Bufalin (10nm) was digoxinlike: it antagonized the vasotonic effect of ouabain (Figs4Cand5), whereas neither MBG nor proscillaridinA exhibited antagonism to ouabain in this protocol (Fig.5, upper blue bars). An alternative protocol for testing the antagonism between CTSs was to increase the MT70constriction by incubating pressurized arteries with 3nmof the variousStrophanthus,Digitalisor bufadienolide CTSs, and then add 10nmdigoxin. effects. CTS action is more complex than previously thought: prolonged subcutaneous administration of ouabain, butnotdigoxin, 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 Itga4 (MT) in isolated, pressurized rat mesenteric small arteries, and Ca2+signalling in primary cultured rat hippocampal neurones. The classic CTSs (0.310 nm) behaved as agonists: all increased MT70(MT at 70 mmHg) and augmented glutamateevoked Ca2+(Fura2) signals. We then tested one CTS in the presence of another. Most CTSs could be divided into ouabainlike (ouabagenin, dihydroouabain (DHO), strophanthidin) or digoxinlike CTS (digoxigenin, digitoxin, bufalin). Within each group, the CTSs were synergistic, but ouabainlike and digoxinlike Eflornithine hydrochloride hydrate CTSs antagonized one another in both assays: For example, the ouabainevoked (3 nm) increases in MT70and neuronal Ca2+signals were both greatly attenuated by the addition of 10 nmdigoxin or 10 nmbufalin, and vice versa. Rostafuroxin (PST2238), a digoxigenin Eflornithine hydrochloride hydrate derivative that displaces3Houabain from Na+,K+ATPase, and attenuates some forms of hypertension, antagonized the effects of ouabain, but not digoxin. SEA0400, a Na+/Ca2+exchanger (NCX) blocker, antagonized the effects of both ouabain and digoxin. CTSs bind to the subunit of pump protomers. Analysis of potential models suggests that,in vivo, Na+pumps function as tetraprotomers (()4) in which the binding of a single CTS to one protomer blocks all pumping activity. The paradoxical ability of digoxinlike CTSs to reactivate the ouabaininhibited complex can be explained by deoligomerization of the tetrameric state. The interactions between these common CTSs may be of considerable therapeutic relevance. == Abbreviations == blood pressure cardiotonic steroids dihydroouabain effective concentration for a halfmaximal effect endogenous ouabain endoplasmic Eflornithine hydrochloride hydrate reticulum glutamate marinobufagenin mass spectroscopy myogenic Eflornithine hydrochloride hydrate tone in arteries pressurized to 70 mmHg Na+/Ca2+exchange(r) passive diameter at 70 mmHg intralumenal pressure plasma membrane rostafuroxin response units surface plasmon resonance sarco/endoplasmic reticulum == Introduction == The classic cardiotonic steroids (CTSs), exemplified by ouabain, digoxin and bufalin, all have a steroid nucleus with a 5 or 6member lactone ring at C17 (Fieser & Fieser,1959; Hoch,1961). They are all, as the class name implies, cardiotonic, i.e. they increase the force of cardiac contraction. They are also all Na+pump (i.e. ATPdependent Na+and K+transport or Na+,K+ATPase) inhibitors (Blanco & Mercer,1998), as discovered 60 years ago (Schatzmann,1953). The minimal functional Na+pump unit consists of paired and Eflornithine hydrochloride hydrate subunits, i.e. an protomer (Vilsenet al.1987; Martin & Sachs,2000), in which is a chaperone and the catalytic () subunit is the only welldocumented CTS receptor (O’Brienet al.1994; Lingrel,2010; Yatimeet al.2011). The enigma of how Na+pump inhibitors exert a cardiotonic effect was solved by the recognition that the Na+/Ca2+exchanger (NCX) is the missing link (Bakeret al.1969). Na+pump inhibition raises the intracellular Na+concentration ([Na+]i). This, in turn, promotes NCXmediated net gain of Ca2+, thereby enhancing contractile activation (Bakeret al.1969; Wier & Hess,1984; Blausteinet al.1998). The fact that nearly all vertebrate cells express Na+pumps with CTS receptors (dog red blood cells are a rare exception; Parker,1973) fostered speculation that there must be an endogenous ligand for this receptor (SzentGyorgi,1953). Indeed, skin glands in certain toads synthesize and secrete bufadienolide CTS (Bagrovet al.2009). A CTS was purified from human plasma and identified, by mass spectroscopy (MS), as ouabain (Hamlynet al.1991), a steroidal rhamnoside originally purified from the plantsStrophanthus gratusandAkocanthera schimperi(Fieser & Fieser,1959; Hoch,1961). Endogenous ouabain (EO) was also purified and identified analytically by MS and nuclear magnetic resonance in bovine adrenals (Tamuraet al.1994; Schneideret.
