Attenuation of apoptotic DNA fragmentation by amiloride.
Amiloride is a K+-sparing diuretic that effectively inhibits the Na+/H+ transporter in the plasma membrane of most mammalian cells. We have examined the effects of amiloride on the progression of apoptosis in HL-60 cells induced by camptothecin (CAM), cycloheximide (CHX), and 20 Gy gamma irradiation. Spectrofluorometric measurements on cell populations showed an inhibition of Na+/H+ transporter activity and a corresponding decrease in intracellular pH following treatment with amiloride alone, or in combination with the apoptosis-inducing agents. Flow cytometric cell cycle analysis, in combination with DNA strand break analysis, indicated that amiloride diminished endonuclease-mediated degradation of nuclear chromatin 3 h following treatment with CAM or CHX, and prevented degradation for 3 h following gamma radiation treatment. Apoptosis-associated DNA degradation was significantly greater for all three agents in the absence of amiloride. Protection from radiation-induced apoptosis was transient, since apoptotic subpopulations were observed, but still at a decreased level, 5 h following irradiation. Amiloride was as effective as zinc, an inhibitor of Ca2+/Mg2+-dependent endonucleases, in reducing or delaying the onset of endonuclease activity. Data presented show that effects of amiloride on membrane Na+/H+ transporter activity and intracellular pH can potentially affect apoptotic signaling cascades, leading to a retardation in the rate of progression to an apoptotic cell death. Results also point to the involvement of intracellular pH and Ca2+ in the regulation of apoptotic endonuclease activity, and the need for a functional Na+/H+ exchanger for the induction of apoptosis.
Cobo
J M
JM
Department of Nutricion y Bromatologia, Universidad de Alcalá de Henares, Madrid, Spain.
Garcia-Cañero
R
R
Valdez
J G
JG
Barrasso
A M
AM
Sailer
B L
BL
Crissman
H A
HA
D000584:Amiloride / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects* / Q000528:radiation effects; D002118:Calcium / Q000502:physiology; D002453:Cell Cycle / Q000502:physiology; D002455:Cell Division / Q000187:drug effects; D053938:DNA Fragmentation / Q000187:drug effects*; D004273:DNA, Neoplasm / Q000032:analysis; D004232:Diuretics / Q000494:pharmacology*; D004586:Electrophoresis; D004720:Endonucleases / Q000378:metabolism; D005434:Flow Cytometry; D018922:HL-60 Cells / Q000166:cytology* / Q000187:drug effects / Q000201:enzymology; D006801:Humans; D017923:Sodium-Hydrogen Exchangers / Q000378:metabolism; D015032:Zinc / Q000494:pharmacology
D004273:DNA, Neoplasm; D004232:Diuretics; D017923:Sodium-Hydrogen Exchangers; D000584:Amiloride; D004720:Endonucleases; D015032:Zinc; D002118:Calcium