Studies of a key protein in the mechanism of the excitation-contraction coupling process of frog skeletal muscle, using phenylglyoxal

Fujino, Sumiko; Satoh, Kumi; Nakaï, T.; Togashi, Kunihiko; Kado, Tetsuo; Fujino, Masako; Arima, Takashi

doi:10.1007/bf01989418

Cited by 3 publications

(9 citation statements)

References 23 publications

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“…Therefore, NORP should play a key role in PIE and must be considered in discussions on the mechanism of the PIE of ouabain. Thus, the present study expands our previous findings by applying affinity chromatography and immunological techniques using anti-NORP MoAB (37).…”

Section: Discussionsupporting

confidence: 82%

“…Isolation of TTM-JSR complexes and solubilization and purification of NORP Isolation of TTM-JSR complexes and solubilization and purification of NORP, the 31.5-kD protein, were performed according to the previously described methods (15,37) except for the additional use of wheat germ agglutinin (WGA) affinity chromatography to remove all the dihydropyridine (DHP) receptor protein and its subunits. WGA affinity chromatography was done according to Curtis and Catterall (38).…”

Section: Recording Of Developed Tensionmentioning

confidence: 99%

“…CH9201) was prepared as an immunoglobulin by the previously described method (37) except for the use of cat cardiac NORP instead of frog skeletal 31.5-kD protein (electrometrin) as the antigen. A 5-to 6-week-old female BALB/c mouse received an intraperitoneal injection of the Con A-sensitive proteins (100 pg of protein) from cat cardiac muscle TTM-JSR in Freund complete adjuvant.…”

Section: Preparation Of the Anti-norp Moabmentioning

confidence: 99%

“…Diaminobenzidine was used as the color reagent for developing the immunoblot (a in C-2 of Fig. 1) as previously reported (37).…”

Section: Western Blot Analysismentioning

confidence: 99%

“…In B, the muscle was first immersed for 60 min in a slightly hypertonic Tyrode solution containing the anti NORP MoAB (10 ug/ml, 2-fold higher than the minimum concentration required for saturation), after which time the normal Tyrode solution was restored. The slightly hypertonic bathing solution was used to increase the accessibility of the MoAB to the TTM by dilating the tubules (37). As is seen in the mechanogram, about 30 min after removal of the MoAB, both twitch and K-contracture tensions decreased markedly without affecting caffeine-induced contracture and resting and action potentials ( Table 1).…”

Section: H -Ouabain Binding To Norp and Autoradiographymentioning

confidence: 99%

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An Immunohistochemical Study of the Significance of a New 31.5-kD Ouabain Receptor Protein Isolated from Cat Cardiac Muscle

Fujino

Fujino²

1995

Japanese Journal of Pharmacology

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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). MATERIALS AND...

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Section: Discussionsupporting

confidence: 82%

Section: Recording Of Developed Tensionmentioning

confidence: 99%

Section: Preparation Of the Anti-norp Moabmentioning

confidence: 99%

“…Diaminobenzidine was used as the color reagent for developing the immunoblot (a in C-2 of Fig. 1) as previously reported (37).…”

Section: Western Blot Analysismentioning

confidence: 99%

Section: H -Ouabain Binding To Norp and Autoradiographymentioning

confidence: 99%

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An Immunohistochemical Study of the Significance of a New 31.5-kD Ouabain Receptor Protein Isolated from Cat Cardiac Muscle

Fujino

Fujino²

1995

Japanese Journal of Pharmacology

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The α2 Isoform of Na,K-ATPase Mediates Ouabain-induced Cardiac Inotropy in Mice

Dostanic

Lorenz

Schultz

et al. 2003

Journal of Biological Chemistry

104

135

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Inhibition of Na,K-ATPase activity by cardiac glycosides is believed to be the major mechanism by which this class of drugs increases heart contractility. However, direct evidence demonstrating this is lacking. Furthermore it is unknown which specific ␣ isoform of Na,K-ATPase is responsible for the effect of cardiac glycosides. Several studies also suggest that cardiac glycosides, such as ouabain, function by mechanisms other than inhibition of the Na,K-ATPase. To determine whether Na,K-ATPase, specifically the ␣2 Na,K-ATPase isozyme, mediates ouabain-induced cardiac inotropy, we developed animals expressing a ouabain-insensitive ␣2 isoform of the Na,K-ATPase using Cre-Lox technology and analyzed cardiac contractility after administration of ouabain. The homozygous knock-in animals were born in normal Mendelian ratio and developed normally to adulthood. Analysis of their cardiovascular function demonstrated normal heart function. Cardiac contractility analysis in isolated hearts and in intact animals demonstrated that ouabain-induced cardiac inotropy occurred in hearts from wild type but not from the targeted animals. These results clearly demonstrate that the Na,K-ATPase and specifically the ␣2 Na,K-ATPase isozyme mediates ouabain-induced cardiac contractility in mice.

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Functional types of single muscle fibers from the depressor mandibulae muscle of R. japonica and their mechanism of excitation-contraction (E-C) coupling. I. Isolation, properties and electron microscopy of a single muscle fiber from the depressor mandibulae muscle.

Takahashi

1994

Jpn J Oral Biol

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Studies of a key protein in the mechanism of the excitation-contraction coupling process of frog skeletal muscle, using phenylglyoxal

Cited by 3 publications

References 23 publications

An Immunohistochemical Study of the Significance of a New 31.5-kD Ouabain Receptor Protein Isolated from Cat Cardiac Muscle

An Immunohistochemical Study of the Significance of a New 31.5-kD Ouabain Receptor Protein Isolated from Cat Cardiac Muscle

The α2 Isoform of Na,K-ATPase Mediates Ouabain-induced Cardiac Inotropy in Mice

Functional types of single muscle fibers from the depressor mandibulae muscle of R. japonica and their mechanism of excitation-contraction (E-C) coupling. I. Isolation, properties and electron microscopy of a single muscle fiber from the depressor mandibulae muscle.

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