Preconditioning by Subinotropic Doses of Ouabain in the Langendorff perfused Rabbit Heart

Abstract

Short exposure to low concentrations of digitalis drugs like ouabain protects the rat heart against ischemia/reperfusion injury through the activation of the Na/K-ATPase/c-Src receptor complex and subsequent stimulation of key intracellular cardioprotective signals. Rat Na/K-ATPase, however, is relatively insensitive to digitalis, and it is not known if similar results could be obtained in species with higher sensitivity. Thus, to determine whether ouabain pretreatment protects against ischemic injury and activates the Na/K-ATPase signaling cascade in a species with a cardiac glycoside sensitivity comparable to humans, the present study was conducted in the rabbit model. In Langendorff-perfused rabbit hearts, 20 min exposure to 500 nM ouabain resulted in positive inotropy as evidenced by a significant increase in +dP/dt, and this increase was accompanied by the activation of several well-characterized downstream mediators of the cardiac Na/K-ATPase receptor pathway, including Src, Akt, ERK1/2, and PKCε. A short (4 min) administration of a sub-inotropic dose of ouabain (100 nM) followed by an 8 min washout before 30 min of global ischemia and 120 min of reperfusion resulted in protection against cell death, as evidenced by a significant decrease in infarct size. These data indicate that ouabain administration activates the Na/K-ATPase signaling cascade and protects against ischemic injury in a species with high cardiac Na/K-ATPase sensitivity.

Role of phosphoinositide 3-kinase IA (PI3K-IA) activation in cardioprotection induced by ouabain preconditioning

“…The left ventricle was powdered and ~100 mg of tissue were added to 1 ml of ice-cold radioimmunoprecipitation assay buffer (RIPA- 50mM Tris-base, 150 mM NaCl, 1% IGEPAL, 0.25% sodium deoxycholate, 1 mM EDTA, pH 7.4) containing 1 mM phenylmethylsulfonyl fluoride, 1 mM sodium orthovanadate, 1 mM NaF, 10 nM okadaic acid, 10 μg/ml aprotinin, 10 μg/ml leupeptin, and homogenized in a 10 ml homogenizer by 15 up-and-down strokes. The resulting lysates were centrifuged at 14,000×g for 15 min [9]. …”

“…This inhibition, which results in subsequent increase in intracellular Na + and increased Na + /Ca 2+ exchange, is required for their well-known cardiac positive inotropic action [1, 2]. Binding of ouabain to the catalytic α-subunit of the Na/KATPase protein complex also initiates a cascade of intracellular signaling events that trigger protection against ischemia-reperfusion (IR) injury [3-9]. By analogy to ischemic preconditioning (IPC) induced by several brief episodes of ischemia before a prolonged ischemia, this protection against IR induced by ouabain is referred to as ouabain preconditioning (OPC).…”

Expression of rat Na-K-ATPase α2 enables ion pumping but not ouabain-induced signaling in α1-deficient porcine renal epithelial cells

“…The findings presented here provide strong evidence that Na-K-ATPase ␣2 differs from ␣1 in its ability to act as a signal transducer, which carries several important implications. We and others have previously shown that activation of the sig- naling pool of Na-K-ATPase ␣1 by cardiotonic steroids is capable of protecting the heart from ischemic reperfusion injury (11,37,38,41). On the other hand, chronic stimulation of the Na-K-ATPase/Src receptor complex by either endogenous or infused cardiotonic steroids increases the generation of reactive oxygen species and induces cardiac hypertrophy and fibrosis in vivo (14,20,21,33,35).…”

Pharmacology of Src family kinases and therapeutic implications of their modulators

“…Src kinase inhibitors such as PP2 have been found to reduce infarct volume and attenuate the increase in tyrosine phosphorylation of NR2A subunit, but not NR2B, after transient focal cerebral ischemia . On the other hand, its protective effect has been suggested by phosphorylation of Akt, p38‐mitogen activated protein kinase (MAPK), and extracellular signal‐regulated kinases 1/2 (ERK1/2) .…”