digitoxigenin and 20-22-dihydrodigitoxigenin

Ten cardiac glycosides (

Topics: Antineoplastic Agents, Phytogenic; Cell Line, Tumor; Digitoxigenin; Drug Screening Assays, Antitumor; Glycosides; Humans; Molecular Structure; Plant Stems; Spectrum Analysis

The steady-state voltage and [Na(+)](o) dependence of the electrogenic sodium pump was investigated in voltage-clamped internally dialyzed giant axons of the squid, Loligo pealei, under conditions that promote the backward-running mode (K(+)-free seawater; ATP- and Na(+)-free internal solution containing ADP and orthophosphate). The ratio of pump-mediated (42)K(+) efflux to reverse pump current, I(pump) (both defined by sensitivity to dihydrodigitoxigenin, H(2)DTG), scaled by Faraday's constant, was -1.5 +/- 0.4 (n = 5; expected ratio for 2 K(+)/3 Na(+) stoichiometry is -2.0). Steady-state reverse pump current-voltage (I(pump)-V) relationships were obtained either from the shifts in holding current after repeated exposures of an axon clamped at various V(m) to H(2)DTG or from the difference between membrane I-V relationships obtained by imposing V(m) staircases in the presence or absence of H(2)DTG. With the second method, we also investigated the influence of [Na(+)](o) (up to 800 mM, for which hypertonic solutions were used) on the steady-state reverse I(pump)-V relationship. The reverse I(pump)-V relationship is sigmoid, I(pump) saturating at large negative V(m), and each doubling of [Na(+)](o) causes a fixed (29 mV) rightward parallel shift along the voltage axis of this Boltzmann partition function (apparent valence z = 0.80). These characteristics mirror those of steady-state (22)Na(+) efflux during electroneutral Na(+)/Na(+) exchange, and follow without additional postulates from the same simple high field access channel model (Gadsby, D.C., R.F. Rakowski, and P. De Weer, 1993. Science. 260:100-103). This model predicts valence z = nlambda, where n (1.33 +/- 0.05) is the Hill coefficient of Na binding, and lambda (0.61 +/- 0.03) is the fraction of the membrane electric field traversed by Na ions reaching their binding site. More elaborate alternative models can accommodate all the steady-state features of the reverse pumping and electroneutral Na(+)/Na(+) exchange modes only with additional assumptions that render them less likely.

Topics: Animals; Axons; Biological Transport, Active; Decapodiformes; Digitoxigenin; Enzyme Inhibitors; Kinetics; Membrane Potentials; Models, Biological; Ouabain; Patch-Clamp Techniques; Potassium Radioisotopes; Sodium; Sodium-Potassium-Exchanging ATPase

The stoichiometry and voltage dependence of the Na/K pump were studied in internally dialyzed, voltage-clamped squid giant axons by simultaneously measuring, at various membrane potentials, the changes in Na efflux (delta phi Na) and holding current (delta I) induced by dihydrodigitoxigenin (H2DTG). H2DTG stops the Na/K pump without directly affecting other current pathways: (a) it causes no delta I when the pump lacks Na, K, Mg, or ATP, and (b) ouabain causes no delta I or delta phi Na in the presence of saturating H2DTG. External K (Ko) activates Na efflux with Michaelis-Menten kinetics (Km = 0.45 +/- 0.06 mM [SEM]) in Na-free seawater (SW), but with sigmoid kinetics in approximately 400 mM Na SW (Hill coefficient = 1.53 +/- 0.08, K1/2 = 3.92 +/- 0.29 mM). H2DTG inhibits less strongly (Ki = 6.1 +/- 0.3 microM) in 1 or 10 mM K Na-free SW than in 10 mM K, 390 mM Na SW (1.8 +/- 0.2 microM). Dialysis with 5 mM each ATP, phosphoenolpyruvate, and phosphoarginine reduced Na/Na exchange to at most 2% of the H2DTG-sensitive Na efflux. H2DTG sensitive but nonpump current caused by periaxonal K accumulation upon stopping the pump, was minimized by the K channel blockers 3,4-diaminopyridine (1 mM), tetraethylammonium (approximately 200 mM), and phenylpropyltriethylammonium (20-25 mM) whose adequacy was tested by varying [K]o (0-10 mM) with H2DTG present. Two ancillary clamp circuits suppressed stray current from the axon ends. Current and flux measured from the center pool derive from the same membrane area since, over the voltage range -60 to +20 mV, tetrodotoxin-sensitive current and Na efflux into Na-free SW, under K-free conditions, were equal. The stoichiometry and voltage dependence of pump Na/K exchange were examined at near-saturating [ATP], [K]o and [Na]i in both Na-free and 390 mM Na SW. The H2DTG-sensitive F delta phi Na/delta I ratio (F is Faraday's constant) of paired measurements corrected for membrane area match, was 2.86 +/- 0.09 (n = 8) at 0 mV and 3.05 +/- 0.13 (n = 6) at -60 to -90 mV in Na-free SW, and 2.72 +/- 0.09 (n = 7) at 0 mV and 2.91 +/- 0.21 (n = 4) at -60 mV in 390 mM Na SW. Its overall mean value was 2.87 +/- 0.07 (n = 25), which was not significantly different from the 3.0 expected of a 3 Na/2 K pump.(ABSTRACT TRUNCATED AT 400 WORDS)

Topics: Animals; Axons; Chemical Phenomena; Chemistry; Decapodiformes; Dialysis; Digitoxigenin; Electrophysiology; Mathematics; Potassium; Potassium Channels; Sodium; Sodium Channels

In many studies of the sodium pump in epithelia, a readily reversible analog of ouabain would be most useful. This would enable studies of pump activity to be made under control and experimental conditions on the same tissue. Of three compounds examined on the basolateral membrane of the isolated epithelia of frog skin, dihydroouabain (DHO) had characteristics very similar to ouabain except that it was apparently much more reversible. DHO (1 mmol/l) inhibited short circuit current (Isc) and transepithelial Na flux (JNa13) in a fashion similar to ouabain. Isc was inhibited from 17.0 +/- 2.5 to 10.2 +/- 1.0 microA/cm2 in 2-4 min while JNa13 was decreased from 16.8 +/- 1.9 to 4.7 +/- 0.8 microA/cm2 in the same time interval. After 60 min of washout, Isc and JNa13 recovered to about 70% of control values and were nearly equal. In another set of experiments, the washout of DHO and ouabain were compared directly on the same tissue. Sodium flux recovered four times faster after removal of DHO when compared to ouabain. Pretreatment of tissues with DHO prior to ouabain greatly increased the rate of Na flux recovery after washout of both drugs suggesting that DHO competes for ouabain sites. These data suggest that DHO can be used as a reversible analog for ouabain in studies of the Na pump in frog skin.

Topics: Animals; Digitoxigenin; In Vitro Techniques; Ion Channels; Ouabain; Rana pipiens; Skin; Sodium; Strophanthidin

The nature of Na+ fluxes in resting and in chemotactic factor-activated human neutrophils was investigated. In resting cells, ouabain-insensitive unidirectional 22Na+ in- and effluxes represented passive electrodiffusional fluxes through ion channels: they were nonsaturable and voltage-dependent (PNa = 4.3 X 10(-9) cm/s). Amiloride (1 mM) had little effect on resting 22Na+ influx (approximately 0.8 meq/liter X min), thereby suggesting a minor contribution of Na+/H+ exchange and a lack of amiloride-sensitive Na+ channels. When neutrophils were exposed to the chemotactic tripeptide N-formyl-methionyl-leucyl-phenylalanine (FMLP, 0.1 microM), 22Na+ influx was stimulated approximately 30-fold (initial rate approximately 22 meq/liter X min). The FMLP-induced 22Na+ influx was saturable with respect to external Na+ (Km 26-35 mM, Vmax approximately 28 meq/liter X min), was electroneutral, and could be competitively inhibited by amiloride (Ki 10.6 microM). From a resting value of approximately 30 meq/liter of cell water, internal Na+ in FMLP-stimulated cells rose exponentially to reach a concentration of approximately 60 meq/liter by 10-15 min. This uptake was blocked by amiloride. FMLP also stimulated the efflux of 22Na+ which followed a single exponential time course (rate coefficient approximately 0.16 min-1). The FMLP-induced 22Na+ fluxes were similar to those observed with 10 microM monensin, a known Na+/H+ exchanging ionophore. The data indicate that FMLP activates an otherwise quiescent, amiloride-sensitive Na+/H+ exchange. Furthermore, all of the FMLP-induced 22Na+ fluxes can be satisfactorily accounted for by transport through the exchanger, leaving little room for an appreciable increase in Na+ conductance.

Topics: Amiloride; Carrier Proteins; Cell Membrane Permeability; Chlorides; Digitoxigenin; Humans; Kinetics; Membrane Potentials; Monensin; N-Formylmethionine Leucyl-Phenylalanine; Neutrophils; Ouabain; Potassium; Sodium; Sodium-Hydrogen Exchangers

Topics: Adrenal Glands; Bufanolides; Digitoxigenin; Iodine Radioisotopes; Radionuclide Imaging

The inotropic potencies of four digitalis genins were studied utilizing cat left atrial strips. The genin concentration required to induce a 50% increase of isometric tension (T50) was found to closely correlate with the degree of displacement (D) of the C(17) side-group carbonyl oxygen from the position of that atom in digitoxigenin. The line of regression was: log T50 = 0.54D - 6.85, r2 = 0.98, P less than 0.008. These observations were related to recently reported cat ventricular Na+, K+ -ATPase inhibitory potencies of the same genins [expressed as 50% inhibitory (I50) concentrations]. I50 correlated strongly with T50: log I50 = 0.78 log T50 - 1.68, r2 = 0.99, P less than 0.003. Thus, the activity of digitalis genins towards their receptor in intact cardiac tissue is closely related to genin carbonyl oxygen position as well as to Na+, K+ -ATPase inhibitory activity. These results further support our earlier conclusions, based upon isolated Na+, K+ -ATPase studies, that the digitalis genin C(17) side-group carbonyl oxygen position versus activity relationship is biologically relevant and may prove to be a useful unifying structural model in the further elucidation of the mechanism of digitalis-receptor interactions.

Topics: Animals; Cardiotonic Agents; Cats; Digitoxigenin; Digoxigenin; Digoxin; Dose-Response Relationship, Drug; Myocardial Contraction; Sodium-Potassium-Exchanging ATPase; Structure-Activity Relationship; X-Ray Diffraction