Magotoshi Morii

The potency of substituted benzimidazoles such as E3810, omeprazole, RO 18-5364 to inhibit gastric H+,K+-ATPase is correlated with the rate of acid-activation of the inhibitor

The half maximal inhibitory concentrations (IC50) of substituted benzimidazoles for the H+, K(+)-ATPase in hog gastric vesicles were measured by using the pyruvate kinase-lactate dehydrogenase-linked system in which hydrolysis of ATP was coupled with the oxidation of NADH. The vesicles were incubated in a solution containing a high concentration of KCl, valinomycin and Mg-ATP, and the intravesicular medium was acidified. The inhibitor was activated in the acidic medium and reacted with SH groups on the luminal (intravesicular) side of the ATPase. The active compound formed in the extravesicular medium (pH 6.11) was quenched by GSH. Under these conditions, IC50 of new compound E3810, 2[(4-(3-methoxypropoxy)-3-methylpyridine-2-yl)methyl-sulfinyl]-1H- benzimidazole sodium salt, was 0.072 microM and that of omeprazole was 0.47 microM at 25 degrees. On the other hand, the rates of formation of active compounds, tetracyclic sulfenamide derivatives, from original substituted benzimidazoles in 0.1 N HCl (k) were determined by measuring optical density at the characteristic wavelengths of the active compounds. There was a good correlation between IC50 and k for various substituted benzimidazoles including E3810, methoxy derivative of E3810, omeprazole, Ro 18-5364, H compound, picoprazole and timoprazole. This fact suggest that the rate of the formation of the acid-activated compound is a main factor determining the potency of the inhibitor.

Upregulation of thromboxane synthase in human colorectal carcinoma and the cancer cell proliferation by thromboxane A2

Tumor growth of colorectal cancers accompanies upregulation of cyclooxygenase‐2, which catalyzes a conversion step from arachidonic acid to prostaglandin H2 (PGH2). Here, we compared the expression levels of thromboxane synthase (TXS), which catalyzes the conversion of PGH2 to thromboxane A2 (TXA2), between human colorectal cancer tissue and its accompanying normal mucosa. It was found that TXS protein was consistently upregulated in the cancer tissues from different patients. TXS was also highly expressed in human colonic cancer cell lines. Depletion of TXS protein by the antisense oligonucleotide inhibited proliferation of the cancer cells. This inhibition was rescued by the direct addition of a stable analogue of TXA2. The present results suggest that overexpression of TXS and subsequent excess production of TXA2 in the cancer cells may be involved in the tumor growth of human colorectum.

Involvement of aquaporin-5 in differentiation of human gastric cancer cells

Litttle is known about the function of aquaporin (AQP) water channels in human gastric cancer. In the upper or middle part of human stomach, we found that expression level of AQP5 protein in intestinal type of adenocarcinoma was significantly higher than that in accompanying normal mucosa. AQP5 was localized in the apical membrane of the cancer cells. On the other hand, both AQP3 and AQP4 were not up-regulated in the adenocarcinoma. To elucidate the role of AQP5 in cancer cells, AQP5 was exogenously expressed in a cell line of poorly differentiated human gastric adenocarcinoma (MKN45). The AQP5 expression significantly increased the proportion of differentiated cells with a spindle shape, the activity of alkaline phosphatase, a marker for the intestinal epithelial cell type of cancer cells, and the expression level of laminin, an epithelial cell marker. Treatment of the MKN45 cells stably expressing AQP5 with HgCl2, an inhibitor of aquaporins, significantly decreased the proportion of differentiated cells and the activity of alkaline phosphatase. Our results suggest that up-regulation of AQP5 may be involved in differentiation of human gastric cancer cells.

Anion and cation channels in the basolateral membrane of rabbit parietal cells.

Ion channels in the basolateral membrane of rabbit parietal cells in isolated gastric glands were studied by the patch clamp technique. Whole-cell current-clamp recordings showed that the membrane potential (Em) changed systematically as a function of the chloride concentrations of the basolateral bathing solution ([Cl−]0), and of the pipette (intracellular) solution. The relationship betweenEm and [Cl−]0 was not affected by additions of histamine, dibutyryl-cAMP, 4-acetoamido-4′-isothiocyanostilbene-2,2′-disulfonic acid and diphenylamine-2-carboxylate. The whole-cell Cl− conductance was insensitive to voltage. In cell-attached and cell-free patch membranes, however, single Cl− channel opening events could not be observed. The value ofEm depended little on the basolateral K+ concentration, but inward-rectifier K+ currents were observed in the whole-cell configuration, activated by hyperpolarizing pulses and inhibited by extracellular Ba2+. In cell-attached and cell-free patches, openings of single inward-rectifier K+ channels and non-selective cation channels were infrequently recorded. Neither cAMP nor Ca2+ activated these cation channels. The single K+ channel conductance was about 230 pS under the symmetrical high K+ conditions and was inhibited by intracellular tetraethylammonium ions (TEA). The non-selective cation channel had a voltage-independent single conductance of 22 pS and was not inhibited by TEA.

Functional association between k+-cl- cotransporter-4 and H+, K+-ATPase in the apical canalicular membrane of gastric parietal cells.

We studied whether K+-Cl- cotransporters (KCCs) are involved in gastric HCl secretion. We found that KCC4 is expressed in the gastric parietal cells more abundantly at the luminal region of the gland than at the basal region. KCC4 was found in the stimulation-associated vesicles (SAV) derived from the apical canalicular membrane but not in the intracellular tubulovesicles, whereas H+,K+-ATPase was expressed in both of them. In contrast, KCC1, KCC2, and KCC3 were not found in either SAV or tubulovesicles. KCC4 coimmunoprecipitated with H+,K+-ATPase in the lysate of SAV. Interestingly the MgATP-dependent uptake of 36Cl- into the SAV was suppressed by either the H+,K+-ATPase inhibitor (SCH28080) or the KCC inhibitor ((R)-(+)-[(2-n-butyl-6,7-dichloro-2-cyclopentyl-2,3-dihydro-1-oxo-1H-inden-5-yl)oxy]acetic acid). The KCC inhibitor suppressed the H+ uptake into SAV and the H+,K+-ATPase activity of SAV, but the inhibitor had no effects on these activities in the freeze-dried leaky SAV. These results indicate that the K+-Cl- cotransport by KCC4 is tightly coupled with H+/K+ antiport by H+,K+-ATPase, resulting in HCl accumulation in SAV. In the tetracycline-regulated expression system of KCC4 in the HEK293 cells stably expressing gastric H+,K+-ATPase, KCC4 was coimmunoprecipitated with H+,K+-ATPase. The rate of recovery of intracellular pH in the KCC4-expressing cells after acid loading through an ammonium pulse was significantly faster than that in the KCC4-non-expressing cells. Our results suggest that KCC4 and H+,K+-ATPase are the main machineries for basal HCl secretion in the apical canalicular membrane of the resting parietal cell. They also may contribute in part to massive acid secretion in the stimulated state.

Specific proton pump inhibitors E3810 and lansoprazole affect the recovery process of gastric secretion in rats differently

After a single subcutaneous administration (30 mg/kg) of proton pump inhibitor 2-[(4-(3-methoxypropoxy)-3-methylpyridin-2-yl)-methylsulfiny l]- 1H-benzimidazole sodium salt (E3810), or lansoprazole in rats, time courses of inhibitory and recovery processes of acid secretion in vivo and pump enzyme activity in isolated microsomes were measured. The acid secretion rate which reflects H+,K(+)-ATPase activity in the secretory canalicular (apical) membrane was compared with that in the microsomal fraction which consists mostly of resting, intracellularly-pooled tubulovesicles. We found that the canalicular pump was first inhibited, followed by slow inhibition of the microsomal pump enzyme activity, with the rate of the latter process depending on the inhibitors. It took 2.5 hr for the half-maximal inhibition of the microsomal pump in E3810-treated rats, and 6 hr in lansoprazole-treated rats. The acid secretion and the microsomal enzyme activity completely recovered within 48 hr after the administration of E3810, but recovered by only 20% even 96 hr after the administration of lansoprazole. Incubation with dithiothreitol of isolated microsomes obtained from E3810-treated rats reactivated the enzyme activity, but not from rats treated with lansoprazole. These results suggest that dissociation of inhibitor from the pump and/or intracellular transport of the pump is affected differently by these inhibitors. Furthermore, it is possible that the half life of the proton pump protein is much longer (greater than 96 hr) than the previously proposed value of 30-48 hr.

The proton pump inhibitor, E3810, binds to the N-terminal half of the α-subunit of gastric H+,K+-ATPase

Abstract E3810 (2-{[4-(3-methoxypropoxy)-3-methylpyridine-2-yl]methylsulphinyl}-1 H -benzimidazole sodium salt), an inhibitor of gastric proton pump (gastric H + ,K + -ATPase), is activated in a luminal acidic environment of gastric glands and binds to a Cys residue of H + ,K + -ATPase on its luminal side. It was found that bound E3810 is transformed into a strongly fluorescent compound by UV-light irradiation (excitation wavelength = 335 nm, emission wavelength = 470 nm). The location of Cys residue bound with E3810 in the α-subunit of hog gastric H + ,K + -ATPase was estimated from the fluorescence labelling and limited tryptic digestion of the enzyme. Tryptic digestion in the presence of Mg-ATP produces N-terminal 67 kDa subfragment which contains the phosphorylation and fluorescein 5′-isothiocyanate binding sites and C-terminal 35 kDa subfragment. Trypsin digestion in the presence of KCl produces N-terminal 42 kDa and C-terminal 56 kDa subfragments. E3810 was found to bind to both N-terminal but not to any of two C-terminal subfragments. Taking the amino acid sequence and topology of this ATPase as well as the fact that the ratio of specific binding sites per α-subunit is one into consideration, the possibility that E3810 specifically binds to Cys 322 residue of hog gastric H + ,K + -ATPase is discussed.

Binding site of omeprazole in hog gastric H+,K+-ATPase

Omeprazole transforms into an active compound in an acidic environment, which is able to modify a sulfhydryl group of gastric H+,K(+)-ATPase. Omeprazole was transformed into a strongly fluorescent molecule by UV-light irradiation (excitation wavelength = 290 nm, emission wavelength = 335 nm). The omeprazole-modified residue of hog H+,K(+)-ATPase was estimated by the fluorescence of the omeprazole moiety and limited tryptic digestion of the enzyme. Among the four main tryptic digested subfragments, omeprazole was bound to the 67, 42 and 32-kDa subfragments, but not to the 52-kDa subfragment. Taking the amino acid sequence of this ATPase into consideration, we propose that omeprazole specifically binds with Cys322 in hog H+,K(+)-ATPase (Cys321 in rat).

Acid Activation of Omeprazole in Isolated Gastric Vesicles, Oxyntic Cells, and Gastric Glands

Omeprazole, a potent inhibitor of gastric hydrogen ion transporting, potassium-stimulated adenosine triphosphatase, was found to be transformed into an SH-reactive strong fluorescent molecule (excitation and emission wavelengths of 370 and 560 nm, respectively) in an acidic medium. The addition of glutathione- or protein-containing sulfhydryl groups such as pepsin to the medium decreased the fluorescence. Also, the increase in the pH of the medium decreased the fluorescence. The fluorescent molecule was identified to be an acid-activated planar cyclic sulfenamide derivative of omeprazole. The transformation was studied in H+-preaccumulated hog gastric vesicles, which contain the hydrogen ion transporting, potassium-stimulated adenosine triphosphatase. The addition of omeprazole to the vesicle suspension induced a rapid increase in the fluorescence intensity, indicating that omeprazole was activated in the intravesicular space. Then, the intensity biphasically decreased with time. The slower small decrease was due to the reaction of the sulfenamide with sulfhydryl group(s) located on the acid secretory side of the hydrogen ion transporting, potassium-stimulated adenosine triphosphatase. Omeprazole was also activated in the acidic lumina of isolated rabbit gastric glands that were stimulated with histamine. Furthermore, direct evidence was obtained from the imaging of the fluorescence that omeprazole was activated in the acidic compartments of the isolated Xenopus oxyntic cell.

Involvement of the H3O+-Lys-164 -Gln-161-Glu-345 Charge Transfer Pathway in Proton Transport of Gastric H+,K+-ATPase

Gastric H+,K+-ATPase is shown to transport 2 mol of H+/mol of ATP hydrolysis in isolated hog gastric vesicles. We studied whether the H+ transport mechanism is due to charge transfer and/or transfer of hydronium ion (H3O+). From transport of [18O]H2O, 1.8 mol of water molecule/mol of ATP hydrolysis was found to be transported. We performed a molecular dynamics simulation of the three-dimensional structure model of the H+,K+-ATPase α-subunit at E1 conformation. It predicts the presence of a charge transfer pathway from hydronium ion in cytosolic medium to Glu-345 in cation binding site 2 (H3O+-Lys-164 -Gln-161-Glu-345). No charge transport pathway was formed in mutant Q161L, E345L, and E345D. Alternative pathways (H3O+-Gln-161-Glu-345) in mutant K164L and (H3O+-Arg-105-Gln-161-Gln-345) in mutant E345Q were formed. The H+,K+-ATPase activity in these mutants reflected the presence and absence of charge transfer pathways. We also found charge transfer from sites 2 to 1 via a water wire and a charge transfer pathway (H3O+-Asn-794 -Glu-797). These results suggest that protons are charge-transferred from the cytosolic side to H2O in sites 2 and 1, the H2O comes from cytosolic medium, and H3O+ in the sites are transported into lumen during the conformational transition from E1PtoE2P.