RESUMEN
A search for the relic neutrinos from all past core-collapse supernovae was conducted using 1496 days of data from the Super-Kamiokande detector. This analysis looked for electron-type antineutrinos that had produced a positron with an energy greater than 18 MeV. In the absence of a signal, 90% C.L. upper limits on the total flux were set for several theoretical models; these limits ranged from 20 to 130 macro nu(e) cm(-2) s(-1). Additionally, an upper bound of 1.2 macro nu(e) cm(-2) s(-1) was set for the supernova relic neutrino flux in the energy region E(nu)>19.3 MeV.
RESUMEN
Small bowel perforation is rarely caused by metastasis from an extra-abdominal malignancy. This report describes three cases of small bowel perforation that occurred secondary to a metastatic tumor. The first case involved a 72-year-old man with malignant lymphoma of the larynx that had been treated with chemo- and radiation therapy; the second involved a 70-year-old man with rhabdomyosarcoma of the mediastinum that had been treated with radiation therapy; and the third involved a 41-year-old man with lung carcinoma that had been treated with surgery 10 months prior to perforation. Each patient presented with acute abdominal pain, had X-ray findings of free air in the abdomen, and underwent limited emergency surgery. Wedge resection and closure of the ileum was performed for the first patient and partial bowel resection with the creation of an intestinal stoma was performed for the second and third patients. In each case, the histologic findings of the resected specimens were consistent with the extra-abdominal primary tumors. Although the patients recovered sufficiently to begin eating and moving about, all three died of cancer or cancer-related complications within 45 days of surgery. We conclude that surgeons should be aware of the poor prognosis of such patients and perform only the minimal surgery required.
Asunto(s)
Enfermedades del Íleon/cirugía , Neoplasias del Íleon/secundario , Perforación Intestinal/cirugía , Enfermedades del Yeyuno/cirugía , Neoplasias del Yeyuno/secundario , Adulto , Anciano , Carcinoma de Células Grandes/patología , Carcinoma de Células Grandes/secundario , Carcinoma de Células Grandes/cirugía , Humanos , Enfermedades del Íleon/patología , Neoplasias del Íleon/patología , Neoplasias del Íleon/cirugía , Íleon/patología , Íleon/cirugía , Perforación Intestinal/patología , Enfermedades del Yeyuno/patología , Neoplasias del Yeyuno/patología , Neoplasias del Yeyuno/cirugía , Yeyuno/patología , Yeyuno/cirugía , Neoplasias Laríngeas/patología , Neoplasias Laríngeas/cirugía , Neoplasias Pulmonares/patología , Neoplasias Pulmonares/cirugía , Linfoma de Células B/patología , Linfoma de Células B/cirugía , Masculino , Neoplasias del Mediastino/patología , Neoplasias del Mediastino/cirugía , Rabdomiosarcoma/patología , Rabdomiosarcoma/secundario , Rabdomiosarcoma/cirugíaRESUMEN
In patients with inflammatory conditions such as infection, cytokines induce the production of C-reactive protein(CRP) and serum amyloid A protein(SAA) in hepatic cells. It has been reported that upon viral infection, the serum SAA level increases by a greater degree than the serum CRP level. Procalcitonin (PCT), the precursor of calcitonin, is a new type of inflammatory marker that is specifically induced by bacterial infection, sepsis and lethal multiple organ failure, but not by viral infection, autoimmune diseases, tumors or surgical stress. To evaluate the immunoluminometric assay(LUMI test PCT; Brahms Diagnostics, Berlin, Germany) procedure for determining the PCT level and to study the clinical significance of the serum PCT level, we determined the serum levels of PCT, CRP and SAA in patients with various inflammatory diseases and normal subjects. The serum PCT level in the normal subjects was < 0.3 ng/ml. Among the patients with inflammatory disease who had a high CRP level(CRP > 20000 micrograms/dl), the PCT level was elevated only in those patients with severe bacterial infection. These results suggest that determining the PCT level may be useful in the differential diagnosis of severe bacterial infection. The patients who had a low CRP level(CRP < 150 micrograms/dl), had a PCT level within the normal range. The patients with autoimmune disease, viral infection, and fungal infection did not have an elevated PCT level.
Asunto(s)
Infecciones Bacterianas/diagnóstico , Calcitonina/sangre , Precursores de Proteínas/sangre , Adulto , Biomarcadores/sangre , Proteína C-Reactiva/análisis , Péptido Relacionado con Gen de Calcitonina , Diagnóstico Diferencial , Femenino , Humanos , Inmunoensayo , Inflamación/diagnóstico , Masculino , Persona de Mediana Edad , Proteína Amiloide A Sérica/análisis , Índice de Severidad de la EnfermedadRESUMEN
To elucidate whether a relationship exists between the site of trauma and severity of acute hyperextension spinal cord injury without bone damage, we examined the clinical features of 25 male and 10 female patients aged 13 to 88 years. None of the patients had vertebral damage such as fracture and dislocation. The site of impact was classified as the buccal, forehead, or mandibular region. The neurological findings were assessed according to Frankel's classification at admission and at follow up after 3 months or more to assess outcome. Eleven patients suffered trauma in the buccal region, one patient in Frankel's grade B, three in grade C, and seven in grade D at admission. All 11 of these patients showed an improvement of one grade or more to an outcome of C in one patient, D in one, and E in nine. Trauma occurred at the forehead region in 18 patients, four in grade B, 10 in grade C, and four in grade D. Improvement was seen at follow up by one grade or more to C in one patient, D in 10, and E in seven. Trauma occurred at the mandibular region in six patients, four in grade B and two in grade C. Four of these patients showed improvement of one grade or more to grade B in one, grade C in four, and grade E in one. Overall, seven patients had poor outcomes, five of whom suffered trauma to the mandibular region, indicating that impact to the mandibular region tends to have an unfavorable clinical outcome. Our findings indicate that the site of trauma greatly influences the severity of hyperextension spinal cord injury.
Asunto(s)
Traumatismos de la Médula Espinal/etiología , Lesiones por Latigazo Cervical/diagnóstico , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Femenino , Estudios de Seguimiento , Humanos , Masculino , Traumatismos Mandibulares/complicaciones , Traumatismos Mandibulares/diagnóstico , Persona de Mediana Edad , Examen Neurológico , Traumatismos de la Médula Espinal/diagnósticoRESUMEN
On the basis of two case reports it is suggested that serious convulsions may occur in patients treated with flucoazole when serum trough concentrations exceed 80 microg/mL. The authors recommend monitoring fluconazole concentrations during high-dose therapy in patients with poor kidney function.
Asunto(s)
Antifúngicos/administración & dosificación , Antifúngicos/efectos adversos , Fluconazol/administración & dosificación , Fluconazol/efectos adversos , Convulsiones/inducido químicamente , Anciano , Antifúngicos/sangre , Antifúngicos/farmacocinética , Candidiasis/tratamiento farmacológico , Esquema de Medicación , Femenino , Fluconazol/sangre , Fluconazol/farmacocinética , Humanos , Masculino , Persona de Mediana Edad , Neumonía/tratamiento farmacológico , Infección de la Herida Quirúrgica/tratamiento farmacológicoRESUMEN
Cardiac output is measured by pulse dye-densitometry using indocyanine-green (ICG). This cardiac output is estimated by correlating with the cardiac output measured by pulmonary artery catheter using thermodilution method. Twenty-four patients scheduled for elective cardiovascular surgeries under general anesthesia were studied. The pulse dye-densitometry monitoring system used was DDG-2001 (Nihon Kohden, Japan). In group A (13 patients: 19 times), ICG was administered from the peripheral vein as bolus doses of 5, 10 or 20 mg (5 mg.ml-1 water solution). In group B (11 patients: 12 times), ICG was administered from the peripheral vein as bolus doses of 20, 10 or 5 mg (5 mg.ml-1 water solution). The correlation (Pearson's correlation coefficient) and precision (the method proposed by Bland and Altman) compared with cardiac output measured by pulmonary artery catheter were examined. Better correlation and precision were recognized after 20 mg ICG injection than 5 or 10 mg ICG injection. In conclusion, the measurement of cardiac output by pulse dye-densitometry with peripheral vein ICG injection was useful using a bolus dose of ICG 20 mg.
Asunto(s)
Gasto Cardíaco , Colorantes , Pruebas de Función Cardíaca/métodos , Verde de Indocianina , Anciano , Colorantes/administración & dosificación , Densitometría/métodos , Femenino , Cardiopatías/fisiopatología , Humanos , Verde de Indocianina/administración & dosificación , Inyecciones Intravenosas , Masculino , Persona de Mediana Edad , Monitoreo Fisiológico/métodos , Reproducibilidad de los ResultadosRESUMEN
OBJECTIVE: We sought to evaluate the effect of iron ion on the absorption of mycophenolate mofetil, which is an immunosuppressive agent. The pharmacokinetics of mycophenolic acid were studied. METHODS: A randomized crossover design with two phases was used. A 7-day washout period separated the two treatment conditions. In the first phase, the volunteers received 1.0 g of mycophenolate mofetil alone (study 1); in the second phase, the volunteers received 1.0 g of mycophenolate mofetil and 2 tablets of iron ion preparations concomitantly (study 2). The serum concentration of mycophenolic acid, which is a pharmacologically active metabolite, was measured by reverse-phase HPLC. RESULTS: The area under the plasma concentration-time curve from 0 to 12 hours and the maximum concentration of mycophenolic acid in study 2 were significantly less than in study 1 (area under the curve, 32.9 +/- 14.7 versus 2.92 +/- 0.883 microg x h/mL, P < .001, maximum concentration, 20.1 +/- 9.21 versus 1.30 +/- 0.367 microg x h/mL, P < .001). CONCLUSIONS: This finding shows that when mycophenolate mofetil and iron ion preparations were administered concomitantly, a remarkable decrease of mycophenolate mofetil absorption was observed. Therefore it seems to be clear that we must avoid the concomitant administration of mycophenolate mofetil and iron ion preparations.
Asunto(s)
Compuestos Ferrosos/efectos adversos , Inmunosupresores/farmacocinética , Absorción Intestinal/efectos de los fármacos , Ácido Micofenólico/análogos & derivados , Ácido Micofenólico/farmacocinética , Adulto , Cationes Bivalentes , Estudios Cruzados , Preparaciones de Acción Retardada , Interacciones Farmacológicas , Femenino , Compuestos Ferrosos/administración & dosificación , Humanos , Masculino , Persona de Mediana Edad , Ácido Micofenólico/sangreRESUMEN
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.
Asunto(s)
ATPasa de Ca(2+) y Mg(2+)/metabolismo , Carbonil Cianuro m-Clorofenil Hidrazona/farmacología , Mucosa Gástrica/enzimología , Ionóforos/farmacología , 4-Cloro-7-nitrobenzofurazano/análogos & derivados , 4-Cloro-7-nitrobenzofurazano/farmacología , Animales , Antibacterianos/farmacología , Transporte Biológico/efectos de los fármacos , Carbonil Cianuro p-Trifluorometoxifenil Hidrazona/farmacología , Activación Enzimática/efectos de los fármacos , Gramicidina/farmacología , Membranas Intracelulares/enzimología , Macrólidos/farmacología , Fosfatidilcolinas/farmacocinética , Fosfatidilcolinas/farmacología , Cloruro de Potasio/farmacología , Cloruro de Sodio/farmacología , Porcinos , Valinomicina/farmacologíaRESUMEN
Hantaan virus (HTN) and Seoul virus (SEO) are members of the genus Hantavirus in the family Bunyaviridae and are causative agents of hemorrhagic fever with renal syndrome. The complete and truncated nucleocapsid proteins (NP) of HTN and SEO were expressed by a recombinant baculovirus system. Antigenic characterization of the NP using monoclonal antibodies (MAbs) indicated that the binding sites for the serotype-specific MAbs were located between amino acids (aa) 155 and 429. A Western blot assay indicated that the serotype-specific epitopes were conformation dependent. An indirect immunofluorescence antibody (IFA) assay with the truncated NP (aa 155 to 429) was able to distinguish convalescent-phase sera from HTN and SEO patients. However, the antibody titers with the truncated NP were lower than those with the whole NP. The truncated NP of SEO (aa 155 to 429) could be used as an enzyme-linked immunosorbent assay (ELISA) antigen, but the truncated NP from HTN lost its reactivity when used for ELISA. The IFA assay using baculovirus-expressed truncated NP as an antigen is a rapid, simple, and safe test for distinguishing between HTN and SEO infections by serotype.
Asunto(s)
Virus Hantaan/clasificación , Infecciones por Hantavirus/diagnóstico , Fiebre Hemorrágica con Síndrome Renal/diagnóstico , Proteínas de la Nucleocápside/inmunología , Orthohantavirus/clasificación , Adulto , Animales , Anticuerpos Monoclonales , Línea Celular , Clonación Molecular/métodos , Ensayo de Inmunoadsorción Enzimática , Epítopos/análisis , Femenino , Técnica del Anticuerpo Fluorescente Indirecta , Infecciones por Hantavirus/sangre , Fiebre Hemorrágica con Síndrome Renal/sangre , Humanos , Insectos , Masculino , Persona de Mediana Edad , Proteínas de la Nucleocápside/biosíntesis , Proteínas de la Nucleocápside/química , Nucleopoliedrovirus , Reacción en Cadena de la Polimerasa/métodos , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/inmunología , Eliminación de Secuencia , Serotipificación , TransfecciónRESUMEN
We found that isolated gastric vesicles contain a novel Mg2+-ATP-dependent phospholipid translocation (flippase) activity. Fluorescence analogue of phosphatidylcholine, 2-(12-(7-nitrobenz-2-oxa-1, 3-diazol-4-yl)amino)dodecanoyl-1-hexadecanoyl-sn-glycero-3- phosphocholine, was ATP-dependently translocated from the outer (cytosolic) to inner (luminal) leaflet of the lipid membrane bilayer of hog gastric vesicles. The translocation was saturable and depended on time and the ATP concentration (Km = 3.1 microM). The basal Mg2+-ATPase activity of gastric vesicles in the absence of K+ showed high (Km = 1.6 microM) and low (Km = 80 microM) affinities for ATP, indicating that the present flippase activity is driven mostly by the high affinity Mg2+-ATPase activity. It required Mg2+ but not K+. Verapamil, which is an inhibitor of mouse mdr2 phosphatidylcholine flippase, did not inhibit the present flippase activity. Isolated sarcoplasmic reticulum vesicles that contain Ca2+-ATPase did not show any flippase activity. Fluorescence analogues of phosphatidylserine and phosphatidylethanolamine were similarly translocated by the gastric flippase. These phospholipid flippase activities were inhibited by 2-methyl-8-(phenylmethoxy)imidazo[1,2-a]pyridine-3-acetonitrile (SCH 28080) (IC50 = 0.14-0.25 microM), a specific K+-ATPase inhibitor of gastric H+,K+-ATPase rich in gastric vesicles. IC50 value for the SCH 28080-inhibitable Mg2+-ATPase activity was about 0.13 microM, indicating that the phospholipid translocation was driven mostly by the SCH 28080-sensitive Mg2+-ATPase activity. Possible physiological roles of flippases were discussed in relation with the gastric acid secretory and cytoprotective mechanisms.
Asunto(s)
Subfamilia B de Transportador de Casetes de Unión a ATP , ATPasa de Ca(2+) y Mg(2+)/metabolismo , Mucosa Gástrica/enzimología , Membrana Dobles de Lípidos/metabolismo , Lípidos de la Membrana/metabolismo , Microsomas/enzimología , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/antagonistas & inhibidores , Transportadoras de Casetes de Unión a ATP/antagonistas & inhibidores , Nucleótidos de Adenina/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Inhibidores Enzimáticos/farmacología , Imidazoles/farmacología , Cinética , Ratones , Especificidad por Sustrato , Porcinos , Verapamilo/farmacologíaRESUMEN
Gastric H+, K+ -ATPase comprised of alpha- and beta-subunits was functionally expressed in an animal cell-line. When glutamic acid (345) of the alpha-subunit was mutated to glutamine, the affinity of K+ decreased 10-fold, indicating that this residue in the 4th transmembrane domain engages in the determination of the K+ affinity. The roles of other residues are also discussed. The number of the binding site of proton pump inhibitors such as omeprazole and E3810 (rabeprazole) is 1, which is contrary to the proposal of 2 or 3 by other researchers. The reason of this discrepancy is explained. The interaction between 2 or 4 alpha-subunits was shown to be necessary for the function of this pump. Finally, recent topics about H+ -ATPase are discussed.
Asunto(s)
ATPasa Intercambiadora de Hidrógeno-Potásio , 2-Piridinilmetilsulfinilbencimidazoles , Aminoácidos , Animales , Bencimidazoles/metabolismo , Sitios de Unión , ATPasa Intercambiadora de Hidrógeno-Potásio/química , ATPasa Intercambiadora de Hidrógeno-Potásio/fisiología , Humanos , Omeprazol/metabolismo , Proteínas , Inhibidores de la Bomba de Protones , Rabeprazol , Estómago/enzimologíaRESUMEN
The H+,K+-ATPase of intact gastric vesicles has two Km values for ATP hydrolysis, 7 and 80 microM. Irradiation of vesicles with ultraviolet light in the presence of 1 mM ATP resulted in K+-ATPase activity that shows only the low affinity ATP binding. The irradiation stimulated or inhibited proton uptake rate compared with control vesicles at high or low ATP concentrations, respectively. The relation between proton uptake rate and K+-ATPase activity at different ATP concentrations was linear with irradiated vesicles and nonlinear with control vesicles. These results indicate that hydrolysis at the high affinity ATP binding site regulates the energy-transport coupling in negative and positive manners at high and low ATP concentrations, respectively. The complete inhibition of K+-ATPase by a specific proton pump inhibitor E3810 (rabeprazole) (2-([4-(3-methoxypropoxy)-3-methylpyridin-2-yl]methylsulf i nyl)-1H-benzimidazole sodium salt) occurred when E3810 bound to half of the alpha-subunit of H+,K+-ATPase in unirradiated vesicles at both 200 and 10 microM ATP, whereas the complete inhibition of proton uptake occurred when E3810 bound to half or a quarter of the alpha-subunit at 200 or 10 microM ATP, respectively. These results suggest that dimeric interaction between the alpha-subunits is necessary for the enzyme activity at all ATP concentrations and that dimeric or tetrameric interaction is necessary for proton transport at high or low ATP concentrations, respectively.
Asunto(s)
Mucosa Gástrica/enzimología , ATPasa Intercambiadora de Hidrógeno-Potásio/metabolismo , 2-Piridinilmetilsulfinilbencimidazoles , Adenosina Trifosfato/metabolismo , Animales , Bencimidazoles/metabolismo , Bencimidazoles/farmacología , Sitios de Unión , Fraccionamiento Celular , Membrana Celular/enzimología , Inhibidores Enzimáticos/metabolismo , Inhibidores Enzimáticos/farmacología , ATPasa Intercambiadora de Hidrógeno-Potásio/química , Cinética , Sustancias Macromoleculares , Omeprazol/análogos & derivados , Inhibidores de la Bomba de Protones , Rabeprazol , PorcinosRESUMEN
E3810 (2-([4-(3-methoxypropoxy)-3-methylpyridine-2-yl]methylsulphinyl )- 1H-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 alpha-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 alpha-subunit is one into consideration, the possibility that E3810 specifically binds to Cys322 residue of hog gastric H+,K(+)-ATPase is discussed.
Asunto(s)
Bencimidazoles/metabolismo , ATPasa Intercambiadora de Hidrógeno-Potásio/metabolismo , Inhibidores de la Bomba de Protones , Estómago/enzimología , 2-Piridinilmetilsulfinilbencimidazoles , Animales , Colorantes Fluorescentes , ATPasa Intercambiadora de Hidrógeno-Potásio/química , Omeprazol/análogos & derivados , Rabeprazol , Porcinos , Tripsina/metabolismo , Rayos UltravioletaRESUMEN
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.
Asunto(s)
Bencimidazoles/farmacología , Ácido Gástrico/metabolismo , Mucosa Gástrica/efectos de los fármacos , Omeprazol/análogos & derivados , Inhibidores de la Bomba de Protones , 2-Piridinilmetilsulfinilbencimidazoles , Adenosina Trifosfatasas/metabolismo , Animales , Proteínas de Transporte de Catión , Mucosa Gástrica/metabolismo , ATPasa Intercambiadora de Hidrógeno-Potásio , Lansoprazol , Masculino , Microsomas/enzimología , Omeprazol/farmacología , Potasio/farmacología , Rabeprazol , Ratas , Ratas Wistar , Factores de TiempoRESUMEN
Excitation-Ca2+ release coupling properties in the heavy microsomal fraction of the rabbit skeletal muscle enriched in triads were investigated by following the same type of approach used for the studies of excitation-contraction coupling in the skinned fiber system. Incubation of the triads with Mg-ATP in a solution containing 150 mM K+, 15.0-37.2 mM Na+, 150-180 mM gluconate-, and 150-200 microM Ca2+ (priming solution) led to (a) the generation of a T-tubule membrane potential making the cytoplasmic side negative, as assessed by potential-dependent uptake of the potential probe [14C]SCN- by triads, and (b) active transport of Ca2+ into the SR moiety. One volume of the primed (viz., polarized and Ca(2+)-loaded) triads was mixed with nine volumes of depolarization solution according to Cl(-)-replacement [Donaldson, S.K.B. (1985) J. Gen. Physiol 86, 501-525; Stephenson, E. W. (1985) J. Gen. Physiol. 86, 813-832] and Na(4)-replacement [Lamb, G.D., & Stephenson, D.G. (1990) J. Physiol. 423, 495-517] protocols used for the induction of contraction in skinned fiber system. The ionic replacement procedure by either protocol produced a rapid release of Ca2+ from SR as determined by stopped-flow fluorometry using fluo-3 as a Ca2+ probe in the presence of BAPTA-calcium buffer. Both the rate constant and the magnitude of Ca2+ release increased with the degree of ionic replacement. The ionic replacement-dependent changes in the release kinetics showed a striking similarity to the voltage-dependent changes of the Ca2+ transient in the intact fiber system.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Calcio/metabolismo , Retículo Sarcoplasmático/metabolismo , Animales , Transporte Biológico , Técnicas In Vitro , Iones , Potenciales de la Membrana , Microsomas/metabolismo , Conejos , SolucionesRESUMEN
Gastric intracellular tubulovesicles fuse with the apical membrane upon histamine stimulation. Disulfide cross-linking of isolated gastric tubulovesicles by cupper-o-phenanthroline (CuP) opened the chloride channel in the vesicles. A functional monoclonal antibody raised against H+,K(+)-ATPase of gastric vesicles inhibited both the enzyme activity and the CuP-induced opening of the chloride channel. This fact indicates that the chloride channel is part of the function of the ATPase. Another evidence which supports the above concept that both the pump and the chloride channel coexist in the same molecule was obtained in this study. The conformation of H+,K(+)-ATPase was changed in the direction of E2 form by incubation with SCH 28080 or low concentrations of K+. SCH 28080 is an H+,K(+)-ATPase specific inhibitor and binds to the high affinity K+ site. Both SCH 28080 and K+ inhibited the channel opening, indicating that the channel opening by the S-S cross-linking depends on the conformational state of the enzyme.
Asunto(s)
Canales de Cloruro/fisiología , ATPasa Intercambiadora de Hidrógeno-Potásio/fisiología , Animales , Antiulcerosos , Histamina/farmacología , Imidazoles/farmacología , Membranas , Potasio , Bombas de Protones , EstómagoRESUMEN
Omeprazole and E3810 were found to inhibit gastric H+,K(+)-ATPase following different biochemical mechanisms. Effects of the specific binding of the inhibitors on the conformational state of the enzyme were studied by measuring the fluorescence of the enzyme labeled with fluorescein 5'-isothiocyanate. The absolute fluorescence level of the omeprazole-bound enzyme was lower than that of the control enzyme, and reduction of S-S cross-linking between the enzyme and omeprazole increased the fluorescence. Addition of K+ into the control vesicle solution quenched the fluorescence (E1-->E2K+). The quench was inhibited in the omeprazole-bound enzyme but not in the E3810-bound enzyme. These results suggest that the omeprazole-bound enzyme has a low fluorescence conformation (E2 form). On the other hand, the conformation of the E3810-bound enzyme was the same as that of the control enzyme (E1 form). Phosphoenzyme formation in the absence of K+ was inhibited in both the E3810- and omeprazole-bound enzymes. Binding of 2',3'-o-(2,4,6-trinitrophenyl)adenosine 5'-triphosphate to the enzyme was equally inhibited by E3810 and omeprazole. K(+)-dependent dephosphorylation from the phosphoenzyme was inhibited in the E3810-bound enzyme but not in the omeprazole-bound enzyme. These experimental results have shown that the inhibition mechanism of H+,K(+)-ATPase by omeprazole was different from that by E3810; the partial reaction that was the most differently affected by the inhibitors was the conformational change from the E2 to E1 form for omeprazole and the luminal K(+)-dependent dephosphorylation for E3810.
Asunto(s)
Bencimidazoles/farmacología , Omeprazol/farmacología , Inhibidores de la Bomba de Protones , 2-Piridinilmetilsulfinilbencimidazoles , Adenosina Trifosfato/análogos & derivados , Adenosina Trifosfato/metabolismo , Animales , Bencimidazoles/metabolismo , Sitios de Unión , Fluoresceína-5-Isotiocianato , ATPasa Intercambiadora de Hidrógeno-Potásio/química , ATPasa Intercambiadora de Hidrógeno-Potásio/metabolismo , Concentración de Iones de Hidrógeno , Técnicas In Vitro , Omeprazol/metabolismo , Potasio/farmacología , Conformación Proteica , Bombas de Protones/efectos de los fármacos , Rabeprazol , Estómago/enzimología , PorcinosRESUMEN
1. Cl- channels in the basolateral membrane of non-stimulated parietal cells in isolated rabbit gastric glands were studied by patch-clamp and noise analysis techniques. 2. Voltage-independent whole-cell currents were recorded from parietal cells equilibrated with Cl(-)-containing solutions. Upon reducing the Cl- concentration of the basolateral bathing solution, the current-voltage curve was rapidly shifted to the right. The reversal potential changed according to changes in the equilibrium potential for Cl-. 3. A Cl- channel blocker, 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB) inhibited whole-cell Cl- currents. The half-maximal inhibitory concentration of NPPB was 300 microM, and the inhibition was reversible (at less than or equal to 200 microM). Another Cl- channel blocker, 4-acetamido-4'-isothiocyanatostilbene-2,2'-dilsulphonic acid or diphenylamine-2-carboxylate was much less effective. 4. The power spectra of whole-cell Cl- current fluctuations contained one Lorentzian component. The single Cl- channel conductance was estimated to be 0.35 pS by the variance noise analysis, which is in agreement with the value obtained by a method of power spectrum analysis (0.29 pS). 5. The addition of arachidonic acid (10 microM) to the basolateral medium markedly increased the whole-cell Cl- current. The combined application of a cyclo-oxygenase inhibitor, indomethacin (50 microM), and a lipoxygenase inhibitor, esculetin (100 microM), increased the Cl- current, whereas the administration of a phospholipase A2 inhibitor, mepacrine (100 microM), significantly decreased the whole-cell Cl- current. 6. A sizeable increase in the whole-cell Cl- current was also induced by a metabolite of arachidonic acid, prostaglandin E2 (10 microM), but not by leukotriene B4 (5 microM) or D4 (10 microM). 7. The present study has shown that small-conductance Cl- channels are present in the basolateral membrane of rabbit parietal cells, and that the channel was functionally regulated by arachidonic acid and prostaglandin E2.
Asunto(s)
Ácido Araquidónico/farmacología , Cloro/metabolismo , Dinoprostona/farmacología , Canales Iónicos/efectos de los fármacos , Células Parietales Gástricas/efectos de los fármacos , Animales , Relación Dosis-Respuesta a Droga , Indometacina/farmacología , Masculino , Potenciales de la Membrana/fisiología , Nitrobenzoatos/farmacología , Quinacrina/farmacología , Conejos , Umbeliferonas/farmacologíaRESUMEN
Substituted benzimidazoles such as omeprazole, E3810 and methoxy E3810 were inhibitors of gastric H+, K(+)-ATPase which is rich in the apical membrane of gastric parietal or oxyntic cells at the secreting state. The acid-activated compounds of omeprazole and methoxy E3810, which have methoxy group at the 5-position in the benzimidazole ring, are fluorescent (excitation wavelength = 370 nm; emission wavelength = 560 nm). The fluorescence disappeared when the activated compounds reacted with the ATPase or glutathione. Using this fluorescence property, the distribution of the intracellular acidic canalicular space in isolated single parietal cells was determined. On the other hand, irradiation with ultraviolet light (335 nm) of the acid-activated compound of E3810 which had been reacted with sulfhydryl group of the ATPase or glutathione resulted in a formation of a fluorescent compound (emission = 470 nm). Using this second fluorescence property, we determined the distribution of the apical membrane of the intracellular canaliculus of isolated single mammalian parietal cells and also the location of the apical membrane on the external surface of newt oxyntic cells.
Asunto(s)
Colorantes Fluorescentes , Ácido Gástrico/metabolismo , Mucosa Gástrica/metabolismo , 2-Piridinilmetilsulfinilbencimidazoles , Adenosina Trifosfatasas/antagonistas & inhibidores , Animales , Bencimidazoles , Membrana Celular/metabolismo , Mucosa Gástrica/citología , Cobayas , ATPasa Intercambiadora de Hidrógeno-Potásio , Técnicas In Vitro , Omeprazol , Células Parietales Gástricas/metabolismo , Conejos , Rabeprazol , SalamandridaeRESUMEN
Heavy sarcoplasmic reticulum vesicles were labeled with the thiol-reacting fluorescent probe N-(7-dimethylamino-4-methyl-4-coumarinyl)maleimide (DACM), and the DACM-labeled foot protein moiety was purified. The fluorescence intensity of the DACM attached to the foot protein decreased by the addition of low (activating) concentrations of ryanodine, while it increased at higher (inhibitory) concentrations, suggesting that the lower fluorescence represents the active state of the foot protein, while the higher fluorescence, its inactive state. Under conditions that induce Ca2+ release from SR (Ca2+ jump, addition of Ca2+ release inducing reagents such as caffeine and polylysine), the fluorescence intensity of the protein-attached DACM decreased rapidly (e.g. k congruent to 70 s-1 under optimum conditions). The initial rate of Ca2+ release from the DACM-labeled SR showed a close correlation with the amplitude of the fluorescence change of the foot protein-attached DACM under variety of conditions; e.g. in the presence of Ca2+, polylysine, ATP, and ruthenium red, etc. The fluorescence change of the foot protein was much faster than Ca2+ release from SR under a variety of conditions of Ca2+ release. We propose that the binding of release triggering reagents to the foot protein induces a rapid conformational change, which in turn regulates Ca2+ release.