Activation of mitogen-activated protein kinase by cholinergic agonists and EGF in human compared with rat cultured conjunctival goblet cells.
OBJECTIVE
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 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.
Horikawa
Yoshitaka
Y
Schepens Eye Research Institute, Harvard Medical School, Boston, Massachusetts 02114, USA.
Shatos
Marie A
MA
Hodges
Robin R
RR
Zoukhri
Driss
D
Rios
Jose D
JD
Chang
Eli L
EL
Bernardino
Carlo R
CR
Rubin
Peter A D
PA
Dartt
Darlene A
DA
D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D000818:Animals; D015153:Blotting, Western; D002217:Carbachol / Q000494:pharmacology*; D002478:Cells, Cultured; D018679:Cholinergic Agonists / Q000494:pharmacology*; D003228:Conjunctiva / Q000166:cytology*; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004815:Epidermal Growth Factor / Q000494:pharmacology*; D066246:ErbB Receptors / Q000037:antagonists & inhibitors / Q000378:metabolism; D020397:Goblet Cells / Q000187:drug effects* / Q000201:enzymology; D006801:Humans; D008297:Male; D008875:Middle Aged; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D018727:Muscarinic Antagonists / Q000494:pharmacology; D010880:Piperidines / Q000494:pharmacology; D011799:Quinazolines; D051381:Rats; D017207:Rats, Sprague-Dawley; D013997:Time Factors; D020032:Tyrphostins / Q000494:pharmacology
D018679:Cholinergic Agonists; D004791:Enzyme Inhibitors; D018727:Muscarinic Antagonists; D010880:Piperidines; D011799:Quinazolines; D020032:Tyrphostins; C101044:RTKI cpd; D004815:Epidermal Growth Factor; C042375:4-diphenylacetoxy-1,1-dimethylpiperidinium; D002217:Carbachol; D066246:ErbB Receptors; D020928:Mitogen-Activated Protein Kinases