epidermal-growth-factor and fura-2-am

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.

Topics: Animals; Blotting, Western; Calcium; Cells, Cultured; Cholinergic Agonists; Conjunctiva; Dose-Response Relationship, Drug; Enzyme Inhibitors; Enzyme-Linked Immunosorbent Assay; Epidermal Growth Factor; ErbB Receptors; Fura-2; Goblet Cells; Male; Microscopy, Fluorescence; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mucin 5AC; Rats; Rats, Sprague-Dawley; RNA, Small Interfering; Signal Transduction

To determine in human corneal epithelial cells (HCECs) the role of protein kinase C (PKC) in mediating epidermal growth factor (EGF)-induced stimulation of store-operated channel (SOC) activity and capacitative calcium entry (CCE).. Single-cell Ca2+ fluorescence imaging of fura2-loaded HCECs was used to evaluate CCE. PKC translocation induced by EGF or PDBu was monitored by Western blot analyses of four different subcellular fractions. Plasma membrane Ca2+ influx was measured by Mn2+ quench rates of fura2-fluorescence. The whole-cell patch clamp configuration was used to determine the SOC activation induced by EGF.. EGF-induced increases in SOC currents through PKC stimulation, since calphostin C inhibited this response. To determine which PKC isoforms mediated EGF-induced increases in CCE, the PKC isoform enrichment of a plasma membrane-containing fraction was determined. From 5 to 30 minutes, its rank order of enrichment was: delta > betaI > alpha approximately epsilon. Preferential PKCdelta and PKCbeta translocation was in accordance with other results showing that rottlerin and hispidin have the highest efficacy in suppressing EGF-induced CCE augmentation. Furthermore, after PKCbeta and PKCdelta siRNA knockdown of gene and protein expression, declines in EGF-induced increases in CCE matched those obtained after exposure to a corresponding selective PKC isoform inhibitor.. EGF-induced PKC stimulation in HCECs mediates SOC activation. This response contributes to CCE, which preferentially depends on PKCdelta and PKCbeta isoform stimulation. This rank order is based on the findings that either selective knockdown of their expression or exposure to PKCdelta and PKCbeta isoform inhibitors elicited the largest declines in EGF-augmented CCE.

Topics: Blotting, Western; Calcium; Calcium Channels; Cell Line; Epidermal Growth Factor; Epithelium, Corneal; Fura-2; Gene Deletion; Humans; Isoenzymes; Microscopy, Fluorescence; Naphthalenes; Patch-Clamp Techniques; Phorbol 12,13-Dibutyrate; Protein Kinase C; Protein Kinase C beta; Protein Kinase C-delta; RNA, Small Interfering; Transfection

Despite recent improvements in intraocular lens (IOL) design, posterior capsule opacification (PCO) arising from lens cell growth remains a major problem. Calcium signaling has been shown to play a major role in driving human lens cell growth, and therefore it is necessary to understand the underlying mechanisms.. Calcium signaling was studied in capsular bags (ex vivo) removed from donors who had undergone earlier cataract surgery. Fresh capsular bags were also produced from intact donor lenses and cultured in serum-free EMEM for up to 8 weeks. Both preparations were loaded with Fura-2, and ratiometric imaging of cytoplasmic calcium was performed using epifluorescence techniques. Changes were monitored in response to 10 microM ATP (adenosine triphosphate), 10 microM acetylcholine, and 10 ng/mL epidermal growth factor (EGF), and data were collected from equatorial, posterior, and anterior regions. Calcium transients were also recorded from anterior epithelial specimens in response to pilocarpine.. All equatorial cells of ex vivo bags responded to ATP and EGF, but not to acetylcholine, and this pattern was maintained in the cultured bags. Posterior capsule cells of both preparations also had similar properties, in which a large proportion of the cells responded to ATP and EGF, but not to acetylcholine. Conversely, most anterior cells of the in vivo bags responded to pilocarpine, whereas no cells in the cultured bags responded. All cells in the fresh anterior epithelium responded to pilocarpine.. Ex vivo capsular bags retain the region-specific calcium-signaling characteristics of the native lens. Apart from losing M1 muscarinic expression properties, the in vitro capsular bags also reflect region-specific signaling properties and therefore provide a good model for the investigation of the contribution of calcium-signaling to PCO.

Topics: Acetylcholine; Adenosine Triphosphate; Aged; Calcium; Calcium Signaling; Epidermal Growth Factor; Epithelial Cells; Fura-2; Humans; Lens Capsule, Crystalline; Muscarinic Agonists; Organ Culture Techniques; Pilocarpine; Receptor Protein-Tyrosine Kinases; Receptors, Purinergic P2

The lens epithelium can be separated into two regions, the nondividing central zone and the equator, the site of all division in the normal lens. In the present study, the distribution of epithelial growth factor (EGF)/epithelial growth factor receptor (EGFR) signaling components was investigated and related to mitotic distribution in the lens.. Anterior and equatorial regions of the native epithelium were prepared separately from donor lenses. In vitro capsular bags were prepared from donor eyes and cultured. Receptor distribution was determined by immunocytochemistry and RT-PCR. Western blot analysis of phospholipase C (PLC)-gamma and extracellular signal-regulated kinase (ERK; total and active) was performed on cell lysates. Function was determined by calcium imaging of Fura-2-AM-loaded cells and also, in the case of capsular bags, by cell growth.. Immunocytochemistry and RT-PCR showed an even distribution of EGFR across the native epithelium. Whole lenses, however, exhibited only a calcium response to EGF (10 ng/mL) at the equatorial region. Western blot analysis demonstrated significantly greater expression of PLCgamma and ERK (total and active) in the equator than in the central region. Addition of EGF increased growth rates of cells in capsular bags and an EGFR inhibitor decreased rates. EGF also induced a calcium response in posterior capsule cells in the capsular bags.. EGFR is evenly distributed across the entire epithelium, whereas related calcium signaling and expression of PLCgamma and ERK have a marked bias to the equator. Therefore, levels of downstream enzyme components rather than changes in receptor expression dictate EGFR signaling output in the normal lens. In the wounded lens (capsular bag) EGFR signaling persists in cells growing on the posterior capsule.

Topics: Blotting, Western; Calcium; Epidermal Growth Factor; Epithelium; ErbB Receptors; Fluorescent Antibody Technique, Indirect; Fura-2; Humans; Lens Capsule, Crystalline; Lens, Crystalline; Mitogen-Activated Protein Kinases; Mitosis; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction; Type C Phospholipases

Endothelin is a novel peptide secreted by endothelial cells, the vasoconstrictor effects of which appear dependent on the activation of phospholipase C. We examined in tissue culture its potential as a growth factor for vascular smooth muscle. In quiescent cultures of rat aortic smooth muscle cells, endothelin rapidly elevated levels of c-fos and c-myc mRNA. Peak effects on c-fos mRNA occurred between 15 and 30 min and were completely gone after 2 h. The elevation in c-fos mRNA was, in part, dependent on protein kinase C, since phorbol myristate acetate (PMA) also elevated c-fos mRNA and further increased c-fos mRNA expression by endothelin, but the effects were not additive. Furthermore, the endothelin-induced elevation in c-fos mRNA was attenuated but not abolished in protein kinase C-depleted cells. Maximum levels of c-myc mRNA occurred between 15 and 30 min after exposing the cells to endothelin and persisted for at least 6 h. The effects of simultaneous addition of endothelin and PMA on c-myc mRNA levels were essentially similar to those observed with c-fos mRNA. [3H]thymidine incorporation into DNA occurred 8 h after exposing the cells to endothelin. The mitogenic effect of endothelin was smaller than that observed with either fetal calf serum or epidermal growth factor and was dependent on both pertussis toxin-insensitive and -sensitive pathways. Sensitivity to the latter pathway did not appear dependent on attenuation of phospholipase C activity, since neither peak intracellular calcium concentrations nor c-fos mRNA levels were reduced in pertussis toxin-treated cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Aorta; Benzofurans; Calcium; Cell Division; Cells, Cultured; DNA Replication; Endothelins; Endothelium, Vascular; Epidermal Growth Factor; Fluorescent Dyes; Fura-2; Kinetics; Muscle, Smooth, Vascular; Peptides; Pertussis Toxin; Rats; Rats, Inbred SHR; RNA; Tetradecanoylphorbol Acetate; Virulence Factors, Bordetella

The aim of this study was to elucidate the cellular mechanisms of action of epidermal growth factor (EGF) inhibition of parietal cell secretion. EGF effects on histamine- and carbachol-stimulated [14C]aminopyrine (AP) uptake and intrinsic factor (IF) secretion were evaluated in isolated rabbit parietal cells. EGF inhibited histamine-stimulated [14C]AP uptake and IF secretion through a reduction in stimulated adenosine 3',5'-cyclic monophosphate (cAMP) levels. EGF decreased the phosphorylation of a cytosolic 30-kDa, histamine-stimulated, cAMP-dependent protein kinase substrate. These effects on histamine-stimulated activation were reversed by pertussis toxin preincubation. EGF inhibited carbachol-stimulated [14C]AP uptake and IF secretion, but did not alter the carbachol-stimulated Ca2+ transient. These results indicate that EGF inhibits histamine-stimulated secretion through the inhibitory Gi guanosine 5'-triphosphate-binding protein and carbachol-stimulated secretion through a mechanism independent of the activation of an increase in intracellular Ca2+.

Topics: Aminopyrine; Animals; Benzofurans; Buffers; Calcium; Carbachol; Colforsin; Cyclic AMP; Cytosol; Epidermal Growth Factor; Fluorescent Dyes; Fura-2; Gastric Acid; Histamine; In Vitro Techniques; Intrinsic Factor; Kinetics; Parietal Cells, Gastric; Phosphorylation; Proteins; Rabbits; Signal Transduction