Biomedical subjects

Taku Nagao

Publications and source records attributed to Taku Nagao.

At least 19 recordsLinked to original sources

PubMed2006

TRPC3 and TRPC6 are essential for angiotensin II-induced cardiac hypertrophy.

Angiotensin (Ang) II participates in the pathogenesis of heart failure through induction of cardiac hypertrophy. Ang II-induced hypertrophic growth of cardiomyocytes is mediated by nuclear factor of activated T cells (NFAT), a Ca(2+)-responsive transcriptional factor. It is believed that phospholipase C (PLC)-mediated production of inositol-1,4,5-trisphosphate (IP(3)) is responsible for Ca(2+) increase that is necessary for NFAT activation. However, we demonstrate that PLC-mediated production of diacylglycerol (DAG) but not IP(3) is essential for Ang II-induced NFAT activation in rat cardiac myocytes. NFAT activation and hypertrophic responses by Ang II stimulation required the enhanced frequency of Ca(2+) oscillation triggered by membrane depolarization through activation of DAG-sensitive TRPC channels, which leads to activation of L-type Ca(2+) channel. Patch clamp recordings from single myocytes revealed that Ang II activated DAG-sensitive TRPC-like currents. Among DAG-activating TRPC channels (TRPC3, TRPC6, and TRPC7), the activities of TRPC3 and TRPC6 channels correlated with Ang II-induced NFAT activation and hypertrophic responses. These data suggest that DAG-induced Ca(2+) signaling pathway through TRPC3 and TRPC6 is essential for Ang II-induced NFAT activation and cardiac hypertrophy.

Angiotensin II↗

PubMed2006

Extracellular ATP counteracts the ERK1/2-mediated death-promoting signaling cascades in astrocytes.

Oxidative stress is the main cause of neuronal death in pathological conditions. Hydrogen peroxide (H(2)O(2)), one of the reactive oxygen species, activates many intracellular signaling cascades including src family and mitogen-activated protein kinases (MAPKs), some of which are critically involved in the induction of cellular damage. We previously showed that H(2)O(2)-induced cell death in astrocytes and adenosine 5(')-triphosphate (ATP), acting on P2Y(1) receptors, had a protective effect. Here, we examined the H(2)O(2)-induced changes in intracellular signaling cascades that promote cell death in astrocytes, showing the molecular mechanisms by which the activation of P2Y(1) receptors counteracts such signals. Although H(2)O(2) activated three MAPKs including ERK1/2, p38, and JNK, only the activation of ERK1/2 participated in the H(2)O(2)-evoked cell death. H(2)O(2) induced a sustained activation of ERK1/2 mainly in the nucleus region, which was well in accordance with the H(2)O(2)-induced cell death. H(2)O(2) also activated the src tyrosine kinase family, which was an upstream signal for ERK1/2. Activation of P2Y(1) receptors by 2methylthio-ADP (2MeSADP) inhibited the H(2)O(2)-evoked activation of src tyrosine kinase, resulting in the inhibition of the phosphorylated-ERK1/2 accumulation in the nucleus. 2MeSADP enhanced the gene expression and activity of protein tyrosine phosphatase (PTP), which was responsible for the inhibition of src tyrosine kinase. Thioredoxin reductase, another cytoprotective gene we previously showed to be upregulated by 2MeSADP, also controlled the activity of PTP. Taken together, ATP, acting on P2Y(1) receptors, upregulates the PTP expression and its activity, which counteracts the H(2)O(2)-promoted death signaling cascades including ERK1/2 and its upstream signal src tyrosine kinase in astrocytes.

Adenosine Diphosphate↗

PubMed2006

Retinoic acids increase P2X2 receptor expression through the 5'-flanking region of P2rx2 gene in rat phaeochromocytoma PC-12 cells.

The P2X2 receptor is a subtype of ionotropic ATP receptor and plays a significant role in regulating fast synaptic transmission in the nervous system. Because the expression level of the P2X2 receptor is known to determine its channel properties and functional interactions with other neurotransmitter channels, elucidating the mechanisms underlying the regulation of P2X2 receptor expression in neuronal cells is important. Here, we identified three motifs that correspond to the retinoic acid response element in the 5'-flanking region of the rat P2X2 gene. In rat pheochromocytoma PC-12 cells, treatment with 9-cis-retinoic acid as well as all-trans-retinoic acid significantly increased the mRNA and protein level of P2X2 receptor. In addition, in PC-12 cells transiently transfected with a luciferase reporter gene driven by the promoter region of the rat P2X2 gene, both 9-cis-retinoic acid and all-trans-retinoic acid increased the luciferase activity, whereas their effects were diminished by truncation of the retinoic acid response elements in the promoter. Furthermore, 9-cis-retinoic acid enhanced the ATP-evoked whole cell currents and intracellular Ca2+- and ATP-evoked dopamine release, indicating the up-regulation of functional P2X2 receptors on the plasma membrane. These results provide the molecular mechanism underlying the transcriptional regulation of P2X2 receptors and suggest that retinoid is an important factor in regulating P2X2 receptors in the nervous system.

5' Flanking Region↗

PubMed2006

"Per cell" normalization method for mRNA measurement by quantitative PCR and microarrays.

BACKGROUND: Transcriptome data from quantitative PCR (Q-PCR) and DNA microarrays are typically obtained from a fixed amount of RNA collected per sample. Therefore, variations in tissue cellularity and RNA yield across samples in an experimental series compromise accurate determination of the absolute level of each mRNA species per cell in any sample. Since mRNAs are copied from genomic DNA, the simplest way to express mRNA level would be as copy number per template DNA, or more practically, as copy number per cell. RESULTS: Here we report a method (designated the "Percellome" method) for normalizing the expression of mRNA values in biological samples. It provides a "per cell" readout in mRNA copy number and is applicable to both quantitative PCR (Q-PCR) and DNA microarray studies. The genomic DNA content of each sample homogenate was measured from a small aliquot to derive the number of cells in the sample. A cocktail of five external spike RNAs admixed in a dose-graded manner (dose-graded spike cocktail; GSC) was prepared and added to each homogenate in proportion to its DNA content. In this way, the spike mRNAs represented absolute copy numbers per cell in the sample. The signals from the five spike mRNAs were used as a dose-response standard curve for each sample, enabling us to convert all the signals measured to copy numbers per cell in an expression profile-independent manner. A series of samples was measured by Q-PCR and Affymetrix GeneChip microarrays using this Percellome method, and the results showed up to 90 % concordance. CONCLUSION: Percellome data can be compared directly among samples and among different studies, and between different platforms, without further normalization. Therefore, "percellome" normalization can serve as a standard method for exchanging and comparing data across different platforms and among different laboratories.

Animals↗

PubMed2006

Utilization of a one-dimensional score for surveying chemical-induced changes in expression levels of multiple biomarker gene sets using a large-scale toxicogenomics database.

A large-scale toxicogenomcis database has now been constructed in the Toxicogenomics Project in Japan (TGP). To facilitate the analytical procedures for such large-scale microarray data, we developed a simple one-dimensional score, named TGP1 which expresses the trend of the changes in expression of biomarker genes as a whole. To evaluate the usefulness of the TGP1 score, microarray data of rat liver and rat hepatocytes deposited in the TGP database were scored for three biomarker gene sets, i.e., carcinogenesis-related, PPARalpha-regulated and glutathione depletion-related gene sets. The TGP1 scoring system gave reasonable results, i.e., the scores for carcinogenesis-related genes were high in omeprazole-, chlorpromazine-, hexachlorobenzene-, sulfasalazine- and Wy-14,643-treated rat livers, that for PPARalpha-regulated genes were high in clofibrate-, Wy-14,643-, gemfibrozil-, benzbromarone- and aspirin-treated rat livers as well as rat hepatocytes, and for glutathione deficiency-related genes were high in omeprazole-, bromobenzene-, acetaminophen- and coumarin-treated rat liver. We concluded that the TGP1 score is useful for surveying the expression changes in multiple biomarker gene sets for a large-scale toxicogenomics database, which would reduce the time of doing conventional multivariate statistical analysis. In addition, the TGP1 score can be applied to screening of compatible biomarker gene sets between rat liver and rat hepatocytes, like PPARalpha-regulated gene sets, which will allow us to develop an appropriate in vitro system for drug safety assessment in vivo.

Animals↗

PubMed2006

Comparison of gene expression profiles among papilla, medulla and cortex in rat kidney.

The aim of this study was to compare gene expression profiles in the different kidney regions as the basis for toxicogenomics. Rat kidney was separated into papilla, medulla and cortex, and total RNA was isolated from these and from the whole slice. Gene expression profiling was performed using Affymetrix Rat Genome 230 2.0 Array. When global normalization was applied, the expression of beta-actin or GAPDH varied among the regions. It was considered that such a comparison could not be made, especially between papilla and other portions, since the production of total mRNA in the former was relatively low. In fact, ANOVA was performed on the gene expression values with global normalization in papilla, medulla, cortex, and whole slice, and the numbers of genes appeared to be the highest in papilla. It was also observed that many genes showed their maximum or minimum in the whole slice, which was theoretically impossible. To overcome the problems associated with global normalization, the "percellome" normalization (a way to obtain the values directly related to the copies of mRNA per cell) was employed to compare the regions. In applying this procedure, probe sets with regional difference in expression were efficiently extracted by ANOVA. When they were sorted by the fold difference to other regions, the higher rank was occupied by genes characteristic of the functions of kidney, i.e., channels, transporters and metabolic enzymes. Some of them were consistent with the literature and were related to pathophysiological phenomena. Comprehensive comparison of data of gene expression in the renal anatomical area will greatly enhance studies of the physiological function and mechanism of toxicity in kidney.

Animals↗

PubMed2006

Profiling of gene expression in rat liver and rat primary cultured hepatocytes treated with peroxisome proliferators.

The Toxicogenomics project has been constructing a large-scale database of about 150 compounds exposed to rat (single dose, 3, 6, 9, 24 hrs and repeated dose for 3, 7, 14 28 days with 3 dose levels) and rat hepatocytes (2, 8, 24 hr with 3 concentrations) and data of transcriptome in liver using GeneChip, and the related toxicological measures are being accumulated. In the present study, the data of three ligands of peroxisome proliferator activated receptor alpha (PPARalpha), i.e., clofibrate, WY-14643 and gemfibrozil in our database were analyzed. Many of the beta-oxidation-related genes were commonly induced in vivo and in vitro, whereas expression changes in genes related to cell proliferation, apoptosis, were detected in vivo (single and repeated dose) but not in vitro. Changes in those related to the immune response, coagulation and the stress response were also detectable exclusively in vivo. Using the genes mobilized in two or three PPARalpha agonists, hierarchical clustering was performed on 32 compounds stored in our database. In the profiling of an in vivo single dose, benzbromarone and aspirin were located in the same cluster of the three PPARalpha agonists. The clustering of in vitro data revealed that benzbromarone, three NSAIDs (aspirin, indomethacin and diclofenac sodium) and valproic acid belonged to the same cluster of PPARalpha agonists, supporting the reports that benzbromarone,valproic acid and some NSAIDs were reported to be PPARalpha agonists. Using the genes commonly up-regulated both in vivo and in vitro, principal component analysis was performed in 32 compounds, and principal component 1 was found to be the convenient parameter to extract PPARalpha agonist-like compounds from the database.

Alanine Transaminase↗

PubMed2006

Gene expression profile in liver of differing ages of rats after single oral administration of acetaminophen.

In order to verify the influence of the rat age on hepatotoxicity, male Sprague-Dawley rats of 6 (young) and 12 (adult) weeks of age were orally administered acetaminophen (APAP), isoniazid (INH), or carbon tetrachloride (CCl4). Liver samples were obtained in a time-course manner, and changes in gene expression examined by an Affymetrix GeneChip. APAP caused more prominent hepatic injury with respect to pathology and blood biochemistry in adults than in young rats, whereas no obvious age-related differences were observed in INH- or CCl4-treated rats. Comparing gene expression in control rats, CYP3A13 was higher and GSTY2c was lower in adults, suggesting that production of the active metabolite of APAP is higher and its detoxification is lower in adults. The total amount of glutathione and total SH in rat liver was found to be higher in adult rats whereas the extent of its reduction by APAP was larger in adults. A detailed analysis of genes showing age-related differences revealed that some of them were different not in their extent but in their time course, i.e., the stress responses occurred earlier in the young than in the adult, resulting in a difference at 24 hr after dosing. These results suggest that the age-related difference in toxicity would be attributed to a higher expression of CYP3A13, producing the active metabolite of APAP as well as the lower expression of the detoxification enzyme, GSTY2c, in adult rats. Furthermore, these differences affect the time course of APAP toxicity. The present study clearly depicts the advantage of the multi-time, multi-dose protocol employed in our project for analyzing the mechanism of toxicity by gene expression profiling.

Acetaminophen↗

PubMed2006

Evaluation of methods for duration of preservation of RNA quality in rat liver used for transcriptome analysis.

In The Toxicogenomics Project, about 150 chemicals are administered to rats, and gene expression in the liver analyzed by Affymetrix GeneChip and stored in the database. As the quality of RNA greatly influences the accuracy of gene expression data, conditions of the storage of the sample are very important. Recently, an RNA stabilization solution, RNAlater, has become commercially available. In this study, the new storage method was compared with the traditional storage method (stored in freezer or liquid nitrogen) under various conditions by looking at the degradation of RNA assessed by its total yield, OD260/280 ratio, 28S/18S ratio, and quantity of beta-actin. It was confirmed that RNAlater preserved the liver tissue sample by maintaining the quality of RNA for one year (in liquid N(2) or -80 degrees C), for 3 days (4 degrees C), or for 2 hr (room temperature) without degradation of RNA. Quality of RNA samples dissolved in buffer RLT and stored at -20 degrees C tended to decrease, but samples stored at -80 degrees C were almost equivalent to those stored in liquid nitrogen. In conclusion, we recommend the following procedure for preservation of liver tissue for extraction of RNA: 1) tissues removed should be put into chilled RNAlater as soon as possible; 2) samples in RNAlater must be stored overnight or longer at 4 degrees C and can be left for as long as 2 weeks without freezing; 3) samples in RNAlater can be stored for at least one year under less than -20 degrees C and 4) samples dissolved in buffer RLT can be preserved at least for one year under -80 degrees C.

Actins↗

PubMed2005

Effect of the difference in vehicles on gene expression in the rat liver--analysis of the control data in the Toxicogenomics Project Database.

The Toxicogenomics Project is a 5-year collaborative project by the Japanese government and pharmaceutical companies in 2002. Its aim is to construct a large-scale toxicology database of 150 compounds orally administered to rats. The test consists of a single administration test (3, 6, 9 and 24 h) and a repeated administration test (3, 7, 14 and 28 days), and the conventional toxicology data together with the gene expression data in liver as analyzed by using Affymetrix GeneChip are being accumulated. In the project, either methylcellulose or corn oil is employed as vehicle. We examined whether the vehicle itself affects the analysis of gene expression and found that corn oil alone affected the food consumption and biochemical parameters mainly related to lipid metabolism, and this accompanied typical changes in the gene expression. Most of the genes modulated by corn oil were related to cholesterol or fatty acid metabolism (e.g., CYP7A1, CYP8B1, 3-hydroxy-3-methylglutaryl-Coenzyme A reductase, squalene epoxidase, angiopoietin-like protein 4, fatty acid synthase, fatty acid binding proteins), suggesting that the response was physiologic to the oil intake. Many of the lipid-related genes showed circadian rhythm within a day, but the expression pattern of general clock genes (e.g., period 2, arylhydrocarbon nuclear receptor translocator-like, D site albumin promoter binding protein) were unaffected by corn oil, suggesting that the effects are specific for lipid metabolism. These results would be useful for usage of the database especially when drugs with different vehicle control are compared.

Animals↗

PubMed2005

5Alpha-bile alcohols function as farnesoid X receptor antagonists.

The farnesoid X receptor (FXR) is a bile acid/alcohol-activated nuclear receptor that regulates lipid homeostasis. Unlike other steroid receptors, FXR binds bile acids in an orientation that allows the steroid nucleus A ring to face helix 12 in the receptor, a crucial domain for coactivator-recruitment. Because most naturally occurring bile acids and alcohols contain a cis-oriented A ring, which is distinct from that of other steroids and cholesterol metabolites, we investigated the role of this 5beta-configuration in FXR activation. The results showed that the 5beta-(A/B cis) bile alcohols 5beta-cyprinol and bufol are potent FXR agonists, whereas their 5alpha-(A/B trans) counterparts antagonize FXR transactivation and target gene expression. Both isomers bound to FXR, but their ability to induce coactivator-recruitment and thereby induce transactivation differed. These findings suggest a critical role for the A-ring orientation of bile salts in agonist/antagonist function.

Cell Line, Tumor↗

PubMed2005

Riccardin C: a natural product that functions as a liver X receptor (LXR)alpha agonist and an LXRbeta antagonist.

Liver X receptors (LXRs) alpha and beta share considerable sequence homology and several functions, respond to the same endogenous and synthetic ligands, and play critical roles in maintaining lipid homeostasis. In this study, liverwort-derived riccardin C (RC) and F (RF) were identified as an LXRalpha agonist/LXRbeta antagonist and an LXRalpha antagonist, respectively. RC and RF bound to LXRs, but had different abilities to recruit a coactivator and thereby induce transactivation. Despite its unique subtype-selective activity, RC enhanced ABCA1 and ABCG1 expression and cellular cholesterol efflux in THP-1 cells. RC may provide a novel tool for identifying subtype-function and drug development.

Biological Products↗

PubMed2005

Thyroid hormone targets matrix Gla protein gene associated with vascular smooth muscle calcification.

Thyroid hormones have marked cardiovascular effects in vivo. However, their direct effects on vascular smooth muscle cells have been unclear. Because thyroid hormones play critical roles in bone remodeling, we hypothesized that they are also associated with vascular smooth muscle calcification, one of the pathological features of vascular sclerosis. To test this hypothesis, we examined the effects of 3',3,5-triiodo-L-thyronine (T3) on the expression of calcification-associated genes in rat aortic smooth muscle cells (RAOSMCs). Quantitative RT-PCRs revealed that a physiological concentration of T3 (15 pmol/L free T3) increased mRNA level of matrix Gla protein (MGP), which acts as a potent inhibitor of vascular calcification in vivo, by 3-fold in RAOSMCs, as well as in cultured human coronary artery smooth muscle cells. In RAOSMCs transiently transfected with a luciferase reporter gene driven by the MGP promoter, T3 significantly stimulated luciferase activity. In addition, RNA interference against thyroid hormone receptor-alpha gene diminished the effect of T3 on MGP expression. Aortic smooth muscle tissues from methimazole-induced hypothyroid rats (400 mg/L drinking water; 4 weeks) also showed a 68% decrease in the MGP mRNA level, as well as a 33% increase in calcium content compared with that from the control euthyroid animals, whereas hyperthyroidism (0.2 mg T3/kg IP; 10 days) upregulated MGP mRNA by 4.5-fold and reduced calcium content by 11%. Our findings suggest that a physiological concentration of thyroid hormone directly facilitates MGP gene expression in smooth muscle cells via thyroid hormone nuclear receptors, leading to prevention of vascular calcification in vivo.

Animals↗

PubMed2005

Galpha12/13-mediated production of reactive oxygen species is critical for angiotensin receptor-induced NFAT activation in cardiac fibroblasts.

Angiotensin II (Ang II) activates multiple signaling pathways leading to hyperplasia of cardiac fibroblasts. Reactive oxygen species (ROS) produced by Ang II stimulation are assumed to play pivotal roles in this process. Here, we show that ROS mediate Ang II-induced activation of nuclear factor of activated T cells (NFAT) in rat cardiac fibroblasts. Ang II-induced NFAT activation was suppressed by diphenyleneiodonium (an NADPH oxidase inhibitor), dominant negative (DN)-Rac, DN-p47(phox), and an inhibitor of Galpha(12/13) (Galpha(12/13)-specific regulator of G protein signaling domain of p115RhoGEF, p115-regulator of G protein signaling (RGS)). Stimulation of Ang II receptor increased the intracellular ROS level in a Rac- and p47(phox)-dependent manner. Because p115-RGS suppressed Ang II-induced Rac activation, Ang II receptor-coupled Galpha(12/13) mediated NFAT activation through ROS production by Rac activation. Ang II-induced nuclear translocation of the green fluorescent protein (GFP)-tagged amino-terminal region of NFAT4 (GFP-NFAT4) was suppressed by p115-RGS or BAPTA but not by diphenyleneiodonium. The expression of constitutively active (CA)-Galpha(12/13), CA-G translocation alpha(13), or CA-Rac increased the nuclear of GFP-NFAT4. These results suggest that NFAT activity is regulated by both Ca(2+)-dependent and ROS-dependent pathways. Furthermore, activation of c-Jun NH(2)-terminal kinase (JNK) induced by Ang II stimulation is required for NFAT activation because Ang II-induced NFAT activation was inhibited by SP600125, a selective JNK inhibitor. These results indicate that Ang II stimulates the nuclear translocation and activation of NFAT by integrated pathways including the activation of Galpha(12/13), Rac, NADPH oxidase, and JNK and that Galpha(12/13)-mediated ROS production is essential for NFAT transcriptional activation.

Active Transport, Cell Nucleus↗

PubMed2005

G alpha 12/13- and reactive oxygen species-dependent activation of c-Jun NH2-terminal kinase and p38 mitogen-activated protein kinase by angiotensin receptor stimulation in rat neonatal cardiomyocytes.

In the present study, we examined signal transduction mechanism of reactive oxygen species (ROS) production and the role of ROS in angiotensin II-induced activation of mitogen-activated protein kinases (MAPKs) in rat neonatal cardiomyocytes. Among three MAPKs, c-Jun NH(2)-terminal kinase (JNK) and p38 MAPK required ROS production for activation, as an NADPH oxidase inhibitor, diphenyleneiodonium, inhibited the activation. The angiotensin II-induced activation of JNK and p38 MAPK was also inhibited by the expression of the Galpha(12/13)-specific regulator of G protein signaling (RGS) domain, a specific inhibitor of Galpha(12/13), but not by an RGS domain specific for Galpha(q). Constitutively active Galpha(12)- or Galpha(13)-induced activation of JNK and p38 MAPK, but not extracellular signal-regulated kinase (ERK), was inhibited by diphenyleneiodonium. Angiotensin II receptor stimulation rapidly activated Galpha(13), which was completely inhibited by the Galpha(12/13)-specific RGS domain. Furthermore, the Galpha(12/13)-specific but not the Galpha(q)-specific RGS domain inhibited angiotensin II-induced ROS production. Dominant negative Rac inhibited angiotensin II-stimulated ROS production, JNK activation, and p38 MAPK activation but did not affect ERK activation. Rac activation was mediated by Rho and Rho kinase, because Rac activation was inhibited by C3 toxin and a Rho kinase inhibitor, Y27632. Furthermore, angiotensin II-induced Rho activation was inhibited by Galpha(12/13)-specific RGS domain but not dominant negative Rac. An inhibitor of epidermal growth factor receptor kinase AG1478 did not affect angiotensin II-induced JNK activation cascade. These results suggest that Galpha(12/13)-mediated ROS production through Rho and Rac is essential for JNK and p38 MAPK activation.

Angiotensin Receptor Antagonists↗

PubMed2003

L-type Ca2+ channels serve as a sensor of the SR Ca2+ for tuning the efficacy of Ca2+-induced Ca2+ release in rat ventricular myocytes.

In cardiac excitation-contraction coupling, Ca2+-induced Ca2+ release (CICR) from ryanodine receptors (RyRs), triggered by Ca2+ entry through the nearby L-type Ca2+ channel, induces Ca2+-dependent inactivation (CDI) of the Ca2+ channel. Aiming at elucidating the physiological role of CDI produced by CICR (CICR-dependent CDI), we investigated the contribution of the CICR-dependent CDI to action potential (AP) waveform and the amount of Ca2+-influx through Ca2+ channels during AP in rat ventricular myocytes. The elimination of the CICR-dependent CDI, by depletion of the SR Ca2+ with thapsigargin, significantly prolonged AP duration (APD). APD changed in parallel with the magnitude of CICR during the recovery of the SR Ca2+ content after transient depletion by caffeine. Such CICR-dependent change of APD persisted under the highly Ca2+ buffered condition where the Ca2+ signalling was restricted to nanoscale domains. Blockers of the Ca2+-dependent Cl- channel or the BK channel did not affect AP waveform. The amount of Ca2+-influx through Ca2+ channels during the SR-depleted type AP waveform, measured in the SR-depleted myocyte, was increased by 40 % over that during the SR-intact type AP waveform measured in the SR-intact myocyte. The protein kinase A stimulation further enhanced the Ca2+-influx during AP under the SR-depleted condition to 70 % of that under the SR-intact condition. These results indicate that the CICR-dependent CDI of L-type Ca2+ channels, under control of the privileged cross-signalling between L-type Ca2+ channels and RyRs, play important roles for monitoring and tuning the SR Ca2+ content via changes of AP waveform and the amount of Ca2+-influx during AP in ventricular myocytes.

Action Potentials↗

PubMed2003

Role of ADP-ribosylation factor 6 (ARF6) in gastric acid secretion.

ADP-ribosylation factor (ARF) proteins are monomeric GTPases that are essential for membrane transport and exocytosis in a number of secretory cells. We investigated ARF6, the activation of which is insensitive to brefeldin A, to determine whether it regulates membrane traffic in gastric parietal cells. ARF6 translocated from cytosol to tubulovesicle in the presence of GTPgammaS, a potential inhibitor of acid secretion in permeabilized cells, whereas under the Mg2+-chelated condition where activity of ARF-GTPase activating protein is inhibited, ARF6 translocated to the apical secretory membrane. Immunohistochemical examination revealed that ARF6 mainly located in parietal cell within the gastric glands, and it translocated from the cytosol to the intracellular canaliculi when the glands were stimulated. These results indicated that the distribution of ARF6 between cytosol and the two different membranes was regulated by its GTPase activity. In cultured gastric glands infected with adenovirus expressing ARF6 Q67L, a mutant lacking GTP hydrolysis activity, gastric acid secretion was inhibited. These results suggest that ARF6 regulates gastric acid secretion in parietal cell and that the GTP hydrolysis cycle of ARF6 is essential for the activation pathway.

ADP-Ribosylation Factor 6↗

PubMed2003

Negative modulation of L-type Ca2+ channels via beta-adrenoceptor stimulation in guinea-pig detrusor smooth muscle cells.

beta-Adrenergic stimulation enhances the activity of L-type Ca(2+) channels through mechanisms mediated by adenosine 3'5'-cyclic monophosphate (cAMP) and protein kinase A in cardiac myocytes. However, in smooth muscle cells, the effect of beta-adrenoceptor stimulation on the L-type Ca(2+) channel activity has been controversial, and the exact mechanism is still unclear. The present study was aimed at elucidating the effect of beta-adrenergic stimulation upon the activity of L-type Ca(2+) channels in guinea-pig detrusor smooth muscle cells. Isoproterenol (0.1-1 microM) inhibited Ba(2+) currents through L-type Ca(2+) channels (I(Ba)). Isoproterenol (0.1 microM) shifted the steady-state inactivation curve to negative voltages by 11 mV without affecting activation curves. The stimulation of cAMP-mediated signal transduction pathway by forskolin, 8-bromoadenosine 3'5'-cyclic monophosphate (8-Br-cAMP), or the intracellular application of cAMP also mimicked the effects of isoproterenol on I(Ba), which was blocked by the inhibition of protein kinase A. These results indicate that, in detrusor smooth muscles, the stimulation of beta-adrenoceptors exerts negative modulation of L-type Ca(2+) channels via cAMP/protein kinase A-dependent mechanism.

Animals↗