• The Korean Journal of Physiology
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• 제29권2호
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• pp.269-277
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• 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.

• 한국임상수의학회지
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• 제7권2호
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• pp.517-519
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• 1990

소 난소로부터 난자를 채취하여 체외성숙과 체외수정을 실시하였다. 난의 성숙배양을 위한 배양액은 소 태아혈청을 첨가한 TCM 199으로 $CO_2$배양기에서 24시간 배양하였다. 체외수정에는 동결융해정액을 사용하였으며 Caffeine과 Ca Ionophore 처리후 매정에 공하였다. 배양난자중 40%가 Metaphase II까지 성숙하였다. 성숙율에 대한 소 태아혈청의 농도변화와 HEPES 첨가여부의 효과는 없었다. 체외수정율과 분할율의 성적은 각각 23.1%와 14.4%였다.

• Journal of Plant Biology
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• 제37권3호
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• pp.271-276
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• 1994

The possibility that 1-aminocyclopropane-1-carboxylic acid (ACC)-uptake may be dependent on the H+-gradient established across the plsma membrane was tested in protoplasts isolated from 2.5 day old mungbean hypocotyls. The ACC-induced ethylene production was inhibited when the H+-gradient was collapsed by the treatment with carbonycyamide-p-trifluro-methoxy-phenylhydrazone (FCCP). Moreover, the treatment with o-vanadate, a specific inhibitor of plasma membrane H+-ATPase, caused the inhibition of ethylene production. The ACC-induced ethylene production was inhibited by the treatemnt with verapamil (Ca2+-channel blocker), or ethylene glycol-bis($\beta$-aminoethyl ether) N, N, N', N'-tetraacetic acid (EGTA) (Ca2+-chelator). In contrast, the ehtylene production was stimulated by the application of A23187 (Ca2+ ionophore). The inhibitory effect of EGTA in the ethylene producton was magnified in the presence of A23187. From these results, we suggest that the external Ca2+ influx to the cytosol resulted in the stimulatin of ACC oxidase activity after ACC-uptake resulting from a H+-gradient across the plasma membrane.

• Molecular & Cellular Toxicology
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• 제4권1호
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• pp.16-21
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• 2008

Cell death of trophoblast, particularly by abnormal release of physiological nitric oxide (NO) has been known to be a causative factor of pre-eclampsia. In the present study, effects of intracellular calcium increase enhancing the activity of NO synthases (neuronal NO synthase, nNOS in this trophoblast cells) on the cell death were examined in a human placental full-term cell line (HT-1). Furthermore, we analyzed the possible mechanisms underlying the augmentation of $Ca^{++}$-mediated NOS activity mediated by protein kinases like PKC, PKA, or CaM-KII. In experiments for cell toxicity, a calcium ionophore (ionomycin $10{\mu}M$) enhanced cell death confirmed by MTT assay, and increased significantly nNOS activity determined with a hemoglobin oxidation assay. This cell death was partially protected by pre-treatment of 7-nitroindazole (7-NI, $10{\mu}M$ and $100{\mu}M$), a nNOS-specific inhibitor. Additionally, $Ca^{++}$-ionophore -induced increase of nNOS activity also was partially normalized by pre-treatment of specific inhibitors of protein kinases, PKC, PKA or CaM-KII. Therefore, we suggest that an increase of calcium influx, leading to the activation of nNOS activity, which in turn may result in the death of trophoblast cells by involvement of signaling mechanisms of protein kinases.

• 한국동물학회지
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• 제38권4호
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• pp.514-522
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• 1995

생쥐 정자의 수정능력 획득과정 및 첨체반응을 전후로 정자표면과 albumin의 상호작용을 조사하였다. 부정소 정자를 체외에서 배양하는 과정에서 정자표면에서 이탈된 단백질의 분석과 함께 FITC-bovine serum albumin으로 정자를 형광염색하여 정자표면에 대한 albumin의 결합양상의 변화를 조사하였다. 90분간 정자를 배양한 수 정자를 제거한 후 농축한 배양액내에 정자 또는 부정소액에서 기원한 여러종의 단백질가 함께 albumin이 다량으로 발견되었다 정자의 체외배양 과정에서 일어나는 albumin의 이탈은 배양액내의 $Ca^2$+과 무관하게 일어났다. BSA-FITC는 정소내 정자의 두부표면에 미약하게 결합한 반면 미부부정소 정자의 첨체표면에는 다량 결합하였다. $Ca^2$+-ionophore인 A23187으로 첨체반응을 유발한 정자의 두부 표면에서는 후첨체부위만이 강하게 염색되었다. 이러한 결과는 정소 및 웅성 생식수관니에서 정자표면에 부착된 albumin이 자성 생식수관을 거치는 동안 이탈됨을 시사하며 이러한 현상은 정자의 수정능력획득과 밀접한 관련이 있는 것으로 사료된다.

• Journal of Ginseng Research
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• 제20권2호
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• pp.168-172
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• 1996

Ginsenoside Rb,, at a concentration of 10 $\mu\textrm{g}$/ml and over, initiated the cycle of oscillation of ion flux in erythrocytes after the cells had been treated with a protonophore, carbonyl cyanide p-trifluoro-methoxyphenyl hydrazone (FCCP) and then with a $Ca^{2+}$ ionophore, A23,3,. Its action was similar to the additional portion of $Ca^{2+}$-ionophore or $Ca^{2+}$ ion to the erythrocytes. Effects of $Rg_1$ and Rf were different from that of Rb,. They did not induce the oscillation. They, however, increased the extracellular $K^{+}$ concentration and pH without returning to the initial state in the erythrocytes processed with FCCP and $A_{23187}$. We established that ginsenosides from 20-(5)-panaxatriol family induced the membrane hyperpolarization in erythrocytes, which was attenuated by the pretreatment of $Rb_1$, a major component of 20-(5)-panaxadiol.

• Bulletin of the Korean Chemical Society
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• 제20권5호
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• pp.556-558
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• 1999

Lead-selective PVC membrane electrodes based on newly synthesized bis(crown ether)s containing 18-crown-6 moiety was prepared using standard PVC membrane composition. In order to monitor lead in environmental samples by lead sensor, especially good selectivity over alkali and alkaline earth metals has to be obtained. Thus, responses of the PVC membrane prepared with new bis(crown ether)s and Fluka ionophore V (crown ether) base to various cations include lead, alkali and alkaline earth metal ions were investigated for their use as a lead sensor. The polymeric liquid membrane based on trans-1 8-crown-6 ether exhibits the best overall potentiometric performances as a lead-selective electrode in terms of a wide linear dynamic range (between 10-6 and 10-2 M, Pb2+), excellent detection limit (less than 10-6 M) and good durability within limited error. The preferences of lead over other cations, such as Ag+, Hg2+, Na+, Ca2+ and even K+ in the aforementioned electrode are much better than the Fluka ionophore V system.

• 한국가축번식학회지
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• 제24권3호
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• pp.281-288
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• 2000

활성화처리를 위해 22시간 성숙된 난자를 5 $\mu$M ionomycin(I)에서 5분, 10 $\mu$M calcium ionophore(Ca)에서 5분, 2 mM 6-dimethylaminopurine(DMAP)에서 3시간 및 10 $\mu\textrm{g}$/$m\ell$ cycloheximide(CH)에서 6시간 동안 단용 또는 병용 처리하였다. 활성화 처리된 난자는 mCR$_1$aa 배양액 내에서 배양되었으며, 배양조건은 5% $CO_2$, 95% air 또는 5% $CO_2$, 5% $O_2$, 90% $N_2$ 이었다. 1 I, Ca DMAP 및 CH에 의해 처리된 난자의 48시간 난할율은 각각, 12.7%, 14.1%, 28.9% 및 22.9%였다. 그러나, 배반포는 형성되지 않았다. 2. I＋DMAP, I＋CH, Ca＋DMAP및 Ca＋CH에 의해 처리된 난자의 48시간 난할율은 각각, 96.9%, 82.1%, 93.1% 및 84.7%였고, 배반포 발생율은 각각, 10.4%, 5.3%, 17.6% 및 7.1%로, I 및 Ca를 이용하여 세포 내 칼슘수준을 증가시킨 후, DMAP로 3시간 동안 배양하였을 때, 배반포 발생율이 유의적으로 높았다(P<0.05). 3. I, Ca, DMAP 및 CH에 의해 단용 처리된 난자의 전핵 발생율은 각각, 5.4%, 3.6%, 28.3% 및 28.8%였다. 그러나, 병용 처리된 난자는 100%의 전핵 형성율을 나타냈다.

• 한국동물학회지
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• 제34권4호
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• pp.460-468
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• 1991

계배 limb bud간충직 연골원성 세포로부터 연골원세포로의 분화과정에서 Ca2+의 역할을 추구해 보기 위하여 Hamburger-Hamilton stage 23/24의 계배 limb bud간충직세포들을 미세배양법으로 배양하면서, Ca2+, calcium ionophore인 A23187, calcium channel blocker인 D600을 각각 농도 및 처리기간을 변화시켜 처리하면서 연골화의 정도를 검정하여, 연골세포 분화에 미치는 Ca2+의 작용양상을 분석하였다. 그 결과 Ca2+은 3 mM의 농도로 배양초기에 처리하였을 때 가장 효과적으로 연골화를 촉진하였으며, A23187(0.05 $\mu$ M) 처리는 세포내로 Ca2+유입을 증가시켜 연골화를 촉진시킨 반면, D6OO(30 $\mu$ M이하) 처리는 세포내로 Ca2+ 유입을 차단시킴으로서 연골화를 억제하는 것으로 나타났다. 이러한 결과에 따른 세포내로의 칼슘 유입 변화는 45Ca로 확인하였다. 그러므로 Ca2+에 의한 간충직세포의 연골세포로의 분화촉진 작용은 Ca2+이 연골화의 응집시기에 세포간의 접촉을 유도할 뿐만 아니라 배양 초기에 Ca2+이 세포 내로 들어감으로써 수반되는 일련의 기작에 의한 것임을 알았다.

• Bulletin of the Korean Chemical Society
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• 제27권6호
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• pp.835-840
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• 2006

Dimethyl 1-(4-nitrobenzoyl)-8-oxo-2,8-dihydro-1H-pyrazolo[5,1-a]isoindole-2,3-dicarboxylate has been used as an ionophore and o-nitrophenyloctyl ether as a plasticizer in order to develop a poly(vinyl chloride)-based membrane electrode for calcium ion detection. The sensors exhibit significantly enhanced response towards calcium(II) ions over the concentration range $8.0{\times}10^{-7}\;1.0{\times}10^{-1}$ M at pH 3.0-11 with a lower detection limit of $5.0 {\times}10^{-7}$ M. The sensors display Nernstian slope of 29.5 ${\pm}$ 0.5 mV per decade for Ca(II) ions. Effects of plasticizers, lipophilic salts and various foreign common ions are tested. It has a fast response time within 10 s over the entire concentration range and can be used for at least 2 months without any divergence in potentials. The proposed electrode revealed good selectivity and response for $Ca^{2+}$ over a wide variety of other metal ions. The selectivity of the sensor is comparable with those reported for other such electrodes. The proposed sensor was successfully applied as an indicator electrode for the potentiometric titration of a Ca(II) solution, with EDTA.