Lc. Wright et al., REGULATION OF THE ACTIVITY AND PHOSPHORYLATION OF THE PLASMA-MEMBRANECA2-ATPASE BY ADRIAMYCIN IN INTACT HUMAN ERYTHROCYTES(), Archives of biochemistry and biophysics, 321(2), 1995, pp. 459-466
We have previously shown in intact human erythrocytes that both the pl
asma membrane Ca2+ pump activity and its phosphorylation can be increa
sed by phorbol-la-myristate 13-acetate (PMA), a known stimulator of pr
otein kinase C. These effects were inhibited by high doses of adriamyc
in (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 m
u M for 1-6 min) decrease the amplitude of the intracellular calcium (
[Ca2+](i)) transient induced by 2.5 mu M CaCl2 and 10 mu M A23187 in i
ntact human erythrocytes, This is reflected by a parallel increase in
Ca2+-ATPase activity in plasma membranes isolated from pretreated inta
ct cells, When 10 mu M ADR and 1 mu M PMA were combined the effects we
re additive, with a maximum decrease in the Ca2+ transient amplitude o
f 50%. A similar effect was seen on the Ca2+-ATPase activities in isol
ated membranes, In erythrocytes labeled with [P-32]orthophosphate 10 m
u M ADR induced a 1.5-fold increase in the phosphorylation of the Ca2 pump and when combined with 1 mu M PMA the phosphorylation was greatl
y enhanced (2.3 times that induced by PMA alone), ADR alone and in com
bination with PMA was found to decrease both P-32 labeling and lipid p
hosphate content of phosphatidylinositol 4,5-bisphosphate (PIP2). This
was accompanied by an increase in the amount of 1,2-diacylglycerol fo
rmed in response to 10 mu M ADR, We conclude that low doses of ADR are
able to stimulate the breakdown of 6-13% of erythrocyte PIP2 by phosp
holipase C at an intracellular calcium concentration of 2.5 mu M, norm
ally regarded as below threshold for phospholipase C activation in ery
throcytes, The diacylglycerol formed appears to stimulate protein kina
se C to activate the Ca-2+ pump and enhance its phosphorylation and Ca
2+ efflux in intact human erythrocytes. (C) 1995 Academic Press, Inc.