Inhibitory Effects of Ginsenoside Metabolites, Compound K and Protopanaxatriol, on GABAC Receptor-Mediated Ion Currents

Ginsenosides, one of the active ingredients of Panax ginseng, show various pharmacological and physiological effects, and they are converted into compound K (CK) or protopanaxatriol (M4) by intestinal microorganisms. CK is a metabolite derived from protopanaxadiol (PD) ginsenosides, whereas M4 is a metabolite derived from protopanaxatriol (PT) ginsenosides. The gamma-aminobutyric acid receptorC (GABAC) is primarily expressed in retinal bipolar cells and several regions of the brain. However, little is known of the effects of ginsenoside metabolites on GABAC receptor channel activity. In the present study, we examined the effects of CK and M4 on the activity of human recombinant GABAC receptor (rho1) channels expressed in Xenopus oocytes by using a 2-electrode voltage clamp technique. In oocytes expressing GABAC receptor cRNA, we found that CK or M4 alone had no effect in oocytes. However, co-application of either CK or M4 with GABA inhibited the GABA-induced inward peak current (IGABA). Interestingly, pre-application of M4 inhibited IGABA more potently than CK in a dose-dependent and reversible manner. The half-inhibitory concentration (IC50) values of CK and M4 were 52.1+/-2.3 and 45.7+/-3.9 microM, respectively. Inhibition of IGABA by CK and M4 was voltage-independent and non-competitive. This study implies that ginsenoside metabolites may regulate GABAC receptor channel activity in the brain, including in the eyes.

Resveratrol Inhibits GABAC rho Receptor-Mediated Ion Currents Expressed in Xenopus Oocytes

Resveratrol is a phytoalexin found in grapes, red wine, and berries. Resveratrol has been known to have many beneficial health effects, such as anti-cancer, neuroprotective, anti-inflammatory, and life-prolonging effects. However, relatively little is known about the effects of resveratrol on the regulation of ligand-gated ion channels. We have previously reported that resveratrol regulates subsets of homomeric ligand-gated ion channels such as those of 5-HT3A receptors. The gamma-aminobutyric acidC (GABAC) receptor is mainly expressed in retinal bipolar cells and plays an important role in visual processing. In the present study, we examined the effects of resveratrol on the channel activity of homomeric GABAC receptor expressed in Xenopus oocytes injected with cRNA encoding human GABAC rho subunits. Our data show that the application of GABA elicits an inward peak current (IGABA) in oocytes that express the GABAC receptor. Resveratrol treatment had no effect on oocytes injected with H2O or with GABAC receptor cRNA. Co-treatment with resveratrol and GABA inhibited IGABA in oocytes with GABAC receptors. The inhibition of IGABA by resveratrol was in a reversible and concentration-dependent manner. The IC50 of resveratrol was 28.9+/-2.8 microM in oocytes expressing GABAC receptor. The inhibition of IGABA by resveratrol was in voltage-independent and non-competitive manner. These results indicate that resveratrol might regulate GABAC receptor expression and that this regulation might be one of the pharmacological actions of resveratrol on the nervous system.

Effects of Protopanaxatriol-Ginsenoside Metabolites on Rat N-Methyl-D-Aspartic Acid Receptor-Mediated Ion Currents

Ginsenosides are low molecular weight glycosides found in ginseng that exhibit neuroprotective effects through inhibition of N-methyl-D-aspartic acid (NMDA) receptor channel activity. Ginsenosides, like other natural compounds, are metabolized by gastric juices and intestinal microorganisms to produce ginsenoside metabolites. However, little is known about how ginsenoside metabolites regulate NMDA receptor channel activity. In the present study, we investigated the effects of ginsenoside metabolites, such as compound K (CK), protopanaxadiol (PPD), and protopanaxatriol (PPT), on oocytes that heterologously express the rat NMDA receptor. NMDA receptor-mediated ion current (INMDA) was measured using the 2-electrode voltage clamp technique. In oocytes injected with cRNAs encoding NMDA receptor subunits, PPT, but not CK or PPD, reversibly inhibited INMDA in a concentration-dependent manner. The IC50 for PPT on INMDA was 48.1+/-4.6 microM, was non-competitive with NMDA, and was independent of the membrane holding potential. These results demonstrate the possibility that PPT interacts with the NMDA receptor, although not at the NMDA binding site, and that the inhibitory effects of PPT on INMDA could be related to ginseng-mediated neuroprotection.

Inhibitory Effects of Quercetin on Muscle-type of Nicotinic Acetylcholine Receptor-Mediated Ion Currents Expressed in Xenopus Oocytes

The flavonoid quercetin is a low molecular weight compound generally found in apple, gingko, tomato, onion and other red-colored fruits and vegetables. Like other flavonoids, quercetin has diverse pharmacological actions. However, relatively little is known about the influence of quercetin effects in the regulation of ligand-gated ion channels. Previously, we reported that quercetin regulates subsets of nicotinic acetylcholine receptors such as alpha3beta4, alpha7 and alpha9alpha10. Presently, we investigated the effects of quercetin on muscle-type of nicotinic acetylcholine receptor channel activity expressed in Xenopus oocytes after injection of cRNA encoding human fetal or adult muscle-type of nicotinic acetylcholine receptor subunits. Acetylcholine treatment elicited an inward peak current (IACh) in oocytes expressing both muscle-type of nicotinic acetylcholine receptors and co-treatment of quercetin with acetylcholine inhibited IACh. Pre-treatment of quercetin further inhibited IACh in oocytes expressing adult and fetal muscle-type nicotinic acetylcholine receptors. The inhibition of IACh by quercetin was reversible and concentration-dependent. The IC50 of quercetin was 18.9+/-1.2 microM in oocytes expressing adult muscle-type nicotinic acetylcholine receptor. The inhibition of IACh by quercetin was voltage-independent and non-competitive. These results indicate that quercetin might regulate human muscle-type nicotinic acetylcholine receptor channel activity and that quercetin-mediated regulation of muscle-type nicotinic acetylcholine receptor might be coupled to regulation of neuromuscular junction activity.

Quercetin Inhibits alpha3beta4 Nicotinic Acetylcholine Receptor-Mediated Ion Currents Expressed in Xenopus Oocytes

Quercetin mainly exists in the skin of colored fruits and vegetables as one of flavonoids. Recent studies show that quercetin, like other flavonoids, has diverse pharmacological actions. However, relatively little is known about quercetin effects in the regulations of ligand-gated ion channels. In the previous reports, we have shown that quercetin regulates subsets of homomeric ligand-gated ion channels such as glycine, 5-HT3A and alpha7 nicotinic acetylcholine receptors. In the present study, we examined quercetin effects on heteromeric neuronal alpha3beta4 nicotinic acetylcholine receptor channel activity expressed in Xenopus oocytes after injection of cRNA encoding bovine neuronal alpha3 and beta4 subunits. Treatment with acetylcholine elicited an inward peak current (IACh) in oocytes expressing alpha3beta4 nicotinic acetylcholine receptor. Co-treatment with quercetin and acetylcholine inhibited IACh in oocytes expressing alpha3beta4 nicotinic acetylcholine receptors. The inhibition of IACh by quercetin was reversible and concentration-dependent. The half-inhibitory concentration (IC50) of quercetin was 14.9+/-0.8 microM in oocytes expressing alpha3beta4 nicotinic acetylcholine receptor. The inhibition of IACh by quercetin was voltage-independent and non-competitive. These results indicate that quercetin might regulate alpha3beta4 nicotinic acetylcholine receptor and this regulation might be one of the pharmacological actions of quercetin in nervous systems.

Screening of virulence gene in golden hamster cheek pouch mucosa carcinomatous change induced by 9,10-dimethylene-1,2-benzanthracene / 中华口腔医学杂志

<p><b>OBJECTIVE</b>To examine and analyze the global gene expression at the different stages of golden hamster cheek pouch mucosa carcinomatous change induced by 9,10-dimethylene-1,2 benzanthracene (DMBA).</p><p><b>METHODS</b>The model of golden hamster cheek pouch squamous cell carcinoma was induced by DMBA. The RNA of normal mucosa, precancerous lesions and squamous cell carcinoma of fresh tissue of golden hamsters was extracted and purified and the cRNA labeled by fluorescent Cy3 synthesized, which respectively hybridized with the agilent rat cDNA microarray containing 41 000 genes-expressed sequence tags, scanning with Agilent G2565AA fluorescence scanner. The Ratio>or=2 and Ratio<or=0.5 were used to determine the threshold value of the differences gene expression and analyze the relevant biological information of the three different stages of cancer. The Venn diagram by Gene Spring 10.0 analysis software was then used to select the genes of continuing expression in the three different stages of cancer. Meanwhile, RT-PCR was used to confirm the correctness of the results of the gene chip.</p><p><b>RESULTS</b>A total of 5255 differentially expressed genes were detected during the process from normal mucosa to the squamous cell carcinoma, of which 2896 was up-regulated and 2359 down-regulated. There were 22 genes that showed continues abnormal expression through the three different stages of carcinomatous change, including 3 up-regulated, 19 down-regulated. The RT-PCR results of Eaf-2 and Ecg-2 were consistent with the gene chip.</p><p><b>CONCLUSIONS</b>The development of oral mucosal squamous cell carcinoma involved a number of abnormal genes. The genes showing continues abnormal expression at different stages of carcinomatous change may be the important pathogenetic ones.</p>

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.

Electromagnetic field change the expression of osteogenesis genes in murine bone marrow mesenchymal stem cells / 华中科技大学学报（医学）（英德文版）

In order to identify the differentially expressing gene of bone marrow mesenchymal stem cells (MSCs) stimulated by electromagnetic field (EMF) with osteogenesis microarray analysis, the bone marrow MSCs of SD rats were isolated and cultured in vitro. The third-passage cells were stimulated by EMFs and total RNA was extracted, purified and then used for the synthesis of cDNA and cRNA. The cRNA of stimulated group and the control group was hybridized with the rat oligo osteogenesis microarray respectively. The hybridization signals were acquired by using X-ray film after chemiluminescent detection and the data obtained were analyzed by employing the web-based completely integrated GEArray Expression Analysis Suite. RT-PCR was used to identify the target genes: Bmp1, Bmp7, Egf and Egfr. The results showed that 19 differentially expressing genes were found between the stimulated group and the control group. There were 6 up-regulated genes and 13 down-regulated genes in the stimulated group. Semi-quantitative RT-PCR confirmed that the expressions of Bmp1, Bmp7 mRNA of the stimulated group were up-regulated (P<0.05) and those of Egf, Egfr were down-regulated (P<0.05). It was suggested that the gene expression profiles of osteogenesis of the bone marrow MSCs were changed after EMF treatment. It is concluded that the genes are involved in skeletal development, bone mineral metabolism, cell growth and differentiation, cell adhesion etc.

Rapid, reliable and inexpensive quality assessment of biotinylated cRNA

The interpretation of oligonucleotide array experiments depends on the quality of the target cRNA used. cRNA target quality is assessed by quantitative analysis of the representation of 5' and 3' sequences of control genes using commercially available Test arrays. The Test array provides an economically priced means of determining the quality of labeled target prior to analysis on whole genome expression arrays. This manuscript validates the use of a duplex RT-PCR assay as a faster (6 h) and less expensive (6 were chosen and classified as degraded cRNAs, and 31 samples with a ß-actin 3'/5' ratio <6 were selected as good quality cRNAs. Blinded samples were then used for the RT-PCR assay. After gel electrophoresis, optical densities of the amplified 3' and 5' fragments of ß-actin were measured and the 3'/5' ratio was calculated. There was a strong correlation (r² = 0.6802) between the array and the RT-PCR ß-actin 3'/5' ratios. Moreover, the RT-PCR 3'/5' ratio was significantly different (P < 0.0001) between undegraded (mean ± SD, 0.34 ± 0.09) and degraded (1.71 ± 0.83) samples. None of the other parameters analyzed, such as i) the starting amount of RNA, ii) RNA quality assessed using the Bioanalyzer Chip technology, or iii) the concentration and OD260/OD280 ratio of the purified biotinylated cRNA, correlated with cRNA quality.

Gene chip analyses reveal differential genetic responses to iron deficiency in rat duodenum and jejunum

Previous studies revealed novel genetic changes in the duodenal mucosa of iron-deprived rats during post-natal development. These observations are now extended to compare the genetic response to iron deficiency in the duodenum versus jejunum of 12-wk-old rats. cRNA samples were prepared from the duodenal and jejunal mucosa of three groups each of control and iron-deficient rats and hybridized with RAE 230A and 230B gene chips (Affymetrix). Stringent data reduction strategies were employed. Results showed that several genes were similarly induced in both gut segments, including DMT1, Dcytb, transferrin receptor 1, heme oxygenase 1, metallothionein, the Menkes copper ATPase (ATP7A), tripartitie motif protein 27, and the sodium-dependent vitamin C transporter. However, a subset of genes showed regulation in only one or the other gut segment. In duodenum only, gastrokine 1, trefoil factor 1 and claudin 2 were induced by iron-deficiency. Other genes previously identified were only regulated in the duodenum. Overall, these studies demonstrate similarities and distinct differences in the genetic response to iron deprivation in the duodenum versus jejunum and provide evidence that more distal gut segments also may play a role in increasing iron absorption in iron-deficiency anemia.

Electrophysiological characterization of long QT syndrome associated mutations V630A and N633S / 中华心血管病杂志

<p><b>OBJECTIVE</b>To identify the electrophysiological properties of long-QT syndrome (LQTS) associated missense mutations in the outer mouth of the HERG potassium channel in vitro.</p><p><b>METHODS</b>Mutations V630A and N633S were constructed by Megaprimer PCR method and cRNA were produced by T7 RNA polymerase. The electrophysiological properties of the mutation were investigated in the Xenopus oocyte heterologous expression system.</p><p><b>RESULTS</b>Coexpression of mutant and wild-type HERG subunits caused a dominant-negative effect, and the currents were significantly decreased. Compared with wild-type HERG channels, V630A and N633S mutations were related to decreased time constants for inactivation for V630A/WT and N633S/WT at all potentials, reduced slope conductance and the voltage dependence of steady-state inactivation was shifted to negative potentials for V630A/WT and N633S/WT.</p><p><b>CONCLUSION</b>Present study shows that LQTS associated missense mutations located in the outer mouth of HERG cause a dominant-negative effect and alterations in steady-state voltage dependence of channel gating of heteromultimeric channels suggesting a reduction in expressional current might be one of the pathophysiologic mechanisms of LQTS.</p>

Microarray Analysis of Oxygen-Glucose-Deprivation Induced Gene Expression in Cultured Astrocytes

Since astrocytes were shown to play a central role in maintaining neuronal viability both under normal conditions and during stress such as ischemia, studies of the astrocytic response to stress are essential to understand many types of brain pathology. The microarray system permitted screening of large numbers of genes in biological or pathological processes. Therefore, the gene expression patterns in the in vitro model of astrocytes following exposure to oxygen-glucose deprivation (OGD) were evaluated by using the microarray analysis. Primary astrocytic cultures were prepared from postnatal Swiss Webster mice. The cells were exposed to OGD for 4 hrs at 37 degrees C prior to cell harvesting. From the cultured cells, we isolated mRNA, synthesized cDNA, converted to biotinylated cRNA and then reacted with GeneChips. The data were normalized and analyzed using dChip and GenMAPP tools. After 4 hrs exposure to OGD, 4 genes were increased more than 2 folds and 51 genes were decreased more than 2 folds compared with the control condition. The data suggest that the OGD has general suppressive effect on the gene expression with the exception of some genes which are related with ischemic cell death directly or indirectly. These genes are mainly involved in apoptotic and protein translation pathways and gap junction component. These results suggest that microarray analysis of gene expression may be useful for screening novel molecular mediators of astrocyte response to ischemic injury and making profound understanding of the cellular mechanisms as a whole. Such a screening technique should provide insights into the molecular basis of brain disorders and help to identify potential targets for therapy.

Co-localization and interaction of organic anion transporter 1 with caveolin-2 in rat kidney

The organic anion transporters (OAT) have recently been identified. Although the some transport properties of OATs in the kidney have been verified, the regulatory mechanisms for OAT's functions are still not fully understood. The rat OAT1 (rOAT1) transports a number of negatively charged organic compounds between the cells and their extracellular milieu. Caveolin (Cav) also plays a role in membrane transport. Therefore, we investigated the protein-protein interactions between rOAT1 and caveolin-2. In the rat kidney, the expressions of rOAT1 mRNA and protein were observed in both the cortex and the outer medulla. With respect to Cav-2, the expressions of mRNA and protein were observed in all portions of the kidney (cortex < outer medulla = inner medulla). The results of Western blot analysis using the isolated caveolae-enriched membrane fractions or the immunoprecipitates by respective antibodies from the rat kidney showed that rOAT1 and Cav-2 co-localized in the same fractions and they formed complexes each other. These results were confirmed by performing confocal microscopy with immunocytochemistry using the primary cultured renal proximal tubular cells. When the synthesized cRNA of rOAT1 along with the antisense oligodeoxynucleotides of Xenopus Cav-2 were co-injected into Xenopus oocytes, the [14C]p-aminohippurate and [3H]methotrexate uptake was slightly, but significantly decreased. The similar results were also observed in rOAT1 over-expressed Chinese hamster ovary cells. These findings suggest that rOAT1 and caveolin-2 are co-expressed in the plasma membrane and rOAT1's function for organic compound transport is upregulated by Cav-2 in the normal physiological condition.

Inhibitory effect of rhynchophylline on human ether-a-go-go related gene channel / 生理学报

We studied the effects of Chinese traditional medicine rhynchophylline (Rhy) on human ether-a-go-go related gene (HERG) channel and characterized the electrophysiological properties of Rhy's pharmacological effect on HERG channel using Xenopus oocytes. Xenopus oocytes were injected with either 23 nl (5.75 ng) HERG cRNA or 23 nl distilled water. Xenopus oocytes were randomly assigned to receive one of the following different concentrations of Rhy: (1) control, (2)10 mumol/L Rhy, (3)100 mumol/L Rhy, (4) 500 mumol/L Rhy, (5) 1 000 mumol/L Rhy, (6) 10 000 mumol/L Rhy. Cell currents were recorded in oocytes. The peak tail currents of HERG channel were inhibited by Rhy. The inhibition was in a dose-dependent manner [IC(50)=(773.4 +/- 42.5) mumol/L]. Experiment with 100 mumol/L Rhy indicated that the degree of HERG blockade showed some voltage dependence (within -40 mV to -20 mV ). Kinetic analyses revealed that Rhy decreased the rate of channel activation. The findings indicate that Rhy inhibits HERG encoded potassium channels. It may underline the molecular mechanism of myocardial electrophysiological characteristics associated with this drug.

Gene Expression Analysis of Murine Primary Microglia Stimulated with LPS using Microarray

BACKGROUND: Since heightened microglial activation was shown to play a role in the pathogenesis of many brain disorders, understanding the molecular mechanisms of microglial activation may lead to new treatment strategies. The microarray system permitted screening of large numbers of genes in biological or pathological processes. Therefore, we evaluated the gene expression pattern during microglial activation using microarray analysis. METHODS: Primary microglial cultures were prepared from postnatal Swiss Webster mice. The cells were activated by lipopolysaccharide (LPS, 10 microgram/ml) for 5 hours prior to cell harvesting. From the cultured cells, we isolated mRNA, synthesized cDNA, converted to biotinylated cRNA and then reacted with GeneChips (Affymetrix MU74A-v2). The data were normalized and analyzed. RESULTS: After microglial activation with LPS, we found >4 fold increases in the expression of 139 genes and >4 fold decreases of 16 genes expression compared with control. Most of the induced or suppressed genes were known to regulate inflammation, immune reactions, injury responses, cell death or survival related mechanisms. CONCLUSIONS: These results suggest that microarray analysis of gene expression may be useful for screening novel molecular mediators of microglial activation and making profound understanding of the cellular mechanisms as a whole. Such screening techniques should provide insights into the molecular basis of brain disorders and help to identify potential targets for therapy.

Expression of Aquaporin-8 in Placenta of Preeclampsia / 대한산부인과학회잡지

OBJECTIVE: This study was to determine whether aquaporin-8, which plays a role as a transcellular water channel, is expressed in human placenta, and to compare the degree of its expression between preeclamptic women and normal pregnant women. METHODS: Placentas were obtained from severely preeclamptic women and normal pregnant women who were delivered babies by cesarean section before the onset of labor in the Chungbuk National University Hospital. In situ hybridization with aquaporin-8 cRNA probe was performed using paraffin-embedded tissue section. Signal of aquaporin-8 expression was observed with light microscope. RESULTS: In situ hybridization demonstrated strong expression of aquaporin-8 mRNA in the placentas of both preeclamptic women and normal pregnant women. The degree of expression was the same in both group. CONCLUSION: Aquaporin-8 in human placenta may not be related to the pathogenesis of preeclampsia.

Protein kinase A-mediated phosphorylation of HERG potassium channels in a human cell line / 中华医学杂志(英文版)

<p><b>OBJECTIVE</b>To investigate the molecular mechanism of human ether-a-go-go-related gene (HERG) potassium channels regulated by protein kinase A (PKA) in a human cell line.</p><p><b>METHODS</b>HERG channels were stably expressed in human embryonic kidney (HEK) 293 cells, and currents were measured with the patch clamp technique. The direct phosphorylation of HERG channel proteins expressed heterologously in Xenopus laevis oocytes was examined by (32)P labeling and immunoprecipitation with an anti-HERG antibody.</p><p><b>RESULTS</b>Elevation of the intracellular cAMP-concentration by incubation with the adenylate cyclase activator, forskolin (10 micromol/L), and the broad range phosphodiesterase inhibitor, IBMX (100 micromol/L), caused a HERG tail current reduction of 83.2%. In addition, direct application of the membrane permeable cAMP analog, 8-Br-cAMP (500 micromol/L), reduced the tail current amplitude by 29.3%. Intracellular application of the catalytic subunit of protein kinase A (200 U/ml) led to a tail current decrease by 56.9% and shifted the activation curve by 15.4 mV towards more positive potentials. HERG WT proteins showed two phosphorylated bands, an upper band with a molecular mass of approximately 155 kDa and a lower band with a molecular mass of approximately 135 kDa, indicating that both the core- and the fully glycosylated forms of the protein were phosphorylated.</p><p><b>CONCLUSIONS</b>PKA-mediated phosphorylation of HERG channels causes current reduction in a human cell line. The coupling between the repolarizing cardiac HERG potassium current and the protein kinase A system could contribute to arrhythmogenesis under pathophysiological conditions.</p>

Functional abnormalities of HERG mutations in long QT syndrome 2 (LQT2)

The chromosome 7-linked long QT syndrome (LQT2) is caused by mutations in the human ether-a-go-go-related gene (HERG) that encodes the rapidly activating delayed rectifier K+ current, IKr, in cardiac myocytes. Different types of mutations have been identified in various locations of HERG channel. One of the mechanisms for the loss of normal channel function is due to membrane trafficking of channel protein. The decreased channel function in some deletion mutants appears to be due to loss of coupling with wild type HERG to form the functional channel as the tetramer. Most of missense mutants with few exceptions could interact with wild type HERG to form functional tetramer and caused dominant negative suppression with co-injection with wild type HERG showing variable effects on current amplitude, voltage dependence, and kinetics of activation and inactivation. Two missense mutants at pore regions of HERG found in Japanese LQT2 (A614V and V630L) showed accentuated inward rectification due to a negative shift in steady-state inactivation and fast inactivation. One mutation in S4 region (R534C) produced a negative shift in current activation, indicating the S4 serving as the voltage sensor and accelerated deactivation. The C-terminus mutation, S818L, could not express the current by mutant alone and did not show dominant negative suppression with co-injection of equal amount of wild type cRNA. Co-injection of excess amount of mutant with wild type produced dominant negative suppression with a shift in voltage dependent activation. Therefore, multiple mechanisms are involved in different mutations and functional abnormality in LQT2. Further characterization with the interactions between various mutants in HERG and the regulatory subunits of the channels (MiRP1 and minK) is to be clarified.

Inhibitory Effects of Structurally Different Neuromuscular Blockers on the Serotonin Type 3 Receptor Expressed in Xenopus Oocytes / 대한마취과학회지

BACKGROUND: The serotonin type 3 receptors are diffusely distributed in both the central and the peripheral nervous system. Physiological and pathophysiological processes thought to be mediated by this receptor include nausea and vomiting, peripheral nociception and central antinociception, conditioned aversion response to drugs, anxiety, and cognition. Because of the structural similarity between the nicotinic acetylcholine receptor and the 5HT3 receptor, we investigated the effects of clinically used neuromuscular blockers on the 5HT3 receptor function related with PONV. METHODS: A cDNA clone encoding the full length murine 5HT3a receptor was subcloned into an oocyte expression vector and 50 ng of cRNA transcribed in vitro injected per oocyte. After 24 72 h incubation, oocytes were placed into a recording chamber continuously perfused with frog Ringer's solution and electrophysiological recordings were obtained by the two electrode voltage clamp technique. Serotonin with or without the various drugs were bath applied by a computer controlled solenoid valve. Peak currents induced by the drug applications were measured and dose responses were obtained. RESULTS: The 5HT3 receptor expression in Xenopus oocyte was identified by the pharmacologic tools. Serotonin induced rapid inward currents, and thus was showed dose-dependent: KD = 2.5 micrometer, Hill coefficiency = 2.09. Inhibition by the neuromuscular blockers showed dose-dependence and their inhibitory potency on 5HT3 receptor (IC50) was in order of d-tubocurarine (0.046 micrometer) > vecuronium (16.32 micrometer) > gallamine (1,169 micrometer). CONCLUSIONS: There was a different inhibitory effect of nicotinic cholinergic antagonists, clinically used neuromuscular blockers, on the 5HT3 receptor and a judicious selection of them might contribute to reducing the incidence of PONV clinically.

Inhibitory Effects of Structurally Different Neuromuscular Blockers on the Serotonin Type 3 Receptor Expressed in Xenopus Oocytes / 대한마취과학회지

BACKGROUND: The serotonin type 3 receptors are diffusely distributed in both the central and the peripheral nervous system. Physiological and pathophysiological processes thought to be mediated by this receptor include nausea and vomiting, peripheral nociception and central antinociception, conditioned aversion response to drugs, anxiety, and cognition. Because of the structural similarity between the nicotinic acetylcholine receptor and the 5HT3 receptor, we investigated the effects of clinically used neuromuscular blockers on the 5HT3 receptor function related with PONV. METHODS: A cDNA clone encoding the full length murine 5HT3a receptor was subcloned into an oocyte expression vector and 50 ng of cRNA transcribed in vitro injected per oocyte. After 24 72 h incubation, oocytes were placed into a recording chamber continuously perfused with frog Ringer's solution and electrophysiological recordings were obtained by the two electrode voltage clamp technique. Serotonin with or without the various drugs were bath applied by a computer controlled solenoid valve. Peak currents induced by the drug applications were measured and dose responses were obtained. RESULTS: The 5HT3 receptor expression in Xenopus oocyte was identified by the pharmacologic tools. Serotonin induced rapid inward currents, and thus was showed dose-dependent: KD = 2.5 micrometer, Hill coefficiency = 2.09. Inhibition by the neuromuscular blockers showed dose-dependence and their inhibitory potency on 5HT3 receptor (IC50) was in order of d-tubocurarine (0.046 micrometer) > vecuronium (16.32 micrometer) > gallamine (1,169 micrometer). CONCLUSIONS: There was a different inhibitory effect of nicotinic cholinergic antagonists, clinically used neuromuscular blockers, on the 5HT3 receptor and a judicious selection of them might contribute to reducing the incidence of PONV clinically.