ABSTRACT-A new 31.5-kD ouabain receptor protein (NORP), which is independent of Na+-K+ ATPase, was recently isolated selectively from transverse tubule membrane-junctional sarcoplasmic reticulum (TTM-JSR) complexes of cat cardiac muscle. We investigated the role of this NORP in cardiac function with special reference to the positive inotropic effect (PIE) of ouabain, preparing and using a monoclonal antibody (MoAB, immunoglobulin) raised against the receptor protein. Electrically stimulated papillary muscles were immersed in a Tyrode solution containing the anti-NORP MoAB (401iM), of which the binding potency was high enough for immunological use, for 60 min and then washed out. Thirty minutes after removal of the MoAB, both twitch and K-contracture were still inhibited, but both resting and action potentials and caffeine-induced contracture were unchanged, indicating that NORP plays a key role in excitation (E)-contraction (C) coupling. The intracellular localization of the protein was investigated by immunohistochemical electron microscopy, and the protein was shown to be located on the TTM, the location being probably its external surface and opposite to feet which occupy the TTM-JSR gap. These results indicate that E-C coupling of cardiac muscle cells is mediated through NORP and that ouabain-PIE occurs through the influence of ouabain on NORP in the E-C coupling process.Keywords: New ouabain receptor protein, Monoclonal antibody, Excitation-contraction Coupling, Immunoelectron microscopy, Cardiac muscle (cat)There have been many studies on the mechanism of the positive inotropic effect (PIE) of cardiac glycosides on cardiac muscle. They demonstrated that glycosides bind to a high affinity site on the Na+-K+ ATPase, but the detailed mechanism is still a matter of speculation (1-14). However, we recently solubilized a novel 31.5-kD ouabain receptor protein (NORP) from cardiac muscle that is independent of the Na+-K+ ATPase of cardiac muscles (15). This protein is extractable almost selectively from the transverse tubule membrane-junctional sarcoplasmic reticulum (TTM-JSR) system, which is regarded as a regulator of mechanical contraction or force-generation in muscle cells (16)(17)(18)(19); and as shown by 3H-ouabain photolabeling, it is probably responsible for ouabain potentiation (15). Although the mechanism of coupling of the electrical events (E) at the TTM with the intracellular Ca-release indispensable for contraction (C) is still not sufficiently understood (17, 18, 20-25), our previous studies (26-36) have suggested that ouabain potentiation is produced by the action of ouabain on the E-C coupling process in the cardiac muscle.In the present study, therefore, we examined the role of NORP in cardiac function with special reference to the PIE of ouabain; i.e., the physiological role and intracellular localization of NORP were investigated with a monoclonal antibody (MoAB) raised against the protein.A preliminary report of some of these results was published previously as an abstract (36).
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