• The Korean Journal of Physiology and Pharmacology
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• 제18권4호
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• pp.297-305
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• 2014

Flavonoids have an ability to suppress various ion channels. We determined whether one of flavonoids, cyanidin-3-glucoside, affects adenosine 5'-triphosphate (ATP)-induced calcium signaling using digital imaging methods for intracellular free $Ca^{2+}$ concentration ([$Ca^{2+}$]i), reactive oxygen species (ROS) and mitochondrial membrane potential in PC12 cells. Treatment with ATP ($100{\mu}M$) for 90 sec induced [$Ca^{2+}$]i increases in PC12 cells. Pretreatment with cyanidin-3-glucoside ($1{\mu}g/ml$ to $100{\mu}g/ml$) for 30 min inhibited the ATP-induced [$Ca^{2+}$]i increases in a concentration-dependent manner ($IC_{50}=15.3{\mu}g/ml$). Pretreatment with cyanidin-3-glucoside ($15{\mu}g/ml$) for 30 min significantly inhibited the ATP-induced [$Ca^{2+}$]i responses following removal of extracellular $Ca^{2+}$ or depletion of intracellular [$Ca^{2+}$]i stores. Cyanidin-3-glucoside also significantly inhibited the relatively specific P2X2 receptor agonist 2-MeSATP-induced [$Ca^{2+}$]i responses. Cyanidin-3-glucoside significantly inhibited the thapsigargin or ATP-induced store-operated calcium entry. Cyanidin-3-glucoside significantly inhibited the ATP-induced [$Ca^{2+}$]i responses in the presence of nimodipine and ${\omega}$-conotoxin. Cyanidin-3-glucoside also significantly inhibited KCl (50 mM)-induced [$Ca^{2+}$]i increases. Cyanidin-3-glucoside significantly inhibited ATP-induced mitochondrial depolarization. The intracellular $Ca^{2+}$ chelator BAPTA-AM or the mitochondrial $Ca^{2+}$ uniporter inhibitor RU360 blocked the ATP-induced mitochondrial depolarization in the presence of cyanidin-3-glucoside. Cyanidin-3-glucoside blocked ATP-induced formation of ROS. BAPTA-AM further decreased the formation of ROS in the presence of cyanidin-3-glucoside. All these results suggest that cyanidin-3-glucoside inhibits ATP-induced calcium signaling in PC12 cells by inhibiting multiple pathways which are the influx of extracellular $Ca^{2+}$ through the nimodipine and ${\omega}$-conotoxin-sensitive and -insensitive pathways and the release of $Ca^{2+}$ from intracellular stores. In addition, cyanidin-3-glucoside inhibits ATP-induced formation of ROS by inhibiting $Ca^{2+}$-induced mitochondrial depolarization.

• 한국의학물리학회지:의학물리
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• 제18권1호
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• pp.42-47
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• 2007

목적: 망간 조영증강 자기공명영상을 이용하여 시신경경로의 활성화를 추적해 보고자 하였다. 대상 및 방법: 뉴질랜드 암컷 흰색 토끼의 망막에 $30{\mu}l$의 $MnSl_2(1mol)$ 용액을 주입한 후 고성능 경사자계 시스템이 장착된 임상용 1.5 기기에서 3D FSPGR 펄스열을 사용하여 고해상도 T1-강조영상을 망간 주입 후 12시간, 24시간, 48시간에 각각 획득한 후 시신경경로의 주요 해부학적 구조물에서의 조영증강을 관찰하였다. 또한 정량적 평가를 위하여 해부학적 위치에 동일한 원형의 관심영역을 정하여 신호강도를 측정 한 후 배경 잡음의 신호강도와의 신호대 잡음비(signal-to-noise ratio)를 구하였다. 결과: 망간 주입 후 시신경 경로상의 주요 구조물들이 조영증강 되었다 조영 증강된 주요 구조물로는 오른쪽 시신경(optic nerve), 시각교차(optic chiasm), 왼쪽 시신경 경로(optic tract), 왼쪽 가쪽 무릎핵(lateral geniculate nucleus), 왼쪽 상구(superior colliculus) 등 오른쪽 망막의 대측(contralateral) 시신경 경로상의 구조물이었으며 동측(ipsilateral) 시신경 경로는 조영증강을 보이지 않았다. 결론: 시신경계의 말단부위인 망막에 $MnCl_2$를 주입한 후 시신경계의 축삭을 통한 망간이온의 전파를 비침습적으로 관찰 할 수 있었다. 이러한 망간이온의 전파는 전압 개폐성 칼슘채널을 통하여 일어나는 것으로 여겨지며 특히 망막에 직접 주입하는 경우 전압 개폐성 칼슘채널의 빠른 이동 경로를 이용하는 것으로 추측된다.

• 대한의생명과학회지
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• 제13권2호
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• pp.119-125
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• 2007

It is widely known that protein tyrosine kinases (PTKs) are involved in controlling many biological processes such as cell growth, differentiation, proliferation, survival and apoptosis. An $\alpha3\beta4$ subunit combination acts as a major functional acetylcholine receptor (nAChRs) in male rat major pelvic ganglion (MPG) neurons, and their activation induces fast inward currents and intracellular calcium increases. Recently it has been reported that the activity of acetylcholine receptors (AChRs) in some neurons can be negatively regulated by PTKs. However, the exact mechanism of regulation of nAChRs by PTKs is poorly understood. Therefore, we examined the potential role particular in nAChR by PTK using electrophysiology and calcium imaging in male rat MPG neurons. ACh induced inward currents and $(Ca^{2+})_i$ increases in MPG neurons, concomitantly. These responses were inhibited by more than 90% in $Na^+$- or $Ca^{2+}$- free solution. $\alpha$-conotoxin AuIB, a selective $\alpha3\beta4$ nAChR blocket, inhibited ACh-induced inward currents. Genistein (10 $\mu$M), a broad-spectrum tyrosine kinase inhibitor, markedly decreased ACh-induced currents and $Ca^{2+}$ transients, whereas 10 $\mu$M genistin, an inactive analogue, had little effect. Overall these data suggest that the activities of $\alpha3\beta4$ AChRs in MPG neurons are positively regulated by PTK. In conclusion, trosine kinase may be one of the key factors in the regulation of $\alpha3\beta4$ nAChRs in rat MPG neurons, which may play an important roles in the autonomic neuronal function such as synaptic transmission, autonomic reflex, and neuronal plasticity.

• The Korean Journal of Physiology and Pharmacology
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• 제17권1호
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• pp.65-71
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• 2013

The transient receptor potential melastatin type 7 (TRPM7) channel is a widely expressed non-selective cation channel with fusion to the C-terminal alpha kinase domain and regarded as a key regulator of whole body $Mg^{2+}$ homeostasis in mammals. However, the roles of TRPM7 during osteoclastogenesis in RAW264.7 cells and bone marrow-derived monocyte/macrophage precursor cells (BMMs) are not clear. In the present study, we investigate the roles of TRPM7 in osteoclastogenesis using methods of small interfering RNA (siRNA), RT-PCR, patch-clamp, and calcium imaging. RANKL (receptor activator of NF-${\kappa}B$ ligand) stimulation did not affect the TRPM7 expression and TRPM7-mediated current was activated in HEK293, RAW264.7, and BMM cells by the regulation of $Mg^{2+}$. Knock-down of TRPM7 by siTRPM7 reduced intracellular $Ca^{2+}$ concentration ($[Ca^{2+}]_i$) increases by 0 mM $[Mg^{2+}]_e$ in HEK293 cells and inhibited the generation of RANKL-induced $Ca^{2+}$ oscillations in RAW264.7 cells. Finally, knock-down of TRPM7 suppressed RANKL-mediated osteoclastogenesis such as activation and translocation of NFATc1, formation of multinucleated cells, and the bone resorptive activity, sequentially. These results suggest that TRPM7 plays an essential role in the RANKL-induced $[Ca^{2+}]_i$ oscillations that triggers the late stages of osteoclastogenesis.

• 대한의생명과학회지
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• 제15권4호
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• pp.319-326
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• 2009

Extracellular ATP elicits diverse physiological effects by binding to the G-protein-coupled P2Y receptors on the plasma membrane. In addition to the short-term effects of extracellular nucleotides on cell functions, there is evidence that such purinergic signalling can have long-term effects on cell proliferation, differentiation and death. The 3T3-L1 cell line derived from mouse embryo is a well-established and commonly utilized in vitro model for adipocytes differentiation and function. However, the distributions and roles of P2Y subtypes are still unknown in the preadipocyte. In this study, we identified the distributions and roles of P2Y subtypes in preadipocyte using $Ca^{2+}$ imaging and realtime PCR. ATP increased the $[Ca^{2+}]_i$ in a concentration-dependent manner. ATP increased $Ca^{2+}$ in absence and/or presence of extracellular $Ca^{2+}$. Suramin, non-selective P2Y blocker, largely blocked the ATP-induced $Ca^{2+}$ response. U73122, a PLC inhibitor, completely inhibited $Ca^{2+}$ mobilization in 3T3-L1 cells. The mRNA expression by realtime PCR of P2Y subtypes was $P2Y_2:P2Y_5:P2Y_6=1.0:12.5:0.3$. In conclusion, we showed that $P2Y_5$ receptor is a dominant purinergic receptor in preadipocytes, and multiple P2Y receptors could involve in differentiation and migration via regulating of intracellular calcium concentration.

• Archives of Pharmacal Research
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• 제27권5호
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• pp.524-530
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• 2004

Epilepsy or the occurrence of spontaneous recurrent epileptiform discharges (SREDs, seizures) is one of the most common neurological disorders. Shift in the balance of brain between excitatory and inhibitory functions due to different types of structural or functional alterations may cause epileptiform discharges. N-Methyl-D-aspartate (NMDA) receptor dysfunctions have been implicated in modulating seizure activities. Seizures and epilepsy are clearly dependent on elevated intracellular calcium concentration ([C $a^{2+}$]$_{i}$ ) by NMDA receptor activation and can be prevented by NMDA antagonists. This perturbed [C $a^{2+}$]$_{i}$ levels is forerunner of neuronal death. However, therapeutic tools of elevated [C $a^{2+}$]$_{i}$ level during status epilepticus (SE) and SREDs have not been discovered yet. Our previous study showed fast inhibition of ginseng total saponins and ginsenoside R $g_3$ on NMDA receptor-mediated [C $a^{2+}$]$_{i}$ in cultured hippocampal neurons. We, therefore, examined the direct modulation of ginseng on hippocampal neuronal culture model of epilepsy using fura-2-based digital $Ca^{2+}$ imaging and neuronal viability assays. We found that ginseng total saponins and ginsenoside R $g_3$ inhibited $Mg^{2+}$ free-induced increase of [C $a^{2+}$]$_{i}$ and spontaneous [C $a^{2+}$]$_{i}$ oscillations in cultured rat hippocampal neurons. These results suggest that ginseng may playa neuroprotective role in perturbed homeostasis of [C $a^{2+}$]$_{i}$ and neuronal cell death via the inhibition of NMDA receptor-induced SE or SREDs.d SE or SREDs..

• Journal of Ginseng Research
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• 제42권4호
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• pp.470-475
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• 2018

Background: It was previously found that Korean Red Ginseng water extract (KRGE) inhibits the histamine-induced itch signaling pathway in peripheral sensory neurons. Thus, in the present study, we investigated whether KRGE inhibited another distinctive itch pathway induced by chloroquine (CQ); a representative histamine-independent pathway mediated by MrgprA3 and TRPA1. Methods: Intracellular calcium changes were measured by the calcium imaging technique in the HEK293T cells transfected with both MrgprA3 and TRPA1 ("MrgprA3/TRPA1"), and in primary culture of mouse dorsal root ganglia (DRGs). Mouse scratching behavior tests were performed to verify proposed antipruritic effects of KRGE and ginsenoside Rg3. Results: CQ-induced $Ca^{2+}$ influx was strongly inhibited by KRGE ($10{\mu}g/mL$) in MrgprA3/TRPA1, and notably ginsenoside Rg3 dose-dependently suppressed CQ-induced $Ca^{2+}$ influx in MrgprA3/TRPA1. Moreover, both KRGE ($10{\mu}g/mL$) and Rg3 ($100{\mu}M$) suppressed CQ-induced $Ca^{2+}$ influx in primary culture of mouse DRGs, indicating that the inhibitory effect of KRGE was functional in peripheral sensory neurons. In vivo tests revealed that not only KRGE (100 mg) suppressed CQ-induced scratching in mice [bouts of scratching: $274.0{\pm}51.47$ (control) vs. $104.7{\pm}17.39$ (KRGE)], but also Rg3 (1.5 mg) oral administration significantly reduced CQ-induced scratching as well [bouts of scratching: $216.8{\pm}33.73$ (control) vs.$115.7{\pm}20.94$ (Rg3)]. Conclusion: The present study verified that KRGE and Rg3 have a strong antipruritic effect against CQ-induced itch. Thus, KRGE is as a promising antipruritic agent that blocks both histamine-dependent and -independent itch at peripheral sensory neuronal levels.

• Maxillofacial Plastic and Reconstructive Surgery
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• 제40권
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• pp.3.1-3.8
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• 2018

Background: In this research article, we evaluate the use of sub-periosteal tunneling (tunnel technique) combined with alloplastic in situ hardening biphasic calcium phosphate (BCP, a compound of β-tricalcium phosphate and hydroxyapatite) bone graft for lateral augmentation of a deficient alveolar ridge. Methods: A total of 9 patients with deficient mandibular alveolar ridges were included in the present pilot study. Ten lateral ridge augmentation were carried out using the sub-periosteal tunneling technique, including a bilateral procedure in one patient. The increase in ridge width was assessed using CBCT evaluation of the ridge preoperatively and at 4 months postoperatively. Histological assessment of the quality of bone formation was also carried out with bone cores obtained at the implant placement re-entry in one patient. Results: The mean bucco-lingual ridge width increased in average from 4.17 ± 0.99 mm to 8.56 ± 1.93 mm after lateral bone augmentation with easy-graft CRYSTAL using the tunneling technique. The gain in ridge width was statistically highly significant (p = 0.0019). Histomorphometric assessment of two bone cores obtained at the time of implant placement from one patient revealed 27.6% new bone and an overall mineralized fraction of 72.3% in the grafted area 4 months after the bone grafting was carried out. Conclusions: Within the limits of this pilot study, it can be concluded that sub-periosteal tunneling technique using in situ hardening biphasic calcium phosphate is a valuable option for lateral ridge augmentation to allow implant placement in deficient alveolar ridges. Further prospective randomized clinical trials will be necessary to assess its performance in comparison to conventional ridge augmentation procedures.

• Restorative Dentistry and Endodontics
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• 제44권1호
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• pp.10.1-10.11
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• 2019

Objectives: The objective of this study was to assess coronal discoloration induced by the following intracanal medicaments: calcium hydroxide (CH), a mixture of CH paste and chlorhexidine gel (CH/CHX), and triple antibiotic paste (3Mix). Materials and Methods: Seventy extracted single-canal teeth were selected. Access cavities were prepared and each canal was instrumented with a rotary ProTaper system. The specimens were randomly assigned to CH, CH/CHX, and 3Mix paste experimental groups (n = 20 each) or a control group (n = 10). Each experimental group was randomly divided into 2 subgroups (A and B). In subgroup A, medicaments were only applied to the root canals, while in subgroup B, the root canals were completely filled with medicaments and a cotton pellet dipped in medicament was also placed in the pulp chamber. Spectrophotometric readings were obtained from the mid-buccal surface of the tooth crowns immediately after placing the medicaments (T1) and at 1 week (T2), 1 month (T3), and 3 months (T4) after filling. The ${\Delta}E$ was then calculated. Data were analyzed using 2-way analysis of variance (ANOVA), 3-way ANOVA, and the $Scheff{\acute{e}}$ post hoc test. Results: The greatest color change (${\Delta}E$) was observed at 3 months (p < 0.0001) and in 3Mix subgroup B (p = 0.0057). No significant color change occurred in the CH (p = 0.7865) or CH/CHX (p = 0.1367) groups over time, but the 3Mix group showed a significant ${\Delta}E$ (p = 0.0164). Conclusion: Intracanal medicaments may induce tooth discoloration. Use of 3Mix must be short and it must be carefully applied only to the root canals; the access cavity should be thoroughly cleaned afterwards.

• The Korean Journal of Physiology and Pharmacology
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• 제28권1호
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• pp.93-103
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• 2024

Satellite glial cells (SGCs), a major type of glial cell in the autonomic ganglia, closely envelop the cell body and even the synaptic regions of a single neuron with a very narrow gap. This structurally unique organization suggests that autonomic neurons and SGCs may communicate reciprocally. Glial Ca²⁺ signaling is critical for controlling neural activity. Here, for the first time we identified the machinery of store-operated Ca²⁺ entry (SOCE) which is critical for cellular Ca²⁺ homeostasis in rat sympathetic ganglia under normal and pathological states. Quantitative realtime PCR and immunostaining analyses showed that Orai1 and stromal interaction molecules 1 (STIM1) proteins are the primary components of SOCE machinery in the sympathetic ganglia. When the internal Ca²⁺ stores were depleted in the absence of extracellular Ca²⁺, the number of plasmalemmal Orai1 puncta was increased in neurons and SGCs, suggesting activation of the Ca²⁺ entry channels. Intracellular Ca²⁺ imaging revealed that SOCE was present in SGCs and neurons; however, the magnitude of SOCE was much larger in the SGCs than in the neurons. The SOCE was significantly suppressed by GSK7975A, a selective Orai1 blocker, and Pyr6, a SOCE blocker. Lipopolysaccharide (LPS) upregulated the glial fibrillary acidic protein and Toll-like receptor 4 in the sympathetic ganglia. Importantly, LPS attenuated SOCE via downregulating Orai1 and STIM1 expression. In conclusion, sympathetic SGCs functionally express the SOCE machinery, which is indispensable for intracellular Ca²⁺ signaling. The SOCE is highly susceptible to inflammation, which may affect sympathetic neuronal activity and thereby autonomic output.