The activation of phospholipase D (PLD) by transforming Ras is well documented. Although two distinct PLD isoforms, PLD1 and PLD2, have been cloned from mammalian cells, it has remained unclear whether both isoenzymes are activated by Ras and, if this is the case, whether they are stimulated by a common mechanism. In the present study we show that expression of transforming Ras in HC11 mouse mammary epithelial cells enhanced the activity of endogenous PLD. Co-expression of Ras with either PLD1b or PLD2 resulted in elevated activities of both PLD isoenzymes in HC11 cells, indicating that transforming Ras was capable of activating both PLD isoforms in vivo. Ras-induced activation of PLD was resistant to the protein kinase C (PKC) inhibitor GF109203X, which preferentially affects conventional- and novel-type PKCs, but sensitive to Ro-31-8220, which inhibits atypical PKCs more effectively. Co-transfection of atypical PKC-iota with either PLD1b or PLD2 led to a selective activation of PLD2 by PKC-iota, whereas PLD1b was not affected. PLD1b, however, was found to be a potent activator of PKC-iota, whereas PLD2 was less effective in this respect. The data suggest that PKC-iota acts upstream of PLD2 and that PLD1b is implicated in the activation of PKC-iota. The data are discussed as indicating a putative signalling cascade comprising Ras-->PLD1b-->PKC-iota-->PLD2. Evidence for the implication of this pathway in the transcriptional regulation of cyclin D1 is also presented.

Retinal pigment epithelial (RPE) cells provide crucial functions for the maintenance of the retinal environment. We investigated the phagocytotic mechanisms of RPE cells evaluating the question whether particle uptake underlies a diurnal rhythm. Additionally, a possible connection of volume regulation and the phagocytotic function of RPE cells was studied. As antiviral nucleoside analogues influence cell-volume-regulating mechanisms, we tested several antiviral drugs. Cultured primary RPE cells and a permanent cell line (ARPE-19) were tested for uptake of europium-labeled microspheres quantified by time-resolved fluorometry. Cells were also exposed to cyclic illumination or continuous light and dark culture conditions. Inhibitors of cytoskeleton (microtubuli, actin) and osmotic swelling were also tested. Ingested FITC-labeled microparticles were found in phagosomes strongly associated which the cytoskeleton as they could not be easily moved by laser tweezer microscopy. Phagocytosis was observed predominately during dark intervals and was reduced by continuous light exposure. The diurnal rhythm of unsynchronized RPE cultures was abolished by microtubule inhibitors although no inhibition of overall particle uptake by cytoskeletal blockers was observed. Hypoosmotic swelling of RPE also decreased phagocytosis. Acyclovir was found inhibitory in ARPE-19 cells, whereas azidothymidine showed a protracted inhibiting activity on primary RPE cells and ganciclovir was inactive in both cell types. The presence of a diurnal rhythm also in culture indicates genetic determination of light-regulated particle uptake. This mechanism appears to be influenced by the regulation of cell volume and microtubule function. Inhibition of RPE function by antiviral drugs is a novel finding and in accordance with interferences of the tested drugs with cellular chloride channels described earlier. It may give a hint towards possible ocular side effects in the long-term use of nucleoside-analogous substances.

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The activation of phospholipase D (PLD) by transforming Ras is well documented. Although two distinct PLD isoforms, PLD1 and PLD2, have been cloned from mammalian cells, it has remained unclear whether both isoenzymes are activated by Ras and, if this is the case, whether they are stimulated by a common mechanism. In the present study we show that expression of transforming Ras in HC11 mouse mammary epithelial cells enhanced the activity of endogenous PLD. Co-expression of Ras with either PLD1b or PLD2 resulted in elevated activities of both PLD isoenzymes in HC11 cells, indicating that transforming Ras was capable of activating both PLD isoforms in vivo. Ras-induced activation of PLD was resistant to the protein kinase C (PKC) inhibitor GF109203X, which preferentially affects conventional- and novel-type PKCs, but sensitive to Ro-31-8220, which inhibits atypical PKCs more effectively. Co-transfection of atypical PKC-ι with either PLD1b or PLD2 led to a selective activation of PLD2 by PKC-ι, whereas PLD1b was not affected. PLD1b, however, was found to be a potent activator of PKC-ι, whereas PLD2 was less effective in this respect. The data suggest that PKC-ι acts upstream of PLD2 and that PLD1b is implicated in the activation of PKC-ι. The data are discussed as indicating a putative signalling cascade comprising Ras → PLD1b →PKC-ι → PLD2. Evidence for the implication of this pathway in the transcriptional regulation of cyclin D1 is also presented.