RESUMO
Introduction: The gastrooesophageal reflux disease (GERD) is a possible cause of chronic cough. The laparoscopic fundoplication is well established in the treatment of GERD. In a retrospective study, the effectivity of this operation on the GERD associated cough was examined and possible preoperative predictive factors concerning the post-surgical therapy effect were characterized. Patients and Methods: 85 patients after laparoscopic fundoplication due to GERD treated with proton pump inhibitors without (RS-H: n = 31) or with associated cough (RS+H: n = 54) were evaluated in a three-month follow-up by data analysis regarding an indication point score from typical symptoms as well as findings (gastroscopy, histology, 24-hour oesophagus pH-metry). Results: For the leading symptoms of heartburn and regurgitation a complete freedom from complaints was reached with 98.8â% of all patients postal-surgically. In the group RS+H 70.4â% of the patients were free of cough after 3 months, other 22.2â% with significant improvement and 7.4â% with unchanged irritant cough. Higher values of the typical reflux symptoms and a therapy resistance to proton pump inhibitors (PPI) were clearly seen in the RS-H patients. The RS+H patients showed less reflux complaints with lower PPI resistance, frequent allergies as well as significantly more often an acid or bitter taste and hoarseness. After further subdivision of the RS+H patients into the subgroups RS>H (mainly reflux, n = 31) and H>RS (mainly cough), the lowest values for heartburn, regurgitation and PPI resistance were found in subgroup H>RS. Diagnostics did not show any significiant differences between the groups although a trend could be seen towards fewer duodenogastric bile reflux, larger hiatus hernias and higher DeMeester scores in RS+H and H>RS. Also smokers, non-allergic asthmatics and polyallergic sufferers with cough profited from the intervention. Conclusion: Patients with reflux-associated respiratory symptoms present an own entity with good PPI therapy response to heartburn, but not to cough. They should be considered more often for surgery. Since the cough symptoms in more than two-thirds of appropriately selected patients disappear in a short time after surgery, laparoscopic antireflux surgery should also be considered from pneumological aspects. There are no individual predictors for the success of antireflux surgery, only the sum of all relevant individual case history and clinical criteria, as they are combined in the used score, can provide a reliable indication for surgery.
Assuntos
Tosse/cirurgia , Fundoplicatura/métodos , Refluxo Gastroesofágico/cirurgia , Laparoscopia/métodos , Adulto , Idoso , Tosse/etiologia , Feminino , Seguimentos , Refluxo Gastroesofágico/diagnóstico , Humanos , Masculino , Pessoa de Meia-Idade , Complicações Pós-Operatórias/etiologiaRESUMO
The importance of K(ATP) channels in stimulus-secretion coupling of ß-cells is well established, although they are not indispensable for the maintenance of glycaemic control. This review article depicts a new role for K(ATP) channels by showing that genetic or pharmacological ablation of these channels protects ß-cells against oxidative stress. Increased production of oxidants is a crucial factor in the pathogenesis of type 2 diabetes mellitus (T2DM). T2DM develops when ß-cells can no longer compensate for the high demand of insulin resulting from excess fuel intake. Instead ß-cells start to secrete less insulin and ß-cell mass is diminished by apoptosis. Both, reduction of insulin secretion and ß-cell mass induced by oxidative stress, are prevented by deletion or inhibition of K(ATP) channels. These findings may open up new insights into the early treatment of T2DM.
Assuntos
Diabetes Mellitus Tipo 2/tratamento farmacológico , Gliclazida/uso terapêutico , Hipoglicemiantes/uso terapêutico , Células Secretoras de Insulina/efeitos dos fármacos , Canais KATP/antagonistas & inibidores , Estresse Oxidativo/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Glicemia/efeitos dos fármacos , Glicemia/metabolismo , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/metabolismo , Feminino , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Gliclazida/farmacologia , Humanos , Hipoglicemiantes/farmacologia , Células Secretoras de Insulina/metabolismo , Masculino , Regulação para CimaRESUMO
AIMS/HYPOTHESIS: Evidence is accumulating that Ca(2+)-regulated K(+) (K(Ca)) channels are important for beta cell function. We used BK channel knockout (BK-KO) mice to examine the role of these K(Ca) channels for glucose homeostasis, beta cell function and viability. METHODS: Glucose and insulin tolerance were tested with male wild-type and BK-KO mice. BK channels were detected by single-cell RT-PCR, cytosolic Ca(2+) concentration ([Ca(2+)](c)) by fura-2 fluorescence, and insulin secretion by radioimmunoassay. Electrophysiology was performed with the patch-clamp technique. Apoptosis was detected via caspase 3 or TUNEL assay. RESULTS: BK channels were expressed in murine pancreatic beta cells. BK-KO mice were normoglycaemic but displayed markedly impaired glucose tolerance. Genetic or pharmacological deletion of the BK channel reduced glucose-induced insulin secretion from isolated islets. BK-KO and BK channel inhibition (with iberiotoxin, 100 nmol/l) broadened action potentials and abolished the after-hyperpolarisation in glucose-stimulated beta cells. However, BK-KO did not affect action potential frequency, the plateau potential at which action potentials start or glucose-induced elevation of [Ca(2+)](c). BK-KO had no direct influence on exocytosis. Importantly, in BK-KO islet cells the fraction of apoptotic cells and the rate of cell death induced by oxidative stress (H(2)O(2), 10-100 µmol/l) were significantly increased compared with wild-type controls. Similar effects were obtained with iberiotoxin. Determination of H(2)O(2)-induced K(+) currents revealed that BK channels contribute to the hyperpolarising K(+) current activated under conditions of oxidative stress. CONCLUSIONS/INTERPRETATION: Ablation or inhibition of BK channels impairs glucose homeostasis and insulin secretion by interfering with beta cell stimulus-secretion coupling. In addition, BK channels are part of a defence mechanism against apoptosis and oxidative stress.
Assuntos
Glucose/metabolismo , Canais de Potássio/metabolismo , Animais , Apoptose/genética , Apoptose/fisiologia , Sobrevivência Celular/genética , Sobrevivência Celular/fisiologia , Células Cultivadas , Eletrofisiologia , Homeostase , Masculino , Camundongos , Camundongos Knockout , Reação em Cadeia da Polimerase , Canais de Potássio/genéticaRESUMO
Pancreatic beta-cells of sulfonylurea receptor type 1 knock-out (SUR1(-/-)) mice exhibit an oscillating membrane potential (V (m)) demonstrating that hyper-polarisation occurs despite the lack of K(ATP) channels. We hypothesize that glucose activates the Na(+)/K(+)-ATPase thus increasing a hyper-polarising current. Elevating glucose in SUR1(-/-) beta-cells resulted in a transient fall in V (m) and [Ca(2+)](c) independent of sarcoplasmic and endoplasmic reticulum Ca(2+)-activated ATPase (SERCA) activation. This was not affected by K(+) channel blockade but inhibited by ATP depletion and by ouabain. Increasing glucose also reduced [Na(+)](c), an effect reversed by ouabain. Exogenously applied insulin decreased [Na(+)](c) and hyper-polarised V (m). Inhibiting insulin signalling in SUR1(-/-) beta-cells blunted the glucose-induced decrease of [Ca(2+)](c). Tolbutamide (1 mmol/l) disclosed the SERCA-independent effect of glucose on [Ca(2+)](c) in wild-type beta-cells. The data show that in SUR1(-/-) beta-cells, glucose activates the Na(+)/K(+)-ATPase presumably by increasing [ATP](c). Insulin can also stimulate the pump and potentiate the effect of glucose. Pathways involving the pump may thus serve as potential drug targets in certain metabolic disorders.
Assuntos
Glucose/farmacologia , Células Secretoras de Insulina/metabolismo , Insulina/farmacologia , ATPase Trocadora de Sódio-Potássio/metabolismo , Trifosfato de Adenosina/fisiologia , Animais , Cálcio/metabolismo , Ativação Enzimática , Insulina/fisiologia , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Camundongos , Transdução de Sinais , ATPase Trocadora de Sódio-Potássio/efeitos dos fármacosRESUMO
In recent years, flower development has emerged as a model system for studying pattern formation in plants. Homeotic mutants with an altered pattern of floral organs have been found in many species. Recently, several of the floral homeotic genes have been isolated; and the mechanisms underlying pattern formation during flower development are beginning to be elucidated.
Assuntos
Plantas/anatomia & histologia , Genes Homeobox , Morfogênese , Mutação , Plantas/genética , Especificidade da EspécieRESUMO
Flowering plants are the most highly evolved and complex organisms within the plant kingdom. The flower consists of several distinct organ systems that are responsible for higher plant reproduction. Cells within specific floral organs differentiate into spores and gametes required by the plant to complete its life cycle. Flower development represents an excellent model for understanding the molecular and physiological processes that control organ differentiation in higher plants. Rapidly emerging gene tagging procedures are facilitating the isolation of genes that control flower morphogenesis.
Assuntos
Plantas/genética , Diferenciação Celular , Regulação da Expressão Gênica , Genes , Meiose , Mitose , Morfogênese , Desenvolvimento VegetalRESUMO
The beginning of modern microbiology can be traced back to the 1870s, and it was based on the development of new concepts that originated during the two preceding centuries on the role of microorganisms, new experimental methods, and discoveries in chemistry, physics, and evolutionary cell biology. The crucial progress was the isolation and growth on solid media of clone cultures arising from single cells and the demonstration that these pure cultures have specific, inheritable characteristics and metabolic capacities. The doctrine of the spontaneous generation of microorganisms, which stimulated research for a century, lost its role as an important concept. Microorganisms were discovered to be causative agents of infectious diseases and of specific metabolic processes. Microscopy techniques advanced studies on microorganisms. The discovery of sexuality and development in microorganisms and Darwin's theory of evolution contributed to the founding of microbiology as a science. Ferdinand Cohn (1828-1898), a pioneer in the developmental biology of lower plants, considerably promoted the taxonomy and physiology of bacteria, discovered the heat-resistant endospores of bacilli, and was active in applied microbiology.
Assuntos
Microbiologia/história , Bactérias/classificação , Bactérias/genética , Fenômenos Fisiológicos Bacterianos , Evolução Biológica , História do Século XVII , História do Século XVIII , História do Século XIX , PolôniaRESUMO
Cells of Rhodopseudomonas capsulata, strain 37b4, leu-, precultivated anaerobically under low light intensity, were exposed to high light intensity (2000 W.m-2). The cells grew with a mass doubling time of 3 h. The synthesis of bacteriochlorophyll (BChl) began after two doublings of cell mass. Reaction center and light-harvesting BChl I (B-875) were the main constituents of the photosynthetic apparatus incorporated into the membrane. The size of the photosynthetic unit (total BChl/reaction center) decreased and light-harvesting BChl I became the dominating BChl species. Concomitant with the appearance of the different spectral forms of BChl the respective proteins were incorporated into the membrane, i.e. the three reaction center polypeptides, the polypeptide associated with light-harvesting BChl I, the two polypeptides associated with BChl II. A polypeptide of an apparent molecular weight of 45 000 was also incorporated. A lowering of the light intensity to 7 W.m-2 resulted in a lag phase of growth for 6 h. Afterwards, the time for doubling of cell mass was 11 h. The concentration of all three BChl complexes (reaction center, light-harvesting BChl I and II complexes)/cell and per membrane protein increased immediately. Also the size of the photosynthetic unit and the amount of intracytoplasmic membranes/cell increased. The activities of photophosphorylation, succinate dehydrogenase, NADH dehydrogenase and NADH oxidation (respiratory chain)/membrane protein are higher in membrane preparations isolated from cells grown at high light intensities than in such preparations from cells grown at low light intensities.
Assuntos
Bacterioclorofilas/biossíntese , Clorofila/análogos & derivados , Membranas Intracelulares/metabolismo , Rodopseudomonas/efeitos da radiação , Luz , NADH Desidrogenase/metabolismo , Fotofosforilação/efeitos da radiação , Rodopseudomonas/crescimento & desenvolvimento , Succinato Desidrogenase/metabolismoRESUMO
The isolation of two native light harvesting bacteriochlorophyl.protein complexes from Rhodopseudomonas capsulata is described. The light harvesting bacteriochlorophyll I (B 875) has been isolated from the blue-green mutant A1a+ lacking both carotenoids and light harvesting bacteriochlorophyll II. Light harvesting bacteriochlorophyll I is associated with a protein (light harvesting band 2) of 12 000 molecular weight. Light harvesting bacteriochlorophyll II complex has been isolated from the mutant Y5 lacking a reaction center and light harvesting bacteriochlorophyll I. Light harvesting bacteriochlorphyll II (B 800 + 850) together with carotenoids is associated with two polypeptides (light harvesting bands 3 and 4) having molecular weights of about 8000 and 10 000 (sodium dodecyl sulfate polyacrylamide gel electrophoresis). A third protein (light harvesting band 1) is in the purified light harvesting II fraction (mol. wt. approx. 14 000), but not associated with bacteriochlorophyll or carotenoids. The amino acid composition of the 3 antenna pigment II proteins is given. The polarity of these proteins was found to be 48%. From the amino acid composition the following molecular weights were calculated band 1: 17 350, band 3: 13 350 and band 4: 10 500.
Assuntos
Proteínas de Bactérias/isolamento & purificação , Bacterioclorofilas/isolamento & purificação , Clorofila/análogos & derivados , Rodopseudomonas , Aminoácidos/análise , Carotenoides/análise , Luz , Peso Molecular , Mutação , Fosfolipídeos/análise , Ligação ProteicaRESUMO
Adrenaline and galanin inhibit insulin release through strikingly similar mechanisms triggered by distinct receptors in pancreatic B cells. In this study we evaluated whether activation of alpha 2-adrenoceptors and galanin receptors use a common or only a similar transduction pathway. The membrane potential of B-cells was measured with intracellular microelectrodes and 86Rb efflux was monitored in normal mouse islets perifused with a medium containing 15 mM glucose. At a maximally effective concentration of 10 microM, adrenaline partially repolarized the membrane, inhibited but did not abolish electrical activity, and caused a decrease in 86Rb efflux (due to a lesser activation of Ca(2+)- and voltage-activated K+ channels). In the presence of 10 microM adrenaline, galanin had no effect on membrane potential, electrical activity and 86Rb efflux. Decreasing the concentration of glucose from 15 to 6 mM repolarized the B-cell membrane to the same extent as did adrenaline but did not prevent galanin from causing an additional hyperpolarization. In contrast to galanin, diazoxide, a selective opener of ATP-sensitive K+ channels still produced a small hyperpolarization and further decrease in 86Rb efflux when added at a low concentration (15 microM) to a medium containing 10 microM adrenaline. At a high concentration (250 microM), diazoxide repolarized the membrane to the resting potential and markedly accelerated 86Rb efflux both in the presence and absence of adrenaline. The non-additivity of the effects of adrenaline and galanin suggests that alpha 2-adrenoceptors and galanin receptors share common targets in pancreatic B-cells.
Assuntos
Epinefrina/farmacologia , Ilhotas Pancreáticas/efeitos dos fármacos , Peptídeos/farmacologia , Radioisótopos de Rubídio/análise , Animais , Diazóxido/farmacologia , Relação Dose-Resposta a Droga , Interações Medicamentosas , Feminino , Galanina , Glucose/farmacologia , Ilhotas Pancreáticas/metabolismo , Potenciais da Membrana/efeitos dos fármacos , CamundongosRESUMO
Nitric oxide (NO) is assumed to contribute to the impairment of B-cell function in type 1 diabetes mellitus (IDDM). In the present paper we show that in mouse B-cells with intact metabolism authentic NO (20 microM) led to a biphasic effect on the K(+)(ATP) current, namely a transient increase and a consecutive almost complete inhibition. This resembles closely the effect that we have observed previously with the NO donor S-nitrosocysteine (SNOC, 1 mM) suggesting that merely NO caused both phases of this effect. We now demonstrate that the rise in the current amplitude was accompanied by a depolarization of the mitochondrial membrane potential DeltaPsi and a concomitant reduction in the ATP/ADP ratio. Thus, it seems likely that the increase in current amplitude is due to the interference of NO with cell metabolism. The subsequent inhibition of the K(+)(ATP) current is assumed to be caused by a direct effect on the channel since K(+)(ATP) single channel current activity measured in excised patches was strongly reduced by authentic NO and SNOC. Our data reveal new insights into the mechanisms underlying the biphasic action of NO on K(+)(ATP) channels in pancreatic B-cells.
Assuntos
Trifosfato de Adenosina/metabolismo , Ilhotas Pancreáticas/efeitos dos fármacos , Óxido Nítrico/farmacologia , Canais de Potássio/metabolismo , S-Nitrosotióis , Difosfato de Adenosina/metabolismo , Animais , Cisteína/análogos & derivados , Cisteína/farmacologia , Feminino , Técnicas In Vitro , Ilhotas Pancreáticas/metabolismo , Potenciais da Membrana/efeitos dos fármacos , Camundongos , Mitocôndrias/efeitos dos fármacos , Doadores de Óxido Nítrico/farmacologia , Compostos Nitrosos/farmacologia , Técnicas de Patch-ClampRESUMO
Photooxidation of endogenous cytochrome(s) c, photoreduction of endogenous chrome(s) b and photobleaching of bacteriochlorophyll have been demonstrated in an in vitro reconstituted system, previously demonstrated to support photophosphorylation. The kinetic responses of these redox reactions to substrate and antimycin A in these particle are characteristic of electron transport processes and stronglysupport the contention that all, or a part of, the oxidative phosphorylation electron transport pathway can be coupled to reaction center, photopigment complex in a manner which supports photophosphorylation. In addition, a succinate-supported light dependent reduction of NAD+ was found.
Assuntos
Citocromos/metabolismo , Fotofosforilação , Rodopseudomonas/metabolismo , Antimicina A/farmacologia , Clorofila/metabolismo , Escuridão , Transporte de Elétrons , Cinética , Luz , OxirreduçãoRESUMO
The photosynthetic green bacterium Chlorobium limicola 6230 has been examined by freeze-fracture electron microscopy to investigate the size, form, distribution and supramolecular architecture of its chlorosomes (chlorobium vesicles) as well as the chlorosome attachment sites on the cytoplasmic membrane. The oblong chlorosomes that underlie the cytoplasmic membrane show a considerable variation in size from about 40 X 70 nm to 100 X 260 nm and exhibit no particular orientation. The chlorosome core, which appears to be hydrophobic in nature, contains between 10 and 30 rod-shaped elements (approx. 10 nm in diameter) surrounded by an unetchable matrix. The rod elements are closely packed and extend the full length of the chlorosome. Separating the chlorosome core from the cytoplasm is a approx. 3 nm thick lipid-like envelope layer, which exhibits no substructure. A 5-6 nm thick, crystalline baseplate connects the chlorosome to the cytoplasmic membrane. The ridges of the baseplate lattice make an angle of between 40 degrees and 60 degrees with the longitudinal axis of the chlorosome and have a repeating distance of approx. 6 nm. In addition, each ridge exhibits a granular substructure with a periodicity of approx. 3.3 nm. The cytoplasmic membrane regions adjacent to the baseplates are enriched in large (greater than 9 nm) intramembrane particles, most of which belong to approx. 10 nm and approx. 12.5 nm particle size categories. Each chlorosome attachment site contains between 20 and 30 very large (greater than 12.0 nm diameter) intramembrane particles. The following interpretive model of a chlorosome is discussed in terms of biophysical, biochemical and structural information reported by others: it is proposed that the bacteriochlorophyll c (BChl c; chlorobium chlorophyll) is located in the rod elements of the core and that it is complexed with specific proteins. The cytoplasm-associated envelope layer is depicted as consisting of a monolayer of galactosyl diacylglycerol molecules. BChl alpha-protein complexes in a planar lattice configuration most likely make up the crystalline baseplate. The greater than 12-nm particles in the chlorosome attachment sites of the cytoplasmic membrane, finally, may correspond to complexes containing a reaction center and non-crystalline light-harvesting BChl alpha. The crystalline nature of the baseplate is consistent with the notion that it serves two functions: besides transferring excitation energy to the reaction centers it could also function as a distributor of this energy amongst the reaction centers.
Assuntos
Bactérias/ultraestrutura , Fenômenos Fisiológicos Bacterianos , Membrana Celular/ultraestrutura , Grânulos Citoplasmáticos/ultraestrutura , Técnica de Fratura por Congelamento , Microscopia Eletrônica , FotossínteseRESUMO
Absorption and fluorescence emission spectra of Rhodopseudomonas capsulata, strains 37b4 (wild type), A1a+ (blue-green mutant strain), Y5 (phototroph negative, having only B-800--850 bacteriochlorophyll-carotenoid-protein complex) at 4 K, 77 K and 300 K were measured. The fluorescence emission at 890 nm of the B-870 bacteriochlorophyll band dominates the emission of other spectral forms of the strains 37b4 and A1a+, while in strain Y5 a fluorescence emission band at 865 nm of the B-850 bacteriochlorophyll dominates. Very little fluorescence was observed at 805 nm. A linear relation between relative fluorescence intensity and the exciting light intensity was observed. The integrated fluorescence yield increased as the temperature was lowered from 300 K to 4 K. The results are discussed in the light of the arrangement of pigment molecules in the membrane and the process of energy migration within the photosynthetic apparatus.
Assuntos
Fluorescência , Rodopseudomonas/metabolismo , Bacterioclorofilas/metabolismo , Transferência de Energia , Mutação , Fotossíntese , Pigmentos Biológicos/metabolismo , Rodopseudomonas/genética , Espectrometria de Fluorescência , Análise Espectral , TemperaturaRESUMO
Absorption, linear dichroism and circular dichroism spectra of Rhodopseudomonas capsulata (wild-type-St. Louis strain, mutant Y5 and mutant Ala+) are particularly sensitive to the nature of the light-harvesting bacteriochlorophyll-carotenoid-protein complexes. Evidence for exciton-type interactions is seen near 855 nm in the membranes from the wild-type and from mutant Y5, as well as in an isolated B-800 + 850 light-harvesting complex from mutant Y5. The strong circular dichroism that reflects these interactions is attenuated more than 10-fold in membranes from the Ala+ mutant, which lacks both B-800 + 850 and colored carotenoids and contains only the B-875 light-harvesting complex. These results lead to the conclusion that these two light-harvesting complexes have significantly different chromophore arrangements or local environments.
Assuntos
Membrana Celular/metabolismo , Proteínas de Membrana/metabolismo , Rodopseudomonas/metabolismo , Dicroísmo Circular , Cinética , Mutação , Conformação Proteica , EspectrofotometriaRESUMO
Measurements of the membrane potential showed that osmotic swelling (-80 mosmol/l) of pancreatic B-cells led to a transient hyperpolarization followed by a more sustained depolarization of the cell membrane. Cell swelling triggers a transient activation of the K+ATP current and of an inward current, carried by Cl-. This current was inhibited by DIDS, D600, and by omission of extracellular Ca2+. The depolarization opens voltage dependent L-type Ca2+ channels, thereby increasing the intracellular Ca2+ activity ([Ca2+]i). This effect was blunted by D600 or abolished by omission of Ca2+. Moreover, osmotic swelling transiently increased the amplitude of the Ca2+ currents. Replacement of NaCl by d-mannitol proved that the observed effects are due to an increase in cell volume and not to a reduction of extracellular Na+ or Cl-. Our results suggest that regulatory volume decrease is achieved by activation of K+ and Cl- currents. The Cl- current is responsible for the previously described depolarization and increase in insulin release induced by osmotic cell swelling.
Assuntos
Insulina/metabolismo , Canais Iônicos/metabolismo , Ilhotas Pancreáticas/metabolismo , Ilhotas Pancreáticas/fisiologia , Trifosfato de Adenosina/metabolismo , Animais , Transporte Biológico , Canais de Cálcio/fisiologia , Canais de Cloreto/metabolismo , Feminino , Líquido Intracelular/metabolismo , Canais Iônicos/fisiologia , Potenciais da Membrana , Camundongos , Camundongos Endogâmicos , Pressão Osmótica , Canais de Potássio/fisiologiaRESUMO
Mouse islets were used to study the effects of inhibitors of cyclooxygenase and lipoxygenase pathways on insulin release, ionic fluxes, and beta-cell membrane potential. The cyclooxygenase inhibitors, Na-salicylate and Na-acetylsalicylate, potentiated glucose-induced insulin release, despite a decrease in Ca influx evidenced by inhibition of the Ca-dependent electrical activity in beta-cells and 45Ca efflux from islets perifused with a medium containing Ca. This paradox can probably be explained by a mobilization of intracellular Ca (acceleration of 45Ca efflux in the absence of Ca) with subsequent activation of K+ channels (acceleration of 86Rb efflux) and repolarization of the membrane. These effects of salicylate could not be ascribed to a change in intracellular pH because they were not mimicked by 2-Cl-benzoate, which has a similar pK as salicylate but increased insulin release by stimulating Ca influx in beta-cells. Among the other cyclooxygenase inhibitors tested, indomethacin caused a slight potentiation of insulin release accompanied by marginal increases in 45Ca efflux and electrical activity, whereas flurbiprofen and ibuprofen were ineffective. Among the lipoxygenase inhibitors, compound BW 755c reversibly decreased glucose-induced insulin release by inhibiting Ca influx in beta-cells, but nordihydroguaiaretic acid had no effect. Inhibitors of arachidonic acid metabolism have effects on ionic fluxes and beta-cell membrane potential, which may explain some of the changes in insulin release they produce.
Assuntos
Ácidos Araquidônicos/metabolismo , Ilhotas Pancreáticas/citologia , Ilhotas Pancreáticas/metabolismo , Salicilato de Sódio/farmacologia , 4,5-Di-Hidro-1-(3-(Trifluormetil)Fenil)-1H-Pirazol-3-Amina/farmacologia , Animais , Aspirina/farmacologia , Cálcio/metabolismo , Permeabilidade da Membrana Celular/efeitos dos fármacos , Células Cultivadas , Inibidores de Ciclo-Oxigenase/farmacologia , Condutividade Elétrica/efeitos dos fármacos , Feminino , Glucose/metabolismo , Glucose/farmacologia , Concentração de Íons de Hidrogênio , Ibuprofeno/farmacologia , Indometacina/farmacologia , Insulina/metabolismo , Ilhotas Pancreáticas/fisiologia , Inibidores de Lipoxigenase/farmacologia , Potenciais da Membrana/efeitos dos fármacos , Camundongos , Microeletrodos , Potássio/metabolismo , Canais de Potássio/efeitos dos fármacos , Canais de Potássio/fisiologia , Radioimunoensaio , Radioisótopos de RubídioRESUMO
This study demonstrates for the first time that the microelectrode array (MEA) technique allows analysis of electrical activity of islets isolated from human biopsies. We have shown before that this method, i.e., measuring beta cell electrical activity with extracellular electrodes, is a powerful tool to assess glucose responsiveness of isolated murine islets. In the present study, human islets were shown to exhibit glucose-dependent oscillatory electrical activity. The glucose responsiveness could be furthermore demonstrated by an increase of insulin secretion in response to glucose. Electrical activity was increased by tolbutamide and inhibited by diazoxide. In human islets bursts of electrical activity were markedly blunted by the Na(+) channel inhibitor tetrodotoxin which does not affect electrical activity in mouse islets. Thus, the MEA technique emerges as a powerful tool to decipher online the unique features of human islets.Additionally, this technique will enable research with human islets even if only a few islets are available and it will allow a fast and easy test of metabolic integrity of islets destined for transplantation.
Assuntos
Hiperglicemia/metabolismo , Insulina/metabolismo , Ilhotas Pancreáticas/metabolismo , Animais , Biópsia , Criança , Estimulação Elétrica , Glucose/metabolismo , Humanos , Hiperglicemia/sangue , Hiperglicemia/tratamento farmacológico , Hiperglicemia/patologia , Hipoglicemiantes/farmacologia , Secreção de Insulina , Ilhotas Pancreáticas/efeitos dos fármacos , Ilhotas Pancreáticas/patologia , Canais KATP/agonistas , Canais KATP/metabolismo , Potenciais da Membrana/efeitos dos fármacos , Moduladores de Transporte de Membrana/farmacologia , Camundongos , Microeletrodos , Pessoa de Meia-Idade , Bloqueadores dos Canais de Sódio/farmacologia , Especificidade da Espécie , Análise Serial de Tecidos , Técnicas de Cultura de TecidosRESUMO
The spatial relationship of the three polypeptides contained in the B800-850 light-harvesting complex of Rhodopseudomonas capsulata has been studied with chemical cross-linking of crude membrane preparations of the phototrophic negative mutant strain Y5. Samples were cross-linked with the cleavable reagent dithiobis (succinimidyl propionate) (1.1 nm chain length) and analyzed by two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis. Membranes labelled with 14C-amino acids were used to determine the compositional stoichiometry of cross-linked products. It was found that the two polypeptides with an apparent Mr of 8000 and 10 000, respectively, that are associated with the pigments bacteriochlorophyll a and carotenoid form homooligomers as well as heterooligomers. The data support the idea that these polypeptides are closely arranged in clusters probably containing at least four of each species. The third subunit with an Mr of 14 000, which is not associated with pigments, was found to be most susceptible to cross-linking and formed homooligomers but no heterooligomers with the other two subunits, and is thus likely to be loosely attached to these clusters. Comparative studies with the phototrophic positive wild type strain indicated that the results found with the phototrophic negative mutant strain Y5 reflect the organization of the B800-850 complex in the membrane of Rhodopseudomonas capsulata. Studies with the isolated B800-850 complex revealed that the sterical arrangement of the three constituent polypeptides in dodecyl dimethylamine-N-oxide containing solutions must be very similar to that in the membrane.
Assuntos
Proteínas de Bactérias/metabolismo , Pigmentos Biológicos/metabolismo , Rodopseudomonas/metabolismo , Reagentes de Ligações Cruzadas , Complexos de Proteínas Captadores de Luz , Proteínas de Membrana/metabolismo , Peso Molecular , Fotossíntese , Complexo de Proteínas do Centro de Reação Fotossintética , Conformação ProteicaRESUMO
The obligatory halophilic, phototrophic bacterium, strain WS 68, grows under anaerobic conditions in the light in a mineral medium with acetate as carbon source and 6 to 20% sodium chloride, or in the same medium semiaerobically in the dark. The cells are vibrio- or spiral-shaped and show a bipolar polytrichous flagellation. The absorption spectrum of membranes, isolated from phototrophically grown cells, shows absorption maxima at 375, 490, 520, 555, 590, 800, 840-845 and 870-875 nm indicating the presence of bacteriochlorophyll a and carotenoids as pigments of the photosynthetic apparatus. The cell surface is covered with a layer of regular arranged particles (center to center distance approximately 10.5 nm). A murein (peptidoglycan) sacculus has not been detected in ultrathin sections, freeze fracture preparations and cells extracted with hot dodecyl sulfate. The intracytoplasmic membranes lie parallel to the cell envelope. The polypeptide patterns of isolated membranes and of the envelope fraction have been demonstrated. The results are discussed in the light of taxonomy and cell organization.