The influence of ouabain on twitch potentiation by anticholinesterases in the phrenic nerve-diaphragm muscles of mice.

1. (+)-Tubocurarine, hexamethonium, atropine, ouabain, and removal of potassium from the bathing medium were examined for their effects on indirectly evoked twitches (IT) of mouse phrenic nerve-diaphragm muscles in the presence or absence of neostigmine. 2. Neostigmine increased the amplitude of IT. The twitch potentiation was reduced by (+)-tubocurarine at low concentrations that had no inhibitory effect on normal IT. Hexamethonium (10-100 microM), but not atropine (0.1-1 microM), partially inhibited the twitch potentiation. Neither hexamethonium nor atropine had an inhibitory effect on IT in the absence of neostigmine. 3. Ouabain (5 microM) abolished the twitch potentiation by neostigmine while having no inhibitory effect on directly evoked twitches in the presence of neostigmine and (+)-tubocurarine together. 4. The potentiating effect of neostigmine was less in a potassium-free bathing solution. The inhibitory effect of ouabain disappeared in this solution. 5. Reinclusion of KCl at 2.5 mM restored both the potentiating effect of neostigmine and the antagonistic effect of ouabain. This reinclusion did not potentiate IT in the absence of neostigmine. 6. An interaction resembling that between ouabain and neostigmine was obtained between ouabain and physostigmine or paraoxon. 7. Both endplate potentials (e.p.ps) and miniature e.p.ps increased in terms of their amplitude and duration in the presence of neostigmine. Ouabain did not reduce the enhanced endplate responses. 8. These results indicate that the potentiation of IT by anticholinesterases may occur via nicotinic receptors which are sensitive to both (+)-tubocurarine and hexamethonium, and that the interaction between anticholinesterases and ouabain depends on the presence of K+. It appears that the mechanisms of twitch potentiation are dependent on the ionic gradients maintained by Na+-K+-ATPase.     

Inhibition by ouabain and veratridine of acetylcholine-evoked phasic contraction in the guinea-pig taenia coli

Summary Inhibitory action of ouabain and veratridine on acetylcholine (ACh)-evoked phasic contraction was examined in the guinea-pig taenia coli. ACh (5×10^–4 m) produced a biphasic (phasic and tonic) contraction. As the phasic contraction, but not the tonic contraction, was resistant to gallopamil (D600, 2 × 10^–7 m), a blocker of voltage-dependent Ca²⁺ channels, all experiments were carried out in the presence of gallopamil at this concentration. Addition of high Ca²⁺ (30 mm) to the solution containing ACh induced a sustained contraction, which increased in the presence of ouabain (10^–5 m), an inhibitor of the Na+-K+ exchange system. On the other hand, the ACh-evoked phasic contraction was suppressed by ouabain 10^–7–(10^–5 m) in a time- and dose-dependent manner. The suppression by ouabain (10^–6 m) of the phasic contraction was transiently potentiated by the addition of veratridine (10^–6 m), an activator of Na⁺ channel. In contrast, the greater suppression by ouabain (10^–5 m) of the contraction was antagonized by amiloride (10^–4 m), a blocker of Na+ channel. This antagonism by amiloride was transiently inhibited in the presence of veratridine. In the absence of ouabain, the amplitude of the phasic contraction was transiently reduced by adding veratridine but was increased by amiloride. In addition, the phasic contraction by ACh increased 80 min after exposure to Na⁺-free isotonic high-K⁺ solution (K⁺, 143 mm), which elicited greater depolarization of the cell membrane. In a fluorescence study with Fura-2, an intracellular free-Ca²⁺ indicator, ACh increased the fluorescence intensity from the tissue by excitative light at 340 nm, which coupled with the phasic contraction. Both the increased fluorescence and tension development were reduced by ouabain. These findings suggest that the ACh-evoked phasic contraction is induced by an increase in intracellular free-Ca²⁺ levels and seems to be inhibited by an elevation of intracellular free-Na⁺ levels. Send offprint requests to: S. Usune at the above address     

The influence of metiamide on ouabain cardiotoxicity.

The effect of metiamide on the cardiotoxicity produced by ouabain was studied in pentobarbital-anesthetized cats. The onset of ouabain-induced ventricular tachycardia and fibrillation was significantly delayed in cats treated with metiamide as compared with cats that did not receive metiamide. Although the mechanism by which metiamide inhibits ouabain toxicity is speculative, the data suggest that histamine H2-receptor blocking agents may be useful as anti-arrhythmic drugs in digitalis cardiotoxicity.     

Effect of ouabain on norepinephrine-induced contractions of isolated rabbit aorta

The acute potentiating effect of ouabain on norepinephrine (NE) induced contractions of isolated rabbit aorta was investigated. Ouabain, at concentrations of 3 X 10(-7) to 10(-5) mol/l potentiated the vasoconstrictor effect of NE as demonstrated by a shift to the left of the NE concentration-response curve in a parallel manner and a concentration-dependent increase in the EC50 ratio. A significant increase in maximal contractility was observed by ouabain at 3 X 10(-6) and 10(-5) mol/l. Removal of the endothelium did not alter the effect of 10(-5) mol/l ouabain. When the NE concentration-response curves were again determined after ouabain had been washed out, it was found that the ouabain-induced increase in EC50 ratios returned to control values, while the increase in maximal contractility did not. The slopes of the NE concentration-response curves were not significantly different in the presence of ouabain than control values, but were higher when obtained after ouabain had been washed out.     

The influence of propranolol on ouabain uptake by the guinea pig heart

The action of propranolol on cardiotoxicity produced by ouabain was studied in intact and isolated guinea pig hearts. The lethal event in the intact guinea pig was ventricular fibrillation; in the isolated heart it was asystole. Myocardial ouabain content at death was significantly higher in isolated preparations than in the intact heart. Propranolol was observed to alter the mode of death in the intact guinea pig from ventricular fibrillation to asystole. The myocardial ouabain content at the time of death was elevated in guinea pigs pretreated with propranolol to the level observed in isolated guinea pig heart preparations. Exposure of the isolated preparation to propranolol prolonged the time to the induction of cardiac rhythm disorders and death without changing myocardial ouabain content. These data suggest that propranolol, by eliminating neural influence transforms the cardiotoxicity of ouabain to an in vitro mechanism. In the isolated heart, propranolol prolongs the time to the induction of cardiac rhythm disorders by inhibiting the myocardial uptake of ouabain.     

Effects of the neuroprotectant lubeluzole on the cytotoxic actions of veratridine,barium, ouabain and 6-hydroxydopamine in chromaffin cells

1. Incubation of bovine adrenal chromaffin cells with veratridine (10–100 μM) during 24 h, caused a concentration-dependent release of the cytosolic lactate dehydrogenase (LDH) into the bathing medium, an indicator of cell death. Lubeluzole or its R(−) enantiomer, {"type":"entrez-nucleotide","attrs":{"text":"R91154","term_id":"958694","term_text":"R91154"}}R91154, did not enhance LDH release. Both lubeluzole and {"type":"entrez-nucleotide","attrs":{"text":"R91154","term_id":"958694","term_text":"R91154"}}R91154 (0.3–10 μM) decreased the veratridine-induced LDH release.
2. Penfluridol did not increase LDH release at concentrations 0.003–1 μM; 3–10 μM increased LDH release to 50–60%, after 24 h exposure. Penfluridol (0.03–0.3 μM) did not protect against the cytotoxic effects of veratridine; at 1 μM, 15% protection was produced. Higher concentrations (3–10 μM) enhanced the cytotoxic effects of veratridine.
3. Ba²⁺ ions caused a concentration-dependent increase of LDH release. This cytotoxic effect was partially prevented by 3 μM lubeluzole and fully counteracted by 1 μM penfluridol. {"type":"entrez-nucleotide","attrs":{"text":"R91154","term_id":"958694","term_text":"R91154"}}R91154 was less potent than lubeluzole and only protected against the lesion induced by 0.5 mM Ba²⁺.
4. Ouabain (10 μM during 24 h) increased LDH release to about 30%. Both lubeluzole (0.3–10 μM) and the lower concentrations of penfluridol (0.003–0.3 μM) prevented the ouabain cytotoxic effects. At higher concentrations (3 μM), penfluridol increased drastically the ouabain cytotoxic effects.
5. 6-Hydroxydopamine (6-OHDA) caused significant cytotoxic effects at 30 and 100 μM. Lubeluzole (3–10 μM) or penfluridol (0.03–0.3 μM) had no cytoprotective effects against 6-OHDA.
6. Lubeluzole (3 μM), {"type":"entrez-nucleotide","attrs":{"text":"R91154","term_id":"958694","term_text":"R91154"}}R91154 (3 μM) and penfluridol (1 μM) blocked the current through Na⁺ channels in voltage-clamped chromaffin cells (I_Na) by around 20–30%. Ca²⁺ current through Ca²⁺ channels (I_Ca) was inhibited 57% by lubeluzole and {"type":"entrez-nucleotide","attrs":{"text":"R91154","term_id":"958694","term_text":"R91154"}}R91154 and 50% by penfluridol. The effects of penfluridol were not washed out, but those of lubeluzole and {"type":"entrez-nucleotide","attrs":{"text":"R91154","term_id":"958694","term_text":"R91154"}}R91154 were readily reversible.
7. Lubeluzole (3 μM) induced reversible blockade of the oscillations of the cytosolic Ca²⁺, [Ca²⁺]_i, in fura-2-loaded cells exposed to 30 or 100 μM veratridine. Penfluridol (1 μM) inhibited those oscillations in an irreversible manner.
8. The results suggest that lubeluzole and its R-isomer caused cytoprotection against veratridine cell damage, by blocking the veratridine stimulated Na⁺ and Ca²⁺ entry, as well as the [Ca²⁺]_i oscillations. The Ba²⁺ and ouabain cytotoxic effects were prevented more efficiently by penfluridol, likely by blocking the plasmalemmal Na⁺/Ca²⁺ exchanger. It remains dubious whether these findings are relevant to the reported neuroprotective action of lubeluzole in stroke; the doubt rests in the stereoselective protecting effects of lubeluzole in in vivo stroke models, as opposed to its lack of stereoselectivity in the in vitro model reported here.

     

The effect on the rat isolated atria of amiodarone in the presence of either ouabain or verapamil

Amiodarone causes a decrease in the rate of contraction of the rat isolated atria and has a negative inotropic action in the paced preparation. Interactions occur between amiodarone and ouabain and amiodarone and verapamil. It is possible that the clinically reported drug interaction with amiodarone may have a component of direct interactions on the myocardium rather than solely changes in plasma protein binding.     

Competitive antagonism by glibenclamide of cromakalim inhibition of twitch contractions in rabbit vas deferens

Electrically induced contractions of the rabbit isolated vas deferens were potentiated by carbachol but inhibited by the K+ channel opener cromakalim. The inhibition by cromakalim could be competitively antagonized by 10(-7)-10(-6) M glibenclamide (pA2 = 7.17) and was also reversed by carbachol, extra K+ or Bay k 8644. The data support the view that smooth muscle membrane depolarization is the cause for carbachol potentiation in rabbit vas deferens and that hyperpolarization by cromakalim is susceptible to blockade by the inhibitor of ATP-sensitive K+ channels, glibenclamide.     

The influence of nanomolar ouabain on vascular pressor responses is modulated by the endothelium

Ouabain has been shown to be an endogenous hormone that is synthesized and released from the adrenal cortex and is present in nanomolar to subnanomolar concentrations in plasma. It has been proposed that endogenous ouabain can increase vascular resistance and induce hypertension. This substance inhibits the Na(+)-pump activity, which leads to intracellular Na+ accumulation and ultimately to increased vascular tone. It is also suggested that circulating ouabain influences the vascular smooth muscle response to vasopressor substances. However, the mechanisms by which low concentrations of ouabain influence the smooth muscle, directly or acting through the endothelium, have not been completely elucidated. We tested the hypothesis that the endothelium exerts a modulatory effect on the actions of ouabain. In these studies, isolated rat-tail vascular bed preparations obtained from normotensive animals were used. The effects of 10 nM ouabain on the reactivity of the vascular smooth muscle to phenylephrine were determined under conditions in which endothelial function was preserved or reduced by endothelial removal and treatment with N(omega)-nitroL-arginine methyl ester (L-NAME) or potassium channel blocker (tetraethylammonium; TEA). Results showed that ouabain enhanced the reactivity to phenylephrine. The enhancement of the reactivity to phenylephrine produced by ouabain was potentiated by deendothelialization and by using TEA, but it was reduced by treatment with L-NAME. The effect of 10 nM ouabain on the functional activity of the Na+,K(+)-adenosine triphosphatase (ATPase) also was evaluated. Na+,K(+)-ATPase activity was reduced after 1-h treatment with ouabain. These results suggested that low concentrations of ouabain reduced the functional activity of the Na+,K(+)-ATPase and stimulated the release of a potassium channel opener, suggesting that the effects of ouabain are partially modulated by the endothelium.     

The positive inotropic drugs DPI 201-106, BDF 9148, and veratridine increase ouabain toxicity and [3H]ouabain binding in guinea pig heart.

The interaction of three positive inotropic compounds, which are modulators of sodium channels, with the cardiac glycoside ouabain was investigated in isolated guinea pig atria. In the presence of DPI 201-106 (3 x 10(-7) M), of its new acetidine derivative BDF 9148 (10(-7) M), or of veratridine (10(-6) M), the threshold ouabain concentration to induce toxicity was lowered by a factor of 2. This effect can be explained by the observation that specific equilibrium [3H]ouabain binding in intact atria was elevated by these compounds in the appropriate concentrations. The binding results were analyzed by means of a previously established model of "positive cooperative ouabain binding to intact myocardium," which describes the relationship between cellular sodium homeostasis and ouabain binding. The extent to which the compounds increased ouabain binding was in good quantitative agreement with the observed shift in the threshold concentration of ouabain toxicity. The increase of specific [3H]ouabain binding is likely initiated by a gain in cytosolic sodium. It takes place at the cellular level only, since a direct enhancement of [3H]ouabain binding to isolated cardiac membranes was not found. In conclusion, at least under some conditions, new inotropic drugs acting as sodium channel modulators can increase the risk of digitalis toxicity.     

The influence of DMAE and its parent substance hemicholinium on intestinal contractions in the dog

The actions of DMAE and its structural prototype hemicholinium (HC-3) were compared with respect to contractility of the small and large intestines of the dog in response to extrinsic (vagus or pelvic) or intrinsic (intramural) nerve stimulation. Contractions of the colon, duodenum, and jejunum to extrinsic nerve stimulation were effectively inhibited by both DMAE and HC-3. Contractions of these structures to intramural nerve stimulation were less susceptible to inhibition by DMAE (especially colonic and duodenal contractions) than to inhibition by HC-3. Neither DMAE nor HC-3 inhibited the contractile responses to ACh. In studies relating responses of the nictitating membrane of the dog to preganglionic cervical sympathetic nerve excitation and blood pressure responses to either peripheral vagal stimulation or bilateral carotid occlusion, HC-3, but not DMAE, appeared to exert some ganglionic blocking action akin to hexamethonium. It was concluded that the mode of action of DMAE and HC-3 on intestinal motor responses was not the same. It was postulated that the sites of action of DMAE were preganglionic parasympathetic nerve endings and specific parasympathetic ganglionic chemoreceptors in the intestine.     

Effects of hypoxia, 2,4-dinitrophenol and ouabain on twitch potentiation elicited following conditioning pulses in the mouse phrenic nerve-diaphragm preparation

Hypoxia is suggested to increase intracellular Ca2+, thereby affecting cellular functions. If this is the case, the effect would be modified by other treatments which also elevate intracellular levels of Ca2+. To test this possibility, in mouse diaphragm preparations, the effects of hypoxia were examined on twitch potentiations after application of neural conditioning pulses which are considered to elevate intracellular Ca2+. The effects were compared with those of 2,4-dinitrophenol (DNP) and ouabain. Hypoxia increased the tension of twitches elicited directly or neurally with 0.1 or 0.5 Hz pulses at 36 degrees C but not at 24 degrees C. Paired pulses to the nerve induced a complex response profile in which the amplitude of the second twitch was enhanced (twitch facilitation). Tetanic pulses (50 Hz) to the nerve were followed by post-tetanic twitches of increased amplitude (post-tetanic twitch potentiation, PTP). Hypoxia little affected the twitch facilitation but abolished PTP at both temperatures. These effects differed from those of DNP and ouabain in some ways. Thus, present experiments indicate that hypoxia selectively influences the process which is responsible for the PTP phenomenon rather than for the twitch facilitation. It is possible that the mechanism by which hypoxia would accumulate intracellular Ca2+ may be included in the process through which PTP occurs.     

The influence of heart rate on ouabain cardiotoxicity in cats with spinal cord transection.

The dose, serum level and ventricular content of ouabain needed to produce cardiotoxicity were examined in control cats, cats with transected spinal cords and cats with transected spinal cords whose heart rates were restored to control values by artificial pacing. The lethal dose of ouabain was higher in cats with transected spinal cords and not paced than it was in the control group. However, the lethal dose of ouabain in spinal-sectioned cats with ventricular pacing was no different from that in controls. However, in both groups of spinal-sectioned cats, death was associated with higher ventricular and serum levels of ouabain than in controls. The ventricular ouabain content of paced animals with transected spinal cords was higher than that of controls and lower than that of unpaced spinal cats. Thus, restoration of heart rate to control levels in spinal animals appeared to accelerate myocardial ouabain uptake. The lower myocardial ouabain content in the spinal-sectioned animals which were paced suggests that pacing sensitizes the heart to cardiotoxicity. Spinal section itself appears to decrease the sensitivity to ouabain partly through a decrease in cardiac rate and partly through a loss of neurogenic influence.     

Comparison of the effects of endothelin-1 and Bay k 8644 on twitch contractions of the field-stimulated rat vas deferens.

Endothelin-1 (ET-1) potentiated both the fast and the slow components of the twitches of the isolated field-stimulated rat vas deferens, whereas Bay k 8644 potentiated only the fast component. Nicardipine tended to decrease the potentiation. ET-1, but not Bay k 8644, caused membrane depolarization and resting tension elevation, which were suppressed by nicardipine. The excitatory junction potentials (EJPs) were augmented by Bay k 8644 and the effect was not changed by nicardipine. ET-1, however, apparently did not augment the EJPs, because of the accompanying depolarization. These results indicate a difference in mechanism of the potentiating effects of ET-1 and Bay k 8644.