• Animal cells and systems
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• 제1권1호
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• pp.77-80
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• 1997

DOantrolene is a primary specific therapeutic drug for prevention and treatment of malignant hyperthermia symptoms. The mechanisms underlying the therapeutic effects of the drug are not well understood. The present study aimed at the characterization of the effects of azumolene, a water soluble dantrolene analogue, on ryanodine binding to sarcoplasmic reticulum (SR) from normal and malign::lnt hyperthermia susceptible (MHS) swine muscles. Characteristics of $[^3H]ryanodine$ binding were clearly different between the two types of SR. Kinetic analysis of eH]ryanodine binding to SR in the presence of $2{\mu}M$ $Ca^{2+}$ showed that association constant $(K_{ryanodine}_7$ is significantly higher in MHS than normal muscle SR $(2.83 vs. 1.32{\times}10^7 M^{-1}$, whereas the maximal ryanodine binding capacity $(B_{max})$ is similar between the two types of SR. Addition of azumolene $(e.g. 400{\mu}M)$ did not significantly alter both $K_{ryanodine}$ and $B_{max}$ of $[^3H]$ryanodine binding in both types of SR, indicating that the azumolene effect was not on the ryanodine binding sites. Addition of caffeine activated $[^3H]$ ryanodine binding in both types of SR, and caffeine sensitivity was significantly higher in MHS muscle SR than normal muscle SR $(K_{caffeine}:3.24 vs. 0.82 {\times} 10^2 M^{-l}). Addition of azumolene $(e.g.400{\mu}M)$ decreased Kcaffeine without significant change in $B_{max}$ in both types of SR suggesting that azumolene competes with caffeine binding site(s). These results suggest that malignant hyperthermia symptoms are caused at least in part by greater sensitivity of the MHS muscle SR to the $Ca^{2+}$ release drug(s), and that azumolene can reverse the symptoms by reducing the drug affinity to $Ca^{2+}$ release channels.

• 대한약리학회지
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• 제31권2호
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• pp.207-217
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• 1995

To investigate the functional and immunological properties of the Ca-release channel in the sarcoplasmic reticulum(SR) of the eel skeletal muscle, $[^3H]ryanodine$ binding, SDS gel electrophoresis, $^{45}Ca\;release$ studies, and immunoblot assay were carried out in the SR of the eel skeletal muscle. Maximal binding sites(Bmax) and $K_D$ values of $[^3H]ryanodine$ for Ca-release channel of the SR of the eel skeletal muscle were $19.44{\pm}1.40\;pmole/mg$ protein and $15.55{\pm}1.69\;nM$, respectively. $[^3H]Ryanodine$ binding to RyR was increased by calcium and AMP. The SR of the eel skeletal muscle has two high molecular weight bands on the SDS PAGE. The mobility of upper band was more slower than the single band of the rabbit skeletal muscle, and that of the lower band was similar with the single band of canine cardiac muscle. Vesicular $^{45}Ca-release$ was activated by calcium. Ca-induced $^{45}Ca-release$ was significantly inhibited by $MgCl_2(2\;mM)$, ruthenium red$(10\;{/mu}M)$ or tetracaine(1 mM), but not by high concentration of calcium itself. AMP-induced $^{45}Ca-release$ was slightly occurred only in the absence of calcium, it was not inhibited by $MgCl_2$ or ruthenium red. Caffeine also increased $^{45}Ca-release$ from the SR vesicles, but it was not affected by $MgCl_2$ or ruthenium red. Polyclonal Ab against rat skeletal muscle RyR is reacted with that of rabbit, but not reacted with that of the eel skeletal muscle. These results suggested that ryanodine receptor of the SR of the eel skeletal muscle is showing some similar properties with that of mammalian skeletal muscle, but might be an another isotype channel having two bands which is less sensitive to AMP, not cross-reacted with antisera against rat RyR, and not inhibited by high concentration of calcium.

• The Korean Journal of Physiology and Pharmacology
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• 제5권6호
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• pp.487-494
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• 2001

During depolarization, extrusion of $Ca^{2+}$ from sarcoplasmic reticulum through forward-mode $Na^+-Ca^{2+}$ exchange was studied in the rat ventricular myocytes patch-clamped in whole-cell configuration. In order to confine the $Ca^{2+}$ responses in a micro-domain by limiting the $Ca^{2+}$ diffusion time, rat ventricular myocytes were dialyzed with high (14 mM) EGTA. $K^+$ current was suppressed by substituting KCl with 105 mM CsCl and 20 mM TEA in the pipette filling solution and by omitting KCl in the external Tyrode solution. $Cl^-$ current was suppressed by adding 0.1 mM DIDS in the external Tyrode solution. During stimulation roughly mimicking action potential, the initial outward current was converted into inward current, $47{\pm}1%$ of which was suppressed by 0.1 mM $CdCl_2.$ 10 mM caffeine increased the remaining inward current after $CdCl_2$ in a cAMP-dependent manner. This caffeine-induced inward current was blocked by $1\;{\mu}M$ ryanodine, $10\;{\mu}M$ thapsigargin, 5 mM $NiCl_2,$ or by $Na^+\;and\;Ca^{2+}$ omission, but not by $0.1\;{\mu}M$ isoproterenol. The $I{\sim}V$ relationship of the caffeine-induced current elicited inward current from -45 mV to +3 mV with the peak at -25 mV. Taken together, it is concluded that, during activation of the rat ventricular myocyte, forward-mode $Na^+-Ca^{2+}$ exchange extrudes a fraction of $Ca^{2+}$ released from sarcoplasmic reticulum mainly by voltage-sensitive release mechanism in a micro-domain in the t-tubule, which is functionally separable from global $Ca^{2+}{_i}$ by EGTA.

• The Korean Journal of Physiology and Pharmacology
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• 제15권6호
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• pp.431-436
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• 2011

Vascular smooth muscle cells can obtain a proliferative function in environments such as atherosclerosis in vivo or primary culture in vitro. Proliferation of vascular smooth muscle cells is accompanied by changes in ryanodine receptors (RyRs). In several studies, the cytosolic $Ca^{2+}$ response to caffeine is decreased during smooth muscle cell culture. Although caffeine is commonly used to investigate RyR function because it is difficult to measure $Ca^{2+}$ release from the sarcoplasmic reticulum (SR) directly, caffeine has additional off-target effects, including blocking inositol trisphosphate receptors and store-operated $Ca^{2+}$ entry. Using freshly dissociated rat aortic smooth muscle cells (RASMCs) and cultured RASMCs, we sought to provide direct evidence for the operation of RyRs through the $Ca^{2+}$- induced $Ca^{2+}$ -release pathway by directly measuring $Ca^{2+}$ release from SR in permeabilized cells. An additional goal was to elucidate alterations of RyRs that occurred during culture. Perfusion of permeabilized, freshly dissociated RASMCs with $Ca^{2+}$ stimulated $Ca^{2+}$ release from the SR. Caffeine and ryanodine also induced $Ca^{2+}$ release from the SR in dissociated RASMCs. In contrast, ryanodine, caffeine and $Ca^{2+}$ failed to trigger $Ca^{2+}$ release in cultured RASMCs. These results are consistent with results obtained by immunocytochemistry, which showed that RyRs were expressed in dissociated RASMCs, but not in cultured RASMCs. This study is the first to demonstrate $Ca^{2+}$ release from the SR by cytosolic $Ca^{2+}$ elevation in vascular smooth muscle cells, and also supports previous studies on the alterations of RyRs in vascular smooth muscle cells associated with culture.

• 대한약리학회지
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• 제32권1호
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• pp.57-66
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• 1996

파충류 골격근의 근소포체에서 칼슘유리 채널의 존재를 밝히고저 뱀 골격근에서 근소포체를 분리하여 SDS-PAGE 전기영동, RyR의 정제, $[^3H]ryanodine$ 결합실험 및 $^{45}Ca$ 유리 실험으로 아래와 같은 결과를 얻었다. 1) 뱀골격근 소포체도 단일 band의 high molecular weight 단백을 가지고 있고, 그 mobility는 포유류 골격근의 것과 유사했다. 2) RyR의 정제과정에서 얻어진 $[^3H]ryanodine$의 peak 결합 분획에서 high molecular weight의 단백분획이 발견되었다. 3) 뱀 골격근 SR vesicles에 대한 $[^3H]ryanodine$의 maximum binding site와 Kd값은 각각 6.36 pmole/mg protein과 17.62nM이었으며, $[^3H]ryanodine$의 특이성 결합은 칼슘과 AMP에 의해 유의성있게 증가되었고 (P<0.005), tetracaine에 의해 억제되지 않았으나 ruthenium red와 $MgCl_2$에 의해 일부만 억제되었다. 4) 근 소포체로부터 $^{45}Ca$ 유리는 낮은 농도의 칼슘 $(1{\sim}10{\mu}M)$과 AMP에 의해 증가되었고 (P<0.05), 고농도의 칼슘 $(300{\mu}M)$, tetracaine, ruthenium red 또는 $MgCl_2$에 의해 억제되었다 (P<0.05). 이상의 실험성적으로 파충류 (뱀)의 골격근에도 칼슘유리 채별이 있어 근 수축시 세포내 칼슘 농도 조절에 관여할 수 있을 것으로 여겨지며, 채널의 기능적 특징 일부가 포유류의 것과 유사한 것으로 사료된다.

• 한국생물물리학회:학술대회논문집
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• 한국생물물리학회 2003년도 정기총회 및 학술발표회
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• pp.66-66
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• 2003

In skeletal muscle cells, depolarization of the transverse tubules (T-tubules) results in Ca$\^$2+/ release from the sarcoplasmic reticulum (SR), leading to elevated cytoplasmic Ca$\^$2+/ and muscle contraction. This process has been known as excitation-contraction coupling (E-C coupling). Several proteins, such as the ryanodine receptor (RyR), triadin, junctin, and calsequestrin (CSQ), have been identified to be involved in the Ca$\^$2＋/ release process. However, the molecular interactions between the SR proteins have not been resolved. In the present study, the mechanisms of interaction between RyRl and triadin have been studied by in vitro protein binding and $\^$45/Ca$\^$2+/ overlay assays. Our data demonstrate that the intraluminal loop II of RyR1 binds to triadin in Ca$\^$2+/-independent manner. Moreover, we could not find any Ca$\^$2+/ binding sites in the loop II region. GST-pull down assay revealed that a KEKE motif of triadin, which was previously identified as a CSQ binding site (Kobayasi et al.,2000 JBC) was also a binding site for RyR1. Our results suggest that the intraluminal loop II of RyR could participate in the RyR-mediated Ca$\^$2+/ release process by offering a direct binding site to luminal triadin.

• The Korean Journal of Physiology and Pharmacology
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• 제14권2호
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• pp.105-111
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• 2010

Inositol 1,4,5-trisphosphate receptors ($InsP_3Rs$) modulate $Ca^{2+}$ release from intracellular $Ca^{2+}$ store and are extensively expressed in the membrane of endoplasmic/sarcoplasmic reticulum and Golgi. Although caffeine and 2-aminoethoxydiphenyl borate (2-APB) have been widely used to block $InsP_3Rs$, the use of these is limited due to their multiple actions. In the present study, we examined and compared the ability of caffeine and 2-APB as a blocker of $Ca^{2+}$ release from intracellular $Ca^{2+}$ stores and $Ca^{2+}$ entry through store-operated $Ca^{2+}$ (SOC) channel in the mouse pancreatic acinar cell. Caffeine did not block the $Ca^{2+}$ entry, but significantly inhibited carbamylcholine (CCh)-induced $Ca^{2+}$ release. In contrast, 2-APB did not block CCh-induced $Ca^{2+}$ release, but remarkably blocked SOC-mediated $Ca^{2+}$ entry at lower concentrations. In permeabilized acinar cell, caffeine had an inhibitory effect on InsP3-induced $Ca^{2+}$ release, but 2-APB at lower concentration, which effectively blocked $Ca^{2+}$ entry, had no inhibitory action. At higher concentrations, 2-APB has multiple paradoxical effects including inhibition of Ins$P_3$-induced $Ca^{2+}$ release and direct stimulation of $Ca^{2+}$ release. Based on the results, we concluded that caffeine is useful as an inhibitor of $InsP_3R$, and 2-APB at lower concentration is considered a blocker of $Ca^{2+}$ entry through SOC channels in the pancreatic acinar cell.

• 약학회지
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• 제58권4호
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• pp.245-249
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• 2014

Murrayafoline-A (1-methoxy-3-methylcarbazole) is a monomeric carbazole alkaloid found in Murraya euchrestifolia HAYATA and Glycosmis stenocarpa. We have recently shown that murrayafoline-A has positive inotropic effect in isolated rat ventricular myocytes. To know possible mechanisms for the positive inotropic effect of murrayafoline-A we examined the effects of murrayafoline-A on in situ behavior of cardiac $Ca^{2+}$ release units ('$Ca^{2+}$ sparks') and sarcoplasmic reticulum (SR) $Ca^{2+}$ loading using confocal $Ca^{2+}$ imaging method in single rat ventricular myocytes. Murrayafoline-A significantly increased the frequency (events/($10^3{\mu}m^2{\cdot}s$)) of $Ca^{2+}$ sparks in a concentration-dependent manner, with an $EC_{50}$ of $28{\pm}6.4{\mu}M$ and a maximal ~twofold change. The $Ca^{2+}$ content in the SR, measured as caffeine (10 mM)-induced $Ca^{2+}$ transient, was significantly increased by murrayafoline-A (${\approx}$116% and ${\approx}$123% of control at 25 and 100 ${\mu}M$, respectively). In addition, murrayafoline-A significantly increased the fractional $Ca^{2+}$ release, suggesting increase in the efficacy of $Ca^{2+}$ release at given SR $Ca^{2+}$ loading. These results suggest that murrayafoline-A may enhance contractility via increase in $Ca^{2+}$ release from the SR through the ryanodine receptors in ventricular myocytes.

• The Korean Journal of Physiology
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• 제24권1호
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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.

• 한국생물물리학회:학술대회논문집
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• 한국생물물리학회 2003년도 정기총회 및 학술발표회
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• pp.25-25
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• 2003

Atrial myocytes have two functionally separate $Ca^{2+}$ release sites: those in peripheral sarcoplasmic reticulum (SR) adjacent to the $Ca^{2+}$ channels of surface membrane and those in central SR not associated with $Ca^{2+}$ channels. Study on the spatio-temporal properties of focal $Ca^{2+}$ releases (“sparks”) occurring spontaneously in central and peripheral sites of voltage-clamped rat atrial myocytes, using rapid two-dimensional (2-D) confocal $Ca^{2+}$ imaging revealed that peripheral and central sparks were similar in size and release time (~300,000 $Ca^{2+}$ ions for=12 ms), but significantly larger and longer than ventricular sparks. Both sites were resistant to Cd$^{2+}$ and inhibited by ryanodine. Peripheral sparks were brighter and flattened against surface membrane, had ~5-fold higher frequency, ~2 times faster diffusion coefficient, and dissipated abruptly. Central sparks, in contrast, occurred less frequently, were elongated along the cellular longitudinal axis, and dissipated slowly. Compound sparks (composed of 2-5 unitary focal releases) aligned longitudinally, occurred more frequently at the center.at the center.