Phospholipase C-γ (PLC-γ) is stimulated by epidermal growth factor via activation of the epidermal growth factor receptors. The PLC inhibitor, 3-nitrocoumarin (3-NC), selectively inhibited PLC-γ in Madin-Darby canine kidney cells without affecting the activity of PLC-β. In contrast, inhibitors of PLC-β, hexadecylphosphocholine and U73122, had no effect on the activity of PLC-γ. Inhibition of PLC-γ by 3-NC was associated with an increase in tight junction permeability across Madin-Darby canine kidney cell monolayers, as evidenced by 3-NC-induced decrease in transepithelial electrical resistance and increase in mannitol flux over a concentration range that was inhibitory to PLC-γ. An analog of 3-NC, 7-hydroxy-3-NC (7-OH-3-NC), which was inactive as an inhibitor of PLC-γ, also had no effect on tight junction permeability. Treatment with 3-NC caused punctate disruption in the cortical actin filaments. The PLC-γ inhibitor, 3-NC, but not the inactive analog, 7-OH-3-NC, caused hyperphosphorylation of the tight junction proteins, occludin, ZO-1, and ZO-2. The serine/threonine kinase inhibitor, staurosporine (50–200 nm), significantly attenuated 3-NC-induced hyperphosphorylation of ZO-2. This corresponded with attenuation by staurosporine of 3-NC-induced increase in tight junction permeability, suggesting a relationship between ZO-2 phosphorylation and tight junction permeability.
This work was supported by PhRMA Foundation in the form of a Predoctoral Fellowship (to P. W.) and by GlaxoWellcome (unrestricted gift).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
