정상 및 무칼륨 식이 백서 신장에서 Na⁺-K⁺-ATPase Subunit Isoform mRNA 발현에 관한 연구

안규윤(Kyu Youn Ahn),김석채(Sug Chae Kim),문 범(Bum Moon),김경근(Kyung Keun Kim),김백윤(Baik Yoon Kim) 대한해부학회 1998 Anatomy & Cell Biology Vol.31 No.3

저칼륨혈증은 여러 조직에서 Na+-K+-ATPase 유전자 발현의 정도를 변화시키며 또한 신장의 속질바깥층집합세관에서는 Na+-K+-ATPase 활성과 α1과 β1 subunit 단백을 증가시키지만 겉질집합세관에서는 유의한 변화가 없다는 것은 잘 알려져 있다. 그러나 집합세관을 위시한 다른 콩팥단위 부위에서의 Na+-K+-ATPase subunit isoform들의 적응반응에 관한 연구는 없는 실정이다. 이에 저자들은 정상 및 무칼륨 식이 백서 신장에서 Na+-K+-ATPase subunit isoform mRNA 발현의 상대적인 양과 분포를 구명하기 위해 Northern 분석과 in situ hybridization 조직화학을 실시하였다. Northern 분석 결과, 대조군의 Na+-K+-ATPase subunit isoform mRNA들은 속질바깥층, 겉질 및 속질속층 순으로 발현되었 으나 실험군에서는 속질바깥층, 속질속층 및 겉질 순으로 발현되었다. α1 mRNA 양은 α2, α3 보다 훨씬 많았고, 각 isoform에 대한 mRNA의 양은 속질속층에서 대조군 보다 실험군에서 2~3배 이상 증가하였으나 겉질과 속질바깥층에서는 두 군간에 유의한 차이가 없었다. In situ hybridization 조직화학 결과에서 각 isoform에 대한 mRNA의 분포는 대조군의 토리쪽세관의 S3 부위, 겉질상행후각, 속질상행후각, 먼쪽곱슬세관 및 전 집합세관에서 관찰되었다. 실험군 백서에서 α1, α3 및 β1 isoform의 반응정도는 속질상행후각, 겉질상행후각, 먼쪽곱슬세관 및 겉질집합세관에서 감소하였으나 속질바깥층집합세관 내층 상부와 속질속층집합세관 상부에서는 현저히 증가하였다. α2 isoform은 속질상행후각에서는 감소하였으나 겉질상행후각, 먼쪽곱슬세관 및 겉질집합세관에서 약간 증가하였고 속질바깥층집합세관 내층 상부와 속질속층집합세관 상부에서는 현저히 증가하였다. 이상의 소견으로 저칼륨혈증에 의해 Na+-K+-ATPase subunit isoform 유전자들의 발현은 속질바깥층집합세관 내층 상부와 속질속층집합세관 상부에서 증가되며, 이들 부위에서 유전자들이 K 이온 보존에 관여하고 있음을 암시해 준다. Chronic hypokalemia alters Na+-K+-ATPase gene expression in several tissues. While it is established that Na+- K+-ATPase activity and α1 and β1 subunit protein levels increase during K depletion in the outer medullary collecting duct (OMCD) and do not significantly change in the cortical collecting duct (CCD), little is known about the adaptive responses of the other isoforms in these other nephron segments. Accordingly, this study was performed to characterize the relative levels of expression and cellular distribution of mRNAs encoding the Na+-K+-ATPase subunit isoforms in normal and K-deprived (2 weeks) rats using the Northern analysis and in situ hybridization (ISH). Isoform specific 32Plabeled cDNA (for Northerns) or digoxigenin labeled cRNA (for ISH) probes were used. In normal rats, the order of expression amounts of all isoforms mRNAs from highest was outer medulla > cortex > inner medulla , and that of K-deprived rats was outer medulla > inner medulla > cortex. α1 mRNA levels were much greater than those of α2 or α3 in cortex, outer and inner medulla. mRNA levels for all isoforms were 2~3 folds greater in inner medulla of K-deprived rats compared to controls. In contrasts, the levels of all isoforms mRNAs in cortex and outer medulla were comparable between the two gruops. By ISH, mRNAs for all isoforms were observed in the S3 segment of proximal tubule, the cortical thick ascending limb (CTAL), medullary thick ascending limb (MTAL), distal convoluted tubule (DCT), connecting tuble (CNT), and the entire collecting duct. Both groups exhibited comparable cellular patterns of labeling, but the signal intensity of K-deprived rats was much greater in the proximal portion of the inner stripe of outer medullary collecting duct (OMCDi) and proximal portion of the inner medullary collecting duct (IMCD), and less in the MTAL compared to controls. The signal intensity of α1, α3, and β1 isoforms was less in the CTAL, DCT, and CCD of K-deprived rats, but α2 isoform was slightly increased. These results suggest that chronic hypokalemia enhances expression of Na+-K+-ATPase subunit isoforms in the proximal portion of OMCDi and proximal IMCD, but not other nephron segments, and that these isoforms may participate in potassium conservation by these segments during potassium deprivation.

인태아(人胎兒) 활액막세포(滑液膜細胞)의 전자현미경적(電子顯微鏡的) 연구(硏究)

윤재룡,전철홍,안규윤,Yoon, Jae-Rhyong,Chun, Cheol-Hong,Ahn, Kyu-Youn 한국현미경학회 1994 Applied microscopy Vol.24 No.1

The development of synovial membrane from knee joint was studied by electron microscope in human fetuses ranging from 20mm to 260mm crown rump length (40days to 30weeks of gestational age). At 40mm fetus, developing synovial tissue was observed in homogenous interzone as a vascular mesenchyme around the periphery. The primitive joint space was appeared after the intermediate layer of the interzone in direct contact with chondrogenic layer at 60mm fetus. Differentiation of the synovial membrane coincided with clarification of the joint cauity. When dilatation of the synovial cavity occurred, the two types of synovial cells were well endowed with rough endoplasmic reticulum. At 100mm fetus, type A cells with a markedly attenuated cytoplasm were found as well as those cells which contained pinocytotic vesicles and vacuoles. By 150-200mm fetuses a majority of the intimal cells were type B. These cells were characterized by abundant rough endoplasmic reticulum and well developed Golgi complex. In contrast, A-type cell had numerous filopodia, pinocytotic vesicles lysosomes, and large vacuoles containing amorphous material. At 260mm fetus, the intimal cells were well developed and plentiful. The most marked difference between the synovial membrane of full-term fetus and adult was the large amount of collagen in the latter. During fetal period, the B-cells were most numerous cell type in the intimal cells. The B-cells were clearly distinguishable from the A-cells by their content of extensive rough endoplasmic reticulum and well developed Golgi complex.

각막신생혈관에 대한 버테포르핀을 이용한 광역학치료의 동물실험

나현주,윤경철,임욱빈,안규윤,서만성,Hyeon-Ju Nah,M,N,Kyung-Chul Yoon,Wook-Bin Im,Kyu-Youn Ahn,Man-Seong Seo 대한안과학회 2005 대한안과학회지 Vol.46 No.4

Purpose: To determine the efficacy of photodynamic therapy (PDT) with verteporfin (Visudyne?, Norvatis Ophthalmics AG, Hettingen, Switzerland), a benzoporphyrin derivative, in the treatment of corneal neo- vascularization (CN) in a rabbit eye model. Methods: CN was induced by placing instrastromal sutures in the cornea. Two weeks after suturing, verteporfin was administrated intravenously and 1 hour later, the right eye (treated group) was exposed to a laser with a 689 nm wavelength, and the left eye was used as the control. The changes in CN were analyzed using biomicroscopy and optical microscopy in twelve rabbits. Results: The mean percentages of the neovascular area in the control and treated groups were 96.4±1.9% and 90.3±3.5% (P=0.009) at three days after the PDT, 88.6±4.6% and 71.6±6.2% (P<0.001) at one week, and 76.8±4.4%와 43.6±15.1% (P<0.001) at two weeks, respectively. Optical microscopy showed significant differences between the control and treated group in terms of the area and number of CN (P<0.05). Conclusions: PDT with verteporfin is a safe and effective procedure for regressing CN. However, a further study will be necessary.

흰쥐 눈물샘에서 Carbonic Anhydrase 발현에 관한 면역조직화학적 연구

안 민(Min Ahn),이송은(Song Eun Lee),남광일(Kwang Il Nam),정채용(Chaeyong Jung),이승원(Seung-won Lee),안규윤(Kyu Youn Ahn),배춘상(Choon Sang Bae),김백윤(Baik Yoon Kim),박성식(Sung Sik Park) 대한해부학회 2008 Anatomy & Cell Biology Vol.41 No.1

흰쥐 안구 밖 눈물샘에서 carbonic anhydrase (CA) 동종효소의 발현 여부를 관찰하고자 흰쥐 눈물샘을 대상으로 CA I, II, IV 및 IX 동종효소에 대한 항체를 사용하여 면역조직 화학 염색을 시행하였고, 이들 조직에서 각각의 CA 동종효 소 단백 발현을 Western blotting 분석을 시행하여 확인하였다. Western blotting 분석 결과 CA II와 IX가 가장 강하게, CA I이 중등도로, CA IV가 가장 약하게 발현되었다. H-E 염색에서 안구 밖 눈물샘은 대롱꽈리샘으로 많은 소엽과 사이막으로 구성되어 있었다. 소엽에는 장액샘꽈리와 사이관 그리고 소집합관들이 관찰되었다. 면역조직화학 염색에서 CA I 반응은 샘꽈리세포에서는 음성이었으나 사이관세포와 소집합관세포에서는 양성 반응을 보였다. CA II 반응은 샘꽈리세포의 경우 샘꽈리에 따라 다양한 양성 반응을 보였으며 양성 부위는 샘꽈리세포의 핵상부 세포질이었다. 사이관과 소집합관에서는 대부분 음성이었으나 일부 사이관과 소집합관은 약한 반응을 보였다. CA IV 반응은 샘꽈리세포에서는 음성이었으나 사이관 세포와 소집합관세포는 강한 양성을 보였다. CA IX 반응은 대부분의 샘꽈리세포에서는 음성이었으나 극히 일부 샘세포에서는 바닥세포막에서 양성 반응을 보였다. 사이관과 소집합관세포에서는 양성 반응을 보였다. 이상의 관찰로 흰쥐 안구 밖 눈물샘에서 샘꽈리세포와 도관세포에 분포하는 CA 동종효소가 다름을 확인하였으며, 눈물샘에서의 전해질 대사는 샘꽈리세포는 주로 CA II에 의해 이루어지고 도관계는 주로 CA I, IV 및 IX에 의해 이루어질 것으로 추측되었다. There are several carbonic anhydrase (CA) isozymes, which differ in their kinetic properties, tissue distribution, and subcellular localization. In this study, the distribution of CA isozymes I, II, IV, and IX was investigated in the rat exorbital lacrimal gland using Western blotting analysis and immunohistochemical staining. In the Western blotting analysis of the rat lacrimal gland, CA II and CA IX were expressed abundantly and CA IV was expressed weakly. Hematoxylin-eosin staining of the exorbital lacrimal gland showed a multilobular tubuloacinar gland composed of acinar and ductal cells. Immunohistochemical reaction revealed no CAI staining in acinar cells and positive staining in intercalated and small duct cells. CA II reactivity was detected in the supranuclear cytoplasm of acinar cells and appeared to vary between acini. The intercalated and collecting duct cells showed weak or no immunoreactivity for CA II. CA IV was detected in the intercalated and collecting duct cells but not at the acinar cells. CA IX was detected in the intercalated and collecting duct cells, and in only a few acinar cells. These results demonstrate the differential distribution of CA isoenzymes in the exorbital lacrimal gland of the rat and suggest that CA II is related mainly to the electrolyte metabolism of tears in the acinar cells and that CAs I, IV, and IX are related to the electrolyte metabolism of tears in the duct cells.

토끼에서 αvβ5 인테그린 항체의 결막하주사에 의한 각막신생혈관 억제효과

윤경철,임성규,오한진,안규윤,김경근,Kyung-Chul Yoon,Seong-Kyu Im,Han-Jin Oh,Kyu-Youn Ahn,Kyung-Keun Kim 대한안과학회 2005 대한안과학회지 Vol.46 No.11

Purpose: To investigate the efficacy of a subconjunctival injection of αvβ5 integrin antibody on corneal angiogenesis induced by chemical epithelial denudation in a rabbit eye model. Methods: One week after debridement by heptanol, rabbits were treated with a subconjunctival injection of αvβ5 integrin antibody or control immunoglobulin G weekly for 2 weeks. Rabbits that did not receive injection after debridement served as the untreated group. The percentage of neovascularized corneal area was calculated by biomicroscopy, and the sectioned area and number of new vessels were calculated by histological examinations. Results: At 7 days after the first injection, αvβ5 integrin antibody-treated eyes had 9.5% (P=0.02) and 6.8% (P=0.03) less neovascularized corneal area than vector-treated eyes and untreated eyes, respectively. At 7 days after the second injection, αvβ5 integrin antibody-treated eyes had 21.1% (P=0.02) and 18.3% (P=0.02) less neovascularized corneal area than vector-treated eyes and untreated eyes, respectively. Light microscopic examination showed a smaller neovascularized corneal area and a reduced number of new vessels in the αv β5 integrin antibody-treated eyes compared to the control eyes. Conclusions: Subconjunctival injection of αvβ5 integrin antibody effectively reduces experimental corneal neovascularization induced by chemical injury, and could be used as a corneal angiogenesis inhibitor in the future.

자간전증 태반의 세포 고사

김혜정(Hye Jung Kim),김윤하(Yoon Ha Kim),안규윤(Kyu Yoon Ahn),남광일(Kwang Il Nam),김석모(Seok Mo Kim),송태복(Tae Bok Song),변지수(Ji Soo Byun) 대한산부인과학회 2001 Obstetrics & Gynecology Science Vol.44 No.4

N/A Objective : Our purpose was to investigate the incidence of placental apoptosis in pregnancies complicated by preeclampsia. Methods : Placental samples were obtained from 15 uncomplicated third-trimester pregnancies and from 17 pregnancies complicated by preeclampsia. TUNEL (terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling) staining and electron microscopy were performed on all placental samples and identified apoptosis within the cells of the placenta. TUNEL positive stained cells were counted at each photograph(five sections were photoragphed at 1,500 magnifications for each sample). We focused on only the syncytiotrophoblast nuclei of the placenta. The number of apoptotic syncytiotrophoblast nuclei identified was expressed as a percentage of total number of syncytiotrophoblast nuclei counted. Results : Quantification of apoptosis (mean±SD) was as follows: normal third trimester (n=15) 0.57±0.47% of cells and preeclampsia third trimester (n=17) 1.41±0.67% of cells. The incidence of apoptosis was significantly increased in placentas from pregnancies with preeclampsia compared with normal placentas (p< 0.01). Conclusion : These results suggest that placental apoptosis may play a role in the pathophysiologic mechanisms of preeclampsia.

저칼륨혈증 흰쥐 신장에서 Akt, p-Akt, ERK 및 p-ERK 단백발현의 변화

배춘상(Choon Sang Bae),조혜정(Hye Jung Cho),안규윤(Kyu Yoon Ahn) 대한체질인류학회 2017 해부·생물인류학 (Anat Biol Anthropol) Vol.30 No.3

저칼륨혈증은 신장의 형태학적 변화와 대사성 알칼리증을 유발시키는 것으로 알려져 있다. 이전 결과들은 저칼륨혈증의 병태생리학적 변화에 K+평형 조절 이온채널, pump 유전자, NF-E2-related factor 2 (Nrf2) 전사유전자를 포함한 다양한 유전자가 관여할 것이라는 가능성을 제시하였다. 이에 본 연구는 칼륨제한 식이 기간에 따른 흰쥐 신장 내 Akt와 p-Akt 및 ERK와 p-ERK의 발현 및 분포의 변화를 Western 분석과 면역조직화학 방법으로 관찰함으로써, 저칼륨혈증이 신장에서 AKT/ERK 인산화에 영향을 미치는지를 확인하였다. Western 분석소견에서 Akt 및 p-Akt 단백질 발현은 칼륨제한 식이가 진행될수록 증가하는 양상을 보였으며, ERK 및 p-ERK 단백질발현은 칼륨제한 식이 2주군에서 정상 식이군에 비해 약간 증가하는 양상을 보였다. 면역조직화학 소견에서 Akt 단백의 면역반응성은 먼쪽곱슬세관, 겉질곧은부분 및 속질곧은부분에서 중등도의 발현을 보였다. 칼륨제한 식이군의 Akt의 면역반응성은 칼륨제한 식이가 진행될수록 바깥속질집합관에서 현저히 증가하였다. 칼륨제한 식이군의 p-Akt의 면역반응성은 칼륨제한 식이 2주군의 먼쪽곱슬세관, 치밀반점과 속질곧은부분에서 증가하였고 토리쪽곱슬세관에서는 중등도의 발현을 보였다. 칼륨제한 식이군의 ERK의 면역반응성은 칼륨제한 식이 2주군과 3주군의 바깥속질집합관에서 현저히 증가하였으며 먼쪽곱슬세관, 겉질집합관에서는 증등도의 증가를 보였다. 칼륨제한 식이군의 p-ERK의 발현부위는 정상식이군과 비교해 차이가 없었으나 면역반응성은 칼륨제한 식이 2주군의 바깥속 질집합관의 핵에서 현저히 증가하였다. 이상의 결과 저칼륨혈증 시 p-Akt의 발현은 칼륨제한 식이가 길어질수록 점진적으로 증가하였지만, p-ERK의 발현은 칼륨제한 식이 2주군에서 현저히 증가되었다. 따라서 저칼륨 상태에서의 Akt 및 ERK 인산화의 촉진은 이온채널 및 이온수송체 유전자 조절뿐만 아니라 세포 내 신호전달에 있어 중요한 역할에 관여할 것임을 시사해 주었다. Hypokalemia causes metabolic alkalosis and morphological changes of the kidney. K+ balance is regulated not only by ion channels or pump gene, but also by various genes including NF-E2-related factor 2 (Nrf2). Previous study suggested the possibility that Akt and ERK kinase may be involved in Nrf2 transcriptional gene activation. In present study, we investigate the alterations of Akt, p-Akt, ERK, p-ERK protein in both normal kidney and K+-deficient diet kidney using Western blot analysis, and immunohistochemisrty. Our western blot data showed that the expression of Akt and p-Akt was increased gradually in K+-depleted diet (from 1W-3W) compared to normal group. The expression of ERK and p-ERK was markedly increased in K+-depleted diet 2W in comparison with normal group. Based on our immunostaining results, Akt protein immunoreactivity was prominently increased in outer medullary collecting duct, especially in K+-depleted diet 2 weeks. The localization of p-Akt proteins in K+-depleted groups was not different from normal group, but the immunoreactivity was significantly increased in distal convoluted tubule, macula densa and outer medullary thick ascending limb in K+-depleted diet 1 and 2 weeks groups. ERK protein immunoreactivity was prominently increased in outer medullary collecting duct, especially in K+-depleted diet 2 and 3 weeks. The localization of p-ERK proteins in K+-depleted groups was not different from normal group, but the immunoreactivity was prominently increased in the nucleus of outer medullary collecting duct especially in K+-depleted diet 2 weeks. Taken together, we suggest that the expression of p-Akt was gradually increased in K+-depleted groups of kidney, but the expression of p-ERK was markedly increased in K+-depleted diet 2 week group. Hence, the promotion of AKT and ERK phosphorylation in hypokalemic condition may be involved in the regulation of ion channels, ion transporters and subsequent intracellular signal transduction.

사람 창자에서 Carbonic Anhydrase 동위효소 발현에 관한 연구

이동진(Dong Jin Lee),남광일(Kwang Il Nam),이승원(Seung Won Lee),안규윤(Kyu Youn Ahn),배춘상(Choon Sang Bae),김백윤(Baik Yoon Kim),박성식(Sung Sik Park) 대한해부학회 2004 Anatomy & Cell Biology Vol.37 No.4

사람 소화관 상피에서 carbonic anhydrase (CA)의 발현 여부를 관찰하고자 샘창자와 주름창자를 대상으로 CAI, CAII, CAIV, CAIX 동위효소에 대한 항체를 사용하여 면역조직화학 염색을 시행하였고, 이들 조직에서 각각의 CA 동위효소 단백 발현을 Western blot을 시행하여 확인하였다. Western blot에서 CAI, IX는 샘창자와 주름창자에서 모두 강하게 발현되었다. CAII는 샘창자와 주름창자의 점막조직에서 강하게 발현되었고, CAIV는 샘창자와 주름창자의 점막조직에서 약하게 발현되었다. 샘창자에서 면역조직화학 염색 결과 CAI은 음성이었다. CAII는 융모원주세포와 점막밑샘세포 및 도관세포에서 양성이었으나 창자샘에서는 음성이었다. CAIV는 융모상피와 창자샘에서는 음성이었으나 점막밑샘에서는 도관세포가 양성반응을 보였다. CAIX는 융모상피는 음성이었으나 창자샘은 양성이었고 점막밑샘에서는 극히 일부의 샘세포에서 양성이었다. 주름창자에서 면역조직화학 염색 결과 CAI과 II는 상피 원주세포와 술잔세포에서 양성이었다. CAIV와 IX는 상피의 원주세포에서만 양성이었다. 이상의 관찰로 샘창자에서 표면상피는 CAII가, 창자샘은 CAIX이, 점막밑샘에서 분비세포는 CAII가, 도관세포는 CAII와 IV가 가장 강하게 발현되고, 주름창자에서 표면상피는 CAI, II, IV, IX 모두 발현되나 창자샘은 모두 음성임을 알 수 있었다. 이상의 결과는 샘창자 점막밑샘은 샘창자에서의 산-염기 균형 조절에 CAII와 IV를 통해 관여할 것임을 시사하였다. The distribution of carbonic anhydrase (CA) isozymes I, II, IV, and IX was investigated in the human duodenum and colon using Western blotting analysis and immunohistochemical staining. A Western blotting analysis revealed an abundant expression of CAI and IX in the duodenum and colon. The expression of CAII and IV was detected in mucosa of duodenum and colon. The degree of expression, however, showed regional difference. The expression of CAII was strong in the duodenum and colon, and that of CAIV was weak in the duodenum and colon. Immunohistochemical staining of duodenum revealed no staining for CAI. CAII was detected at the columnar cells of surface epithelium and secretory cells and ductal cells of Brunner’s gland. CAIV was detected at the ductal cells of Brunner’s gland. CAIX was detected at the cells of intestinal gland and rare cells of the Brunner’s gland. Immunohistochemical staining of colon showed a positive reaction of CAI and II at the columnar and goblet cells of surface epithelium, and CAIV and IX at the columnar cells of surface epithelium. These results demonstrate the differential distribution of CA isozymes in duodenum and colon, and suggest that Brunner’s gland may contribute the control of acid-base balance in the duodenal lumen by secreting bicarbonate ion catalyzed by CAII and IV.

흰쥐 침샘에서 Carbonic Anhydrase 동위효소 IV와 IX의 분포에 관한 면역조직화학적 연구

조태영(Tae Young Cho),이송은(Song Eun Lee),남광일(Kwang Il Nam),정채용(Chaeyong Jung),안규윤(Kyu Youn Ahn),배춘상(Choon Sang Bae),김백윤(Baik Yoon Kim),박성식(Sung Sik Park) 대한해부학회 2009 Anatomy & Cell Biology Vol.42 No.4

침샘에서 carbonic anhydrase(CA) 동위효소의 발현 여부를 관찰하고자 흰쥐 귀밑샘과 턱밑샘을 대상으로 CAs I, II, IV 및 IX 동위효소에 대한 항체를 사용하여 면역조직화학 염색을 시행하였고, 이들 조직에서 각각의 CA 동위효소 단백발현을 Western blot 분석을 시행하여 확인하였다. Western blot 분석 결과 귀밑샘은 CAs I, II와 IX는 강하게 발현되었고 CA IV는 미약하게 발현되었다. 턱밑샘에서는 CAs I과 II는 강하게 발현되었으나 Cs IX는 미약하게 발현하게 발현되었고 CA IV는 발현되지 않았다. H-E 염색에서 귀밑샘은 장액샘꽈리와 도관으로 구성되어 있었으며 턱밑샘은 샘의 대부분을 차지하는 고유턱밑샘형소엽과 점액샘꽈리와 장액반달로 구성된 혀밑샘형 소엽으로 되어 있었다. 면역조직화학 염색에서 귀밑샘은 CA IV, IX 및 I의 반응은 사이관, 줄무늬관 및 소엽사이관 같은 도관세포에서 양성을 보이고 샘꽈리세포에서는 음성이었다. CA II 반응은 도관세포와 샘꽈리세포에서 양성이었다. 턱밑샘의 고유턱밑샘형 소엽에서 CA IV, IX 및 I의 반응은 도관세포에서는 양성 반응을 보였으나 샘꽈리세포에서는 음성이었다. CA II 반응은 도과세포와 장액샘꽈리세포에서 모두 양성이었다. 턱밑샘의 혀밑샘형 소엽에서 CAs IV, IX 및 I 반응은 도관세포에서는 양성을 보였으나 샘꽈리세포에서는 음성이었다. CA II 반응은 도과세포와 장액반달세포에서 양성이었으나 점액샘꽈리세포에서는 음성이었다. 이상의 관찰로 흰쥐 귀밑샘과 턱밑샘에 분포하는 CA 동위 효소의 분포를 확인하였으며, 침 도관세포에서의 전해질 대사는 CAs I과 II뿐 아니라 CAs IV와 IX도 관여할 것으로 추측되었다. This study presents distribution of carbonic anhydrase(CA) isozymes IV and IX, membrane associated forms, and CA I and II, cytoplasmic forms, in rat parotid and submandibular glands using Western blot analysis and immunohistochemical staining. Western blot analysis demonstrated that CAs I, II and IX were found to be abundantly expressed, but CA IV was weakly expressed in parotid gland. Submandibular gland expressed abundant CAs I and II, weak CA IX, and undetectable level of CA IV. In hematoxylin-eosin staining, parotid gland was entirely composed of serous acini and their ducts while submandibular gland was mixed population of serous and mucous lobules. Most of lobules (submandibular gland proper type) contained mostly serous acini and their ducts with granular convoluted duct. Some lobules (sublingual gland type) contained mostly mucous acini with serous demilune and their ducts without granular convoluted duct. In parotid gland, CAs IV and IX were immunolocalized in duct cells and not in serous acinar cells. Immunoreactivity for CAs I and II was also detectavle in duct cells. Serous acinar cells were positive for CA II, and negative for CA I. In submandibular gland, CAs IV and IX were immunolocalized in duct cells but not in acinar cells of both types of lobules. Immunoreactivity for CAs I and II was also detectable in duct cells of both types of lobules. Cells of serous acini and serous demilune were positive for CA II, and negative for CA I. Mucous cells were negative for both CAs I and II. These results demonstrate the distribution of CA isoenzymes in parotid and submandibular glands of the rat, and suggest CAs IV and IX as well as CAs I and II are related to electrolytes metabolism of saliva in duct cells.

Dexamethasone 투여 흰쥐 콩팥 토리쪽세관의 형태학적 적응반응

이송은(Song Eun Lee),남광일(Kwang Il Nam),배춘상(Choon Sang Bae),김백윤(Baik Yoon Kim),박성식(Sung Sik Park),안규윤(Kyu Youn Ahn) 대한해부학회 2003 Anatomy & Cell Biology Vol.36 No.1

체내 glucocorticoid의 증가는 콩팥 세관에서 H+의 분비와 HCO3-의 재흡수를 증가시켜서 대사성 알칼리증을 일으킨다. 토리쪽세관에서 HCO3-의 재흡수는 첨부세포막의 H+-ATPase와 Na+/H+ exchanger (NHE-3), 바닥쪽세포막의 Na+/HCO3 - cotransporter-1 (NBC-1)에 의해 일어난다고 알려져 있다. 본 연구는 흰쥐 콩팥토리쪽세관에서 dexamethasone 투여에 의한 NHE-3와 NBC-1 단백 발현의 변화를 면역조직화학적 방법으로, 토리쪽세관 상피세포의 형태학적 변화를 전자현미경으로 관찰하였다. 면역조직화학 소견에서 대조군의 NHE-3에 대한 강한 면역반응성은 토리쪽세관 S3 부위의 첨부세포막과 솔가장자리에서 관찰되었으며, S1과 S2 부위에서는 중등도로 관찰되었다. NBC-1에 대한 강한 면역반응성은 S1과 S2 부위의 바닥쪽세포막에서만 관찰되었다. Dexamethasone 투여 실험군에서 NHE-3에 대한 면역반응성은 대조군과 같이 토리쪽세관의 S1, S2와 S3 부위에서 관찰되었으나 발현 정도는 상당히 증가되어 관찰되었다. NBC-1에 대한 면역반응성은 대조군과 같이 S1과 S2 부위에서 관찰되었으며, 발현 정도는 현저히 증가하였다. 전자현미경 관찰결과, 대조군에서 S1과 S2 부위는 첨부세포질내에 많은 공포와 소포가 관찰되었고, S3 부위는 S1과 S2 부위에 비해 솔가장자리의 길이가 길고, 세포질내 사립체의 수와 크기가 적었다. Dexamethasone 투여군에서는 대조군에 비해 S1과 S2 부위에서는 첨부세포막의 관소포와 소포가 증가하였으며, 솔가장자리 미세융모의 길이와 수가 증가하였다. 바닥쪽세포막의 주름은 현저히 증가되었고, 세포질내 사립체의 수가 증가되었다. S3부위에서는 솔가장자리미세융모의 수와 길이 그리고 사립체의 수가 증가되었지만, 바닥쪽세포막은 대조군과 비슷한 양상이었다. 이상의 결과로 장기간 glucocorticoid의 체내 농도의 증가는 콩팥 토리쪽세관에서의 NHE-3와 NBC-1 단백발현을 증가시켜 HCO3- 재흡수를 촉진시키며, 이러한 적응반응은 토리쪽세관 상피세포의 미세형태학적인 변화에 기인할 가능성을 시사하였다. Excess accumulation of glucocorticoid increases acid secretion and HCO3- reabsorption in the kidney. Reabsorption of HCO3-, which almost occurs at the proximal tubule, is mediated Na+/H+ exchanger-3 (NHE-3) and H+-ATPase on the apical membrane and the Na+/ HCO3- cotransporter-1 (NBC-1) on the basolateral membrane. Impact of glucocorticoid was investigated by immunohistochemistry and electron microscopy to correlate the changes with the effect of in vivo dexamethasone treatment for the rat kidney proximal tubule. In a control group, immunoreactivity of NHE-3 was detected in the apical membrane and the brush borders of S1, S2 and S3 segments of the proximal tubule. Immunoreactivity of NBC-1 was detected in the basolateral membrane of S1 and S2 segments of the proximal tubule. Immunoreactivity of NHE-3 and NBC-1 protein was more pronounced in dexamethasone treated groups than the control group. Dexamethasone 1 mg/kg caused most intense immunoreactivity for NHE-3 and NBC-1 protein, however, 0.01 mg/kg and 0.1 mg/kg produced less intense immunoreactivity with no appreciable differences between these lower doses of dexamethasone groups. By electron microscopy, the tubular cells of S1 segment of the control group revealed numerous mitochondria, endocytic apparatuses, lysosomes and many basal cytoplasmic processes. In dexamethasone treated groups, the cells of S1 and S2 segments of the proximal tubule had more mitochodria and more basolateral invaginations and had an increased number of more elongated microvilli, compared with the control group. The cells of the S3 segment of the control group showed scant lateral interdigitations and had a few smaller mitochondria. The cells of the S3 segment of dexamethasone treated groups had many mitochodria and an increased number of microvilli in the brush border, but revealed no difference of basolateral invaginations among the different groups of dexamethasone. These results indicate that prolonged administration of excess glucocorticoid increases NHE-3 and NBC-1 protein, and the up-regulation of these proteins could result in increased HCO3- reabsorption in the rat renal proximal tubules. It also suggests that these adaptive responses closely correlate to morphological alterations of proximal tubular epithelial cells.