Rapid Measurement of NH₃ and Weak Acid Permeation Through Liposomes and Renal Proximal Tubule Membranes

Bae. Hae-Rahn,Suh. Duck-Joon,Lee. Sang-Ho 대한생리학회 1994 대한생리학회지 Vol.28 No.2

Using the methods of stopped-flow and epifluorescence microscopy with entrapped fluorophore, membrane permeability of NH₃ and weak acids in liposomes, renal brush border (BBMV) and basolateral membrane vesicles (BLMV), and primary culture cells from renal proximal tubule was measured. Permeability coefficient (cm/sec) of NH₃ was (2.9 X 10^-2 in phosphatidylcholine liposome 25℃), 5.9 X 10^-2 in renal proximal tubule cell (37℃), 4.0 X 10^-2 and 2.4 X 10^-2 in BBMV and BLMV (25℃), respectively. Formic acid has the highest permeability coefficient among the weak acids tested, which was 4.9 X 10^-3 in liposome, 5.0 X 10^-3 in renal proximal tubule cell, 9.1 X 10^-3 in BBMV and 3.8 X 10^-3 in BLMV. There was a linear relationship between external concentration of nonionized formic acid and initial rate of flux of formic acid in liposome, and the slope coincided with the value of permeability coefficient of formic acid measured in pH 7.0. These results show that techniques of stopped-flow and epifluorescence microscopy with entrapped fluorophore provide the precise method of measurement of very rapid transport of nonelectrolytes through membranes with the advantages of instantaneous mixing effect, good resolution time and easy manipulation.

Rapid Measurement of $NH_3$ and Weak Acid Permeation Through Liposomes and Renal Proximal Tubule Membranes

Bae, Hae-Rahn,Suh, Duck-Joon,Lee, Sang-Ho The Korean Physiological Society 1994 대한생리학회지 Vol.28 No.2

Using the methods of stopped-flow and epifluorescence microscopy with entrapped fluorophore, membrane permeability of $NH_3$ and weak acids in liposomes, renal brush border (BBMV) and basolateral membrane vesicles (BLMV), and primary culture cells from renal proximal tubule was measured. Permeability coefficient (cm/sec) of $NH_3$ was $(2.9{\times}10^{-2}$ in phosphatidylcholine liposome $25^{\circ}C)$, $5.9{\times}10^{-2}$ in renal proximal tubule cell $(37^{\circ}C)$, $4.0{\times}10^{-2}\;and\;2.4{\times}10^{-2}$ in BBMV and BLMV $(25^{\circ}C)$, respectively. Formic acid has the highest permeability coefficient among the weak acids tested, which was $4.9{\times}10^{-3}$ in liposome, $5.0{\times}10^{-3}$ in renal proximal tubule cell, $9.1{\times}10^{-3}$ in BBMV and $3.8{\times}10^{-3}$ in BLMV. There was a linear relationship between external concentration of nonionized formic acid and initial rate of flux of formic acid in liposome, and the slope coincided with the value of permeability coefficient of formic acid measured in pH 7.0. These results show that techniques of stopped-flow and epifluorescence microscopy with entrapped fluorophore provide the precise method of measurement of very rapid transport of nonelectrolytes through membranes with the advantages of instantaneous mixing effect, good resolution time and easy manipulation.

Serum Levels of MCP - 1 and IL - 8 in patients with Atherosclerosis and with Acute Ischemic Stroke and Aspirin Inhibits the Secretion of MCP - 1 and IL - 8 on HUVEC through the NF - k B Pathway

Cha,Jae Kwan,Bae,Hae Rahn,Jung,Min Ho 한국지질학회 2001 韓國脂質學會誌 Vol.11 No.1

Regulation of AQP-4 Water Channel Expression in the Brain during Development and by Ischemia

Jung, Jin-Sup,Kim, Hae-Gyu,Bae, Hae-Rahn,Suh, Duk-Joon,Park, Hwan-Tae,Lee, Sang-Ho The Korean Society of Pharmacology 1997 The Korean Journal of Physiology & Pharmacology Vol.1 No.5

Water transport is mediated by two distinct pathways, diffusional and channel-mediated water transport. The first molecular water channel was identified from human erythrocytes in 1992. Genetically-related proteins from other mammalian tissues have subsequently been identified to transport water, and the group is referred to as th "Aquaporins". Aquaporin-4 (AQP4) is most abundant in the brain, which may be involved in CSF reabsorption and osmoregulation. However, ontogeny and regulatory mechanisms of AQP4 channels have not been reported. Northern blot analysis showed that AQP4 mRNA began to be expressed in the brain just before birth and that its expression gradually increased by PN7 and then decreased at adult level. AQP4 was expressed predominantly in the ependymal cells of ventricles in newborn rats. And then its expression decreased in ependymal cells and increased gradually in other regions including supraoptic and paraventricular nuclei. AQP4 is also expressed in the subfornical organ, in which the expression level is not changed after birth. Cryogenic brain injury did not affect expression of AQP4 mRNA, while ischemic brain injury decreased it. Osmotic water permeability of AQP4 channel expressed in Xenopus oocytes was inhibited by the pretreatment of BAPTA/AM and calmidazolium, a $Ca^{2+}/Calmodulin$ kinase inhibitor, in a dose-dependent manner. These results indicate that the expression and the function of AQP4 channel are regulated by developmental processes and various pathophysiological conditions. These results will contribute to the understanding of fluid balance in the central nervous system and the osmoregulatory mechanisms of the body.

쥐 대동맥 평활근 장력조절에 대한 Ehanol의 효과

서덕준,배혜란,정동근,이중희 대한순환기학회 2001 Korean Circulation Journal Vol.31 No.2

The Effect of Topiramate on Status Epilepticus-Induced Neurotoxicity in Immature Mouse Brain

Sang Soo Park(박상수),Hae Rahn Bae(배혜란),Kyu Geun Hwang(황규근) 대한소아신경학회 2006 대한소아신경학회지 Vol.14 No.2

목 적 : 미성숙 뇌는 경련에 대한 감수성, 경련의 특징 및 항간질약제의 반응성 등에서 성숙 뇌와 다르다. 출생 초기의 간질 경련과 항간질약제의 사용이 이 후 뇌 성숙 과정에 어떠한 영향을 주는지가 소아신경학 분야의 주요 관심사이다. 이에 본 연구자는 미성숙 쥐에서 간질중첩증을 유발시킨 후 신경세포의 손상 및 신경 흥분성 정도를 성숙 뇌와 비교하고 항간질약제인 topiramate가 신경세포 손상 및 뇌 성숙 과정에 미치는 효과를 알아보고자 하였다. 방 법 : 생후 14일된 미성숙 쥐에 kainate를 주입하여 간질중첩증을 유발시킨 뒤 실험군은 1주 혹은 1달 동안 topiramate를 투여한 뒤 뇌를 적출하여, 간질중첩증으로 인한 지속적인 신경흥분성은 western blot을 이용하여 glutamate 및 GABA 수용체의 발현 차이로 관찰하였고, 신경세포 손상 유발 정도는 공촛점현미경을 이용하여 TUNEL 및 HE 염색으로 관찰하였다. 결 과 : 미성숙 쥐에서 간질중첩증 유발 1개월 후 해마의 유의한 세포손상과 구조적 변화를 관찰하였다. 간질중첩증으로 인해 GluR2에 비해 GluR1의 발현이 현저히 증가하여 신경세포 흥분독성이 유발될 수 있는 Ca²+ 투과성 AMPA 수용체의 형성이 증가되었다. Topiramate 치료군에서는 간질중첩증으로 인해 유발된 GluR1의 발현 증가가 억제되었다. GABAB 수용체는 간질중첩증 1개월 후 유의한 변화가 없었으나, GABAA 수용체의 발현은 현저히 증가되었으며 이는 topiramate 처리에 의해 억제되었다. Topiramate 치료군에서는 간질중첩증으로 인해 유발된 해마 CA1 및 CA3 부위의 세포 손상과 구조적 변화가 감소됨으로써, topiramate는 미성숙 뇌에서 간질중첩증으로 인한 해마 손상을 보호하는 효과가 있었다. 결 론 : 이상의 결과를 종합하면 topiramate는 미성숙 쥐의 GluR1와 GABAA수용체의 발현을 조절함으로써 신경보호작용을 가지는 것으로 보인다. Purpose : This study was performed to elucidate that status epilepticus (SE) induces longterm neuronal damages in an immature brain and to evaluate that topiramate (TPM) has a protective effect. Metho ds : We investigated the changes in a subtype expression of glutamate and gammaamino butyric acid (GABA) receptors, and the structural integrity due to cell losses in the mouse pup hippocampus after SE using an immunoblot and confocal microscopy. Resu lts : SE induced significant cell losses with structural changes in the hippocampus 1 month later. SE up-regulated the glutamate receptor1 (GluR1) expression with an increased ratio of GluR1 to glutamate recptor2(GluR2), leading to the formation of Ca²+ permeable α-amino-3-hydroxy-5-methyl-4-isoxazoleepropionic acid (AMPA) receptors for the enhanced neurotoxicity. TPM prevented the SE-induced GluR1 expression. The expression of GABAA receptors was highly increased 1 month after SE, whereas that of GABAB receptors was not changed. The TPM treatment attenuated SE-induced upregulation of GABAA receptors. SE induced significant cell losses and disruption of structural integrity in the hippocampus CA1 and CA3 regions, but the TPM treatment for 1 month in developing brains reduced the SEinduced hippocampal damage. Conclusio n : TPM has a neuroprotective action, which might be mediated by the modulation of GluR1 and GABAA receptors.

춘계학술대회 기념호: 구연 7: Serum Levels of MCP-1 and IL-8 in patients with Atherosclerosis and with Acute Ischemic Stroke and Aspirin Inhibits the Secretion of MCP-1 and IL-8 on HUVEC through the NF-k B Pathway

Jae Kwan Cha,Hae Rahn Bae,Min Ho Jung 한국지질동맥경화학회 2001 韓國脂質學會誌 Vol.11 No.1

The Role of Aquaporin-4 in Cerebral Edema Formation after Focal Cerebral Ischemia in Rats

Song, Young-Jin,Bae, Hae-Rahn,Ha, Se-Un,Huh, Jae-Taeck The Korean Neurosurgical Society 2007 Journal of Korean neurosurgical society Vol.41 No.1

Objective : To elucidate the role of aquaporin-4[AQP4] in cerebral edema formation, we studied the expression and subcellular localization of AQP4 in astrocytes after focal cerebral ischemia. Methods : Cerebral ischemia were induced by permanent middle cerebral artery[MCA] occlusion in rats and estimated by the discoloration after triphenyltetrazolium chloride[TTC] immersion. Change of AQP4 expression were evaluated using western blot. Localization of AQP4 was assessed by confocal microscopy and its interaction with ${\alpha}-syntrophin$ was analyzed by immunoprecipitation. Results : After right MCA occlusion, the size of infarct and number of apoptotic cells increased with time. The ratio of GluR1/GluR2 expression also increased during ischemia. The polarized localization of AQP4 in the endfeet of astrocytes contacting with ventricles, vessels and pia mater was changed into the diffuse distribution in cytoplasm. The interactions of AQP4 and Kir with ${\alpha}-syntrophin$, an adaptor of dystrophin complex, were disrupted by cerebral ischemia. Conclusion : The deranged spatial buffering function of astrocytes due to mislocalized AQP4/Kir4.1 channel as well as increased assembly of $Ca^{2+}$ permeable AMPA receptors might contribute to the development of edema formation and the excitotoxic neuronal cell death during ischemia.

P2 Receptor-mediated Inhibition of Vasopressin-stimulated Fluid Transport and cAMP Responses in AQP2-transfected MDCK Cells

Kim, Yang-Hoo,Choi, Young-Jin,Bae, Hae-Rahn,Woo, Jae-Suk The Korean Society of Pharmacology 2009 The Korean Journal of Physiology & Pharmacology Vol.13 No.1

We cultured canine kidney(MDCK) cells stably expressing aquaporin-2(AQP2) on collagen-coated permeable membrane filters and examined the effect of extracellular ATP on arginine vasopressin(AVP)-stimulated fluid transport and cAMP production. Exposure of cell monolayers to basolateral AVP resulted in stimulation of apical to basolateral net fluid transport driven by osmotic gradient which was formed by addition of 500 mM mannitol to basolateral bathing solution. Pre-exposure of the basolateral surface of cell monolayers to ATP(100 ${\mu}M$) for 30 min significantly inhibited the AVP-stimulated net fluid transport. In these cells, AVP-stimulated cAMP production was suppressed as well. Profile of the effects of different nucleotides suggested that the $P2Y_2$ receptor is involved in the action of ATP. ATP inhibited the effect of isoproterenol as well, but not that of forskolin to stimulate cAMP production. The inhibitory effect of ATP on AVP-stimulated fluid movement was attenuated by a protein kinase C inhibitor, calphostin C or pertussis toxin. These results suggest that prolonged activation of the P2 receptors inhibits AVP-stimulated fluid transport and cAMP responses in AQP2 transfected MDCK cells. Depressed responsiveness of the adenylyl cyclase by PKC-mediated modification of the pertussis-toxin sensitive $G_i$ protein seems to be the underlyihng mechanism.

Molecular Analysis of AQP2 Promoter. I. cAMP-dependent Regulation of Mouse AQP2 Gene

Mi Young Park,Yong Hwan Lee,Hae Rahn Bae,Ryang Hwa Lee,Sang Ho Lee,Jin Sup Jung 대한생리학회-대한약리학회 1999 The Korean Journal of Physiology & Pharmacology Vol.3 No.2

<P> To determine molecular mechanisms of Aquaporin-CD (AQP2) gene regulation, the promoter region of the AQP2 gene was examined by transiently transfecting a promoter-luciferase reporter fusion gene into mouse renal collecting duct cell lines such as mIMCD-3, mIMCD-K2, and M-1 cells, and NIH3T3 mouse embryo fibroblast cells. PCR-Southern analysis reveals that mIMCD-3 and mIMCD-K2 cells express AQP2, but M-1 and NIH3T3 cells do not, and that the treatment with cpt-cAMP (400μM) or forskolin/ isobutylmethylxanthine (IBMX) increased the AQP2 expression in IMCD cells. In both IMCD and NIH3T3 cells, the constructs containing the promoter of AQP2 gene showed promoter activities, indicating lack of tissue-specific element in the 1.4 kb 5 -flanking region of the mouse AQP2 gene. Luciferase activity in the IMCD cells transfected with the construct containing 5-flanking region showed responsiveness to cpt-cAMP, indicating that the 1.4 kb 5 -flanking region contains the element necessary for the regulatory mechanism by cAMP. The promoter-luciferase constructs which do not have a cAMP-responsible element (CRE) still showed the cAMP responsiveness in IMCD cells, but not in NIH3T3 cells. Increase in medium osmolarity did not affect AQP2 promoter activity in mIMCD-K2 cells. These results demonstrate that AQP2 gene transcription is increased with cAMP treatment through multiple motifs including CRE in the 5 -flanking region of the gene <I>in vitro</I>, and the regulatory mechanism may be important for <I>in vivo</I> regulation of AQP2 expression.