• 한국발생생물학회지:발생과생식
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• 제15권1호
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• pp.31-39
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• 2011

Change in intracellular $Ca^{2+}$-concentration ($[Ca^{2+}]_i$) is an essential event for egg activation and further development. $Ca^{2+}$ ion is originated from intracellular $Ca^{2+}$-store via inositol 1,4,5-triphosphate receptor and/or $Ca^{2+}$ influx via $Ca^{2+}$ channel. This study was performed to investigate whether changes in $Ca^{2+}$/calmodulin dependent protein kinase II (CaM KII) activity affect $Ca^{2+}$ influx during artificial egg activation with ethanol using $Ca^{2+}$ monitoring system and whole-cell patch clamp technique. Under $Ca^{2+}$ ion-omitted condition, $Ca^{2+}$-oscillation was stopped within 30 min post microinjection of porcine sperm factor, and ethanol-induced $Ca^{2+}$ increase was reduced. To investigate the role of CaM KII known as an integrator of $Ca^{2+}$- oscillation during mammalian egg fertilization, CaM KII activity was tested with a specific inhibitor KN-93. In the eggs treated with KN-93, ethanol failed to induce egg activation. In addition, KN-93 inhibited inward $Ca^{2+}$ current ($I_{Ca}$) in a time-dependent manner in whole-cell configuration. Immunostaining data showed that the voltage-dependent $Ca^{2+}$ channels were distributed along the plasma membrane of mouse egg and 2-cell embryo. From these results, we suggest that $Ca^{2+}$ influx during fertilization might be controlled by CaM KII activity.

• BMB Reports
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• 제54권10호
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• pp.516-521
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• 2021

Although arginase primarily participates in the last reaction of the urea cycle, we have previously demonstrated that arginase II is an important cytosolic calcium regulator through spermine production in a p32-dependent manner. Here, we demonstrated that rhaponticin (RPT) is a novel medicinal-plant arginase inhibitor and investigated its mechanism of action on Ca²⁺-dependent endothelial nitric oxide synthase (eNOS) activation. RPT was uncompetitively inhibited for both arginases I and II prepared from mouse liver and kidney. It also inhibited arginase activity in both aorta and human umbilical vein endothelial cells (HUVECs). Using both microscope and FACS analyses, RPT treatments induced increases in cytosolic Ca²⁺ levels using Fluo-4 AM as a calcium indicator. Increased cytosolic Ca²⁺ elicited the phosphorylations of both CaMKII and eNOS Ser1177 in a time-dependent manner. RPT incubations also increased intracellular L-arginine (L-Arg) levels and activated the CaMKII/AMPK/Akt/eNOS signaling cascade in HUVECs. Treatment of L-Arg and ABH, arginase inhibitor, increased intracellular Ca²⁺ concentrations and activated CaMKII-dependent eNOS activation in ECs of WT mice, but, the effects were not observed in ECs of inositol triphosphate receptor type 1 knockout (IP3R1^-/-) mice. In the aortic endothelium of WT mice, RPT also augmented nitric oxide (NO) production and attenuated reactive oxygen species (ROS) generation. In a vascular tension assay using RPT-treated aortic tissue, cumulative vasorelaxant responses to acetylcholine (Ach) were enhanced, and phenylephrine (PE)-dependent vasoconstrictive responses were retarded, although sodium nitroprusside and KCl responses were not different. In this study, we present a novel mechanism for RPT, as an arginase inhibitor, to increase cytosolic Ca²⁺ concentration in a L-Arg-dependent manner and enhance endothelial function through eNOS activation.

• 약학회지
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• 제41권6호
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• pp.773-781
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• 1997

The activity of $Ca^{2+}$calmodulin (CaM)-dependent protein kinase Ia (CaM kinase Ia) is shown to be regulated through direct phosphorylation by CaM kinase I kinase (CaMK IK). In the present study, three distinct CaMKIK peaks were separated from Q-Sepharose colunm chromatography of pig brain homogenate using a Waters 650 Protein Purification System. The purified CaMKIK from the major peak potently and rapidly enhanced CaM kinase Ia activity, reaching a maximal stimulation within 2min at the concentrations of 12-15nM. The activated state of CaM kinase Ia is characterized by a markedly enhanced $V_{max}4 as well as significantly decreased $K_m\;and\;K_a$ values toward peptide substrate and CaM, respectively. These observations suggest the activation process of CaM kinase Ia. The phosphorylation of CaM kinase Ia by CaMKIK may induce its conformational change responsible for the alterations in the kinetic properties, which ultimately leads to the rapid enzyme activation.

• The Korean Journal of Physiology and Pharmacology
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• 제6권4호
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• pp.187-191
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• 2002

Tamoxifen, an antiestrogen, has previously been shown to induce apoptosis in HepG2 human hepatoblastoma cells through activation of the pathways independent of estrogen receptors, i.e., intracellular $Ca^{2+}$ increase and generation of reactive oxygen species (ROS). However, the mechanism of tamoxifen to link increased intracellular $Ca^{2+}$ to ROS generation is currently unknown. Thus, in this study we investigated the possible involvement of calmodulin, a $Ca^{2+}$ activated protein, and $Ca^{2+}$/calmodulin-dependent protein kinase II in the above tamoxifen-induced events. Treatment with calmodulin antagonists (calmidazolium and trifluoroperazine) or specific inhibitors of $Ca^{2+}$/calmodulin-dependent protein kinase II (KN-93 and KN-62) inhibited the tamoxifen-induced apoptosis in a dose-dependent manner. In addition, these agents blocked the tamoxifen-induced ROS generation in a concentration-dependent fashion, which was completely suppressed by intracellular $Ca^{2+}$ chelation. These results demonstrate for the first time that, despite of its well-known direct calmodulin-inhibitory activity, tamoxifen may generate ROS and induce apoptosis through indirect activation of calmodulin and $Ca^{2+}$/calmodulin-dependent protein kinase II in HepG2 cells.

• 한국발생생물학회지:발생과생식
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• 제24권4호
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• pp.297-306
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• 2020

Repetitive changes in the intracellular calcium concentration ([Ca²⁺]i) triggers egg activation, including cortical granule exocytosis, resumption of second meiosis, block to polyspermy, and initiating embryonic development. [Ca²⁺]i oscillations that continue for several hours, are required for the early events of egg activation and possibly connected to further development to the blastocyst stage. The sources of Ca²⁺ ion elevation during [Ca²⁺]i oscillations are Ca²⁺ release from endoplasmic reticulum through inositol 1,4,5 tri-phosphate receptor and Ca²⁺ ion influx through Ca²⁺ channel on the plasma membrane. Ca²⁺ channels have been characterized into voltage-dependent Ca²⁺ channels (VDCCs), ligand-gated Ca²⁺ channel, and leak-channel. VDCCs expressed on muscle cell or neuron is specified into L, T, N, P, Q, and R type VDCs by their activation threshold or their sensitivity to peptide toxins isolated from cone snails and spiders. The present study was aimed to investigate the localization pattern of N and P/Q type voltage-dependent calcium channels in mouse eggs and the role in fertilization. [Ca²⁺]i oscillation was observed in a Ca²⁺ contained medium with sperm factor or adenophostin A injection but disappeared in Ca²⁺ free medium. Ca²⁺ influx was decreased by Lat A. N-VDCC specific inhibitor, ω-Conotoxin CVIIA induced abnormal [Ca²⁺]i oscillation profiles in SrCl₂ treatment. N or P/Q type VDC were distributed on the plasma membrane in cortical cluster form, not in the cytoplasm. Ca²⁺ influx is essential for [Ca²⁺]i oscillation during mammalian fertilization. This Ca²⁺ influx might be controlled through the N or P/Q type VDCCs. Abnormal VDCCs expression of eggs could be tested in fertilization failure or low fertilization eggs in subfertility women.

• 약학회지
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• 제45권4호
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• pp.419-425
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• 2001

The neuronal cell death induced by excess glutamate (Glu) has been implicated in many acute and chronic neurodegenerative diseases including cerebral ischemia. Glu-induced elevation of intra-cellular $Ca^{2+}$ plays a critical role in the excitotoxicity, partly through the activation of a variety of $Ca^{2+}$ dependent enzymes. In the present study, we investigated the Glu-induced modulation of $Ca^{2+}$/calmodulin-dependent protein kinase IV (CaMK IV), a multifunctional enzyme abundantly present in the nuclei of neurons. The exposure of cultured rat cortical neurons to $100{\mu}$M Glu for 3 min dramatically increased CaMK IV activity up to 4.5-fold of the control-treated enzyme activity. The activation was very rapid, reaching peak at 3 min and then declined gradually. Under the same experimental conditions, time-dependent acute and delayed neuronal cell death was observed. Immunoblot analyses using specific antibodies showed that the expressions of CaMK IV and $CaMKK_{\alpha}$ were time-dependently modulated by Glu. Taken together, these results imply that the modulation of CaMK IV activity by Glu may be involved in the cascade of events resulting in neuronal cell death in cortical cultures.

• The Korean Journal of Physiology and Pharmacology
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• 제21권2호
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• pp.241-249
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• 2017

Plasma membrane hyperpolarization associated with activation of $Ca^{2+}$-activated $K^+$ channels plays an important role in sperm capacitation during fertilization. Although Slo3 (slowpoke homologue 3), together with the auxiliary ${\gamma}^2$-subunit, LRRC52 (leucine-rich-repeat-containing 52), is known to mediate the pH-sensitive, sperm-specific $K^+$ current KSper in mice, the molecular identity of this channel in human sperm remains controversial. In this study, we tested the classical $BK_{Ca}$ activators, NS1619 and LDD175, on human Slo3, heterologously expressed in HEK293 cells together with its functional interacting ${\gamma}^2$ subunit, hLRRC52. As previously reported, Slo3 $K^+$ current was unaffected by iberiotoxin or 4-aminopyridine, but was inhibited by ~50% by 20 mM TEA. Extracellular alkalinization potentiated hSlo3 $K^+$ current, and internal alkalinization and $Ca^{2+}$ elevation induced a leftward shift its activation voltage. NS1619, which acts intracellularly to modulate hSlo1 gating, attenuated hSlo3 $K^+$ currents, whereas LDD175 increased this current and induced membrane potential hyperpolarization. LDD175-induced potentiation was not associated with a change in the half-activation voltage at different intracellular pHs (pH 7.3 and pH 8.0) in the absence of intracellular $Ca^{2+}$. In contrast, elevation of intracellular $Ca^{2+}$ dramatically enhanced the LDD175-induced leftward shift in the half-activation potential of hSlo3. Therefore, the mechanism of action does not involve pH-dependent modulation of hSlo3 gating; instead, LDD175 may modulate $Ca^{2+}$-dependent activation of hSlo3. Thus, LDD175 potentially activates native KSper and may induce membrane hyperpolarization-associated hyperactivation in human sperm.

• 대한의생명과학회지
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• 제23권3호
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• pp.251-260
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• 2017

Platelet activation is essential at the sites of vascular injury, which leads to hemostasis through adhesion, aggregation, and secretion process. However, potent and continuous platelet activation may be an important reason of circulatory disorders. Therefore, proper regulation of platelet activation may be an effective treatment for vascular diseases. In this research, inhibitory effects of cordycepin (3'-deoxyadenosine) on platelet activation were determined. As the results, cordycepin increased cAMP and cGMP, which are intracellular $Ca^{2+}$-antagonists. In addition, cordycepin reduced collagen-elevated $[Ca^{2+}]_i$ mobilization, which was increased by a cAMP-dependent protein kinase (PKA) inhibitor (Rp-8-Br-cAMPS), but not a cGMP-protein kinase (PKG) inhibitor (Rp-8-Br-cGMPS). Furthermore, cordycepin increased $IP_3RI$ ($Ser^{1756}$) phosphorylation, indicating inhibition of $IP_3$-mediated $Ca^{2+}$ release from internal store via the $IP_3RI$, which was strongly inhibited by Rp-8-Br-cAMPS, but was not so much inhibited by Rp-8-Br-cGMPS. These results suggest that the reduction of $[Ca^{2+}]_i$ mobilization is caused by the cAMP/A-kinase-dependent $IP_3RI$ ($Ser^{1756}$) phosphorylation. In addition, cordycepin increased the phosphorylation of VASP ($Ser^{157}$) known as PKA substrate, but not VASP ($Ser^{239}$) known as PKG substrate. Cordycepin-induced VASP ($Ser^{157}$) phosphorylation was inhibited by Rp-8-Br-cAMPS, but was not inhibited by Rp-8-Br-cGMPS, and cordycepin inhibited collagen-induced fibrinogen binding to ${\alpha}IIb/{\beta}_3$, which was increased by Rp-8-Br-cAMPS, but was not inhibited by Rp-8-Br-cGMPS. These results suggest that the inhibition of ${\alpha}IIb/{\beta}_3$ activation is caused by the cAMP/A-kinase-dependent VASP ($Ser^{157}$) phosphorylation. In conclusion, these results demonstrate that inhibitory effects of cordycepin on platelet activation were due to inhibition of $[Ca^{2+}]_i$ mobilization through cAMP-dependent $IP_3RI$ ($Ser^{1756}$) phosphorylation and suppression of ${\alpha}IIb/{\beta}_3$ activation through cAMP-dependent VASP ($Ser^{157}$) phosphorylation. These results strongly indicated that cordycepin might have therapeutic or preventive potential for platelet activation-mediated disorders including thrombosis, atherosclerosis, myocardial infarction, or cardiovascular disease.

• Archives of Pharmacal Research
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• 제24권6호
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• pp.546-551
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• 2001

We have previously shown that, in circular muscle cells of the lower esophageal sphincter (LES) isolated by enzymatic digestion, contraction in response to maximally effective doses of acetylcholine (ACh) or Inositol Triphosphate ($IP_3$) depends on the release of $Ca^{2+}$ from intracellular stores and activation of a $Ca6{2+}$-calmodulin (CaM)-dependent pathway. On the contrary, maintenance of LES tone, and response to low doses of ACh or $IP_3$ depend on a protein kinase C (PKC) mediated pathway. In the present investigation, we have examined requirements for $Ca6{2+}$ regulation of the interaction between CaM- and PKC-dependent pathways in LES contraction. Thapsigargin (TG) treatment for 30 min dose dependently reduced ACh-induced contraction of permeable LES cells in free $Ca6{2+}$ medium. ACh-induced contraction following the low level of reduction of $Ca6{2+}$ stores by a low dose of TG ($10^{-9}{\;}M$) was blocked by the CaM antagonist, CCS9343B but not by the PKC antagonists chelerythrine or H7, indicating that the contraction is CaM-dependent. After maximal reduction in intracellular $Ca{2+}$ from $Ca6{2+}$stores by TG ($10^{-6}{\;}M$), ACh-induced contraction was blocked by chelerythrine or H7, but not by CCS9343B, indicating that it is PKC-dependent. In normal $Ca^{2+}$medium, the contraction by ACh after TG ($10^{-9}{\;}M$) treatment was also CaM-dependent, whereas the contraction by ACh after TG ($10^{-9}{\;}M$) treatment was PKC-dependent. We examined whether PKC activation was inhibited by activated CaM. CCS 7343B Inhibited the CaM-induced contraction, but did not inhibit the DAC-induced contraction. CaM inhibited the DAC-induced contraction in the presence of CCS 9343B. This inhibition by CaM was $Ca{2+}$dependent. These data are consistent with the view that the switch from a PKC-dependent pathway to a CaM dependent pathway can occur and can be regulated by cytosolic $Ca{2+}$ in the LES.

• Molecules and Cells
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• 제21권1호
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• pp.121-128
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• 2006

Maitotoxin (MTX) is known as one of the most potent marine toxins involved in Ciguatera poisoning, but intracellular signaling pathways caused by MTX was not fully understood. Thus, we have investigated whether intracellular reactive oxygen species (ROS) are involved in MTX-induced cellular responses in human umbilical vein endothelial cells. MTX induced a dose-dependent increase of intracellular [$Ca^{2+}$]. MTX stimulated the production of intracellular ROS in a dose- and time-dependent manner, which was suppressed by BAPTA-AM, an intracellular $Ca^{2+}$ chelator. Ionomycin also elevated the ROS production in a dose-dependent manner. MTX elevated transamidation activity in a time-dependent manner and the activation was largely inhibited by transfection of tissue transglutaminase siRNA. The activation of tissue transglutaminase and ERK1/2 by MTX was suppressed by BAPTA-AM or ROS scavengers. In addition, MTX-induced cell death was significantly delayed by BAPTA-AM or a ROS scavenger. These results suggest that [$Ca^{2+}$]-dependent generation of intracellular ROS, at least in part, play an important role in MTX-stimulated cellular responses, such as activation of tTGase, ERK phosphorylation, and induction of cell death, in human umbilical vein endothelial cells.