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Role and localization of the new ouabain receptor protein (31.5 kD) were studied by using its monoclonal AB. The protein may be a key protein in the E-C coupling process in the cat cardiac muscle.

To obtain further evidence concerning the intracellular action of ouabain, we observed the effect of this compound on contractures evoked by rapid cooling in the presence of caffeine (rapid cooling caffeine contracture; RCCC), in cardiac muscles under conditions of a Ca deprived medium. The experiments were carried out at 20 degrees C using cat papillary muscles and frog ventricle strips, with the exception of cooling (2 degrees C). The muscles were first immersed in normal solution, electrically driven, then immersed in 1 mM EDTA solution for 6 min. The strips were then immersed for 10 min in a caffeine solution prepared by eluting a no-Ca solution containing 50 mM caffeine through Dowex A-1, Ca-chelating resin. Finally, the caffeine solution was exchanged with fresh caffeine solution of 2 degrees C to evoke to RCCC. Ouabain (1 X 10(-7)--1 X 10(-5) M) markedly potentiated RCCC. An ouabain-induced increase of RCCC did not appear in the resting strips unless the tissues were electrically stimulated. 3H-ouabain occupied a considerable cellular space (0.55) at the appearance of the ouabain potentiation of RCCC (14C-inulin space; 0.20). The appearance of the ouabain potentiation of RCCC was independent of changes in 22Na, Ca2+ and ATP contents in the strips. A possible mechanism of the potentiating effect of ouabain on contraction is discussed.

The present study was performed to determine the effect of ouabain on Ca release from fragmented sarcoplasmic reticulum (FSR) isolated from cat cardiac muscles. The results clearly demonstrate that ouabain potentiates the Ca release from FSR by changing the ionic environment.

In this article, we describe a possible mechanism of ouabain potentiation in heart based on the following findings in cardiac and skeletal muscles of various species. (1) In heart ventricle muscles of frog and guinea pig, the ouabain potentiation is produced without an effect on Ca influx. In both frog and cat heart ventricle muscles, ouabain potentiates the rapid cooling contracture with or without caffeine in a Ca-deprived medium. It follows, therefore, that the ouabain potentiation is produced by an "intracellular" mechanism. (2) In crab single muscle fibers, contractile responses such as twitch, potassium-induced contracture, caffeine-induced contracture, and water-induced contracture are remarkably potentiated if ouabain is present within the fibers by microinjection, whereas the situation is reversed if the drug is given extracellularly. (3) The ouabain potentiated the Ca release from fragmented sarcoplasmic reticulum (FSR) isolated from cat, guinea pig, and frog heart and from skeletal muscles as a result of the procedures used, such as changing the ionic environment. (4) In frog, cat, and guinea pig heart ventricle muscles, a reduction of contractility as a result of pretreatment with urea--Ringer's was completely cancelled by ouabain almost without influencing the membrane depolarization. Based on these findings and others, the deduction was made that the positive inotropic effect of cardiac glycosides on the heart is brought about by potentiation of contraction - Ca release from the intracellular store sites, namely the sarcoplasmic reticulum.

A new glycoprotein of 31,500 dalton, which has a high affinity for ouabain, and is independent of (Na+-K+)-ATPase, was solubilized from transverse tubule membrane and junctional sarcoplasmic reticulum complexes (TTM-JSR) of cat cardiac muscle. This protein could be extracted only in small amounts from sarcolemma-plasma membrane (SLM-PL) fragments, suggesting that it indeed originates from the TTM-JSR.