Comparison of Digitalis Sensitivities of Na/K-ATPases from Human and Pig Kidneys.

Digitalis drugs are selective inhibitors of the plasma membrane Na/K-ATPase. There are many studies on molecular mechanisms of digitalis interaction with purified pig kidney enzyme, with the tacit assumption that it is a good model of human kidney enzyme. However, previous studies on crude or recombinant human kidney enzymes are limited, and have not resulted in consistent findings on their digitalis sensitivities.

Ouabain prevents pathological cardiac hypertrophy and heart failure through activation of phosphoinositide 3-kinase α in mouse.

Background: Use of low doses of digitalis to prevent the development of heart failure was advocated decades ago, but conflicting results of early animal studies dissuaded further research on this issue. Recent discoveries of digitalis effects on cell signal pathways prompted us to reexamine the possibility of this prophylactic action of digitalis. The specific aim of the present study was to determine if subinotropic doses of ouabain would prevent pressure overload-induced cardiac remodeling in the mouse by activating phosphoinositide 3-kinase α (PI3Kα).

Digitalis-induced cell signaling by the sodium pump: on the relation of Src to Na(+)/K(+)-ATPase.

In addition to performing its essential transport function, the sodium pump also activates multiple cell signaling pathways in response to digitalis drugs such as ouabain. Based mainly on cell-free studies with mixtures of purified Src kinase and Na(+)/K(+)-ATPase, a well-advocated hypothesis on how ouabain initiates the activation of signaling pathways is that there is a preexisting physiological complex of inactive Src bound to the α-subunit of Na(+)/K(+)-ATPase, and that ouabain binding to this subunit disrupts the bound Src and activates it. Because of the published disagreements of the results of such cell-free experiments of two other laboratories, our aim was to attempt the resolution of these discrepancies.

Comparative properties of caveolar and noncaveolar preparations of kidney Na+/K+-ATPase.

To evaluate previously proposed functions of renal caveolar Na(+)/K(+)-ATPase, we modified the standard procedures for the preparation of the purified membrane-bound kidney enzyme, separated the caveolar and noncaveolar pools, and compared their properties. While the subunits of Na(+)/K(+)-ATPase (α,β,γ) constituted most of the protein content of the noncaveolar pool, the caveolar pool also contained caveolins and major caveolar proteins annexin-2 tetramer and E-cadherin. Ouabain-sensitive Na(+)/K(+)-ATPase activities of the two pools had similar properties and equal molar activities, indicating that the caveolar enzyme retains its ion transport function and does not contain nonpumping enzyme.

Interactions of K+ATP channel blockers with Na+/K+-ATPase.

Two K(+) (ATP) channel blockers, 5-hydroxydecanoate (5-HD) and glyburide, are often used to study cross-talk between Na(+)/K(+)-ATPase and these channels. The aim of this work was to characterize the effects of these blockers on purified Na(+)/K(+)-ATPase as an aid to appropriate use of these drugs in studies on this cross-talk. In contrast to known dual effects (activating and inhibitory) of other fatty acids on Na(+)/K(+)-ATPase, 5-HD only inhibited the enzyme at concentrations exceeding those that block mitochondrial K(+) (ATP) channels.

Interaction of SDS with Na+/K+-ATPase: SDS-solubilized enzyme retains partial structure and function.

Because nearly all structure/function studies on Na(+)/K(+)-ATPase have been done on enzymes prepared in the presence of SDS, we have studied previously unrecognized consequences of SDS interaction with the enzyme. When the purified membrane-bound kidney enzyme was solubilized with SDS or TDS concentrations just sufficient to cause complete solubilization, but not at concentrations severalfold higher, the enzyme retained quaternary structure, exhibiting alpha,alpha-, alpha,beta-, beta,beta-, and alpha,gamma-associations as detected by chemical cross-linking. The presence of solubilized oligomers was confirmed by sucrose density gradient centrifugation.