June 2003
Volume 44, Issue 6
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Cornea  |   June 2003
Activation of Mitogen-Activated Protein Kinase by Cholinergic Agonists and EGF in Human Compared with Rat Cultured Conjunctival Goblet Cells
Author Affiliations
  • Yoshitaka Horikawa
    From the Schepens Eye Research Institute and
    Department of Ophthalmology, Asahikawa Medical College, Asahikawa, Japan; and the
  • Marie A. Shatos
    From the Schepens Eye Research Institute and
    Department of Ophthalmology, Harvard Medical School, Boston, Massachusetts; the
  • Robin R. Hodges
    From the Schepens Eye Research Institute and
    Department of Ophthalmology, Harvard Medical School, Boston, Massachusetts; the
  • Driss Zoukhri
    From the Schepens Eye Research Institute and
    Department of Ophthalmology, Harvard Medical School, Boston, Massachusetts; the
  • Jose D. Rios
    From the Schepens Eye Research Institute and
  • Eli L. Chang
    Department of Ophthalmology, Harvard Medical School, Boston, Massachusetts; the
    Massachusetts Eye and Ear Infirmary, Boston, Massachusetts.
  • Carlo R. Bernardino
    Department of Ophthalmology, Harvard Medical School, Boston, Massachusetts; the
    Massachusetts Eye and Ear Infirmary, Boston, Massachusetts.
  • Peter A. D. Rubin
    Department of Ophthalmology, Harvard Medical School, Boston, Massachusetts; the
    Massachusetts Eye and Ear Infirmary, Boston, Massachusetts.
  • Darlene A. Dartt
    From the Schepens Eye Research Institute and
    Department of Ophthalmology, Harvard Medical School, Boston, Massachusetts; the
Investigative Ophthalmology & Visual Science June 2003, Vol.44, 2535-2544. doi:10.1167/iovs.02-1117
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      Yoshitaka Horikawa, Marie A. Shatos, Robin R. Hodges, Driss Zoukhri, Jose D. Rios, Eli L. Chang, Carlo R. Bernardino, Peter A. D. Rubin, Darlene A. Dartt; Activation of Mitogen-Activated Protein Kinase by Cholinergic Agonists and EGF in Human Compared with Rat Cultured Conjunctival Goblet Cells. Invest. Ophthalmol. Vis. Sci. 2003;44(6):2535-2544. doi: 10.1167/iovs.02-1117.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

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.

methods. 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 M3 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.

Conjunctival goblet cells are the primary source for the mucous component of the tear film, which serves to protect the cornea and conjunctiva from bacterial infection 1 and to facilitate the occurrence of the smooth refractive surface necessary for clear vision. The goblet cells’ main function is to synthesize, store, and secrete mucin. 2 They are present in a wide variety of tissues: nasal mucosa, trachea, bronchi, bronchioles, small intestine, colon, and pancreatic ducts. 3 4 5 The epithelium comprising the conjunctiva is classified as a nonkeratinizing, stratified squamous epithelium consisting of several layers. 6 Goblet cells, which are highly specialized epithelial cells, are located in the apical surface of the conjunctiva, interspersed among the layers of stratified epithelium. 7 8  
Depending on the species and the tissue, the release of mucin granules from goblet cells may be mediated by distinct mechanisms in response to nerve stimulation, inflammatory processes, changes in temperature, or osmolarity. 2 Cholinergic agonists have been shown to stimulate mucin secretion in goblet cells of the small intestine, 9 trachea, 10 pancreatic duct, 5 and rabbit nictitating membrane. 11 Cholinergic agonists also play a major role in stimulating conjunctival goblet cell secretion. We have shown that parasympathetic and sympathetic nerves surround the basolateral membranes of goblet cells in rat conjunctiva, and stimulation of the afferent sensory nerves in the cornea triggers mucin secretion from goblet cells, through efferent parasympathetic nerves in the conjunctiva. 12 13 In addition, we have found that addition of exogenous carbachol, an analogue of the parasympathetic neurotransmitter acetylcholine that acts through the M2 and M3 muscarinic receptors, and vasoactive intestinal peptide (VIP), a peptidergic parasympathetic neurotransmitter, stimulate conjunctival goblet cell secretion from rat conjunctiva. 14 15 We have found that cholinergic agonist activation of p42/p44 mitogen-activated protein kinase (MAPK) induces mucin secretion in rat conjunctival tissue. 16 We have also shown that M2 and M3 muscarinic receptors are present in rat conjunctiva and M1, M2, and M3 muscarinic receptors are present in cultured rat goblet cells. 15 17  
Mitogen-activated protein kinases (MAPKs) are proline-directed serine-threonine protein kinases that have important functions as mediators of cellular responses to a variety of extracellular stimuli. Three subgroups of the MAPK superfamily have been clearly identified: the extracellularly responsive kinases (p42/44 MAPK or extracellular signal-regulated kinase 1/2 [ERK1/2]), the c-Jun N-terminal kinases (p46/54JNK), and the p38MAPK. 18 p42/p44 MAPK activation is mediated through a number of signaling molecules including growth factor receptor tyrosine kinases and protein kinase C (PKC). Growth factor receptor tyrosine kinases such as EGF activate p42/p44 MAPK. On ligand binding, the EGF receptor becomes autophosphorylated and recruits the adapter molecules, Shc and Grb2, which bind to tyrosine-phosphorylated residues on the receptor by virtue of their SH2 domains. Formation of the EGF receptor-Shc-Grb2 complex leads to activation of a guanine nucleotide exchange factor, mSOS, which in turn activates the low-molecular-weight G-protein, p21ras. This in turn brings about activation of the upstream components of the p42/p44 MAPK cascade, Raf and MEK, ultimately leading to the stimulation of p42/p44 MAPK. 
Another mechanism of activation of the EGFR involves activation of p60src by the focal adhesion kinase Pyk2, which is activated by Ca2+ and PKC. We have found that cholinergic agonists activate p42/p44 MAPK through activation of Pyk2 and Src that transactivate the EGFR. 17  
Our laboratory has recently developed methods by which both rat and human goblet cells can be isolated and grown in vitro. 19 20 Despite differences in their growth patterns, both rat and human goblet cells, similar to their respective counterparts in vivo, react positively with alcian blue–periodic acid Schiff reagent and with either Helix pomatia (HPA) and/or Ulex europaeus (UEA-1) lectins. Cultured goblet cells from both species positively express the goblet cell–specific markers cytokeratin-7 and the mucin MUC5 AC. Furthermore, both rat and human goblet cells secrete mucin into their growth medium. We have also identified the presence of the EGF receptors EGFR, ErbB2, and ErbB3 in cell lysates of cultured human goblet cells and have observed an increase in both rat and human goblet cell proliferation after addition of EGF to serum-deprived cultures. 
To ascertain whether the signaling mechanisms used by cholinergic agonists and EGF are the same in both human and rat goblet cells, we determined whether cholinergic agonists and EGF activate MAPK by transactivation of the EGFR in goblet cells cultured from both human and rat conjunctiva. We then compared cholinergic and EGF activation of MAPK in human conjunctival goblet cells with that in rat conjunctival goblet cells. We conclude that the signaling pathways studied in rat and human goblet cultured goblet cells are similar and that cultured rat goblet cells can be used as a model for studying human goblet cells. 
Materials and Methods
Materials
RPMI-1640 culture medium, l-glutamine, penicillin-streptomycin, Hanks’ balanced salt solution, and typsin-EDTA solution were obtained from BioWhittaker (Walkersville, MD) and fetal bovine serum (FBS) from HyClone Laboratories (Logan, UT). Tissue culture flasks (Falcon), pipettes, and other routine plastics were obtained from BD Labware (Franklin Lakes, NJ). EGF was purchased from Upstate Biotechnology, Inc. (Lake Placid, NY), carbamylcholine chloride (carbachol) and 4-diphenylacetoxy-N-(2-chloroethyl)-piperidine hydrochloride (4-DAMP mustard) from Sigma (St. Louis, MO), and 4-(3-chloroanilino)-6,7-dimethoxy-quinazoline (tyrphostin AG1478) from Biomol (Plymouth Meeting, PA). Mouse anti-p42/p44 MAPK and mouse anti-phospho- p42/p44 MAPK antibodies and horseradish peroxidase (HRP)-conjugated anti-mouse IgG were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). All the reagents for Western blotting were purchased from Bio-Rad Laboratories (Hercules, CA). The chemiluminescence reagents for visualization of immunoreactive bands were from Pierce (Rockford, IL). 
Culture of Conjunctival Goblet Cells
All experiments conformed to the guidelines established by the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and were approved by the Schepens Eye Research Institute Animal Care and Use Committee. Male adult Sprague–Dawley rats weighing between 250 and 300 g were obtained from Taconic Farms (Germantown, NY). Rats were anesthetized for 1 minute in CO2 and decapitated and both eyes surgically removed. The nictitating membranes and the fornix areas of the conjunctiva were excised and immediately placed into Hanks’ balanced salt solution containing 3× penicillin-streptomycin (300 μg/mL), as described previously. 19  
Pieces of normal human conjunctiva were removed from patients (ages, 37–88 years) during oculoplastic surgery for ptosis, ectropion, or entropion. In all cases, tissue that would normally be discarded was donated by the patient for research. Only normal-appearing tissue was used. These tissues were obtained from three of the authors (PADR, ELC, CRB) at the Massachusetts Eye and Ear Infirmary (Boston, MA). Informed consent was obtained from each patient according to the tenets of the Declaration of Helsinki, and the Institutional Review Board of the Massachusetts Eye and Ear Infirmary approved the procedure. 
Rat and human cultures were established as previously described. 19 20 Briefly, rat and human tissues ware finely minced into 1-mm3 pieces that were anchored onto scored culture dishes. One piece of tissue was anchored per tissue culture well. The culture dishes contained just enough medium to cover the bottom of the dish so that the tissue would receive nutrients through surface tension. Cell medium used to feed explants and culture goblet cells consisted exclusively of RPMI 1640 medium supplemented with 10% heat-inactivated FBS, 2 mM l-glutamine, and 100 μg penicillin-streptomycin. Both rat and human explants were refed every 2 days with the medium and were grown under routine culture conditions of 95%O2−5% CO2 at 37°C. Once cells began to grow from the explant in approximately 4 to 6 days, the tissue piece was removed from the dish to inhibit the rampant growth of connective tissue cells. At this juncture, only cultured cells exhibiting goblet cell morphology were continued. When necessary, nongoblet cells were removed from the culture dish by scraping with a rubber policeman. 
Western Blot Analysis
Cultured cells were maintained for 48 hours in serum-free medium and then incubated with the cholinergic agonist carbachol or EGF at various concentrations and for various times. In selected experiments, before stimulation, cells were incubated with the EGFR inhibitor, AG1478 (10−7 M) or the muscarinic M3 receptor inhibitor, 4-DAMP (10−5 M) for 10 minutes. After incubation, the medium was removed and iced-cold RIPA buffer plus proteinase inhibitors (10 mM Tris-HCl [pH 7.4], containing 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholic acid, 0.1% SDS, 1 mM EDTA, 10 mg/mL phenylmethylsulfonyl fluoride, 5 U/mL aprotinin, and 100 nM sodium orthovanadate) was added to terminate the reaction. Cells were collected, sonicated for 3 seconds, and centrifuged at 2000g for 30 minutes at 4°C. A portion of the supernatant was suspended in sample buffer and boiled for 5 minutes. The remainder of the supernatant was assayed for protein content to load an equal amount of protein. Equal amounts of protein were separated by SDS–polyacrylamide gel electrophoresis (SDS-PAGE) on 10% acrylamide gels. Proteins were then transferred to nitrocellulose membranes, blocked in 5% dried milk in TBST (10 mM Tris-HCl [pH 8.0], 150 mM NaCl, and 0.05% Tween-20) for 1 hour, and incubated with a mouse anti-p42 MAPK antibody (1:500) for 1 hour or mouse anti-phospho-p42/p44 MAPK antibody (1:100) for 2 hours at room temperature. Membranes were washed three times in TBST and incubated with HRP-conjugated anti-mouse IgG (1:2000) for 30 minutes at room temperature. Immunoreactive bands were visualized by the enhanced chemiluminescence method. 
Data Presentation and Statistical Analysis
The bands obtained from Western blot experiments were analyzed on computer with NIH Image (available by ftp from zippy.nimh.nih.gov/ or from http://rsb.info.nih.gov/nih-image/; developed by Wayne Rasband, National Institutes of Health, Bethesda, MD). The values for the two bands for phosphorylated p42/p44 MAPK were analyzed together to determine the amount of phosphorylated MAPK. These data were normalized to the amount of total MAPK, as determined with an antibody to total p42 MAPK. Data are expressed as percentage of increase above basal value, which was normalized to 1. 16 21 Data are expressed as mean ± SEM and analyzed by Student’s t-test for paired data. P < 0.05 was considered to represent statistically significant differences. 
Results
Activation of MAPK by Cholinergic Agonists
We have shown that the cholinergic agonist carbachol activates p42/p44 MAPK from cultured rat goblet cells in a concentration-dependent manner, with a statistically significant maximum increase of 164% ± 11% at 10−4 M, after a 10-minute incubation. 17 In cultured human goblet cells, carbachol similarly increased p42/p44 MAPK activity with a maximum of 160% ± 10% at a concentration of 10−4 M (Fig. 1) . The increase was statistically significant at 10−4 and 10−3 M carbachol (P = 0.02 and 0.01, respectively). 
Carbachol (10−4 M) also increased p42/p44 MAPK activation in both human and rat cultured goblet cells in a time-dependent manner, increasing phospho-p42/p44 MAPK to a maximum of 165% ± 5% in human goblet cells (P = 0.006) and 157% ± 9% in rat goblet cells (P = 0.004) after 10 minutes (Fig. 2) . By 30 minutes, phospho-p42/p44 MAPK activation had returned to basal levels in both rat and human cells. We did not determine MAPK activation at times between 10 and 30 minutes. These results suggest that cholinergic activates MAPK in a similar manner in both human and rat cultured conjunctival goblet cells. 
Activation of MAPK by EGF
When incubated for 5 minutes, EGF activated p42/p44 MAPK in human goblet cells in a concentration-dependent manner increasing pMAPK to a maximum of 226% ± 19% at 10−8 M EGF (P = 0.021, Fig. 3 ). MAPK activation was also significantly increased at 10−7 M EGF (P = 0.03). Similarly, we previously showed that EGF, with a 10-minute incubation, significantly increased MAPK activation in rat goblet cells by 156% ± 14%, 178% ± 17%, and 155% ± 9% at 10−9, 10−8, and 10−7 M EGF, respectively. 17  
EGF (10−8 M) activated p42/p44 MAPK in human goblet cells in a time-dependent manner, increasing it significantly with a maximum of 239% ± 24% (P = 0.03) at 5 minutes (Fig. 4A) . EGF (10−8 M) also significantly activated MAPK after 10 minutes (P = 0.02). Similarly, EGF (10−8 M) significantly activated MAPK in rat goblet cells with a maximum of 177% ± 17% (P = 0.02) at 5 minutes and significantly activated MAPK at 10 and 30 minutes (P = 0.04 and 0.04, respectively, Fig. 4B ). We did not determine MAPK activation at times between 10 and 30 minutes. Unlike carbachol stimulation, p42/p44 MAPK was still activated after a 30-minute stimulation with EGF in both human and rat cells. These results suggest that EGF activates MAPK in a similar manner in both human and rat cultured conjunctival goblet cells. 
MAPK Activation by Carbachol’s Activation of Muscarinic Receptors
To determine whether the effects of carbachol on MAPK are mediated through muscarinic receptors in the cultured goblet cells, they were preincubated for 10 minutes with the M1/M3 antagonist, 4-DAMP (10−5 M) before stimulation with carbachol (10−4 M). As shown in Figure 5 , carbachol, after a 10-minutes incubation, significantly increased p42/p44 MAPK activity over basal levels in both human and rat goblet cells (P = 0.0004 and 0.03, respectively). Carbachol-induced activity of p42/p44 MAPK was completely inhibited by 4-DAMP in both human and rat cultured conjunctival goblet cells (P = 0.0005 and 0.008, respectively). 4-DAMP alone had no effect on basal p42/p44 MAPK activity. These results suggest that cholinergic agonists stimulated M1 and M3 receptors to activate MAPK in both human and rat cultured conjunctival goblet cells. 
Carbachol Transactivates the EGFR to Activate MAPK
We previously found that cholinergic agonists transactivated the EGFR to activate MAPK in rat conjunctival pieces. 17 To determine whether transactivation of the EGFR plays a role in MAPK activation in goblet cells, cultured conjunctival goblet cells were preincubated for 10 minutes with the inhibitor of the tyrosine kinase activity of the EGFR, tyrphostin AG1478 (10−7 M) before stimulation with EGF (10−8 M) for 5 minutes or carbachol (10−4 M) for 10 minutes, EGF significantly increased p42/p44 MAPK activity above basal levels in both human (P = 0.04, Fig. 6A ) and rat (P = 0.02, Fig. 6B ) goblet cells. EGF-induced activation of p42/p44 MAPK was completely inhibited by AG1478 in both human and rat cultured conjunctival goblet cells (P = 0.009 and 0.004, respectively Fig. 6 ). Carbachol significantly increased p42/p44 MAPK activity above basal levels in both human (P = 0.004, Fig. 7A ) and rat (P = 0.04, Fig. 7B ) goblet cells. Carbachol-induced stimulation of p42/p44 MAPK was completely inhibited by AG1478 in both human and rat cultured conjunctival goblet cells (P = 0.005 and 0.011, respectively, Fig. 7 ). AG1478 alone did not increase p42/p44 MAPK activity over basal levels (Figs. 6 7) . These results suggest that cholinergic agonists and EGF activate MAPK by transactivation of the EGFR in both human and rat cultured conjunctival goblet cells. 
Discussion
Conjunctival goblet cells synthesize and secrete the gel-forming mucin MUC5AC. 22 The amount of this mucin in the tear film is dependent on the total number of goblet cells present in the conjunctiva, the percentage of goblet cells responding to a given stimuli, and the rate of mucin synthesis. In diseases of the mucous layer of the tear film, these processes can be altered. The diseases that result from these alterations can be classified into diseases of mucous overproduction such as atopic keratoconjunctivitis and of mucous deficiency such as Stevens-Johnson syndrome, neurotrophic keratitis and ocular cicatricial pemphigoid. 23 24 25 To study goblet cell dysfunction that occurs in these diseases, it is critical to first understand goblet cell function under normal conditions. To date, goblet cell function has been studied only in conjunctival tissue, which contains several different cell types. 15 26 As a result, the role of goblet cells in a given function must be extrapolated. In the present study, we show, by using cells in primary culture, that goblet cell functions can be studied directly. In particular, we show that EGF and cholinergic agonists activate p44/p42 MAPK in human goblet cells grown in primary culture from pieces of conjunctiva removed during surgery and in rat goblet cells grown from conjunctival pieces. Second, we demonstrate that goblet cells grown from rat conjunctiva respond, with respect to p44/p42 MAPK activation, almost identically with cells grown from human conjunctiva. Thus, rat goblet cells are a useful and appropriate model to study human goblet cell function. 
Previously, our laboratory has found that cholinergic agonists activate MAPK in whole rat conjunctival tissue leading to mucin secretion, which is mediated by M1, M2, and M3 muscarinic receptors. In addition, we have shown that cholinergic agonists also transactivate the EGFR. 17 As mentioned previously, the conjunctiva contains several cell types including goblet cells, stratified squamous cells, fibroblasts, and endothelial cells. Thus, measurement of p42/p44 MAPK activity in whole tissue does not indicate in which cell type(s) activation of p42/p44 MAPK has occurred. In the present study we have demonstrated that goblet cells contain p42/p44 MAPK that can be activated by cholinergic agonists and EGF as in rat conjunctiva. 
Activation of p42/p44 MAPK in rat cultured cells is similar to p42/p44 MAPK activation in whole rat conjunctival tissue. Cholinergic agonists and EGF stimulate p42/p44 MAPK with comparable concentration dependencies in both cultured cells and whole tissue. 17 These effects are mediated through the muscarinic and EGF receptors, respectively. Cholinergic agonists transactivate the EGF receptor in both rat cultured goblet cells and rat conjunctiva. Similarly, cholinergic agonists and EGF stimulated p42/p44 MAPK from human cultured goblet cells in a time- and concentration-dependent manner almost the same as in cells cultured from rat conjunctiva. In short, p42/p44 MAPK activation in rat cultured goblet cells is similar to that seen in whole rat conjunctiva and both are similar to activation of p42/p44 MAPK in goblet cells from human conjunctiva. 
Carbachol and EGF stimulation of p42/p44 MAPK activity occurred within a similar time frame, with maximum activation occurring by 10 minutes. However, EGF-stimulated activation of p42/p44 MAPK was sustained for as long as 30 minutes (the longest time point measured), whereas carbachol-stimulated activation had returned to basal levels by this time. This suggests that EGF activation of MAPK could initiate different cellular processes from cholinergic agonists. The effects of growth factors such as EGF are usually considered long-term effects that regulate cellular processes such proliferation, migration, and differentiation. In contrast, the effects of cholinergic agonists are usually short-term effects such as secretion and muscle contraction. 27 28 It has been shown that EGF stimulates goblet cell proliferation in conjunctiva, intestine, and pancreas. 19 20 29 30 31 The fact that EGF stimulated proliferation in cultured conjunctival goblet cells may account for the differences in time courses between EGF and cholinergic agonists. Future experiments are necessary to clarify this hypothesis. 
We conclude that, 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. 
 
Figure 1.
 
Effect of concentration on carbachol-induced MAPK activation in human conjunctival goblet cells. Cultured cells were incubated with varying concentrations of carbachol (Cch) for 10 minutes and sonicated, and proteins were separated by SDS-PAGE. Western blot analysis was performed with an antibody to phosphorylated p42/p44 MAPK and total p42 MAPK. Inset: a representative blot. Densitometric analyses were then performed. Data represent the mean ± SEM of results in three independent experiments. *Statistically significant compared with basal activation.
Figure 1.
 
Effect of concentration on carbachol-induced MAPK activation in human conjunctival goblet cells. Cultured cells were incubated with varying concentrations of carbachol (Cch) for 10 minutes and sonicated, and proteins were separated by SDS-PAGE. Western blot analysis was performed with an antibody to phosphorylated p42/p44 MAPK and total p42 MAPK. Inset: a representative blot. Densitometric analyses were then performed. Data represent the mean ± SEM of results in three independent experiments. *Statistically significant compared with basal activation.
Figure 2.
 
Effect of time on carbachol-induced MAPK activation in human and rat conjunctival goblet cells. Cultured human (A) and rat (B) goblet cells were incubated with carbachol (10−4 M) for various times and sonicated, and proteins were separated by SDS-PAGE. Western blot analysis was performed using an antibody to phosphorylated p42/p44 MAPK and total p42 MAPK. Densitometric analyses were then performed. The data represent the mean ± SEM of results in three (human) or five (rat) independent experiments. *Statistically significant compared with t = 0.
Figure 2.
 
Effect of time on carbachol-induced MAPK activation in human and rat conjunctival goblet cells. Cultured human (A) and rat (B) goblet cells were incubated with carbachol (10−4 M) for various times and sonicated, and proteins were separated by SDS-PAGE. Western blot analysis was performed using an antibody to phosphorylated p42/p44 MAPK and total p42 MAPK. Densitometric analyses were then performed. The data represent the mean ± SEM of results in three (human) or five (rat) independent experiments. *Statistically significant compared with t = 0.
Figure 3.
 
Effect of concentration on EGF-induced MAPK activation in human conjunctival goblet cells. Cultured cells were incubated with various concentrations of EGF for 10 minutes and sonicated, and proteins were separated by SDS-PAGE. Western blot analysis was performed with an antibody to phosphorylated p42/p44 MAPK and total p42 MAPK. Inset: representative blot. Densitometric analyses were then performed. The data represent the mean ± SEM of results in three independent experiments. *Statistically significant compared with basal activation.
Figure 3.
 
Effect of concentration on EGF-induced MAPK activation in human conjunctival goblet cells. Cultured cells were incubated with various concentrations of EGF for 10 minutes and sonicated, and proteins were separated by SDS-PAGE. Western blot analysis was performed with an antibody to phosphorylated p42/p44 MAPK and total p42 MAPK. Inset: representative blot. Densitometric analyses were then performed. The data represent the mean ± SEM of results in three independent experiments. *Statistically significant compared with basal activation.
Figure 4.
 
Effect of time on EGF-induced MAPK activation in human and rat conjunctival goblet cells. Cultured human (A) and rat (B) goblet cells were incubated with EGF (10−8 M) for various times and sonicated, and proteins were separated by SDS-PAGE. Western blot analysis was performed with an antibody to phosphorylated p42/p44 MAPK and total p42 MAPK. Densitometric analyses were then performed. The data represent the mean ± SEM of results in three (human) or five (rat) independent experiments. *Statistically significant compared with t = 0.
Figure 4.
 
Effect of time on EGF-induced MAPK activation in human and rat conjunctival goblet cells. Cultured human (A) and rat (B) goblet cells were incubated with EGF (10−8 M) for various times and sonicated, and proteins were separated by SDS-PAGE. Western blot analysis was performed with an antibody to phosphorylated p42/p44 MAPK and total p42 MAPK. Densitometric analyses were then performed. The data represent the mean ± SEM of results in three (human) or five (rat) independent experiments. *Statistically significant compared with t = 0.
Figure 5.
 
Effect of 4-DAMP on carbachol-induced MAPK activation in human and rat conjunctival goblet cells. Human (A) and rat (B) cultured goblet cells were preincubated with selective M1/M 3 muscarinic antagonist 4-DAMP (10−5 M) for 10 minutes and then stimulated with carbachol (Cch, 10−4 M) for 10 minutes. The goblet cells were sonicated, and proteins were separated by SDS-PAGE. Western blot analysis was performed with an antibody to phosphorylated p42 MAPK and total p42 MAPK. Densitometric analyses were then performed. The data represent the mean ± SEM of results in three independent experiments. *Statistically significant compared with basal activation; †statistically significant compared with Cch alone.
Figure 5.
 
Effect of 4-DAMP on carbachol-induced MAPK activation in human and rat conjunctival goblet cells. Human (A) and rat (B) cultured goblet cells were preincubated with selective M1/M 3 muscarinic antagonist 4-DAMP (10−5 M) for 10 minutes and then stimulated with carbachol (Cch, 10−4 M) for 10 minutes. The goblet cells were sonicated, and proteins were separated by SDS-PAGE. Western blot analysis was performed with an antibody to phosphorylated p42 MAPK and total p42 MAPK. Densitometric analyses were then performed. The data represent the mean ± SEM of results in three independent experiments. *Statistically significant compared with basal activation; †statistically significant compared with Cch alone.
Figure 6.
 
Effect of AG1478 on EGF-induced MAPK activation in human and rat conjunctival goblet cells. Human (A) and rat (B) cultured conjunctival goblet cells were preincubated with the EGF receptor inhibitor AG1478 (10−7 M) for 10 minutes and then stimulated with EGF (10−8 M) for 5 minutes. The goblet cells were sonicated, and proteins were separated by SDS-PAGE. Western blot analysis was performed with an antibody to phosphorylated p42/p44 MAPK and total p42 MAPK. Densitometric analyses were then performed. The data represent the mean ± SEM of results in three independent experiments. *Statistically significant compared with basal activation; †statistically significant compared with EGF alone.
Figure 6.
 
Effect of AG1478 on EGF-induced MAPK activation in human and rat conjunctival goblet cells. Human (A) and rat (B) cultured conjunctival goblet cells were preincubated with the EGF receptor inhibitor AG1478 (10−7 M) for 10 minutes and then stimulated with EGF (10−8 M) for 5 minutes. The goblet cells were sonicated, and proteins were separated by SDS-PAGE. Western blot analysis was performed with an antibody to phosphorylated p42/p44 MAPK and total p42 MAPK. Densitometric analyses were then performed. The data represent the mean ± SEM of results in three independent experiments. *Statistically significant compared with basal activation; †statistically significant compared with EGF alone.
Figure 7.
 
Effect of AG1478 on carbachol-induced MAPK activation in human and rat conjunctival goblet cells. Human (A) and rat (B) cultured conjunctival goblet cells were preincubated with the EGF receptor inhibitor AG1478 (10−7 M) for 10 minutes and then stimulated with carbachol (Cch, 10−4 M) for 10 minutes. The goblet cells were sonicated, and proteins were separated by SDS-PAGE. Western blot analysis was performed using an antibody to phosphorylated p42/p44 MAPK and total p42 MAPK. Densitometric analyses were then performed. The data represent the mean ± SEM of results in three independent experiments. *Statistically significant compared with basal activation; †statistically significant compared with Cch alone.
Figure 7.
 
Effect of AG1478 on carbachol-induced MAPK activation in human and rat conjunctival goblet cells. Human (A) and rat (B) cultured conjunctival goblet cells were preincubated with the EGF receptor inhibitor AG1478 (10−7 M) for 10 minutes and then stimulated with carbachol (Cch, 10−4 M) for 10 minutes. The goblet cells were sonicated, and proteins were separated by SDS-PAGE. Western blot analysis was performed using an antibody to phosphorylated p42/p44 MAPK and total p42 MAPK. Densitometric analyses were then performed. The data represent the mean ± SEM of results in three independent experiments. *Statistically significant compared with basal activation; †statistically significant compared with Cch alone.
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Figure 1.
 
Effect of concentration on carbachol-induced MAPK activation in human conjunctival goblet cells. Cultured cells were incubated with varying concentrations of carbachol (Cch) for 10 minutes and sonicated, and proteins were separated by SDS-PAGE. Western blot analysis was performed with an antibody to phosphorylated p42/p44 MAPK and total p42 MAPK. Inset: a representative blot. Densitometric analyses were then performed. Data represent the mean ± SEM of results in three independent experiments. *Statistically significant compared with basal activation.
Figure 1.
 
Effect of concentration on carbachol-induced MAPK activation in human conjunctival goblet cells. Cultured cells were incubated with varying concentrations of carbachol (Cch) for 10 minutes and sonicated, and proteins were separated by SDS-PAGE. Western blot analysis was performed with an antibody to phosphorylated p42/p44 MAPK and total p42 MAPK. Inset: a representative blot. Densitometric analyses were then performed. Data represent the mean ± SEM of results in three independent experiments. *Statistically significant compared with basal activation.
Figure 2.
 
Effect of time on carbachol-induced MAPK activation in human and rat conjunctival goblet cells. Cultured human (A) and rat (B) goblet cells were incubated with carbachol (10−4 M) for various times and sonicated, and proteins were separated by SDS-PAGE. Western blot analysis was performed using an antibody to phosphorylated p42/p44 MAPK and total p42 MAPK. Densitometric analyses were then performed. The data represent the mean ± SEM of results in three (human) or five (rat) independent experiments. *Statistically significant compared with t = 0.
Figure 2.
 
Effect of time on carbachol-induced MAPK activation in human and rat conjunctival goblet cells. Cultured human (A) and rat (B) goblet cells were incubated with carbachol (10−4 M) for various times and sonicated, and proteins were separated by SDS-PAGE. Western blot analysis was performed using an antibody to phosphorylated p42/p44 MAPK and total p42 MAPK. Densitometric analyses were then performed. The data represent the mean ± SEM of results in three (human) or five (rat) independent experiments. *Statistically significant compared with t = 0.
Figure 3.
 
Effect of concentration on EGF-induced MAPK activation in human conjunctival goblet cells. Cultured cells were incubated with various concentrations of EGF for 10 minutes and sonicated, and proteins were separated by SDS-PAGE. Western blot analysis was performed with an antibody to phosphorylated p42/p44 MAPK and total p42 MAPK. Inset: representative blot. Densitometric analyses were then performed. The data represent the mean ± SEM of results in three independent experiments. *Statistically significant compared with basal activation.
Figure 3.
 
Effect of concentration on EGF-induced MAPK activation in human conjunctival goblet cells. Cultured cells were incubated with various concentrations of EGF for 10 minutes and sonicated, and proteins were separated by SDS-PAGE. Western blot analysis was performed with an antibody to phosphorylated p42/p44 MAPK and total p42 MAPK. Inset: representative blot. Densitometric analyses were then performed. The data represent the mean ± SEM of results in three independent experiments. *Statistically significant compared with basal activation.
Figure 4.
 
Effect of time on EGF-induced MAPK activation in human and rat conjunctival goblet cells. Cultured human (A) and rat (B) goblet cells were incubated with EGF (10−8 M) for various times and sonicated, and proteins were separated by SDS-PAGE. Western blot analysis was performed with an antibody to phosphorylated p42/p44 MAPK and total p42 MAPK. Densitometric analyses were then performed. The data represent the mean ± SEM of results in three (human) or five (rat) independent experiments. *Statistically significant compared with t = 0.
Figure 4.
 
Effect of time on EGF-induced MAPK activation in human and rat conjunctival goblet cells. Cultured human (A) and rat (B) goblet cells were incubated with EGF (10−8 M) for various times and sonicated, and proteins were separated by SDS-PAGE. Western blot analysis was performed with an antibody to phosphorylated p42/p44 MAPK and total p42 MAPK. Densitometric analyses were then performed. The data represent the mean ± SEM of results in three (human) or five (rat) independent experiments. *Statistically significant compared with t = 0.
Figure 5.
 
Effect of 4-DAMP on carbachol-induced MAPK activation in human and rat conjunctival goblet cells. Human (A) and rat (B) cultured goblet cells were preincubated with selective M1/M 3 muscarinic antagonist 4-DAMP (10−5 M) for 10 minutes and then stimulated with carbachol (Cch, 10−4 M) for 10 minutes. The goblet cells were sonicated, and proteins were separated by SDS-PAGE. Western blot analysis was performed with an antibody to phosphorylated p42 MAPK and total p42 MAPK. Densitometric analyses were then performed. The data represent the mean ± SEM of results in three independent experiments. *Statistically significant compared with basal activation; †statistically significant compared with Cch alone.
Figure 5.
 
Effect of 4-DAMP on carbachol-induced MAPK activation in human and rat conjunctival goblet cells. Human (A) and rat (B) cultured goblet cells were preincubated with selective M1/M 3 muscarinic antagonist 4-DAMP (10−5 M) for 10 minutes and then stimulated with carbachol (Cch, 10−4 M) for 10 minutes. The goblet cells were sonicated, and proteins were separated by SDS-PAGE. Western blot analysis was performed with an antibody to phosphorylated p42 MAPK and total p42 MAPK. Densitometric analyses were then performed. The data represent the mean ± SEM of results in three independent experiments. *Statistically significant compared with basal activation; †statistically significant compared with Cch alone.
Figure 6.
 
Effect of AG1478 on EGF-induced MAPK activation in human and rat conjunctival goblet cells. Human (A) and rat (B) cultured conjunctival goblet cells were preincubated with the EGF receptor inhibitor AG1478 (10−7 M) for 10 minutes and then stimulated with EGF (10−8 M) for 5 minutes. The goblet cells were sonicated, and proteins were separated by SDS-PAGE. Western blot analysis was performed with an antibody to phosphorylated p42/p44 MAPK and total p42 MAPK. Densitometric analyses were then performed. The data represent the mean ± SEM of results in three independent experiments. *Statistically significant compared with basal activation; †statistically significant compared with EGF alone.
Figure 6.
 
Effect of AG1478 on EGF-induced MAPK activation in human and rat conjunctival goblet cells. Human (A) and rat (B) cultured conjunctival goblet cells were preincubated with the EGF receptor inhibitor AG1478 (10−7 M) for 10 minutes and then stimulated with EGF (10−8 M) for 5 minutes. The goblet cells were sonicated, and proteins were separated by SDS-PAGE. Western blot analysis was performed with an antibody to phosphorylated p42/p44 MAPK and total p42 MAPK. Densitometric analyses were then performed. The data represent the mean ± SEM of results in three independent experiments. *Statistically significant compared with basal activation; †statistically significant compared with EGF alone.
Figure 7.
 
Effect of AG1478 on carbachol-induced MAPK activation in human and rat conjunctival goblet cells. Human (A) and rat (B) cultured conjunctival goblet cells were preincubated with the EGF receptor inhibitor AG1478 (10−7 M) for 10 minutes and then stimulated with carbachol (Cch, 10−4 M) for 10 minutes. The goblet cells were sonicated, and proteins were separated by SDS-PAGE. Western blot analysis was performed using an antibody to phosphorylated p42/p44 MAPK and total p42 MAPK. Densitometric analyses were then performed. The data represent the mean ± SEM of results in three independent experiments. *Statistically significant compared with basal activation; †statistically significant compared with Cch alone.
Figure 7.
 
Effect of AG1478 on carbachol-induced MAPK activation in human and rat conjunctival goblet cells. Human (A) and rat (B) cultured conjunctival goblet cells were preincubated with the EGF receptor inhibitor AG1478 (10−7 M) for 10 minutes and then stimulated with carbachol (Cch, 10−4 M) for 10 minutes. The goblet cells were sonicated, and proteins were separated by SDS-PAGE. Western blot analysis was performed using an antibody to phosphorylated p42/p44 MAPK and total p42 MAPK. Densitometric analyses were then performed. The data represent the mean ± SEM of results in three independent experiments. *Statistically significant compared with basal activation; †statistically significant compared with Cch alone.
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