Effect of protein kinase C and Ca(2+) on p42/p44 MAPK, Pyk2, and Src activation in rat conjunctival goblet cells.

Conjunctival goblet cells synthesize and secrete mucins onto the ocular surface to lubricate it and protect it from bacterial infections. Mucin secretion is under neural control, and cholinergic agonists released from parasympathetic nerves are major stimuli of this secretion. The signal transduction pathways these agonists use to stimulate secretion involve activating protein kinase C (PKC) and increasing intracellular [Ca(2+)] to activate the non-receptor kinases Pyk2 and p60Src (Src) to transactivate the EGF receptor. Transactivation of the EGF receptor activates a kinase cascade culminating in the activation of p42/p44 MAPK (MAPK) and ultimately that leads to secretion of high molecular weight glycocongujates (HMWGC), including mucins. To further examine the roles of PKC and Ca(2+) in the activation of MAPK, Pyk2, and Src in mucin secretion, rat conjunctival pieces and cultured goblet cells were incubated with the PKC activator phorbol myristate acid (PMA), the cholinergic agonist carbachol, or the calcium ionophore, ionomycin for varying times. Conjunctival pieces were preincubated with PKC inhibitors 10min prior to addition of carbachol (10(-4)M) for 10min. The amount of phosphorylated (activated) MAPK, Pyk2 and Src was determined by Western blotting techniques using antibodies specific to the phosphorylated forms of each kinase. PMA significantly increased the activation of MAPK, Pyk2, and Src in a time and concentration-dependent manner. PMA-stimulated MAPK activity was completely inhibited by the EGF receptor inhibitor AG1478 (10(-7)M). Carbachol-stimulated MAPK activity was inhibited by three PKC inhibitors, calphostin C, chelethyrine, and staurosporine. Ionomycin (10(-6)M)-stimulated MAPK activity was inhibited 66% by AG1478 (10(-7)M). Ionomycin also significantly increased Pyk2 and Src in time dependent manner. PKC and ionomycin also activated p42/p44 MAPK, Pyk2, and Src in cultured conjunctival goblet cells. We conclude that PKC and intracellular Ca(2+) activate Pyk2 and Src and phosphorylate the EGF receptor leading to stimulation of MAPK in conjunctival goblet cells.

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.

VEGF164(165) as the pathological isoform: differential leukocyte and endothelial responses through VEGFR1 and VEGFR2.

PURPOSE: Vascular endothelial growth factor (VEGF) induces angiogenesis and vascular permeability and is thought to be operative in several ocular vascular diseases. The VEGF isoforms are highly conserved among species; however, little is known about their differential biological functions in adult tissue. In the current study, the inflammatory potential of two prevalent VEGF isoform splice variants, VEGF(120(121)) and VEGF(164(165)), was studied in the transparent and avascular adult mouse cornea. METHODS: Controlled-release pellets containing equimolar amounts of VEGF(120) and VEGF(164) were implanted in corneas. The mechanisms underlying this differential response of VEGF isoforms were explored. The response of VEGF in cultured endothelial cells was determined by Western blot analysis. The response of VEGF isoforms in leukocytes was also investigated. RESULTS: VEGF(164) was found to be significantly more potent at inducing inflammation. In vivo blockade of VEGF receptor (VEGFR)-1 significantly suppressed VEGF(164)-induced corneal inflammation. In vitro, VEGF(165) more potently stimulated intracellular adhesion molecule (ICAM)-1 expression on endothelial cells, an effect that was mediated by VEGFR2. VEGF(164) was also more potent at inducing the chemotaxis of monocytes, an effect that was mediated by VEGFR1. In an immortalized human leukocyte cell line, VEGF(165) was found to induce tyrosine phosphorylation of VEGFR1 more efficiently. CONCLUSIONS: Taken together, these data identify VEGF(164(165)) as a proinflammatory isoform and identify multiple mechanisms underlying its proinflammatory biology.

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.

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.

Cholinergic agonists transactivate EGFR and stimulate MAPK to induce goblet cell secretion.

Conjunctival goblet cells are the primary source of mucins in the mucous layer, the innermost layer of the tear film. Conjunctival goblet cell mucin secretion is under neural control because exogenous addition of parasympathetic agonists stimulates goblet cell secretion. To elucidate the intracellular signal pathways used by cholinergic agonists to stimulate goblet cell mucin secretion, we determined whether p42/p44 mitogen-activated protein kinase (MAPK) is activated during cholinergic agonist-stimulated mucin secretion. Rat conjunctiva was removed, preincubated with or without antagonists, and stimulated with the cholinergic agonist carbachol (10(-4) M). Carbachol statistically significantly stimulated the phosphorylation of MAPK in a time- and concentration-dependent manner. U-0126, an inhibitor of MAPK activation, completely inhibited both the activation of MAPK and goblet cell secretion stimulated by carbachol. The M(1) muscarinic antagonist pirenzepine, the M(2) muscarinic antagonist gallamine, and the M(1)/M(3) muscarinic receptor antagonist N-(3-chloropropyl)-4-piperidinyl diphenylacetate (4-DAMP) also inhibited carbachol-stimulated MAPK activation. Increasing the intracellular Ca(2+) concentration with a Ca(2+) ionophore increased MAPK activation, and chelation of extracellular Ca(2+) inhibited carbachol-stimulated activation. Carbachol also increased tyrosine phosphorylation of Pyk2, p60Src, and the epidermal growth factor receptor (EGFR). The Src inhibitor PP1 and the EGFR inhibitor AG-1478 completely inhibited carbachol-stimulated MAPK activation. AG-1478 also inhibited goblet cell secretion. We conclude that carbachol transactivates the EGFR to activate MAPK, leading to conjunctival goblet cell secretion. In addition, carbachol also activates Pyk2 and p60Src that could play a role in the transactivation of the EGFR.

Detection of drusen in the fellow eye of Japanese patients with age-related macular degeneration using scanning laser ophthalmoscopy.

OBJECTIVE: To study the prevalence of drusen in the fellow eye of Japanese patients with age-related macular degeneration (AMD). DESIGN: Retrospective, cross-sectional study. PARTICIPANTS: Seventeen eyes of 17 Japanese patients with unilateral AMD. MAIN OUTCOME MEASURES: To compare the frequency of drusen based on photography and scanning laser ophthalmoscopy with confocal and ring apertures and a diode laser (780 nm). RESULTS: Using the scanning laser ophthalmoscope (SLO) with a ring aperture, drusen were detected clearly as in topographic imaging. In the fellow eyes of the study patients with AMD, photography showed drusen in 10 cases (58.8%); however, SLO imaging detected drusen in 15 cases (88.2%). The number of drusen detected using SLO imaging was significantly greater than when using photography (P < 0.05). CONCLUSIONS: Scanning laser ophthalmoscope imaging is superior to photography for detecting drusen in the fellow eyes of Japanese patients with unilateral AMD. The prevalence of drusen in the fellow eye of Japanese patients with AMD is much higher than previously speculated.

Inhibitory effect of bucillamine on laser-induced choroidal neovascularization in rats.

PURPOSE: We investigated the inhibitory effects of bucillamine on formation of laser-induced choroidal neovascularization (CNV) in a rat model. METHODS: Bucillamine administration (approximately 150 mg/kg/day) was started 1 week before photocoagulation and continued to the end of the study. Control groups received drinking water. Two weeks after photocoagulation, choroidal neovascularization development was evaluated using simultaneous fluorescein and indocyanine green angiography, and the maximal thickness of the lesions was measured histologically. RESULTS: The incidence of CNV formation was 99.5 +/- 0.2% [mean +/- standard deviation (SD)] in control rats and 64.3 +/- 15.1% with bucillamine (P < 0.01). Histological study showed that the thickness of the CNV lesions was 23.4 +/- 6.5 microm (mean +/- SD) in the bucillamine-treated rats, which was significantly decreased compared to that in controls (60.8 +/- 9.2 microm) (P < 0.01). CONCLUSIONS: Our results suggest that bucillamine may inhibit the development of laser-induced CNV in rats.

Inhibitory effects of bucillamine on increased blood-retinal barrier permeability in streptozotocin-induced diabetic rats.

PURPOSE: To investigate the effect of bucillamine for prevention of increasing blood-retinal barrier (BRB) permeability in streptozotocin (STZ)-induced diabetic rats. METHODS: The groups included control and STZ-induced diabetic rats treated with or without bucillamine. Six months after intervention, the concentrations of reduced and oxidative glutathione (GSH and GSSG) in the retina were measured biochemically. In addition, vitreous fluorescein, which leaks from the vessels after intravenous injection of fluorescein sodium, was measured to evaluate BRB permeability. To evaluate the scavenging ability against the reactive oxygen species (ROS) in vitro, the second-order rate constant for the reaction of bucillamine with ROS was estimated from the kinetics based on the rate constant for the reaction of ROS. RESULTS: The BRB permeability was significantly higher (p = 0.01) in diabetic rats not treated with bucillamine, and bucillamine inhibited the BRB permeability. The GSH concentration and the GSH/GSSG ratio in the retinas decreased in diabetic rats not treated with bucillamine; bucillamine inhibited the decrease of the GSH concentrations. The ROS scavenging activity of bucillamine was similar with that of GSH. CONCLUSIONS: In diabetic retinas, oxidative stress might increase, which may be one of the causes of BRB breakdown. The antioxidant effects of bucillamine might take part in inhibition of increased permeability of the BRB in diabetes.