Ouabain–digoxin antagonism in rat arteries and neurones

Key points ‘Classic’ cardiotonic steroids (CTSs) all inhibit Na + ,K + ‐ATPase (Na + pumps) and exert cardiotonic and vasotonic effects. Nevertheless, prolonged ouabain, but not digoxin, administration induces hypertension; moreover, digoxin antagonizes the hypertensinogenic effect of ouabain. To examine acute ouabain–digoxin interactions, we tested these and related CTSs on myogenic tone (MT) in pressurized rat mesenteric small arteries and glutamate‐evoked Ca 2+ transients in primary cultured rat hippocampal neurones. The CTSs (0.3–10 n m ) all augmented MT at 70 mmHg and Ca 2+ signals, but separated into two functional groups according to whether they were ouabain‐ or digoxin‐like. CTSs within each group were synergistic, but between groups, were antagonistic to one another in both assays. Na + pump αβ protomers may function as tetraprotomers ((αβ) 4 ) with quarter‐site reactivity; simultaneous ouabain‐ and digoxin‐like molecule binding promotes tetraprotomer disaggregation, enabling partial protomer reactivation. These results may reveal why some patients respond poorly to digoxin therapy, and why Na + pumps may be a novel target for therapeutic development.Abstract ‘Classic’ cardiotonic steroids (CTSs) such as digoxin and ouabain selectively inhibit Na + ,K + ‐ATPase (the Na + pump) and, via Na + /Ca 2+ exchange (NCX), exert cardiotonic and vasotonic effects. CTS action is more complex than previously thought: prolonged subcutaneous administration of ouabain, but not digoxin, induces hypertension, and digoxin antagonizes ouabain's hypertensinogenic effect. We studied the acute interactions between CTSs in two indirect assays of Na + pump function: myogenic tone (MT) in isolated, pressurized rat mesenteric small arteries, and Ca 2+ signalling in primary cultured rat hippocampal neurones. The ‘classic’ CTSs (0.3–10 n m ) behaved as ‘agonists’: all increased MT 70 (MT at 70 mmHg) and augmented glutamate‐evoked Ca 2+ (Fura‐2) signals. We then tested one CTS in the presence of another. Most CTSs could be divided into ouabain‐like (ouabagenin, dihydroouabain (DHO), strophanthidin) or digoxin‐like CTS (digoxigenin, digitoxin, bufalin). Within each group, the CTSs were synergistic, but ouabain‐like and digoxin‐like CTSs antagonized one another in both assays: For example, the ouabain‐evoked (3 n m ) increases in MT 70 and neuronal Ca 2+ signals were both greatly attenuated by the addition of 10 n m digoxin or 10 n m bufalin, and vice versa. Rostafuroxin (PST2238), a digoxigenin derivative that displaces 3 H‐ouabain from Na + ,K + ‐ATPase, and attenuates some forms of hypertension, antagonized the effects of ouabain, but not digoxin. SEA0400, a Na + /Ca 2+ exchanger (NCX) blocker, antagonized the effects of both ouabain and digoxin. CTSs bind to the α subunit of pump αβ protomers. Analysis of potential models suggests that, in vivo , Na + pumps function as tetraprotomers ((αβ) 4 ) in which the binding of a single CTS to one protomer blocks all pumping activity. The paradoxical ability of digoxin‐like CTSs to reactivate the ouabain‐inhibited complex can be explained by de‐oligomerization of the tetrameric state. The interactions between these common CTSs may be of considerable therapeutic relevance.