Regulation of the activity and phosphorylation of the plasma membrane Ca(2+)-ATPase by adriamycin in intact human erythrocytes.
We have previously shown in intact human erythrocytes that both the plasma membrane Ca2+ pump activity and its phosphorylation can be increased by phorbol-12-myristate 13-acetate (PMA), a known stimulator of protein kinase C. These effects were inhibited by high doses of adriamycin (L. C. Wright et al., 1993, Arch. Biochem. Biophys. 306, 277-284). We now show that low doses of adriamycin (ADR) (maximum effect at 10 microM for 1-6 min) decrease the amplitude of the intracellular calcium ([Ca2+]i) transient induced by 2.5 microM CaCl2 and 10 microM A23187 in intact human erythrocytes. This is reflected by a parallel increase in Ca(2+)-ATPase activity in plasma membranes isolated from pretreated intact cells. When 10 microM ADR and 1 microM PMA were combined the effects were additive, with a maximum decrease in the Ca2+ transient amplitude of 50%. A similar effect was seen on the Ca(2+)-ATPase activities in isolated membranes. In erythrocytes labeled with [32P]orthophosphate 10 microM ADR induced a 1.5-fold increase in the phosphorylation of the Ca2+ pump and when combined with 1 microM PMA the phosphorylation was greatly enhanced (2.3 times that induced by PMA alone). ADR alone and in combination with PMA was found to decrease both 32P labeling and lipid phosphate content of phosphatidylinositol 4,5-bisphosphate (PIP2). This was accompanied by an increase in the amount of 1,2-diacylglycerol formed in response to 10 microM ADR. We conclude that low doses of ADR are able to stimulate the breakdown of 6-13% of erythrocyte PIP2 by phospholipase C at an intracellular calcium concentration of 2.5 microM, normally regarded as below threshold for phospholipase C activation in erythrocytes. The diacylglycerol formed appears to stimulate protein kinase C to activate the Ca2+ pump and enhance its phosphorylation and Ca2+ efflux in intact human erythrocytes.
Wright
L C
LC
Department of Pharmacy, University of Sydney, NSW, Australia.
Chen
S
S
Roufogalis
B D
BD
D000001:Calcimycin / Q000494:pharmacology; D002118:Calcium / Q000097:blood; D000252:Calcium-Transporting ATPases / Q000097:blood* / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D004317:Doxorubicin / Q000494:pharmacology*; D004910:Erythrocyte Membrane / Q000187:drug effects / Q000201:enzymology* / Q000378:metabolism; D004912:Erythrocytes / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008563:Membrane Lipids / Q000097:blood; D010743:Phospholipids / Q000097:blood; D010766:Phosphorylation; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D008563:Membrane Lipids; D010743:Phospholipids; D000001:Calcimycin; D004317:Doxorubicin; D000252:Calcium-Transporting ATPases; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium