• 약학회지
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• 제50권6호
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• pp.409-414
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• 2006

Although mammalian sperms are cryopreserved for in vitro fertilization a process of cryopreservation decreases the fertility. Acrosome reaction requires depolarization-induced Ca$^{2+}$ influx and Ca$^{2+}$ releases from the Ca$^{2+}$ stores. To examine whether the cellular Ca$^{2+}$ mobilization is altered by a sperm cryopreservation we compared cytosolic Ca$^{2+}$ signals between fresh and cryopreserved pig sperms using confocal Ca$^{2+}$ imaging. The magnitudes of depolarization induced Ca$^{2+}$ increases were significantly smaller in cryopreserved sperms. Exposures to 10 mM caffeine or 5 ${\mu}$M thapsigargin elicited less Ca$^{2+}$ increases in the cryopreserved sperms compared to fresh sperms. In addition, progesterone-trig-gered Ca$^{2+}$ rises, that are thought to enhance acrosome reaction, were completely abolished in the cryopreserved sperms. These results suggest that storage and(/or) release of Ca$^{2+}$ from the intracellular Ca$^{2+}$ stores in pig sperms are significantly impaired by the process of cryopreservation.

• The Korean Journal of Physiology and Pharmacology
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• 제26권3호
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• pp.219-225
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• 2022

Glucagon like peptide-1 (GLP-1) released from enteroendocine L-cells in the intestine has incretin effects due to its ability to amplify glucose-dependent insulin secretion. Promotion of an endogenous release of GLP-1 is one of therapeutic targets for type 2 diabetes mellitus. Although the secretion of GLP-1 in response to nutrient or neural stimuli can be triggered by cytosolic Ca²⁺ elevation, the stimulus-secretion pathway is not completely understood yet. Therefore, the aim of this study was to investigate the role of reverse Na⁺/Ca²⁺ exchanger (rNCX) in Ca²⁺ entry induced by muscarinic stimulation in NCI-H716 cells, a human enteroendocrine GLP-1 secreting cell line. Intracellular Ca²⁺ was repetitively oscillated by the perfusion of carbamylcholine (CCh), a muscarinic agonist. The oscillation of cytosolic Ca²⁺ was ceased by substituting extracellular Na⁺ with Li⁺ or NMG⁺. KB-R7943, a specific rNCX blocker, completely diminished CCh-induced cytosolic Ca²⁺ oscillation. Type 1 Na⁺/Ca²⁺ exchanger (NCX₁) proteins were expressed in NCI-H716 cells. These results suggest that rNCX might play a crucial role in Ca²⁺ entry induced by cholinergic stimulation in NCI-H716 cells, a GLP-1 secreting cell line.

• 대한의생명과학회지
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• 제15권4호
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• pp.273-279
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• 2009

Cordycepin (3'-deoxyadenosine) is an adenosine analogue isolated from Cordyceps militaris, and it has been used as an anti-cancer and anti-inflammation ingredient in traditional Chinese medicine. We investigated the effects of cordycepin on human platelet aggregation induced by thapsigargin, and determined the cytosolic free $Ca^{2+}$ levels ($[Ca^{2+}]_i$), an aggregation-stimulating factor. Cordycepin significantly inhibited thapsigargin-induced platelet aggregation. Its inhibitory effect was continually sustained at the maximal aggregation concentration of thapsigargin. The thapsigargin-induced $[Ca^{2+}]_i$ were clearly reduced by cordycepin in the presence of exogenous $CaCl_2$ or extracellular $Ca^{2+}$-chelator (EDTA). These results suggest that cordycepin inhibited thapsigargin-induced $Ca^{2+}$-influx from extracellular domain and thapsigargin-induced $Ca^{2+}$-mobilization from intracellular $Ca^{2+}$ storage. Accordingly, our data demonstrated that cordycepin may have a beneficial effect on platelet aggregation-mediated thrombotic diseases by inhibiting a $[Ca^{2+}]_i$-elevation.

• Journal of Chest Surgery
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• 제25권4호
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• pp.347-355
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• 1992

The precise mechanism of the Excitation-Contraction Coupling is still uncertain. But the concept that Ca2+ induced Ca2+ release [CICR] from the Ryanodine receptor in the sarcoplasmic reticulum [foot structure] may play a major role in E-C coupling has been widely accepted since 1970`s. It is believed that increased cytosolic Ca2+ followed by CICR is main contributor for E-C coupling of striated muscle. Resulting phenomena of ischemic /post-reperfusion myocyte is increased cytosolic Ca2+, even to the absence of Ca2+ in reperfusate. So intracellular inhibitor to CICR might prevent the ischemic and reperfusion damage of myocardial cells. The relatively purified foot protein, especially heavy sarcoplasmic reticulum rich, of the skeletal muscle was incorporated into the black lipid bilayer [Phosphatidyl ethanolamine: Phosphatidyl serine=l: 1]. Under the steady state of membrane potential [+20 mV], ionic current through Ryanodine receptor was measured with Cs+ as charge carrier. In the cis chamber [Cytoplasmic side], Mg2+ strongly inhibited CICR of Ryanodine receptor[Kd=6.2 nM]. In conclusion, naturally existing intracellular free Mg2+ can inhibit CICR from intracellular Ca2+ reservior [heavy SR]. So post-ischemic or post-reperfusing myocardium could be preserved using additional free Mg2+ in cardioplegic solution or reperfusate, otherwise the optimal concentration is undetermined.

• 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.

• BMB Reports
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• 제28권6호
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• pp.499-503
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• 1995

In our previous studies (Chae et al., 1990; Chae et a1., 1993), we found that a phytochrome signal was clearly connected with the change in cytosolic free $Ca^{2+}$ concentration ($[Ca^{2+}]_i$) in oat cells. It was determined that the $[Ca^{2+}]_i$ change occured both by mobilization out of the intracellular $Ca^{2+}$ store and by influx from the medium. The specific aim of this work is to elucidate the processes connecting $Ca^{2+}$ mobilization and influx. The cells treated with thapsigargin (increasing $[Ca^{2+}]_i$ by inhibition of the $Ca^{2+}$-ATPase in the calcium pool) in the presence of external $Ca^{2+}$ showed the same increasing pattern (sustained increasing shape) of $[Ca^{2+}]_i$ as that measured in animal cells. Red light irradiation after thapsigargin treatment did not increase $[Ca^{2+}]_i$ These results suggest that thapsigargin also acts specifically in the processes of mobilization and influx of $Ca^{2+}$ in oat cells. When the cells were treated with TEA ($K^+$ channel blocker), changes in $[Ca^{2+}]_i$ were drastically reduced in comparison with that measured in the absence of TEA. The results suggest that the change in $[Ca^{2+}]_i$ due to red light irradiation is somehow related with $K^+$ channel opening to change membrane potential. The membrane potential change due to $K^+$ influx might be the critical factor in opening a voltage-dependent calcium channel for $Ca^{2+}$ influx.

• The Korean Journal of Physiology and Pharmacology
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• 제2권4호
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• pp.419-425
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• 1998

To clarify the effect of extracellular ATP in cultured C6 glioma cells, ATP-induced cytosolic free calcium ($[Ca^{2+}]_i$) mobilization and cell proliferation were investigated. ATP-induced $[Ca^{2+}]_i$ increased in a dose-dependent manner $(10^{-7}\;M{\sim}10^{-3}\;M)$. ATP-induced $[Ca^{2+}]_i$ increases were slightly slowed in extracellular calcium-free conditions especially in sustained phase. ATP-induced $[Ca^{2+}]_i$ increment was also inhibited by the pretreatment of U73122, a phospholipase C (PLC) inhibitor, in a time-dependent manner. Suramin, a putative $P_{2Y}$ receptor antagonist, dose-dependently weakened ATP-induced $[Ca^{2+}]_i$ mobilization. Significant increases in cell proliferation were observed at 2, 3, and 4 days after ATP was added. Stimulated cell proliferation was also observed with adenosine at days 2 and 3. This cell proliferation was significantly inhibited by the treatment with suramin. Ionomycin also stimulated cell proliferation in a concentration-dependent manner. In conclusion, we suggest that extracellular ATP stimulates C6 glioma cell proliferation via intracellular free calcium mobilization mediated by purinoceptor.

• 대한약리학회지
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• 제24권2호
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• pp.197-201
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• 1988

본 연구에서는 세포내의 칼슘이온과 결합하여 형광을 발하는 형광색소 fura 2를 이용하여 세포내의 칼슘이은 농도를 측정함과 동시에 장력을 측정하였으며 이에 대한 카페인과 고농도의 칼륨의 영향을 정토하였다. 72.7 mM의 칼륨 이온은 장력과 세포내 칼슘이온농도에서 각각 지속적 인 증가를 보여주었으며, 20 mM의 카페인은 일과성의 빠른 세포내의 칼슘이은농도의 증가에 이어 감소를 보여주었으나 기본치 보다는 높았다. 그러나, 장력에 있어서는 카페인은 일과성의 증가에 이어 기본 장력보다 낮은 감소를 보여주었다. 한편 칼슘이온 제거 용액에서 칼륨이온은 세포내의 갈륨이온 농도도 장력도 증가 시키지 못하였으나 카페인은 일과성의 세포내의 칼륨이온 농도와 장책의 증가를 보여주었다. 이상과 결과로부터 rat 대동맥에서의 고농도의 칼륨이은에 의한 수축은 세포 딴으로 부터의 칼슘이온 유입에 의한 것이며 반면에 카페인에 의한 수축은 세포내의 칼슘이온저장부위로 부터의 칼슘이온 유리에 의한 것임을 시사한다. 또한, 카페인은 세포내의 수축 단백의 칼슘 이온에 대한 감수성을 저하시키는 듯하다.

• 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.

• 한국생물물리학회:학술대회논문집
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• 한국생물물리학회 2003년도 정기총회 및 학술발표회
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• pp.43-43
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• 2003

Large-conductance $Ca^{2+}$-actived $K^{+}$ channels ($BK_{Ca}$ channels) play a key role in setting the pace of contractile activity in muscle and are involved in the regulation of neurotransmitter release in neuron. $BK_{Ca}$ channels are activated by depolarizing membrane potential and the elevated level of intracellular calcium. Using yeast-two hybrid assay, we have identified a novel protein interacting with the cytosolic carboxyl terminus of rSlo, the brain isoform of rat large-conductance $Ca^{2+}$-activated $K^{+}$ channel $\alpha$-subunit. The novel gene encodes 51 kDa protein and is named as SIRK(rSlo-interacting RGS-like protein). SIRK is expressed in various tissues and localized in the cytosolic and the membrane fraction. Biochemical and immunological studies indicated that SIRK physically interacted with the cytosolic region of rSlo. To investigate whether SIRK can modulate the activity of rSlo, GFP-fused SIRK and rSlo were transiently transfected into COS-7 cells and the effects of SIRK was studied using electrophysiological means. We concluded that the overexpression of SIRK alters the surface expression of rSlo channel with only a limited effect on the biophysical characteristics of the channel.the channel.