• The Korean Journal of Physiology and Pharmacology
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• 제1권5호
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• pp.565-572
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• 1997

In the freshly isolated rat nephron, the effect of endothelin-1, -2 and -3 (ET-1, -2 and -3) on cytosolic free calcium concentration ($[Ca^{2+}]_i$) was determined using the fluorescent indicator Fura-2/AM. $[Ca^{2+}]_i$ increase was investigated in 9 parts of the single nephron including glomerulus (Glm), $S_1,\;S_2,\;S_3$, cortical and medullary thick ascending limb and cortical (CCT) and outer medullary collecting tubule (OMCT). Endothelins increased $[Ca^{2+}]_i$ in Glm (ET-1; $127{\pm}17%$, ET-2; $93{\pm}5%$, ET-3; $169{\pm}17%$), CCT (ET-1; $30{\pm}6%$, ET-2; $38{\pm}19%$, ET-3; $158{\pm}18%$) and OMCT (ET-1; $197{\pm}11%$, ET-2; $195{\pm}11%$, ET-3; $215{\pm}37%$) at 10-7 M. In OMCT, ET-1 and ET-2 increased $[Ca^{2+}]_i$ in a dose-dependent manner ($10^{-10}{\sim}10^{-6}$ M). To the contrary, ET-3-induced $[Ca^{2+}]_i$ rise was begun from $10^{-12}$ M. BQ-123Na, an antagonist of ETA receptor, at $10^{-4}$ M inhibited about 30% of $[Ca^{2+}]_i$ rise induced by ET-1 and -3. Binding experiments using $[^{125}I]ET-3$ showed the existence of $ET_B$ receptor in OMCT. This binding was replaced by ET-1, ET-2 or ET-3 by the almost same degree but not by angiotensin II or vasopressin.

• Journal of Microbiology
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• 제34권3호
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• pp.263-263
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• 1996

Infection of fish cells with IHNV resulted in gradual increase in cytosolic free $Ca^{2+}$ concentration $([Ca^{2+}]_i)$ in CHSE, gradual decrease in $[Ca^{2+}]_i$ in FHM, and no significant change in RTG cells. The degree of $[Ca^{2+}]_i$ increase or decrease was dependent on the amount of infectious virus, and these $[Ca^{2+}]_i$ variations were maximal at 16 hours after virus infection (p. i.) in both cell lines. When the fish cells were infected with inactivated IHNV, evident variation in $[Ca^{2+}]_i$ was not observed. Thus, infectivity of IHNV appears to correlate with changes in $[Ca^{2+}]_i$ in virus-infected cells. These IHNV-induced $[Ca^{2+}]_i$ changes were partially blocked by cycloheximide, but not affected by cordycepin. It seems to be that virus-induced $Ca^{2+}$ variations were more related with protein synthesis than RNA synthesis. Various $Ca^{2+}$ related drugs were used in search for the mechanisms of the $[Ca^{2+}]_i$, changes following IHNV infection of CHSE cells. Decreasing extracellular $Ca^{2+}$ concentration or blocking $Ca^{2+}$ influx from extracellular media inhibited the IHNV-induced increase in $[Ca^{2+}]_i$, in CHSE cells. Similar results were obtained with intracellular $Ca^{2+}$ blockers. Thus it is suggested that both the extracellular and the intracellular $Ca^{2+}$ sources are important in IHNV-induced $[Ca^{2+}]_i$ increase in CHSE cells.

• 미생물학회지
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• 제36권4호
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• pp.306-311
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• 2000

Herpes simplex virus type-1 (HSV-1)의 감염에 따른 세포내 유리 칼슘농도의 변화에 대한 실험을 수행한 결과, HSV-1이 Vero 세포에 감염한 후 4시간째에 세포내 칼슘농도가 최대로 감소한 것을 알았으며 이러한 세포내 유리 칼슘농도의 감소는 감염성 바이러스의 양에 따라 커지며, 유전자 발현 억제제의 처리나 바이러스의 불활성화에 의해 극복되었다. 따라서 바이러스의 유전자발현이 세포내 유리 칼슘농도의 감소에 중요한 역할을 한다는 것을 알 수 있다. 또한 Vero 세포에 바이러스를 감염시키고 미세소관 안정제인 taxol을 처리하여 4 시간째의 세포내 유리 칼슘농도의 감소가 극복된다는 사실로부터 바이러스이 유전자 물질의 이동에는 미세소관이 관여한다는 것을 알 수 있었다. 이와 같은 실험 결과로부터 Vero 세포에서 HSV-1에 의해 유도되는 세포내 유리칼슘 농도의 감소는 HSV-1 증식과 밀접한 관계를 가진다고 생각된다.

• Preventive Nutrition and Food Science
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• 제6권1호
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• pp.43-50
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• 2001

The effects of cardiac ischemia-reperfusion and $\beta$-adrenergic stimulation on metabolic function and energetics were investigated in Lan gendorff-perfused spontaneously hypertensive rat (SHR) hearts. Sarcoplasmic reticulum {TEX}$Ca^{2+}${/TEX}-dependent ATPase and cardiac lactate dehydrogenase (LDH) are additionally studied. The perfusion medium (1.0 mM {TEX}$Ca^{2+}${/TEX}) contained 5 mM glucose(+5 U/L insulin) and 2 mM pyruvate as substrates. Global ischemia was induced by reducing perfusion pressure of 100 to 40 cm {TEX}$H_{2}${/TEX}O, followed by 20 min reperfusin. Isoproterenol (ISO, 1$\mu$M) was infused for 10 min. Coronary vascular resistance and myocardial oxygen consumption ({TEX}$MVO_{2}${/TEX}) of SHR were increased in parallel with enhanced venous lactate during ischemia and reperfusion compared to those of Sprague Dawley (SD) hearts. Although ischemia-induced increase in venous lactate and combined adenosine plus inosine was abolished, coronary vasodilation produced in SD during reperfusion. In SHR, depressed reactive hyperemia was associated with a fall in cardiac ATP and CrP/Pi ratio and a rise in intracellular lactate/Pyruvate ratio. On the other hand, ISO produced coronary functional hyperemia and an increase in {TEX}$MVO_{2}${/TEX}. However, these responses were less than those in SHR hearts. The ATPase activity of SHR was attenuated in free {TEX}$Ca^{2+}${/TEX} concentrations used under basal condition and with ISO compared to that of SD. Venous lactate output and cardiac LDH activity were augmented in SHR as influenced by ISO. These results demonstrate that coronary reactive and functional hyperemia was dpressed in SHR, which cold be explained by alterations in the cytosolic phosphorylation potential and the cytosolic redox state manipulated by LDH, and by abnormal free calcium handling.

• Asian Pacific Journal of Cancer Prevention
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• 제15권4호
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• pp.1551-1556
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• 2014

Background: Transient receptor potential melastatin 8 (TRPM8), a principle membrane receptor involved in calcium ion influx and cell signal transduction, has been found to be up-regulated in some cancer types, including melanomas. Efficiency of menthol, an agonist of TRPM8, in killing melanoma cancer cells has been reported previously, but the mechanisms remain unclear. We here determined whether in vitro cytotoxic effects of menthol on A-375 human malignant melanoma cells might be related to TRPM8 transcript expression. Materials and Methods: The $PrestoBlue^{(R)}$ cell viability assay was used to assess the in vitro cytotoxic effect of menthol after 24h of treatment. RT-PCR was used to quantify TRPM8 transcript expression levels in normal and menthol-treated cells. Cell morphology was observed under inverted phase contrast light microscopy. Results: TRPM8 transcript expression was found at low levels in A-375 cells and down-regulated in a potentially dose-dependent manner by menthol. Menthol exerted in vitro cytotoxic effects on A-375 cells with an $IC_{50}$ value of 11.8 ${\mu}M$, which was at least as effective as 5-fluorouracil ($IC_{50}=120{\mu}M$), a commonly applied chemotherapeutic drug. Menthol showed no dose-dependent cytotoxicity on HeLa cells, a TRPM8 non-expressing cell line. Conclusions: The cytotoxic effects on A-375 cells caused by menthol might be related to reduction of the TRPM8 transcript level. This suggests that menthol might activate TRPM8 to increase cytosolic $Ca^{2+}$ levels, which leads to cytosolic $Ca^{2+}$ imbalance and triggers cell death.

• Biomolecules & Therapeutics
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• 제23권5호
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• pp.421-427
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• 2015

Imperatorin has been known to exert many biological functions including anti-inflammatory activity. In this study, we investigated the inhibitory effects of imperatorin on the production of inflammatory mediators in mouse bone marrow-derived mast cells (BMMC). Imperatorin inhibited degranulation and the generation of eicosanoids (leukotriene $C_4$ ($LTC_4$) and prostaglandin $D_2$ ($PGD_2$) in IgE/antigen (Ag)-stimulated BMMC. To elucidate the molecular mechanism involved in this process, we investigated the effect of imperatorin on intracellular signaling in BMMC. Biochemical analyses of the IgE/Ag-mediated signaling pathway demonstrated that imperatorin dramatically attenuated degranulation and the production of 5-lipoxygenase-dependent $LTC_4$ and cyclooxygenase-2-dependent $PGD_2$ through the inhibition of intracellular calcium influx/phospholipase $C{\gamma}1$, cytosolic phospholipase $A_2$/mitogen-activated protein kinases and/or nuclear factor-${\kappa}B$ pathways in BMMC. These results suggest that the effects of imperatorin on inhibition of degranulation and eicosanoid generation through the suppression of multiple steps of IgE/Ag-mediated signaling pathways would be beneficial for the prevention of allergic inflammation.

• The Korean Journal of Physiology and Pharmacology
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• 제19권4호
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• pp.373-382
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• 2015

Fura-2 analogs are ratiometric fluoroprobes that are widely used for the quantitative measurement of [$Ca^{2+}$]. However, the dye usage is intrinsically limited, as the dyes require ultraviolet (UV) excitation, which can also generate great interference, mainly from nicotinamide adenine dinucleotide (NADH) autofluorescence. Specifically, this limitation causes serious problems for the quantitative measurement of mitochondrial [$Ca^{2+}$], as no available ratiometric dyes are excited in the visible range. Thus, NADH interference cannot be avoided during quantitative measurement of [$Ca^{2+}$] because the majority of NADH is located in the mitochondria. The emission intensity ratio of two different excitation wavelengths must be constant when the fluorescent dye concentration is the same. In accordance with this principle, we developed a novel online method that corrected NADH and Fura-2-FF interference. We simultaneously measured multiple parameters, including NADH, [$Ca^{2+}$], and pH/mitochondrial membrane potential; Fura-2-FF for mitochondrial [$Ca^{2+}$] and TMRE for ${\Psi}_m$ or carboxy-SNARF-1 for pH were used. With this novel method, we found that the resting mitochondrial [$Ca^{2+}$] concentration was $1.03{\mu}M$. This $1{\mu}M$ cytosolic $Ca^{2+}$ could theoretically increase to more than 100 mM in mitochondria. However, the mitochondrial [$Ca^{2+}$] increase was limited to ${\sim}30{\mu}M$ in the presence of $1{\mu}M$ cytosolic $Ca^{2+}$. Our method solved the problem of NADH signal contamination during the use of Fura-2 analogs, and therefore the method may be useful when NADH interference is expected.

• The Korean Journal of Physiology and Pharmacology
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• 제22권2호
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• pp.215-223
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• 2018

Intracellular $Ca^{2+}$ mobilization is closely linked with the initiation of salivary secretion in parotid acinar cells. Reactive oxygen species (ROS) are known to be related to a variety of oxidative stress-induced cellular disorders and believed to be involved in salivary impairments. In this study, we investigated the underlying mechanism of hydrogen peroxide ($H_2O_2$) on cytosolic $Ca^{2+}$ accumulation in mouse parotid acinar cells. Intracellular $Ca^{2+}$ levels were slowly elevated when $1mM\;H_2O_2$ was perfused in the presence of normal extracellular $Ca^{2+}$. In a $Ca^{2+}-free$ medium, $1mM\;H_2O_2$ still enhanced the intracellular $Ca^{2+}$ level. $Ca^{2+}$ entry tested using manganese quenching technique was not affected by perfusion of $1mM\;H_2O_2$. On the other hand, $10mM\;H_2O_2$ induced more rapid $Ca^{2+}$ accumulation and facilitated $Ca^{2+}$ entry from extracellular fluid. $Ca^{2+}$ refill into intracellular $Ca^{2+}$ store and inositol 1,4,5-trisphosphate ($1{\mu}M$)-induced $Ca^{2+}$ release from $Ca^{2+}$ store was not affected by $1mM\;H_2O_2$ in permeabilized cells. $Ca^{2+}$ efflux through plasma membrane $Ca^{2+}-ATPase$ (PMCA) was markedly blocked by $1mM\;H_2O_2$ in thapsigargin-treated intact acinar cells. Antioxidants, either catalase or dithiothreitol, completely protected $H_2O_2-induced$ $Ca^{2+}$ accumulation through PMCA inactivation. From the above results, we suggest that excessive production of $H_2O_2$ under pathological conditions may lead to cytosolic $Ca^{2+}$ accumulation and that the primary mechanism of $H_2O_2-induced$ $Ca^{2+}$ accumulation is likely to inhibit $Ca^{2+}$ efflux through PMCA rather than mobilize $Ca^{2+}$ ions from extracellular medium or intracellular stores in mouse parotid acinar cells.

• Molecules and Cells
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• 제43권11호
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• pp.909-920
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• 2020

Cytosolic Ca²⁺ levels ([Ca²⁺]_c) change dynamically in response to inducers, repressors, and physiological conditions, and aberrant [Ca²⁺]_c concentration regulation is associated with cancer, heart failure, and diabetes. Therefore, [Ca²⁺]_c is considered as a good indicator of physiological and pathological cellular responses, and is a crucial biomarker for drug discovery. A genetically encoded calcium indicator (GECI) was recently developed to measure [Ca²⁺]_c in single cells and animal models. GECI have some advantages over chemically synthesized indicators, although they also have some drawbacks such as poor signal-to-noise ratio (SNR), low positive signal, delayed response, artifactual responses due to protein overexpression, and expensive detection equipment. Here, we developed an indicator based on interactions between Ca²⁺-loaded calmodulin and target proteins, and generated an innovative GECI sensor using split nano-luciferase (Nluc) fragments to detect changes in [Ca²⁺]_c. Stimulation-dependent luciferase activities were optimized by combining large and small subunits of Nluc binary technology (NanoBiT, LgBiT:SmBiT) fusion proteins and regulating the receptor expression levels. We constructed the binary [Ca²⁺]_c sensors using a multicistronic expression system in a single vector linked via the internal ribosome entry site (IRES), and examined the detection efficiencies. Promoter optimization studies indicated that promoter-dependent protein expression levels were crucial to optimize SNR and sensitivity. This novel [Ca²⁺]_c assay has high SNR and sensitivity, is easy to use, suitable for high-throughput assays, and may be useful to detect [Ca²⁺]_c in single cells and animal models.

• Journal of Chest Surgery
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• 제32권7호
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• pp.603-612
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• 1999

연구배경: 짧은 기간 동안 허혈-재관류를 반복(ischemic preconditioning, IP)할 경우 후속되는 보다 긴 기간 동 안의 허혈에 대하여 재관류시 심근의 수축기능 회복이 증가, 심근괴사 범위 감소 등의 심근보호효과가 있음 은 여러 가지 동물실험으로 밝혀졌으며 인간의 심장에서도 유사한 효과가 나타나는 것으로 보고되고 있다. 최근 칼슘이 매개가 되어 protein kinase C(PKC)의 활성화가 일어남으로서 IP효과가 나타날 것이라는 실험결 과들이 제시되고 있으나 논란이 많다. 본 연구에서는 적출 토끼심장을 이용하여 칼슘이 심근세포내의 PKC 활성도에 어\ulcorner 영향을 미치는가를 연구하고자 하였다. 대상 및 방법: 적출관류 흰토끼 심장을 이용하여 관 류를 차단하는 방법으로 전체허혈을 유도하였으며 전체허혈(5분), 재관류(10분)를 1회 실시하여 IP를 유도하 고 45분 동안 전체허혈후 120분 동안 재관류를 실시하였다(IP군, n=13). 허혈 대조군(n=10)에서는 IP없이 45 분 동안 전체허혈후 120분 동안 재관류를 실시하였다. 칼슘투여군에서는 5분 동안 허혈후 10분 동안 10 (n=10) 또는 20 mM(n=11)의 칼슘을 포함한 관류액으로 관류하고 이어서 45분 동안 전체허혈과 120분 동안 재관류를 실시하였다. 전 실험 기간 동안 좌심실기능, 관혈류를 측정하였으며 실험 종료 후 PKC-specific peptide와 32P-${\gamma}$-ATP incorporation으로 PKC활성도(nmol/g tissue)를 측정하였다. 심근괴사 크기는 1% tetra zolium chloride로 염색하여 형태계측하였다. 결과: IP를 실시한 결과, LVDP(left ventricular developed pressure), 심근수축력, 관혈류 등은 허혈 대조군에 비하여 현저히 증가하였으며(p<0.05) 이완말기압의 상승폭은 저하되 었고(p<0.05) 심근괴사 크기는 38%에서 20%로 감소하였다(p<0.05). 칼슘투여군에서는 LVDP, 심근수축력, 관 혈류 등에는 허혈 대조군에 비하여 큰 차이가 없거나 오히려 저하되었으나 심근괴사 크기는 19~23%로 현 저히 감소하였다(p<0.05). 세포질분획의 PKC활성도(nmol/g tissue)는 IP군, 칼슘투여군에서 각각 5.98$\pm$0.57, 6.30$\pm$0.24(20 mM 칼슘 전처치군), 4.19$\pm$0.39(10 mM 칼슘 전처치군)로 기준(7.31$\pm$0.31)에 비하여 특히 10 mM 칼슘 전처치군에서 유의하게 감소하였으며(p<0.01), 세포막분획의 PKC활성도는 각각 4.00$\pm$0.14, 2.50$\pm$ 0.31, 4.02$\pm$0.70으로 기준(1.84$\pm$0.21)에 비하여 IP군과 10 mM 칼슘 전처치군에서 유의하게 증가하였다 (p<0.05). 그러나 허혈대조군에서는 두 분획 모두 기준선과 비교하여 큰 차이가 없었다. 결론: 이상으로 적출 관류 토끼심장에서 장시간 동안의 허혈전 높은 농도의 칼슘으로 전처치하면 허혈후 재관류시 심근기능의 회 복증가는 기대하기 어려우나 IP와 유사한 심근괴사 범위 감소효과가 있으며 이러한 효과는 아마도 칼슘의 매개에 따라 PKC활성화가 일어남으로써 나타나는 것으로 생각된다.