Key pointsr '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.r 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.r The CTSs (0.3-10 nM) 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.r 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.r 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 nM) 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 nM) increases in MT 70 and neuronal Ca 2+ signals were both greatly attenuated by the addition of 10 nM digoxin or 10 nM 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) of a single CTS to one protomer blocks all pumping activity. The paradoxical ability of digoxin-like CTSs to reactivate the ouabain-inhibited com...
