• Biomedical Science Letters
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• v.13 no.3
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• pp.197-206
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• 2007

P2X receptors are membrane-bound ion channels that conduct $Na^+,\;K^+$, and $Ca^{2+}$ in response to ATP and its analogs. There are seven subunits identified so far ($P2X_1-P2X_7$). $P2X_2$ receptors are known to be expressed in a wide range of organs including brains and adrenal grands. PC12 cells are originated from adrenal grand and differentiated by nerve growth factor or pituitary adenylate cyclase activating poly peptide (PACAP). Previous studies indicate that $P2X_2$ receptor activation in PC12 cells couples to $Ca^{2+}-dependent$ release of catecholamine and ATP. It is known that acidic pH potentiates ATP currents at $P2X_2$ receptors. This leads to a hypothesis that $P2X_2$ receptors may play an important role in PC12 cell differentiation, one of the characteristics of which is neurite outgrowth, induced by the hormones under lower pH. In the present study, we isolated several clones which potentiate neurite outgrowth by PACAP in acidic pH (6.8), but not in alkaline pH (7.6). RT-PCR and electrophysiology data indicate that these clones express only functional $P2X_2$ receptors in the absence or presence of PACAP for 3 days. Potentiation of neurite outgrowth resulted from PACAP (100 nM) in acidic pH is inhibited by the two P2X receptor antagonists, suramin and PPADS ($100\;{\mu}M)$ each), and exogenous exprerssion of ATP-binding mutant $P2X_2$ receptor subunit ($P2X_2[K69A]$). However, acid sensing ion channels (ASICs) are not involved in PACAP-induced neurite outgrowth potentiation in lower pH since treatments of an inhibitor of ASICs, amyloride ($10\;{\mu}M$), did not give any effects to neurite extension. The vesicular proton pump ($H^+-ATPase$) inhibitor, bafilomycin (100 nM), reduced neurite extension indicating that ATP release resulted from $P2X_2$ receptor activation in PC12 cells is needed for neurite outgrowth. These were confirmed by activation of mitogen activated protein kinases, such as ERKs and p38. These results suggest roles of ATP and $P2X_2$ receptors in hormone-induced cell differentiation or neuronal synaptogenesis in local acidic environments.

• The Korean Journal of Physiology and Pharmacology
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• v.5 no.5
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• pp.397-405
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• 2001

The ryanodine receptor, a $Ca^{2+}$ release channel of the sarcoplasmic reticulum (SR), is responsible for the rapid release of $Ca^{2+}$ that activates cardiac muscle contraction. In the excitation-contraction coupling cascade, activation of SR $Ca^{2+}$ release channel is initiated by the activity of sarcolemmal $Ca^{2+}$ channels, the dihydropyridine receptors. Previous study showed that the relaxation defect of diabetic heart was due to the changes of the expressional levels of SR $Ca^{2+}$ATPase and phospholamban. In the diabetic heart contractile abnormalities were also observed, and one of the mechanisms for these changes could include alterations in the expression and/or activity levels of various $Ca^{2+}$ regulatory proteins involving cardiac contraction. In the present study, underlying mechanisms for the functional derangement of the diabetic cardiomyopathy were investigated with respect to ryanodine receptor, and dihydropyridine receptor at the transcriptional and translational levels. Quantitative changes of ryanodine receptors and the dihydropyridine receptors, and the functional consequences of those changes in diabetic heart were investigated. The levels of protein and mRNA of the ryanodine receptor in diabetic rats were comparable to these of the control. However, the binding capacity of ryanodine was significantly decreased in diabetic rat hearts. Furthermore, the reduction in the binding capacity of ryanodine receptor was completely restored by insulin. This result suggests that there were no transcriptional and translational changes but functional changes, such as conformational changes of the $Ca^{2+}$ release channel, which might be regulated by insulin. The protein level of the dihydropyridine receptor and the binding capacity of nitrendipine in the sarcolemmal membranes of diabetic rats were not different as compared to these of the control. In conclusion, in diabetic hearts, $Ca^{2+}$ release processes are impaired, which are likely to lead to functional derangement of contraction of heart. This dysregulation of intracellular $Ca^{2+}$ concentration could explain for clinical findings of diabetic cardiomyopathy and provide the scientific basis for more effective treatments of diabetic patients. In view of these results, insulin may be involved in the control of intracellular $Ca^{2+}$ in the cardiomyocyte via unknown mechanism, which needs further study.

• The Korean Journal of Physiology and Pharmacology
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• v.20 no.1
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• pp.9-14
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• 2016

Adenosine 3',5'-cyclic monophosphate (cAMP) participates in the regulation of numerous cellular functions, including the $Na^+-K^+$-ATPase (sodium pump). Ouabain, used in the treatment of several heart diseases, is known to increase cAMP levels but its effects on the atrium are not understood. The aim of the present study was to examine the effect of ouabain on the regulation of atrial cAMP production and its roles in atrial endothelin-1 (ET-1) secretion in isolated perfused beating rabbit atria. Our results showed that ouabain ($3.0{\mu}mol/L$) significantly increased atrial dynamics and cAMP levels during recovery period. The ouabain-increased atrial dynamics was blocked by KB-R7943 ($3.0{\mu}mol/L$), an inhibitor for reverse mode of $Na^+-Ca^{2+}$ exchangers (NCX), but did not by L-type $Ca^{2+}$ channel blocker nifedipine ($1.0{\mu}mol/L$) or protein kinase A (PKA) selective inhibitor H-89 ($3.0{\mu}mol/L$). Ouabain also enhanced atrial intracellular cAMP production in response to forskolin and theophyline ($100.0{\mu}mol/L$), an inhibitor of phosphodiesterase, potentiated the ouabain-induced increase in cAMP. Ouabain and 8-Bromo-cAMP ($0.5{\mu}mol/L$) markedly increased atrial ET-1 secretion, which was blocked by H-89 and by PD98059 ($30{\mu}mol/L$), an inhibitor of extracellular-signal-regulated kinase (ERK) without changing ouabain-induced atrial dynamics. Our results demonstrated that ouabain increases atrial cAMP levels and promotes atrial ET-1 secretion via the mitogen-activated protein kinase (MAPK)/ERK signaling pathway. These findings may explain the development of cardiac hypertrophy in response to digitalis-like compounds.

• The Korean Journal of Physiology and Pharmacology
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• v.12 no.3
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• pp.101-109
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• 2008

The aim of the present study was to examine the effects of ketamine, a dissociative anesthetics, on secretion of catecholamines (CA) secretion evoked by cholinergic stimulation from the perfused model of the isolated rat adrenal gland, and to establish its mechanism of action, and to compare ketamine effect with that of thiopental sodium, which is one of intravenous barbiturate anesthetics. Ketamine ($30{\sim}300{\mu}M$), perfused into an adrenal vein for 60 min, dose- and time-dependently inhibited the CA secretory responses evoked by ACh (5.32 mM), high $K^+$ (a direct membrane-depolarizer, 56 mM), DMPP (a selective neuronal nicotinic NN receptor agonist, $100{\mu}M$) and McN-A-343 (a selective muscarinic M1 receptor agonist, $100{\mu}M$). Also, in the presence of ketamine ($100{\mu}M$), the CA secretory responses evoked by veratridine (a voltage-dependent $Na^+$ channel activator, $100{\mu}M$), Bay-K-8644 (an L-type dihydropyridine $Ca^{2+}$ channel activator, $10{\mu}M$), and cyclopiazonic acid (a cytoplasmic $Ca^{2+}$-ATPase inhibitor, $10{\mu}M$) were significantly reduced, respectively. Interestingly, thiopental sodium ($100{\mu}M$) also caused the inhibitory effects on the CA secretory responses evoked by ACh, high $K^+$, DMPP, McN-A-343, veratridine, Bay-K-8644, and cyclopiazonic acid. Collectively, these experimental results demonstrate that ketamine inhibits the CA secretion evoked by stimulation of cholinergic (both nicotinic and muscarinic) receptors and the membrane depolarization from the isolated perfused rat adrenal gland. It seems likely that the inhibitory effect of ketamine is mediated by blocking the influx of both $Ca^{2+}$ and $Na^+$ through voltage-dependent $Ca^{2+}$ and $Na^+$ channels into the rat adrenal medullary chromaffin cells as well as by inhibiting $Ca^{2+}$ release from the cytoplasmic calcium store, which are relevant to the blockade of cholinergic receptors. It is also thought that, on the basis of concentrations, ketamine causes similar inhibitory effect with thiopental in the CA secretion from the perfused rat adrenal medulla.

• Applied Microscopy
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• v.19 no.2
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• pp.119-137
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• 1989

It has been shown in this and earlier investigation that the turtle bladder mucosa has three main cell types on their mucosal surface. They are the granular cells, ${\alpha}$ CA cells, and ${\beta}$ CA cells. The three major transport mechanisms that occurs in the turtle bladder are sodium reabsorption, proton secretion, and bicarbonate secretion. In the present work the trans-port mechanisms by bladder epithelial cells of freshwater turtle, Pseudemys scripta, are summarized as follows. 1. The granular cells play an important role in sodium transport, while the ${\alpha}$ and ${\beta}$ CA cells do not appear to play a determining role in sodium transport. 2. It appears that the active sodium transport in the granular cells occurs in two-step process, implying that first, sodium diffuses into the cells, followed by an energy-dependent efflux step, which is catalyzed by the ouabain-sensitive Na-K ATPase. 3. The ${\alpha}$ type of CA cells are responsible for the proton secretion using the proton pump on the apical plasma membrane, while the ${\beta}$ type of CA cells are believed to be responsible for bicarbonate secretion. 4. When looked at under freeze-fracture electron microscopy, the apical plasma membrane of ${\alpha}$ cells have a characteristic population of rod-shaped intramembranous particles which are believed to be components of the proton pumps. Conversely, ${\beta}$ type of CA cells show rod-shaped particles in their basolateral plasma membranes, which is consistent with the proton absorptive, bicarbonate secretory mechanism. 5. In the turtle bladder, the ${\alpha}$ and ${\beta}$ type of cells are believed to be both responsible for proton transport, but in opposite directions.

• Korean Journal of Veterinary Research
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• v.36 no.3
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• pp.551-563
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• 1996

This study investigated the properties of primary cultured proximal tubule cells in hormonally defined(insulin, transferrin, and hydrocortisone), serum-free medium or 10% serum-supplemented medium. The growth rate of the primary cultured proximal tubule cells was lower in the hormonally defined, serum-free medium than in the 10% serum- supplemented medium(p < 0.05), while the activities of brush border marker enzymes, alkaline phosphatase(AP), leucine aminopeptidase(LAP), and y-glutamyl transpeptidase(${\gamma}$-GTP) were increased(p < 0.05). The activities of these enzymes, however, decreased with the lapse of incubation time to 50-70% after 6 days culture compared to those of the freshly-prepared proximal tubules. The enzymatic activities of the primary cultured proximal tubul cells on 6, 9, 12, and 15 days of culture were significantly increased in the hormonally defined, serum-free medium compared to the 10% serum-supplemented medium(p < 0.05). The functional differentiation of the primary culture was examined by observing multicellular domes of the confluent monolayer, which is indicative of transepithelial solute transport. The dome formation by the proximal tubule cultures occurred at a higher frequency in the hormonally defined, serum-free medium than in the 10% serum-supplemented medium(p < 0.05). Upon electron microscopic examination, an increased density of the brush border was observed in the hormonally defined, serum-free medium compared to the cells grown in 10% serum-supplemented medium. The activities of $Na^+$glucose cotransporter($^{14}C$-a-MG uptake), $Na^+$phosphate cotransportere($^{32}P$ uptake) and $Na^+$ transporter($^{22}Na^+$ uptake) in the brush border membrane, and of $Na^+/K^+$-ATPase($^{86}Rb$ uptake) in the basolateral membrane were significantly stimulated in the hormonally defined, serum-free medium than in 10% serum-supplemented medium(p < 0.05). In conclusion, the primary cultured proximal tubule cells grown in the hormonally defined, serum-free medium demonstrated a slower growth rate, but the functions of cell were enhanced.

• Parasites, Hosts and Diseases
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• v.27 no.3
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• pp.161-170
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• 1989

The proteases of Toxoplasma gcndii were purified partially and characterisrd for some biochemical properties including various chromatographic patterns, major catalytic classes, and conditions to promote the activity of these enzymes. When Toxoplasma extract was incubated with 3H-casein at various pH, peak hydrolysis of casein was observed at pH 6.0 and at pH 8.5. Proteasfs working at pH 6.0 and at pH 8.5 were purified partially by conventional methods of chromatographies of DE52 anion rxchange, Sephadex G-200 gel permeation, and hydroxylapatite chromatography. Partially purified enzymes were tested by site-specific inhibitors and promotorf. The protease working at pH 6.0 was inactivated by iodoacetamide with LDso of 10-5 M and promoted by dithiothreitol, while the protease working at pH 8.5 was inhibited by phenylmethylsulfonyl fluoride with LD50 of 10-5 M and was Promoted by ATP (excess ATP beyond 2 mM inhibited the activity reversely). The protease of pH 8.5 had the activity of ATPase which might exert the energy to its action. Therefore the former was referred to as a cysteinyl acid protease and the latter, ATP-dependent neutral serine protease.

• Applied Biological Chemistry
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• v.40 no.6
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• pp.567-571
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• 1997

Numbers of chemical agents which have been shown to inhibit either auxin signal transduction pathway or ethylene formation in plant cells were applied to cut flower stems of snapdragon (Antirrhinum majus L.) and their effects on the postharvest gravitropic response were studied. The chemical treatments were done by submerging either the stem base or the top part of cut flower, which involves the gravistimulus-sensitive region, for 1 h at $25^{\circ}C$. When the chemicals were supplied from the cut stem base, the gravitropic upward bending of flower stalks kept horizontally after the treatments with 20 mM CDTA or 10 mM $CoCl_2$ was comparable to that of the untreated control, but o-vanadate showed a certain degree of effectiveness for suppressing the bending response. In contrast, the direct application of those agents to the gravitropically sensitive region of cut flowers in the presence of 0.01% Triton X-100 resulted in a substantial reduction of the gravitropic response. In the case of 20 mM $CoCl_2$ treatment, almost total elimination of gravitropism without any significant deterioration of flower quality was observed. The results indicate the possibility of preparation of a protocol involving $CoCl_2$ and a proper surfactant for commercial use to suppress the gravitropic response of cut flowers during postharvest storage and transportation.

• Journal of Life Science
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• v.14 no.2
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• pp.325-330
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• 2004

The RAD3 gene of Saccharomyces cerevisiae is required for excision repair and is essential for cell viability. RAD3 encoded protein possesses a single stranded DNA-dependent ATPase and DNA and DNA-RNA helicase activities. To examine the extent of conservation of structure and function of RAD3 during eukaryotic evolution, the RAD3 homolog gene was isolated by screening of genomic DNA library. The isolated gene was designated as HRD3 (Homologue of RAD3 gene). The over-expressed HRD3 protein was estimated to be a 75 kDa in size which is in good agreement with the estimated by the nucleotide sequence of the cloned gene. Two-dimensional gel electrophoresis showed that a number of other protein spots dramatically disappeared when the HRD3 protein was overexpressed. The overexpressed RAD3 protein showed a toxicity in E. coli host, suggesting that this protein may be involved in the inhibition of protein synthesis and/or degradation of host protein. To determine which part of HRD3 gene contributes to the toxicity in E. coli, various fusion plasmids containing a partial sequence of HRD3 and lac'Z gene were constructed. These results suggest that the C-terminal domain of HRD3 protein may be important for both toxic effect in E. coli and for its role in DNA repair in S. pombe.

• Parasites, Hosts and Diseases
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• v.23 no.2
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• pp.269-284
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• 1985

The metabolism of Entamoeba histolytica would be affected by various environmental factors, and alteration of the environment was known to afEect the fine structure of 5. histolytica. The present study was designed electronmicroscopically to investigate the ultrastructure and enzyme activities in the aEonic and conventional strains of 5. histolytica. The trophozoites of axenically cultivated HK-9 strain and conventional YS-27 and YS-49 strains of 5. histolytica were collected and liKed with 4% paraformaldehyde/0.1M cacodylate buffier(pH 74), After washing them by centrifugation, 1% warm agar was added in the sediment. Solidified agar with the trophozoites was cut into $lmm^3$ cubes, and incubated in the various substrates to observe enzyme activities. Then, the specimen was post-fixed with 3% glutaraldehyde/0.1M cacodylate buffer (PH 7.4) and 1% osmium tetroBide/0.1M cacodylate buffier (pH 7.4) , dehydrated in ascending ethanol series and embedded in epoxy resin. These were sectioned on an ultramicrotome and observed with a transmission electronmicroscope. The procedures for the observation of the fine structure were same as the above, except for the incubation in the substrate. The sections were stained with uranyl acetate and lead citrate. For the observation of the surface of the amoebae, scanning-electronmicroscopy was carried out. The results obtained in the present study are summarized as follows: 1. The fuzzy coat around double-layered plasma membrane of 5. histolytica was more irregularly and densely distributed in the conventional strains (YS-27, YS-49 strains) than in the axonic strain (HK-9 strain). 2. The endosomes, button bodies and chromatin material were surrounded by a double-layered nuclear membrane having scattered nuclear fores. The paranuclear body, mono- or double-layered vacuoles, vacuolar membrane whorls, rosette-like cylindrical bodies, aggregation of cylindrical bodies and helical bodies were found in the cytoplasm of the amoebae. Helical bodies and glycogen granules were generally abundant, while a few smooth endoplasmic reticula were observed in the cytoplasm. 3. Alkaline phosphatase activity was mainly demonstrated in the plasma membrane, limiting membranes of vacuoles and smooth endoplasmic reticula. ATPase activity was observed in the nucleus, limiting membranes of vacuoles and vacuolar membrane whorls. 4. Acid phosphatase activity was commonly demonstrated in the limiting membranes an contents of vacuoles, Iysosome-like organelles, plasma membrane and the button bodies in the nucleus. The activity was more weakly demonstrated in the HK-9 strain than in the other conventional strains of 5. histolytica. No peroBidase activity was observed in the amoeba strains employed in the present study. 5. With a scanning electron-microscope, no distinct structural differences were observed between the amoeba strains. All the trophozoite forms of the amoebae showed crater-like depressions and rugged features on the outer surface.