Ventricular arrhythmias related to antibiotic usage in dogs.

In dogs, pretreatment with the macrolide antibiotic tylosin (5 milligrams per day per kilogram of body weight) increased the incidence of ventricular tachycardia and fibrillation during acute myocardial ischemia. Another group received a dose of acetyl strophanthidin which was nontoxic in controls, but which resulted in a ventricular arrhythmia in six of seven animals on antibiotic treatment. Enhancement of loss of potassium ion from the myocardium by the antibiotic was presumed to be related to the altered cardiac rhythm.

Concentration dependence of active potassium transport in the human red blood cell in the presence of inhibitors.

The active potassium influx in the human red blood cell is inhibited by strophanthidin, ethacrynic acid, and MK-870 (a new diuretic), and the degree of inhibition is greater at low concentrations of extracellular potassium than at high. In the case of ethacrynic acid, potassium appears to diminish the rate of combination of the drug with the transport system. The kinetic behavior of the active potassium influx in the presence of the inhibitors strophanthidin and ethacrynic acid is consistent with a model in which the binding of potassium at one of the potassium-sensitive sites in the transport system reduces the affinity of the system for the drug, and binding of a second potassium ion further reduces the affinity. It is not possible to distinguish between the sites on the basis of the studies presented here.

Influence of the thyroid state on the intrinsic contractile properties and energy stores of the myocardium.

The intrinsic contractile properties of isolated cat papillary muscles and myocardial high energy phosphate stores were examined at three levels of thyroid activity and correlated with hemodynamic measurements in the intact animal. In addition, the relationship of thyroid state to endogenous norepinephrine stores and myocardial responsiveness to certain inotropic interventions were studied. In muscles from hyperthyroid cats, the velocity of shortening and the rate of tension development were markedly augmented, while duration of active state was decreased, compared to euthyroid muscles. These findings occurred in the presence and absence of intact norepinephrine stores and over a wide range of temperature and contraction frequency. The opposite changes occurred in muscles from hypothyroid cats. Isometric tension was slightly higher in muscles from hyperthyroid and lower in muscles from hypothyroid cats. The inotropic response to both norepinephrine and strophanthidin varied inversely with the level of thyroid state and allowed all three groups of muscles to reach a common ceiling of isometric tension regardless of thyroid state. Creatine phosphate and adenosine triphosphate stores were intact at all three levels of thyroid state. Thus, the level of thyroid activity profoundly affects the intrinsic contractile state of cardiac muscle, independent of both norepinephrine stores and alterations in high energy phosphate stores, and, in addition, modifies the responsiveness of cardiac muscle to inotropic agents.

Reversal of ouabain and acetyl strophanthidin effects in normal and failing cardiac muscle by specific antibody.

Isolated cat right ventricular papillary muscles were used to study the effects of antibodies with high affinity for ouabain and acetyl strophanthidin on myocardium exposed to these cardioactive steroids. Antibodies with average intrinsic affinity constants for ouabain and acetyl strophanthidin of the order of 10(8) M(-1) were raised in rabbits challenged by repeated injection of a conjugate of ouabain covalently linked to a poly D,L-alanyl derivative of human serum albumin. Effects were assessed in terms of time-course and extent of inotropy reversal, influence of experimentally induced ventricular failure, digitalis-antibody concentration relations, influence of digitalis-antibody complex on response to additionally added digitalis, and relation of antibody effects on digitalis-induced automaticity and contracture to reversal of inotropy. Specific antibody (but not control antibody) in 1.1-1.5-fold molar excess over cardioactive steroid concentrations blocked positive inotropic effects of ouabain and acetyl strophanthidin, and gradually reversed established contractile effects of these agents with a mean time for half-reversal of ouabain-induced inotropy of 124+/-6 (SEM) min and 37+/-3 min for half-reversal of acetyl strophanthidin-induced inotropy. Papillary muscles from cats with right ventricular failure induced by chronic pulmonary artery constriction responded similarly. Both normal and failing muscles returned to but not below levels of contractility existing before cardioactive steroid exposure, and time for half-reversal of inotropy by antibody was significantly shorter than time for half-reversal after removal of ouabain or acetyl strophanthidin by muscle bath washout alone. Presence of ouabain- or acetyl strophanthidin-antibody complex did not alter the myocardial contractile response to subsequently added cardioactive steroids. Spontaneous automaticity occurring as a toxic response to ouabain or acetyl strophanthidin in eight muscles was rapidly reversed by specific antibody at a time when positive inotropic effects were still fully manifest. Early contracture was also reversed by specific antibody. These studies provide further support for the concept that cardiac glycoside-specific antibodies are capable of reversing established cellular effects of cardioactive steroids.

Digitalis-induced increase in aortic regurgitation and the contrasting effect of glucagon in the sedated dog.

The hemodynamic and phasic ascending aortic flow changes induced by acetylstrophanthidin and glucagon were studied in closed-chest sedated dogs with aortic regurgitation. While the positive inotropic effect of both agents was reflected in an increase in peak rate of rise of left ventricular pressure, acetylstrophanthidin increased aortic regurgitation, while glucagon decreased it. With the former, left ventricular end-diastolic pressure rose from 20+/-6 to 27+/-6 mm Hg (P < 0.005), but fell from 18+/-4 to 11+/-3 mm Hg (P < 0.001) with glucagon. Acetylstrophanthidin increased systemic vascular resistance, aortic diastolic pressure, and diastolic regurgitant flow rate, and, heart rate and the duration of regurgitation per beat and per minute being unchanged, regurgitant flow per beat increased 32+/-15% (P < 0.001). Glucagon decreased regurgitant flow per beat 27+/-14% (P < 0.001) because of abbreviation of diastole associated with tachycardia, and because of reduction in regurgitant flow rate. Despite tachycardia, the duration of regurgitation per minute was unchanged, and the small fall in regurgitant blood flow per minute was not significant, but this pertained in the face of 47% increase in effective cardiac output (P < 0.001). In contrast, acetylstrophanthidin increased regurgitant flow per minute 28+/-14% (P < 0.001) without change in effective cardiac output. The increase in cardiac contractility, tachycardia, and systemic vasodilatation induced by glucagon preferentially enhanced forward blood flow, which led to reduction in left ventricular volume overload, while it increased cardiac output. Contrarily, acetylstrophanthidin increased aortic regurgitation and, despite its inotropic effect, increased left ventricular volume overload without an increase in cardiac output.

Sodium extrusion by internally dialyzed squid axons.

A method has been developed which allows a length of electrically excitable squid axon to be internally dialyzed against a continuously flowing solution of defined composition. Tests showed that diffusional exchange of small molecules in the axoplasm surrounding the dialysis tube occurred with a half-time of 2-5 min, and that protein does not cross the wall of the dialysis tube. The composition of the dialysis medium was (mM): K isethionate 151, K aspartate 151, taurine 275, MgCI(2) 4-10, NaCl 80, KCN 2, EDTA 0.1, ATP 5-10, and phosphoarginine 0-10. The following measurements were made: resting Na influx 57 pmole/cm(2)sec (n = 8); resting potassium efflux 59 pmole/ cm(2)sec (n = 4); stimulated Na efflux 3.1 pmole/cm(2)imp (n = 9); stimulated K efflux 2.9 pmole/cm(2)imp (n = 3); resting Na efflux 48 pmole/cm(2)sec (n = 18); Q(10) Na efflux 2.2 (n = 5). Removal of ATP and phosphoarginine from the dialysis medium (n = 4) or external application of strophanthidin (n = 1) reversibly reduced Na efflux to 10-13 pmole/cm(2)sec. A general conclusion from the study is that dialyzed squid axons have relatively normal passive permeability properties and that a substantial fraction of the Na efflux is under metabolic control although the Na extrusion mechanism may not be working perfectly.

Strophanthidin-sensitive components of potassium and sodium movements in skeletal muscle as influenced by the internal sodium concentration.

"Low sodium" muscles were prepared which contained around 5 mmoles/kg fiber of intracellular sodium. "High sodium" muscles containing between 15 and 30 mmoles/kg fiber of intracellular sodium were also prepared. In low sodium muscles application of 10(-5)M strophanthidin reduced potassium influx by about 5%. Potassium efflux was unaffected by strophanthidin under these conditions. In high sodium muscles, 10(-5)M strophanthidin reduced potassium influx by 45% and increased potassium efflux by 70%, on the average. In low sodium muscles sodium efflux was reduced by 25% during application of 10(-5)M strophanthidin while in high sodium muscles similarly treated, sodium efflux was reduced by about 60%. Low sodium muscles showed a large reduction in sodium efflux when sodium ions in the Ringer solution were replaced by lithium ions. The average reduction in sodium efflux was 4.5-fold. Of the amount of sodium efflux remaining in lithium. Ringer's solution, 40% could be inhibited by application of 10(-5)M strophanthidin. The total sodium efflux from low sodium muscles exposed to Ringer's solution in which lithium had been substituted for sodium ions for a period of 1 hr can be fractionated as 78% Na-for-Na interchange, 10% strophanthidin-sensitive sodium pump, and 12% residual sodium efflux. It is concluded that large strophanthidin-sensitive components of sodium and potassium flux can be expected only at elevated sodium concentrations within the muscle cells.

The dual effect of lithium ions on sodium efflux in skeletal muscle.

Sartorius muscle cells from the frog were stored in a K-free Ringer solution at 3 degrees C until their average sodium contents rose to around 23 mM/kg fiber (about 40 mM/liter fiber water). Such muscles, when placed in Ringer's solution containing 60 mM LiCl and 50 mM NaCl at 20 degrees C, extruded 9.8 mM/kg of sodium and gained an equivalent quantity of lithium in a 2 hr period. The presence of 10(-5)M strophanthidin in the 60 mM LiCl/50 mM NaCl Ringer solution prevented the net extrusion of sodium from the muscles. Lithium ions were found to enter muscles with a lowered internal sodium concentration at a rate about half that for entry into sodium-enriched muscles. When sodium-enriched muscles labeled with radioactive sodium ions were transferred from Ringer's solution to a sodium-free lithium-substituted Ringer solution, an increase in the rate of tracer sodium output was observed. When the lithium-substituted Ringer solution contained 10(-5)M strophanthidin, a large decrease in the rate of tracer sodium output was observed upon transferring labeled sodium-enriched muscles from Ringer's solution to the sodium-free medium. It is concluded that lithium ions have a direct stimulating action on the sodium pump in skeletal muscle cells and that a significantly large external sodium-dependent component of sodium efflux is present in muscles with an elevated sodium content. In the sodium-rich muscles, about 23% of the total sodium efflux was due to strophanthidin-insensitive Na-for-Na interchange, about 67% being due to strophanthidin-sensitive sodium pumping.

Interaction of external K, Na, and cardioactive steroids with the Na-K pump of the human red blood cell.

The interaction of extracellular Na (Na(o)), K (K(o)), and strophanthidin with the Na-K pump of the human red blood cell has been investigated. Inhibition by submaximal concentrations of strophanthidin rapidly reaches a level which does not increase further over a relatively long period of time. Under these circumstances, it is possible to apply a steady-state kinetic analysis to the interaction of Na(o), K(o), and strophanthidin with the pump. In Na-free solutions, strophanthidin increases the apparent K(1/2) of the pump for K(o), but does not change the form of the relation between the reciprocal of the active K influx ((i)M(K) (P-1)) and the reciprocal of [K(o)] ([K(o)](-1)); the relation both in the presence and absence of strophanthidin is adequately described by a straight line. In solutions containing Na, strophanthidin changes the form of the curve describing the relation between (i)M(K) (P-1) vs. [K(o)](-1); the curve becomes more parabolic in solutions containing strophanthidin. The rate of ouabain binding to K-free cells has also been measured; in the absence of K, the rate of binding is unaffected by Na(o). The data are considered in terms of a simple kinetic model. The findings can be explained if it is supposed that at low external K the form of the pump combined with one Na(o) is more likely to combine with strophanthidin than is the uncombined form of the pump. The uncombined form of the pump is more likely to combine with K even at very low K(o) than with strophanthidin.

Sodium fluxes in internally dialyzed squid axons.

The effects which alterations in the concentrations of internal sodium and high energy phosphate compounds had on the sodium influx and efflux of internally dialyzed squid axons were examined. Nine naturally occurring high energy phosphate compounds were ineffective in supporting significant sodium extrusion. These compounds were: AcP, PEP, G-3-P, ADP, AMP, GTP, CTP, PA, and UTP.(1) the compound d-ATP supported 25-50% of the normal sodium extrusion, while ATP supported 80-100%. The relation between internal ATP and sodium efflux was nonlinear, rising most steeply in the range 1 to 10 microM and more gradually in the range 10 to 10,000 microM. There was no evidence of saturation of efflux even at internal ATP concentrations of 10,000 microM. The relation between internal sodium and sodium efflux was linear in the range 2 to 240 mM. The presence of external strophanthidin (10 microM) changed the sodium efflux to about 8-12 pmoles/cm(2) sec regardless of the initial level of efflux; this changed level was not altered by subsequent dialysis with large concentrations of ATP. Sodium influx was reduced about 50 % by removal of either ATP or Na and about 70 % by removing both ATP and Na from inside the axon.

An electrogenic sodium pump in Limulus ventral photoreceptor cells.

A hyperpolarization can be recorded intracellularly following either a single bright light stimulus or the intracellular injection of Na(+). This after-hyperpolarization is abolished by bathing in 5 x 10(-6)M strophanthidin or removal of extracellular K(+). Both treatments also lead to a small, rapid depolarization of the dark-adapted cell. When either treatment is prolonged, light responses can still be elicited, although with repetitive stimuli the responses are slowly and progressively diminished in size. The rate of diminution is greater for higher values of [Ca(++)](out); with [Ca(++)](out) = 0.1 mM, there is almost no progressive diminution of repetitive responses produced by either K(+)-free seawater or strophanthidin. We propose that an electrogenic Na(+) pump contributes directly to dark-adapted membrane voltage and also generates the after-hyperpolarizations, but does not directly generate the receptor potential. Inhibition of this pump leads to intracellular accumulation of sodium ions, which in turn leads to an increase in intracellular Ca(++) (provided there is sufficient extracellular Ca(++)). This increase in intracellular calcium probably accounts for the progressive decrease in the size of the receptor potential seen when the pump is inhibited.

Fractionation of sodium effux in frog sartorius muscles by strophanthidin and removal of external sodium.

The influence of strophanthidin, ouabain, and the removal of external sodium on the sodium efflux from frog sartorius muscle was measured. In freshly dissected muscles strophanthidin and ouabain in maximally effective concentrations reduced the efflux of sodium by about 50%. Of the sodium efflux which is strophanthidin-insensitive about 75% is inhibited after complete replacement of external sodium by lithium. In the absence of strophanthidin replacement of external sodium by lithium, calcium, or magnesium produces an initial rise in the sodium efflux, followed by a fall in the efflux as the exposure of the muscles to sodium-free media is continued. When the muscles are exposed for prolonged periods in sodium-free media, the fraction of internal sodium lost per minute is higher when returned to normal Ringer fluid than it was initially. The activation of sodium efflux by external sodium after long periods in sodium-free solutions is partly strophanthidin-sensitive and partly strophanthidin-insensitive. The internal sodium concentration is an important factor in these effects. The effects of temperature on the sodium efflux were also measured. Above 7 degrees C the Q(10) of both the strophanthidin-sensitive and strophanthidin-insensitive sodium efflux is about 2.0. Below 7 degrees C the strophanthidin-insensitive sodium efflux has a Q(10) of about 7.4.

Fluorometric measurement of pyridine nucleotide reduction in the giant axon of the squid.

By monitoring the fluorescence of the isolated giant axon of the squid Loligo pealei, it was possible to follow changes in its oxidation-reduction state as caused by the action of anoxia, cyanide, Amytal, and azide. The response to oxygen depletion was very rapid, the NAD within the axon being 90% reduced within 1-2 min. Cyanide and Amytal gave essentially similar results, although somewhat longer periods of time elapsed during their onset and washout periods. The extent of NAD reduction was essentially the same under conditions of anoxia and treatment with cyanide and Amytal. Azide was less effective in this respect, and at comparatively high levels of concentration (25-50 mM) gave values of 40% or less of the reduction observed with the other inhibitors. The application of ouabain and strophanthidin gave no observable NAD reduction. Variations in the time required to consume given quantities of dissolved oxygen before and after stimulation indicated an increase of 10-20% in oxygen uptake rate associated with activity, although this figure appeared to be a function of the surface-to-volume ratio of the axon. A biochemical analysis of axoplasm for oxidized and reduced pyridine nucleotide was made. Fluorometric examination of centrifuged axoplasm indicated that the NAD-NADH was largely confined to the mitochondria of the axon.

Effects of intracellular adenosine-5'-diphosphate and orthophosphate on the sensitivity of sodium efflux from squid axon to external sodium and potassium.

A study was made of sodium efflux from squid giant axon, and its sensitivity to external K and Na. When sodium efflux from untreated axons was strongly stimulated by K(o), Na(o) was inhibitory; when dependence on K(o) was low, Na(o) had a stimulatory effect. Incipient CN poisoning or apyrase injection, which produces high intracellular levels of ADP(1) and P(i), rendered sodium efflux less dependent on external K and more dependent on external Na. Injection of ADP, AMP, arginine, or creatine + creatine phosphokinase, all of which raise ADP levels without raising P(i) levels, had the same effect as incipient CN poisoning. P(i) injection had no effect on the K sensitivity of sodium efflux. Axons depleted of arginine and phosphoarginine by injection of arginase still lost their K sensitivity when the ATP:ADP ratio was lowered and regained it partially when the ratio was raised. Rough calculations show that sodium efflux is maximally K(o)-dependent when the ATP:ADP ratio is about 10:1, becomes insensitive to K(o) when the ratio is about 1:2, and is inhibited by K(o) when the ratio is about 1:10. Deoxy-ATP mimicked ADP when injected into intact axons. Excess Mg, as well as P(i), inhibited both strophanthidin-sensitive and strophanthidin-insensitive sodium efflux. An outline is presented for a model which might explain the effects of ADP, P(i) and deoxy-ATP.

Cardenolide analogues. 4. (20R)- and (20S)-Cardanolides: on the roles of the 20(22)-ene and 14beta-hydroxyl in genin activity.

(20R)-20,22-Dihydrodigitoxigenin (3a) and (20S)-20,22-dihydrodigitoxigenin (3b) were isolated from (20R,S)-20,22-dihydrodigitoxigenin (3) by three fractional crystallizations each from ethyl acetate. The two diastereomers have distinct NMR spectra and similar (Na+,K+)ATPase inhibitory activities (I50 = 1.1-1.4 X 10(-5) M)--about 1/100 as active as digitoxigenin (1). Their activity compared with other cardenolide analogues suggests a passive geometric role for the 20(22) double bond in eliciting (Na+,K+)ATPase inhibition, keeping the lactone carbonyl in the proper orientation. (20S)-3 beta,14 beta-Dihydroxy-22-methylene-5 beta,14 beta-cardanolide (7a) was then synthesized from 3a, and (20R)-3 beta,14 beta-dihydroxy-22-methylene-5 beta,14 beta-cardanolide (7b) from 3b. They were found to be equivalently active in inhibiting (Na+,K+)ATPase, with I50 values of 7.0 x 10(-5) M. Although it has been usually believed that the 14 beta-hydroxyl of cardenolides increases binding to the receptor, 2b (the 14-ene derivative of 7b) was more than twice as active (I50 = 3.0 X 10(-5)) than either 7a or 7b.

Role of catecholamines in the central mechanism of emetic response induced by peruvoside and ouabain in cats.

1 Peruvoside, (a glycoside obtained from the plant, Thevetia neriifolia Juss) and ouabain produce emesis in cats. Vomiting is not produced by these drugs in animals pretreated with catecholamine depleting drugs like reserpine, tetrabenazine or syrosingopine. Chloropromazine hydrochloride, mepyramine maleate, or BOL-148 administered intravenously or intracerebro-ventricularly do not afford protection.2 Phenoxybenzamine produces partial protection against peruvoside-induced emesis.3 Haloperidol (1 mg/kg i.v.) prevents vomiting induced by peruvoside or ouabain. Intracerebroventricularly administered haloperidol is ineffective.4 Cats pretreated with SKF-525-A, are not protected by haloperidol. Animals pretreated with phenobarbitone in a dose of 25 mg/kg for a week were protected by haloperidol, 250 mug/kg i.e. one quarter of the effective antiemetic dose in normal cats.5 It is postulated that catecholamines are involved in the mechanism of vomiting induced by cardiac gycosides. Further, a metabolite of haloperidol seems to be responsible for its effective antiemetic action.

Cardiotonic steroids: correlation of sodium-potassium adenosine triphosphate inhibition and ion transport in vitro with inotropic activity and toxicity in dogs.

1. A new series of cardiotonics based on five steroid nuclei has been evaluated for inhibition of Na-+/K-+-ATPase and Rb uptake by red blood cells, and for inotropic activity and toxicity in dogs. Structure-activity relationships are discussed. 2. The in vitro tests can be used satisfactorily to predict inotropic activity, but not toxicity or therapeutic ratio. 3. Although compounds with greatly improved therapeutic ratios relative to ouabain and tolusin have been obtained, they proved to be strongly emetic in the conscious dog.

Reversible inhibition of (Na+ + K+)-ATPase with a cardiac glycoside.

The effect of a semisynthetic cardiac glycoside, Actinogen (Ay22241), on Na+ + K+ - ATPase was studied. Ay22241 was found to be as an effective inhibitor of the enzyme as ouabain, Ay22241 inhibition was a time dependent process and was completely reversible. While ouabain inhibition was also time dependent, it was only partially reversible. This reversibility with Ay22241 should make it a useful tool in studying the mode of action of cardiac glycosides.

The association with receptors regulates the Na+,K(+)-ATPase inhibitory potency of some cardioactive steroids.

The onset of inhibition of Na+,K(+)-ATPase from guinea-pig myocardium was quantified with pseudo-first-order rate constants in a series of 14 cardioactive steroids. From these data the association and dissociation rate constants of the steroid-receptor complex were calculated. It was then found that the association of the steroids with receptors but not the dissociation of the steroid-receptor complex determined the largely different inhibitory potencies. Consistent with this finding, at equieffective steroid concentrations the rates of inhibition varied only slightly. The correlation of the association rate with the hydrophobicity of the compounds suggests that hydrophobic interactions facilitate the access of the steroid to the receptor. A conformational transition of the vicinity of the receptor subsequent to the formation of the steroid-receptor complex seems to alter the hydrophobic properties of the receptor environment to make the dissociation rate independent from hydrophobicity.

Effects of AY-22,241 (Actodigin) on electrical and mechanical activity of cardiac tissues.

AY-22,241 (Actodigin) is a new rapid-acting semisynthetic cardiotonic steroid. In experiments on contractility of cat papillary muscle, Actodigin (2 times 10(-7) to 4 times 10(-6) M) produced a dose-dependent positive inotropic effect, a marked increase in the maximum rate of force development, and no change in resting tension. Electrophysiologic studies performed with microelectrode techniques on isolated Purkinje fibers superfused with Tyrode solution, revealed dose-dependent decreases in resting membrane potential, action potential amplitude and duration, and the maximum rate of rise of phase 0 (Vmax). Purkinje fibers superfused with extracorporeally circulated blood from a donor dog receiving 0.075 mg/kg/min Actodigin showed small decreases in resting membrane potential preceding the onset of donor premature ventricular contractions. Progressive decreases in resting membrane potential, action potential amplitude and duration, and Vmax accompanied donor ventricular tachycardia. All effects were rapidly reversible, and compared to ouabain, equi-inotropic concentrations of Actodigin caused significantly less electrophysiologic toxicity.