• Journal of the Korean Magnetic Resonance Society
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• v.19 no.2
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• pp.74-82
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• 2015

Ryanodine receptor (RyR) is one of the two major $Ca^{2+}$ channels in membranes of intracellular $Ca^{2+}$ stores and is found in sarcoplasmic reticulum (SR), endoplasmic reticulum (ER). RyR1 is also the major calmodulin-binding protein of sarcoplasmic reticulum membranes. Residues 4064-4210 in the RyR1 polypeptide chain has similar primary sequence with calmodulin (CaM) and was designated as CaM-like domain (CaMLD). When expressed as a recombinant peptide, CaMLD showed several CaM-like properties in previous studies. Still, previous studies of CaMLD were focused on protein-protein interactions rather than its own properties. Here, we studied the expression of CaMLD and its sub-domains corresponding to each lobe of CaM in Escherichia coli. CaMLD could be obtained only as inclusion body, and it was refolded using urea solubilization followed by dialysis. Using spectroscopic approaches, such as NMR, circular dichroism, and gel filtration experiment, we found that the refolded CaMLD exists as nonspecific aggregate, even though it has alpha helical secondary structure. In comparison, the first half of CaMLD (R4061-4141) could be obtained as natively soluble protein with thioredoxin fusion. After the removal of the fusion tag, it exhibited folded and helical properties as shown by NMR and circular dichroism experiments. Its oligomeric status was different from CaMLD, existing as dimeric form in solution. However, the second half of the protein could not be obtained as soluble protein regardless of fusion tag. Based on these results, we believe that CaMLD, although similar to CaM in sequence, has quite different physicochemical properties and that the second half of the protein renders it the aggregative properties.

• Exercise Science
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• v.26 no.3
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• pp.229-237
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• 2017

PURPOSE: This study was to investigate the Glycolysis mediated sarcoplasmic reticulum (SR) $Ca^{2+}$ signal regulates mitochondria $Ca^{2+}$ during skeletal muscle contraction by using glycolysis inhibitor. METHODS: To examine the effect of Glycolysis inhibitor on SR and mitochondria $Ca^{2+}$ content, we used skeletal muscle fiber from gastrocnemius muscle. 2-deoxy glucose and 3-bromo pyruvate used as glycolysis inhibitor, it applied to electrically stimulated muscle contraction experiment. Intracellular $Ca^{2+}$ content, SR, mitochondria $Ca^{2+}$ level and mitochondria membrane potential (MMP) was detected by confocal microscope. Mitochondrial energy metabolism related enzyme, citric acid synthase activity also examined for mitochondrial function during the muscle contraction. RESULTS: Treatment of 2-DG and 3BP decreased the muscle contraction induced SR $Ca^{2+}$ increase however the mitochondria $Ca^{2+}$ level was increased by treatment of inhibitors and showed and overloading as compared with the control group. Glycolysis inhibitor and thapsigargin treatment showed a significant decrease in MPP of skeletal muscle cells compared to the control group. CS activity significantly decreased after pretreatment of glycolysis inhibitor during skeletal muscle contraction. These results suggest that regulation of mitochondrial $Ca^{2+}$ levels by glycolysis is an important factor in mitochondrial energy production during skeletal muscle contraction CONCLUSIONS: These results suggest that mitochondria $Ca^{2+}$ level can be regulated by SR $Ca^{2+}$ level and glycolytic regulation of intraocular $Ca^{2+}$ signal play pivotal role in regulation of mitochondria energy metabolism during the muscle contraction.

• The Korean Journal of Zoology
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• v.12 no.4
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• pp.114-122
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• 1969

Electron microscopic examination of the liver and heart tissues of methylene blue-treated rats before gamma-irradiation was observed in this study. 1. It was observed severe alteration and degeneration of organelles: accumulation of glycogen particles, severe swollen mitochondria, and broken endoplasmic reticulum in liver tissue of saline-treated rat(control) opposed by emthylene blue-treated rat at 64 and 212 hours following gamma-irradiation. 2. Heart muscles of both methylene blue-treated and saline-treated rats showed no significant alterations, but it was observed that slightly elongated mitochondria with broken cristae and some of vacuoles as well as increased glycogen particles in sarcoplasmic reticulum at 212 hours following gamma-irradiation. 3. It may be considered that methylene blue greatly reduces the sensitivities of rats to gamma-irradiation.

• The Korean Journal of Pharmacology
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• v.30 no.1
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• pp.117-124
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• 1994

Phospholamban is the regulator of $Ca^{2+}-ATPase$ in cardiac sarcoplasmic reticulum(SR). The mechanism of regulation appears to involve inhibition by dephosphorylated phospholamban. Phosphorylation of phospholamban relieves this inhibition. Recently, there has been a report that the cytoplasmic domain (amino acids 1-25) of phospholamban is insufficient to inhibit the $Ca^{2+}$ pump. To explore the domains of phospholamban responsible for $Ca^{2+}-ATPase$ inhibitory activity, we examined the effect of a synthetic phospholamban peptide consisting of amino acid residues 1-25 on $Ca^{2+}$ uptake by reconstituted skeletal SR $Ca^{2+}-ATPase$. The $Ca^{2+}-ATPase$ of skeletal SR was purified and reconstituted in proteoliposomes containing phosphatidylcholine (PC) or phosphatidylcholine: phosphatidylserine (PC:PS). Inclusion of a phospholamban peptide in PC proteoliposomes was associated with significant inhibition of the initial rates of $Ca^{2+}$ uptake at pCa 6.0, and phosphorylation of this peptide by the catalytic subunit of cAMP-dependent protein kinase reversed the inhibitory effect on the $Ca^{2+}$ pump. Similar effects of phospholamban peptide were also observed using PC:PS proteoliposomes. Based on these results, we could conclude that the cytoplasmic domain of phospholamban, containing the phosphorylation sites, by itself is sufficient to inhibit the $Ca^{2+}$ pump of SR.

• The Korean Journal of Physiology and Pharmacology
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• v.9 no.2
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• pp.87-94
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• 2005

It is not clear whether $Ca^{2+}-induced$ $Ca^{2+}$ release from the sarcoplasmic reticulum (SR) is involved in the regulation of atrial natriuretic peptide (ANP) release. Previously, we have shown that nifedipine increased ANP release, indicating that $Ca^{2+}$ entry via voltage-gated L-type $Ca^{2+}$ channel activation decreases ANP release. The purpose of the present study was two-fold: to define the role of SR $Ca^{2+}$ release in the regulation of ANP release and whether $Ca^{2+}$ entry via L-type $Ca^{2+}$ channel is prerequisite for the SR-related effect on ANP release. Experiments were performed in perfused beating rabbit atria. Ryanodine, an inhibitor of SR $Ca^{2+}$ release, increased atrial myocytic ANP release ($8.69{\pm}3.05$, $19.55{\pm}1.09$, $27.31{\pm}3.51$, and $18.91{\pm}4.76$% for 1, 2, 3, and $6{\mu}M$ ryanodine, respectively; all P<0.01) with concomitant decrease in atrial stroke volume and pulse pressure in a dose-dependent manner. In the presence of thapsigargin, an inhibitor of SR $Ca^{2+}$ pump, ryanodine-induced increase in ANP release was not observed. Thapsigargin attenuated ryanodine-induced decrease in atrial dynamic changes. Blockade of L-type $Ca^{2+}$ channel with nifedipine abolished ryanodine-induced increase in ANP release ($0.69{\pm}5.58$% vs. $27.31{\pm}3.51$%; P＜0.001). In the presence of thapsigargin and ryanodine, nifedipine increased ANP release and decreased atrial dynamics. These data suggest that $Ca^{2+}$-induced $Ca^{2+}$ release from the SR is inversely involved in the regulation of atrial myocytic ANP release.

• The Korean Journal of Physiology
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• v.24 no.1
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• pp.77-90
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• 1990

There have been conflicting reports concerning the effect of Panax ginseng on the contractility of vascular smooth muscle, i.e., Panax ginseng extract has been reported to cause relaxation, contraction or to have no effect on the tension of vascular smooth muscle. A further investigation of $Ca^{++}$ stores which supply $Ca^{++}$ for contraction of vascular smooth muscle is needed to understand the underlying mechanisms of this conflicting effect of ginseng alcohol extract (GAE). The present study was intended to examine the sources of calcium mobilized for contraction of vascular smooth muscle by GAE. Aortic ring preparations were made from the rabbit thoracic aorta and endothelial cells were removed from the ring. The contractility of the aortic ring was measured under various experimental conditions and $Ca^{++}$ flux across the membrane of aortic ring and the sarcoplasmic reticulum and mitochondria were measured with a calcium selective electrode. The result were summarized as follows; 1) At low concentration of extracellular $Ca^{++}$, GAE increased the contractility of vascular smooth muscle in dose-dependent fashion except high concentration $Ca^{++}$ (1 mM). 2) In the presence of ryanodine, GAE still increased contractility of vascular smooth muscle as much as control group, but in the presence of caffeine, GAE increased it significantly. i.e. Their effects seemed to be additive. 3) In the presence of verapamil+lanthanum, and verapamil+lanthanum+ryanodine, the contractility of the vascular smooth muscle was decreased, but a dose dependent increase in vascular tension was still demonstrated by GAE although total tension was low. 4) GAE increased $Ca^{++}$ efflux from vascular smooth muscle cells, but have no effect on $Ca^{++}$ influx. 5) GAE increased $Ca^{++}$ efflux from sarcoplasmic reticulum and mitochondria vesicles. From the above results, it may be concluded that GAE increased the release of $Ca^{++}$ from sarcoplasmic reticulum, mitochondria or other intracellular $Ca^{++}$ stores of vascular smooth muscle, but it does not increase $Ca^{++}$ influx across the plasma membrane.

• The Korean Journal of Physiology and Pharmacology
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• v.5 no.5
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• pp.397-405
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• 2001

The ryanodine receptor, a $Ca^{2+}$ release channel of the sarcoplasmic reticulum (SR), is responsible for the rapid release of $Ca^{2+}$ that activates cardiac muscle contraction. In the excitation-contraction coupling cascade, activation of SR $Ca^{2+}$ release channel is initiated by the activity of sarcolemmal $Ca^{2+}$ channels, the dihydropyridine receptors. Previous study showed that the relaxation defect of diabetic heart was due to the changes of the expressional levels of SR $Ca^{2+}$ATPase and phospholamban. In the diabetic heart contractile abnormalities were also observed, and one of the mechanisms for these changes could include alterations in the expression and/or activity levels of various $Ca^{2+}$ regulatory proteins involving cardiac contraction. In the present study, underlying mechanisms for the functional derangement of the diabetic cardiomyopathy were investigated with respect to ryanodine receptor, and dihydropyridine receptor at the transcriptional and translational levels. Quantitative changes of ryanodine receptors and the dihydropyridine receptors, and the functional consequences of those changes in diabetic heart were investigated. The levels of protein and mRNA of the ryanodine receptor in diabetic rats were comparable to these of the control. However, the binding capacity of ryanodine was significantly decreased in diabetic rat hearts. Furthermore, the reduction in the binding capacity of ryanodine receptor was completely restored by insulin. This result suggests that there were no transcriptional and translational changes but functional changes, such as conformational changes of the $Ca^{2+}$ release channel, which might be regulated by insulin. The protein level of the dihydropyridine receptor and the binding capacity of nitrendipine in the sarcolemmal membranes of diabetic rats were not different as compared to these of the control. In conclusion, in diabetic hearts, $Ca^{2+}$ release processes are impaired, which are likely to lead to functional derangement of contraction of heart. This dysregulation of intracellular $Ca^{2+}$ concentration could explain for clinical findings of diabetic cardiomyopathy and provide the scientific basis for more effective treatments of diabetic patients. In view of these results, insulin may be involved in the control of intracellular $Ca^{2+}$ in the cardiomyocyte via unknown mechanism, which needs further study.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.31 no.4
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• pp.553-559
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• 1998

Electron micrographs of muscle of plaice killed instantly by spiking at the head clearly showed the A-band, I-band, Z-line and M-line in muscle strips, whereas these bands could not be distinguished from each other in electrically stimulated plaice muscle strips. As the electrical stimulation time increased, the continuity of Z-line disrupted rapidly. Electron microscopic observation showed that sarcoplasmic reticulum (SR) in unstimulated plaice had natural triad structures between Z-line. However these structures were disrupted in the electrically stimulated sample. These structures were clearly observed after storage for 10 hrs at $5^{\circ}C$ from all the samples. In association with the $Ca^{2+}$ translocation, $Ca^{2+}$-pyroantimonate deposits were observed at the inner part of SR immediately after spiked the plaice, However, $Ca^{2+}$-pyroantimonate deposits in electrically stimulated plaices were observed in the muscle strips and this phenomenon was clearly observed when electrical stimulation was prolonged.

• YAKHAK HOEJI
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• v.31 no.3
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• pp.140-148
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• 1987

The effects of orally administered ginseng ethanol extract on the calcium release from sarcoplasmic reticulum (SR) calcium pool and on the calcium content in the rat heart perfused with the Langendorff apparatus. The total amount of calcium released from SR calcium pool and the total calcium content in the rat heart were significantly decreased by 43% and 26%, respectively compared with the control.

• Proceedings of the Korean Biophysical Society Conference
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• 2001.06a
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• pp.46-46
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• 2001

Evidence has suggested that an anion channel blocker, 4, 4'-diisothiocyanatostilbene-2, 2' disulfonic acid (DIDS) could trigger Ca release from skeletal sarcoplasmic reticulum (SR) by binding to a 30 kDa SR protein. Since the high molecular weight $Ca^{2＋}$ release channel (CRC)/ryanodine receptor (RyR) is the main SR protein that conducts $Ca^{2＋}$ efflux in skeletal muscles, the relationship between CRC and the 30kDa protein remains to be elucidated.(omitted)