Immunolocalization of lacrimal gland PKC isoforms. Effect of phorbol esters and cholinergic agonists on their cellular distribution.
In previous studies, we showed that lacrimal gland acini express three isoforms of protein kinase C (PKC): PKCalpha,-delta, and -epsilon. In the present study, we report the identification of two other PKC isoforms, namely PKCmu and -iota/lambda. Using immunofluorescence techniques, we showed that these isoforms are differentially located. PKCalpha and -mu showed the most prominent membrane localization, whereas PKCdelta, -epsilon and -iota/lambda were mainly cytosolic. Using cell fractionation and western blotting techniques, we showed that the phorbol ester, phorbol 12,13-dibutyrate (PdBu, 10(-6) M), translocated all PKC isoforms, except PKCiota/lambda, from the soluble fraction into the particulate fraction. The effect was maximum at 5 min and persisted at 10 min. PKCepsilon was the most responsive to PdBu reaching almost maximal translocation at a PdBu concentration as low as 10(-9) M. The cholinergic agonist, carbachol (10(-5) and 10(-3) M), induced translocation which was transient for PKCdelta, and -mu, but persisted for 10 min for PKCepsilon. Carbachol did not translocate PKCalpha and, like PdBu, did not translocate PKCiota/lambda. We concluded that lacrimal gland PKC isoforms are differentially localized and that they translocate differentially in response to phorbol esters and cholinergic agonists.
Zoukhri
D
D
Schepens Eye Research Institute and Department of Ophthalmology, Harvard Medical School, 20 Staniford Street, Boston, MA 02114, USA.
Hodges
R R
RR
Willert
S
S
Dartt
D A
DA
D000818:Animals; D015153:Blotting, Western; D002217:Carbachol / Q000494:pharmacology*; D002273:Carcinogens / Q000494:pharmacology*; D002458:Cell Fractionation; D004591:Electrophoresis, Polyacrylamide Gel; D019084:Fluorescent Antibody Technique, Indirect; D007527:Isoenzymes / Q000378:metabolism*; D007765:Lacrimal Apparatus / Q000187:drug effects / Q000201:enzymology*; D008297:Male; D018721:Muscarinic Agonists / Q000494:pharmacology*; D015240:Phorbol 12,13-Dibutyrate / Q000494:pharmacology*; D011493:Protein Kinase C / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar
D002273:Carcinogens; D007527:Isoenzymes; D018721:Muscarinic Agonists; D015240:Phorbol 12,13-Dibutyrate; D002217:Carbachol; D011493:Protein Kinase C