Additive positive inotropic effects of milrinone,ouabain and calcium in diseased human ventricular myocardium

Summary The interactions of milrinone, ouabain and calcium on force of contraction in isolated, contracting human papillary muscle strips were measured. Milrinone (EC₅₀, 8 × 10^–5 M) increased force of contraction maximally by 2.8±0.8 mN at 5 × 10^–4M; significantly less than either ouabain (1 × 10^–7M; 4.8±0.5 mN increase) or calcium (15 mM; 6.2±0.6 mN increase). A submaximal, but not a maximal, inotropic effect of ouabain could be increased by the addition of milrinone; in contrast, both ouabain and calcium increased the maximal inotropic effect of milrinone by 1.7±0.2 mN and 2.7±0.3 mN, respectively. The combined inotropic effect of milrinone with either ouabain of 4.2±0.3 mN or calcium of 5.6±0.4 mN was not different from that with calcium or ouabain alone. We conclude that further positive inotropic effects should be expected when digitalis is given to patients with congestive heart failure who are already optimally treated with milrinone.Abbreviations K _D dissociation constant - (Na⁺ + K⁺)-ATPase sodium and potassium activated adenosine triphosphatase (EC 3.6.1.3) This study was carried out using human heart samples provided by: Prof. E. Kreuzer, Prof. B. Kemkes, Dr. C. Weinhold and their colleagues, Herzchirurgische Klinik der Universität, Klinikum Großhadern, München     

Ion content and ion fluxes in the smooth muscle cells of the longitudinal layer of the guinea-pig's vas deferens

Summary The intracellular K⁺, Cl^– and Na⁺ concentrations in the vas deferens cells of the guinea-pig have been estimated from the total ion content, the sorbitol space and the dry wt/wet wt ratio. The exchange of K⁺, Cl^– and Na⁺ was studied by following the uptake and the efflux of these ions with radioactive isotopes. The following steady state flux values have been calculated;m _K, 2.8 p-moles x cm^–2 x sec^–1;m _Cl, 13.2 p-moles x cm^–2 x sec^–1 andm _Na, 7.4 p-moles x cm^–2 x sec^–1. The values for the permeability constants calculated by the constant field assumptions from the ion fluxes are forP _K 8.9×10^–8 cm/sec;P _Cl 9.7×10^–8 cm/sec and forP _Na 2.1×10^–8 cm/sec. The introduction of these values and of the concentrations in the Goldman equation give a resting potential of –30 mV. This value is only half of the value of the measured resting potential. One of the possible explanations for the discrepancy between the measured resting potential and the calculated one is that the resting potential of these smooth muscle cells is partly a diffusion potential and partly due to the operation of an electrogenic sodium pump.This work was supported in part by the N.F.W.O. and F.G.W.O. of Belgium     

Paradox response of frog muscle membrane to changes in external potassium

Applying conventional microelectrode technique the anomalous behaviour of membrane potential in response to changes in [K⁺]_o was demonstrated in normal and cevadine-treated muscles bathed in Cl^–-free medium. Partial repolarization of the cevadine-depolarized membrane and reappearance of the slow membrane potential oscillation (SMPO) were induced by elevating [K⁺]_o from 2.5 mM to 10–20 mM. Both effects were reversed by return to 2.5 mM [K⁺]_o. The K-induced repolarization was markedly reduced by 20 mM Cs⁺, but not by 0.1 mM ouabain, 1 mM 4-aminopyridine, or 1 mM diethyl-pyrocarbonate. The elevation of [K⁺]_o failed to repolarize muscle fibers that had been depolarized only to a small extent. No K-induced repolarization has been observed in Cl^–-containing fluid. In cevadine-free experiments the omission of potassium from the extracellular space in Cl^–-free solution hyperpolarized some of the fibers, while depolarized others. Strong electrical stimuli applied in zero K-zero Cl solution turned all the fibers into depolarized state; on returning to 2.5 mM [K⁺]_o complete repolarization was achieved in most of the fibers. It has been concluded that the paradox response of the muscle membrane to changes in [K⁺]_o can be attributed to the K-dependent conductance changes of the inward rectifier K channel providing an explanation for the plateau-formation of SMPO and for the existence of two stable levels of membrane potential of the skeletal muscle bathed in Cl^–-free medium.     

Voltage dependence of the Na−K ATPase: measurements of ouabain-dependent membrane current and ouabain binding in oocytes ofXenopus laevis

Ouabain- or dihydroouabain(DHO)-sensitive membrane currents and binding of³H-ouabain were investigated under voltage-clamp conditions in full-grown prophase-arrested oocytes ofXenopus laevis. (1) The ouabainsensitive current is outwardly directed and usually exhibits a maximum at about +20 mV. The occurrence of the maximum is not affected by application of blockers for passive K⁺ currents. In the presence of Ba²⁺ as a K⁺ channel blocker, theK ₁ value for inhibition of the Na–K ATPase by ouabain is reduced by an order of magnitude, the number of binding sites is not affected. In K⁺-free solution (which inhibits the normal reaction cycle of the Na–K ATPase), addition of DHO has no significant effect on the remaining currents. (2) The voltage-dependence of the ouabain-sensitive current can be modulated. Reduction of extracellular Na⁺ increases the pump current at the resting potential and reduces the positive slope of the I–V curve. Simultaneously, the number of binding sites for ouabain is reduced by about 25%. Seasonal variations of an unknown factor affect the negative slope. (3) Modulation of the voltage-clamp conditions has no effect on the number of binding sites for ouabain, on current inhibition by ouabain, or on ouabain binding at different concentrations of the inhibitor. It is concluded that the ouabain-sensitive current is not significantly affected by passive permeabilities and that its current-voltage dependence reflects the voltage dependence of current generated by the Na–K pump. Since ouabain binding, also at non-maximal binding concentrations, is not affected by membrane potential, steps that affect the probability of the E₂P state should be voltage-insensitive.Major parts of this work are included in the thesis by B.S.     

Cardiac glycoside tolerance in cultured chicken heart muscle cells — A dose-dependent phenomenon

Summary In cultured heart muscle cells from 10–13 day-old chicken embryos, the effects of acute (4 h) and chronic (3 days) exposure of the cells to varying concentrations of ouabain have been studied. In these cells, the cardiac glycoside ouabain binds to a specific cardiac glycoside receptor (K_D=4 × 10^–7 M; 750,000 receptors/cell). Binding to this receptor results in inhibition of active Na⁺/K⁺-transport [EC₅₀ for active (⁸⁶Rb⁺ + K⁺)-influx=4 × 10^–6 M], and in an increase in beating velocity (positive inotropic effect;; EC₅₀=4 × 10^–7 M); toxic signs (arrhythmias) appear at concentrations 6 × 10^–7 M. During exposure of the cells to 3 × 10^–6 M ouabain for 3 days, tolerance develops with respect to both the positive inotropic and the toxic effect. The mechanism underlying this tolerance is identified as an increase in the number of active sodium pump molecules per cell, while the binding properties of the cardiac glycoside receptor remain unchanged. The development of cardiac glycoside tolerance is only observed in the presence of severe impairment of Na⁺/K⁺-homeostasis, due to cardiac glycoside-induced inhibition of active Na⁺/K⁺-transport. This, however, only occurs in the presence of toxic (receptor occupation 60%), but not in the presence of positive inotropic, non-toxic (receptor occupation 20–60%), ouabain concentrations. We conclude that the development of cardiac glycoside tolerance during long-term treatment in patients with heart failure should not occur with submaximal dose regimens, when toxic signs (arrhythmias) are absent.Abbreviations CGP-12177 (4-3-tert-butylamino-2-hydroxy-propoxy)-benzimidazol-2-one hydrochloride) - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulphonic acid Some of the results were presented at the IXth European Congress of Cardiology, July 1984, Düsseldorf, Germany [49]     

Modulation of delayed rectifier K+ channel activity by external K+ ions inXenopus axon

The effect of external K⁺ ions upon the activation of delayed rectifier K⁺ channels was studied in demyelinated amphibian nerve fibres by means of the patch-clamp technique. In external 105 mM K⁺ solution (high-K_o) macroscopic K⁺ currents activated at more negative potentials (–15 mV) than in external Ringer (2.5 mM K⁺). Since the rapid substitution of external Ringer with high-K_o solution at holding potentials of –70 mV and –60 mV directly activated K⁺ currents, it is concluded that K⁺ ions interact with a binding site outside the channel that facilitates channel activation. At –70 mV an increase in external K⁺ concentration from 5 mM to 10, 20, 50 and 105 mM gradually increased the open probability of the channels. Although Rb⁺ ions were less permeant through the channels, they were more potent in their interaction with the binding site and shifted K⁺ channel activation to more negative potentials. In contrast, external Cs⁺ ions had only a weak effect on the binding site. Thus, external K⁺ ions at physiological concentrations modulate the activation of delayed rectifier K⁺ channels at potentials between –90 mV and –60 mV.     

Inhibitory action of norepinephrine on sodium transport in vascular smooth muscle cells in culture

Cultured vascular smooth muscle cells from porcine aortas incubated in Na⁺-free medium rapidly release their intracellular Na⁺ contents (Na_i) (23±4% of baseline after 60 min incubation, mean ± SEM of 18 experiments). Total Na_i release was inhibited by 35–40% after addition of ouabain and by 60–70% after addition of ouabain + bumetanide. Norepinephrine inhibited ouabain and bumetanide-sensitives Na⁺ efflux with an IC₅₀ of about 10^–9–10^–8 M. Addition of the alpha-adrenergic agonist phenylephrine (10 M) to the cells mimicked the inhibitory action of norepinephrine on Na_i release. Conversely, the beta-adrenergic agonist isoproterenol was without effect on Na_i release. Simultaneous addition of 10 M norepinephrine and the alpha-adrenergic antagonist phentolamine prevented any effect of norepinephrine on the rate of Na_i decline. In A-10 cultured vascular smooth muscle cells, the alpha-adrenergic agonist phenylephrine (10 M) inhibited 40.0±8.1% of ouabain-sensitive Rb⁺ influx and 70.7±6.9% of bumetanide-sensitive Rb⁺ influx (mean ± SEM of three experiments). 50% inhibition of bumetanide-sensitive Rb⁺ influx was obtained with about 5×10^–7 M of phenylephrine. Our results show that in vascular smooth muscle cells a [Na⁺, K⁺, Cl^–]-cotransport system is able to catalyze outward Na⁺ movements (in Na⁺-free media) of a similar order of magnitude to those of the Na⁺, K⁺ pump and that alpha-adrenergic stimulation markedly inhibits Na⁺ efflux (and Rb⁺ influx) through these two transport systems.     

Relation between extracellular [K+], membrane potential and contraction in rat soleus muscle: modulation by the Na+-K+ pump

An increased extracellular K⁺ concentration ([K⁺]₀) is thought to cause muscle fatigue. We studied the effects of increasing [K⁺]₀ from 4 mM to 8–14 mM on tetanic contractions in isolated bundles of fibres and whole soleus muscles from the rat. Whereas there was little depression of force at a [K⁺]₀ of 8–9 mM, a further small increase in [K⁺]₀ to 11–14 mM resulted in a large reduction of force. Tetanus depression at 11 mM [K⁺]_o was increased when using weaker stimulation pulses and decreased with stronger pulses. Whereas the tetanic force/resting membrane potential (E _M) relation showed only moderate force depression with depolarization from –74 to –62 mV, a large reduction of force occurred whenE _M fell to –53 mV. The implications of these relations to fatigue are discussed. Partial inhibition of the Na⁺-K⁺ pump with ouabain (10^–6 M) caused additional force loss at 11 mM [K⁺]₀. Salbutamol, insulin, or calcitonin gene-related peptide all stimulated the Na⁺-K⁺ pump in muscles exposed to 11 mM [K⁺ ₀] and induced an average 26–33% recovery of tetanic force. When using stimulation pulses of 0.1 ms, instead of the standard 1.0-ms pulses, force recovery with these agents was 41–44% which was significantly greater (P < 0.025). Only salbutamol caused any recovery ofE _M (1.3 mV). The observations suggest that the increased Na⁺ concentration difference across the sarcolemma, following Na⁺-K⁺ pump stimulation, has an important role in restoring excitability and force.     

(Na+K+)-activated ATPase in human cornea

Summary Distribution and principal characteristics of (Na⁺K⁺)-activated ATPase in human cornea were investigated.(Na⁺K⁺)-ATPase was present in both epithelium and endothelium, whereas the corneal stroma did not exhibit significant enzyme activity.In homogenates specific activity of the (Na⁺K⁺)-ATPase was 2.3-fold higher in endothelium than in epithelium. Calculation of total enzyme activity revealed a 6.1-fold higher content of (Na⁺K⁺)-ATPase in the epithelium.In the epithelium a 7-fold enrichment of (Na⁺K⁺)-ATPase compared to the homogenate was obtained in the 150–1500×g _av fraction. Maximum enrichment in the endothelium was 3.5-fold and was achieved in the 1500–2500×g _av fraction. Both fractions showed, however, the same specific activity.The pH-optimum of (Na⁺K⁺)-ATPase in the 150–1500×g _av fraction ranged from 8.0–8.2 in both epithelium and endothelium.In the epithelial 150–1500×g _av fraction the apparentK _m-values were 4.0 mM for Na⁺, 2.8 mM for K⁺ and 0.12 mM for Mg²⁺ · ATP in equimolar concentrations.The inhibition constant of epithelial (Na⁺K⁺)-ATPase for ouabain was determined asK _i=3.3×10^–7 M.The present data support the view that control of corneal hydration in man is a function of both endothelium and epithelium.     

Human colonic (Na++K+)-ATPase and specific ouabain binding are not influenced by lipoxygenase products or superoxide radicals

Summary The effects of lipoxygenase products (5-, 12-, 15-HETE, LTB₄) and superoxide radicals on human colonic (Na⁺+K⁺)-ATPase and specific ouabain binding were measured. No significant inhibition in concentrations up to 3 × 10^–5 M was observed. The results are discussed with regard to a possible role of lipoxygenase products and radicals in the pathogenesis of water and electrolyte disturbances in various diarrheal states including inflammatory bowel disease.Abbrevations IBD Inflammatory bowel disease - HETE Hydroxytetraenoic acid - LTB₄ Leukotriene B₄ Supported by DFG (Er 65/4-4)     

Occurrence and properties of a (Na+−K+)-activated ATPase in the mucosa of the rat intestine

Summary The existence of an ouabain-sensitive (Na⁺–K⁺)-activated ATPase system has been demonstrated in the total intestine of the rat. The (Na⁺–K⁺)-ATPase activity was about 10–15% of the total ATPase in 4 equal parts of the small intestine; in the colon about 35% of the total ATPase was (Na⁺–K⁺)-activated ATPase. The highest (Na⁺–K⁺)-ATPase activity has been observed in the first and second part of the small intestine, while in the colon the activity was 2–4 times higher than in the ileum.The (Na⁺–K⁺)-ATPase of rat colon required both Na⁺ (K _m=8.3 mmoles/l) and K⁺ (K _m=0.6 mmoles/l). Maximal activation of the (Na⁺–K⁺)-ATPase system required 2 mM Mg²⁺ at an ATP concentration of 2 mM. The pH optimum for (Na⁺–K⁺)-ATPase of rat colon was 7.5, while the Mg²⁺-activated ATPase activity had a pH optimum of 8.6. The (Na⁺–K⁺)-ATPase was inhibited by ouabain (pI ₅₀=3.6).The relation between the differences in (Na⁺–K⁺)-ATPase activity and Na⁺-absorption on different parts of the intestine is discussed.     

The Ba2+ block of the ATP-sensitive K+ current of mouse pancreaticβ-cells

We studied the block of whole-cell ATP-sensitive K⁺ (K_ATP) currents in mouse pancreatic-cells produced by external Ba²⁺. Ba²⁺ produced a time- and voltage-dependent block of K_ATP currents, both the rate and extent of the block increasing with hyperpolarization. With 5.6 mM [K⁺]_o, the relationship between the steady-state KATP current and [Ba²⁺]_o, was fit by the Hill equation with aK _d of 12.5 ± 2.8 M at –123 mV and of 0.18 ± 0.02 mM at –62 mV The Hill coefficient (n) was close to 1 at all potentials indicating that binding of a single Ba²⁺ ion is sufficient to block the channel. When [K⁺]_o was raised to 28 mM the K_d was little changed (12.4 ± 4.1 gM at –123 mV 0.27 ± 0.05 mM at –62 mV) and n was unaffected, suggesting that K⁺ does not interact with the Ba²⁺ binding site. The kinetics of Ba²⁺ block were slow, 10 M Ba²⁺ blocking the K_ATP current with a time constant of 20 ms at –123 mV in 28 mM [K⁺]_o. The blocking rate constant was calculated as 1.7 mM^–1 ms^–1 and the unblocking rate as 0.02 ms^–1, at –123 mV The data are discussed in terms of a model in which Ba²⁺ binds to a site at the external mouth of the channel to inhibit the K_ATP channel.     

O2 consumption,aerobic glycolysis and tissue phosphagen content during activation of the NA+/K+ pump in rat portal vein

Oxygen consumption, lactate production and tissue contents of ATP, phosphocreatine (PCr) and lactate were measured following readdition of K⁺ to K⁺-depleted rat portal veins, in order to study the energy turnover associated with Na⁺/K⁺ pumping. During incubation in K⁺-free medium at 37° C spontaneous contractions disappeared in 10–20 min. Readdition of K⁺ (5.9 mM) after 40 min K⁺-free incubation caused hyperpolarization of the cell membrane for the first 5–10 min and then gradual depolarization with return of spontaneous action potentials and contractions by 10–20 min. During the first 4–6 min after K⁺ readdition aerobic lactate production was about doubled and then gradually returned to the original level (0.17 mol/min g) at about 20 min. The increase in glycolytic rate was prevented by 1 mM ouabain. In contrast, O₂ consumption (in K⁺-free medium, 0.38 mol/min g) rose by about 10% when K⁺ was added and this increase lasted about 5 min. By 8 min after K⁺ addition the increased glycolysis and oxidative phosphorylation had accounted for each about the same amount of extra ATP generation over that extrapolated from the steady rate before K⁺ addition. The average total increase in ATP turnover in the first 8 min was 15%. During this period there was no change in the cellular content of ATP, PCr, or extractable ADP. The results indicate that Na⁺/K⁺ pumping utilizes a relatively small share of the total energy turnover in the vascular smooth muscle but is to a large extent dependent on aerobic glycolysis and therefore a major site of carbohydrate usage.     

Negative inotropic effects of aldosterone antagonists in isolated human and guinea-pig ventricular heart muscle

Summary The effects of K⁺-canrenoate (Aldactone^® pro inj.) and its metabolite canrenone on isometric force of contraction were measured in isolated guinea-pig and human papillary muscle preparations driven electrically at a frequency of 1 Hz. In guinea-pig hearts both substances exerted a concentration-dependent negative inotropic effect; the IC₅₀ of K⁺-canrenoate and canrenone were 129±22 µmol l^–1 (n=5) and 85±11 µmol l^–1 (n=12), respectively. At the maximally tested concentration canrenone (250 µmol l^–1) and K⁺-canrenoate (1,000 µmol l^–1) reduced force of contraction by 68±4% (n=12) and 83±3% (n=5), respectively. The negative inotropic effects of canrenone and K⁺-canrenoate were not affected by 10 µmol l^–1 atropine. The negative inotropic effect of canrenone was also not affected by 14 µmol l^–1 aldosterone, but canrenone (10 µmol l^–1) diminished the maximal positive inotropic effect of dihydro-ouabain from 554±75% (n=4) to 269±39% (n=4) of the predrug value.In human heart muscles K⁺-canrenoate and canrenone also exerted a concentration-dependent negative inotropic effect. K⁺-canrenoate (1,000 µmol l^–1) and canrenone (250 µmol l^–1) reduced force of contraction by 57±7% (n=8) and 67±2% (n=6), respectively. A positive inotropic effect of both substances was never observed.It is concluded that the improvement of cardiac performance after application of aldosterone antagonists observed in patients cannot be explained by a direct effect on the heart. K⁺-canrenoate and canrenone are devoid of any direct cardiotonic action. Instead, K⁺-canrenoate and canrenone have direct negative inotropic effects at high concentrations.Abbreviations Aldactone^® pro inj Aldactone^® pro injection - g gramm - Hz Hertz - IC₅₀ concentration of drugs which produce 50% inhibition of force of contraction - i.v. intravenous - min minutes - mm millimeter - mm s^–1 millimeter per second - mmol l^–1 millimolar - mg milligramm - mN milli newton - ms millisecond - SC 8109 spironolactone derivative - SEM standard error of the mean - TRIS Tris(hydroxymethyl)-aminomethan - v/v volume per volume - µmol l^–1 micromolar This work was supported by the Deutsche Forschungsgemeinschaft     

Quantitative localization of Na-K pump sites in the frog sacculus

Summary The Na-K pump site distribution within the macula, perimacula, and wall epithelia of the sacculus in the frog inner ear was examined with quantitative [³H]ouabain autoradiography. Excised tissue was incubated for 10–30 min (23 ° C) in micromolar concentrations of high specific activity [³H]ouabain (14–70 Ci mT^–1, 5–15 Ci mmor^–1), washed for 30 min (4 ° C), then rapidly frozen (–175 ° C) and processed for light and electron microscope autoradiography. Control experiments based on (1) high K⁺ (50 mm) in the incubation and (2) low specific activity [³H]Jouabain (1 mM, 0.013–0.025 Ci mmol^–1) indicated negligible nonspecific binding of the [³H]ouabain.Measurable levels of specific [³H]ouabain binding occurred in all saccular regions examined. Binding was localized to the basolateral cell membranes with no detectable binding to the apical membranes. [³H]ouabain binding across the apical-basal axis of the saccule macular epithelium was nonuniform. Binding was low in the apical region, rose to a peak in the middle two-thirds, and then fell again close to the basement membrane. Electron microscope autoradiography suggested that this peak was due to ouabain binding to nerve terminals. Denervation of the sacculus eliminated the peak in [³H]ouabain binding and quantitative grain density analysis revealed that 45% of the Na-K pumps within the saccule macula were located on the nerve terminals.Na-K pump site density per unit volume was estimated by quantitative grain density analysis and the following values were obtained (sites m^–3 × 10³, means ± S.E.M.): saccule macula, 1.9 ± 0.2; saccule perimacula, 1.1 ± 0.1; saccule wall, 2.3 ± 0.3. Stereological analysis of conventionally fixed tissue was used to estimate overall plasma membrane surface area per unit volume (S _v). Na-K pump site densities per unit membrane area for the various regions were calculated by combining the autoradiographical and Stereological data. The following values were obtained (sites m^–2 ± 25%): saccule macula, 2500; saccule perimacula, 2500. Values for individual cells within the macula (sites m^–2 ± 25%) were: hair cells, 3000; nerve terminals, 3000; supporting cells, 1500.     

The mechanism of electrodiffusive K+ transport in leaky epithelia and some of its consequences for anion transport

The ventricular membrane of the epithelium from the choroid plexus ofNecturus maculosus was probed with double-barrelled ion-selective microelectrodes while the ventricular bathing solution was changed abruptly. The transient states induced by increasing the external concentration of K⁺ or by the application of ouabain showed that the passive movements of K⁺ across the ventricular membrane were electro-diffusive and could be described by the Goldmann equation and one constant permeability (P _K) of 24×10^–6 cm s^–1. The passive efflux was balanced by a ouabain-sensitive influx.P _K was different in different steady states; when the cell was acidified by half a pH unit by increasing CO₂ in the bathing solutions from 1 to 5%, thenP _K decreased to 13×10^–6 cm s^–1. Removal of Cl^– from the bathing solution increasedP _K by 50% and reduction of Cl^– transport by furosemide did not alterP _K, consequently major movements of K⁺ were independent of Cl^– movements. Depolarizations of the cell caused an increase in the cellular HCO ₃ ^– concentration due to an electrodiffusive permeability (P _HCO3), the value of which was estimated to 17×10^–6 cm s^–1.     

K+-induced swelling of vestibular dark cells is dependent on Na+ and Cl− and inhibited by piretanide

Epithelial cell height was measured in order to estimate the cell volume of dark cells from the ampullae of the semicircular canal of the gerbil. Under control conditions, addition of 10^–4 mol/l piretanide, 10^–5 mol/l 5-nitro-2(3-phenylpropylamino)-benzoic acid (NPPB), 5 mmol/l barium or 10^–3 mol/l quinidine had no significant effect on cell height. Addition of 10^–4 mol/l NPPB or 10^–3 mol/l ouabain led to a small significant decrease in cell height which was not reversible. Substitution of Na⁺ by N-methyl-d-glucamine or of Cl^– by gluconate led to a significant and reversible reduction in cell height. Isotonic elevation of [K⁺] from 3.6 to 25 mmol/l in a PO₄-buffered, HCO₃-free solution led to an increase in cell height from 5.8±0.1 (SEM) to 8.7±0.2 (n= 62) during the first 40 s. During prolonged exposure to elevated [K⁺] (3–5 min; n=19), some tissue samples underwent a regulatory volume decrease. K⁺-induced swelling was absent in both isotonic Cl^– -free and isotonic Na⁺-free solutions and was inhibited by the loop diuretic piretanide (10^–5 and 10^–4 mol/l) or by the (Na⁺+ K⁺) ATPase inhibitor ouabain (10^–3 mol/l) or by 10^–4mol/l NPPB. After the removal of ouabain or 10^–4 mol/l NPPB, K⁺-induced swelling under control conditions was enhanced and was less reversible as compared to control conditions before the experiment. K⁺-induced swelling was not altered by NPPB (10^–5 mol/l) or barium (5 mmol/l); however, barium slowed shrinking upon return of [K⁺] to control level. In the presence of 10^–3 mol/l quinidine, K⁺-induced swelling was enhanced and not reversible. These data suggest that dark cells from the semicircular canal possess an Na⁺2Cl^–K⁺ cotransporter as a solute uptake mechanism and a solute efflux mechanism which is sensitive to barium and inhibited by quinidine.     

Diminished toxicity of ouabain in the hypertrophied rat heart

The responsiveness to ouabain of hypertrophied rat hearts has been investigated either in vivo using an isolated Langendorff rat heart perfused at various external calcium concentrations, or in vitro on purified sarcolemma vesicles. (i) The physiological study shows that at 0.25 mM CaCl₂, the positive inotropic effect of 10^–5 M ouabain was diminished in hypertrophied hearts (p<0.02). At 0.5 mM CaCl₂, the drug has no effect in controls, but it has a slight positive inotropic effect in hypertrophied hearts. At 2.50 mM CaCl₂, ouabain has a negative inotropic effect accompanied by extrasystoles in controls, but in hypertrophied hearts it still has a positive inotropic effect and is not arrhythmogenic. (ii) After the pretreatment of the hearts with 2.5 mM CaCl₂, the responsiveness of the (Na⁺, K⁺)-ATPase activity to ouabain was studied: the sarcolemma from hypertrophied heart contains half as many low affinity forms of (Na⁺, K⁺)-ATPase for ouabain (35%±6) than in controls (80%±2). Assuming that the low affinity forms are responsible for the toxic effect, these data correlate well with some of the physiological findings and suggest that the diminished toxicity for ouabain in hypertrophied hearts rather reflects a modification of the properties of the (Na⁺, K⁺)-ATPases than a change in the myocardial calcium metabolism.Supported by a grant from Caisse Nationale d'Assurances Maladie des Travailleurs Salariés (1986)     

Effect of amiloride on electrolyte transport parameters of the main duct of the rabbit mandibular salivary gland

We have studied the response of the rabbit mandibular main duct perfused in vitro to luminally administered amiloride. The half-maximal inhibitory concentrations (K_I) when the duct was bathed in Cl solutions were: for net Na⁺ transport, 3×10^–6 mol l^–1; for transepithelial potential difference, 6×10^–6 mol l^–1; and for transepithelial conductance, 3×10^–7 mol l^–1. Substitution of the impermeant SO ₄ ^2– anion for Cl^– changed the K_I for conductance to 3×10^–6 mol l^–1. Within Cl^–-containing media, the time course of the amiloride effect on potential difference showed an early rapid fall of 10 mV with a half-time 2 s, followed by a slower depolarization of 9 mV, and the conductance change followed the slower component of the potential change. In SO ₄ ^2– -containing media, the potential difference and conductance changes followed time courses similar to one another. Finally, experiments on the effect of serosal applications of ouabain revealed that, although, in general, ouabain reduced resistance, it caused an increase in resistance in those ducts where the initial resistance was low. We conclude that: i) luminal Na⁺ transport occurs via amiloride-sensitive, conductive Na⁺ channels; ii) the Cl^– conductance is the major determinant of transepithelial conductance; iii) the first phase of the potential response is due to blocking of the Na⁺ conductive channels, whilst the slow phase reflects secondary inhibition of an electrogenic Na⁺ pump; and iv) duct resistance changes are secondary to alterations in intracellular Cl^– concentration.     

Membrane electrical properties of vascular smooth muscle from the guinea pig superior mesenteric artery

Some electrical membrane properties of an isolated small artery, namely, the superior mesenteric artery of the guinea pig, were studied by intracellular microelectrodes. The mean resting membrane potential (E _m) was –54 mV. The average slope of theE _m vs. log [K]_o curve (between 10 and 100 mM [K]_o) was 32 mV/decade, and the curve extrapolated to a [K]_i of 160 mM. The ratio of Na⁺ permeability to K⁺ permeability (P _Na/P _K) at 4.0 mM [K]_o calculated from the Goldman constant-field equation (assuming Cl^– to be passively distributed) was 0.18 (E _m=–46 mV after a 5 min exposure to ouabain to suppress any electrogenic pump potential). The normal input resistance (R _in) averaged 8.5 m. Choline substitution for Na⁺ or amiloride, an agent known to depressP _Na, hyperpolarized the muscle to about –63 mV without a significant change inR _in. Ba²⁺ (0.5 mM) depolarized the muscle to –37 mV, increasedR _in to 15 m, and produced spontaneous action potentials in this normally quiescent artery; tetraethylammonium (TEA, 5 mM) enabled large overshooting action potentials to be produced upon stimulation. Glutamate of NO ₃ ^– substitution for Cl^– produced an initial depolarization followed by a return to the original resting potential within 10 min; readdition of 25 mM Cl^– transiently hyperpolarized the muscle markedly, followed by a return to the originalE _m. These data indicate that Cl^– is passively distributed and does not contribute to the steady-state resting potential in this vascular muscle. The data also suggest that the relatively lowE _m in this arterial muscle is not due to a low [K]_i, but is due to a highP _Na/P _K ratio, presumably related to a low K⁺ conductance (g _K). Since Ba²⁺ and TEA⁺ are known to decrease restingg _K and K⁺ activation, the data also suggest that K⁺ activation could inhibit action potential generation.