Cholinergic-induced Ca2+ elevation in rat lacrimal gland acini is negatively modulated by PKCdelta and PKCepsilon.
OBJECTIVE
To investigate the role of protein kinase C (PKC) in cholinergic agonist-induced Ca2+ elevation in lacrimal gland acini.


METHODS
Lacrimal gland acini were prepared by collagenase digestion, and changes in intracellular Ca2+ ([Ca2+]i) were measured using fura-2 as a fluorescent probe.


RESULTS
Preactivation of PKC by phorbol 12-myristate 13-acetate (PMA), or inhibition of protein phosphatase type 1/2A (PP1/2A) by calyculin A, decreased both the [Ca2+]i transient and the plateau of [Ca2+]i induced by increasing concentrations of carbachol, a cholinergic agonist. Staurosporine, an inhibitor of PKC, completely reversed the effect of PMA. Inhibition of the Ca(2+)-independent PKC isoforms PKCdelta and -epsilon, but not the Ca(2+)-dependent isoform PKCalpha substantially reversed the inhibitory effect of PMA on cholinergic agonist-induced Ca2+ elevation. The inhibitory effect of PMA was obtained only in the presence of extracellular Ca2+, suggesting that PKC inhibits the influx of Ca2+. PMA completely inhibited the cholinergic agonist-induced plateau of [Ca2+]i. PMA and calyculin A decreased both the [Ca2+]i transient and the plateau of [Ca2+]i induced by thapsigargin, further supporting the idea that PKC modulates the entry of Ca2+.


CONCLUSIONS
In the lacrimal gland, agonist-induced changes in [Ca2+]i are negatively regulated by PKC-dependent phosphorylation of a target protein(s) that is sensitive to PP1/2A.
Zoukhri
D
D
Schepens Eye Research Institute and the Department of Ophthalmology, Harvard Medical School, Boston, Massachusetts 02114, USA. zoukhri@vision.eri.harvard.edu
Hodges
R R
RR
Sergheraert
C
C
Dartt
D A
DA
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002217:Carbachol / Q000037:antagonists & inhibitors / Q000494:pharmacology*; D018679:Cholinergic Agonists / Q000494:pharmacology*; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D016257:Fura-2 / Q000378:metabolism; D007527:Isoenzymes / Q000378:metabolism*; D007765:Lacrimal Apparatus / Q000187:drug effects* / Q000378:metabolism; D008297:Male; D008387:Marine Toxins; D010080:Oxazoles / Q000494:pharmacology; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism*; D051745:Protein Kinase C-delta; D051744:Protein Kinase C-epsilon; D051381:Rats; D017208:Rats, Wistar; D019311:Staurosporine / Q000494:pharmacology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D019284:Thapsigargin / Q000494:pharmacology
D018679:Cholinergic Agonists; D004791:Enzyme Inhibitors; D007527:Isoenzymes; D008387:Marine Toxins; D010080:Oxazoles; D019284:Thapsigargin; C059041:calyculin A; D002217:Carbachol; C497260:Prkcd protein, rat; C497257:Prkce protein, rat; D011493:Protein Kinase C; D051745:Protein Kinase C-delta; D051744:Protein Kinase C-epsilon; D010749:Phosphoprotein Phosphatases; D019311:Staurosporine; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium; D016257:Fura-2