Inhibiting cell volume regulation with ecto-protein kinase C in HL-60 cells
Grill, Thomas Joseph
MetadataShow full item record
Regulation of cell volume is a process common to plants, animals, and microorganisms. This process is controlled by many molecules, proteins, and channels within the cell. The effects of adding exogenous protein kinase C-[alpha] (PKC-[alpha]) and adenosine triphosphate (ATP) to cells undergoing the processes of apoptotic volume decrease (AVD) and regulatory volume decrease (RVD) were characterized in this study. The processes of AVD and RVD were induced in HL-60 cells by exposing them to UV irradiation or by swelling the cells in hypotonic medium. These two processes were inhibited by addition of PKC-[alpha] and ATP at specific time points. When PKC-[alpha] and ATP are added to cells undergoing AVD an inhibition of a decrease in cell size was observed, a normal step in apoptotic cells. At the time points of 3.5 and 4.5 hours after 2.5 minutes of UV irradiation cells treated with PKC-[alpha] (60 ng/well) and ATP (100 [mu]M) showed an 81% inhibition and a complete inhibition respectively. When added to cells undergoing RVD, cells were unable to regulate their volume from a swollen to a normal size. When PKC-[alpha] (30 ng/well) and ATP (100 [mu]M) were added to the system at 30 minutes, the cells showed a complete inhibition of the process at 45 minutes. One way ecto-PKC could inhibit cell shrinkage is by inhibiting the opening of K + channels in a manner similar to K + channel inhibitors. Erythromycin (10 [mu]g/ml), quinidine (100 [mu]M), ergtoxin (20 nM), and E-4031 (0.25 [mu]M) were all shown to inhibit a decrease in cell volume when tested under apoptotic conditions. Erythromycin had an 83% inhibition; quinidine showed a complete inhibition; ergtoxin had an inhibition of 54%; and E-4031 showed an inhibition of 56%. E-4031 showed a complete inhibition in the process of RVD when tested under hypotonic conditions. An ethidium homodimer (EHD) assay was also performed to assess cellular damage using quinidine (100 [mu]M) and erythromycin (10 [mu]g/ml), both showing a significant inhibition. When the PKC inhibitors PKC [19-31] inhibitor peptide, bisindolylmaleimide, and polymyxin B were tested, the former two showed a statistically significant difference in cell size compared to cells treated with UV, PKC, and ATP. The effects of ecto-PKC-[alpha] required extracellular ATP. No effects of ecto-PKC-[alpha] were observed if ATP was substituted with ADP, GTP, and ATP[gamma]-s. Taken together, these data fulfill the criteria for the inhibitor effects observed by adding exogenous PKC-[alpha] and ATP on the processes of AVD and RVD. It provides strong evidence ecto-PKC-[alpha] becomes activated and acts catalytically, which may affect K + channels in HL-60 cell membranes resulting in their closure. It is our belief that it is this step that inhibits the cells from decreasing in volume due to their inability to allow ions and water to exit.