DIFFERENTIAL EFFECTS OF OUABAIN AND 2,4‐DINITROPHENOL ON CONTRACTILE TENSION OF AND ON SODIUM AND CALCIUM EFFLUX FROM FROG HEART VENTRICULAR STRIPS
Authors: OCAMPO MARÍA CONCEPCIÓN , ORREGO F. 
Published: July 2012

The efflux, from heart ventricular strips of Rana pipiens, of sodium (22Na) and calcium (45Ca) was measured simultaneously

22Na efflux could be resolved into two first order kinetic components: kI − 0.105 min−1, thought to represent efflux from the extracellular space, and kII = 0.0182 min−1 representing efflux from the cells

45Ca efflux was also resolved into an extracellular component, kI = 0.1216 min−1; and an intracellular one, kII = 0.0102 min−1. 45Ca kII was greatly increased by 2,4‐dinitrophenol (DNP), but unchanged by caffeine. This suggests that it represents a mitochondrial calcium compartment

22Na kII was not changed by DNP. This indicates that, at the time of DNP addition, 22Na was passively bound to undefined intracellular components

Ouabain (10−6 m) decreased 45Ca efflux (kII) 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

Ouabain (0.25 to 4μM) did not decrease the kII 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

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 μM), or in ‘calcium‐free’ Ringer with 0.1 mM of the calcium chelator ethyleneglycol bis (β‐aminoethylether) N,N'‐tetraacetic acid (EGTA)

In Ringer, the inotropic effect of ouabain was already observed at 5 −10 s after steroid addition, even with the lowest concentration tested (0.25 μM), while signs of toxicity appeared only after 15 min in 4 μM ouabain, the highest concentration used

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 4μM 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

Incubation of the strips in EGTA‐Ringer also reduced contractile force to about 2% of that in Ringer, and 4 μM 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

Caffeine (20 mM) induced, in strips previously incubated for 1 h in 4.4 mM calcium Ringer solution plus 10−6M ouabain, a marked initial contracture, that relaxed spontaneously, and was followed by slow waves of contracture. This was not observed if the strips were incubated, prior to caffeine, in 4.4 mM calcium Ringer without ouabain, or in 1.1 mM calcium Ringer solution that contained 10−6m ouabain

Based on these findings, a hypothesis that can explain the inotropic effect of cardioactive steroids is presented.