효모에서 SHC1 유전자의 이온 농도 조절에 의한 세포내 pH 항상성 유지

하승길,전준철,최의열 한국미생물학회 2002 미생물학회지 Vol.38 No.3

출아효모는 주변 환경 pH의 커다란 변화 속에서 적응할 수 있는 효과적인 체계를 지니고 있으며 SHC1 유전자는 알칼리 pH 조건에서 세포의 성장에 필요한 유전자 중에 하나임을 확인하였다. SHC1 유전자의 세포내 pH 조절 기작을 보다 구체적으로 알아보기 위하여 이 유전자가 소실된 돌연변이주를 제조하였다. 성장률의 차이가 나타나는 원인을 세포 내부의 pH 완충능력 결여에 의한 것으로 추측하고 pH 감수성 형광물질인 C.SNARE를 사용하여 외부 pH의 변화에 따른 세포 내부의 pH를 측정하였다. 알칼리 pH 완충효과는 소실 돌연변이의 경우는 야생종 대비 70% 수준을 보였다. 또한 pH 조절에 관여하는 효모세포 내부의 $Na^{+}$과 $K^{+}$의 농도를 원자흡광계를 사용하여 조사한 바, $K^{+}$ 이온의 경우에는 돌연변이주에 비하여 야생형 세포내에 더 많이 존재하는 것으로 나타났으나 $Na^{+}$ 이온의 경우는 별다른 차이점을 보이지 않았다. 이러한 결과는 $K^{+}$ 이온의 조절이 효모에서 세포내 pH조절 기작에 중요하며 SHC1 유전자는 이 $K^{+}$ 이온의 세포내 농도 유지에 관여하고 있다는 것을 제시해 주었다. Budding yeasts maintain an effective system to regulate intracellular pH in response to environmental pH fluctuation. In a previous study we reported that SHC1 plays a role in cell growth at alkaline condition, not at acid pH. We constructed a null mutant deleted an entire open reading frame for SHC1. To test whether the retardation in cell growth was caused by the absence of intracellular pH buffering capacity, we measured intracellular pH with a pH-sensitive fluorescent dye, C.SNARE. The intracellular pH of the mutant cell was much higher than that of wild-type cells, indicating that the mutant cells lack some types of buffering capacity. We also investigated the level of $Na^+ and K^+$ content with atomic mass spectroscopy after alkali shock. Wild-type cell showed a higher level of intracellular K^+$ content, whereas there was no difference in $Na^+$ level. The result suggested that K^+$ is more important in the regulation of intracellular pH in yeasts.

$Na^+/H^+$ exchanger와 $HCO_3^-$ transporter에 의한 흰쥐 타액선 선세포내 pH 조절

박동범,서정택,손흥규,이종갑,Park, Dong-Bum,Seo, Jeong-Taeg,Sohn, Heung-Kyu,Lee, Jong-Gap 대한소아치과학회 1998 大韓小兒齒科學會誌 Vol.25 No.2

Intracellular pH (pHi) plays an important role in the regulation of cellular processes by influencing the acitivity of various enzymes in cells. Therefore, almost every type of mammalian cell possesses an ability to regulate its pHi. One of the most prominent mechanisms in the regulation of pHi is $Na^+/H^+$ exchanger. This exchanger has been known to be activated when cells are stimulated by the binding of agonist to the muscarinic receptors. Therefore, the aims of this study were to compare the rates of $H^+$ extrusion through $Na^+/H^+$ exchanger before and during muscarinic stimulation and to investigate the possible existence of $HCO_3^-$ transporter which is responsible for the continuous supply of $HCO_3^-$ ion to saliva. Acinar cells were isolated from the rat mandibular salivary glands and loaded with pH-sensitive fluoroprobe, 2', 7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein(BCECF), for 30min at room temperature. Cells were attached onto the coverglass in the perfusion chamber and the changes in pHi were measured on the iverted microscope using spectrofluorometer. 1. By switching the perfusate from $HCO_3^-$-free to $HCO_3^-$-buffered solution, pHi decreased by $0.39{\pm}0.02$ pH units followed by a slow increase at an initial rate of $0.04{\pm}0.007$ pH units/min. The rate of pHi increase was reduced to $0.01{\pm}0.002$ pH units/min by the simultaneous addition of 1 mM amiloride and $100{\mu}M$ DIDS. 2. An addition and removal of $NH_4^+$ caused a decrease in pHi which was followed by an increase in pHi. The increase of pHi was almost completely blocked by 1mM amiloride in $HCO_3^-$-free perfusate which implied that the pHi increase was entired dependent on the activation of $Na^+/H^+$ exchanger in $HCO_3^-$-free condition. 3. An addition of $10{\mu}M$ carbachol increased the initial rate of pHi recovery from $0.16{\pm}0.01$ pH units/min to $0.28{\pm}0.03pH$ units/min. 4. The initial rate of pHi decrease induced by 1mM amiloride was also increased by the exposure of the acinar cells to $10{\mu}M$ carbachol ($0.06{\pm}0.008pH$ unit/min) compared with that obtained before carbachol stimulation ($0.03{\pm}0.004pH$ unit/min). 5. The intracellular buffering capacity ${\beta}1$ was $14.31{\pm}1.82$ at pHi 7.2-7.4 and ${\beta}1$ increased as pHi decreased. 6. The rate of $H^+$ extrusion through $Na^+/H^+$ exchanger was greatly enhanced by the stimulation of the cells with $10{\mu}M$ carbachol and there was an alkaline shift in the activity of the exchanger. 7. An intrusion mechanism of $HCO_3^-$ was identified in rat mandibular salivary acinar cells. Taken all together, I observed 3-fold increase in $Na^+/H^+$ exchanger by the stimulation of the acinar cells with $10{\mu}M$ carbachol at pH 7.25. In addition, I have found an additional mechanism for the regulation of pHi which transported $HCO_3^-$ into the cells.

Na+/H+ exchanger와 HCO-₃Transporter에 의한 흰쥐 타액선 선세포내 pH 조절

서정택,박동범,손흥규,이종갑 大韓小兒齒科學會 1998 大韓小兒齒科學會誌 Vol.25 No.2

Intracellular pH (pHi) plays an important role in the regulation of cellular processes by influencing the activity of various enzymes in cells. Therefore, almost every type of mammalian cell possesses an ability to regulate its pHi. One of the most prominent mechanisms in the regulation of pHi is Na+/H+ exchanger. This exchanger has been known to be activated when cells are stimulated by the binding of agonist to the muscarinic receptors. Therefore, the aims of this study were to compare the rates of H+ extrusion through Na+/H+ exchanger before and during muscarinic stimulation and to investigate the possible existence of HCO-₃ transporter which is responsible for the continuous supply of HCO-₃ ion to saliva. Acinar cells were isolated from the rat mandibular salivary glands and loaded with pH-sensitive fluoroprobe, 2', 7' -bis(2-carboxyethyl)-5(6)-carboxyfluorescein(BCECF), for 30min at room temperature. Cells were attached onto the coverglass in the perfusion chamber and the changes in pHi were measured on the iverted microscope using spectrofluormeter. 1. By switching the perfusate from HCO-₃-free to HCO-₃-buffered solution, pHi decreased by 0.39±0.02 pH units followed by a slow increase at an initial rate of 0.04±0.007 pH units/min. the rate of pHi increase was reduced to 0.01±0.002 pH units/min by the simultaneous addition of 1 mM amilorede and 100μM DIDS. 2. An addition and removal of NH+₄ caused a decrease in pHi which was followed by an increase in pHi. The increase of pHi was almost completely blocked by 1mM amiloride in HCO-₃-free perfusate which implied that the pHi increase was entired dependent on the activation of Na+/H+ exchanger in HCO-₃=free condition. 3. An addition of 10μM carbachol increased the initial rate of pHi recovery from 0.16±0.01 pH units/min to 0.28±0.03pH units/min. 4. The initial rate of pHi decrease induced by 1mM amilorede was also increased by the exposure of the acinar cells 10μM carbachol(0.06±0.008pH unit/min) compared with that obtained before carbachol sitmulation (0.03±0.004pH unit/min). 5. The intracellular buffering capacity β1 was 14.31±1.82 at pHi 7.2-7.4 and β1 increased as pHi decreased. 6. The rate of H+ extrusion through Na+/H+ exchanger was greatly enhanced by the stimulation of the cells with 10μM carbachol and there was an alkaline shift in the activity of the exchanger. 7. An intrusion mechanism of HCO-₃ was identified in rat mandibular salivary acinar cells. Taken all together, I observed 3-ford increased in Na+/H+ exchanger by the stimulation of the acinar cells with 10μM carbachol at pH 7.25. In addition, I have found an additional mechanism for the regulation of pHi which transported HCO-₃ into the cells.

Acidic pH-activated $Cl^-$ Current and Intracellular $Ca^{2+}$ Response in Human Keratinocytes

Park, Su-Jung,Choi, Won-Woo,Kwon, Oh-Sang,Chung, Jin-Ho,Eun, Hee-Chul,Earm, Young-E,Kim, Sung-Joon The Korean Society of Pharmacology 2008 The Korean Journal of Physiology & Pharmacology Vol.12 No.4

The layers of keratinocytes form an acid mantle on the surface of the skin. Herein, we investigated the effects of acidic pH on the membrane current and $[Ca^{2+}]_c$ of human primary keratinocytes from foreskins and human keratinocyte cell line (HaCaT). Acidic extracellular pH ($pH_e{\leq}5.5$) activated outwardly rectifying $Cl^-$ current ($I_{Cl,pH}$) with slow kinetics of voltage-dependent activation. $I_{Cl,pH}$ was potently inhibited by an anion channel blocker 4,4'-diisothiocyanostilbene-2,2'-disulphonic acid (DIDS, 73.5% inhibition at 1${\mu}$M). $I_{Cl,pH}$ became more sensitive to $pH_e$ by raising temperature from $24^{circ}C$ to $37^{circ}C$. HaCaT cells also expressed $Ca^{2+}$-activated $Cl^-$ current ($I_{Cl,Ca}$), and the amplitude of $I_{Cl,Ca}$ was increased by relatively weak acidic $pH_e$ (7.0 and 6.8). Interestingly, the acidic $pH_e$ (5.0) also induced a sharp increase in the intracellular [$Ca^{2+}$] (${\triangle}[Ca^{2+}]_{acid}$) of HaCaT cells. The ${\triangle}[Ca^{2+}]_{acid}$ was independent of extracellular $Ca^{2+}$, and was abolished by the pretreatment with PLC inhibitor, U73122. In primary human keratinocytes, 5 out of 28 tested cells showed ${\triangle}[Ca^{2+}]_{acid}$. In summary, we found $I_{Cl,pH}$ and ${\triangle}[Ca^{2+}]_{acid}$ in human keratinocytes, and these ionic signals might have implication in pathophysiological responses and differentiation of epidermal keratinocytes.

Acidic pH-activated Cl<SUP>−</SUP> Current and Intracellular Ca<SUP>2+</SUP> Response in Human Keratinocytes

Su Jung Park,Won Woo Choi,Oh Sang Kwon,Jin Ho Chung,Hee Chul Eun,Yung E Earm,Sung Joon Kim 대한생리학회-대한약리학회 2008 The Korean Journal of Physiology & Pharmacology Vol.12 No.4

The layers of keratinocytes form an acid mantle on the surface of the skin. Herein, we investigated the effects of acidic pH on the membrane current and [Ca²⁺]_c of human primary keratinocytes from foreskins and human keratinocyte cell line (HaCaT). Acidic extracellular pH (pH_e≤5.5) activated outwardly rectifying Cl⁻ current (I_Cl,pH) with slow kinetics of voltage-dependent activation. I_Cl,pHwas potently inhibited by an anion channel blocker 4,4`-diisothiocyanostilbene-2,2`-disulphonic acid (DIDS, 73.5% inhibition at 1ՌM). I_Cl,pHbecame more sensitive to pH_e by raising temperature from 24^oC to 37^oC. HaCaT cells also expressed Ca²⁺-activated Cl⁻ current (I_Cl,Ca), and the amplitude of I_Cl,Ca was increased by relatively weak acidic pH_e (7.0 and 6.8). Interestingly, the acidic pH_e (5.0) also induced a sharp increase in the intracellular [Ca²⁺] (⁘[Ca²⁺]_acid) of HaCaT cells. The ⁘[Ca²⁺]_acid was independent of extracellular Ca²⁺, and was abolished by the pretreatment with PLC inhibitor, U73122. In primary human keratinocytes, 5 out of 28 tested cells showed ⁘[Ca²⁺]_acid. In summary, we found I_Cl,pH and ⁘[Ca²⁺]_acid in human keratinocytes, and these ionic signals might have implication in pathophysiological responses and differentiation of epidermal keratinocytes.

기니픽 유두근에서 α₁-adrenoceptor 자극에 의한 세포내 pH와 Na⁺ 증가는 Na⁺-H⁺ 교환기를 경유

김진상,Kim, Jin-sang 대한수의학회 1995 大韓獸醫學會誌 Vol.35 No.2

The effect of ${\alpha}_1$-adrenoceptor(${\alpha}_1$-AR) stimulation on intracellular pH($pH_i$), $Na^+$ activity($a_{Na}{^i}$) and contractility were investigated in isolated papillary muscles of euthyroid or hyperthyroid guinea pig with conventional microelectrode, $Na^+$ or $H^+$-selective microelectrodes, and tension transducer. Stimulation of the ${\alpha}_1$-AR by phenylephrine produced a decrease in $a_{Na}{^i}$ in euthyroid preparations. This decrease in $a_{Na}{^i}$ was abolished in presence of PKC activator, phorbol dibutyrate, and increased contrary to decrease. Phenylephrine also increased $a_{Na}{^i}$ in hyperthyroid ones. However, phenylrephtine produced an increase in $pH_i$ in both euthyroid and hyperthyroid ones. These changes were blocked by prazosin, an antagonist of ${\alpha}_1$-AR. These findings suggest that the changes in $a_{Na}{^i}$ and $pH_i$ are mediated by a stimulation of $Na^+-H^+$ exchange via ${\alpha}_1$-AR stimulation. This study focused on the increase in $a_{Na}{^i}$, $pH_i$ and contractility. The increase in $pH_i$ was blocked by amiloride or EIPA, $Na^+-H^+$ exchange inhibitors. Therefore, the increase in $a_{Na}{^i}$ and $pH_i$ mediated by ${\alpha}_1$-AR appeared to be due to an influx of $Na^+$ and a reduction of $H^+$ through $Na^+-H^+$ exchange. This study also revealed that the increase in $pH_i$ and $a_{Na}{^i}$ might be related to the sustained positive inotropic response. The $a_{Na}{^i}$ increase may contribute to the intracellular $Ca^{2+}$ through the $Na^+-Ca^{2+}$ exchange, and the $pH_i$ increase could cause an increase in the $Ca^{2+}$ sensitivity of myofilaments and may augment the ${\alpha}_1$-AR-mediated positive inotropic response.

pH-modulated Ion Channels in Acinar Cells

Lee, Jong-Heun,Park, Kyungpyo,Oh, Seog-Bae,Kim, Joong-Soo Korean Academy of Oral Biology and the UCLA Dental 1997 International Journal of Oral Biology Vol.22 No.1

The mechanism for the H^+ interaction with Ca^(2+)-activated channels in acinar cells is not clear. In the present work, the effects of intracellular pH(pH_i) on Ca^(2+)-activated Cl^- and K^+ currents in rat submaxillary and lacrimal acinar cells were examined, respectively. Cl^- currents were recorded by conventional whole cell patch-clamp methods using micropipettes filled with NMDG-Cl (K^(+)-free solution) in NMDG-Cl(Na^(+)-free) bath solutions. Intracellular pH was varied by using electrode solutions with pH at 6.8, 7.3 and 7.8 At pH 6.8, there was no obvious time-dependent activation or inactivation. At pH 7.3, there also was no time-dependent activation or inactivation, except at the large depolarizing potential at 80 and 100 mV. However, at pH 7.8, large inward currents inactivated with time at hyperpolarizing potentials and the time-dependent outward currents at depolarizing potentials were observed. At this pH, I-V relationship displayed outward rectification, which is a result of the time-dependent activation of currents at depolarizing potentials. Increasing pH from 6.8 to 7.8 was also found to increase the open probability(Po) of K^+ channel in inside-out mode. Our results suggest that an increase in pH_i help sustain both Cl^- and K^+ channel activities during prolonged secretory activities.

Acidic pH-activated Cl- Current and Intracellular Ca²+ Response in Human Keratinocytes

박수정,최원우,권오상,정진호,은희철,Yung E Earm,김성준 대한약리학회 2008 The Korean Journal of Physiology & Pharmacology Vol.12 No.4

The layers of keratinocytes form an acid mantle on the surface of the skin. Herein, we investigated the effects of acidic pH on the membrane current and [Ca²+]c of human primary keratinocytes from foreskins and human keratinocyte cell line (HaCaT). Acidic extracellular pH (pHe≤5.5) activated outwardly rectifying Cl− current (ICl,pH) with slow kinetics of voltage-dependent activation. ICl,pH was potently inhibited by an anion channel blocker 4,4`-diisothiocyanostilbene-2,2`-disulphonic acid (DIDS, 73.5% inhibition at 1μM). ICl,pH became more sensitive to pHe by raising temperature from 24℃ to 37℃. HaCaT cells also expressed Ca²+-activated Cl− current (ICl,Ca), and the amplitude of ICl,Ca was increased by relatively weak acidic pHe (7.0 and 6.8). Interestingly, the acidic pHe (5.0) also induced a sharp increase in the intracellular [Ca²+] (△[Ca²+]acid) of HaCaT cells. The △[Ca²+]acid was independent of extracellular Ca²+, and was abolished by the pretreatment with PLC inhibitor, U73122. In primary human keratinocytes, 5 out of 28 tested cells showed △[Ca²+]acid. In summary, we found ICl,pH and △[Ca²+]acid in human keratinocytes, and these ionic signals might have implication in pathophysiological responses and differentiation of epidermal keratinocytes.

유체 압력에 의한 흰쥐 심실근세포 pH의 감소 및 Na+ -H+ 교환체의 활성화

김준철(Joon-Chul Kim),우선희(Sun-Hee Woo) 대한약학회 2011 약학회지 Vol.55 No.3

An increase in ventricular pressure can alter cardiac excitation and contraction. Recent report has demonstrated that fluid pressure (FP) suppresses L-type Ca2+ current with acceleration of the current inactivation in ventricular myocytes. Since the L-type Ca2+ channels known to be regulated by intracellular pH (pHi), this study was designed to explore whether pressurized fluid flow affects pHi in isolated rat ventricular myocytes. A flow of pressurized (~16 dyne/cm2) fluid, identical to that bathing the myocytes, was applied onto single myocytes, and intracellular H+ concentration was monitored using confocal H+ imaging. FP significantly decreased pHi by 0.07±0.01 pH units (n=16, P<0.01). Intracellular acidosis enhances the activity of Na+-H+ exchanger (NHE). Therefore, we examined if the NHE activity is increased by FP using the NHE inhibitor, HOE642. Although HOE642 did not alter pHi in control conditions, it decreased pHi in cells pre-exposed to FP, suggesting enhancement of NHE activity by FP. In addition, FP-induced intracellular acidosis was larger in cells pre-treated with HOE642 than in cells under the control conditions. These results suggest that FP induces intracellular acidosis and that NHE may contribute to extrude H+ during the FP-induced acidosis in rat ventricular myocytes.

Block Copolymer-Templated Mineralization for pH-Responsive Robust Nanocarriers of 5-Fluorouracil

최기현,이상천,민경현 한국고분자학회 2014 Macromolecular Research Vol.22 No.3

We describe a novel synthetic method to prepare calcium carbonate (CaCO3)-mineralized nanoparticlesfor the intracellular delivery of an anticancer drug, 5-fluorouracil (5-FU). The 5-FU-loaded mineralized nanoparticles(FU-CaCO3-MNPs) were prepared by block copolymer (poly(ethylene glycol)-b-poly(L-aspartic acid) (PEG-PAsp))-templated mineralization in the presence of calcium cations (Ca2+), carbonate anions (CO32-), and negatively charged5-FU. The mineralization produced hybrid nanoparticles consisting of 5-FU-loaded CaCO3 cores and PEG shells. The mineralized CaCO3 cores of the nanoparticles could stably hold loaded 5-FU by maintaining the structuralrobustness, and the outer corona of PEG provided colloidal stability to the nanoparticles in serum solutions. The 5-FUrelease from the FU-CaCO3-MNPs at physiological pH (pH 7.4) was efficiently inhibited, whereas at an endosomalpH (pH 5.0), the 5-FU release was facilitated. Fluorescence microscopic analysis showed that FU-CaCO3-MNPs weretaken up by the MCF-7 breast cancer cells, and transferred into acidic endosomes to dissolve the CaCO3 core. Finally, the5-FU release was facilitated to inhibit the proliferation of cancer cells. These mineralized hybrid nanoparticles mayserve as a useful candidate for enhanced intracellular delivery of many ionic anticancer drugs.