Interdependence of Ion Transport and the Action of Ouabain in Heart Muscle

The inotropic dose‐response curve of ouabain in rat cardiac ventricular strips exceeded a concentration range of two decades (1 × 10−7 M to 3 × 10−5 M) displaying an intermediate plateau phase. In guinea‐pig ventricular strips the inotropic ouabain concentrations spanned only one decade (1 × 10−7 M‐1 × 10−6 M). Ouabain‐intoxication in guinea‐pig ventricular strips occurring at 3 × 10−6 M consisted of arrhythmia and contracture, while in rat ventricular strips at the toxic concentration of 1 × 10−4 M only a progressive increase in diastolic tension was observed. By means of atomic absorption spectroscopy the ouabain‐induced loss of cellular potassium and gain of sodium in rat ventricular strips was detected only at concentrations of ouabain higher than 10−4 M. Ouabain reduced the activity of Na/K‐ATPase prepared from rat and guinea‐pig cardiac ventricles to half of its maximum at 6.5 × 10−5 M in rat and 1.0 × 10−6 M in guinea‐pig, rat heart Na/K‐ATPase thus being about 60 fold less sensitive towards ouabain. Specific [3H]‐ouabain binding to membrane suspensions prepared from rat and guinea‐pig ventricles was characterized by a similar affinity in rat (KD = 4 × 10−8 M) and guinea‐pig (KD = 13 × 10−8 M). The number of ouabain binding sites in rat membranes was only about 10% of the number found in guinea‐pig membranes. In rat the presence of additional ouabain‐binding with low affinity and high capacity seemed possible, but could not be verified for methodological reasons. In the light of the biochemical results and binding data, the wider range of ouabain concentration exerting a positive inotropic effect in the rat may be attributed to the existence in the latter of two populations of receptors with different affinities for ouabain and different capacities. In contrast, in the guinea‐pig, there is a single population. Nevertheless it is probable that all the receptors in both species are part of the Na/K‐ATPase complex and mediate a positive inotropic effect after ouabain‐binding in an identical manner.

Section: Methodsmentioning

confidence: 99%

“…Effect ofouabain on the cellularK-and Na-content Ouabain intoxication in guinea-pig hearts has been shown to be parallelled by deterioration of the cellular electrolyte homeostasis (Bentfeld et al, 1977;Busse et al, 1979).…”

Section: Effect Ofouabain On Theforce Ofcontractionmentioning

confidence: 99%

Action of ouabain on rat heart: comparison with its effect on guinea‐pig heart

Herzig¹,

Mohr²

1984

“…Consequently, ouabain binding to intact cells (at low subtherapeutic concentrations, e.g. 10-8moll-1) is enhanced by raising intracellular [Na'] (Bentfeld et al, 1977;Temma & Akera, 1982;Kennedy et al, 1983). Alternatively, a compound reducing the 'Na-load' of the cell would be expected to reduce ouabain binding to intact cells.…”

Section: Discussionmentioning

confidence: 99%

“…Preparation ofsarcolemmal membranes Sarcolemmal membranes were isolated from guinea-pig ventricles according to the method of Bers et al (1986 Bentfeld et al (1977).…”

Section: Methodsmentioning

confidence: 99%

Differential effect of R 56865 on ouabain binding to isolated sarcolemma and intact atrial tissue of guinea‐pig

Heers

Scheufler

Wilhelm

et al. 1991

R 56865 (N‐[1‐[4‐(4‐fluorophenoxy)‐butyl]‐4‐piperidinyl]‐N‐methyl‐2‐benzothiazolamine) is a compound known to antagonize cardiac glycoside intoxication. Therefore, the effect of the compound on ouabain binding to intact cardiac tissue as well as cardiac membrane preparations was investigated. The binding of ouabain to highly purified sarcolemmal membranes was not influenced by R 56865 1 × 10−6 mol l−1 (ouabain: KD = 1.3 × 10−7 mol l−1, Bmax = 160 pmol mg−1; ouabain + R 56865: KD = 1.4 × 10−7 mol l−1, Bmax = 168 pmol mg−1). In contrast to the results in purified membranes, the binding of ouabain (10−8 mol l−1 to 5 × 10−7 mol l−1) to intact atria was significantly reduced. Ouabain, 5 × 10−7 mol l−1, led to a transient positive inotropic effect of about 220% followed by a developing negative inotropic effect after 3 h. R 56865, 10−7 mol l−1, led to a maximal positive inotropic effect of about 290% also followed by a delayed decline of contractile force. A tenfold higher concentration of R 56865 led to sustained positive inotropic effect of about 250% in the same time interval. The different effects of R 56865 on ouabain binding in subcellular preparations and intact tissue do not support the view that R 56865 interferes directly with the action of ouabain on Na/K‐ATPase. An indirect effect, which may be mediated by a lowered intracellular sodium load is discussed.

“…However, measurement of intracellular sodium concentration ([Na]i), both chemically (Tuttle, Witt & Farah, 1961;Bentfeld, Lullmann, Peters & Proppe, 1977;GhyselBurton & Godfraind, 1979) or with sodium-sensitive microelectrodes (Ellis, 1977;Deitmer & Ellis, 1978) has shown that low steroid concentrations, while increasing contractile force, do not increase [Na], and may even decrease it. This does not refute the possibility that (as first suggested by Akera, Bennett, Olgaard & Brody, 1976) highly localized or transient increases in [Nal], that cannot be detected at present, do occur, but raises a reasonable doubt as to whether inhibition of the sodium pump is a prerequisite for the positive inotropic action of steroids (see also Okita, 1977).…”

Section: Introductionmentioning

confidence: 99%

Differential Effects of Ouabain and 2,4‐dinitrophenol on Contractile Tension of and on Sodium and Calcium Efflux From Frog Heart Ventricular Strips

Ocampo

Orrego

1981

1 The efflux, from heart ventricular strips of Rana pipiens, of sodium (22Na) and calcium (45Ca) was measured simultaneously.2 22Na efflux could be resolved into two first order kinetic components: k, = 0.105 min'l, thought to represent efflux from the extracellular space, and k,l = 0.0182 min-l representing efflux from the cells.3 45Ca efflux was also resolved into an extracellular component, k, =0.1216 min'; and an intracellular one, kl, = 0.0102 min l. 45Ca k,, was greatly increased by 2,4-dinitrophenol (DNP), but unchanged by caffeine. This suggests that it represents a mitochondrial calcium compartment. 4 22Na kl, was not changed by DNP. This indicates that, at the time of DNP addition, 22Na was passively bound to undefined intracellular components. 5 Ouabain (10-6 M) decreased 45Ca efflux (k,,) initially but at later periods slightly increased it. The former effect is thought to be due to an action at the plasma membrane level, while the latter probably represents an increased exchangeability of mitochondrial calcium. The same effects were always found when ouabain was applied at different times of strip superfusion. 6 Ouabain (0.25 to 4 pM) did not decrease the k,, of 22Na efflux. Kinetic reasons are presented which indicate that, in this preparation, the activity of the sodium pump may be too fast to be measured by means of 22Na efflux, therefore these findings do not necessarily mean that ouabain does not inhibit active sodium transport. 7 The time course of the inotropic effect of ouabain was also studied in ventricular strips of Rana pipiens heart that were stimulated at 0.2 Hz with biphasic, 2 ms pulses of supramaximal intensity, and incubated in Ringer solution containing 1.1 mm calcium, or in 'calcium-free' Ringer (residual calcium: 5.2 pM), or in 'calcium-free' Ringer with 0.1 mM of the calcium chelator ethyleneglycol bis (0-aminoethylether) N,N'-tetraacetic acid (EGTA).8 In Ringer, the inotropic effect of ouabain was already observed at 5-10s after steroid addition, even with the lowest concentration tested (0.25 pM), while signs of toxicity appeared only after 15 min in 4 piM ouabain, the highest concentration used.9 When the strips were incubated in 'calcium-free' Ringer solution, force of contraction decayed to 1-2% of that in 1.1 mm calcium. Addition of 4pAM ouabain to these hypodynamic strips led to a progressive increase in contractile force of up to 300%, that started after a 50 s latency period. No signs of toxicity were observed. 10 Incubation of the strips in EGTA-Ringer also reduced contractile force to about 2% of that in Ringer, and 4 piM ouabain also increased force of contraction by approximately the same amount as seen in 'calcium-free' Ringer, but the effect began after a 10 min latency period. The concentration of calcium ion (Ca2+) in the extracellular space of strips incubated in EGTA-Ringer, was approximately 800 fold lower than in Ringer, and 60 fold lower than in 'calcium-free' Ringer solution. 11 Caffeine (20mM) induced, in strips previously incubated for 1 h in 4.4 mm cal...