ABSTRACT
1. Cibenzoline, (+/-)-2-(2,2-diphenylcyclopropyl-2-imidazoline succinate, has been clinically used as one of the Class I type antiarrhythmic agents and also reported to block ATP-sensitive K(+) channels in excised membranes from heart and pancreatic beta cells. In the present study, we investigated if this drug inhibited gastric H(+),K(+)-ATPase activity in vitro. 2. Cibenzoline inhibited H(+),K(+)-ATPase activity of permeabilized leaky hog gastric vesicles in a concentration-dependent manner (IC(50): 201 microM), whereas no effect was shown on Na(+),K(+)-ATPase activity of dog kidney (IC(50): >1000 microM). Similarly, cibenzoline inhibited H(+),K(+)-ATPase activity of HEK-293 cells (human embryonic kidney cell line) co-transfected with rabbit gastric H(+),K(+)-ATPase alpha- and beta-subunit cDNAs (IC(50): 183 microM). 3. In leaky gastric vesicles, inhibition of H(+),K(+)-ATPase activity by cibenzoline was attenuated by the addition of K(+) (0.5 - 5 mM) in a concentration-dependent manner. The Lineweaver-Burk plot of the H(+),K(+)-ATPase activity shows that cibenzoline increases K(m) value for K(+) without affecting V(max), indicating that this drug inhibits H(+),K(+)-ATPase activity competitively with respect to K(+). 4. The inhibitory effect of H(+),K(+)-ATPase activity by cibenzoline with normal tight gastric vesicles did not significantly differ from that with permeabilized leaky gastric vesicles, indicating that this drug reacted to the ATPase from the cytoplasmic side of the membrane. 5. These findings suggest that cibenzoline is an inhibitor of gastric H(+),K(+)-ATPase with a novel inhibition mechanism, which inhibits gastric H(+),K(+)-ATPase by binding its K(+)-recognition site from the cytoplasmic side.
Subject(s)
Gastric Mucosa/enzymology , H(+)-K(+)-Exchanging ATPase/metabolism , Imidazoles/metabolism , Potassium Channel Blockers/metabolism , Proton Pump Inhibitors , Adenosine Triphosphate/pharmacology , Animals , Binding Sites , Cell Line , Cells, Cultured , Cytoplasm/metabolism , Cytoplasmic Vesicles/drug effects , Dogs , Dose-Response Relationship, Drug , Gastric Mucosa/drug effects , H(+)-K(+)-Exchanging ATPase/genetics , Humans , Imidazoles/pharmacology , Inhibitory Concentration 50 , Kidney/cytology , Kidney/enzymology , Kidney/metabolism , Potassium/metabolism , Potassium/pharmacology , Potassium Channel Blockers/pharmacology , Rabbits , Swine , TransfectionABSTRACT
The sixth transmembrane (M6) segment of the catalytic subunit plays an important role in the ion recognition and transport in the type II P-type ATPase families. In this study, we singly mutated all amino acid residues in the M6 segment of gastric H(+),K(+)-ATPase alpha-subunit with alanine, expressed the mutants in HEK-293 cells, and studied the effects of the mutation on the functions of H(+),K(+)-ATPase; overall K(+)-stimulated ATPase, phosphorylation, and dephosphorylation. Four mutants, L819A, D826A, I827A, and L833A, completely lost the K(+)-ATPase activity. Mutant L819A was phosphorylated but hardly dephosphorylated in the presence of K(+), whereas mutants D826A, I827A, and L833A were not phosphorylated from ATP. We found that almost all of these amino acid residues, which are important for the function, are located on the same side of the alpha-helix of the M6 segment. In addition, we found that amino acids involved in the phosphorylation are located exclusively in the cytoplasmic half of the M6 segment and those involved in the K(+)-dependent dephosphorylation are in the luminal half. Several mutants such as I821A, L823A, T825A, and P829A partly retained the K(+)-ATPase activity accompanying the decrease in the rate of phosphorylation.
Subject(s)
H(+)-K(+)-Exchanging ATPase/chemistry , Stomach/enzymology , Alanine , Amino Acid Sequence , Cell Line , H(+)-K(+)-Exchanging ATPase/physiology , Humans , Molecular Sequence Data , Mutagenesis , Protein Subunits , Structure-Activity Relationship , TemperatureABSTRACT
Gastric H+,K+-ATPase consists of alpha- and beta-subunits. The catalytic alpha-subunit contains a very unique structure consisting of lysine and glycine clusters, KKK(or KKKK)AG(G/R)GGGK-(K/R)K, in the amino-terminal cytoplasmic region. This structure is well conserved in all gastric H+,K+-ATPases from different animal species, and was postulated to be the site controlling the access of cations (or proton) to its binding site. In this report, we studied the role of this unique structure by expressing several H+,K+-ATPase mutants of the alpha-subunit together with the wild-type beta-subunit in HEK-293 cells. Even after replacing all the positively-charged amino acid residues (six lysines and one arginine) in the cluster with alanine or removing all the glycine residues in the cluster, the mutants preserved the H+,K+-ATPase activity, and showed similar affinity for ATP and K+ as well as similar pH profiles as those of wild-type H+,K+-ATPase, indicating that the cluster is not indispensable for H+,K+-ATPase activity and not directly involved in determination of the affinity for cation (proton).
Subject(s)
Gastric Mucosa/enzymology , Glycine/chemistry , H(+)-K(+)-Exchanging ATPase/chemistry , H(+)-K(+)-Exchanging ATPase/metabolism , Lysine/chemistry , Adenosine Triphosphate/pharmacology , Amino Acid Sequence , Cations/pharmacokinetics , Cell Line , Cell Membrane/enzymology , DNA, Complementary , Enzyme Activation/drug effects , Enzyme Activation/genetics , Gene Expression Regulation, Enzymologic , Glycine/genetics , H(+)-K(+)-Exchanging ATPase/genetics , Humans , Hydrogen-Ion Concentration , Kidney/cytology , Lysine/genetics , Molecular Sequence Data , Mutagenesis, Site-Directed/physiology , Potassium/pharmacokinetics , Protein Structure, Tertiary , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , TransfectionABSTRACT
The effects of 9,11-epithio-11,12-methano-thromboxane A(2) (STA(2)), a stable thromboxane A(2) analogue, and carbachol on colonic Ca(2+)-activated K(+) channels were studied. In indo-1-loaded single cells in isolated rat colonic crypts, both STA(2) (0.1 microM) and carbachol (10 microM) transiently increased intracellular free Ca(2+) concentration ([Ca(2+)](i)) by 136 and 155 nm, respectively. In whole-cell current-clamp experiments of the colonic crypt cells with Cl(-)-free solutions, carbachol (10 microM) hyperpolarized the cell by 19.7 mV, while STA(2) (0.1 microM) did not affect the membrane potential. In the isolated colonic mucosa that was permeabilized mucosally by a monovalent ionophore nystatin in the presence of a serosally directed K(+) gradient, carbachol (10 microM) transiently elicited K(+) current, but STA(2) (0.1 microM) did not. These results indicate that STA(2) elevates [Ca(2+)](i) in rat colonic crypt cells but does not activate basolateral Ca(2+)-activated K(+) channels.
Subject(s)
Calcium/metabolism , Colon/metabolism , Potassium Channels/metabolism , Thromboxane A2/analogs & derivatives , Thromboxane A2/pharmacology , Animals , Carbachol/pharmacology , Cholinergic Agonists/pharmacology , Colon/cytology , Colon/drug effects , Female , In Vitro Techniques , Intestinal Mucosa/cytology , Intestinal Mucosa/drug effects , Intestinal Mucosa/metabolism , Ionophores/pharmacology , Membrane Potentials/drug effects , Membrane Potentials/physiology , Nystatin/pharmacology , Patch-Clamp Techniques , Rats , Rats, WistarABSTRACT
The antitumor drug irinotecan clinically causes severe diarrhea as a side effect. Thromboxane A(2) (TXA(2)), released by irinotecan, has been shown to be a novel physiological stimulant of Cl(-) secretion in the rat colon. Herein, we examined the effect of loperamide, an antidiarrhea drug, on Cl(-) secretion induced by irinotecan; 9, 11-epithio-11,12-methano-thromboxane A(2) (STA(2)), a stable TXA(2) analog; and prostaglandin E(2) (PGE(2)) by using isolated mucosae of the rat colon. In the presence of atropine, loperamide in a concentration-dependent manner inhibited the Cl(-) secretion induced by irinotecan, STA(2), and PGE(2). However, the drug inhibited more effectively the irinotecan- and STA(2)-induced secretion (IC(50) = 0. 7 and 1.2 microM, respectively) than the PGE(2)-induced secretion (IC(50) = 23 microM). Naloxone, an opiate antagonist, did not affect the antisecretory action of loperamide. Similar to the case for loperamide, W-7, a specific calmodulin antagonist, inhibited more effectively the STA(2)-induced Cl(-) secretion (IC(50) = 5 microM) than the PGE(2)-induced secretion (IC(50) = 36 microM). W-5, a low-affinity calmodulin antagonist (a dechlorinated control analog of W-7), also inhibited the STA(2)-induced secretion, but this effect was much less than that of W-7. STA(2)-induced increase in the intracellular free Ca(2+) concentration of single colonic crypt cells was not affected by loperamide. We suggest that loperamide efficiently inhibits the TXA(2)-induced secretion by blocking the calmodulin system in the colonic epithelium. The present results may explain why coadministration of loperamide with irinotecan is clinically efficient for avoiding the irinotecan-induced side effect of diarrhea.
Subject(s)
Antidiarrheals/pharmacology , Chlorides/metabolism , Colon/drug effects , Loperamide/pharmacology , Thromboxane A2/antagonists & inhibitors , Animals , Calcium/metabolism , Camptothecin/analogs & derivatives , Camptothecin/pharmacology , Colon/metabolism , Dinoprostone/pharmacology , Female , In Vitro Techniques , Irinotecan , Naloxone/pharmacology , Rats , Rats, Wistar , Thromboxane A2/analogs & derivatives , Thromboxane A2/pharmacologyABSTRACT
Thromboxane A(2) is a novel endogenous secretagogue of Cl(-) secretion in the distal colon. Here, we examined if the Cl(-) secretion caused by platelet-activating factor (PAF; 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) is mediated by thromboxane A(2) production using isolated mucosae of the rat colon. Furosemide (100 microM) and 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB; 300 microM) completely inhibited PAF (10 microM)-induced increase in short-circuit current (Isc) across the mucosa, indicating that PAF caused a Cl(-) secretion in the rat colon. A selective thromboxane A(2) receptor antagonist (sodium(E)-11-[2-(5, 6-dimethyl-1-benzimidazolyl)-ethylidene]-6,11-dihydrobenz[b, e]oxepine-2-carboxylate monohydrate; KW-3635), and a selective thromboxane synthase inhibitor (sodium 4-[alpha-hydroxy-5-(1-imidazolyl)-2-methylbenzyl]-3, 5-dimethylbenzoate dihydrate; Y-20811) inhibited the PAF-induced Cl(-) current in a concentration-dependent manner. The IC(50) values of KW-3635 and Y-20811 were 2.1 and 0.5 microM, respectively. 30 microM KW-3635 and 1 microM Y-20811 inhibited the PAF response by 92% and 83%, respectively. These inhibitors did not affect the prostaglandin E(2)-induced increase in Isc. A 5-lipoxygenase-activating protein inhibitor (3-[1-(p-chlorobenzyl)-5-(isopropyl)-3-t-butylthioindol-2-yl]-2, 2-dimethyl-propanoic acid sodium; MK-886) (5 microM) did not affect the PAF-induced Cl(-) current. The present study suggests that the PAF-induced Cl(-) secretion in the rat colonic mucosa is mainly mediated by a release of thromboxane A(2).
Subject(s)
Chlorides/physiology , Colon/metabolism , Platelet Activating Factor/pharmacology , Thromboxane A2/physiology , 5-Lipoxygenase-Activating Proteins , Animals , Benzimidazoles/pharmacology , Benzoxepins/pharmacology , Carrier Proteins/antagonists & inhibitors , Chlorides/metabolism , Colon/drug effects , Dinoprostone/pharmacology , Enzyme Inhibitors/pharmacology , Female , Imidazoles/pharmacology , In Vitro Techniques , Indoles/pharmacology , Intestinal Mucosa/drug effects , Intestinal Mucosa/metabolism , Lipoxygenase Inhibitors/pharmacology , Membrane Proteins/antagonists & inhibitors , Platelet Activating Factor/antagonists & inhibitors , Platelet Aggregation Inhibitors/pharmacology , Rats , Rats, Wistar , Receptors, Prostaglandin E/antagonists & inhibitors , Receptors, Prostaglandin E, EP1 Subtype , Receptors, Thromboxane/antagonists & inhibitors , Secretory Rate/drug effects , Thromboxane A2/biosynthesis , Thromboxane-A Synthase/antagonists & inhibitorsABSTRACT
By means of a functional expression system and site-directed mutagenesis, we analyzed the role of the putative K(+)-binding site, Glu-345, located in the fourth transmembrane segment of the gastric H(+),K(+)-ATPase alpha-subunit. In the present study, we used several mutants, with alanine, isoleucine, leucine, glutamine, valine, lysine, and aspartic acid instead of Glu-345, and analyzed the H(+),K(+)-ATPase partial reactions of the mutants to determine the precise role of this residue. All the mutants except E345Q exhibited no H(+),K(+)-ATPase activity. The E345Q mutant showed 3-times higher affinity for ATP. This mutation shifted the optimum pH toward a more alkaline one. The E345A, E345I, E345L, E345V as well as E345Q mutants were phosphorylated with ATP as in the case of the wild-type H(+),K(+)-ATPase, whereas the E345K mutant was not phosphorylated. The E345Q mutant was dephosphorylated in the presence of K(+), but its affinity for K(+) was significantly lower than that of the wild type. The E345A, E345I, E345L, and E345V mutants did not exhibit sensitivity to K(+) in the dephosphorylation step below 3 mM K(+). Therefore, Glu-345 is important for the conformational change induced by K(+), especially in the dephosphorylation step in which K(+) reacts with the enzyme from the luminal side with high affinity and accelerates the release of inorganic phosphate. The glutamic acid in the fourth transmembrane segment is conserved, and was found to be involved in the cation-induced conformational change in H(+),K(+)-ATPase as well as Na(+),K(+)-ATPase and Ca(2+)-ATPase, however, the precise roles of the side chain in the function were different.
Subject(s)
Gastric Mucosa/enzymology , H(+)-K(+)-Exchanging ATPase/chemistry , Adenosine Triphosphate/chemistry , Amino Acids/genetics , Animals , Binding Sites , Cell Fractionation , Cell Line , Cell Membrane/chemistry , Cell Membrane/metabolism , DNA Mutational Analysis , Glutamic Acid/genetics , Glutamine/metabolism , H(+)-K(+)-Exchanging ATPase/genetics , H(+)-K(+)-Exchanging ATPase/metabolism , Humans , Hydrogen-Ion Concentration , Membrane Proteins/chemistry , Membrane Proteins/metabolism , Mutagenesis, Site-Directed , Phosphorylation , Point Mutation , Potassium/metabolism , Protein Conformation , Rabbits , TransfectionABSTRACT
Gastric H(+),K(+)-ATPase consists of alpha and beta-subunits. The alpha-subunit is the catalytic subunit, and the beta-subunit is a glycoprotein stabilizing the alpha/beta complex in the membrane as a functional enzyme. There are seven putative N-glycosylation sites on the beta-subunit. In this study, we examined the roles of the carbohydrate chains of the beta-subunit by expressing the alpha-subunit together with the beta-subunit in which one, several, or all of the asparagine residues in the N-glycosylation sites were replaced by glutamine. Removing any one of seven carbohydrate chains from the beta-subunit retained the H(+),K(+)-ATPase activity. The effects of a series of progressive removals of carbohydrate chains on the H(+),K(+)-ATPase activity were cumulative, and removal of all carbohydrate chains resulted in the complete loss of H(+), K(+)-ATPase activity. Removal of any single carbohydrate chain did not affect the alpha/beta assembly; however, little alpha/beta assembly was observed after removal of all the carbohydrate chains from the beta-subunit. In contrast, removal of three carbohydrate chains inhibited the surface delivery of the beta-subunit and the alpha-subunit assembled with the beta-subunit, indicating that the surface delivery mechanism is more dependent on the carbohydrate chains than the expression of the H(+),K(+)-ATPase activity and alpha/beta assembly.
Subject(s)
Gastric Mucosa/enzymology , Glycoproteins/metabolism , H(+)-K(+)-Exchanging ATPase/metabolism , Animals , COS Cells , Cell Compartmentation , Gene Expression Regulation, Enzymologic , Glycoproteins/genetics , Glycosylation , H(+)-K(+)-Exchanging ATPase/genetics , Models, Molecular , Protein Processing, Post-Translational , Protein Structure, Tertiary , Rabbits , Recombinant Proteins/metabolismABSTRACT
We produced an immortalized colonic epithelial cell line, MCE301, using fetal mice transgenic for the temperature-sensitive simian virus 40 large T-antigen gene. MCE301 cells showed epithelial-like morphology and maintained tight connections with neighboring cells. The cells grew at a permissive temperature (33 degrees C), but the growth of the cells was significantly prevented at the nonpermissive temperature (39 degrees C). The cells expressed large T-antigen at 33 degrees C but not at 39 degrees C. MCE301 cells were not transformed, as judged by the absence of anchorage-independent growth in soft agar gel and lack of tumor formation in nude mice. Electron microscopic studies showed that the cells formed microvilli-like structures on the cell surface and junctional complexes such as tight junctions and desmosomes between the cells. The cells expressed cytosketal (acidic cytokeratins and actin), basement membrane (laminin and collagen type IV) and junctional complex proteins (ZO-1 and desmoplakin I + II), as judged by specific antibodies. Fetal bovine serum, epidermal growth factor, insulin-like growth factor and insulin significantly increased the cell growth at 33 degrees C. Moreover, MCE301 cells expressed colonic mucin Muc2 mRNA as demonstrated by reverse transcriptase-polymerase chain reaction, indicating that the cells originate from mucus-secreting cells. Alkaline phosphatase, a brush border-associated enzyme, was detected in the cells. Sodium butyrate (2 mM), an inducer of cellular differentiation, markedly elevated alkaline phosphatase activity. Thus, the present mouse colonic epithelial cell line MCE301 possessing these unique characteristics should provide a useful in vitro model of colonic epithelium.
Subject(s)
Antigens, Viral, Tumor/genetics , Colon/cytology , Epithelial Cells/cytology , Intestinal Mucosa/cytology , Simian virus 40/genetics , Alkaline Phosphatase/metabolism , Animals , Carcinogenicity Tests/methods , Cell Line , Cells, Immobilized , Growth Substances/physiology , Mice , Mice, Transgenic , Microvilli/ultrastructure , Mucin-2 , Mucins/biosynthesis , TemperatureABSTRACT
Recently, a gastric Mg(2+)-ATP-dependent phospholipid flippase was found. Here, the effects of ionophores and monovalent cations on the gastric flippase were examined. We found that translocation of the fluorescent analogue of phosphatidylcholine was inhibited by valinomycin in the presence of K(+). The inhibition depended on both the concentrations of valinomycin and K(+). Valinomycin did not inhibit translocation in the absence of K(+). Protonophores, carbonylcyanide-m-chlorophenylhydrazone (CCCP) and carbonylcyanide-p-(trifluoromethoxy)phenylhydrazone (FCCP), accelerated translocation by 190-270%. These increases were completely abolished by 2-methyl-8-(phenylmethoxy)imidazo-[1, 2-a]pyridine-3-acetonitrile (SCH 28080), a gastric flippase inhibitor. Since these protonophores did not affect the Mg(2+)-dependent ATPase activity that is responsible for phospholipid translocation by the flippase, the coupling ratio of the amount of transported phospholipids/the amount of hydrolyzed ATP was variable and seemed to depend on the state of the membrane bilayer, for example fluidity. Inhibition by the valinomycin-K(+) complex was abolished in the presence of CCCP or FCCP, indicating the valinomycin-K(+)-CCCP(FCCF) ternary complex did not inhibit the flippase.
Subject(s)
Ca(2+) Mg(2+)-ATPase/metabolism , Carbonyl Cyanide m-Chlorophenyl Hydrazone/pharmacology , Gastric Mucosa/enzymology , Ionophores/pharmacology , 4-Chloro-7-nitrobenzofurazan/analogs & derivatives , 4-Chloro-7-nitrobenzofurazan/pharmacology , Animals , Anti-Bacterial Agents/pharmacology , Biological Transport/drug effects , Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone/pharmacology , Enzyme Activation/drug effects , Gramicidin/pharmacology , Intracellular Membranes/enzymology , Macrolides/pharmacology , Phosphatidylcholines/pharmacokinetics , Phosphatidylcholines/pharmacology , Potassium Chloride/pharmacology , Sodium Chloride/pharmacology , Swine , Valinomycin/pharmacologyABSTRACT
Cl(-) channels in the basolateral membrane of parietal cells within isolated rat gastric glands were studied by the whole-cell patch-clamp technique. The membrane potential (E(m)) of non-stimulated parietal cells changed as a function of the basolateral extracellular Cl(-) concentration (46 mV per decade), but E(m) did not change significantly as a function of the K(+) concentration. The extracellular addition of prostaglandin E(2) (PGE(2); 10 microM) increased the whole-cell Cl(-) current. A bifunctional prostaglandin EP3 agonist/EP1 antagonist, 5(Z)-7-[(1S, 2S,3S,5R)-3-(trans-beta-styren)sulfonamido-6,6-dimethylbi cyclo-(3.1. 1)hept-2-yl]-5-heptenoic acid (ONO-NT-012; 10 microM), also increased the Cl(-) current. Basal Cl(-) currents and the PGE(2)- and ONO-NT-012-increased Cl(-) currents were voltage-independent and inhibited by a Cl(-) channel blocker, 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB), at 500 microM. The single Cl(-) channel conductance was estimated to be 0.29 picosiemens (pS) by variance noise analysis. Both PGE(2) and ONO-NT-012 increased intracellular free Ca(2+) concentration in the fura-2-loaded parietal cell transiently. The present study has shown that housekeeping sub-pS Cl(-) channels are present in the basolateral membrane of rat parietal cell, and that the channels are regulated positively by PGE(2) via the EP3 receptor.
Subject(s)
Chloride Channels/metabolism , Dinoprostone/physiology , Intracellular Membranes/metabolism , Parietal Cells, Gastric/metabolism , Animals , Artifacts , Bridged Bicyclo Compounds/pharmacology , Chloride Channels/antagonists & inhibitors , Male , Membrane Potentials/physiology , Osmolar Concentration , Patch-Clamp Techniques , Rats , Rats, Wistar , Styrenes/pharmacologyABSTRACT
Gastric H(+),K(+)-ATPase consists of alpha-subunit with 10 transmembrane domains and beta-subunit with a single transmembrane domain. We constructed cDNAs encoding chimeric beta-subunits between the gastric H(+),K(+)-ATPase and Na(+),K(+)-ATPase beta-subunits and co-transfected them with the H(+),K(+)-ATPase alpha-subunit cDNA in HEK-293 cells. A chimeric beta-subunit that consists of the cytoplasmic plus transmembrane domains of Na(+),K(+)-ATPase beta-subunit and the ectodomain of H(+),K(+)-ATPase beta-subunit assembled with the H(+),K(+)-ATPase alpha-subunit and expressed the K(+)-ATPase activity. Therefore, the whole cytoplasmic and transmembrane domains of H(+),K(+)-ATPase beta-subunit were replaced by those of Na(+),K(+)-ATPase beta-subunit without losing the enzyme activity. However, most parts of the ectodomain of H(+),K(+)-ATPase beta-subunit were not replaced by the corresponding domains of Na(+), K(+)-ATPase beta-subunit. Interestingly, the extracellular segment between Cys(152) and Cys(178), which contains the second disulfide bond, was exchangeable between H(+),K(+)-ATPase and Na(+), K(+)-ATPase, preserving the K(+)-ATPase activity intact. Furthermore, the K(+)-ATPase activity was preserved when the N-terminal first 4 amino acids ((67)DPYT(70)) in the ectodomain of H(+),K(+)-ATPase beta-subunit were replaced by the corresponding amino acids ((63)SDFE(66)) of Na(+),K(+)-ATPase beta-subunit. The ATPase activity was abolished, however, when 4 amino acids ((76)QLKS(79)) in the ectodomain of H(+),K(+)-ATPase beta-subunit were replaced by the counterpart ((72)RVAP(75)) of Na(+),K(+)-ATPase beta-subunit, indicating that this region is the most N-terminal one that discriminates the H(+),K(+)-ATPase beta-subunit from that of Na(+), K(+)-ATPase.
Subject(s)
H(+)-K(+)-Exchanging ATPase/metabolism , Sodium-Potassium-Exchanging ATPase/metabolism , Stomach/enzymology , Amino Acid Sequence , Binding Sites , Gene Expression , H(+)-K(+)-Exchanging ATPase/chemistry , H(+)-K(+)-Exchanging ATPase/genetics , Membranes/enzymology , Models, Molecular , Molecular Sequence Data , Precipitin Tests , Protein Conformation , Recombinant Fusion Proteins/metabolism , Sodium-Potassium-Exchanging ATPase/chemistry , Sodium-Potassium-Exchanging ATPase/geneticsABSTRACT
To clarify the presence of thromboxane A2 (TXA2) receptor in the colonic epithelium, we examined the effect of 9,11-epithio-11, 12-methano-thromboxane A2 (STA2), a stable analogue of TXA2, on intracellular free Ca2+ concentration ([Ca2+]i) of indo-1-loaded single cells in isolated rat colonic crypts by laser confocal microscopy. STA2 increased [Ca2+]i in a concentration-dependent manner with a transient peak phase and a subsequent plateau phase. The EC50 values at peak and plateau phases were 1 and 32 nM, respectively. The STA2-induced increase in [Ca2+]i was completely blocked by two selective TXA2 receptor antagonists, KW-3635 and ONO-3708. These antagonists did not affect both the basal [Ca2+]i and the carbaco-induced increase in [Ca2+]i. Prostaglandin E2 did not increase [Ca2+]i. These results indicate that the STA2-elicited increase in [Ca2+]i is mediated specifically by a TXA2 receptor in colonic crypt cells This is the first report showing the presence of a TXA2 receptor that is associated with Ca2+ mobilization in the colon.
Subject(s)
Calcium/metabolism , Colon/drug effects , Receptors, Thromboxane/metabolism , Animals , Benzimidazoles/pharmacology , Benzoxepins/pharmacology , Colon/cytology , Colon/metabolism , Female , In Vitro Techniques , Rats , Rats, Wistar , Receptors, Thromboxane/agonists , Thromboxane A2/analogs & derivatives , Thromboxane A2/pharmacologyABSTRACT
2-Methyl-8-(phenylmethoxy)imidazo(1,2-a)pyridine-3acetonitrile+ ++ (SCH 28080) is a K+ site inhibitor specific for gastric H+,K+-ATPase and seems to be a counterpart of ouabain for Na+,K+-ATPase from the viewpoint of reaction pattern (i.e. reversible binding, K+ antagonism, and binding on the extracellular side). In this study, we constructed several chimeric molecules between H+,K+-ATPase and Na+,K+-ATPase alpha-subunits by using rabbit H+,K+-ATPase as a parental molecule. We found that the entire extracellular loop 1 segment between the first and second transmembrane segments (M1 and M2) and the luminal half of the M1 transmembrane segment of H+, K+-ATPase alpha-subunit were exchangeable with those of Na+, K+-ATPase, respectively, preserving H+,K+-ATPase activity, and that these segments are not essential for SCH 28080 binding. We found that several amino acid residues, including Glu-822, Thr-825, and Pro-829 in the M6 segment of H+,K+-ATPase alpha-subunit are involved in determining the affinity for this inhibitor. Furthermore, we found that a chimeric H+,K+-ATPase acquired ouabain sensitivity and maintained SCH 28080 sensitivity when the loop 1 segment and Cys-815 in the loop 3 segment of the H+,K+-ATPase alpha-subunit were simultaneously replaced by the corresponding segment and amino acid residue (Thr) of Na+,K+-ATPase, respectively, indicating that the binding sites of ouabain and SCH 28080 are separate. In this H+, K+-ATPase chimera, 12 amino acid residues in M1, M4, and loop 1-4 that have been suggested to be involved in ouabain binding of Na+, K+-ATPase alpha-subunit are present; however, the low ouabain sensitivity indicates the possibility that the sensitivity may be increased by additional amino acid substitutions, which shift the overall structural integrity of this chimeric H+,K+-ATPase toward that of Na+,K+-ATPase.
Subject(s)
Enzyme Inhibitors/pharmacology , Imidazoles/pharmacology , Ouabain/pharmacology , Proton Pump Inhibitors , Recombinant Fusion Proteins/antagonists & inhibitors , Stomach/enzymology , Amino Acid Sequence , Animals , Base Sequence , Cell Line , DNA Primers , H(+)-K(+)-Exchanging ATPase/genetics , Humans , Molecular Sequence Data , Mutagenesis, Site-Directed , Rabbits , Recombinant Fusion Proteins/genetics , Sequence Homology, Amino AcidABSTRACT
Prostaglandin E2 stimulates a nitric oxide/cyclic GMP (NO/cGMP) pathway which activates basolateral Cl- channels in rabbit gastric parietal cells. We examined whether the NO/cGMP pathway protects parietal cells from ethanol (EtOH)-induced cytotoxicity, using a parietal cell-rich suspension purified from rabbit gastric mucosa. Cytotoxicity was assayed by measuring the release of a fluorescent dye from the cells. N2,O2-dibutyryl guanosine 3',5'-cyclic monophosphate (DBcGMP) showed a concentration-dependent protective effect against EtOH-induced cytotoxicity. The half-maximal effect of DBcGMP was observed at 24 microM. DBcGMP in a concentration-dependent manner opened the basolateral Cl- channels of parietal cells, the EC50 value being 44 microM. The EtOH-induced cytotoxicity decreased as the Cl- concentration of medium decreased. A 30-s treatment with 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB), an inhibitor of the Cl- channel, had a cytotoxic effect which was not prevented by pre-incubation with DBcGMP. The cytotoxic effects of EtOH and NPPB were additive and the NPPB effects did not depend on the medium Cl- concentration. The present study showed that cGMP protects the gastric parietal cell from EtOH-induced cytotoxicity, and this cytoprotection is related to basolateral Cl- channel activity in the plasma membrane via an unknown mechanism(s).
Subject(s)
Cyclic GMP/pharmacology , Ethanol/adverse effects , Parietal Cells, Gastric/drug effects , Animals , Calcium Channel Blockers/pharmacology , Chloride Channels/drug effects , Chlorides/pharmacology , Dibutyryl Cyclic GMP/pharmacology , Dose-Response Relationship, Drug , Male , Membrane Potentials/drug effects , Nitrobenzoates/adverse effects , Nitroprusside/pharmacology , Parietal Cells, Gastric/cytology , Parietal Cells, Gastric/pathology , Patch-Clamp Techniques , Rabbits , Vasodilator Agents/pharmacology , ortho-Aminobenzoates/pharmacologyABSTRACT
The regulatory mechanism of the plasma membrane Na+/Ca2+-exchanger in isolated rat hepatocytes was studied using microspectrofluorometry and 45Ca2+ uptake methods. Exposure of single hepatocytes to low-Na+ solutions induced an increase in the intracellular Ca2+ concentration ([Ca2+]i) which depended on the presence of extracellular Ca2+. 2-[2-[4-(4-nitrobenzyloxy)phenyl]ethyl]isothiourea methanesulfonate (KB-R7943), a novel selective inhibitor of Na+/Ca2+-exchangers, inhibited the initial rate of [Ca2+]i increase induced by exposure to the low-Na+ solution (IC50 = 2 microM). KB-R7943 also reduced the initial rate of 45Ca2+ uptake (IC50 = 4 microM). The increase in [Ca2+]i induced by exposure to the low-Na+ solution was inhibited by pre-incubation with 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7, 50 microM), but not with N-[2-(methylamino)ethyl]-5-isoquinolinesulfonamide (H-8, 60 microM) or a tyrosine kinase inhibitor, genistein (100 microM). Furthermore, taurocholate and phorbol-12,13-dibutyrate, both of which activate protein kinase C, promoted the increase in [Ca2+]i. These [Ca2+]i increases were sensitive to KB-R7943. Our results indicate that the Na+/Ca2+-exchanger is up-regulated via protein kinase C. The activity of Na+/Ca2+-exchangers is not evident under normal physiological conditions, suggesting that the exchanger may be activated under pathophysiological conditions.
Subject(s)
Liver/drug effects , Protein Kinase C/physiology , Sodium-Calcium Exchanger/antagonists & inhibitors , Thiourea/analogs & derivatives , Animals , Calcium/metabolism , Calcium/pharmacokinetics , Calcium Channel Blockers/pharmacology , Diltiazem/pharmacology , Extracellular Space/chemistry , Extracellular Space/metabolism , Liver/cytology , Male , Phosphorylation/drug effects , Rats , Rats, Sprague-Dawley , Sodium/pharmacology , Sodium-Calcium Exchanger/metabolism , Thiourea/pharmacology , Up-Regulation , Verapamil/pharmacologyABSTRACT
A guinea pig cDNA encoding the putative colonic H+-K+-ATPase alpha-subunit (T. Watanabe, M. Sato, K. Kaneko, T. Suzuki, T. Yoshida, and Y. Suzuki; GenBank accession no. D21854) was functionally expressed in HEK-293, a human kidney cell line. The cDNA for the putative colonic H+-K+-ATPase was cotransfected with cDNA for either rabbit gastric H+-K+-ATPase or Torpedo Na+-K+-ATPase beta-subunit. In both expressions, Na+-independent, K+-dependent ATPase (K+-ATPase) activity was detected in the membrane fraction of the cells, with a Michaelis-Menten constant for K+ of 0.68 mM. The expressed K+-ATPase activity was inhibited by ouabain, with its IC50 value being 52 microM. However, the activity was resistant to Sch-28080, an inhibitor specific for gastric H+-K+-ATPase. The ATPase was not functionally expressed in the absence of the beta-subunits. Therefore, it is concluded that the cDNA encodes the catalytic subunit (alpha-subunit) of the colonic H+-K+-ATPase. Although the beta-subunit of the colonic H+-K+-ATPase has not been identified yet, both gastric H+-K+-ATPase and Na+-K+-ATPase beta-subunits were found to act as a surrogate for the colonic beta-subunit for the functional expression of the ATPase. The present colonic H+-K+-ATPase first expressed in mammalian cells showed the highest ouabain sensitivity in expressed colonic H+-K+-ATPases so far reported (rat colonic in Xenopus oocytes had an IC50 = 0.4-1 mM; rat colonic in Sf9 cells had no ouabain sensitivity).
Subject(s)
Colon/enzymology , H(+)-K(+)-Exchanging ATPase/biosynthesis , Intestinal Mucosa/enzymology , Animals , Cell Line , Cell Membrane/enzymology , Gastric Mucosa/enzymology , Guinea Pigs , H(+)-K(+)-Exchanging ATPase/genetics , H(+)-K(+)-Exchanging ATPase/metabolism , Humans , Kidney , Kinetics , Molecular Sequence Data , Ouabain/pharmacology , Potassium Chloride/pharmacology , Rabbits , Rats , Recombinant Proteins/biosynthesis , Recombinant Proteins/metabolism , Torpedo , TransfectionABSTRACT
1. A camptothecin derivative, irinotecan (Cpt-11), is a topoisomerase I inhibitor and has a strong activity against a broad range of human cancer. One of the side-effects of this drug is diarrhoea. Here, we tried to determine the mediator of the irinotecan-induced Cl- secretion which may underlie this diarrhoea, using isolated mucosae of rat distal colon. 2. Irinotecan increased Cl- secretory current in a concentration-dependent manner across the mucosa, set between Ussing chambers. Thromboxane A2 (TXA2) has not been reported to date as a physiological stimulant of Cl- secretion in the distal colon. However, the major part of the present irinotecan-induced current was inhibited by selective thromboxane A2 receptor antagonists (KW-3635 and ONO-3708), and a selective thromboxane synthase inhibitor (Y-20811). In fact, we found that irinotecan stimulated the release of TXA2 in a concentration-dependent manner from the isolated mucosa into the bathing solutions. 3. Furthermore, 9,11-epithio-11,12-methano-thromboxane A2 (STA2), a stable analogue of TXA2, induced Cl- secretion, which was almost completely inhibited by the TXA2 receptor antagonists. 4. In single cells of isolated crypts, STA2 depolarized the cell and increased the membrane conductance, indicating that STA2 opened the apical Cl- channel of the crypt cells. 5. We conclude, therefore, that the irinotecan-induced endogenous TXA2 is a novel stimulant of the Cl- secretion from the crypt cells of distal colon.
Subject(s)
Camptothecin/analogs & derivatives , Chloride Channels/drug effects , Chlorides/metabolism , Colon/metabolism , Intestinal Mucosa/metabolism , Thromboxane A2/metabolism , Animals , Antineoplastic Agents, Phytogenic/pharmacology , Benzimidazoles/pharmacology , Benzoxepins/pharmacology , Camptothecin/pharmacology , Camptothecin/toxicity , Carbachol/pharmacology , Colon/drug effects , Cyclooxygenase Inhibitors/pharmacology , Dinoprostone/pharmacology , Female , Imidazoles/pharmacology , Indomethacin/pharmacology , Intestinal Mucosa/drug effects , Irinotecan , Membrane Potentials/physiology , Nitrobenzoates/pharmacology , Patch-Clamp Techniques , Rats , Rats, Wistar , Receptors, Thromboxane/antagonists & inhibitors , Tetrodotoxin/pharmacology , Thromboxane A2/analogs & derivatives , Thromboxane A2/pharmacology , Thromboxane-A Synthase/antagonists & inhibitorsABSTRACT
A compound, SCH 28080 (2-methyl-8-(phenylmethoxy)imidazo [1,2-a]pyridine-3-acetonitrile), reversibly inhibits gastric and renal ouabain-insensitive H+,K+-ATPase, but not colonic ouabain-sensitive H+,K+-ATPase. By using the functional expression system and site-directed mutagenesis, we analyzed the putative binding sites of SCH 28080 in gastric H+,K+-ATPase alpha-subunit. It was previously reported that the binding site of SCH 28080, which is a K+-site inhibitor specific for gastric H+,K+-ATPase, was in the first extracellular loop between the first and second transmembrane segments of the alpha-subunit; Phe-126 and Asp-138 were putative binding sites. However, we found that all the mutants in the first extracellular loop including Phe-126 and Asp-138 retained H+, K+-ATPase activity and sensitivity to SCH 28080. Therefore, amino acid residues in the first extracellular loop are not directly involved in the SCH 28080 binding nor indispensable for the H+, K+-ATPase activity. Here we propose a candidate residue that is important for the binding with SCH 28080, Glu-822 in the sixth transmembrane segment. Mutations of Glu-822 to Asp and Ala (mutants termed E822D and E822A, respectively) decreased the ATPase activity to about 45% and 35% of the wild-type enzyme, respectively, while the mutations to Gln and Leu abolished the activity. Mutant E822A showed a significantly lower affinity for K+ than the wild-type enzyme, indicating that Glu-822 is involved in determining the affinity for K+. The sensitivity of mutant E822D to SCH 28080 was 8 times lower than that of the wild-type enzyme. The counterpart of Glu-822 in gastric H+,K+-ATPase is Asp in Na+,K+-ATPase and other colonic ouabain-sensitive H+,K+-ATPase, which are insensitive to SCH 28080. These results suggest that Glu-822 is one of important sites that bind with SCH 28080.
Subject(s)
Enzyme Inhibitors/pharmacology , H(+)-K(+)-Exchanging ATPase/genetics , Imidazoles/pharmacology , Stomach/enzymology , Adenosine Triphosphate/metabolism , Amino Acid Sequence , Aspartic Acid/metabolism , Binding Sites/genetics , Cell Line , DNA Mutational Analysis , Glutamine/metabolism , H(+)-K(+)-Exchanging ATPase/metabolism , Humans , Molecular Sequence Data , Mutagenesis, Site-Directed , Phenylalanine/metabolism , Potassium/metabolism , Protein Folding , Protein Structure, Secondary , Proton Pump Inhibitors , Sequence AlignmentABSTRACT
The intracellular Ca2+ concentration ([Ca2+]i) in single isolated rat hepatocytes was measured using fura-2. Extracellular ATP induced La(3+)-sensitive and verapamil-insensitive Ca2+ influx together with Ca2+ release from intracellular Ca2+ stores. Incubation of hepatocytes with 2 microM thapsigargin produced a large prolonged increase in [Ca2+]i, which was insensitive to both 100 microM La3+ and 40 microM verapamil. Incubation with 1 mM dibutyryl cAMP increased [Ca2+]i in the presence of extracellular Ca2+ but did not in the absence of extracellular Ca2+, indicating that dibutyryl cAMP induces Ca2+ influx which was found to be sensitive to both La3+ and verapamil, without mobilizing Ca2+ from the intracellular pools. This study shows the presence of at least 3 different Ca2+ influx pathways in the plasma membrane: that is, 1) the ATP-induced, La(3+)-sensitive and verapamil-insensitive pathway; 2) the thapsigargin-induced, La(3+)-insensitive and verapamil-insensitive pathway; and 3) the cAMP-induced, La(3+)-sensitive and verapamil-sensitive pathway.