NADPH Oxidase Mediates β-Amyloid Peptide-Induced Neuronal Death in Mouse Cortical Cultures / 전남의대학술지

β-Amyloid peptide (Aβ) is the main component of senile plaques in patients with Alzheimer's disease, and is known to be a main pathogenic factor of the disease. Recent evidence indicates that activation of NADPH oxidase (NOX) in microglia or astrocytes may be a source of Aβ-induced reactive oxygen species (ROS). We investigated the role of neuronal NOX in Aβ-induced neuronal death in mouse mixed cortical cultures. Cell death was assessed by measuring lactate dehydrogenase efflux to bathing media 24 or 48 hr after exposure to Aβ₂₅₋₃₅, a fragment of Aβ with an equivalent neurotoxic effect. Aβ₂₅₋₃₅ induced neuronal death in concentration- and time- dependent manners with apoptotic features. Neuronal death was significantly attenuated, not only by anti-apoptotic drugs, such as z-VAD-fmk and cycloheximide, but also by antioxidants, such as trolox, ascorbic acid, and epigallocatethin gallate. We also demonstrated that treatment with 20 µM Aβ₂₅₋₃₅ increased fluorescent signals in mixed cortical cultures, but produced only weak signals in pure astrocyte cultures in the presence of 2',7'-dichlorofluorescin diacetate (DCF-DA), an indicator for intracellular ROS. Increased DCF-DA fluorescence was markedly inhibited, not only by trolox, but also by selective NOX inhibitors, such as apocynin and AEBSF. Western blot analyses revealed that Aβ₂₅₋₃₅ increased the expression of gp91phox, a main subunit of NOX in cells. The above antioxidants, apocynin, and AEBSF significantly attenuated neuronal death induced by Aβ₂₅₋₃₅. Furthermore, the gp91phox-specific siRNA-based knockdown of NOX significantly inhibited neuronal death. These results suggest that activation of neuronal NOX is involved in Aβ25-35-induced neuronal death.

Alteration of Akt, p-Akt, ERK, and p-ERK Proteins Expression in the Kidney of Hypokalemic Rat / 체질인류학회지

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.

NADPH Oxidase Mediates β-Amyloid Peptide-Induced Neuronal Death in Mouse Cortical Cultures / 전남의대학술지

β-Amyloid peptide (Aβ) is the main component of senile plaques in patients with Alzheimer's disease, and is known to be a main pathogenic factor of the disease. Recent evidence indicates that activation of NADPH oxidase (NOX) in microglia or astrocytes may be a source of Aβ-induced reactive oxygen species (ROS). We investigated the role of neuronal NOX in Aβ-induced neuronal death in mouse mixed cortical cultures. Cell death was assessed by measuring lactate dehydrogenase efflux to bathing media 24 or 48 hr after exposure to Aβ₂₅₋₃₅, a fragment of Aβ with an equivalent neurotoxic effect. Aβ₂₅₋₃₅ induced neuronal death in concentration- and time- dependent manners with apoptotic features. Neuronal death was significantly attenuated, not only by anti-apoptotic drugs, such as z-VAD-fmk and cycloheximide, but also by antioxidants, such as trolox, ascorbic acid, and epigallocatethin gallate. We also demonstrated that treatment with 20 µM Aβ₂₅₋₃₅ increased fluorescent signals in mixed cortical cultures, but produced only weak signals in pure astrocyte cultures in the presence of 2',7'-dichlorofluorescin diacetate (DCF-DA), an indicator for intracellular ROS. Increased DCF-DA fluorescence was markedly inhibited, not only by trolox, but also by selective NOX inhibitors, such as apocynin and AEBSF. Western blot analyses revealed that Aβ₂₅₋₃₅ increased the expression of gp91phox, a main subunit of NOX in cells. The above antioxidants, apocynin, and AEBSF significantly attenuated neuronal death induced by Aβ₂₅₋₃₅. Furthermore, the gp91phox-specific siRNA-based knockdown of NOX significantly inhibited neuronal death. These results suggest that activation of neuronal NOX is involved in Aβ25-35-induced neuronal death.

Expression and Cellular Localization of Psx Gene in Rat Placenta / 체질인류학회지

Homeobox genes seem to play critical roles in regulating morphogenesis, patterning, organogenesis, and differentiation. They have the conserved sequence that codes the DNA-binding domain called homeodomain. The expression and cellular localization of rPsx mRNA in rat placenta during placental development were examined by in situ hybridization histochemistry at different embryonic stages (Embryonic days 7.5~16.5). rPsx mRNA was first detected in chorionic ectoderm of placenta at E 10.5. This transcript was localized in labyrinth trophoblast and trophoblast giant cells at E 11.5. Hybridization signals were observed in labyrinth trophoblast, spongiotrophoblast, and trophoblast giant cells at E 12.5, E 13.5, and E 14.5. At E 15.5, hybridization signal was detected in labyrinth trophoblast and spongiotrophoblast but not in trophoblast giant cells. Hybridization signal was only detected in labyrinth trophoblast at E 16.5. rPsx mRNA was not detected in decidua and any tissues of the embryo from E 7.5 to E 9.5 of gestations. From these results, a new rPsx homeobox gene is first expressed at E 10.5 and detected in chorionic ectoderm, labyrinth trophblast, spongiotrophoblast and trophoblast giant cells of the placenta. This gene may play a critical role in differentiation and development of trophoblast cells.

Enhanced Expression of Carbonic anhydrase II in Hypokalemic Rat Kidney / 체질인류학회지

A number of acid-base or electrolyte disorders are associated with decreased or increased HCO3- reabsorption in the renal tubules. The present study was to examine the alterations of expression and distribution of Carbonic anhydrase II in the kidneys of normal and potassium-depleted rats using Western blot analysis and immuno-histochemistry. Western blot analysis demonstrated that CA II protein, ~30 kDa at molecular mass, was abundantly expressed in normal group. All potassium-depleted groups showed slightly increased CA II protein compared to normal group. In control group, immunoreactivity of CA II protein was detected in the entire collecting duct. Signal intensity was prominent in the intercalated cells and weak in the principal cells of the cortical collecting ducts. In potassium-depleted groups, the pattern of cellular labeling of CA II protein was identical to that of normal group, but the signal intensity was decreased in cortical collecting duct, markedly increased in the inner stripe of outer medullary and inner medullary collecting ducts, and unchanged in the outer stripe of outer medullary collecting duct. These results suggest that chronic hypokalemia impact the expression pattern of CA II protein depending the portion of the collecting duct.

Effects of Pilocarpine-induced Seizures on the Neuronal Cell Death, Mossy Fiber Sprouting and Neurogensis in the Hippocampus of Mice / 체질인류학회지

Present study was performed to delineate the inter-relationship among neuronal death, mossy fiber sprouting (MFS) and neurogenesis in hippocampal formation of pilocarpine-treated mice. Status epilepticus was induced by intraperitoneal administration of 300 mg/kg pilocarpine in male ICR and C57BL/6 mouse. The severity of seizure was evaluated using 5 grades of Racine scales for first 4 hr after pilocarpine injection. Fluro-Jade C (FJC) staing, NeoTimm's staining and immunohistochemistry for BrdU were employed to evaluate neuronal cell death, MFS and neurogenesis, respectively. All animals in the present study induced seizures over grade 3 of Racine scale by pilocarpine injection. ICR mice show higher seizure severity (mean Racine scale; 4.37) than C57BL/6 mice do (mean Racine scale; 3.22), while the latency times for the first seizure over Racine scale grade 3 are from 15 min to 20 min and showed no difference between the 2 strains. In ICR mouse, numerous FJC-positive cells in hilus of hippocampus were detected at 4 h after pilocarpine injection, while they were not detected at that time in C57BL/6 mouse. The number of FJC-positive neuronal cells, which were densely found in the pyramidal layer of CA1, CA3 and hilus polymorphic regions of hippocampus, reached peak at 3 days after injection and then few cells were found at 7 days after injection in both strains. In control animals, BrdU positive cells in dentate subgranular layer which represent the hippocampal neurogenesis were more numerous in C57BL/6 than in ICR. The number of BrdU positive cells significantly increased at 2 days after pilocarpine injection and reached the peak at 8 days after injection and returned to control level at 15 day after injection in both strains. The percent increase of the BrdU positive cell was more prominent in ICR mouse. MFS was found at 2 weeks after the injection and the intensity of MFS was getting strong at 4 weeks after injection. There was no differences in MFS grading between 2 strains. These results suggest that there are some inter-relationships among the seizure severity, hippocampal neuronal cell death and hippocampal neurogenesis, but they don't have any significant relationships with the mossy fiber sprouting from dentate granule cells.

Neuroprotection by Valproic Acid in Mouse Models of Permanent and Transient Focal Cerebral Ischemia

Valproic acid (VPA) is a well-known anti-epileptic and mood stabilizing drug. A growing number of reports demonstrate that VPA is neuroprotective against various insults. Despite intensive efforts to develop new therapeutics for stroke over the past two decades, all treatments have thus far failed to show clinical effect because of treatment-limiting side effects of the drugs. Therefore, a safety-validated drug like VPA would be an attractive candidate if it has neuroprotective effects against ischemic insults. The present study was undertaken to examine whether pre- and post-insult treatments with VPA protect against brain infarct and neurological deficits in mouse transient (tMCAO) and permanent middle cerebral artery occlusion (pMCAO) models. In the tMCAO (2 hr MCAO and 22 hr reperfusion) model, intraperitoneal injection of VPA (300 mg/kg, i.p.) 30 min prior to MCAO significantly reduced the infarct size and the neurological deficit. VPA treatment immediately after reperfusion significantly reduced the infarct size. The administration of VPA at 4 hr after reperfusion failed to reduce the infarct size and the neurological deficit. In the pMCAO model, treatment with VPA (300 mg/kg, i.p.) 30 min prior to MCAO significantly attenuated the infarct size, but did not affect the neurological deficit. Western blot analysis of acetylated H3 and H4 protein levels in extracts from the ischemic cortical area showed that treatment with VPA increased the expression of acetylated H3 and H4 at 2 hrs after MCAO. These results demonstrated that treatment with VPA prior to ischemia attenuated ischemic brain damage in both mice tMCAO and pMCAO models and treatment with VPA immediately after reperfusion reduced the infarct area in the tMCAO model. VPA could therefore be evaluated for clinical use in stroke patients.

HOXB13 is co-localized with androgen receptor to suppress androgen-stimulated prostate-specific antigen expression / 대한해부학회지

During the prostate cancer (PCa) development and its progression into hormone independency, androgen receptor (AR) signals play a central role by triggering the regulation of target genes, including prostate-specific antigen. However, the regulation of these AR-mediated target genes is not fully understood. We have previously demonstrated a unique role of HOXB13 homeodomain protein as an AR repressor. Expression of HOXB13 was highly restricted to the prostate and its suppression dramatically increased hormone-activated AR transactivation, suggesting that prostate-specific HOXB13 was a highly potent transcriptional regulator. In this report, we demonstrated the action mechanism of HOXB13 as an AR repressor. HOXB13 suppressed androgen-stimulated AR activity by interacting with AR. HOXB13 did neither bind to AR responsive elements nor disturb nuclear translocation of AR in response to androgen. In PCa specimen, we also observed mutual expression pattern of HOXB13 and AR. These results suggest that HOXB13 not only serve as a DNA-bound transcription factor but play an important role as an AR-interacting repressor to modulate hormone-activated androgen receptor signals. Further extensive studies will uncover a novel mechanism for regulating AR-signaling pathway to lead to expose new role of HOXB13 as a non-DNA-binding transcriptional repressor.

Immunohistochemical Localizaion of Carbonic Anhydrase Isozymes IV and IX in Rat Salivary Gland / 대한해부학회지

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 detectable 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.

Alterations of Aquaporin mRNA and Protein Expression in Hypokalemic Rat Kidney / 대한해부학회지

There has been a general agreement that potassium depletion causes metabolic alkalosis and substantial morphological changes in kidney structure, and is associated with renal functional abnormalities, including a decrease in urinary concentrating ability. The present study was to examine the alterations of expression and distribution of AQP-1, 2, 3 and 4 mRNAs and proteins in the kidneys of normal and K-depleted rats using RT-PCR, Western blot analysis, and immunohistochemistry. Predicted size of AQP-1, 2, 3, and 4 mRNAs was 119, 822, 539, and 642 bp, respectively. AQP-1 mRNA expression was gradually decreased in K-depleted rats, particularly LK 2W. AQP-2, 3 mRNAs were markedly decreased in K-depleted rats. AQP-4 mRNA expression was markedly increased in K-depleted rats, particularly LK 2W. Western blot analysis demonstrated that AQP-1 protein expression was only decreased in LK 3D and others were comparable with normal rat. AQP-2, 3 proteins expression was markedly decreased in K-depleted rats, compared with normal rat. But, AQP-4 protein expression was markedly increased in K-depleted rats, particularly LK 3W. In immunohistochemistry, AQP-1 was detected in the apical membranes of proximal tubules and thin limb of Henle loop. In potassium-depleted kidney, the pattern of cellular labeling and signal intensity of AQP-1 protein is identical to that of normal rat. AQP-2 was detected in apical region and cytoplasm of the principal cells of entire collecting duct. In potassium-depleted kidney, the pattern of cellular labeling of AQP-2 protein is identical to that of normal rat, but signal intensity is markedly decreased. AQP-3 was detected in the bosolateral plasma membrane of principal cells of entire collecting duct. In potassium-depleted kidney, the pattern of cellular labeling of AQP-3 protein is identical to that of normal rat, but signal intensity is markedly decreased. AQP-4 was detected in the bosolateral plasma membrane of principal cells of entire collecting duct. In potassium-depleted kidney, the pattern of cellular labeling of AQP-4 protein is identical to that of normal rat, but signal intensity is markedly increased in outer and inner medullary collecting ducts. In summary, these results demonstrate that chronic hypokalemia shows the different expression pattern of AQP-1, 2, 3, and 4 mRNAs and proteins. These results suggest that a decrease in urinary concentrating ability is a major factor in the decreased AQP-2, 3 expression, and that is partly compensated by increased expression of AQP-4.

Expression and Localization of General Transcription Factor IIA Gene in Hypokalemic Rat / 대한해부학회지

Potassium balance in chronic hypokalemia is regulated by ion channels, ion transporters, and various related genes. We isolated general transcription factor IIA (GTF IIA) gene using a DNA chip microassay, a useful method in cloning genes. Northern analysis and in situ hybridization (ISH) were carried out to analyze the expression and localization of GTF IIA mRNA in rat in relation to the amount of potassium in the diet. Isoform-specific 32P-labeled cDNA (Northern analysis) or digoxigenin-labeled cRNA (ISH) probes were used. Northern analysis demonstrated that GTF IIA mRNA was expressed abundantly in testis; modestly in heart, kidney, lung, adrenal gland, liver, and spleen; and weakly in brain, distal colon, duodenum, salivary gland, and stomach. In potassium-restricted animals, GTF IIA expression was decreased in the kidney, adrenal gland, and spleen, but expression was restored to normal levels in L3w. The expression level in the lung was decreased in L3d and L2w, and increased in L1w and L3w. ISH showed that mRNA for the GTF IIA gene was detected in the distal convoluted tubule, S3 segment of the proximal tubule, and cortical collecting duct in the normal group. In potassium-restricted groups, the hybridization signal was detected in the distal convoluted tubule, S3 segment of the proximal tubule, and entire collecting tubule. The signal intensity of the outer and inner medullary collecting ducts was higher in the potassium-restricted group than in the normal group but was decreased in the distal convoluted tubule and S3 segment of the proximal tubule. In the normal group, mRNA of the GTF IIA gene was detected in the zona glomerulosa cells of the adrenal gland, lymphocytes of the marginal zone, germinal center of the spleen, and bronchial epithelium and lymphocytes of the lung. mRNA for the GTF IIA gene was also detected in the cells of the basal portion of the intestinal glands of the distal colon and stomach, and in spermatogonia and spermatocytes of the seminiferous tubule. These results suggest that expression of GTF IIA differs between various tissues and that increased expression of the GTF IIA gene in the outer and inner medullary collecting ducts of the hypokalemic kidney might regulate the ion transporter genes in these segments.

Immunohistochemical Distribution of Carbonic Anhydrase in Rat Exorbital Lacrimal Gland / 대한해부학회지

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.

Application of Tyramide Signal Amplification (TSA) Both to Biochip Platform and to the Immunoelectron Microscopy to Label Proteins within the Organelle / 대한해부학회지

The tyramide signal amplification (TSA) technique, based on the ability of HRP to catalyze the deposition of tyramide onto the surrounding proteins, has been proved to detect scarce tissue antigens. In this study we applied this technique to a biochip platform and an immunocytochemistry at the electron microscopic level. First, in the optical fluorescence sensing, the signal was amplified by Dako Envision(TM) (goat anti-mouse immunoglobulins IgG conjugated to peroxidase labelled-dextran polymer) and tyramide-Cy3, which was then compared to the non-amplified control using goat antimouse IgG-Cy3 conjugate instead. The result showed that the tyramide method produced a more sensitive signal than the control method. Secondly, in the pre-embedding immunocytochemistry, we investigated to see whether it is possible to label proteins within a organelle in the cell using the TSA method. The signal was amplified by a primary antibody, a biotinylated secondary antibody, streptavidin-HRP, biotinyl-tyramide, and streptavidinnanogold followed by silver enhancement and gold toning. Then, this protocol was compared to the non-amplified or simple protocol that does not include the steps of streptavidin-HRP and biotinyl-tyramide. With the TSA protocol, the labeling for a membrane bound antigen (gp100) that is known to be exclusively localized to melanosomes in melanocyte, was tested in a melanoma cell line (G361) and found to be highly sensitive and more enhanced than with the simple protocol. Moreover, the gold particles were well localized to the subcellular structures or melanosomes both in the TSA and simple protocols, which indicates that resolution of the signals remains high. Control experiment with omission of the primary antibody demonstrated that background levels or nonspecific bindings are negligible. This result showed that the TSA method can be successfully applied to label the intra-organelle protein that is known to be labeled only in the specific fixation condition with the optimal permeability.

Regulations of Bicarbonate Ions in Pregnant Rat Kidney / 대한해부학회지

The marked hemodynamic and hormonal changes of normal pregnancy are associated with striking alterations in renal physiology involving structure, dynamics, tubular function, and volume homeostasis. A number of acid-base or electrolyte disorders are associated with decreased or increased HCO3-reabsorption in the renal tubules. The present study was to examine the alterations of expression and distribution of Na+/HCO3-cotransporter (NBC), Na+/H+ exchanger-3 (NHE-3), and carbonic anhydrase I and II (CA I, II) proteins in the kidneys of non-pregnant (NP) and pregnant rats using Western blot analysis and immunohistochemistry. Sprague-Dawley female rats were studied on days 10 (P 10), 12 (P 12), 14 (P 14), 17 (P 17), and 19 (P 19) of pregnancy. Western blot analysis demonstrated that the expression of NBC, ~110 kDa at molecular mass, was increased in pregnant rats, particularly P 12, compared with NP rat. The expression of NHE-3, ~83 kDa at molecular mass, was increased in pregnant rats, particularly P 12 and P 14. The expression of CA I, ~30 kDa at molecular mass, was decreased in pregnant rats, particularly P 14, but, CA II protein, ~30 kDa at molecular mass, was similar NP rat. In immunohistochemistry, strong immunoreactivity of NBC of NP rat was exclusively detected in the basolateral membranes of S1 and S2 segment of proximal tubules whereas not in S3 segment. In pregnant rats, the pattern of cellular labeling of NBC was identical to that of NP rat, but signal intensity was increased, particularly P 12. In NHE-3, strong immunoreactivity was detected in apical membranes and brush borders of S3 segments and moderate in S1 and S2 segments. In pregnant rats, the pattern of cellular labeling was identical to that of NP rat, but the signal intensity was increased, particularly P 12 and P 14. Expression of CA I and II proteins was detected in entire collecting duct. Signal intensity was prominent in type A intercalated cells and moderate in type B intercalated cells. In pregnant rats, the pattern of cellular labeling of CA I and II proteins was identical to that of non-pregnant rat, but the signal intensity of CA I was decreased in cortical collecting duct, particularly P 14 and CA II was identical to that of NP rat. These results suggest that the regulation of NBC and NHE-3 expressions in the proximal tubules and CA I expression in cortical collecting duct may maintain HCO3-concentration during the pregnancy.

Immunohistochemical Study on the Distribution of Carbonic Anhydrase Isozymes in Pancreatic Islet of Rat / 대한해부학회지

The distribution of carbonic anhydrase (CA) isoenzymes I, II, IV, and IX was investigated in pancreatic islet of the rat using Western blotting analysis and immunohistochemistry. Western blotting analysis demonstrated strong CAI and II expression, but weak CAIV and no CAIX expression. Immunohistochemical reaction of pancreatic islet revealed no staining for CAI and II. CAIV was detected in the peripheral cells of the islet. CAIX was detected in the peripheral cells and occasional in the centrally located cells. Signals for CAIV were observed at the plasma membrane and/or in the cytoplasm of islet cells. Location of CAIV in the A cells was confirmed by subjecting serial sections of pancreas to staining for CAIV and glucagon, which showed colocalization in the A cells. Immunohistochemical staining of pancreatic acinus revealed abundant staining for CAI in interacinar blood vessels and CAII in ductal and acinar cells. These results demonstrate the differential distribution of CA isoenzymes in pancreatic islet, and suggest that A cells of pancreatic islet might contain both CAIV and IX.

Immunohistochemical Study on the Distribution of Carbonic Anhydrase Isozymes in Rat Small Intestine / 대한해부학회지

Carbonic anhydrase catalizes the reversible hydration of carbonic dioxide and participate in various biological processes. There are several isozymes and differ in their kinetic properties, tissue distribution and subcellular localization. The expression of carbonic anhydrase isozymes in digestive tract vary according to animal species and region of the tract. The distribution of carbonic anhydrase (CA) isozymes I, II, IV and IX was investigated in various portions of the rat small intestine using Western blotting analysis and immunohistochemical staining. Western blotting analysis of rat small intestine revealed that CAI was found to be abundantly expressed throughout the small intestine. Expression of CAII in duodenum was much higher than that in jejunum and ileum. Expression of CAIV and IX was found to be weak throughout the small intestine. Immunohistochemical reaction revealed no staining of CAI in all parts of small intestine except blood vessels. CAII was detected at the supranuclear cytoplasm of surface epithelium, but not in intestinal gland. Staining intensity was most strong in the proximal duodenum. CAIV was detected at the apical surface of epithelial cells of villi, and showed most strong staining intensity in the terminal ileum. CAIX was detected at the surfcae epithelium, cells of intestinal gland and Brunner's gland, and the positive reaction was confined to the supranuclear cytoplasm. CAIX differed from CAII in tissue distribution, but subcellular localization of CAIX and II were the same. These results indicate that the surface epithelium of small intestine express CAII, IV and IX, intestinal gland and Brunner's gland express CAIX, and suggest that CAIX may somewhat contribute the control of acid-base balance in the small intestine.

Effect of Maternal Thyroxine Treatment on the Postnatal Development of Brain-derived Neurotrophic Factor-containing Neuron in the Brain of Pups of Alcohol Abused Mother / 대한해부학회지

Maternal alcohol abuse is thought to be the common cause of mental retardation. Especially, continuous alcohol consumption during critical period of brain development induce fetal alcohol effects. In this study, the authors investigated the effects of maternal alcohol drinking on the postnatal changes of BDNF contents and patterns of BDNF-containing neuron in neonatal rat brain, and, the influence of maternal thyroxine treatment on the brain of pups of alcohol abused mother. Pregnant rats were divided into three groups. Alcohol-fed group (n=4) received 35 calories of liquid alcohol diet daily from gestation day 6; control pair-fed group (n=4) was fed a liquid diet in dextrin replaced alcohol isocalorically; alcohol+T4 group (n=4) received 35 calories liquid alcohol diet and exogenous thyroxine (5 microgram/kg/day) subcutaneously. The amount of BDNF was significantly higher in the alcohol+T4 group as compared to the alcohol group at P7, P14 and P21, especially, alcohol+T4-exposed pups showed a significant increase of BDNF at P7. The decrease in BDNF was found in alcohol group compared to control pair-fed group at all ages. In alcohol+T4 group, BDNF-containing Purkinje cells exhibited mature pattern and monolayer arrangement at P14. Alcohol+T4 group showed mature pattern and numerical increase of BDNF-containing cells in cerebral cortex, hypothalamus and hippocampus at P7. The BDNF immunoreactivity of hippocampus continued to show prominent configuration in alcohol+T4 group at P28. These results indicate that the increase of the BDNF-containing neurons and BDNF amount in pups of thyroxinesupplemented alcohol-exposed dams as compared to control pair-fed and alcohol-exposed pups at P7, presumably suggest the early postnatal growth stimulatory effect of the exogenously supplemented thyroxine. Therefore, the increase of BDNF synthesis caused by maternal administration of exogenous thyroxine may ameliorate fetal alcohol effects, one of the ill effects as a result of the dysthyroid state following maternal alcohol abuse.

Adaptive Responses to Dexamethasone Treatment in Rat Kidney Proximal Tubules / 대한해부학회지

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 3 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.

Effect of Insulin Therapy on Vagina in Diabetic Rat / 대한해부학회지

The most commonly reported sexual problems in diabetic women are sexual arousal disorder and a lack of vaginal lubrication. The aims of this study were to investigate the vaginal structural changes and expressions of TGF-beta1, Ec-NOS and estrogen receptor alphaby histochemistry, immunohistochemistry and Western blot analysis in diabetic and insulin-treated diabetic rats. The mean blood glucose levels were significantly increased in the diabetic rats (453+/-88.4 mg/dL)compared to the control group (79+/-6 mg/dL)and insulin-treated diabetic rats (56.7+/-0.6 mg/dL).The vaginal wall in control rat showed 6~11 layered stratified squamous epithelial lining and submucosal smooth muscle, connective tissue and vasculatures. In diabetic rat, the vaginal epithelium was reduced to 2~6 layers and the submucosal vasculatures were decreased n size and number.Collagen fibers were increased and irregularly distorted arrangement. Insulin-treated diabetic rat showed similar morphologic features as control rat.In diabetic rat, TGF-beta1 expression was upregulated by 1.65 times and Ec-NOS expression was 40% downregulated compared to control and insulin-treated diabetic rats in Western blot analysis. In control and insulin-treated diabetic rats, TGF-beta1 immunoreactivity was detected in fibroblasts and the collagen fibers, Ec-NOS immunoreactivity in the endothelial cells of blood vessels, and estrogen receptor alphaimmunoreactivity in the basal and intermediate cell layers of stratified squamous epithelium, smooth muscle fibers, and nerve fibers. In diabetic rat, expression of TGF-beta1, Ec-NOS, and estrogen receptor alphawas exhibited comparable cellular patterns of labeling, but signal intensity was increased in TGF-beta1 and decreased in Ec-NOS and estrogen receptor alpha. These results suggest that vaginal tissue fibrosis in diabetes mellitus may be caused by altered expression of TGF-beta1, NOS and estrogen. It also mplies that sexual arousal disorder and lack of vaginal lubrication in the diabetic women could be protected or delayed by controlling blood glucose levels.

Alterations of Cyclooxygenase-1, 2 Expression in Hypokalemic Rat Kidney / 대한해부학회지

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 metabolic alkalosis and affects the expression of the several ion transporters in rats. The present study was to examine the alterations of expression and distribution of COX-1, 2 mRNAs and proteins in the kidneys of normal and K-depleted rats using RT-PCR, Western blot analysis, and immunohistochemistry. Predicted size of COX-1 mRNA was 306 bp. It's expression was increased in K-depleted rats, particularly LK 2W, but decreased in LK 3D. Predicted size of COX-2 mRNA was 356 bp and it's expression was increased in K-depleted rats, particularly LK 2W. Western blot analysis demonstrated that COX-1 protein, ~70 kDa at molecular mass, was increased in potassium-depleted rats, particularly LK 2W and decreased in LK 3D, compared with normal rat. COX-2 protein, ~72 kDa at molecular mass, was only increased in LK 3D and others were comparable with normal rat. In immunohistochemistry, COX-1 was detected in entire collecting duct, intraglomerular mesangial cells, arterial endothelial cells, medullary interstitial cells, papillary epithelial cells, and pelvic epithelium. Signal intensity of the collecting duct was more increased toward the papillary tip. In K-depleted rat, the pattern of cellular labeling of COX-1 protein was identical to that of normal rat. However, the signal intensity of LK 3D was only decreased in cortical and outer medullary collecting duct and that of LK 2W was increased particularly in the inner stripe of outer medullary collecting duct and proximal 1/3 inner medullary collecting duct. Immunoreactivity of COX-2 of normal rat was detected in the cortical thick ascending limb and macula densa. In K-depleted rat, the pattern of cellular labeling of COX-2 protein was identical to that of normal rat, but the signal intensity was only increased in LK 3D rat. These results suggest that chronic hypokalemia enhances the expression of COX-1, 2 mRNAs and proteins and the regulation of K reabsorption depends on COX-1 rather than COX-2 by the portions of expression.