• The Korean Journal of Physiology
• /
• v.29 no.2
• /
• pp.269-277
• /
• 1995

Membrane vesicles were prepared by differential centrifugation from epithelial cells of porcine trachea. Total activity of microsomal ATPases was measured spectrophotometrically by a coupled enzyme assay. The steady-state activity of the enzyme was $329{\pm}10$ nmol/min mg protein. Thapsigargin, a specific antagonist of intracellular $Ca^{2+}-ATPase$, inhibited about 50% of the activity, leaving $178{\pm}18\;nmol/min .mg$ protein (n=6), indicating that the $Ca^{2+}-ATPase$ is one of the major microsomal ATPases. The microsomes used in this study appeared to be tight-sealed vesicles since they showed saturation in $^{45}Ca^{2+}$ uptake experiments. Inositol 1,4,5-trisphosphate $InsP_{3}, 4\;{\mu}M$, an agonist of $InsP_{3}$-sensitive $Ca^{2+}$ release channel ($InsP_{3}$, receptor), and Ca-ionophore A23187 $(10\;{\mu}M)$ induced $^{45}Ca^{2+}$ releases of 20% and 50% of stored $^{45}Ca^{2+}$, respectively. The addition of $(10\;{\mu}M\;InsP_{3}$ also increased the microsomal ATPase activity from $282{\pm}8$ nmol/min mg protein to $334{\pm}21$ nmol/min . mg protein in the intact vesicles. Similar increase in the activity was observed by making microsomes leaky (uncoupling) using the Ca-ionophore A23187. ;$InsP_{3}-induced$ effects were blocked by either thapsigargin or heparin suggesting that: 1) the $InsP_{3}-induced$ increase in ATPase activity is mediated by microsomal $Ca^{2+}-ATPase$, and 2) dissipation of $Ca^{2+}$ gradient across the microsomal membrane is responsible for the $InsP_{3}-induced$ effect. In order to test the dependence of the $Ca^{2+}-ATPase$ activity on the activity of $InsP_{3}-induced$ the activity of ATPases was monitored in various concentrations of free $Ca^{2+}$ using $EGTA-Ca^{2+}$ buffers. The $Ca^{2+}$-dependent biphasic change is the well-known character of $InsP_{3} receptor but not of microsomal $Ca^{2+}-ATPase$ in non-excitable cells; however, the activity of microsomal ATPase appeared biphasic and a maxim진 activity of $397{\pm}36nmol/min\;.mg$ protein was obtained in the solution containing 100 nM free $Ca^{2+}$. Below or above this concentration, the activity of ATPases was lower. These results strongly support a positive correlation of microsomal $Ca^{2+}-ATPase$ to the $InsP_{3}$ receptors in epithelial microsomes.

• The Korean Journal of Pharmacology
• /
• v.32 no.3
• /
• pp.417-424
• /
• 1996

The present study was done to examine the involvement of protein kinase C and protein tyrosine kinase in intracellular $Ca^{2+}$ mobilization in C5a-stimulated neutrophils. Although protein kinase C inhibitors, staurosporine and H-7 inhibited intracellular $Ca^{2+}$ release in C5a-stimulated neutrophils, they did not affect $Ca^{2+}$ influx across the plasma membrane and elevation of $[Ca^{2+}]_i$ C5a-induced intracellular $Ca^{2+}$ release and $Ca^{2+}$ influx were inhibited by protein tyrosine kinase inhibitors, genistein and methyl-2,5-dihydroxycinnamate. ADP-evoked elevation of $[Ca^{2+}]_i$ was inhibited by genistein and methyl-2,5-dihydroxycinnamate but was not affectd by staurosporine and H-7. Genistein and methyl-2,5-dihydroxycinnamate reduced the store-regulated $Ca^{2+}$ influx in thapsigargin-treated neutrophils, while the effect of staurosporine and H-7 was not detected. When neutrophils were preincubated wih phorbol 12-myristate 13-acetate, the stimulatory effect of C5a on the elevation of $[Ca^{2+}]_i$ was reduced. These results suggest that protein tyrosine kinase may be involved in control of intracellular $Ca^{2+}$ release and $Ca^{2+}$ influx across the plasma membrane in C5a-activated neutrophils.

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

• Journal of Genetic Medicine
• /
• v.12 no.1
• /
• pp.25-30
• /
• 2015

Purpose: Charcot-Marie-Tooth disease (CMT) is a peripheral neuropathy mainly divided into CMT type 1 (CMT1) and CMT2 according to the phenotype and genotype. Although molecular pathologies for each genetic causative have not been revealed in CMT2, the correlation between cell death and accumulation of misfolded proteins in the endoplasmic reticulum (ER) of Schwann cells is well documented in CMT1. Establishment of in vitro models of ER stress-mediated Schwann cell death might be useful in developing drug-screening systems for the treatment of CMT1. Materials and Methods: To develop high-throughput screening (HTS) systems for CMT1, we generated cell models using transient expression of mutant proteins and chemical induction. Results: Overexpression of wild type and mutant peripheral myelin protein 22 (PMP22) induced ER stress. Similar results were obtained from mutant myelin protein zero (MPZ) proteins. Protein localization revealed that expressed mutant PMP22 and MPZ proteins accumulated in the ER of Schwann cells. Overexpression of wild type and L16P mutant PMP22 also reduced cell viability, implying protein accumulation-mediated ER stress causes cell death. To develop more stable screening systems, we mimicked the ER stress-mediated cell death in Schwann cells using ER stress inducing chemicals. Thapsigargin treatment caused cell death via ER stress in a dose dependent manner, which was measured by expression of ER stress markers. Conclusion: We have developed genetically and chemically induced ER stress models using Schwann cells. Application of these models to HTS systems might facilitate the elucidation of molecular pathology and development of therapeutic options for CMT1.

• Journal of Life Science
• /
• v.31 no.4
• /
• pp.430-441
• /
• 2021

Sesquiterpene lactones (STLs) are terpenoids found mostly in the Asteraceae family and are known for their strong cytotoxic properties, among other notable bioactivities. Some STLs, such as artemisinin and mipsagargin, are already commercially available and are used to fight malaria and tumor growth, respectively. Although the interest in STLs was low for a time after their discovery due to their toxic nature, past decades have witnessed a soar in STL-based studies focused on developing novel pharmaceuticals via chemical diversification. These studies have reported several promising physiological effects for STLs, including lower toxicity and diverse modes of action, and have demonstrated the antimicrobial, antioxidant, hepatoprotective, antiviral, antiprotozoal, phytotoxic, antitumor, and antiaging properties of STLs. STLs are mainly considered as valuable natural molecules for the fight against cancer since most STLs induce death of different types of cancer cells, as shown by in vitro and in vivo studies. Some STLs can also enhance the effects of drugs that are already in clinical use. Medicinal chemists use various STLs as starting molecules for the synthesis of new STLs or different bioactive compounds. All these developments warrant future research to provide more information on STLs, their bioactivities, and their mode of action. In this context, this review has summarized the bioactivities of some of the widely studied STLs, namely artemisinin, costunolide, thapsigargin, arglabin, parthenolide, alantolactone, cynaropicrin, helenalin, and santonin.

• Journal of Life Science
• /
• v.31 no.7
• /
• pp.662-670
• /
• 2021

Interstitial cells of Cajal (ICCs) are the pacemaking cells in the gastrointestinal (GI) muscles that generate the rhythmic oscillation in membrane potentials known as slow waves. In the present study, we investigated the effects of mirtazapine, a noradrenergic and serotonergic antidepressant, on pacemaking potential in cultured ICCs from the murine small intestine. The whole-cell patch-clamp configuration was used to record pacemaker potential in cultured ICCs. Mirtazapine induced pacemaker potential depolarizations in a concentration-dependent manner in the current clamp mode. Y25130 (a 5-HT3 receptor antagonist), RS39604 (a 5-HT4 receptor antagonist), and SB269970 (a 5-HT7 receptor antagonist) had no effects on mirtazapine-induced pacemaker potential depolarizations. Also, methoctramine, a muscarinic M₂ receptor antagonist, had no effect on mirtazapine-induced pacemaker potential depolarizations, whereas 4-diphenylacetoxy-N-methyl-piperidine methiodide (4-DAMP), a muscarinic M₃ receptor antagonist, inhibited the depolarizations. When guanosine 5'-[β-thio] diphosphate (GDP-β-S; 1 mM) was in the pipette solution, mirtazapine-induced pacemaker potential depolarization was blocked. When an external Ca²⁺ free solution or thapsigargin, a Ca²⁺-ATPase inhibitor of the endoplasmic reticulum, was applied, the generation of pacemaker potentials disappeared, and under these conditions, mirtazapine induced pacemaker potential depolarizations. In addition, protein kinase C (PKC) inhibitor, calphostin C, and chelerythrine inhibited mirtazapine-induced pacemaker potential depolarizations. These results suggest that mirtazapine regulates pacemaker potentials through muscarinic M₃ receptor activation via a G protein-dependent and an external or internal Ca²⁺-independent PKC pathway in the ICCs. Therefore, mirtazapine can control GI motility through ICCs.

• International Journal of Oral Biology
• /
• v.35 no.3
• /
• pp.129-135
• /
• 2010

Recent studies have implicated reactive oxygen species (ROS) as determinants of the pathological pain caused by the activation of peripheral neurons. It has not been elucidated, however, how ROS activate the primary sensory neurons in the pain pathway. In this study, calcium imaging was performed to investigate the effects of NaOCl, a ROS donor, on the intracellular calcium concentration ($[Ca^{2+}]i$) in acutely dissociated dorsal root ganglion (DRG) neurons. DRG was sequentially treated with 0.2 mg/ml of both protease and thermolysin, and single neurons were then obtained by mechanical dissociation. The administration of NaOCl then caused a reversible increase in the $[Ca^{2+}]i$, which was inhibited by pretreatment with phenyl-N-tertbuthylnitrone (PBN) and isoascorbate, both ROS scavengers. The NaOCl-induced $[Ca^{2+}]i$ increase was suppressed both in a calcium free solution and after depletion of the intracellular $Ca^{2+}$ pool by thapsigargin. Additionally, this increase was predominantly blocked by pretreatment with the transient receptor potential (TRP) antagonists, ruthenium red ($50\;{\mu}M$) and capsazepine ($10\;{\mu}M$). Collectively, these results suggest that an increase in the intracellular calcium concentration is produced from both extracellular fluid and the intracellular calcium store, and that TRP might be involved in the sensation of pain induced by ROS.

• BMB Reports
• /
• v.45 no.8
• /
• pp.482-487
• /
• 2012

To identify the novel inhibitors of endoplasmic reticulum stress-induced cell death, we performed a high throughput assay with a chemical library containing a total of 3,280 bioactive small molecules. Cyclosporine A and bromocriptine were identified as potent inhibitors of thapsigargiin-induced cell death (cut-off at $4{\sigma}$ standard score). However, U74389G, the potent inhibitor of lipid peroxidation had lower activity in inhibiting cell death. The inhibition effect of cyclosporine A and bromocriptine was specific for only thapsigargin-induced cell death. The mechanism of inhibition by these compounds was identified as modification of the expression of glucose regulated protein-78 (GRP-78/Bip) and inhibition of phosphorylation of p38 mitogen activated protein kinase (MAPK). However, these compounds did not inhibit the same events triggered by tunicamycin, which was in agreement with the cell survival data. We suggest that the induction of protective unfolded protein response by these compounds confers resistance to cell death. In summary, we identified compounds that may provide insights on cell death mechanisms stimulated by ER stress.

• Journal of Physiology & Pathology in Korean Medicine
• /
• v.27 no.1
• /
• pp.112-117
• /
• 2013

We studied the modulation of pacemaker activities by Samchulkunbi-tang (SCKB) in cultured interstitial cells of Cajal (ICC) from murine small intestine with the whole-cell patch-clamp technique. Externally applied SCKB produced membrane depolarization in the current-clamp mode. The pretreatment with $Ca^{2+}$-free solution and thapsigargin, a $Ca^{2+}$-ATPase inhibitor in endoplasmic reticulum, abolished the generation of pacemaker potentials and suppressed the SCKB-induced action. The application of flufenamic acid (a nonselective cation channel blocker) abolished the generation of pacemaker potentials by SCKB. However, the application of niflumic acid (a chloride channel blocker) did not inhibit the generation of pacemaker potentials by SCKB. In addition, the membrane depolarizations were inhibited by not only GDP-${\beta}$-S, which permanently binds G-binding proteins, but also U-73122, an active phospholipase C inhibitor. These results suggest that SCKB modulates the pacemaker activities by nonselective cation channels and external $Ca^{2+}$ influx and internal $Ca^{2+}$ release via G-protein and phospholipase C-dependent mechanism. Therefore, the ICC are targets for SCKB and their interaction can affect intestinal motility.

• Journal of Food Hygiene and Safety
• /
• v.19 no.3
• /
• pp.161-166
• /
• 2004

We investigated the antiplatelet effect of a newly synthesized guanidine derivative KR-32558, a sodium-hydrogen exchanger-1 (NHE-1) inhibitor, together with the elucidation of the possible mechanisms of action. KR-32558 concentration -dependently inhibited the aggregation of washed rabbit platelets induced by collagen (10 ${\mu}g/ml$) with an $IC_{50}$ value of 85.9 ${\mu}M$, but with much weaker potency against aggregation induced by thapsigargin (0.5 ${\mu}M$) or A23187 (5 ${\mu}M$). And had no effect on platelet aggregation induced by arachidonic acid (100 ${\mu}M$), thrombin (0.05 U/ml) and U46619 (1 ${\mu}M$) up to 100 ${\mu}M$. KR-32558 completely inhibited the $[Ca^{2+}]_i$ mobilization induced by collagen at concentration of 100${\mu}iM$. Taken together, these observation suggest that KR-32558 selectively inhibited collagen-mediated platelet aggregation by blocking the cytoplasmic calcium mobilization in addition to NHE-1 inhibition.