RESUMEN

Previous data indicated that diquat-mediated protein oxidation (protein carbonyl formation) occurs through multiple pathways, one of which is lipid dependent, and the other, lipid independent. Studies reported here investigated potential mechanisms of the lipid-independent pathway in greater detail, using bovine serum albumin as the target protein. One hypothesized mechanism of protein carbonyl formation involved diquat-dependent production of H2O2, which would then react with site-specifically bound ferrous iron as proposed by Stadtman and colleagues. This hypothesis was supported by the inhibitory effect of catalase on diquat-mediated protein carbonyl formation. However, exogenous H2O2 alone did not induce protein carbonyl formation. Hydroxyl radical-generating reactions may result from the H2O2-catalyzed oxidation of ferrous iron, which normally is bound to protein in the ferric state. Therefore, the possible reduction of site-specifically bound Fe3+ to Fe2+ by the diquat cation radical (which could then react with H2O2) was also investigated. The combination of H2O2 and an iron reductant, ascorbate, however, also failed to induce significant protein carbonyl formation. In a phospholipid-containing system, an ADP:Fe2+ complex induced both lipid peroxidation and protein carbonyl formation; both indices were largely inhibitable by antioxidants. There was no substantial ADP:Fe(2+)-dependent protein carbonyl formation in the absence of phospholipid under otherwise identical conditions. Based on the lipid requirement and antioxidant sensitivity, these data suggest that ADP:Fe(2+)-dependent protein carbonyl formation occurs through reaction of BSA with aldehydic lipid peroxidation products. The precise mechanism of diquat-mediated protein carbonyl formation remains unclear, but it appears not to be a function of H2O2 generation or diquat cation radical-dependent reduction of bound Fe3+.

Asunto(s)

Cromanos/toxicidad , Diquat/toxicidad , Fosfolípidos/fisiología , Piperazinas/toxicidad , Proteínas/metabolismo , Adenosina Difosfato/análogos & derivados , Adenosina Difosfato/metabolismo , Animales , Antioxidantes/toxicidad , Herbicidas/toxicidad , Peróxido de Hidrógeno/metabolismo , Peroxidación de Lípido/efectos de los fármacos , Masculino , Microsomas Hepáticos/efectos de los fármacos , Microsomas Hepáticos/enzimología , NADPH-Ferrihemoproteína Reductasa/metabolismo , Oxidación-Reducción/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Sustancias Reactivas al Ácido Tiobarbitúrico/metabolismo

RESUMEN

Ferritin is an iron storage protein that is regulated at the transcriptional and transcriptional levels, resulting in a complex mixture of tissue- and condition-specific isoforms. The protein shell of ferritin is composed of 24 subunits of two types (heavy or light), which are encoded by two distinct and independently regulated genes. In the present studies, the isoform profile for lung ferritin differed from other tissues (liver, spleen, and heart) as determined by isoelectric focusing (IEF) and polyacrylamide gel electrophoresis (PAGE). Lung ferritin was composed of equal amounts of heavy and light subunits. Differences in isoform profiles were the result of tissue-specific differential expression of the ferritin subunit genes as demonstrated by Northern blot analyses. Like heart ferritin, lung ferritin exhibited a low iron content that did not increase extensively in response to iron challenge, which contrasts with ferritins isolated from liver or spleen. When animals were exposed to hyperoxic conditions (95% oxygen for up to 60 h), ferritin heavy subunit mRNA levels did not markedly change at any of the investigated time points. In contrast, ferritin light subunit mRNA increased severalfold in response to hyperoxic exposure. Investigation of the cytoplasmic distribution of ferritin mRNA showed that a substantial portion was associated with the ribonucleoprotein (RNP) fraction of the cytosol, suggesting that a pool of untranslated ferritin mRNA exists in the lung. Upon hyperoxic insult, all ferritin light subunit mRNA pools (RNP, monosomal, polysomal) were elevated, although a specific shift from RNP to polysomal pools was not evident. Therefore, the increase in translatable ferritin mRNA in response to hyperoxia resulted from transcriptional rather than specific translational activation. The observed pattern of light chain-specific transcriptional induction of ferritin is consistent with the hypothesis that hyperoxic lung injury is at least partially iron mediated.

Asunto(s)

Ferritinas/genética , Ferritinas/metabolismo , Hiperoxia/genética , Hiperoxia/metabolismo , Lesión Pulmonar , Pulmón/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Animales , Ferritinas/química , Expresión Génica , Hierro/metabolismo , Masculino , Conformación Proteica , Ratas , Ratas Sprague-Dawley

RESUMEN

In a previous report on diquat-dependent oxidative damage in rat hepatic microsomes, protein oxidation, as measured by protein carbonyl (PC) formation, was observed in addition to lipid peroxidation (LP). Both phenomena were antioxidant sensitive. Inhibition of PC formation was somewhat surprising given the proposed mechanism of metal-catalyzed protein oxidation. Studies reported here examined diquat-dependent PC formation in greater detail. In rat hepatic microsomes, diquat-dependent thiobarbituric acid-reactive substances (TBARS) and PC formation were time and concentration dependent. In this system, LP was inhibited completely by U-74006F or U-78517G, whereas PC formation was inhibited only partially by these antioxidants. In an essentially lipid-free system consisting of purified rat hepatic cytochrome P450 reductase, BSA and an NADPH-generating system, PC formation was also observed, but was not antioxidant-sensitive. Under these conditions, minimal diquat-dependent TBARS formation was observed. The observation of relative antioxidant insensitivity is consistent with H2O2 (generated during the diquat redox cycle) catalyzing protein oxidation via a site-specific, metal-catalyzed mechanism. Thus, different pathways would appear to be involved in diquat-dependent PC formation in lipid-containing and lipid-free systems. Carbon tetrachloride induces LP following reductive activation to the trichloromethyl free radical, a pathway not directly involving H2O2 generation. In the microsomal system, CCl4 induced TBARS and PC formation, both of which were completely inhibitable by antioxidants. Taken together, these data suggest that diquat induces PC formation by lipid-dependent (antioxidant-sensitive) and lipid-independent (antioxidant-insensitive) pathways. In microsomes, both pathways contribute to diquat-dependent PC formation. Data for the lipid-independent pathway are consistent with the mechanism of metal-catalyzed protein oxidation proposed by Stadtman and colleagues (reviewed in Free Radic Biol Med 9: 315-325, 1990), while the lipid-dependent pathway is likely secondary to LP itself--via a Michael-type addition reaction between hydroxyalkenals and protein sulfhydryl groups, amino groups or other protein nucleophiles. The latter pathway is also responsible for carbon tetrachloride-dependent PC formation. Additional studies are in progress to further characterize the lipid-independent mechanism.

Asunto(s)

Diquat/toxicidad , Peroxidación de Lípido/efectos de los fármacos , Proteínas/química , Animales , Antioxidantes/farmacología , Tetracloruro de Carbono/toxicidad , Cromanos/farmacología , Hierro/química , Masculino , Microsomas Hepáticos/efectos de los fármacos , Oxidación-Reducción , Piperazinas/farmacología , Pregnatrienos/farmacología , Ratas , Ratas Endogámicas F344 , Sustancias Reactivas al Ácido Tiobarbitúrico/análisis

RESUMEN

S-(1,2-dichlorovinyl)-L-cysteine (DCVC)-induced nephrotoxicity in vivo was investigated in New Zealand White rabbits. A primary emphasis in these studies was further characterization of DCVC-induced nephrotoxicity using a variety of serum and urinary analytes, including sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Additionally, the role of oxidative injury was assessed to address the dichotomy between reports indicating that such a mechanism is important in vivo and those indicating that such mechanisms do not contribute substantially to the mechanism of effects observed in vitro. Urine was collected prior to and at 8 and 24 hr after iv administration of DCVC. Serum was collected 15 min prior to and 24 hr after DCVC administration. Rabbits were euthanized 24 hr post-DCVC administration, and kidneys were fixed in formalin and further processed for light microscopic examination. DCVC (10 mg/kg, iv) induced a 45-50-fold increase in total urinary protein excretion, a 10-15-fold increase in urinary N-acetyl-beta-D-glucosaminidase concentration, plus a marked glucosuria by 24 hr postadministration. Additionally, DCVC increased serum creatinine levels by about 2-fold, with a trend toward increased blood urea nitrogen. SDS-PAGE analysis of rabbit urine confirmed the clinical finding of marked proteinuria in DCVC-treated animals, which in contrast to previously reported data was due to the presence of both low and high molecular weight proteins. Antioxidants had no significant effect on DCVC-dependent renal injury, nor was there evidence for DCVC-induced lipid peroxidation, as measured by either thiobarbituric acid-reactive substances or a commercial assay for malondialdehyde and hydroxalkenals.(ABSTRACT TRUNCATED AT 250 WORDS)

Asunto(s)

Cisteína/análogos & derivados , Riñón/patología , Riñón/fisiopatología , Peroxidación de Lípido/fisiología , Proteinuria/inducido químicamente , Acetilglucosaminidasa/análisis , Animales , Antioxidantes/farmacología , Cisteína/toxicidad , Electroforesis en Gel de Poliacrilamida , Riñón/efectos de los fármacos , Peroxidación de Lípido/efectos de los fármacos , Masculino , Consumo de Oxígeno/efectos de los fármacos , Proteinuria/metabolismo , Proteinuria/patología , Conejos

RESUMEN

U-78875 [imidazo[1,5-a]quinoxalin-4(5H)-one, 3-(5-cyclopropyl-1,2,4-oxadiazol-3-yl)-5-(1-methylethyl)] belongs to a series of imidazoquinoxaline derivatives, recently discovered ligands with high affinity for benzodiazepine receptors. In this study, we have examined the drug and its analogs for their modes of interaction with the receptors, with a particular emphasis on finding molecular determinants for their functional properties. Changes in the substituents on N5 and C6 of the heterocyclic ring produced no major effects on binding characteristics but yielded drugs of widely varying efficacy (antagonist to full agonist), measured as gamma-aminobutyric acid (GABA)-mediated 36Cl- uptake and t-butylbicyclophosphoro[35S]thionate binding in rat cerebrocortical membranes. The relative binding affinity and efficacy of the analogs measured in brain membranes were similar to those in cloned GABAA receptors of the alpha 1 beta 2 gamma 2 (type I) and alpha 3 beta 2 gamma 2 (type II) subtypes. The imidazoquinoxalines showed no marked subtype selectivity. Their Ki value against [3H]flunitrazepam binding for type I was only 2-3 times lower than that for type II, and their rank order for agonistic activity was the same in the two subtypes, measured as GABA-mediated Cl- currents in human kidney cells (A293) expressing the subtypes of GABAA receptors. According to computational modeling of the drugs using both molecular and quantum mechanics, the agonistic activity of the imidazoquinoxaline derivatives depends on the presence of a bulky alkyl substituent at N5 and the deformation of the substituted portion of the otherwise planar ring system induced by a bulky moiety at N5 or C6. With a fixed N5 substituent (isopropyl), the relative efficacy in the brain membranes, as well as in the cloned receptors, appeared to be dependent on the degree of the ring deformation. This out-of-plane portion of the imidazoquinoxalines can be assigned to the general region occupied by the 5-phenyl group of diazepam and other agonistic functional groups of several nonbenzodiazepine ligands. It seems that this region, apparently common to various agonistic ligands, interacts with an agonistic pocket in type I and type II subtypes of the benzodiazepine receptors in the brain. Our results also provide direct support for the view that the agonists and nonagonists share largely overlapping binding regions in the benzodiazepine receptor, which has been proposed earlier from in vivo efficacy measurements of other series of ligands.

Asunto(s)

Compuestos Bicíclicos Heterocíclicos con Puentes , Quinoxalinas/farmacología , Receptores de GABA-A/efectos de los fármacos , Animales , Ansiolíticos/farmacología , Unión Competitiva , Compuestos Bicíclicos con Puentes/metabolismo , Corteza Cerebral/metabolismo , Canales de Cloruro , Cloruros/farmacocinética , Cloro , Clonación Molecular , Interacciones Farmacológicas , Electrofisiología , Flunitrazepam/metabolismo , Antagonistas de Receptores de GABA-A , Cinética , Masculino , Proteínas de la Membrana/efectos de los fármacos , Proteínas de la Membrana/fisiología , Modelos Químicos , Conformación Molecular , Oxadiazoles/metabolismo , Oxadiazoles/farmacología , Quinoxalinas/metabolismo , Radioisótopos , Ratas , Ratas Endogámicas , Receptores de GABA-A/fisiología , Relación Estructura-Actividad , Sinaptosomas/metabolismo , Tritio

RESUMEN

The synthesis of N-substituted benzimidazole (H(+)-K+)-ATPase or proton-pump inhibitors is described. These compounds were prepared to function as prodrugs of the parent N-H compound and evaluated for their ability to inhibit gastric (H(+)-K+)-ATPase and gastric acid secretion. The prodrugs reported rely on either in vivo esterase hydrolysis for liberation of the parent compound (type I and type II) or require an acid environment for release of the active drug (type III and type IV). The N-(acyloxy)alkyl-substituted benzimidazoles 9, 11, and 24 showed improved chemical stability in the solid state and in aqueous solutions when compared to their parent N-H compounds. When given orally, 24 was found to be twice as potent as omeprazole in both the Shay rat and inactivation of gastric (H(+)-K+)-ATPase in the rat. The N-ethoxy-1-ethyl-substituted benzimidazoles 48-50 were found equally as effective as the N-H compound for inhibition of rat (H(+)-K+)-ATPase activity. In the Shay rat 48 at 10 mg/kg was approximately twice as active as parent timoprazole.

Asunto(s)

Adenosina Trifosfatasas/antagonistas & inhibidores , Bencimidazoles/síntesis química , Ácido Gástrico/metabolismo , Profármacos/síntesis química , 2-Piridinilmetilsulfinilbencimidazoles , Animales , Bencimidazoles/metabolismo , Bencimidazoles/farmacología , Fenómenos Químicos , Química , Estabilidad de Medicamentos , Esterasas/metabolismo , Femenino , Mucosa Gástrica/enzimología , ATPasa Intercambiadora de Hidrógeno-Potásio , Concentración de Iones de Hidrógeno , Masculino , Estructura Molecular , Omeprazol/farmacología , Conejos , Ratas , Ratas Endogámicas , Porcinos

RESUMEN

We have explored the functional significance of various drug-induced changes in t-[35S]butylbycyclophosporothionate (TBPS) binding to gamma-aminobutyric acidA (GABAA) receptors by comparing them with the actions of the drugs on GABA-induced 36Cl- uptake in rat cerebrocortical membrane preparations. In the presence of micromolar concentrations of GABA, various benzodiazepine receptor agonists, 3 alpha-21-dihydroxy-5 alpha-pregnan-20-one, and pentobarbital inhibited [35S]TBPS binding, whereas ethyl-beta-carboline-3carboxylate (beta-CCE), an inverse agonist, stimulated it, in general agreement with earlier reports [Mol. Pharmacol. 23:326-336 (1983); Mol. Pharmacol. 30:218-225 (1986)]. The drug-induced changes in [35S]TBPS binding, after normalization with respect to the corresponding action of diazepam, were closely related to the relative ability of the drugs to affect 36Cl- uptake, with a correlation coefficient of 0.98 and a slope of 0.85. Upon abolishment of GABA action by the use of bicuculline, however, all the tested drugs stimulated [35S]TBPS binding to various degrees, and their relative changes displayed a lower correlation coefficient of 0.69, with a slope of 2. In particular, the effects of the anesthetic steroid and pentobarbital on [35S]TBPS binding were markedly altered by GABA, which at 2 microM increased not only their maximal effects, but also their half-maximal concentrations severalfold. On the other hand, GABA did not significantly affect these parameters for diazepam under our experimental conditions. Also, the GABA-independent changes in [35S]TBPS binding produced by various benzodiazepine receptor agonists matched reasonably well the actions of the drugs on 36Cl- uptake, with a correlation coefficient of 0.85 and a slope of 1.0. These data suggest more pronounced functional coupling of the GABA sites with those for the steroid and the barbiturate, as compared with the benzodiazepine site. It appears that the degree of [35S]TBPS binding in the presence of GABA closely reflects the functional state of GABAA receptors and may be useful for characterization of allosteric interactions between various sites on the receptors.

Asunto(s)

Compuestos Bicíclicos Heterocíclicos con Puentes , Compuestos Bicíclicos con Puentes/metabolismo , Corteza Cerebral/metabolismo , Cloruros/metabolismo , Receptores de GABA-A/metabolismo , Ácido gamma-Aminobutírico/metabolismo , Animales , Bicuculina/farmacología , Carbolinas/farmacología , Canales de Cloruro , Desoxicorticosterona/análogos & derivados , Desoxicorticosterona/farmacología , Diazepam/farmacología , Etanol/farmacología , Flumazenil/farmacología , Técnicas In Vitro , Ligandos , Proteínas de la Membrana/efectos de los fármacos , Proteínas de la Membrana/metabolismo , Pentobarbital/farmacología , Pregnanolona/análogos & derivados , Pregnanolona/farmacología , Ratas , Receptores de GABA-A/efectos de los fármacos , Sinaptosomas/metabolismo

RESUMEN

Muscimol-induced 36Cl- uptake in rat cerebrocortical synaptoneurosomes was reduced upon exposure of the membrane sacs to low Na+ media. This Na+ requirement led us to examine the role of the Na(+)-dependent gamma-aminobutyric acid (GABA) transport system in 36Cl- uptake. Incubation of the synaptoneurosomes with nipecotic acid, a specific inhibitor of the GABA transport system, for 10 min increased the level of endogenous external GABA from less than 10 to 150 microM and induced the same signs of desensitization as observed with high muscimol-treated synaptoneurosomes; a marked reduction of muscimol-induced 36Cl- uptake and an appearance of a slow bicuculline-sensitive 36Cl- uptake, probably due to a continuous recovery of a population of GABAA receptors from desensitization. Similar results were obtained upon dissipation of Na+ electrochemical gradient across the membranes by inhibition of Na+, K(+)-ATPase with ouabain or by blocking energy metabolism with azide or N-ethylmaleimide. We propose that the Na(+)-dependent GABA transport system, its operation being dependent on inwardly directed Na+ electrochemical gradient, is responsible for scavenging endogenous GABA released from the synaptoneurosomes, and thus prevents desensitization of GABAA receptors.

Asunto(s)

Corteza Cerebral/metabolismo , Cloruros/farmacocinética , Prolina/análogos & derivados , Receptores de GABA-A/metabolismo , Sodio/fisiología , Sinaptosomas/metabolismo , Ácido gamma-Aminobutírico/farmacocinética , Animales , Corteza Cerebral/efectos de los fármacos , Cloruros/fisiología , Masculino , Muscimol/farmacología , Inhibidores de la Captación de Neurotransmisores/farmacología , Ácidos Nipecóticos/farmacología , Ratas , Ratas Endogámicas , Receptores de GABA-A/efectos de los fármacos , Sinaptosomas/efectos de los fármacos , Ácido gamma-Aminobutírico/fisiología

RESUMEN

Functional interactions between steroidal anesthetics and gamma-aminobutyric acidA (GABAA) receptors have been examined with 36Cl- uptake measurements in rat cerebrocortical synaptoneurosomes. The primary effect of the steroids was to enhance the affinity of GABA for its receptors without much effect on the maximal uptake rate; the ED50 for GABA decreased from 66.4 +/- 5.7 to 8.9 +/- 1.2 microM in the presence of 20 microM 3 alpha,21-dihydroxy-5 alpha-pregnan-20-one. Stimulation of 36Cl- uptake by high concentrations of the anesthetic steroid in the absence of exogenous GABA was not due to direct stimulation of GABAA receptors, as currently proposed, but is due to enhanced action of endogenous GABA, inasmuch as the steroid markedly increases GABA affinity for the receptors. Typically, endogenous GABA was maintained at near 1 microM by a Na(+)-dependent GABA transport system in the synaptoneurosomes. Elevation of its level with nipecotic acid, a specific inhibitor of the GABA transport system, or reduction with GABase, a GABA-scavenging system, increased or decreased, respectively, the steroid-induced bicuculline-sensitive 36Cl- uptake. At low concentrations of GABA (less than 2 microM), the stimulatory effect of 3 alpha,21-dihydroxy-5 alpha-pregnan-20-one was markedly potentiated by pentobarbital but antagonized by 3 alpha,21-dihydroxy-5 alpha-pregnan-20-one, a partial agonist of higher affinity. These observations, along with the structure-activity relationships of steroid analogs, strongly suggest the existence of a specific binding site for the steroids in GABAA receptors and led us to propose a minimal model in which two key common functional groups of anesthetic steroids, 3 alpha-OH- and 17 beta-polar substituents, interact with GABAA receptors (probably through hydrogen bondings) while their hydrophobic backbone remains in contact with the fatty acyl chains of membrane phospholipids.

Asunto(s)

Anestésicos/farmacología , Desoxicorticosterona/análogos & derivados , Progesterona/análogos & derivados , Prolina/análogos & derivados , Receptores de GABA-A/efectos de los fármacos , Animales , Cloruros/metabolismo , Desoxicorticosterona/farmacología , Técnicas In Vitro , Cinética , Ácidos Nipecóticos/farmacología , Pentobarbital/farmacología , Pregnanolona , Progesterona/farmacología , Ratas , Receptores de GABA-A/metabolismo , Membranas Sinápticas , Ácido gamma-Aminobutírico/farmacología

RESUMEN

ConA-sepharose and polylysine-agarose beads effectively bound detergent-solubilized GABAA receptors from rat cerebrocortical membranes. The immobilized receptors showed a single class of high affinity binding sites specific for flunitrazepam or muscimol and displayed GABA-stimulated flunitrazepam binding. Maximal binding capacities of the ConA-immobilized receptor for the ligands were about three times greater than those of the polylysine-immobilized receptors. The relative affinities for each of the ligands were not affected by the method of receptor immobilization. The dissociation constants for muscimol of these immobilized receptors were somewhat dependent on the solubilizing agents used, but were considerably lower than those measured using extensively dialyzed rat cerebrocortical membranes.

Asunto(s)

Receptores de GABA-A/metabolismo , Animales , Corteza Cerebral/metabolismo , Concanavalina A/metabolismo , Flunitrazepam/metabolismo , Técnicas In Vitro , Ligandos , Muscimol/metabolismo , Polilisina/metabolismo , Ratas , Solubilidad , Ácido gamma-Aminobutírico/metabolismo

RESUMEN

Limited tryptic digestion of fluorescein isothiocyanate (FITC)-labeled (H+-K+)-ATPase from rat resting light gastric membranes produced a soluble 27-kDa polypeptide which retained the fluorescence of the parent enzyme. Its production was markedly enhanced in the presence of an amphiphilic detergent, Zwittergent 3-14, which potently inhibits the ATPase activity. This increase is probably due to protection of certain tryptic cleavage sites through conformational changes of the membrane enzyme by the detergent. The NH2-terminal sequence of the 27-kDa polypeptide corresponded exactly to that beginning at Asn-369 of the cDNA-deduced primary structure of the rat ATPase. The presence of the phosphorylation site, Asp-385, and FITC-labeled Lys-517, which is known to be a part of the ATP-binding site, indicates that the 27-kDa polypeptide contains a major cytoplasmic portion of (H+-K+)-ATPase. Interestingly, the polypeptide was stained with periodate-Schiff's base, indicating its glycoprotein nature. The carbohydrate group attached to the polypeptide seems to include at least an N-linked high-mannose moiety, since the polypeptide showed Con A binding activity as detected with a Con A-biotin/avidin-peroxidase assay on nitrocellulose transblots. Also, its Con A binding activity was inhibited by excess methyl alpha-D-mannopyranoside and disappeared upon treatment of the polypeptide with endoglycosidase H and N-glycanase. Further tryptic action converted the 27-kDa polypeptide to 2 smaller FITC-labeled polypeptides of 25 and 15 kDa, which lost 18 and 96 amino acid residues, respectively, from the NH2 terminus of the parent polypeptide.(ABSTRACT TRUNCATED AT 250 WORDS)

Asunto(s)

Adenosina Trifosfatasas/aislamiento & purificación , Carbohidratos/aislamiento & purificación , Secuencia de Aminoácidos , Animales , Sitios de Unión , Concanavalina A , Fluoresceína-5-Isotiocianato , Fluoresceínas , ATPasa Intercambiadora de Hidrógeno-Potásio , Técnicas In Vitro , Masculino , Manosa/aislamiento & purificación , Membranas/enzimología , Datos de Secuencia Molecular , Fragmentos de Péptidos/aislamiento & purificación , Ratas , Ratas Endogámicas , Estómago/enzimología , Tiocianatos , Tripsina

RESUMEN

Rat stimulated heavy gastric membranes enriched with (H+-K+)-ATPase, a marker for the apical membrane of the parietal cell, displayed a 32P-histone-dephosphorylating activity which appeared to be physically copurified with, but functionally independent of, the ATPase. The protein phosphatase activity was optimal at pH 7.5 and was inhibited by fluoride (50 mM), inorganic phosphate (50 mM), and p-chloromercuribenzoate (0.1 mM), but was insensitive to vanadate (1 mM). The 32P-phosphoproteins in the heavy gastric membranes were also dephosphorylated, apparently by their own membrane-bound phosphatase in the presence of Mg2+ at millimolar concentrations, which is likely to enhance membrane-membrane interaction. Heavy gastric membrane vesicles incubated with Mg2+ (2 mM) exhibited no alterations in K+-dependent ATP-hydrolyzing activity, Cl permeability, and protein and lipid compositions, but irreversibly lost the ATP, K+-dependent H+-pumping activity. Since valinomycin, a K+-specific ionophore, restored the intravesicular acidifying activity and an inhibitor of the protein phosphatase, inorganic phosphate, largely blocked the Mg2+-induced change in the membrane transport function, it is reasonable to propose that the phosphatase action on certain membrane proteins, possibly the putative K+ transporter or regulatory proteins, selectively decreases K+-conductance in the apical membranes of gastric parietal cells.

Asunto(s)

Adenosina Trifosfatasas/metabolismo , Mucosa Gástrica/enzimología , Membranas Intracelulares/enzimología , Microsomas/enzimología , Fosfoproteínas Fosfatasas/metabolismo , Animales , Calcio/farmacología , ATPasa Intercambiadora de Hidrógeno-Potásio , Cinética , Magnesio/farmacología , Masculino , Lípidos de la Membrana/metabolismo , Fosfolípidos/metabolismo , Ratas , Ratas Endogámicas , Valinomicina/farmacología

RESUMEN

K+- and ATP-dependent H+-accumulation in rat heavy gastric membrane vesicles enriched with (H+-K+)-ATPase was markedly stimulated by amphiphiles like lysophosphatidylcholine and Zwittergent 3-14 at concentrations of 10(-5) M. Their stimulatory effect was dependent on K+-concentration in the medium and was abolished by SCH 28,080, a specific inhibitor of (H+-K+)-ATPase. Lysophosphatidylcholine at the optimal dose (3 X 10(-5) M) showed dual effects on K+-dependent membrane functions; it stimulated the rate of K+-uptake by nearly 60%, but partially inhibited SCH 28,080-sensitive and K+-dependent ATP-hydrolysis (about 20% reduction). These data indicate that H+-pumping through (H+-K+)-ATPase in the inside-out gastric membrane vesicles was facilitated by the stimulatory effect of lysophosphatidylcholine on membrane K+-transport in spite of its partial inhibition of ATP-hydrolysis. It appears that the rate limiting step for operation of the ATPase is the availability of K+ ions in the luminal side of the pump. We propose that ionic amphiphiles may modulate K+-transport in rat heavy gastric membranes through specific interactions with the putative K+-transporter.

Asunto(s)

Mucosa Gástrica/metabolismo , Lisofosfatidilcolinas/farmacología , Potasio/metabolismo , ATPasas de Translocación de Protón/análisis , Animales , Transporte Biológico Activo/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Cinética , Masculino , Membranas/enzimología , Compuestos de Amonio Cuaternario , Ratas , Ratas Endogámicas , Rubidio/metabolismo , Estómago/enzimología

RESUMEN

Treatment of rats with cycloheximide 1 h before carbachol dose-dependently reduced the secretagogue-stimulated gastric acid secretion in pylorus ligated rats, and partially blocked carbachol- or histamine-induced activation of rat gastric (H+ + K+)-ATPase which includes translocation of reserve intracellular (H+ + K+)-ATPase into the apical membrane of the parietal cells and induction of a KCl pathway. Time-course studies showed that the drug was effective only when administered at least 30 min before the secretagogues. Puromycin showed the same effect as cycloheximide. Pulse labelling studies with [35S]methionine led to identification of two most actively synthesized polypeptides in rat gastric mucosa; the proteins of 38,000 and 14,000 molecular weight. The larger polypeptide was identified as rat pepsinogen. The identity of the smaller protein is not known yet. We suggest that synthesis of nascent polypeptide(s) is required for certain steps of the acid secretory process leading to the activation of the acid pump.

Asunto(s)

Carbacol/farmacología , Cicloheximida/farmacología , Ácido Gástrico/metabolismo , Jugo Gástrico/efectos de los fármacos , Biosíntesis de Péptidos , Adenosina Trifosfatasas/biosíntesis , Animales , ATPasa Intercambiadora de Hidrógeno-Potásio , Cinética , Masculino , Microsomas/enzimología , Peso Molecular , Péptidos/aislamiento & purificación , Biosíntesis de Proteínas/efectos de los fármacos , Puromicina/farmacología , Ratas , Ratas Endogámicas

RESUMEN

Resting rat light gastric membranes prepared through 2H2O and Percoll gradient centrifugations were enriched not only with (H+-K+)-ATPase and K+ transport activity (Im, W. B., Blakeman, D. P., and Davis, J. P. (1985) J. Biol. Chem. 260, 9452-9460), but also with a K+-independent, ATP-dependent H+-pumping activity. This intravesicular acidification has been ascribed to an oligomycin-insensitive H+-ATPase which differed from (H+-K+)-ATPase in several respects. The H+-ATPase is electrogenic, apparently of lower capacity, required a lower optimal ATP concentration (4 microM for the H+-ATPase and 500 microM for (H+-K+)-ATPase), of lower sensitivity to vanadate and sulfhydryl agents such as p-chloromercuribenzoate and N-ethylmaleimide, and insensitive to SCH 28,080, a known competitive inhibitor of (H+-K+)-ATPase with respect to K+. Operation of the H+-ATPase, however, appeared to interfere with the K+ transport activity in the light gastric membranes, probably through development of intravesicular positive membrane potential; for example, micromolar levels of Mg2+-ATP fully inhibited K+ uptake and stimulated K+ efflux as measured with 86Rb+. Involvement of (H+-K+)-ATPase in the K+ transport is not likely, since the inhibitory effect of Mg2+-ATP continued even after removal of the nucleotide with an ATP-scavenging system. Moreover, nigericin, an electroneutral H+/K+ exchanger, could bypass the inhibitory effect of Mg2+-ATP and equilibrate the membrane vesicles with 86Rb+ while valinomycin, an electrogenic K+ ionophore, could not. Finally, the H+-ATPase could possibly be involved in the acid secretory process, since its H+-pumping activity was removed from the light gastric membrane fraction upon carbachol treatment, along with the K+ transport and (H+-K+)-ATPase activities. We have speculated that the H+-ATPase is responsible for maintaining the K+-permeable intracellular membrane vesicles acidic and K+ free during the resting state of acid secretion and may contribute to basal acid secretion.

Asunto(s)

Adenosina Trifosfatasas/metabolismo , Adenosina Trifosfato/metabolismo , Mucosa Gástrica/metabolismo , Membranas Intracelulares/metabolismo , ATPasas de Translocación de Protón/metabolismo , Animales , ATPasa Intercambiadora de Hidrógeno-Potásio , Membranas Intracelulares/efectos de los fármacos , Cinética , Masculino , Microsomas/efectos de los fármacos , Microsomas/metabolismo , Cloruro de Potasio/farmacología , Ratas , Rubidio/metabolismo , Valinomicina/farmacología , Vanadatos , Vanadio/farmacología

RESUMEN

Gastric heavy microsomal membranes highly enriched in (H+-K+)-ATPase were obtained from cimetidine- or carbachol-treated rats through 2H2O and Percoll gradient centrifugations. Both the resting (cimetidine-treated) and the stimulated (carbachol-treated) heavy membranes which presumably represent the apical membrane of gastric parietal cells were enriched with the polypeptides of 81,000 and 45,000 besides that of 93,000 representing (H+-K+)-ATPase. No apparent differences could be detected between the resting and the stimulated heavy membranes in their polypeptide profiles or their specific activity of (H+-K+)-ATPase. Nevertheless, the level of 86RbCl uptake was greater in the stimulated than the resting heavy microsomal membrane vesicles. The light gastric microsomes which abound in intracellular tubulovesicles containing reserve (H+-K+)-ATPase as isolated from cimetidine-treated rats were similarly purified with respect to (H+-K+)-ATPase. The purified light gastric membranes were largely devoid of the polypeptides of 81,000 and 45,000 found in the heavy gastric membranes. These observations further support the current hypothesis that secretagogues bring about changes in the environment of (H+-K+)-ATPase and induce KCl permeability in the apical membrane of the parietal cells, although at present we have been unable to identify the polypeptide(s) responsible for the KCl pathway.

Asunto(s)

Adenosina Trifosfatasas/metabolismo , Fraccionamiento Celular/métodos , Microsomas/ultraestructura , Estómago/ultraestructura , Actinas/metabolismo , Animales , Carbacol/farmacología , Permeabilidad de la Membrana Celular/efectos de los fármacos , Centrifugación por Gradiente de Densidad , Cimetidina/farmacología , Deuterio , Fundus Gástrico , ATPasa Intercambiadora de Hidrógeno-Potásio , Membranas Intracelulares/enzimología , Membranas Intracelulares/ultraestructura , Masculino , Peso Molecular , Péptidos/metabolismo , Cloruro de Potasio/metabolismo , Ratas , Ratas Endogámicas , Estómago/efectos de los fármacos , Valinomicina/farmacología

RESUMEN

A population of gastric membrane vesicles of high K+ permeability and of lower density than endoplasmic tubulovesicles containing (H+-K+)-ATPase was detected in gastric mucosal microsomes from the rat fasted overnight. The K+-transport activity as measured with 86RbCl uptake had a Km for Rb+ of 0.58 +/- 0.11 mM and a Vmax of 13.7 +/- 1.9 nmol/min X mg of protein. The 86Rb uptake was reduced by 40% upon substituting Cl- with SO2-4 and inhibited noncompetitively by ATP and vanadate with a Ki of 3 and 30 microM, respectively; vanadate also inhibited rat gastric (H+-K+)-ATPase but with a Ki of 0.03 microM. Carbachol or histamine stimulation decreased the population of the K+-permeable light membrane vesicles, at the same time increased K+-transport activity in the heavy, presumably apical membranes of gastric parietal cells, and enabled the heavy microsomes to accumulate H+ ions in the presence of ATP and KCl without valinomycin. The secretagogue-induced shift of K+ permeability was blocked by cimetidine, a H2-receptor antagonist. Four characteristics of the K+ permeability as measured with 86RbCl were common in the resting light and the carbachol-stimulated heavy microsomes; (a) Km for +Rb, (b) anion sensitivity (Cl- greater than SO2-4), (c) potency of various divalent cations (Hg2+, Cu2+, Cd2+, and Zn2+) to inhibit Rb+ uptake, and (d) inhibitory effect of ATP, although the nucleotide sensitivity was latent in the stimulated heavy microsomes. The Vmax for 86RbCl uptake was about 10 times greater in the resting light than the stimulated heavy microsomes. These observations led us to propose that secretagogue stimulation induces the insertion of not only the tubulovesicles containing (H+-K+)-ATPase, but also the light membrane vesicles containing KCl transporter into the heavy apical membranes of gastric parietal cells.

Asunto(s)

Adenosina Trifosfatasas/metabolismo , Mucosa Gástrica/metabolismo , Microsomas/metabolismo , Potasio/metabolismo , Animales , Carbacol/farmacología , Cloruros/metabolismo , Cimetidina/farmacología , ATPasa Intercambiadora de Hidrógeno-Potásio , Histamina/farmacología , Membranas Intracelulares/metabolismo , Cinética , Masculino , Proteínas de la Membrana/aislamiento & purificación , Proteínas de la Membrana/metabolismo , Microsomas/efectos de los fármacos , Microsomas/ultraestructura , Peso Molecular , Ratas , Ratas Endogámicas , Rubidio/metabolismo , Termodinámica

RESUMEN

Omeprazole (5-methoxy-2-[[(4-methoxy-3,5- dimethylpyridinyl)methyl]sulfinyl]-1H-benzimidazole) appeared to inhibit gastric (H+-K+)-ATPase by oxidizing its essential sulfhydryl groups, since the gastric ATPase inactivated by the drug in vivo or in vitro recovered its K+-dependent ATP hydrolyzing activity upon incubation with mercaptoethanol. Biological reducing agents like cysteine or glutathione, however, were unable to reverse the inhibitory effect of omeprazole. Moreover, acidic environments enhanced the potency of omeprazole. For example, in vivo pretreatment of rats with carbachol, a secretagogue, enhanced the activity of omeprazole to inhibit gastric (H+-K+)-ATPase, while pretreatment with cimetidine, an antisecretory agent, reduced its potency. In vitro, lowering pH of incubation media from 7.4 to 5.0 improved the ability of omeprazole to inhibit hog gastric (H+-K+)-ATPase almost 60-fold. The inhibitory effect of the drug was accompanied by a dose-dependently decreased amount of free sulfhydryl groups in the isolated hog gastric membranes. The chemical reactivity of omeprazole with mercaptans is also consistent with the biological action of omeprazole. The drug, only under acidic conditions, reacted with a stoichiometric amount of ethyl mercaptan (or beta-mercaptoethanol) to produce regio-isomers of N-sulfenylated omeprazole sulfide (5-methoxy-2[[(4-methoxy-3,5- dimethyl-2-pyridinyl)methyl]thio]-1- or 3-(ethylthio)benzimidazole). The N-sulfenylated compound reacted at neutral pH with another stoichiometric amount of ethyl mercaptan to produce omeprazole sulfide quantitatively. The gastric polypeptides of 100 kilodaltons representing (H+-K+)-ATPase in the rat gastric mucosa or isolated hog gastric membranes were covalently labeled with [14C]omeprazole. The radioactive label bound to the ATPase, however, could not be displaced by mercaptoethanol under the identical conditions where the ATPase activity was fully restored. These observations suggest that the essential sulfhydryl groups which reacted with omeprazole did not form a stable covalent bond with the drug, but rather that they further reacted with adjacent sulfhydryl groups to form disulfides which could be reduced by mercaptoethanol.

Asunto(s)

Adenosina Trifosfatasas/antagonistas & inhibidores , Bencimidazoles/farmacología , Mucosa Gástrica/enzimología , Compuestos de Sulfhidrilo/metabolismo , Adenosina Trifosfato/metabolismo , Aminopirina/metabolismo , Animales , Membrana Celular/enzimología , Relación Dosis-Respuesta a Droga , ATPasa Intercambiadora de Hidrógeno-Potásio , Concentración de Iones de Hidrógeno , Cinética , Espectroscopía de Resonancia Magnética , Masculino , Omeprazol , Cloruro de Potasio/metabolismo , Protones , Porcinos , Valinomicina/farmacología

RESUMEN

We have examined the interaction of omeprazole, a gastric antisecretory agent, with endogenous or exogenous sulfhydryl compounds in isolated rabbit gastric glands. The glands exposed to omeprazole (2 microM for 50 min) could recover acid secretory response to dibutyryl-cAMP upon addition of dithiothreitol, cysteine or glutathione. Washing the omeprazole-exposed glands free of the extracellular drug also led to a similar recovery of the acid secretory response. Depletion of cellular glutathione with 2-cyclohexen-1-one had no considerable effect on the secretory response of the glands to dibutyryl-cAMP, but prevented the reversal of the antisecretory effect of omeprazole upon washing or adding exogenous cysteine. Also, the antisecretory potency of omeprazole increased several fold in the glutathione-depleted glands. These observations indicate that cellular glutathione is essential to reactivate the omeprazole-modified enzyme(s), possibly (H+ + K+)-ATPase, in acid secretory process and led us to propose that omeprazole is an agent reacting with sulfhydryl groups.

Asunto(s)

Bencimidazoles/farmacología , Mucosa Gástrica/efectos de los fármacos , Compuestos de Sulfhidrilo/farmacología , Animales , Ciclohexanonas/farmacología , Cisteína/farmacología , Ácido Gástrico/metabolismo , Mucosa Gástrica/metabolismo , Glutatión/metabolismo , Glutatión/farmacología , Técnicas In Vitro , Masculino , Omeprazol , Conejos

RESUMEN

Subcutaneous administration of omeprazole, a gastric antisecretory agent belonging to the family of substituted benzimidazoles, brought about a dose-dependent decrease in gastric mucosal (H+-K+)-ATPase activity in the rat. The dose which inhibited 50% of the enzyme activity was 1 mg/kg from dose-response profiles obtained 3 h after the drug dosing. Duration profiles of the drug at 10 mg/kg showed that its ATPase-inhibitory effect reached the maximum in 2 h with 80% reduction of the enzyme activity. The gastric mucosal level of the ATPase activity remained to be maximally inhibited for 12 h and returned to a normal level with a half time of about 20 h. The return of the enzyme activity, however, was blocked by treatment with cycloheximide, an inhibitor of protein synthesis. These observations indicate that omeprazole irreversibly inactivates gastric (H+-K+)-ATPase in vivo.

Asunto(s)

Adenosina Trifosfatasas/antagonistas & inhibidores , Antiulcerosos/farmacología , Bencimidazoles/farmacología , Mucosa Gástrica/enzimología , Animales , Cicloheximida/farmacología , Relación Dosis-Respuesta a Droga , Mucosa Gástrica/efectos de los fármacos , ATPasa Intercambiadora de Hidrógeno-Potásio , Masculino , Omeprazol , Ratas , Ratas Endogámicas , Factores de Tiempo