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Cholinergic-induced Ca2+ elevation in rat lacrimal gland acini is negatively modulated by PKCdelta and PKCepsilon
D Zoukhri1, R R Hodges, C Sergheraert
1Schepens Eye Research Institute and the Department of Ophthalmology, Harvard Medical School, Boston, Massachusetts 02114, USA. zoukhri@vision.eri.harvard.edu
Purpose:
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.