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.

$Na^+/H^+$ 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.

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.

Altered Regulation of type 3 Na+/H+ exchanger, type 1 Na+/HCO3- cotransporter, and Na+, K+-ATPase in the Kidney of Rats with Experimental Rhabdomyolysis

( Seong Kwon Ma ),( Eun Hui Bae ),( Jong Un Lee ),( Sun Young Kim ),( Sung Zoo Kim ),( Ki Chul Choi ),( Soo Wan Kim ) 대한전해질학회 2007 Electrolytes & Blood Pressure Vol.5 No.2

Metabolic acidosis was shown to correlate with deterioration of renal function in patients with rhabdomyolysis. The present study was aimed to investigate whether the changes of type 3 Na+/H+ exchanger (NHE3), type 1 Na+/HCO3- cotransporter (NBC1), and Na+,K+-ATPase α1 subunit may play a role in the pathogenesis of metabolic acidosis in glycerol-induced experimental rhabdomyolysis. Male Sprague-Dawley rats were deprived of fluid intake for 24 hours, and then were injected with 50% glycerol in normal saline (10 mL/kg, intramuscularly). At 24 hours after the glycerol injection, rats were sacrificed by decapitation. Control rats were injected with normal saline. The protein expression of NHE3, NBC1 and Na+,K+-ATPase α1 subunit was determined in the cortex of the kidney by immunoblotting and immunohistochemistry. Following the treatment of glycerol, creatinine clearance was significantly decreased, and high anion gap metabolic acidosis developed. In the experimental group, the expression of Na+,K+-ATPase α1 subunit was significantly decreased in the cortex of the kidney. On the contrary, the expression of NHE3 and NBC1 was significantly increased. Immunohistochemical analyses confirmed the immunoblotting data. In conclusion, the coordinate up-regulation of NHE3 and NBC1 may play an adaptive role against the metabolic acidosis in glycerol-induced rhabdomyolysis.

Effects of ${\alpha}_1-Adrenergic$ Stimulation on Membrane Potential, Twitch Force, Intracellular $Na^+,\;and\;H^+$ Activity in Hyperthyroid Guinea Pig Ventricular Muscle

김진상,채수완,조규박,Kim Jin-Sang,Chae Soo-Wan,Cho Kyu-Park The Korean Society of Pharmacology 1995 대한약리학잡지 Vol.31 No.1

갑상선 기능 항진증 심장에서의 ${\beta}-adrenoceptor$의 역할은 잘 알려져 있으나 ${\alpha}-adrenoceptor$에 대해서는 알려져 있지 않은 바, 저자 등은 갑상선 기능 항진증 기니픽 심장의 유두근에서 일반 미세 전극과 이온-선택적 미세 전극을 이용하여 세포내 $Na^+$과 $H^+$ 활성도에 대한 phenylephrine의 영향을 연구하였다. Phenylephrine ($10^{-5}$ 또는 $3{\times}10^{-5}M$)에 의한 ${\alpha}_1-adrenoceptor$ 자극은 다양한 활동전위의 변동, 수축기 막전위의 과분극 ($1.5{\pm}0.1mM$), 세포내 활성도 증가 ($0.4{\pm}0.15mM$), 현저한 수축력 증가 ($220{\pm}15%$) 그리고 세포내 pH의 증가 ($0.06{\pm}0.002\;unit$)를 일으켰고, 이와 같은 변동이 prazosin과 atenolol에 의해 차단되었다. 그래서 이들 효과가 ${\alpha}_1-adrenoceptor$를 경유함을 알 수 있었고, 역시, 세포내 $Na^+$ 활성도와 수축력 증가 효과가 $Na^{+}-H^{+}$ 교환기 억제제인 ethylisopropylamiloride로 차단됨으로 보아 ${\alpha}_1-adrenoceptor$ 자극은 $Na^{+}-H^{+}$ 교환기를 자극하여 세포내 $a^{i}_{Na}$와 pH를 증가시킴을 시사한다. 이는 ${\alpha}_1-adrenoceptor$ 자극에 의한 세포내 $Na^+$감소와 초기 수축력 감소 효과를 나타내는 정상 기니픽 심장과는 매우 다른 결과로 갑상선 기능 항진증 심장에서 ${\alpha}_1-adrenoceptor$는 매우 중요한 기능을 갖고 있음을 의미한다. The roles of ${\beta}-adrenoceptor$ were well known in hyperthyroidal heart, but not with ${\alpha}-adrenoceptor$. So we studied the effects of phenylephrine on membrane potential, intracellular sodium activity ($a^{i}_{Na}$), twitch force, and intracellular pH ($pH_i$) by continuous intracellular recordings with ion-selective and conventional microelectrodes in the papillary muscles of hyperthyroid guinea pig heart. ${\alpha}_1-adrenoceptor$ stimulation by phenylephrine (10^{-5}\;or\;3{\times}10^{-5}M$) produced the following changes: variable changes in action potential duration, a hyperpolarization ($1.5{\pm}0.1mM$) of the diastolic membrane potential, an increase in $a^{i}_{Na}\;(0.4{\pm}0.15mM)$, a stronger positive inotropic effect ($220{\pm}15%$), an increase in $pH_i\;(0.06{\pm}0.002\;unit)$. These changes were flocked by prazosin and atenolol. This indicated that the changes in membrane potential, $a^{i}_{Na}$ twitch force, and $pH_i$ are mediated by a stimulation of the ${\alpha}_1-adrenoceptor$. Ethylisopropylamiloride ($10^{-5}$) also blocked the increase in $a^{i}_{Na}$ and twitch force. On the other hand, strophanthidin, tetrodotoxin, $Cs^+$ or verapamil did not block the increase in $a^{i}_{Na}$ and twitch force. Thus, it was suggested that ${\alpha}_1-adrenoceptor$ stimulation increased $a^{i}_{Na}\;and\;pH_i$ by stimulation of $Na^{+}-H^{+}$ exchange, thereby allowing intracellular alkalinization and $a^{i}_{Na}$ increase. These results were very different from euthyroidal heart which showed ${\alpha}_1-adrenoceptor$-induced decrease in $a^{i}_{Na}$ and initial negative inotropic effect. From the above results, it was concluded that ${\alpha}_1-adrenoceptor$ had a important role in hyperthy-roidal heart.

갑상선 기능 항진 기니픽 심근에서 α₁-Adrenergic 수용체 자극이 막전위, 수축력 및 세포내 Na⁺과 H⁺ 활성도에 미치는 영향

김진상(Jin-Sang Kim),채수완(Soo-Wan Chae),조규박(Kyu-Park Cho) 대한약리학회 1995 대한약리학잡지 Vol.31 No.1

갑상선 기능 항진증 심장에서의 β-adrenoceptor의 역할은 잘 알려져 있으나 α-adrenoceptor에 대해서는 알려져 있지 않은 바, 저자 등은 갑상선 기능 항진증 기니픽 심장의 유두근에서 일반 미세 전극과 이온-선택적 미세 전극을 이용하여 세포내 Na⁺과 H⁺ 활성도에 대한 phenylephrine의 영향을 연구하였다. Phenylephrine (10^-5 또는 3 × 10^-5M)에 의한 α₁-Adrenoceptor 자극은 다양한 활동전위의 변동, 수축기 막전위의 과분극 (1.5 ± 0.1mM), 세포내 활성도 증가 (0.4 ± 0.15mM), 현저한 수축력 증가 (220 ± 15%) 그리고 세포내 pH의 증가 (0.06 ± 0.002 unit)를 일으켰고, 이와 같은 변동이 prazosin과 atenolol에 의해 차단되었다. 그래서 이들 효과가 α₁-Adrenoceptor를 경유함을 알 수 있었고, 역시, 세포내 Na⁺ 활성도와 수축력 증가 효과가 Na⁺-H⁺ 교환기 억제제인 ethylisopropylamiloride로 차단됨으로 보아 α₁-Adrenoceptor 자극은 Na⁺-H⁺ 교환기를 자극하여 세포내 aⁱ_Na와 pH를 증가시킴을 시사한다. 이는 α₁-Adrenoceptor 자극에 의한 세포내 Na⁺감소와 초기 수축력 감소 효과를 나타내는 정상 기니픽 심장과는 매우 다른 결과로 갑상선 기능 항진증 심장에서 α₁-Adrenoceptor는 매우 중요한 기능을 갖고 있음을 의미한다. The roles of β-adrenoceptor were well known in hyperthyroidal heart, but not with α-adrenoceptor. So we studied the effects of phenylephrine on membrane potential, intracellular sodium activity (aⁱ_Na), twitch force, and intracellular pH (pH_i) by continuous intracellular recordings with ion-selective and conventional microelectrodes in the papillary muscles of hyperthyroid guinea pig heart. α₁-Adrenoceptor stimulation by phenylephrine (10^-5 or 3 × 10^-5M) produced the following changes: variable changes in action potential duration, a hyperpolarization (1.5 ± 0.1mM) of the diastolic membrane potential, an increase in aⁱ_Na (0.4 ± 0.15mM), a stronger positive inotropic effect (220 ± 15%), an increase in pH_i (0.06 ± 0.002 unit). These changes were flocked by prazosin and atenolol. This indicated that the changes in membrane potential, aⁱ_Na twitch force, and pH_i are mediated by a stimulation of the α₁-Adrenoceptor. Ethylisopropylamiloride (10^-5) also blocked the increase in aⁱ_Na and twitch force. On the other hand, strophanthidin, tetrodotoxin, Cs⁺ or verapamil did not block the increase in aⁱ_Na and twitch force. Thus, it was suggested that α₁-Adrenoceptor stimulation increased aⁱ_Na and pH_i by stimulation of Na⁺-H⁺ exchange, thereby allowing intracellular alkalinization and aⁱ_Na increase. These results were very different from euthyroidal heart which showed α₁-Adrenoceptor-induced decrease in aⁱ_Na and initial negative inotropic effect. From the above results, it was concluded that α₁-Adrenoceptor had a important role in hyperthy-roidal heart.

Differential Glycosylation of Na^+/H^+ Exchanger Isoform 1 (NHE1) in Rat Parotid and Sublingual Glands

Park, Keerang,Rechardson, Linda A.,Dharmini Pathmanathan,Lantz, Heather M.,Melvin, James E. Korean Academy of Oral Biology and the UCLA Dental 1999 International Journal of Oral Biology Vol.24 No.2

We characterized the giycosylation pattern of rat Na^+/H^+ exchanger isoform 1 (NHE1) in two distinct types of salivary glands, the serous parotid gland and the mucous sublingual gland. Northern blot analyses showed that the transcript size of NHE1 in both glands was 4.8 kb. However, affinity-purified polyclonal NHE1 antibody detected a 90-95 kDa protein in the crude membrane protein fraction from sublingual glands, whereas NHE1 from parotid gland membrane was 90-100 kDa. Treatment of NHE1proteins with n-glycosidase F, O-glycosidase. Neuraminidase and Endoglycosidase H demonstrated that NHE1 in both types of glands has N-linked, but not O-linked glycosylation. NHE1 in parotid glands was Endoglycosidase H-sensitive indicating the presence of high mannose-type or hybrid-type N-linked oligosaccharides. In contrast, NHE1 in sublingual glands was insensitive to Endoglycosidase H. These results suggest that glycosylation of NHE1 is salivary gland-specific.

급성 척수허혈 및 재관류손상에 대한 Na^+/H^+ 교환억제제(DMA)의 보호효과 : 가토 척수의 허혈 및 재관류손상 모형에 의한 실험적 연구 An Experimental Study in Spinal Cord Ischemia and Reperfusion Injury Model

고정관,박형주,이길노 순천향의학연구소;Soonchunhyang Medical Research Institute 2000 Journal of Soonchunhyang Medical Science Vol.6 No.1

Spinal cord injury after operations on the descending thoracic and thoracoabdominal aorta remains a persistant clinical problem. Previous attemps to decrease the risk of this devastating complication by lowering the rate of metabloism of the spinal cord have met with varying success. We hypothesized that the tolerance of the spinal cord to an ischemic insult could be improved by means of Na^+/H^+ exchange inhibitor(DMA). DMA prevent intracellular Na^+ influx and maintain intracellular acid pH during ischemia by blocking the Na^+/H^+ exchange system. Additionally, subsequent Ca^++ influx during reperfusion is prevented by blocking the Na^+/Ca^++ exchange as well. It is well established that not only the intracellular acidosis during ischemia prolongs cellular survival but almost all of the reperfusion injury is associated with intracellular Ca^++ overload. Twenty four New zealnad white rabbits underwent 30 minutes of isolated infrarenal aortic occlusion after heparin anticoagulation. Clamps were placed both below the left renal vein and above the aortic bifurcation. In 12 rabbit(Amiloride Group), a bolus of amiloride(3㎎/㎏) intravenous injection and a bolus of diluted amiloride(100uM) was infused the isolated aortic segment immediately after crossclamping and immediately before release of clamping. In another control group(12 rabbits), the aortic segment was flushed with normothermic saline in a fashion identical to that of the study group. The aortic clamps were removed after 30 minutes, the abdomen was closed, and the animals were allowed to recover from anesthesia. Spinal cord function was assessed 72 hours after operation by the Talov's scale. All animals were put to death at 72 hours after operation and spinal cords were harvested for MDA and histologic analysis. The spinal cord function of all Amiloride group animals were fully intact with Talov's scores of 3-5; control group animals were all paraplegic with Talov's score of 0-2(p<0.001, student t-test). MDA level in Amiloride group was 0.046±0.06 and control group was 0.065±0.01(p<0.0001, student t-test). Histologic examination of spinal cords from Amiloride gropup rabbits revealed a little evidence of cord injury, whereas spinal cords from control group had evidence of extensive cord injuty with central gray necrosis, axonal swelling, dissolution of Nissl substance, and astrocyte and macrophage infiltration. Systemic and regional infusion of the crossclamped infrarenal rabbit aorta with Na^+/H^+ exchange inhibitor(DMA) nearly complete prevented paraplegia in our model despite a 30-minute ischemic insult.

기니픽심근에서 Na-H 교환기 억제제가 무스카린 수용체 흥분에 의한 세포내 Na활성도 증가에 미치는 영향

이재백,채수완,황룡 의과학연구소 1991 全北醫大論文集 Vol.15 No.2

It has been known that carbachol increases intracullular sodium activity(a_N^i_a) and twich force. However the mechanism of a_N^i_a increase is not clear. The a_N^i_a twich force and membrane potenital guinea pig papillary muscle were measured simultaneously during exposure to carbachol(3X10exp(-4)M) and subsquent washout in the absence and presence of selective NaH exchange inhibitor, hexamethylene amiloride, or low extracellular pH(6.6) condition. The results are : 1. Hexamethylene amiloride(30㎛) inhibited NH_4Cl(10mM) induced transient decrease of a_N^i_a following marked increase of a_N^i_a which indicates that hexamethylene amiloride is a Na-H exchange inhibitor of heart muscle. 2. Carbachol induced a_N^i_a increase in quiescent and beating ventricular muscle were inhibited by hexamethylene amiloride. 3. Low extracelluar pH(6.6) inhibited carbachol indeced a_N^i_a increase in quiescent and beating ventricular muscle. 4. Low extracellular pH increased twitch force, increased action potential duration and intracellular sodium activity, and depolarized diastolic membrance potental. From above results, it is suggested that carbachol increase a_N^i_a via Na-H exchange and Na-H exchange system of ventricular muscle which is different form the action of muscarinic receptor stimulation in Purkinje fibers.

저칼륨혈증 신장에서 HCO₃- 조절에 관한 연구

김지혜(Ji Hye Kim),조혜정(Hye Jung Cho),배미옥(Mi Ok Bae),박정진(Jeong Jin Park),안규윤(Kyu Youn Ahn) 대한해부학회 2004 Anatomy & Cell Biology Vol.37 No.4

산-염기 평형장애나 전해질 불균형은 신요세관에서 HCO₃- 재흡수가 증가되거나 감소되는 것과 연관이 있다고 알려져 있다. 칼륨 제한시 대사성 알칼리증을 유발시킨다는 것은 연구자간에 일치하나 이와 관련된 Na+/H+ exchanger (NHE-3) 와 Na+/ HCO₃- cotransporter (NBC)에 대한 연구는 주로 기능적인 것에 국한되어 있고 또한 연구자 간에도 차이가 있다. 본 연구는 흰쥐 신장에서 칼륨 제한식이 시기에 따라 NHE-3와 NBC-1의 신장내 발현 및 분포의 변화를 Western 분석과 면역조직화학적 방법으로 관찰하였다. Western 분석 소견에서 NHE-3 단백은 83 KDa 정도이고 정상 식이군에서 상당량 발현되었으며 칼륨 제한 식이군에서 정상 식이군에 비해 유의한 증가를 보였다. NBC-1 단백은 110 KDa 정도이고 정상 식이군에 비해 칼륨 제한 식이군에서 증가하였으며 특히 칼륨 제한 식이 1주에서 유의한 증가를 보였다. 면역조직화학 소견에서 NHE-3 단백은 토리쪽세관 상피세포 중 S3 부위에서 강하게 발현되었고, S1, S2 부위에서는 중등도로 발현되었다. 면역반응성은 토리쪽 세관세포의 첨부세포막과 솔가장자리에서 관찰되었다. 칼륨 제한 식이군에서 면역반응 부위는 정상 식이군과 차이가 없었으나 면역반응성은 Western 분석 소견에서와 같이 현저히 증가하였다. NBC-1 단백은 토리쪽세관 S1, S2 부위에서는 강하게 발현되었으나, S3 부위에서는 발현되지 않았다. 면역반응성은 토리쪽세관세포의 기저외세포막에서 관찰되었다. 칼륨 제한 식이군에서 면역반응 부위는 정상 식이군과 차이가 없었으나 면역반응성은 Western 분석 소견에서와 같이 칼륨제한 식이 1주에서 현저히 증가하였다. 이상의 소견은 칼륨 제한시 신장 토리쪽세관에서 NHE-3와 NBC-1의 단백 발현 증가가 혈액내로 HCO₃- 의 재흡수를 항진시켜 대사성 알칼리증을 유지할 것으로 생각되었다. A number of acid-base or electrolyte disorders are associated with decreased or increased HCO₃- reabsorption in the renal tubules. There has been a general agreement that potassium depletion induces and maintains metabolic alkalosis in rats. However, many researchers have approached such issue only from functional studies to investigate Na+/H+ exchanger (NHE-3) and Na+/HCO₃- cotransporter (NBC) activity which closely relates to potassium depletion. In addition the results obtained vary according to their researchers. Thus the present study was employed Western blot analysis and immunohistochemistry together, to examine the alterations of expression and distribution of NHE-3 and NBC-1 with reference to HCO₃- reabsorption in the kidneys of rats fed potassium free diets according to the periods. Western blot analysis demonstrated that NHE-3 protein, ~83 kDa at molecular mass, was abundantly expressed in normal group. All potassium-depleted groups showed significantly increased NHE-3 protein compared to normal group. NBC-1 protein, ~110 kDa at molecular mass, was moderately expressed in normal group. All potassium-depleted groups had much higher amounts of the protein than normal group. There was a highly increased amount of NBC-1 protein especially in K-depleted 1 week group. Immunohistochemistry showed positive immunoreactivity of NHE-3 in the apical membranes and brush borders of proximal renal tubule cells. Its reactivity was most prominent in the S3. S1 and S2 had moderate immunoreactivity. Potassium-depleted groups had an identical pattern of cellular labeling of NHE-3 protein compared with that of normal group. However the signal intensity of NHE-3 protein in potassium-depleted groups was much higher than that of normal group. Immunoreactivity of NBC-1 was observed exclusively in the basolateral plasma membranes of proximal tubule cells. There was a strong reactivity in the S1 and S2, whereas S3 did not show any reactivity. Potassium-deprived rats exhibited an identical pattern of cellular labeling of NBC-1 protein compared with that of normal rats. However, the signal intensity of NBC-1 protein was markedly increased in potassium-deprived rats. These results suggest that increased NHE-3 and NBC-1 expression resulted from potassium depletion in the renal proximal tubules, enhances HCO3- reabsorption and consequently maintains metabolic alkalosis.