• The Korean Journal of Physiology and Pharmacology
• /
• v.18 no.4
• /
• pp.297-305
• /
• 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.

• Toxicological Research
• /
• v.11 no.2
• /
• pp.181-185
• /
• 1995

To know the mechanism of biphenyl dimethyl dicarboxylate (DDB) in the protection of chemically induced hepatotoxicity, the activity of glutamic pyruvic tran.saminase (GPT) and the level of lipid peroxidation metabolite (malondialdehyde, MDA) and ATP content in hepatocytes were determined in serum and primarily cultured hepatocytes. For in vibo study, rats were pretreated with DDB (300 mg/ kg, p.o.)for 7 days. DDB pretreatment efficiently reduced the elevation of serum GPT activity induced by carbon tetrachloride (1.6 ml/kg, s.c.) and acetaminophen administration (1500 mg/kg, i.p.). In ex vivo study, hepatocytes were isolated from the rats pretreated with DDB (300 mg/kg, p.o.)for 7 days and cultured for 12 hrs before inducing cytotoxicity with chemicals. The MDA formation and the GPT release induced by adriamycin $(1\times10^{-4} mg/ml)$ and cisplatin $(2\times10^{-4} mg/ml)$ were markedly decreased in the hepatocytes from the rats pretreated with DDB as compared to vehicle only. However, DDB pretreatment did not prevent the decrease of ATP contents of hepatocytes induced by cisplatin and adriamycin. In in vitro experiment, DDB was pretreated in primary cultured hepatocytes for 3 days. DDB enhanced the decreases of ATP contents induced by cisplatin and adriamycln. These results suggest that DDB may protect the hepatocytes from injury induced by hepatotoxlcants through inhibiting the lipid peroxidation.

• BMB Reports
• /
• v.42 no.3
• /
• pp.166-171
• /
• 2009

Hsp70s assist in the process of protein folding through nucleotide-controlled cycles of substrate binding and release by alternating from an ATP-bound state in which the affinity for substrate is low to an ADP-bound state in which the affinity for substrate is high. It has been long recognized that the two-domain structure of Hsp70 is critical for these regulated interactions. Therefore, it is important to obtain information about conformational changes in the relative positions of Hsp70 domains caused by nucleotide binding. In this study, analytical ultracentrifugation and dynamic light scattering were used to evaluate the effect of ADP and ATP binding on the conformation of the human stress-induced Hsp70.1 protein. The results of these experiments showed that ATP had a larger effect on the conformation of Hsp70 than ADP. In agreement with previous biochemical experiments, our results suggest that conformational changes caused by nucleotide binding are a consequence of the movement in position of both nucleotide- and substrate-binding domains.

• Biomedical Science Letters
• /
• v.7 no.3
• /
• pp.127-131
• /
• 2001

This study was undertaken to examine the effect of amino acids on anoxia-induced cell injury in rabbit renal cortical slices. In order to induce anoxic cell injury, slices were exposed to a 100% $N_2$ atmosphere and control slices were exposed to 100% $O^2$. Irreversible cell injury was estimated by measuring lactate dehydrogenase (LDH) release and alterations in renal cell function were examined by measuring p-aminohippurate (PAH) uptake. Anoxia caused the increase in LDH release in a time-dependent manner. Glycine and glutathione almost completely prevented anoxia-induced LDH release. Of amino acids tested, glycine and alanine exerted the protective effect against anoxia-induced cell injury. However, asparagine with amide side chain, leucine and valine with hydrocarbon side chain, and basic amino acids (lysine, histidine, and arginine) were not effective. Anoxia-induced inhibition of PAM uptake was prevented by glycine. ATP content was decreased by anoxia, which was not affected by glycine. Anoxia-induced depletion of glutathione was significantly prevented by glycine. These results suggest that neutral amino acids with simple structure exert the Protective effect against anoxia-induced cell injury the involvement of specific interaction of amino acids and cell structure.

• Biomedical Science Letters
• /
• v.13 no.3
• /
• pp.197-206
• /
• 2007

P2X receptors are membrane-bound ion channels that conduct $Na^+,\;K^+$, and $Ca^{2+}$ in response to ATP and its analogs. There are seven subunits identified so far ($P2X_1-P2X_7$). $P2X_2$ receptors are known to be expressed in a wide range of organs including brains and adrenal grands. PC12 cells are originated from adrenal grand and differentiated by nerve growth factor or pituitary adenylate cyclase activating poly peptide (PACAP). Previous studies indicate that $P2X_2$ receptor activation in PC12 cells couples to $Ca^{2+}-dependent$ release of catecholamine and ATP. It is known that acidic pH potentiates ATP currents at $P2X_2$ receptors. This leads to a hypothesis that $P2X_2$ receptors may play an important role in PC12 cell differentiation, one of the characteristics of which is neurite outgrowth, induced by the hormones under lower pH. In the present study, we isolated several clones which potentiate neurite outgrowth by PACAP in acidic pH (6.8), but not in alkaline pH (7.6). RT-PCR and electrophysiology data indicate that these clones express only functional $P2X_2$ receptors in the absence or presence of PACAP for 3 days. Potentiation of neurite outgrowth resulted from PACAP (100 nM) in acidic pH is inhibited by the two P2X receptor antagonists, suramin and PPADS ($100\;{\mu}M)$ each), and exogenous exprerssion of ATP-binding mutant $P2X_2$ receptor subunit ($P2X_2[K69A]$). However, acid sensing ion channels (ASICs) are not involved in PACAP-induced neurite outgrowth potentiation in lower pH since treatments of an inhibitor of ASICs, amyloride ($10\;{\mu}M$), did not give any effects to neurite extension. The vesicular proton pump ($H^+-ATPase$) inhibitor, bafilomycin (100 nM), reduced neurite extension indicating that ATP release resulted from $P2X_2$ receptor activation in PC12 cells is needed for neurite outgrowth. These were confirmed by activation of mitogen activated protein kinases, such as ERKs and p38. These results suggest roles of ATP and $P2X_2$ receptors in hormone-induced cell differentiation or neuronal synaptogenesis in local acidic environments.

• YAKHAK HOEJI
• /
• v.43 no.6
• /
• pp.809-817
• /
• 1999

The effect of 2-(4-cyanophenyl)amino-1,4-naphthalenedione-3-pyridinium perchlorate (PQ5) on pla-telet aggregation and its action mechanisms were investigated with rat platelet. PQ5 inhibited the platelet aggregation induced by collagen ($6{\;}{\mu\textrm{g}}/ml$), thrombin (0.4 U/ml) and A23187 ($3{\mu}M$) in concentration-dependent manner with $IC_{50}$ values of 5.50, 25.89 and $37.12{\;}{\mu}M$, respectively. PQ5 also significantly reduced the thromboxane $A_2$ (TXA2) formation in a concentration dependent manner. The collagen-induced arachidonic acid (AA) release in [-3H]-AA incorporated platelet, an indication of the phospholipase $A_2$ activity, was decreased by PQ5 pretreatment PQ5 significantly inhibited the activity of thormboxane synthase only at high concentration ($100{\mu}M$), but did not affect the cyclooxygenase activity at all. Collagen-induced ATP release was significantly reduced by PQ5. Calcium-induced platelet aggregation experiment suggests that the elevation of intracellular free $Ca^{2+}$ concentration ($[Ca^{2+}]_i$) by collagen stimulation is decreased by the pretreatment of PQ5, which is due to the inhibition of calcium release from intracellular store and influx from outside of the cell. PQ5 did not showed the effect of anticoagulation as prothrombin time (PT) or activated partial thromboplastin time (APTT). Form these results, it is suggested that PQ5 exerts its antiplatelet activity through the inhibition of the intracellular $Ca^{2+}$ mobilization and the decrease of the $TXA_2$ synthesis.

• Proceedings of the PSK Conference
• /
• 2000.10a
• /
• pp.171.2-172
• /
• 2000

• Proceedings of the PSK Conference
• /
• 2000.04a
• /
• pp.138-138
• /
• 2000

• The Korean Journal of Physiology and Pharmacology
• /
• v.8 no.2
• /
• pp.95-100
• /
• 2004

Ischemic preconditioning (IPC) has been accepted as a heart protection phenomenon against ischemia and reperfusion (I/R) injury. The activation of ATP-sensitive potassium $(K_{ATP})$ channels and the release of myocardial nitric oxide (NO) induced by IPC were demonstrated as the triggers or mediators of IPC. A common action mechanism of NO is a direct or indirect increase in tissue cGMP content. Furthermore, cGMP has also been shown to contribute cardiac protective effect to reduce heart I/R-induced infarction. The present investigation tested the hypothesis that $K_{ATP}$ channels attenuate DNA strand breaks and oxidative damage in an in vitro model of I/R utilizing rat ventricular myocytes. We estimated DNA strand breaks and oxidative damage by mean of single cell gel electrophoresis with endonuclease III cutting sites (comet assay). In the I/R model, the level of DNA damage increased massively. Preconditioning with a single 5-min anoxia, diazoxide $(100\;{\mu}M)$, SNAP $(300\;{\mu}M)$ and 8-(4-Chlorophenylthio)-guanosine-3',5'-cyclic monophosphate (8-pCPT-cGMP) $(100\;{\mu}M)$ followed by 15 min reoxygenation reduced DNA damage level against subsequent 30 min anoxia and 60 min reoxygenation. These protective effects were blocked by the concomitant presence of glibenclamide $(50\;{\mu}M)$, 5-hydroxydecanoate (5-HD) $(100\;{\mu}M)$ and 8-(4-Chlorophenylthio)-guanosine-3',5'-cyclic monophosphate, Rp-isomer (Rp-8-pCPT-cGMP) $(100\;{\mu}M)$. These results suggest that NO-cGMP-protein kinase G (PKG) pathway contributes to cardioprotective effect of $K_{ATP}$ channels in rat ventricular myocytes.

• Journal of Acupuncture Research
• /
• v.18 no.4
• /
• pp.143-151
• /
• 2001

Objective : This study was undertaken to determine whether Scutellaria balicalensis Georgi (SbG) extract exerts the protective effect against oxidant-induced alterations in organic cation transport in the renal proximal tubule. Methods : Organic cation transport was estimated by examining alterations in tetraethylammon - ium(TEA) uptake in rabbit renal cortical slices. The slices were treated with 0.2 mM tBHP for 60 min at $37^{\circ}C$. tBHP caused an inhibition in TEA uptake by renal cortical slices. Such an effect was accompanied by depressed Na+-K+-ATPase activity and ATP depletion. tBHP also induced a significant increase in LDH release. Results : SbG prevented tBHP-induced inhibition of TEA uptake in a dose-dependent manner at the concentration ranges of 0.05-0.1%. tBHP-induced inhibition of Na+-K+-ATPase activity and ATP depletion were significantly prevented by 0.05% SbG. tBHP-induced LDH release also was blocked by SbG. tBHP caused a significant increase in lipid peroxidation and its effect was prevented by SbG. Conclusion : These results suggest that SbG prevents oxidant-induced alterations in organic cation transport in rabbit renal cortical slices. Such protective effects of SbG may be attributed to inhibition of peroxidation of membrane lipid.