Endogenous ouabain changes rapidly in humans and dogs upon physical exercise and is under the control of epinephrine and angiotensin II. Hence, the steroid acts as a rapidly acting hormone. A search for a specific binding globulin for cardiac glycosides in bovine plasma resulted in the identification of the d allotype of the micro chain of IgM whose hydrophobic surfaces interact with cardiotonic steroids and cholesterol. Such IgM complexes might be involved in the hepatic elimination of cardiotonic steroids. Thus, differences in the signaling cascade starting at Na(+),K(+)-ATPase must explain any differences in the action of ouabain and digoxin in the genesis of arterial hypertension.

Searching for a binding protein in blood, which may be involved in the specific transport of cardiac glycosides to their receptor sites on the sodium pump, we isolated a cardiac glycoside-binding protein (

Ouabain, that has been isolated from bovine adrenals and hypothalamus, is a new cardiotonic steroid hormone, which is either synthesized in the adrenals or stored there after it has absorbed from the diet. Little is known in vivo which events may lead to the release of ouabain into blood. Moreover, a binding protein for cardiotonic steroids exists in blood, which binds cardiac glycosides with high affinity. It may affect the action of endogenous ouabain on heart and circulation, but the physiological function of this protein is unclear. To realize, which physiological stimuli in vivo may affect blood concentrations of endogenous ouabain and which function the cardiotonic binding protein may have in modulating ouabain effects, the effect of physical exercise on endogenous ouabain was studied and the tissue distribution of its binding protein was investigated. We found that endogenous ouabain changes rapidly in blood upon physical exercise and behaves like expected for a hormone of circulation. The cardiotonic steroid binding globulin shows the highest concentration in the kidney, which suggests that sodium pumps of the kidney are protected against its inhibition by ouabain which would lead not only to natriuresis but also to a deleterious loss of glucose, amino acids and phosphate. (Hypertens Res 2000; 23 Suppl: S93-S98)

To test the hypothesis that Na + /K + -ATPase works as an (ab) 2 -diprotomer with interacting catalytic a-subunits, tryptic digestion of pig kidney enzyme, that had been inactivated with substitution-inert MgATP complex analogues, was performed. This led to the demonstration of coexisting C-terminal Na + -like 80-kDa as well as K + -like 60-kDa peptides and N-terminal 40-kDa peptides of the a-subunit. To localize the ATP binding sites on tryptic peptides, studies with radioactive MgATP complex analogues were performed: Co(NH 3 ) 4 -8-N 3 -ATP specifically modified the E 2 ATP (low affinity) binding site of Na + /K + -ATPase with an inactivation rate constant (k 2 ) of 12 £ 10 23´m in 21 at 37 8C and a dissociation constant (K d ) of 207^28 mm. Tryptic digestion of the [g + -ATPase is an integral membrane protein which transports Na + and K + ions against an electrochemical gradient; such transport is presumably connected to an oscillation of the enzyme between two major conformational states, namely, the E 1 Na + and the E 2 K + conformations and these E 1 and E 2 states probably have different affinities for ATP. The pumping is often considered to evolve from a conformational change of a single ATP site of the catalytic a-subunit existing, either as a high affinity E 1 ATP site (from which Na + export starts by phosphorylation) or as a low affinity E 2 ATP site (which is involved in K + import) [1]. A number of studies seem to refute the formerly favoured idea that Na + /K + pumping is the result of the conformational change of a single catalytic a-subunit and its single ATP binding site as proposed by the so-called Albers±Post model. Hence, this model excludes simultaneously existing and cooperating ATP binding sites [2,3] and the finding that specific labelling of the E 1 ATP or the E 2 ATP binding sites does not block labelling and partial activities of the other remaining empty site [4±9]. In addition, the demonstration of superphosphorylation', i.e. more than 2 mol phosphate incorporated per catalytic a-subunit [10], as well as the observation of a phosphorylation from, P i , during Na + -ATPase activity [11] is consistent with a model in which Na + /K + -ATPase is phosphorylated at both ATP binding sites [12,13].All of the above data support the conclusion that Na + /K + transport involves the interaction and cooperation of two ATP binding sites residing on two different catalytic a-subunits [14,15]. Consistent with this, by Fo Èrster energy transfer studies, we recently differentiated between an E 1 ATP site labelled by fluorescein isothiocyanate and an E 2 ATP site labelled by erythrosine isothiocyanate and determined that the two ATP binding sites are 6.45 nm apart. Moreover, the distance between the ouabain binding sites labelled by fluorescent ouabain derivatives is 4.9 nm. These distances are obviously too large to assume that both ATP and ouabain binding sites are on the same catalytic a-subunit [16]. Hence, it was concluded that Na + / K + -ATPase works as an (ab) 2 diprotomer of cooperating as...

A material crossreacting with antibodies against the bufadienolide proscillaridin A and inhibiting the sodium pump was found in human blood plasma. The concentration of the material with a retention time similar to ouabain in a reversed phase HPLC correlated to systolic blood pressure and pulse pressure. Affinity purification of this compound from bovine adrenals resulted in the isolation of a compound with molecular mass of 600 Da that was not identical with ouabain. Consistent with the postulate that endogenous ouabain and proscillaridin A immunoreactivities may belong to a new class of cardiotonic steroid hormones, a protein of Mr = 60 kDa has been found in bovine serum by affinity-labeling with N-hydroxysuccimidyl digoxigenin-3-O-methylcarbonyl-epsilon-aminocaproate.