Effects of ouabain on human bronchial muscle in vitro

The effects of ouabain, an inhibitor of the plasmalemmal Na⁺/K⁺-ATPase activity, were examined in human isolated bronchus. Ouabain produced concentration-dependent contraction with –logEC₅₀=7.16±0.11 and maximal effect of 67±4% of the response to acetylcholine (1 mM). Ouabain (10 M)-induced contraction was epithelium-independent and was not depressed by inhibitors of cyclooxygenase and lipoxygenase, antagonists of muscarinic, histamine H₁-receptors and -adrenoceptors, or neuronal Na⁺ channel blockade. The inhibition of ouabain contraction in tissues bathed in K⁺-free medium, and the inhibition by ouabain of the K⁺-induced relaxation confirm that the contractile action of ouabain is mediated by inhibition of Na⁺/K⁺-ATPase. Furthermore, depolarization (16.4±0.9 mV) was observed in human isolated bronchus by intracellular microelectrode recording. Ouabain (10 M)-induced contractions were abolished by a Ca²⁺-free solution but not by blockers of L-type Ca²⁺ channels. In human cultured bronchial smooth muscle cells, ouabain (10 M) produced a sustained increase in [Ca²⁺]_i (116±26 nM) abolished in Ca²⁺-free medium. Incubation with a Na⁺-free medium or amiloride (0.1 mM) markedly inhibited the spasmogenic effect of ouabain thus suggesting the role of Na⁺/Ca²⁺ exchange in ouabain contraction while selective inhibitors of Na⁺/H⁺-antiport, Na⁺/K⁺/Cl^–-antiport, or protein kinase C had no effect. Ouabain (10 M) failed to increase inositol phosphate accumulation in human bronchus. Ouabain (10 M) did not alter bronchial responsiveness to acetylcholine or histamine but inhibited the relaxant effects of isoprenaline, forskolin, levcromakalim, or sodium nitroprusside. These results indicate that ouabain acts directly to produce contraction of human airway smooth muscle that depends on extracellular Ca²⁺ entry unrelated to L-type channels and involving the Na⁺/Ca²⁺-antiporter.     

Cardiac glycosides: Correlations among Na+, K+-ATPase,sodium pump and contractility in the guinea pig heart

Summary Relationships among positive inotropic response to cardiac glycosides, Na⁺,K⁺-ATPase inhibition and monovalent cation pump activities were studied using paced Langendorff preparations of guinea-pig heart. Na⁺,K⁺-ATPase activity was estimated from the initial velocity of (³H)-ouabain binding in ventricular homogenates, and cation pump activity from ouabain-sensitive ⁸⁶Rb uptake of ventricular slices. These parameters were assayed in control, ouabain- or digitoxintreated hearts either at the time of inotropic response to the cardiac glycosides or during the course of drug washout. Development and loss of the inotropic response during ouabain or digitoxin perfusion and washout was accompanied by reduction and subsequent recovery of the initial ouabain binding velocity, respectively. If homogenates from glycoside-treated hearts were incubated at 37°C for 10 min during ouabain-binding studies, the levels of binding were not different from those of control hearts, indicating a rapid dissociation of the glycosides from cardiac Na⁺,K⁺-ATPase in this species. Despite differences in the time course of the loss of inotropic responses produced by ouabain or digitoxin, the relationship between Na⁺,K⁺-ATPase inhibition and inotropic responses were similar. Inotropic responses to digitoxin during perfusion, and subsequent los during washout, also were accompanied by a reduction and subsequent recovery of ⁸⁶Rb uptake. A correlation between inhibition of cation pump activity and positive inotropy has hitherto not been demonstrated. Thus, it appears that with cardiac glycosides, a relationship exists among contractility, cardiac Na⁺,K⁺-ATPase and monovalent cation pump activities.     

Role of Na+-K+ ATPase enzyme in vascular response of goat ruminal artery

Objective:

To study the role of Na⁺, K⁺- ATPase enzyme in the vascular response of goat ruminal artery.

Materials and Methods:

Ruminal artery was obtained in chilled aerated modified Krebs-Henseleit solution (KHS) from a local slaughterhouse and transported in ice for further processing. The endothelium intact arterial ring was mounted in a thermostatically controlled (37 ± 0.5°C) organ bath containing 20 ml of modified KHS (pH 7.4) bubbled with oxygen (95%) and CO₂ (5%) under 2g tension. An equilibration of 90 min was allowed before addition of drugs into the bath. The responses were recorded isometrically in an automatic organ bath connected to PowerLab data acquisition system. In order to examine intact functional endothelium, ACh (10 μM) was added on the 5-HT (1.0 μM) - induced sustained contractile response. Similarly, functional characterization of Na⁺, K⁺-ATPase activity was done by K⁺-induced relaxation (10 μM-10 mM) in the absence and presence of ouabain (0.1 μM/ 0.1 mM), digoxin (0.1 μM) and barium (30 μM).

Results:

ACh (10⁻⁵ M) did not produce any relaxing effect on 5-HT-induced sustained contractile response suggesting that vascular endothelium has no significant influence on the activation of sodium pump by extracellular K⁺ in ruminal artery. Low concentration of Ba²⁺ (30 μM) (IC₅₀: 0.479 mM) inhibited K⁺-induced relaxation suggesting K_ir (inward rectifier) channel in part had role in K⁺-induced vasodilatation in ruminal artery. Vasorelaxant effect of KCl (10 μM-10 mM) in K⁺-free medium is also blocked by ouabain (0.1 μM and 0.1 mM) (IC₅₀:0.398 mM and IC₃₅: 1.36 mM), but not by digoxin (0.1 μM) (IC₅₀ 0.234 mM) suggesting that ouabain sensitive Na⁺, K⁺-ATPase isoform is present in the ruminal artery.

Conclusion:

In the goat ruminal artery functional regulation of sodium pump is partly mediated by K⁺ channel and ouabain sensitive Na⁺, K⁺ ATPase.     

Effects of glibenclamide and tetracaine on 86Rb+ fluxes in mouse pancreatic β-cells

Summary Potassium transport was measured in -cell-rich islets from ob/ob-mice using the K⁺-analogue ⁸⁶Rb⁺. Both tetracaine (0.1 mM) and glibenclamide (0.1 M) reduced the oubain-resistant ⁸⁶Rb⁺ influx but did not significantly affect the oubain-sensitive portion (Na⁺/K⁺ pump). Tetracaine (0.5–1 mM) or glibenclamide (0.2 mM) decreased the ⁸⁶Rb⁺ equilibrium content and glibenclamide (1 M) transiently reduced the ⁸⁶Rb⁺ efflux rate but 0.1 mM tetracaine had only a slight effect on this flux rate. The results suggest that a change in ouabain-resistant (passive) K⁺ fluxes, but not the Na⁺/K⁺ pump, is involved in stimulation of insulin secretion by glibenclamide and tetracaine. Both drugs may exert similar effects on the -cell plasma membrane.     

Azelastine and allergen transduction signal in MC9 mast cells

Summary MC9 mast cells, sensitized with monoclonal IgE antibody specific for 2,4-dinitrophenyl (DNP) group, were exposed to DNP-BSA and the pH and cytosolic calcium signals were recorded by using the fluorescent probes BCECF and Fura-2 respectively. DNP-BSA induced cell alkalinization was fully inhibited by azelastine with IC₅₀ (1.6±0.5 mol/l, mean±SEM, n = 5) similar to that required to inhibit histamine release (1.4 mol/l), Conversely, high azelastine concentrations (> 100 mol/l) were required to inhibit DNP-BSA-dependent cell calcium mobilization (IC₅₀200 mol/l, n = 3). Amiloride, but not the H₁ histamine antagonist pyrilamine, was able to inhibit the DNP-BSA induced pH signal. In acidified mast cells, azelastine potently inhibited Na⁺:H⁺ exchange activity (IC₅₀ = 7.7±3.6 × 10^–6 M, mean±SEM, n = 3). Conversely, in mouse spleen lymphocytes azelastine was unable to inhibit the amiloride-sensitive pH signal induced by concanavalin A. In conclusion, the inhibition of histamine release by azelastine is not due to an interference with the cytosolic calcium signal. Conversely, azelastine potently antagonized the allergen-dependent Na⁺: H⁺ exchange activation, suggesting an action on the protein kinase C signaling pathway. Correspondence to: R. P. Garay at the above address     

Correlation between inhibition of (Na+, K+)-membrane-ATPase and positive inotropic activity of cardenolides in isolated papillary muscles of guinea pig

Summary Concentrations of 17 cardenolides, cardenolide glucuronides and sulfates producing halfmaximal inhibition of (Na⁺, K⁺)-membrane-ATPase from different organs and animal species were determined in vitro. In addition the concentrations that increased the contractility of guinea pig isolated papillary muscles to a particular level were investigated. Comparisons between ATPase-inhibiting and positive inotropic cardiac activities showed extensive parallelism: the correlation coefficients after log/log transformation were between 0.92 and 0.97. The same close correlations are found if dissociation constants of cardenolide receptor complexes and concentrations causing ⁸⁶Rb-uptake inhibition in human erythrocytes are examined.The concentrations necessary for inhibition of (Na⁺, K⁺)-membrane-ATPase of the guinea pig heart and the concentrations required to achieve a defined positive inotropic effect in guinea pig papillary muscle showed a log/log correlation coefficient of 0.97 (P<0.001). In both tests the potencies covered more than three orders of magnitude. The results support Repke's hypothesis on the digitalis receptor.     

Steroid-like compounds in Chinese medicines promote blood circulation via inhibition of Na^＋/K^＋- ATPase

Aim:

To examine if steroid-like compounds found in many Chinese medicinal products conventionally used for the promotion of blood circulation may act as active components via the same molecular mechanism triggered by cardiac glycosides, such as ouabain.

Methods:

The inhibitory potency of ouabain and the identified steroid-like compounds on Na⁺/K⁺-ATPase activity was examined and compared. Molecular modeling was exhibited for the docking of these compounds to Na⁺/K⁺-ATPase.

Results:

All the examined steroid-like compounds displayed more or less inhibition on Na⁺/K⁺-ATPase, with bufalin (structurally almost equivalent to ouabain) exhibiting significantly higher inhibitory potency than the others. In the pentacyclic triterpenoids examined, ursolic acid and oleanolic acid were moderate inhibitors of Na⁺/K⁺-ATPase, and their inhibitory potency was comparable to that of ginsenoside Rh2. The relatively high inhibitory potency of ursolic acid or oleanolic acid was due to the formation of a hydrogen bond between its carboxyl group and the Ile322 residue in the deep cavity close to two K⁺ binding sites of Na⁺/K⁺-ATPase. Moreover, the drastic difference observed in the inhibitory potency of ouabain, bufalin, ginsenoside Rh2, and pentacyclic triterpenoids is ascribed mainly to the number of hydrogen bonds and partially to the strength of hydrophobic interaction between the compounds and residues around the deep cavity of Na⁺/K⁺-ATPase.

Conclusion:

Steroid-like compounds seem to contribute to therapeutic effects of many cardioactive Chinese medicinal products. Chinese herbs, such as Prunella vulgaris L, rich in ursolic acid, oleanolic acid and their glycoside derivatives may be adequate sources for cardiac therapy via effective inhibition on Na⁺/K⁺-ATPase.     

Inhibition of Na+, K+-ATPase in rat renal proximal tubules by dopamine involved DA-1 receptor activation

Summary Endogenous kidney dopamine (DA) causes natriuresis and diuresis, at least partly, via inhibition of proximal tubular Na⁺,K⁺-ATPase. The present study was done to identify the dopamine receptor subtype(s) involved in dopamine-induced inhibition of Na⁺,K⁺-ATPase activity. Suspensions of renal proximal tubules from Sprague-Dawley rats were incubated with dopamine, the DA-1 receptor agonist fenoldopam or the DA-2 receptor agonist SK&F 89124 in the presence or absence of either the DA-1 receptor antagonist SCH 23390 or the DA-2 receptor antagonist domperidone. Dopamine and fenoldopam (10^–5 to 10^–8 mol/1) produced a concentration-dependent inhibition of Na⁺,K⁺-ATPase activity. However, SK&F 89124 failed to produce any significant effect over the same concentration range. Incubation with fenoldopam (10^–5 to 10^–8 mol/1) in the presence of SK&F 89124 (10^–6 mol/l) inhibited Na+,K+-ATPase activity to a degree similar to that with fenoldopam alone. Furthermore, DA-induced inhibition of Na⁺,K⁺-ATPase activity was attenuated by SCH 23390, but not by domperidone. Since -adrenoceptor activation is reported to stimulate Na⁺,K⁺-ATPase activity and, at higher concentrations, dopamine also acts as an a-adrenoceptor agonist, the potential opposing effect from -adrenoceptor activation on DA-induced inhibition of Na⁺,K⁺-ATPase activity was investigated by using the -adrenoceptor blocker phentolamine. We found that, in the lower concentration range (10^–5 to 10^–7 mol/1), dopamine-induced inhibition of Na⁺,K⁺-ATPase activity in the presence of phentolamine was similar in magnitude to that observed with dopamine alone. However, at the highest concentration used (10^–4 mol/1), dopamine produced a significantly larger degree of inhibition of Na⁺,K⁺-ATPase activity in the presence of phentolamine. These results indicate that the DA-1 dopamine receptor subtype, but not the DA-2 receptor subtype, is involved in dopamine-mediated inhibition of Na⁺,K⁺-ATPase. At higher concentrations of dopamine, the DA-1 receptor-mediated inhibitory effect on Na⁺,K⁺-ATPase activity may be partly opposed by a simultaneous -adrenoceptor-mediated stimulation of the activity of this enzyme.     

Demonstration of a Na+: Mg2+ exchange in human red cells by its sensitivity to tricyclic antidepressant drugs

Summary Iminodibenzyl-, iminostilbene-, dibenzocycloheptadiene-, dibenzooxepine- and dibenzothiepine-derivatives of tricyclic antidepressant drugs were able to inhibit Na⁺-stimulated Mg²⁺ efflux in human erythrocytes at concentrations of 10^–5–10^–3 mol/l. Tricyclic antidepressant drugs belonging to other chemical groups, non-tricyclic antidepressant drugs and phenothiazines were less potent inhibitors (IC₅₀ of 10^–4 mol/l or higher).Imipramine and dothiepine, the most potent compounds, inhibited the Mg" carrier with IC₅₀ of 2.5 and 4 × 10^–5 mol/1 respectively. These IC₅₀ are of similar order of magnitude to those of some classical transport inhibitors (such as furosemide for the [Na⁺K⁺,Cl^–]-cotransport system). In addition, these concentrations of imipramine and dothiepine were free of: i) side effects on other erythrocyte Na and K⁺ transport pathways (with the exception of a slight inhibition of Ca²⁺-sensitive K⁺-channels and [Na⁺,K⁺,Cl^–]- and [K⁺,Cl^–]-cotransport systems) and ii) toxic effects on the membrane leak for divalent or monovalent cations. Therefore, we selected imipramine as an useful tool for investigating fluxes catalyzed by the Na⁺-stimulated Mg²⁺ carrier.Imipramine was tested on the initial rate of ouabain and bumetanide-resistant net Na⁺ influx in Na⁺-depleted, Mg²⁺-loaded erythrocytes. The compound was able to inhibit a Na⁺ influx of about 300–500 mol (l · cells × h)^–1 with an IC₅₀ of about 3 x 10^–5 mol/1. This imipramine-sensitive Na⁺ influx was coupled with an imipramine-sensitive Mg²⁺ efflux in a stoichiometry of 3.03±0.34 (mean±SEM of 7 experiments).Abbreviations MOPS 4-morpholinopropanesulfonic acid - PCMBS p-chloromercuribenzenesulfonate - EGTA ethylene glycol bis-(beta-aminoethyl ether)N,NNN-tetraacetic acid - Tris tris(hydroxymethyl)aminomethane Send offprint requests to R. Garay at the above address     

Biochemical basis for the low sensitivity of the rat heart to digitalis

Summary The concentration of cardiac glycosides to produce positive inotropic effects in the rat heart is markedly higher than that in other species. Such a low digitalis sensitivity of the rat heat is attributed to the low affinity of cardiac Na⁺, K⁺-ATPase for digitalis in this species. In the present study the biochemical cause which is responsible for the formation of the unstable complex between the glycosides and Na⁺, K⁺-ATPase or positive inotropic, receptor in the rat heart was examined using Na⁺, K⁺-ATPase preparations obtained from rat hearts, guinea-pig hearts and rat brains as well as isolated, electrically stimulated atrial preparations obtained from these animals. Monensin, which alters transmembrane Na⁺ movements without interacting with the cardiotonic sites on Na⁺, K⁺-ATPase, had equivalent potencies in guinea-pig and rat hearts. Cassaine, which lacks a lactone ring but interacts with cardiotonic sites on Na⁺, K⁺-ATPase, increased the force of contraction in guinea-pig hearts at low, but in rat hearts only at high, concentrations. AY-22,241 (Actodigin) and prednisolone-3,20-bisguanylhydrazone (PBGH) bind to cardiotonic sites on Na⁺, K⁺-ATPase and had a similar spectrum as cassaine in these two species. Actodigin has an altered lactone ring resulting in a marked reduction of the inotropic potency, and PBGH is devoid of this structure. With the latter agent, the rabbit was as insensitive as the rat, although both rabbit and guinea-pig are equally sensitive to digitalis. K⁺ delayed the development of the positive inotropic action of ouabain with a minimal effect on the plateau response in guinea-pig hearts. In rat hearts, however, K⁺ markedly lowered the plateau response without affecting the time course of the response. These results indicate that the low sensitivity of the rat heart to digitalis is due to a difference in the glycoside binding sites on Na⁺, K⁺-ATPase; but the difference cannot be explained by the lack of a lactone ring complementary binding sites. The difference seems to result from the absence of lipid barrier which regulates the rate of release of cardiac glycosides from their binding sites on Na⁺, K⁺-ATPase.This work was supported by U.S. Public Health Service grant, HL-16052 and by the Michigan Heart Association     

Magnesium lithospermate B extracted from Salvia miltiorrhiza elevats intracellular Ca2＋ level in SHSY5Y cells

Aim:

To examine if magnesium lithospermate B (MLB), a potent inhibitor of Na⁺/K⁺-ATPase, leads to the elevation of intracellular Ca²⁺ level as observed in cells treated with cardiac glycosides.

Methods:

Viability of SH-SY5Y neuroblastoma cells treated with various concentrations of ouabain or MLB was measured. Intracellular Ca²⁺ levels were visualized using Fluo4-AM (fluorescent dye) when cells were treated with ouabain or MLB in the presence or absence of KB-R7943 (Na⁺/Ca²⁺ exchanger inhibitor) and 2-APB (IP₃ receptor antagonist). Molecular modeling was conducted for the docking of ouabain or MLB to Na⁺/K⁺-ATPase. Changes of cell body and dendrite morphology were monitored under a microscope.

Results:

severe toxicity was observed in cells treated with ouabain of concentration higher than 1 μmol/L for 24 h while no apparent toxicity was observed in those treated with MLB. Intracellular Ca²⁺ levels were substantially elevated by MLB (1 μmol/L) and ouabain (1 μmol/L) in similar patterns, and significantly reduced in the presence of KB-R7943 (10 μmol/L) or 2-APB (100 μmol/L). Equivalent interaction with the binding cavity of Na⁺/K⁺-ATPase was simulated for ouabain and MLB by forming five hydrogen bonds, respectively. Treatment of ouabain (1 μmol/L), but not MLB (1 μmol/L), induced dendritic shrink of SH-SY5Y cells.

Conclusion:

Comparable to ouabain, MLB leads to the elevation of intracellular Ca²⁺ level presumably via the same mechanism by inhibiting Na⁺/K⁺-ATPase. The elevated Ca²⁺ levels seem to be supplied by Ca²⁺ influx through the reversed mode of the Na⁺/Ca²⁺ exchanger and intracellular release from endoplasmic reticulum.     

Effect of sugar positions in ginsenosides and their inhibitory potency on Na+/K+-ATPase activity

Aim:

To determine whether ginsenosides with various sugar attachments may act as active components responsible for the cardiac therapeutic effects of ginseng and sanqi (the roots of Panax ginseng and Panax notoginseng) via the same molecular mechanism triggered by cardiac glycosides, such as ouabain and digoxin.

Methods:

The structural similarity between ginsenosides and ouabain was analyzed. The inhibitory potency of ginsenosides and ouabain on Na⁺/K⁺-ATPase activity was examined and compared. Molecular modeling was exhibited for the docking of ginsenosides to Na⁺/K⁺-ATPase.

Results:

Ginsenosides with sugar moieties attached only to the C-3 position of the steroid-like structure, equivalent to the sugar position in cardiac glycosides, and possessed inhibitory potency on Na⁺/K⁺-ATPase activity. However, their inhibitory potency was significantly reduced or completely abolished when a monosaccharide was linked to the C-6 or C-20 position of the steroid-like structure; replacement of the monosaccharide with a disaccharide molecule at either of these positions caused the disappearance of the inhibitory potency. Molecular modeling and docking confirmed that the difference in Na⁺/K⁺-ATPase inhibitory potency among ginsenosides was due to the steric hindrance of sugar attachment at the C-6 and C-20 positions of the steroid-like structure.

Conclusion:

The cardiac therapeutic effects of ginseng and sanqi should be at least partly attributed to the effective inhibition of Na⁺/K⁺-ATPase by their metabolized ginsenosides with sugar moieties attached only to the C-3 position of the steroid-like structure.     

Differential effects of organotin compounds on voltage-gated potassium currents in lymphocytes and neuroblastoma cells

Effects of organotin compounds were studied on voltage-gated K⁺ current in whole-cell voltage clamped lymphocytes and in NlE-115 neuroblastoma cells. In human peripheral blood lymphocytes the immunotoxic compounds dibutyltinchloride (DBT, 2.5 M) and triphenyltinchloride (TPhT, 2.5 M) decrease the peak amplitude of the K⁺ current and prolong time to peak. Tributyltinchloride (TBT, 2.5 M) decreases the K⁺ current to a greater extent than DBT and TPhT, without affecting the time to peak. The neurotoxic organotin compound trimethyltinchloride (TMT, 2.5 M) does not affect the voltage-gated K⁺ current in lymphocytes. Similar effects of DBT were observed in freshly isolated and PHA-activated human lymphocytes and with rat thymocytes. On the other hand, in mouse NIE-115 neuroblastoma cells, none of the organotin compounds altered the voltage-dependent K⁺ current.In human lymphocytes DBT affects both the peak amplitude and the time to peak of the K⁺ current in a concentration-dependent manner. At the maximum concentration of 10 M tested, the peak amplitude of the K⁺ current was reduced to 22 ± 4% of the control current. The IC₅₀ and slope factor for block of the peak outward current by DBT amounts to 6.7 ± 0.4 M, and 2.7 ± 0.4, respectively. The delay in K⁺ current activation does not saturate. At 10 M DMT increases the time to peak to 332 ± 12% of the control value. The present results suggest that the effects by DBT originate from two separate interactions with the voltage-gated K⁺ channel at the extracellular site of the membrane: a direct effect on the closed K⁺ channel causing a delay in current activation and a membrane-related effect causing inhibition of the K⁺ current. The differential effects of the organotin compounds may relate to their differential toxicological action.     

Comparison of the effluxes of 42K+ and 86Rb+ elicited by cromakalim (BRL 34915) in tonic and phasic vascular tissue

Summary The cromakalim-induced effluxes of ⁴²K⁺ and ⁸⁶Rb⁺ were compared in rat aortic segments and in guinea-pig portal vein. In both vessels, low concentrations of cromakalim (0.1 M) increased the permeability to ⁸⁶Rb⁺ 3–4 times less than that to ⁴²K⁺; at 10 M the difference was about a factor of 1.3–2. In rat aorta, the threshold concentration of cromakalim for ⁴²K⁺ efflux was 0.03 M; with ⁸⁶Rb⁺ as the tracer ion it was 0.1 M. At similar concentrations, cromakalim relaxed the tension of aortic segments precontracted with 23 mM KCl (IC₅₀ = 0.06 ± 0.01 M). However, no concomitant increase in ⁴²K⁺ or ⁸⁶Rb⁺ efflux could be detected from this stimulated preparation at these concentrations. In guinea-pig portal vein, ⁴²K⁺ efflux measurements were performed in the presence and absence of the dihydropyridine Ca²⁺ entry blocker PN 200-110 (isradipine) yielding comparable results. In the presence of PN 200-110, where spontaneous activity and the K⁺ efflux associated with it were abolished, the threshold concentration of cromakalim for ⁴²K⁺ efflux was 0.02 M as compared to 0.06 M for ⁸⁶Rb⁺ efflux. In the absence of PN 200-110, spontaneous activity of the portal vein was inhibited by 70% and 90% at these concentrations. In double isotope experiments, the K⁺ channel inhibitor tetraethylammonium did not discriminate between the effluxes of ⁴²K⁺ and ⁸⁶Rb⁺ stimulated by cromakalim.It is concluded that in the two vascular tissues examined, cromakalim increased the permeability to ⁴²K⁺ more than to ⁸⁶Rb⁺, the difference being more marked at low cromakalim concentrations. The use of ⁴²K⁺ as the tracer ion narrows the apparent gap between the concentrations of cromakalim which elicit vasorelaxant effects and those which induce an observable increase in K⁺ permeability; however a significant difference persists.Part of the data was presented at the Winter Meeting of the British Pharmacological Society London 1988 [Br J Pharmacol 93 (1988) p 19] Send offprint requests to U. Quasi at the above address     

The relationship between Na+, K+-ATPase inhibition and cardiac glycoside-induced arrhythmia in dogs

Summary In order to determine if there is a relationship between Na⁺, K⁺-ATPase inhibition and cardiac glycoside-induced arrhythmia, the time course of the onset and offset of the arrhythmia induced by the semi-synthetic glycoside, actodigin, and the enzyme activity during arrhythmia and following reversion to normal sinus rhythm was studied in the intact, anesthetized dog. An infusion of actodigin (AY 22,241) at the rate of 0.1 mol/kg/min for 30 min induced a severe and persistent arrhythmia within 13.1±1.2 min in 9 dogs. Upon termination of the actodigin infusion, the arrhythmia spontaneously converted to sinus rhythm within 17.5±2.3 min. Left ventricular tissue was taken from dogs sacrificed at the peak of the actodigin-induced arrhythmic periods or from the dogs that were allowed to recover from the actodigin-induced arrhythmia. These samples were homogenized and the membrane-containing fraction was passed through a Millipore filter. The membrane fraction trapped in the filter was the assayed for Na⁺+K⁺ stimulate, Mg²⁺ dependent ATPase activity. The results showed that, in comparison to the time matched control dogs, the cardiac microsomes prepared from the arrhythmic dogs had a markedly reduced Na⁺, K⁺-ATPase activity. On the other hand, actodigin-treated dogs that were allowed to recover from the arrhythmic episode had Na⁺, K⁺-ATPase activity that was not significantly different from the control values.The amount of ³H-actodigin bound by the cardiac muscle microsomal fraction was also investigated. The microsomes from left ventricle were isolated with a slight modification of the method of Dutta et al. (1968). The microsomal binding of ³H-actodigin was maximum at 30 min (26.6 pmol/mg protein) when the sample was prepared from the dogs at the peak of the arrhythmic effect. However, the binding was significantly reduced (11.5 pmol/mg protein) in the microsomal fraction from hearts that had returned to sinus rhythm. These data provide direct evidence that inhibition of Na⁺, K⁺-ATPase and cardiac glycosideinduced arrhythmia may have some cause and effect relationship.This investigation was supported in part by the United States Public Health Services Research Grant HE 07051 and The Central Ohio Heart Association GrantA report of this study has been presented in the spring meetings of FASEB, April, 1974, Atlantic City, New Jersey and submitted by J. H. Zavecz in partial fulfillment of the requirements for the degree of Doctor of Philosophy at the Ohio State University     

Dehydro-digitoxosides of digitoxigenin and digoxigenin: Binding to beef heart (Na++K+)-ATPase in relation to unchanged digitoxosides

Summary Dehydro-digitoxosides are metabolites of digitalis glycosides. In order to study their possible biological activity their affinity to (Na⁺+K⁺)-activated ATPase was determined and compared with unchanged glycosides. Based on the dissociation constants of glycoside-enzyme-complexes, the affinity of the dehydro-digitoxosides ranged in the same order of magnitude as that of the native glycosides. Comparing mono-, bis-, and tris-digitoxosides of digitoxigenin (dt-1, dt-2, dt-3) and of digoxin (dg-1, dg-2, dg-3) with the corresponding dehydrodigitoxosides (3-dehydro-dt-1, 9-dehydro-dt-2, 15-dehydro-dt-3, 3-dehydro-dg-1 and 9-dehydro-dg-2, respectively) the dehydro-digitoxosides had lower affinities to the enzyme. The highest dissociation constants (K _D)were found for 3-dehydro-dt-1 and 3-dehydro-dg-1. The half maximal inhibition of (Na⁺+K⁺)-ATPase activity (I₅₀) corresponded to affinity measurements in all but two cases: dehydro-dt-3 and dehydro-dt-2 showed very low I₅₀ values.     

Differential electrophysiologic and inotropic effects of phenylephrine in atrial and ventricular heart muscle preparations from rats

Summary Stimulation of ₁-adrenoceptors evokes a different pattern of inotropic responses in atrial and ventricular heart muscle preparations from rats. The inotropic effects are accompanied by different changes in membrane potential. In an attempt to clarify the question whether or to which extent these events are causally related, the effects of phenylephrine on force of contraction, transmembrane potential, Ca²⁺ current (I_Ca) and K⁺ currents were comparatively studied in either tissue.In atrial preparations, phenylephrine 10 mol/l caused an increase in force of contraction, a marked prolongation of the action potential duration and a depolarization of the membrane at rest. In the ventricle, however, the addition of phenylephrine 10 mol/l produced first a decline in force of contraction associated with a hyperpolarization of the membrane and a reduction in the action potential duration. These changes were followed by an increase in force,of contraction and a slight prolongation of the action potential, whereas the resting membrane potential remained increased. The hyperpolarization was eliminated in the presence of ouabain 100 mol/l.In enzymatically isolated atrial and ventricular myocytes, the whole-cell voltage clamp technique was used to study membrane currents on exposure to phenylephrine. Phenylephrine 30 mol/l did not affect the magnitude of I_Ca in either cell type. Transient and steady state K⁺ outward currents, however, were significantly diminished to a similar extent in atrial and in ventricular myocytes.It is concluded that the positive inotropic effect of ₁-adrenoceptor stimulation in the rat atrium is related to an increase in action potential duration and a decrease in resting membrane potential due to a decrease in K⁺ currents. In the ventricle, phenylephrine additionally activates the Na⁺/K⁺ pump thereby hyperpolarizing the membrane. The rapid onset of pump stimulation seems to overwhelm, in the beginning, the phenylephrine-induced decrease in K⁺ conductance and therefore to evoke a transient negative inotropic effect.It is assumed that phenylephrine can alter the intracellular Ca²⁺ concentration due to changes in the action potential duration. The way how Ca 2+ enters the cell remains speculative, since direct changes of Ica were not detected. The more complicated changes in membrane potential in the ventricle suggest that also other mechanisms for the positive inotropic response to phenylephrine must be considered. Send offprint requests to H. Nawrath at the above address     

Pertussis toxin inhibits noradrenaline accumulation by bovine adrenal medullary chromaffin cells

Summary Bovine adrenal medullary chromaffin cells maintained in tissue culture accumulated [³H]-noradrenaline by a high affinity, Na⁺-dependent, desipramine-sensitive process. The accumulation was linear with time (1–90min) and had an apparent K_m of 0.52±0.24 mol/l and V_max of 1.70±0.48 pmol/(10⁵ cells · 15 min). Pretreatment of the cells with the ADP-ribosylating agent pertussis toxin resulted in a reduction in the V_max, [0.81±0.39 pmol/(10⁵cells · 15 min)] but no significant change in the apparent affinity (K_m = 0.42±0.07 mol/l). This inhibition of [³H]noradrenaline accumulation was distinct from that produced by the vesicular transport inhibitor reserpine. Pertussis toxin inhibition probably did not arise through an indirect action on the Na⁺-gradient because while, as expected, Na⁺, K⁺-ATPase inhibition reduced [³H]noradrenaline accumulation, pertussis toxin pretreatment always caused a further significant reduction even in the presence of maximally effective concentrations of ouabain. Stimulation of the CAMP-protein kinase A system by forskolin or 8-bromocyclic AMP also caused a reduction in [³H] noradrenaline accumulation but again pertussis toxin pretreatment always resulted in a further reduction. Thus, the data provide evidence for a pertussis toxin-sensitive element in the catecholamine accumulation process and are consistent with an action at a site directly associated with the transporter itself rather than with an indirect action via secondary processes.Correspondence to S. J. Bunn at the above address     

Quabain: Temporal relationship between the inotropic effect and the in vitro binding to,and dissociation from, (Na++K+)-activated ATPase

Summary The time course of the inotropic response to ouabain in Langendorff preparations was compared with that of the in vitro ATP-dependent (³H)-ouabain binding to cardiac (Na⁺+K⁺)-activated ATPase preparations, and subsequent dissociation, to determine the temporal relationship between the inotropic response and (Na⁺+K⁺)-activated ATPase inhibition.Species differences were minimal either in the onset of inotropic response or the (³H)-ouabain binding. The rates of both loss of the inotropic response to ouabain during washout and the dissociation of the ouabain-enzyme complex, however, were rapid in guinea pig and rabbit (relatively ouabain-insensitive species) and slow in cat and dog (ouabain-sensitive species). The half-time of the loss of the inotropic response was similar to the half-time of the dissociation of the ouabain-enzyme complex in each species.Since ATP-dependent binding of cardiac glycosides has been related to enzyme inhibition, it was concluded that the time course of the inotropic response to ouabain parallels the time course of (Na⁺+K⁺)-activated ATPase inhibition, and that the dissociation of ouabain from the enzyme may terminate the inotropic response.A part of this study was presented at the Fifth Annual Meeting of the International Study Group for Research in Cardiac Metabolism, Winnipeg, Manitoba, June, 1972.     

Distribution of cardiac glycosides in heart and brain of dogs and their affinity to the (Na++K+)-ATPase

Summary The tissue distribution after repeated intravenous administration of tritium-labelled digoxin, -methyldigoxin and ouabain was examined in heart and brain of 6 beagle dogs. In addition, the (Na⁺+K⁺)-ATPase activity was measured in various heart and brain areas, and its affinity to the cardiac glycosides was determined. The glycoside concentrations in the atria are lower than in the ventricles, and the left heart areas show higher concentrations than the right areas. Significant differences in the (Na⁺+K⁺)-ATPase activity or its binding capacity in the various heart areas, which could be responsible for this characteristic distribution pattern, were not found. In agreement with its greater lipid-solubility, -methyldigoxin shows a higher accumulation in the brain than digoxin and ouabain. However, while -methyldigoxin is evenly distributed throughout all brain areas, concentration differences are found for digoxin and ouabain in the telencephalon, cerebellum and brain stem. This characteristic distribution of the more polar glycosides may be partly determined by the different structure of the capillaries in the central nervous system. In addition, the binding affinities for digoxin and ouabain also differ in the various crude brain preparations. In the diencephalon, pons, cerebellum and medulla the dissociation constants as a reciprocal measure of the binding affinity were lower for digoxin with 7.5 to 9.9×10^–9 than in the telencephalon, mesencephalon and spinal cord with dissociation constants of 1.1 to 1.45×10^–8 M. Since, in these brain areas higher glycoside concentrations per g wet weight were also measured, the glycoside accumulation in the various brain areas could be dependent on the higher receptor affinity of these brain areas. On the other hand, the binding affinities for -methyldigoxin were the same in all brain areas, with a mean dissociation constant of 1.45×10^–8 M.