Abstract
Epidermal growth factor (EGF) inhibits carbachol-induced chloride secretion in T(84) colonic epithelial cells and has been shown to activate phosphatidylinositol (PI) 3-kinase, leading to inhibition of a basolateral potassium conductance. We asked whether the inhibitory effect of EGF on secretion is due to activation of specific isoforms of protein kinase C (PKC) by PI 3-kinase. Western analysis revealed that PKCalpha, gamma, epsilon, eta, mu, lambda/iota, and zeta were expressed in T(84) cells. Ro318220 (an inhibitor active against PKCepsilon, 10 micrometer) but not Gö6983 (an inhibitor active against PKCzeta, 10 micrometer) reversed the inhibitory effect of EGF (100 ng/ml) on carbachol-stimulated chloride secretion. EGF induced the rapid translocation of PKCepsilon from the cytoplasm to the membrane. Wortmannin (50 micrometer) and LY294002 (20 nm), which are PI 3-kinase inhibitors that by themselves had no effect on PKCepsilon activity, significantly suppressed PKCepsilon translocation activated by EGF. LY294002 also reversed the inhibitory action of EGF on chloride secretion. PI (3,4)P(2) increased membrane-associated PKCepsilon and reduced carbachol-induced (86)Rb(+) efflux. Antisense oligonucleotides against PKCepsilon decreased PKCepsilon mass and prevented the inhibitory effect of EGF on carbachol-induced (86)Rb(+) efflux. Thus, the inhibitory effect of EGF on carbachol-induced chloride secretion involves the activation of PKCepsilon mediated by PI 3-kinase. Our findings contribute to the understanding of the cellular mechanisms that control chloride secretion.
Highlights
The ion transport pathways comprising the chloride secretory mechanism of T84 cells, a line of human colonic epithelial cells with a crypt cell phenotype, have been well defined (3, 4)
It had no significant effect on CCh-induced chloride secretion alone, addition of Ro318220 significantly reversed the inhibitory effect of Epidermal growth factor (EGF) on CCh-stimulated chloride secretion (Fig. 2B)
These data suggest that protein kinase C (PKC) is unlikely to mediate the inhibitory effect of EGF on chloride secretion
Summary
The ion transport pathways comprising the chloride secretory mechanism of T84 cells, a line of human colonic epithelial cells with a crypt cell phenotype, have been well defined (3, 4). The inhibitory effect of EGF on carbachol-induced chloride secretion involves the activation of PKC⑀ mediated by PI 3-kinase. Our purpose was to examine whether the inhibitory effect of EGF on CCh-induced chloride secretion is due to the activation of novel and/or atypical isoforms of PKC.
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