75, are present in the adult rat lacrimal gland. METHODS: RT-PCR was performed on RNA isolated from male rat lacrimal gland, using oligonucleotides specific to each NT and NTr. The presence of NT and NTr protein, was determined by Western blot analysis of lacrimal gland homogenate or membranes. The location of NTs and NTrs was determined by immunofluorescence histochemistry. Western blot analyses and immunofluorescence microscopy were performed using primary rabbit polyclonal antibodies raised against NTs and NTrs. RESULTS: RT-PCR showed positive bands at the appropriate sizes for NGF, BDNF, NT3, and NT4, and for the receptors TrkA, TrkB, TrkC, and p75. Western blot analysis confirmed these results, showing that the lacrimal gland expresses NGF, BDNF, NT3, and NT4 as well as the NTrs TrkA, TrkB, and TrkC and the p75 protein. NGF, BDNF, NT3, and NT4 were localized in the lacrimal gland acini with differing cellular distributions, whereas TrkA, TrkB, and TrkC, were localized in myoepithelial cell and ductal cell membranes. The protein p75 was expressed only on myoepithelial cell membranes. CONCLUSIONS: Members of the neurotrophin family of growth factors and their receptors are present in rat lacrimal gland, which suggests a role for NTs and their receptors in the lacrimal gland.

Animals↗