Cardiotonic steroids differentially affect intracellular Na+ and [Na+]i/[K+]i-independent signaling in C7-MDCK cells.
Recently, we reported that ouabain kills renal epithelial and vascular endothelial cells independently of elevation of the [Na(+)](i)/[K(+)](i) ratio. These observations raised the possibility of finding cardiotonic steroids (CTS) that inhibit the Na(+),K(+) pump without attenuating cell survival and vice versa. To test this hypothesis, we compared CTS action on Na(+),K(+) pump, [Na(+)](i) content, and survival of Madin-Darby canine kidney cells. At a concentration of 1 microM, ouabain and other tested cardenolides, as well as bufadienolides such as bufalin, cinobufagin, cinobufotalin, and telobufotoxin, led to approximately 10-fold inhibition of the Na(+),K(+) pump, a 2-3-fold decrease in staining with dimethylthiazol-diphenyltetrazolium (MTT), and massive death indicated by detachment of approximately 80% of cells and caspase-3 activation. In contrast, Na(+),K(+) pump inhibition and elevation of [Na(+)](i) seen in the presence of 3 microM marinobufagenin (MBG) and marinobufotoxin did not affect MTT staining and cell survival. Inhibition of the Na(+),Rb(+) pump in K(+)-free medium was not accompanied by a decline of MTT staining and cell detachment but increased sensitivity to CTS. In K(+)-free medium, half-maximal inhibition of (86)Rb influx was observed in the presence of 0.04 microM ouabain and 0.1 microM MBG, whereas half-maximal detachment and decline of MTT staining were detected at 0.03 and 0.004 microM of ouabain versus 10 and 3 microM of MBG, respectively. Both ouabain binding and ouabain-induced [Na(+)](i),[K(+)](i)-independent signaling were suppressed in the presence of MBG. Thus, our results show that CTS exhibit distinctly different potency in Na(+),K(+) pump inhibition and triggering of [Na(+)](i)/[K(+)](i)-independent signaling, including cell death.
Akimova
Olga A
OA
Centre de recherche, Centre hospitalier de l'Université de Montréal, Montreal, Quebec H2W 1T7, Canada.
Bagrov
Alexei Y
AY
Lopina
Olga D
OD
Kamernitsky
Alexey V
AV
Tremblay
Johanne
J
Hamet
Pavel
P
Orlov
Sergei N
SN
D000818:Animals; D002301:Cardiac Glycosides / Q000494:pharmacology*; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D004285:Dogs; D011188:Potassium / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects*; D012964:Sodium / Q000378:metabolism*; D000254:Sodium-Potassium-Exchanging ATPase / Q000037:antagonists & inhibitors*
D002301:Cardiac Glycosides; D012964:Sodium; D000254:Sodium-Potassium-Exchanging ATPase; D011188:Potassium