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1.
Hum Exp Toxicol ; 28(4): 231-44, 2009 Apr.
Article in English | MEDLINE | ID: mdl-19734275

ABSTRACT

We have constructed a large-scale transcriptome database of rat liver treated with various drugs. In an effort to identify a biomarker for the diagnosis of elevated total bilirubin (TBIL) and direct bilirubin (DBIL), we extracted 59 probe sets of rat hepatic genes from the data for seven typical drugs, gemfibrozil, phalloidin, colchicine, bendazac, rifampicin, cyclosporine A, and chlorpromazine, which induced this phenotype from 3 to 28 days of repeated administration in the present study. Principal component analysis (PCA) using these probes clearly separated dose- and time-dependent clusters in the treated groups from their controls. Eighteen more drugs in the database, reported to elevate TBIL and DBIL, were estimated by PCA using these probe sets. Of these, 12 drugs, that is methapyrilene, thioacetamide, ticlopidine, ethinyl estradiol, alpha-naphthylisothiocyanate, indomethacin, methyltestosterone, penicillamine, allyl alcohol, aspirin, iproniazid, and isoniazid were also separated from the control clusters, as were the seven typical drugs causing elevation of TBIL and DBIL. The principal component 1 (PC1) value showed high correlation with TBIL and DBIL. In the cases of colchicine, bendazac, chlorpromazine, gemfibrozil, and phalloidin, the possible elevation of TBIL and DBIL could be predicted by expression of these genes 24 h after single administration. We conclude that these identified 59 probe sets could be useful to diagnose the cause of elevation of TBIL and DBIL, and that toxicogenomics would be a promising approach for prediction of this type of toxicity.


Subject(s)
Bilirubin/biosynthesis , Chemical and Drug Induced Liver Injury/genetics , Gene Expression Profiling , Liver/drug effects , Liver/metabolism , Animals , Blood Chemical Analysis , Chemical and Drug Induced Liver Injury/metabolism , Dose-Response Relationship, Drug , Glucuronic Acid/metabolism , Male , Oligonucleotide Array Sequence Analysis , Pharmaceutical Preparations/metabolism , Principal Component Analysis , Rats , Rats, Sprague-Dawley , Signal Transduction/drug effects
2.
Hum Exp Toxicol ; 27(1): 23-35, 2008 Jan.
Article in English | MEDLINE | ID: mdl-18480146

ABSTRACT

One expected result from toxicogenomics technology is to overcome the barrier because of species-specific differences in prediction of clinical toxicity using animals. The present study serves as a model case to test if the well-known species-specific difference in the toxicity of coumarin could be elucidated using comprehensive gene expression data from rat in-vivo, rat in-vitro, and human in-vitro systems. Coumarin 150 mg/kg produced obvious pathological changes in the liver of rats after repeated administration for 7 days or more. Moreover, 24 h after a single dose, we observed minor and transient morphological changes, suggesting that some early events leading to hepatic injury occur soon after coumarin is administered to rats. Comprehensive gene expression changes were analyzed using an Affymetrix GeneChip approach, and differentially expressed probe sets were statistically extracted. The changes in expression of the selected probe sets were further examined in primary cultured rat hepatocytes exposed to coumarin, and differentially expressed probe sets common to the in-vivo and in-vitro datasets were selected for further study. These contained many genes related to glutathione metabolism and the oxidative stress response. To incorporate human data, human hepatocyte cultured cells were exposed to coumarin and changes in expression of the bridging gene set were examined. In total, we identified 14 up-regulated and 11 down-regulated probe sets representing rat-human bridging genes. The overall responsiveness of these genes to coumarin was much higher in rats than humans, consistent with the reported species difference in coumarin toxicity. Next, we examined changes in expression of the rat-human bridging genes in cultured rat and human hepatocytes treated with another hepatotoxicant, diclofenac sodium, for which hepatotoxicity does not differ between the species. Both rat and human hepatocytes responded to the marker genes to the same extent when the same concentrations of diclofenac sodium were exposed. We conclude that toxicogenomics-based approaches show promise for overcoming species-specific differences that create a bottleneck in analysis of the toxicity of potential therapeutic treatments.


Subject(s)
Anticoagulants/toxicity , Chemical and Drug Induced Liver Injury/pathology , Coumarins/toxicity , Toxicogenetics , Algorithms , Animals , Anti-Inflammatory Agents, Non-Steroidal/toxicity , Chemical and Drug Induced Liver Injury/genetics , Data Interpretation, Statistical , Diclofenac/toxicity , Gene Expression/drug effects , Gene Expression/physiology , Hepatocytes/drug effects , Hepatocytes/pathology , Hepatocytes/ultrastructure , Humans , Male , Microscopy, Electron, Scanning , Oligonucleotide Array Sequence Analysis , Rats , Rats, Sprague-Dawley , Risk Assessment , Species Specificity
3.
Am J Physiol Gastrointest Liver Physiol ; 281(3): G786-97, 2001 Sep.
Article in English | MEDLINE | ID: mdl-11518691

ABSTRACT

Gastric vesicles purified from acid-secreting rabbit stomach display K(+) permeability manifested by the valinomycin-independent proton pumping of H(+)-K(+)-ATPase as monitored by acridine orange quenching. This apparent K(+) permeability is attenuated by the treatment of the membrane with 5 mM Mg(2+), and this phenomenon has been attributed to membrane-bound phosphoprotein phosphatase. However, with the exception of the nonspecific inhibitor pyrophosphate, protein phosphatase inhibitors failed to inhibit the loss of K(+) permeability. Preincubation of the membrane with neomycin, a phospholipase C inhibitor, surrogated the effect of Mg(2+), whereas another inhibitor, U-73122, did not. Phosphatidylinositol 4,5-bisphosphate (PIP(2)) restored the attenuated K(+) permeability by treatment with either Mg(2+) or neomycin. Furthermore, either phosphatidylinositol bound to phosphatidylinositol transfer protein or phosphatidylinositol 4,5,6-trisphosphate (PIP(3)) surrogated the effect of PIP(2). Mg(2+) and neomycin reduced K(+) permeability in the membrane as determined by Rb(+) influx and K(+)-dependent H(+) diffusion. Treatment with Mg(2+) reduced the contents of PIP(2) and PIP(3) in the membrane. These results suggest that PIP(2) and/or PIP(3) maintain K(+) permeability, which is essential for proton pumping in the apical membrane of the secreting parietal cell.


Subject(s)
Gastric Mucosa/metabolism , H(+)-K(+)-Exchanging ATPase/metabolism , Membrane Proteins , Phosphatidylinositols/metabolism , Potassium/metabolism , Secretory Vesicles/metabolism , Acridine Orange , Aminoglycosides , Animals , Anti-Bacterial Agents/pharmacology , Carrier Proteins/metabolism , Cations, Divalent/pharmacology , Enzyme Inhibitors/pharmacology , Gastric Mucosa/chemistry , Macromolecular Substances , Magnesium/pharmacology , Permeability/drug effects , Phosphatidylinositol 4,5-Diphosphate/analysis , Phosphatidylinositol 4,5-Diphosphate/pharmacology , Phosphatidylinositol Phosphates/pharmacology , Phospholipid Transfer Proteins , Phosphoprotein Phosphatases/antagonists & inhibitors , Rabbits , Rubidium/pharmacokinetics , Secretory Vesicles/chemistry , Spectrometry, Fluorescence , Type C Phospholipases/antagonists & inhibitors
4.
J Biol Chem ; 276(30): 28171-8, 2001 Jul 27.
Article in English | MEDLINE | ID: mdl-11384966

ABSTRACT

When isolated rabbit gastric glands were permeabilized with digitonin, they lost their ability to secrete acid, as monitored by [14C]aminopyrine accumulation, and they never recovered by supplement with cytosol prepared from gastric mucosa. However, the permeabilized glands elicited acid secretion when brain cytosol was supplemented. Fractionation of gastric cytosol by gel filtration revealed that the fraction at 30 kDa stimulated permeabilized glands by itself, whereas the 200-kDa fraction potently inhibited brain cytosol-stimulated acid secretion. Brain cytosol contained only the former stimulatory factor. With further gel filtration, the 30-kDa activator was separated into two components, 20 kDa (peak 1) and 1.8 kDa (peak 2), both of which are necessary for full activity. We purified peak 1 from bovine brain, and phosphatidylinositol transfer protein (PITP) was identified as the main component of the activity. The stimulating activity in brain and gastric mucosa correlated with the contents of PITP, and recombinant PITP mimicked the effect of peak 1, suggesting that PITP is one of the essential components in gastric acid secretion. When gastric glands were stimulated, the inhibitory activity, but not stimulatory activity, in the cytosol was increased. This suggests a regulatory mechanism such as stimulation translocates the inhibitory component from the secretory site on the membrane to cytosol. These results demonstrate a high degree of usefulness for our present model, the reconstituted digitonin-permeabilized gastric glands.


Subject(s)
Carrier Proteins/chemistry , Cytosol/metabolism , Digitonin/pharmacology , Gastric Acid/metabolism , Gastric Mucosa/metabolism , Membrane Proteins , Phosphotransferases , Animals , Bacterial Outer Membrane Proteins/isolation & purification , Bacterial Outer Membrane Proteins/metabolism , Brain/metabolism , Carrier Proteins/isolation & purification , Cattle , Chromatography, Agarose , Chromatography, Gel , Dose-Response Relationship, Drug , Lipoproteins/isolation & purification , Lipoproteins/metabolism , Phosphatidylinositols/metabolism , Phospholipid Transfer Proteins , Protein Isoforms , Rabbits , Rats , Stomach/drug effects , Stomach/physiology
5.
Am J Physiol Cell Physiol ; 280(1): C155-65, 2001 Jan.
Article in English | MEDLINE | ID: mdl-11121387

ABSTRACT

Acid secretion in isolated rabbit gastric glands was monitored by the accumulation of [(14)C]aminopyrine. Stimulation of the glands with carbachol synergistically augmented the response to dibutyryl cAMP. The augmentation persisted even after carbachol was washed out and was resistant to chelated extracellular Ca(2+) and to inhibitors of either protein kinase C or calmodulin kinase II. Cytochalasin D at 10 microM preferentially blocked the secretory effect of carbachol and its synergism with cAMP, whereas it had no effect on histamine- or cAMP-stimulated acid secretion within 15 min. Cytochalasin D inhibited the carbachol-stimulated intracellular Ca(2+) concentration ([Ca(2+)](i)) increase due to release from the Ca(2+) store. Treatment of the glands with cytochalasin D redistributed type 3 inositol 1,4,5-trisphosphate receptor (the major subtype in the parietal cell) from the fraction containing membranes of large size to the microsomal fraction, suggesting a dissociation of the store from the plasma membrane. These findings suggest that intracellular Ca(2+) release by cholinergic stimulation is critical for determining synergism with cAMP in parietal cell activation and that functional coupling between the Ca(2+) store and the receptor is maintained by actin microfilaments.


Subject(s)
1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine/analogs & derivatives , Carbachol/pharmacology , Cyclic AMP/metabolism , Gastric Acid/metabolism , Gastric Mucosa/drug effects , Gastric Mucosa/metabolism , Stomach/drug effects , 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine/pharmacology , Actins/metabolism , Aminopyrine/metabolism , Animals , Bucladesine/pharmacology , Calcium/metabolism , Calcium Channels/drug effects , Calcium Channels/metabolism , Calcium Signaling/drug effects , Calcium Signaling/physiology , Calcium-Calmodulin-Dependent Protein Kinases/antagonists & inhibitors , Calcium-Calmodulin-Dependent Protein Kinases/metabolism , Carbon Radioisotopes , Cytochalasin D/pharmacology , Cytoskeleton/drug effects , Cytoskeleton/metabolism , Drug Interactions/physiology , Enzyme Inhibitors/pharmacology , Gastric Mucosa/cytology , Histamine/pharmacology , In Vitro Techniques , Indoles/pharmacology , Inositol 1,4,5-Trisphosphate Receptors , Maleimides/pharmacology , Parietal Cells, Gastric/drug effects , Parietal Cells, Gastric/metabolism , Parietal Cells, Gastric/ultrastructure , Protein Kinase C/antagonists & inhibitors , Protein Kinase C/metabolism , Rabbits , Receptors, Cytoplasmic and Nuclear/drug effects , Receptors, Cytoplasmic and Nuclear/metabolism , Stomach/cytology
6.
Jpn J Pharmacol ; 83(3): 225-32, 2000 Jul.
Article in English | MEDLINE | ID: mdl-10952071

ABSTRACT

We determined the effect of 1-cis diltiazem, the enantiomer of diltiazem (d-cis isoform), on the energy metabolism of isolated guinea pig hearts during ischemia-reperfusion. We used 31P-NMR to measure the high-energy phosphate content and intracellular pH (pHi) during global ischemia for 30 min followed by reperfusion for 30 min. Before ischemia, the left ventricular developed pressure (LVDP) was reduced less by 10 microM l-cis diltiazem than by 3 microM diltiazem or 500 nM nifedipine. However, 10 microM l-cis diltiazem preserved the intracellular ATP content during ischemia and reperfusion, reduced the end-diastolic pressure increase during ischemia and reperfusion, and restored LVDP after reperfusion. Nifedipine at 50 nM, which reduced the LVDP more than 10 microM l-cis diltiazem, showed no cardioprotective effect. Ten micromolar l-cis diltiazem and 3 microM diltiazem, but neither 50 nor 500 nM nifedipine, reduced the pHi decrease that occurred 25 or 30 min after the onset of ischemia. Therefore, l-cis diltiazem has a cardioprotective effect on ischemic and reperfused myocardium and is less cardiodepressive than diltiazem and nifedipine. The effect of l-cis diltiazem during ischemia and reperfusion involves energy preservation, which is probably independent of its Ca2+-channel blocking action.


Subject(s)
Diltiazem/pharmacology , Heart/drug effects , Myocardial Ischemia , Myocardial Reperfusion , Adenosine Triphosphate/metabolism , Animals , Calcium Channel Blockers/pharmacology , Guinea Pigs , Heart/physiopathology , Hydrogen-Ion Concentration , Magnetic Resonance Spectroscopy , Myocardial Contraction , Nifedipine/pharmacology , Phosphocreatine/metabolism , Phosphorus Isotopes
7.
Biochem Biophys Res Commun ; 269(1): 137-42, 2000 Mar 05.
Article in English | MEDLINE | ID: mdl-10694490

ABSTRACT

We investigated the role of the 27-kDa heat shock protein (HSP27) in cardiac protection using Langendorff-perfused rat hearts. After preconditioning (a single episode of 5 min global ischemia followed by 5 min of reperfusion), HSP27 redistributed from the cytosol to the sarcomere and recovery of the contractile function, after 40 min of global ischemia and 50 min of reperfusion, was significantly enhanced. Both SB203580, a p38 MAP kinase inhibitor, and bisindolylmaleimide I, a protein kinase C inhibitor, prevented the effects of preconditioning. Both 2-chloro-N(6)-cyclopentyladenosine (adenosine A1 agonist) and anisomycin (activator of p38 MAP kinase and c-jun N-terminal kinase) mimicked preconditioning. These results suggest that activation of protein kinase C followed by activation of p38 MAP kinase elicits translocation of HSP27 to the sarcomere, a process which may be involved in the cardioprotective mechanism afforded by ischemic preconditioning in rat heart.


Subject(s)
Heat-Shock Proteins , Ischemic Preconditioning, Myocardial , Mitogen-Activated Protein Kinases , Myocardium/metabolism , Neoplasm Proteins/metabolism , Sarcomeres/metabolism , Animals , Biological Transport, Active/drug effects , Calcium-Calmodulin-Dependent Protein Kinases/antagonists & inhibitors , Cytosol/metabolism , Enzyme Inhibitors/pharmacology , HSP27 Heat-Shock Proteins , Heart/drug effects , Imidazoles/pharmacology , Immunohistochemistry , In Vitro Techniques , Indoles/pharmacology , Male , Maleimides/pharmacology , Perfusion , Protein Kinase C/antagonists & inhibitors , Pyridines/pharmacology , Rats , Rats, Sprague-Dawley , Sarcomeres/drug effects , p38 Mitogen-Activated Protein Kinases
8.
J Biol Chem ; 275(15): 11164-73, 2000 Apr 14.
Article in English | MEDLINE | ID: mdl-10753923

ABSTRACT

We previously reported a 120-kDa phosphoprotein that translocated from cytosol to the apical membrane of gastric parietal cells in association with stimulation of HCl secretion. To determine the molecular identity of the protein, we performed molecular cloning and expression of the protein. Immunoblot analysis showed that this protein was highly enriched in tissues that secrete water, such as parietal cell, choroid plexus, salivary duct, lacrimal gland, kidney, airway epithelia, and chorioretinal epithelia. We named this protein "parchorin" based on its highest enrichment in parietal cells and choroid plexus. We obtained cDNA for parchorin from rabbit choroid plexus coding a protein consisting of 637 amino acids with a predicted molecular mass of 65 kDa. The discrepancy in size on 6% SDS-polyacrylamide gel electrophoresis is considered to be due to its highly acidic nature (pI = 4.18), because COS-7 cells transfected with parchorin cDNA produced a protein with apparent molecular mass of 120 kDa on 6% SDS-polyacrylamide gel electrophoresis. Parchorin is a novel protein that has significant homology to the family of chloride intracellular channels (CLIC), especially the chloride channel from bovine kidney, p64, in the C-terminal 235 amino acids. When expressed as a fusion protein with green fluorescent protein (GFP) in the LLC-PK1 kidney cell line, GFP-parchorin, unlike other CLIC family members, existed mainly in the cytosol. Furthermore, when Cl(-) efflux from the cell was elicited, GFP-parchorin translocated to the plasma membrane. These results suggest that parchorin generally plays a critical role in water-secreting cells, possibly through the regulation of chloride ion transport.


Subject(s)
Body Water/metabolism , Chloride Channels/analysis , Chloride Channels/isolation & purification , Choroid Plexus/chemistry , Parietal Cells, Gastric/chemistry , Phosphoproteins/isolation & purification , Amino Acid Sequence , Animals , Base Sequence , Chloride Channels/genetics , Chloride Channels/physiology , Chlorides/metabolism , Cloning, Molecular , Immunohistochemistry , Molecular Sequence Data , Phosphoproteins/genetics , Rabbits
9.
Biochim Biophys Acta ; 1452(3): 243-53, 1999 Dec 09.
Article in English | MEDLINE | ID: mdl-10590313

ABSTRACT

Gastrin stimulates cell proliferation through the CCK(B) receptor coupled to Gq-protein, whereas the m3 muscarinic receptor, which also couples to Gq, has no trophic effects. In order to elucidate the cause of the difference, we stably transfected CHO cells with human CCK(B) and m3 receptors. Stimulation of the CCK(B), but not the m3 receptor increased cell growth. Activation of MAP kinase via the m3 receptor was to the same extent as that via CCK(B), indicating that there is an initial signaling common to both receptors. Stimulation of either receptor induced a transient increase in [Ca(2+)](i) followed by a sustained plateau phase. After 2 h of stimulation, the [Ca(2+)](i) response to the m3 receptor disappeared, whereas that to the CCK(B) receptor remained as a [Ca(2+)](i) oscillation. Removal of extracellular Ca(2+), which abolished [Ca(2+)](i) oscillation, completely inhibited DNA synthesis via CCK(B). When the C-terminal part of the CCK(B) receptor was truncated, the trophic effect as well as the [Ca(2+)](i) response after 2 h of stimulation disappeared, whereas the chimeric CCK(B) receptor with the C-terminal region of the m3 receptor preserved its ability to elicit both DNA synthesis and [Ca(2+)](i) oscillation. These results suggest that desensitization might be a principal determinant of cell proliferation, and the persistence of the [Ca(2+)](i) response as [Ca(2+)](i) oscillation could be essential for this type of signal transduction.


Subject(s)
Calcium/metabolism , Receptors, Cholecystokinin/metabolism , Amino Acid Sequence , Animals , CHO Cells , Calcium/analysis , Carbachol/pharmacology , Cell Division/drug effects , Cell Line , Cricetinae , DNA/biosynthesis , Enzyme Activation/drug effects , Gene Expression , Humans , Mitogen-Activated Protein Kinases/metabolism , Molecular Sequence Data , Mutation , Receptor, Muscarinic M3 , Receptors, Cholecystokinin/chemistry , Receptors, Cholecystokinin/genetics , Receptors, Muscarinic/metabolism , Signal Transduction , Sincalide/pharmacology , Transfection
10.
Eur J Pharmacol ; 385(2-3): 225-30, 1999 Dec 03.
Article in English | MEDLINE | ID: mdl-10607880

ABSTRACT

We have previously demonstrated that treatment with L-cis diltiazem reduced cardiac infarct size in vivo. To examine the effect of L-cis diltiazem on Ca(2+) overload induced by ischemia/reperfusion, we used a model for Ca(2+) overload produced by metabolic inhibition in isolated guinea pig myocytes. Intracellular Ca(2+) concentration ([Ca(2+)](i)) was quantified by fura-2 fluorescence microscopy and Ca(2+) overload was induced by inclusion of 1 microM of carbonyl cyanide m-chrolophenylhydrazone (CCCP) for 40 min treatment followed by washout for 30 min. This treatment caused a large [Ca(2+)](i) elevation as well as a sustained contracture of the cardiomyocytes. The increase was suppressed by 10 microM of 2-[2-[4-(4-nitrobenzyloxy) phenyl] ethyl] isothiourea methanesulphonate (KB-R7943), a specific inhibitor of the Na(+)/Ca(2+) exchanger, but not by nitrendipine (10 microM). L-cis Diltiazem (10 microM) attenuated the [Ca(2+)](i) increase, suggesting that L-cis diltiazem elicits a cardioprotective effect via attenuation of the [Ca(2+)](i) increase induced by metabolic inhibition and energy repletion.


Subject(s)
Calcium Channel Blockers/pharmacology , Calcium/metabolism , Diltiazem/pharmacology , Heart Ventricles/drug effects , Animals , Carbonyl Cyanide m-Chlorophenyl Hydrazone/pharmacology , Cell Size/drug effects , Diltiazem/chemistry , Dose-Response Relationship, Drug , Energy Metabolism/drug effects , Guinea Pigs , Heart Ventricles/cytology , Heart Ventricles/metabolism , Male , Nicardipine/pharmacology , Nickel/pharmacology , Nitrendipine/pharmacology , Sodium-Calcium Exchanger/antagonists & inhibitors , Stereoisomerism , Thiourea/analogs & derivatives , Thiourea/pharmacology , Uncoupling Agents/pharmacology
11.
Jpn J Pharmacol ; 80(4): 319-25, 1999 Aug.
Article in English | MEDLINE | ID: mdl-10496332

ABSTRACT

l-cis Diltiazem, an optical isomer of diltiazem, protects against myocardial dysfunction in vitro, whereas its Ca2+ channel blocking activity is about 100 times less potent than that of diltiazem. However, there is no evidence that l-cis diltiazem actually protects against ischemia/reperfusion injury in vivo. To assess this, we employed an anesthetized rabbit model, where the left circumflex artery was occluded for 15 min and reperfused for 360 min. Treatment with diltiazem before and during ischemia (bolus 200 microg/kg and 15 microg/kg per minute for 25 min, i.v.; 575 microg/kg total) showed slightly depressed hemodynamic parameters, while l-cis diltiazem (1150 microg/kg) had no effect. Treatment with l-cis diltiazem produced a high recovery of the thickening fraction and limited the infarct size in a dose-dependent manner. Furthermore, the treatment with l-cis diltiazem (1150 microg/kg) or diltiazem (575 microg/kg) 5 min before reperfusion also limited the infarct size, but not after reperfusion. These results suggest that l-cis diltiazem affects some events in the onset of reperfusion, independently of Ca2+-channel-blocking action. Our observations are the first to show that l-cis diltiazem demonstrated its cardioprotective action in the ischemic rabbit heart in vivo.


Subject(s)
Diltiazem/pharmacology , Heart/drug effects , Myocardial Infarction/pathology , Myocardial Ischemia/pathology , Reperfusion Injury/prevention & control , Anesthesia , Animals , Cardiovascular Agents/pharmacology , Dose-Response Relationship, Drug , Hemodynamics/drug effects , Isomerism , Male , Rabbits , Reperfusion Injury/pathology , Time Factors
12.
Biochem Pharmacol ; 58(8): 1349-59, 1999 Oct 15.
Article in English | MEDLINE | ID: mdl-10487539

ABSTRACT

Omeprazole and pantoprazole are known to be irreversible, SH-acting inhibitors of gastric H+,K+-adenosine triphosphatase (H+,K+-ATPase). Both drugs concentration-dependently and pH-dependently inhibited K+-dependent p-nitrophenyl phosphatase (K+-pNPPase) activity in purified rabbit gastric microsomes. The potency of omeprazole was about three times that of pantoprazole in the pH ranges tested. Both drugs also inhibited acid secretion, as determined by [14C]aminopyrine accumulation in isolated rabbit gastric glands, with the potency ratio being about 5 (omeprazole over that of pantoprazole). Under conditions in which acid secretion was inhibited completely by the drugs, the total K+-pNPPase activity in the digitonin-permeabilized glands was scarcely reduced, showing an apparent discrepancy between the acid secretion and the proton pump activity. The isolated glands were stimulated with secretagogues for 30 min in the presence of the inhibitors, homogenized, and then separated into fractions in which K+-pNPPase activity was measured. Omeprazole exclusively inhibited the activity in the low-speed fraction, which was rich in the apical membranes, whereas pantoprazole did not inhibit activity in any fraction. When the time of treatment with the inhibitors was increased up to 5 hr, the inhibition of the total K+-pNPPase activity in the glands reached a plateau at an inhibition rate lower than 50% within 2 hr. This suggested that no continuous recycling of the proton pump was occurring during stimulation. The inhibitory effect of both drugs on the permeabilized gland preparation was less potent than that on the purified enzyme, especially at the higher pH, and it appeared to be partially reversible. The extent of the reduction in potency was more prominent for pantoprazole. It is concluded that a lower amount of proton pump activity needs to be inhibited by pantoprazole than by omeprazole to achieve the same extent of acid secretion inhibition. This appears to be due to the nature of pantoprazole, i.e. the requirement of low pH for activation and the partial reversibility of the inhibition.


Subject(s)
4-Nitrophenylphosphatase/metabolism , Benzimidazoles/pharmacology , Omeprazole/pharmacology , Proton Pump Inhibitors , Sulfoxides/pharmacology , 2-Pyridinylmethylsulfinylbenzimidazoles , 4-Nitrophenylphosphatase/drug effects , Acids/metabolism , Animals , Enzyme Inhibitors/pharmacology , Gastric Mucosa/drug effects , Gastric Mucosa/enzymology , H(+)-K(+)-Exchanging ATPase/metabolism , Microsomes/drug effects , Microsomes/enzymology , Pantoprazole , Rabbits , Stomach/enzymology
13.
Am J Physiol ; 277(3): G736-44, 1999 09.
Article in English | MEDLINE | ID: mdl-10484402

ABSTRACT

We established a beta-escin-permeabilized gland model with the use of rabbit isolated gastric glands. The glands retained an ability to secrete acid, monitored by [14C]aminopyrine accumulation, in response to cAMP, forskolin, and histamine. These responses were all inhibited by cAMP-dependent protein kinase inhibitory peptide. Myosin light-chain kinase inhibitory peptide also suppressed aminopyrine accumulation, whereas the inhibitory peptide of protein kinase C or that of calmodulin kinase II was without effect. Guanosine-5'-O-(3-thiotriphosphate) (GTPgammaS) abolished cAMP-stimulated acid secretion concomitantly, interfering with the redistribution of H+-K+-ATPase from tubulovesicles to the apical membrane. To identify the targets of GTPgammaS, effects of peptide fragments of certain GTP-binding proteins were examined. Although none of the peptides related to Rab proteins showed any effect, the inhibitory peptide of Arf protein inhibited cAMP-stimulated secretion. These results demonstrate that our new model, the beta-escin-permeabilized gland, allows the introduction of relatively large molecules, e.g., peptides, into the cell, and will be quite useful for analyzing signal transduction of parietal cell function.


Subject(s)
Escin/pharmacology , Gastric Mucosa/drug effects , Gastric Mucosa/metabolism , Aminopyrine/antagonists & inhibitors , Animals , Biological Transport , Enzyme Inhibitors/pharmacology , Escin/chemistry , GTP-Binding Proteins/chemistry , Gastric Acid/metabolism , Guanosine 5'-O-(3-Thiotriphosphate)/pharmacology , H(+)-K(+)-Exchanging ATPase/metabolism , In Vitro Techniques , Peptide Fragments/pharmacology , Peptides/pharmacology , Permeability , Protein Kinases/chemistry , Rabbits
14.
J Membr Biol ; 168(3): 209-20, 1999 Apr 01.
Article in English | MEDLINE | ID: mdl-10191355

ABSTRACT

When rabbit isolated gastric glands were stimulated via the cyclic AMP pathway, a phosphorylated protein band of about 120 kDa (pp120) was markedly increased in the apical membrane-rich fraction, concomitant with an increase in the amount of H,K-ATPase and the phosphorylation of the cytoskeletal protein ezrin in the same fraction. The cytosolic fraction, but not other membrane fractions, also contained a protein with common features to the membrane-bound pp120, i.e., comigration in two-dimensional gels with a pI of approximately 4.5, anomalous mobility in SDS-PAGE, reactivity to antibodies, and phosphorylation, indicating that these two proteins were identical. The possibility that pp120 is vinculin was completely excluded. Using antibody against pp120, this protein was found to be almost exclusively in the gastric parietal cell. Biochemical and immunohistochemical analyses suggest that pp120 exists mainly in the cytosol, and that a small part of the protein binds to the apical membrane when the parietal cell is stimulated via the cyclic AMP pathway. In the presence of histone, purified pp120 produced phosphorylation on pp120 as well as histone. The inhibitor profile of this kinase activity is not consistent with any known kinase. We conclude that pp120 is closely associated with a new type of kinase, and translocates from cytosol to the apical membrane when the parietal cell is stimulated.


Subject(s)
1-Methyl-3-isobutylxanthine/pharmacology , Bucladesine/pharmacology , Cyclic AMP/physiology , Histamine/pharmacology , Parietal Cells, Gastric/metabolism , Phosphoproteins/metabolism , Protein Kinases/metabolism , Protein Processing, Post-Translational/drug effects , Second Messenger Systems/drug effects , Animals , Biological Transport , Cell Membrane/enzymology , Cell Polarity , H(+)-K(+)-Exchanging ATPase/metabolism , Microsomes/enzymology , Molecular Weight , Parietal Cells, Gastric/drug effects , Phosphoproteins/isolation & purification , Phosphorylation/drug effects , Rabbits , Rats , Rats, Wistar , Vinculin/isolation & purification , Vinculin/metabolism
15.
Eur J Pharmacol ; 365(1): 77-89, 1999 Jan 15.
Article in English | MEDLINE | ID: mdl-9988126

ABSTRACT

In order to examine some possibly misleading conclusions of the pharmacological analysis of the signal transduction pathways of gastric acid secretion, we evaluated various agents including inhibitors of protein kinase C, cyclic AMP-dependent protein kinase, phospholipase C, phospholipase A2, lipoxygenase, casein kinase, calmodulin, myosin light chain kinase, tyrosine kinase, anion exchanger, and protein phosphatase; and activators of protein kinase C. Among them, the cyclic AMP-dependent protein kinase inhibitor N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinylsulfonamide (H-89), the phospholipase A2 inhibitor 2-(p-amylcinnamoyl)amino-4-chlorobenzoic acid (ONO-RS-082), three myosin light chain kinase inhibitors (1-(5-iodonaphthalene-1-sulfonyl)-1H-hexahydro-1,4-diazepine (ML-7), 1-(5-chloronaphthalene-1-sulfonyl)-1H-hexahydro-1,4-diazepine (ML-9), and wortmannin), the anion exchanger inhibitor 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), the phospholipase C inhibitor neomycin, and most known calmodulin antagonists strongly inhibited [14C]aminopyrine accumulation, an indicator of acid secretion, in isolated rabbit gastric glands stimulated by N6,2'-O-dibutyryl-cyclic AMP. ONO-RS-082, calmidazolium, and DIDS inhibited H+,K+-ATPase. Most of the chemicals with antisecretory activity showed protonophore-like activity in gastric microsomes as well as in the mitochondria. It is concluded that H-89, ONO-RS-082, ML-7, ML-9, neomycin, and all calmodulin antagonists tested so far should not be used as tools to analyze gastric acid secretion.


Subject(s)
Aristolochic Acids , Parietal Cells, Gastric/physiology , Signal Transduction , 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid/pharmacology , Adenosine Triphosphate/pharmacology , Aminobenzoates/pharmacology , Animals , Benzoquinones/pharmacology , Bucladesine/pharmacology , Calmodulin/antagonists & inhibitors , Cell Membrane/drug effects , Cell Membrane/metabolism , Chlorobenzoates , Cinnamates/pharmacology , Enzyme Inhibitors/pharmacology , Gastric Acid/metabolism , Gastric Mucosa/drug effects , Gastric Mucosa/metabolism , H(+)-K(+)-Exchanging ATPase/drug effects , H(+)-K(+)-Exchanging ATPase/metabolism , Hydrogen-Ion Concentration , In Vitro Techniques , Mitochondria/drug effects , Mitochondria/metabolism , Myosin-Light-Chain Kinase/antagonists & inhibitors , Neomycin/pharmacology , Parietal Cells, Gastric/drug effects , Phenanthrenes/pharmacology , Phospholipases A/antagonists & inhibitors , Phospholipases A2 , Phosphoprotein Phosphatases/antagonists & inhibitors , Protein Kinase Inhibitors , Protons , Rabbits , Type C Phospholipases/antagonists & inhibitors , ortho-Aminobenzoates
16.
Jpn J Pharmacol ; 78(2): 147-59, 1998 Oct.
Article in English | MEDLINE | ID: mdl-9829618

ABSTRACT

We examined the validity of the high K+ condition, widely used as a model to investigate the mechanism of acid secretion, employing isolated rabbit gastric glands. When Na+ in the nutrient solution was replaced with K+, the acid secretion monitored by the accumulation of 14C-aminopyrine increased K+-concentration-dependently. Stimulation of carbachol or dbcAMP was also greatly enhanced by K+. The responses to forskolin, phorbol ester, gastrin and submaximal dose of histamine were potentiated in the high K+ condition, but the responses to isobutylmethylxanthine, calyculin A and the maximal dose of histamine were not. The augmented response to carbachol was not dependent on [Ca2+]o. Replacement of Na+ with any other ions, including Rb+, Cs+, Li+, tetramethylammonium and choline, increased the basal aminopyrine ratio. Most of these ions were also effective in enhancing the response to dbcAMP, but failed to augment the effect of carbachol. Replacement of Na+ with K+, Rb+, Cs+ and Li+ transiently elicited acid secretion in the isolated mouse whole stomach. These results suggest that the high K+ is not a simple model for the direct activation of the proton pump, but the augmentation consists of two components, i.e., the combined effects of low Na+ and high K+. The former augments basal and cAMP-stimulated acid production, whereas the latter augments acid production by any agonist except calyculin A.


Subject(s)
Cations/pharmacology , Gastric Acid/metabolism , Potassium/pharmacology , Sodium/pharmacology , 1-Methyl-3-isobutylxanthine/pharmacology , Aminopyrine/metabolism , Animals , Atropine/pharmacology , Bucladesine/pharmacology , Calcium/pharmacology , Carbachol/pharmacology , Carcinogens/pharmacology , Cimetidine , Cyclic AMP/metabolism , Dose-Response Relationship, Drug , Electric Stimulation , Enzyme Inhibitors/pharmacology , Gastric Mucosa/drug effects , Gastric Mucosa/metabolism , Histamine H2 Antagonists/pharmacology , In Vitro Techniques , Male , Mice , Muscarinic Agonists/pharmacology , Muscarinic Antagonists/pharmacology , Omeprazole/pharmacology , Rabbits , Rotenone/pharmacology , Stomach/drug effects , Stomach/innervation , Tetradecanoylphorbol Acetate/pharmacology , Uncoupling Agents/pharmacology , Vagus Nerve/physiology
17.
Biochem Biophys Res Commun ; 251(2): 576-9, 1998 Oct 20.
Article in English | MEDLINE | ID: mdl-9792815

ABSTRACT

We investigated the possible changes in the distribution of HSP27 after a brief hypoxia-reoxygenation stress in the rat myoblast cell line, H9c2, as a model of ischemic preconditioning. Cells were exposed to 4 cycles of 5 min. of hypoxia and 5 min. of reoxygenation. In the normoxic condition, HSP27 was exclusively found in the cytosolic fraction. After the hypoxia-reoxygenation cycle, HSP27 redistributed to the cytoskeletal fraction, which was blocked by 10 microM SB 203580, a specific inhibitor of p38 MAP kinase. Cells treated with the repetitive hypoxia-reoxygenation developed resistance against cell death induced by hypoxia for 24 hours. The changes in localization of HSP27 found in the present study may reflect the mechanism of preconditioning in the cardiac myocyte.


Subject(s)
Cell Hypoxia/physiology , Cytoskeleton/metabolism , Heat-Shock Proteins/metabolism , Mitogen-Activated Protein Kinases , Animals , Calcium-Calmodulin-Dependent Protein Kinases/antagonists & inhibitors , Cell Line , Cell Survival , Cloning, Molecular , Cytoskeleton/drug effects , Enzyme Inhibitors/pharmacology , Imidazoles/pharmacology , Ischemic Preconditioning , Liver/metabolism , Muscles , Pyridines/pharmacology , Rats , Recombinant Fusion Proteins/metabolism , Reverse Transcriptase Polymerase Chain Reaction , p38 Mitogen-Activated Protein Kinases
19.
J Pharmacol Exp Ther ; 282(3): 1379-88, 1997 Sep.
Article in English | MEDLINE | ID: mdl-9316850

ABSTRACT

In order to elucidate the role of phospholipase C (PLC) in gastric acid secretion, we used U73122, a commonly employed specific inhibitor of receptor-mediated PLC, and its negative control, U73343. Although 10 microM U73122 inhibited the increase in [Ca++]i induced by U46619 in rabbit platelets, Ca++ transients in the rabbit parietal cells elicited by histamine and carbachol were both resistant to the inhibitor. U73122 augmented the acid secretion of isolated gastric glands stimulated by histamine, carbachol and dbcAMP, possibly through its indirect Ca++-releasing effect on the intracellular calcium store. U73122 potently inhibited K+-p-nitrophenylphosphatase without affecting overall H+,K+-ATPase activity. On the other hand, the negative control, U73343, strongly inhibited the acid secretion stimulated by all agonists tested. The inhibitory effect was also evident on digitonin-permeabilized glands and on the proton gradient of gastric vesicles. U73343 itself is not a proton pump inhibitor, so it was considered a protonophore. In conclusion, the widely used PLC-inhibitor, U73122, and its negative control, U73343, are both useless as tools for analyzing the role of PLC in rabbit parietal cells. The former is ineffective on gastric PLC and works as an intracellular calcium releaser, and the latter works as a protonophore.


Subject(s)
Estrenes/pharmacology , Parietal Cells, Gastric/drug effects , Phosphodiesterase Inhibitors/pharmacology , Pyrrolidinones/pharmacology , Type C Phospholipases/antagonists & inhibitors , Animals , Calcium/metabolism , Gastric Acid/metabolism , In Vitro Techniques , Parietal Cells, Gastric/metabolism , Rabbits , Sodium-Potassium-Exchanging ATPase/drug effects
20.
Jpn J Pharmacol ; 75(1): 33-42, 1997 Sep.
Article in English | MEDLINE | ID: mdl-9334884

ABSTRACT

In Chinese hamster ovary cells stably expressing the cloned human cholecystokinin (CCK)B/ gastrin receptor, cholecystokinin octapeptide (CCK-8) evoked increases in [Ca2+]i monitored by digitized video imaging of fura-2 fluorescence ratios. At concentrations around 10 pM, CCK-8 elicited [Ca2+]i oscillations, which were blocked by elimination of extracellular Ca2+, by a phospholipase C inhibitor, U-73122, by a protein kinase C inhibitor, H7, as well as by phospholipase A2 (PLA2) inhibitors, ONO-RS-082 and aristolochic acid. At higher concentrations, CCK-8 induced a single biphasic [Ca2+]i rise consisting of a large peak followed by a lower sustained plateau, while the response turned into [Ca2+]i oscillation when the extracellular Ca2+ was eliminated or a PLA2 inhibitor was included. CCK-8 stimulated the release of arachidonic acid, and this was inhibited by aristolochic acid. Arachidonic acid caused an increase in [Ca2+]i which was dependent upon extracellular Ca2+. These results suggest that the activation of PLA2 might be involved, at least in part, in the Ca2+ influx that maintains the sustained plateau phase of [Ca2+]i as well as the [Ca2+]i oscillation when CCKB receptors are stimulated.


Subject(s)
Calcium/metabolism , Receptors, Cholecystokinin/genetics , Animals , Arachidonic Acid/metabolism , Arachidonic Acid/pharmacology , CHO Cells , Calcium/physiology , Calcium Channels/drug effects , Clone Cells/physiology , Cricetinae , Exocrine Glands/drug effects , Exocrine Glands/metabolism , Gastric Mucosa/drug effects , Gastric Mucosa/metabolism , Gastrins/pharmacology , Gastrointestinal Agents/pharmacology , Humans , Phospholipases A/physiology , Phospholipases A2 , Protein Kinase C/physiology , Rabbits , Receptor, Cholecystokinin B , Recombinant Proteins/genetics , Signal Transduction/drug effects , Sincalide/pharmacology , Transfection
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