A contribution to the study on the way of action of digitalis on the myocardial flber of the frog’s heart

Abstract

The study of the effects of Digitoxin upon the proteic structures of the myocardial fiber of the frog constitutes the object of these experiences.

Frogs whose weight varied between 20 and 30 grams were used. The injection of Digitaline Nativelle, was always done in the dorsal lymphatic sac of the frog, using 0.005 mg. as minimal letal dose per gram of weight. A smaller dose was used for digitalization.

Equal groups of normal, digitalised and intoxicated frogs were used. Next the heart of each one of the groups was macerated separately and their proteic fractioning was done following Szent Gyorgy’s technique.

Immediately after 0.5 c. c. was taken from each fíltrate and placed in a vessel, adding zinc sulfate and sodium hydroxide N/10 as a deproteinization method. By this procedure a zinc hydroxide gel is formed which retains within it’s mesh the totality or the greater portion of the proteins of the myocardial fiber of the frog.

The contents on the vessels were filtered once again and the integral filtrate was taken to the Beckman ultraviolet band spectrophotometer, Model DU.

Absorption 0 for each wave length corresponds to the dry trial. The wave length use were: 275 and 290 and 300, fundamentaly.

Posteriorly we reproduced «in vitro» the preceding experiment.

Finally we studied the electrophoretic behavior of the filtrates that were obtained from the normal and the intoxicated macerated hearts without doing their deproteinization ; and the images of said filtrates were photographed through an electronic microscope.

The enclosed tables allow us to sustain that the ultraviolet light absorption is maximal on the aqueous extract of normal heart and minimal on that of the intoxicated. The absorption of the KC1 extracts was significant only on the digitalized heart.

The «in vitro» reproduction of the first of these experiments provides facts which are comparable to those found «in vivo».

From the results obtained in the experiments the following considerations are suggested:

That actin is a protein of such molecular size, which permits its escape through the mesh of the zinc hydroxide gel in a greater quantity than myosin and actomyosin of bigger molecules. This explains the greater absorption of ultraviolet light by the aqueous extract of normal hearts than by the KC1 0.6 M extract of the same.

The fact that, on the aqueous extracts of digitalized or intoxicated hearts the absorption is lower than in the case of normal hearts, can only be explained by one of these two theories:

1. Digitoxin favors the polymerization of actin, consequently longer chains of actin are formed.


2. Digitoxin favors the union of actin and myosin, consequently lowering the values of free actin and increasing those of actomyosin.

In the first case, the lower absorption is explained by the greater difficulty which zinc hydroxide offers to the passage of actin F. For form G, this difficulty is smaller.

In the second case, the explanation is obvious, because upon lowering the number of molecules of free actin the absorption by the aqueous extract of intoxicated heart mus diminish forcibly.

The consideration of the results obtained with the digitalized hearts, and its comparison with those obtained in the case of intoxication, make us suppose that both things probably happen.

The polymerization of actin by the digitalis action explains the lower absorption by the extracts of intoxicated hearts. On the other hand the greater absorption by the KC1 0.6 M extracts, in the case of digitalized hearts indicates, at least apparently. a faster and more ample combination of actin and myosin under the influence of digitalis; that is, a numerical increase of actomyosin. The result of this numerical increase of actomyosin is a greater escape of actomyosin through the gel and a greater absorption in the case of digitalization. In the intoxication there probably takes place a transformation of actomyosin G into actomyosin F which would explain the absence of absorption by the KC1 extracts of intoxicated hearts.

In the included plates, obtained with the electronic microscope, the following facts are clearly apreciated:

In the aqueous extract of normal heart the actin molecules remain dispersed or at least in a state of greater dispersion than the actin molecules in the aqueous extracts of intoxicated hearts. In the latter the actin molecules appear agglomerated, aggregated, explaining the lower absorption found on this extract. The agglomeration of actin facilitates its movilization by zinc hydroxide gel.

In the KC1 0.6 M extracts a greater number of proteic molecules are observed, although of smaller size, in case of digitalization, which corresponds unquestionably to the absorption data obtained with the Beckman.

The electrophoretic behavior of the various extracts confirms these facts.