• The Korean Journal of Physiology and Pharmacology
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• v.28 no.4
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• pp.335-344
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• 2024

Diphenyleneiodonium (DPI) has been widely used as an inhibitor of NADPH oxidase (Nox) to discover its function in cardiac myocytes under various stimuli. However, the effects of DPI itself on Ca²⁺ signaling and contraction in cardiac myocytes under control conditions have not been understood. We investigated the effects of DPI on contraction and Ca²⁺ signaling and their underlying mechanisms using video edge detection, confocal imaging, and whole-cell patch clamp technique in isolated rat cardiac myocytes. Application of DPI suppressed cell shortenings in a concentration-dependent manner (IC₅₀ of ≅0.17 µM) with a maximal inhibition of ~70% at ~100 µM. DPI decreased the magnitude of Ca²⁺ transient and sarcoplasmic reticulum Ca²⁺ content by 20%-30% at 3 µM that is usually used to remove the Nox activity, with no effect on fractional release. There was no significant change in the half-decay time of Ca²⁺ transients by DPI. The L-type Ca²⁺ current (I_Ca) was decreased concentration-dependently by DPI (IC₅₀ of ≅40.3 µM) with ≅13.1%-inhibition at 3 µM. The frequency of Ca²⁺ sparks was reduced by 3 µM DPI (by ~25%), which was resistant to a brief removal of external Ca²⁺ and Na⁺. Mitochondrial superoxide level was reduced by DPI at 3-100 µM. Our data suggest that DPI may suppress L-type Ca²⁺ channel and RyR, thereby attenuating Ca²⁺-induced Ca²⁺ release and contractility in cardiac myocytes, and that such DPI effects may be related to mitochondrial metabolic suppression.

• Journal of Plant Biotechnology
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• v.2 no.3
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• pp.135-142
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• 2000

The activity of promoter sequences was evaluated in garlic cells using the $\beta$-glucuronidase (GUS) gene as a reporter. Histochemical GUS assay indicated transient GUS activity in leaf, callus and root cells 48 hours after particle bombardment transformation. Quantitative fluorometric assays in extracts of transformed leaves demonstrated that the CsVMV promoter induced the highest level of gene expression, which was, on average, ten fold the level induced by CaMV35S and by the Arabidopsis Act2 promoters and two fold the level expression observed with a construct containing a double CaMV35S plus the untranslated leader sequence from AMV. No activity or very low levels were observed when cells were transformed with plasmids rontaining the typical monocot promoters, Actl, from rice or the Ubi-1, from maize. The green fluorescent protein (GFP) was also tested as a marker gene for garlic transformation. Intense fluorescence was observed in leaf, callus and root cells transformed with a construct containing the gfp gene under control of the CaMV35 Promoter. No fluorescence was detected when the gfp was under control of the Ubi-1 promoter.

• The Korean Journal of Physiology and Pharmacology
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• v.23 no.5
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• pp.345-356
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• 2019

Docosahexaenoic acid (DHA), an omega-3-fatty acid, modulates multiple cellular functions. In this study, we addressed the effects of DHA on human umbilical vein endothelial cell calcium transient and endothelial nitric oxide synthase (eNOS) phosphorylation under control and adenosine triphosphate (ATP, $100{\mu}M$) stimulated conditions. Cells were treated for 48 h with DHA concentrations from 3 to $50{\mu}M$. Calcium transient was measured using the fluorescent dye Fura-2-AM and eNOS phosphorylation was addressed by western blot. DHA dose-dependently reduced the ATP stimulated $Ca^{2+}$-transient. This effect was preserved in the presence of BAPTA (10 and $20{\mu}M$) which chelated the intracellular calcium, but eliminated after withdrawal of extracellular calcium, application of 2-aminoethoxy-diphenylborane ($75{\mu}M$) to inhibit store-operated calcium channel or thapsigargin ($2{\mu}M$) to delete calcium store. In addition, DHA ($12{\mu}M$) increased ser1177/thr495 phosphorylation of eNOS under baseline conditions but had no significant effect on this ratio under conditions of ATP stimulation. In conclusion, DHA dose-dependently inhibited the ATP-induced calcium transient, probably via store-operated calcium channels. Furthermore, DHA changed eNOS phosphorylation suggesting activation of the enzyme. Hence, DHA may shift the regulation of eNOS away from a $Ca^{2+}$ activated mode to a preferentially controlled phosphorylation mode.

• The Korean Journal of Physiology and Pharmacology
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• v.24 no.3
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• pp.277-286
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• 2020

Polycystic kidney disease 2-like-1 (PKD2L1), also known as polycystin-L or TRPP3, is a non-selective cation channel that regulates intracellular calcium concentration. Calmodulin (CaM) is a calcium binding protein, consisting of N-lobe and C-lobe with two calcium binding EF-hands in each lobe. In previous study, we confirmed that CaM is associated with desensitization of PKD2L1 and that CaM N-lobe and PKD2L1 EF-hand specifically are involved. However, the CaM-binding domain (CaMBD) and its inhibitory mechanism of PKD2L1 have not been identified. In order to identify CaM-binding anchor residue of PKD2L1, single mutants of putative CaMBD and EF-hand deletion mutants were generated. The current changes of the mutants were recorded with whole-cell patch clamp. The calmidazolium (CMZ), a calmodulin inhibitor, was used under different concentrations of intracellular. Among the mutants that showed similar or higher basal currents with that of the PKD2L1 wild type, L593A showed little change in current induced by CMZ. Co-expression of L593A with CaM attenuated the inhibitory effect of PKD2L1 by CaM. In the previous study it was inferred that CaM C-lobe inhibits channels by binding to PKD2L1 at 16 nM calcium concentration and CaM N-lobe at 100 nM. Based on the results at 16 nM calcium concentration condition, this study suggests that CaM C-lobe binds to Leu-593, which can be a CaM C-lobe anchor residue, to regulate channel activity. Taken together, our results provide a model for the regulation of PKD2L1 channel activity by CaM.

• Proceedings of the Korean Biophysical Society Conference
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• 1998.06a
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• pp.37-37
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• 1998

Effects of endothelin-l (ET-l) on contraction, $Ca^{2＋}$ transient and L -type $Ca^{2＋}$ current ( $I_{Ca,L}$) were investigated in single ventricular myocytes isolated from guinea-pig hearts. ET-l at concentrations of 5 and 10 nM produced a biphasic pattern of inotropism： a first decrease in contraction by 34.4$\pm$2.5 ％ of the control followed by a sustained increase in contraction by 66.6$\pm$8.4% (mean$\pm$S.E.M., n=9).(omitted)

• Journal of Korean Biological Nursing Science
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• v.1 no.1
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• pp.25-41
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• 1999

Physiological activity of osteoblast including bone formation is known to be closely related to the increase of intracellular $Ca^{2+}$ activity($[Ca^{2+}]_i$) in osteoblast. $Ca^{2+}$ is an important intracellular messenger in diverse cellular functions, and regulation of its level is mediated by the transmembrane $Ca^{2+}$ movement via $Ca^{2+}$ channels, $Na^+-Ca^{2+}$ exchange, and by intracellular $Ca^{2+}$ movement through the intracellular stores. The purpose of this study is to investigate how the intracellular $Ca^{2+}$ is regulated in osteoblast-like cells(OLCs) by measuring $Ca^{2+}$ activity with cell imaging technique. OLCs were isolated from femur and tibia of neonatal rats, and cultured for 7 days. Cultured OLCs were loaded with a $Ca^{2+}$-sensitive fluorescent dye, Fura-2, and fluorescence images were monitored with a cooled CCD camera. The images were processed and analyzed with an image analyzing software. The results were as follows. (1) $[Ca^{2+}]_i$ of OLC decreased as the $Ca^{2+}$ concentration in the superfusing Tyrode solution was lowered. When $Na^+$ concentration in the superfusing solution was decreased, $[Ca^{2+}]_i$ increased.. These suggest that $Ca^{2+}$ flux occurs via the $Na^+-Ca^{2+}$ exchange mechanism. (2) When $Na^+$ in the superfusing solution was removed. a transient $Ca^{2+}$, increase($Ca^{2+}$ spike) was occasionally observed. However, $Ca^{2+}$ spike was not observed after adding 1 ${\mu}M$ thapsigargin. This implies that the generation of $Ca^{2+}$ spike is mediated by the release of $Ca^{2+}$ from endoplasmic reticulum(ER). (3) As the $Ca^{2+}$ concentration in the superfusing solution was raised, the frequency of 0mM $Na^+$-induced $Ca^{2+}$ spike increased, suggesting that $Ca^{2+}$-induced $Ca^{2+}$ release(CICR) mechanism exists. (4) After $[Ca^{2+}]_i$ was decreased with the superfusion of $Ca^{2+}$-free solution containing thapsigargin, the recovery of $[Ca^{2+}]_i$ with reperfusion of 2.5mM $Ca^{2+}$ solution transiently exceeded the control level, suggesting that the depletion of $Ca^{2+}$ in ER induces $Ca^{2+}$ influx from extracellular medium via store-operated $Ca^{2+}$ influx(SOCI) mechanism. (5) $[Ca^{2+}]_i$ was not affected by the superfusion of 25mM $K^+$ Tyrode solution. These results suggest that intracellular $Ca^{2+}$ activity in osteoblast is regulated by transmembrane $Ca^{2+}$ flux via $Na^+-Ca^{2+}$ exchange, $Ca^{2+}$ release from the internal store (ER) via $Ca^{2+}$-induced $Ca^{2+}$ release, and store-operated $Ca^{2+}$ influx across the cell membrane.

• Proceedings of the Korean Vacuum Society Conference
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• 2010.02a
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• pp.310-310
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• 2010

Electronic and photovoltaic characteristics of two sensitizers (TA-BTD-CA and TA-BTD-St-CA), composed of a different $\pi$-conjugation in the linker group, have been investigated by theoretical and experimental methods. The electronic structure, transition dipole moment and oscillator strengths of two sensitizers have been scrutinized by using density functional theory (DFT) and time-dependent DFT (TD-DFT) method. The LUMO level and the oscillator strength of TA-BTD-St-CA was higher than that of TA-BTD-CA, which may facilitate the electron injection process as well as increase the absorption coefficient. The relative efficiencies of the electron injection from the excited sensitizer to nanocrystalline TiO2 and SnO2 films have also been investigated by nanosecond transient absorption spectroscopy. The relative electron injection efficiency of TA-BTD-St-CA exhibited similar injection efficiency for two different semiconductors. However, in the case of TA-BTD-CA sensitizer, electron injection into SnO2 was approximately three times larger than that into TiO2. This enhancement of electron injection of TA-BTD-CA for the SnO2 is due to the increment of the driving force caused by positive shift of conduction band of semiconductor, which was also confirmed from the investigation for the photovoltaic characteristics according to the electrolyte additive, such as LiI additive.

• BMB Reports
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• v.37 no.1
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• pp.114-121
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• 2004

Ion channels of the transient receptor potential (TRP) superfamily are non-selective cationic channels with six transmembrane domains. The TRP channel made its first debut as a light-gated $Ca^{2+}$ channel in Drosophila. Recently, research on animal sensation in Drosophila disclosed other members of the TRP family that are required for touch sensation and hearing as well as the sensation of painful stimuli.

• The Korean Journal of Physiology
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• v.19 no.2
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• pp.101-111
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• 1985

Transient inward current (TI) was studied by the two micro-electrode voltage clamp technique in the sinoatrial node of the rabbit. The author confirmed that in $10^{-6}$ M ouabain TI was found in the SA node and investigated the effects of ions, $(Na^+,\;K^+,\;Ca^{2+})$, $\beta-agonist$ (isoprenaline), local anesthetics (quinidine, lidocaine) and Ca-blockers ($Co^{2+}$, verapamil, diltiazem) on the TI recorded during depolarizing voltage clamp pulses to -40 and -20 mV. The results obtained were as follows ; 1) $10^{-6}M$ ouabain increased the frequency of sinus action potential and decreased the amplitude, especially overshoot of action potential. TI was induced by the depolarizing voltage clamp Pulses and the magnitude of the slow inward current (isi) decreased and the time course was slowed by the same depolarizing pulses. 2) 30% $Na^{+}$ and 24mM $K^+$ decreased by $10^{-6}M$ ouabain and 6 mM $Ca^{2+}$ and $10^{-7}M$ isoprenaline increased TI, $i_{si}$ and current oscillations. 3) Quinidine $(5\times10^{-7}M)$ reduced TI and $i_{si}$ but lidocaine $(10^6\;-10^5M)$ didn't reduced or increase TI. Current oscillations increased and isi decreased by lidocaine. 4) Ca-blockers decreased the amplitude and the frequency of sinus action potential. TI and $i_{si}$ decreased significantly but were not abolished completely at the concentrations used in this experiment. Verapamil and diltiazem had inhibitory action on TI in $2\times10^{-7}M$ concentration and showed very slow recovery after wasting out with normal Tyrode solution.

• International Journal of Oral Biology
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• v.31 no.2
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• pp.45-51
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• 2006

R-type($Ca_v2.3$) calcium channel contributes to pain sensation in peripheral sensory neurons. Six isoforms of $Ca_v2.3$ that result from combinations of presence or deletion of three inserts(insert I and insert in the II-III loop, and insert III in N-terminal regions) have been demonstrated to be present in different mammalian tissues. However, the molecular basis of $Ca_v2.3$ in trigeminal ganglion(TG) neurons is not known. In the present study, we determined which isoforms of $Ca_v2.3$ are expressed in rat TG neurons using the RT-PCR analysis. Whole tissue RT-PCR analyses revealed that only two isoforms, $Ca_v2.3a$ and $Ca_v2.3e$, were present in TG neurons. From single-cell RT-PCR, we found that $Ca_v2.3e$ rather than $Ca_v2.3a$ was the major isoform expressed in TG neurons, and $Ca_v2.3e$ was preferentially detected in small-sized neurons that express nociceptive marker, transient receptor potential vanilloid 1(TRPV1). Our results suggest that $Ca_v2.3e$ in trigeminal neurons may be a potential target for the pain treatment.