• The Korean Journal of Physiology and Pharmacology
• /
• v.9 no.1
• /
• pp.29-35
• /
• 2005

To prove the buffering contribution of mitochondria to the increase of intracellular $Ca^{2+}$ level ($[Ca^{2+}]_i$) via background nonselective cation channel (background NSCC), we examined whether inhibition of mitochondria by protonophore carbonylcyanide m-chlorophenylhydrazone (CCCP) affects endothelial $Ca^{2+}$ entry and $Ca^{2+}$ buffering in freshly isolated rabbit aortic endothelial cells (RAECs). The ratio of fluorescence by fura-2 AM ($R_{340/380}$) was measured in RAECs. Biological state was checked by application of acetylcholine (ACh) and ACh ($10{\mu}M$) increased $R_{340/380}$ by $1.1{\pm}0.15$ ($mean{\pm}S.E.$, n=6). When the external $Na^+$ was totally replaced by $NMDG^+$, $R_{340/380}$ was increased by $1.19{\pm}0.17$ in a reversible manner (n=27). $NMDG^+$-induced $[Ca^{2+}]_i$ increase was followed by oscillatory decay after $[Ca^{2+}]_i$ reached the peak level. The increase of $[Ca^{2+}]_i$ by $NMDG^+$ was completely suppressed by replacement with $Cs^+$. When $1{\mu}M$ CCCP was applied to bath solution, the ratio of $[Ca^{2+}]_i$ was increased by $0.4{\pm}0.06$ (n=31). When $1{\mu}M$ CCCP was used for pretreatment before application of $NMDG^+$, oscillatory decay of $[Ca^{2+}]_i$ by $NMDG^+$ was significantly inhibited compared to the control (p＜0.05). In addition, $NMDG^+-induced$ increase of $[Ca^{2+}]_i$ was highly enhanced by pretreatment with $2{\mu}M$ CCCP by $320{\pm}93.7$%, compared to the control ($mean{\pm}S.E.$, n=12). From these results, it is concluded that mitochondria might have buffering contribution to the $[Ca^{2+}]_i$ increase through regulation of the background NSCC in RAECs.

• The Korean Journal of Physiology and Pharmacology
• /
• v.10 no.1
• /
• pp.25-30
• /
• 2006

Lysophosphatidylcholine (LPC), which accumulates in atherosclerotic arteries, has been reported to inhibit endothelium-dependent relaxation (EDR) in many different species. However, the underlying mechanism of LPC-induced inhibition of EDR is still uncertain. In the present study, we measured simultaneously both isometric tension and cytosolic free $Ca^{2+}$ ($[Ca^{2+}]_i$) in rabbit carotid strips, and examined the effect of LPC on tension and $[Ca^{2+}]_i$. In carotid strips with intact-endothelium, high $K^+$ (70 mM) increased both tension and $[Ca^{2+}]_i$, and cumulative addition of acetylcholine (ACh) from 0.1 to $10{\mu}M$ induced dose dependent increase of $[Ca^{2+}]_i$ with concomitant relaxation. In the presence of L-NAME (0.1 mM), ACh increased $[Ca^{2+}]_i$ without affecting the amplitude of high $K^+-induced$ tension. These ACh-induced change of $[Ca^{2+}]_i$ and tension was abolished by removal of endothelium or 10 nM 4-DAMP (muscarinic receptor antagonist) pretreatment. Pretreatment of LPC ($10{\mu}M$) inhibited ACh ($10{\mu}M$)-induced change of tension and $[Ca^{2+}]_i$ in endothelium-intact carotid artery. On the other hand, LPC had no effect on ACh-induced change of tension and $[Ca^{2+}]_i$ in endothelium denuded artery. In $Ca^{2+}$-free external solution, ACh transiently increased $[Ca^{2+}]_i$, and pretreatment of LPC significantly inhibited ACh-induced transient $[Ca^{2+}]_i$ change. Based on the above results, it may be concluded that LPC inhibits the ACh-induced $[Ca^{2+}]_i$ change through inhibition of $Ca^{2+}$ mobilization in vascular endothelial cells, resulting in decreased production of NO and concomitant inhibition of endotheliumdependent vascular relaxation.

• The Korean Journal of Physiology and Pharmacology
• /
• v.6 no.2
• /
• pp.93-99
• /
• 2002

Effects of oxidized low-density lipoprotein (ox-LDL), $1-{\alpha}-stearoyl-lysophosphatidylcholine$ (LPC), on intracellular $Ca^{2+}$ concentration were examined in mouse endothelial cells by measuring intracellular $Ca^{2+}$ concentration $([Ca^{2+}]_i)$ with fura 2-AM and reverse transcription-polymerase chain reaction (RT-PCR). LPC increased $[Ca^{2+}]_i$ under the condition of 1.5 mM $[Ca^{2+}]_o$ but did not show any effect under the nominally $Ca^{2+}-free$ condition. Even after the store depletion with $30{\mu}M$ 2,5-di-tert- butylhydroquinone (BHQ) or $30{\mu}M$ ATP, LPC could still increase the $[Ca^{2+}]_i$ under the condition of 1.5 mM $[Ca^{2+}]_o.$ The time required to increase [$Ca{2+}$]i (about 1 minute) was longer than that for ATP-induced $[Ca^{2+}]_i$ increase $(10{\sim}30\;seconds).$ LPC-induced $[Ca^{2+}]_i$ increase was completely blocked by $1{\mu}M\;La^{3+}.$ Transient receptor potential channel(trpc) 4 mRNA was detected with RT-PCR. From these results, we suggest that LPC increased $[Ca^{2+}]_i$ via the increase of $Ca^{2+}$ influx through the $Ca^{2+}$ routes which exist in the plasma membrane.

• The Korean Journal of Physiology and Pharmacology
• /
• v.9 no.6
• /
• pp.341-346
• /
• 2005

The mechanism underlying oxidant-induced intracellular $Ca^{2+}$ ($[Ca^{2+}]_i$) increase was studied in cultured bovine aortic endothelial cells (BAECs) using fura-2 AM. In the presence of 2 mM extracellular $Ca^{2+}$, the application of DTBNP ($20{\mu}M$), a membrane-permeable oxidant, caused an increase in $[Ca^{2+}]_i$, and DTT (2 mM) as a reductant completely reversed the effect of DTBNP. The $[Ca^{2+}]_i$ increase induced by DTBNP was also observed in an extracellular $Ca^{2+}$-free/2 mM EGTA solution, indicating the release of $Ca^{2+}$ from intracellular store(s). After endoplasmic reticulum was depleted by an $IP_3$-generating agonist, ATP ($30{\mu}M$) or an ER $Ca^{2+}$ pump inhibitor, thapsigargin ($1{\mu}M$), DTBNP-stressed BAECs showed an increase of $[Ca^{2+}]_i$ in $Ca^{2+}$-free/2 mM EGTA solution. Ratio-differences before and after the application of DTBNP after pretreatment with ATP or thapsigargin were $0.42{\pm}0.15$ and $0.49{\pm}0.07$, respectively (n=7), which are significantly reduced, compared to the control value of $0.72{\pm}0.07$ in a $Ca^{2+}$-free/2 mM EGTA solution. After the protonophore CCCP ($10{\mu}M$) challenge to release mitochondrial $Ca^{2+}$, the similar result was obtained. Ratio-difference before and after the application of DTBNP after pretreatment with CCCP was $0.46{\pm}0.09$ (n=7). Simultaneous application of thapsigargin and CCCP completely abolished the DTBNP-induced $[Ca^{2+}]_i$ increase. The above results together indicate that the increase of $[Ca^{2+}]_i$ by DTBNP resulted from the release of $Ca^{2+}$ from both endoplasmic reticulum and mitochondria.

• The Korean Journal of Physiology and Pharmacology
• /
• v.13 no.1
• /
• pp.27-32
• /
• 2009

The effects of oxidized low-density lipoprotein(OxLDL) and its major lipid constituent lysophosphatidylcholine(LPC) on $Ca^{2+}$ entry were investigated in cultured human umbilical endothelial cells(HUVECs) using fura-2 fluorescence and patch-clamp methods. OxLDL or LPC increased intracellular $Ca^{2+}$ concentration($[Ca^{2+}]_i$), and the increase of $[Ca^{2+}]_i$ by OxLDL or by LPC was inhibited by $La^{3+}$ or heparin. LPC failed to increase $[Ca^{2+}]_i$ in the presence of an antioxidant tempol. In addition, store-operated $Ca^{2+}$ entry(SOC), which was evoked by intracellular $Ca^{2+}$ store depletion in $Ca^{2+}$-free solution using the sarcoplasmic reticulum $Ca^{2+}$ pump blocker, 2, 5-di-t-butyl-l,4-benzohydroquinone(BHQ), was further enhanced by OxLDL or by LPC. Increased SOC by OxLDL or by LPC was inhibited by U73122. In voltage-clamped cells, OxLDL or LPC increased $[Ca^{2+}]_i$ and simultaneously activated non-selective cation(NSC) currents. LPC-induced NSC currents were inhibited by 2-APB, $La^{3+}$ or U73122, and NSC currents were not activated by LPC in the presence of tempol. Furthermore, in voltage-clamped HUVECs, OxLDL enhanced SOC and evoked outward currents simultaneously. Clamping intracellular $Ca^{2+}$ to 1 ${\mu}M$ activated large-conductance $Ca^{2+}$-activated $K^+(BK_{ca})$ current spontaneously, and this activated $BK_{ca}$ current was further enhanced by OxLDL or by LPC. From these results, we concluded that OxLDL or its main component LPC activates $Ca^{2+}$-permeable $Ca^{2+}$-activated NSC current and $BK_{ca}$ current simultaneously, thereby increasing SOC.

• BMB Reports
• /
• v.50 no.6
• /
• pp.323-328
• /
• 2017

Lysophosphatidylcholine (LPC) is a major phospholipid component of oxidized low-density lipoprotein (ox-LDL) and is implicated in its atherogenic activity. This study investigated the effects of LPC on cell viability, intracellular calcium homeostasis, and the protective mechanisms of chlorogenic acid (CGA) in human umbilical vein endothelial cells (HUVECs). LPC increased intracellular calcium ($[Ca^{2+}]_i$) by releasing $Ca^{2+}$ from intracellular stores and via $Ca^{2+}$ influx through store-operated channels (SOCs). LPC also increased the generation of reactive oxygen species (ROS) and decreased cell viability. The mRNA expression of Transient receptor potential canonical (TRPC) channel 1 was increased significantly by LPC treatment and suppressed by CGA. CGA inhibited LPC-induced $Ca^{2+}$ influx and ROS generation, and restored cell viability. These results suggested that CGA inhibits SOC-mediated $Ca^{2+}$ influx and ROS generation by attenuating TRPC1 expression in LPC-treated HUVECs. Therefore, CGA might protect endothelial cells against LPC injury, thereby inhibiting atherosclerosis.

• BMB Reports
• /
• v.54 no.10
• /
• pp.516-521
• /
• 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.

• Journal of Chest Surgery
• /
• v.36 no.12
• /
• pp.887-893
• /
• 2003

External stimuli increases intracellular (IC) $Ca^{2+}$, which increases extracellular (EC) $K^{+}$. To verify $K^{+}$ effects on the vascular contraction, we performed an experiment using mouse aortic endothelial cell. Meterial and Method: We examined the mouse aortic contractility changes as we measured the IC $Ca^{2+}$ change and ionic current by using the voltage clamp technique under different conditions such as: increasing EC $K^{+}$, removing endothelial cell, giving L-NAME (N-nitro-L-arginine methyl ester) which suppress nitric oxide formation, Ouabain which control N $a^{+}$ - $K^{+}$ pump and N $i^{2+}$ which repress N $a^{+}$-C $a^{2+}$ exchanger Result: When we increased EC $K^{+}$ from 6 to 12 mM, there was no change in aortic contractility. Aorta contracted with more than 12 mM of EC $K^{+}$. Ace-tylcholine (ACh) induced relaxation was inhibited with EC $K^{+}$ from 6 to 12 mM, but was not found after de-endothelialization or L-NAME treatment. ATP or ACh increased IC $Ca^{2+}$ in cultured endothelium. After maximal increase of IC $Ca^{2+}$, increasing EC $K^{+}$ from 6 to 12 mM made IC $Ca^{2+}$ decrease and re-decreasing EC $K^{+}$ to 6 mM made IC $Ca^{2+}$ increase. Ouabain and N $i^{2+}$ masked the inhibitory effect of endothelium dependent relaxation by increased EC $K^{+}$. Conclusion: These data indicate that increase in EC $K^{+}$ relaxes vascular smooth muscle and reduces $Ca^{2+}$ in the endothelial cells which inhibit endothelium dependent relaxation. This inhibitory mechanism may be due to the activation of N $a^{+}$- $K^{+}$ pump and N $a^{+}$-C $a^{2+}$ exchanger. $a^{+}$-C $a^{2+}$ exchanger.r.

• Biomolecules & Therapeutics
• /
• v.17 no.2
• /
• pp.175-180
• /
• 2009

Taurine is the most abundant free amino acid in the retina and transported into retina via taurine transporter (TauT) at the inner blood-retinal barrier (iBRB). In the present study, we investigated whether the taurine transport at the iBRB is regulated by oxidative stress or disease-like state in a conditionally immortalized rat retinal capillary endothelial cell line (TR-iBRB) used as an in vitro model of iBRB. First, [$^3H$]taurine uptake and efflux by TR-iBRB were regulated in the presence of extracellular $Ca^{2+}$. [$^3H$]Taurine uptake was inhibited and efflux was enhanced under $Ca^{2+}$ free condition in the cells. In addition, oxidative stress inducing agents such as tumor necrosis factor-$\alpha$ (TNF-$\alpha$), lipopolysaccharide (LPS), diethyl maleate (DEM) and glutamate increased [$^3H$]taurine uptake and decreased [$^3H$]taurine efflux in TR-iBRB cells. Whereas, 3-morpholinosydnonimine (SIN-1), which is known to NO donor decreased [$^3H$]taurine uptake. Lastly, TR-iBRB cells exposed to high glucose (25 mM) medium and the [$^3H$]taurine uptake was reduced about 20% at the condition. Also, [$^3H$]taurine uptake was decreased by cytochalasin B, which is known to glucose transport inhibitor. In conclusion, taurine transport in TR-iBRB cells is regulated diversely at extracellular $Ca^{2+}$, oxidative stress and hyperglycemic condition. It suggested that taurine would play a role as a retinal protector in diverse disease states.

• The Korean Journal of Physiology and Pharmacology
• /
• v.8 no.6
• /
• pp.313-317
• /
• 2004

The present study were designed to characterize the action mechanisms of acetylcholine (ACh)-induced endothelium-dependent relaxation in arteries precontracted with high $K^+$(70 mM). For this, we simultaneously measured both muscle tension and cytosolic free $Ca^{2+}$ concentration $([Ca^{2+}]_i)$, using fura-2, in endothelium-intact, rabbit carotid arterial strips. In the artery with endothelium, high $K^+$ increased both $[Ca^{2+}]_i$ and muscle tension whereas ACh $(10{\mu}M)$ significantly relaxed the muscle and increased $[Ca^{2+}]_i$. In the presence of $N^G$-nitro-L-arginine (L-NAME, 0.1 mM), ACh increased $[Ca^{2+}]_i$ without relaxing the muscle. In the artery without endothelium, high $K^+$ increased both $[Ca^{2+}]_i$ and muscle tension although ACh was ineffective. 4-DAMP (10 nM) or atropine $(0.1{\mu}M)$ abolished ACh-induced increase in $[Ca^{2+}]_i$ and relaxation. The increase of $[Ca^{2+}]_i$ and vasorelaxation by ACh was siginificantly reduced by either $3{\mu}M$ gadolinium, $10{\mu}M$ lanthanum, or by $10{\mu}M$ SKF 96365. These results suggest that in rabbit carotid artery, ACh-evoked relaxation of 70 mM $K^+$-induced contractions appears to be mediated by the release of NO. ACh-evoked vasorelaxation is mediated via the $M_3$ subtype, and activation of the $M_3$ subtype is suggested to stimulate nonselective cation channels, leading to increase of $[Ca^{2+}]_i$ in endothelial cells.