Cl- and regulation of pH by MDCK-C11 cells

The interaction between H+ extrusion via H+-ATPase and Cl- conductance was studied in the C11 clone of MDCK cells, akin to the intercalated cells of the collecting duct. Cell pH (pHi) was measured by fluorescence microscopy using the fluorescein-derived probe BCECF-AM. Control recovery rate measured after a 20 mM NH4Cl acid pulse was 0.136 ± 0.008 pH units/min (dpHi/dt) in Na+ Ringer and 0.032 ± 0.003 in the absence of Na+ (0 Na+). With 0 Na+ plus the Cl- channel inhibitor NPPB (10 æM), recovery was reduced to 0.014 ± 0.001 dpHi/dt. 8-Br-cAMP, known to activate CFTR Cl- channels, increased dpHi/dt in 0 Na+ to 0.061 ± 0.009 and also in the presence of 46 nM concanamycin and 50 æM Schering 28080. Since it is thought that the Cl- dependence of H+-ATPase might be due to its electrogenic nature and the establishment of a +PD (potential difference) across the cell membrane, the effect of 10 æM valinomycin at high (100 mM) K+ was tested in our cells. In Na+ Ringer, dpHi/dt was increased, but no effect was detected in 0 Na+ Ringer in the presence of NPPB, indicating that in intact C11 cells the effect of blocking Cl- channels on dpHi/dt was not due to an adverse electrical gradient. The effect of 100 æM ATP was studied in 0 Na+ Ringer solution; this treatment caused a significant inhibition of dpHi/dt, reversed by 50 æM Bapta. We have shown that H+-ATPase present in MDCK C11 cells depends on Cl- ions and their channels, being regulated by cAMP and ATP, but not by the electrical gradient established by electrogenic H+ transport.

Efeito de uma semana de esomeprazol associado a claritromicina e amoxicilina para a erradicação do helicobacter pylori e cicatrização da úlcera duodenal estudo aberto, não randomizado, não controlado e multicêntrico / Effect of 1-week treatment with esomeprazole plus clarithromycin and amoxicillin to eradicate Helicobacter pylori, and to heal duodenal ulcer. Open, non-randomized, non-controlled, multicenter study

A úlcera péptica é causada, na sua maioria, por infecção pelo Helicobacter pylori, sendo portanto recomendada sua erradicação. Um dos tratamentos da erradicação mais frequentemente utilizado é o esquema tríplice baseado em inibidor de bomba protônica associado a dois antimicrobianos. O inibidor da bomba de prótons freqüentemente é continuado por mais três semanas com monoterapia, após o esquema de erradicação, para assegurar cicatrização da úlcera e alívio dos sintomas, em pacientes com úlcera duodenal ativa. O esomeprazol é um dos isômeros ópticos dos composto racêmico omeprazol e, assim, tem o mesmo mecanismo de ação, porém com área sob a curva (AUC) maior e menor variabilidade polimórfica em seu metabolismo hepático. Assim, é possível melhor eficácia clínica do esomeprazol, com efeito inibitório mais duradouro e profundo sobre a secreção ácida gástrica durante o período de 24 horas e menor variação interindividual na acidez gástrica. Vários trabalhos publicados mostraram que o tratamento de erradicação do H. Pylori com esquema tríplice baseado em esomeprazol, por uma semana, tem a mesma eficácia na cicatrização da úlcera duodenal que o tratamento tríplice baseado em omeprazol, por uma semana, continuado por três semanas com omeprazol. Este estudo busca confirmar esses dados em centros brasileiros. O objetivo primário deste estudo foi avaliar as taxas de cicatrização em pacientes com úlcera duodenal, após uma semana de tratamento de erradicação do H. pylori, seguida por quatro semanas de observação sem tratamento. Os pacientes receberam tratamento de erradicação com a terapia tríplice esomeprazol 20mg bid + amoxilina 1.000mg bid + claritromicina 500mg bid, administrados por sete dias. Os objetivos segundários foram: avaliar as taxas de erradicação do H. Pylori; determinar a freqÜência e intensidade de sintomas como azia/pirose e dor epigástrica; avaliar a segurança e a tolerabilidade do esomeprazol em combinação com claritomicina e amoxicilina. O estudo foi aberto, não randonizado, não controlado, participando 12 centros no Brasil. No total, 144 pacientes foram analidados quando a eficácia clínica (análise por protocolo - PP). Os pacientes foram acompanhados por cinco semanas com duas visitas clínicas agendadas durante esse período. A dor epigástrica estava presente em 97,2por cento(140/144) dos pacientes no inicio do estudo

Efeito do hormônio tireoidiano sobre a expressão do RNAm da proteína desacopladora de protóns 3 (UCP3) em miocárdio e músculo esquelético de ratos / Effect of thyroid hormone on UCP-3 mRNA expression in rat heart and skeletal muscle

As proteínas desacopladoras de prótons (UCPs) pertencem à família dos transportadores mitocondriais H+/ácidos graxos. A UCP1 é responsável pela termogênese, mas o exato papel fisiológico da UCP2 e UCP3 ainda não está estabelecido. Ratos foram induzidos ao hipertireoidismo, através da administração de LT3, associado ou não à captopril, propranolol ou clenbuterol. O tratamento com LT3 resultou em aumento estatisticamente significativo do mRNA da UCP3 em miocárdio e músculo esquelético e esse efeito não foi alterado por nenhuma das medicações usadas concomitantemente, nem quando o LT3 foi administrado conjuntamente ao clenbuterol. Os efeitos do hormônio tireoideano sobre a expressão da UCP3, não são dependentes da angiotensina II, nem do sistema β-adrenérgico, refletindo uma ação direta do T3 sobre a expressão da UCP3 / Thyroid hormones stimulate UCP3 expression in skeletal muscle. We examined whether thyroid hormone-induced changes in UCP3 mRNA expression are related to directs effects of T3 or reflect secondary effects of the hormone through stimulation of renin-angiotensin or -adrenergic systems. Hyperthyroidism was produced by injections of T3 with or without concomitant treatment with either captopril, propranolol or clenbuterol. T3 resulted in a significant increase in the relative abundance of UCP3 in heart and skeletal muscle (P < 0.05), and the effect was not altered by captopril or propanolol. There was no synergistic or additive effect of T3 and clenbuterol on UCP3 mRNA expression in skeletal muscle. We conclude that the effect of T3 on UCP3 expression in cardiac and skeletal muscle is not dependent on either angiotensin II or the -adrenergic system and probably reflects a direct action of the hormone on UCP3 gene expression...

Effect of the lipophilic o-naphthoquinone CG 10-248 on rat liver mitochondria structure and function

CG 10-248 (3,4-dihydro-2,2 dimethyl-9-chloro-2H-naphtho[1,2b]pyran-5,6-dione), a beta-lapachone analogue, modified the ultrastructure of rat liver mitochondria in vitro, in the absence of added oxidizable substrates. The condensed mitochondrial state was replaced by the orthodox or swollen state to a significant degree. The number of modified mitochondria depended on incubation time and quinone concentration, in the 25-100 microM range. Under the same experimental conditions, mitochondrial respiration was uncoupled as indicated by the increase in the rate of succinate oxidation by controlled mitochondria in metabolic state "4" (not in state "3"), and by the activation of latent F0F1-ATP synthase. Taking into account structural similarities, the results reported here may be valid for other o-naphthoquinones, such as beta-lapachone.

Cell Biology of H+ Transport in Epithelia

Cell homeostasis of H+ ions has been an object of wide interest in the last two decades, which has led to extended knowledge about a considerable number of membrane transport mechanisms responsible for keeping cell pH within physiological limits. Among these mechanisms the most important are Na+/H+ exchange, the vacuolar H+-ATPase, the H+-K+-ATPase, Cl-/HCO3 - exchange and Na+/HCO3 - cotransport. The present review covers both cellular function and molecular aspects of these transporters, starting from a discussion of the methods used for the determination of cell pH and epithelial H+ transport, and analysing their molecular constitution, cloning and known isoforms, as well as their functional role in the maintenance of cell pH and epithelial transport.

Effect of glandular kallikrein on distal bicarbonate transport. Role of basolateral Cl-/HCO3- exchanger and vacuolar H(+)-ATPase

The luminal membrane of collecting duct cells, specially the intercalated cells, is normally exposed to active kallikrein. This is due to the specific localization of renal kallikrein in the connecting tubule cells. We have previously reported inhibition of distal bicarbonate secretion by renal kallikrein. The present study was performed to evaluate the participation of basolateral Cl-/HCO3- exchanger and luminal H(+)-ATPase activity of cortical collecting duct segments (CCD) in the mechanism involved in the inhibition of bicarbonate secretion induced by the enzyme. The effect of orthograde injections of 1 microgram/ml (250 U/6.3 mg) pig pancreatic kallikrein, in the absence and presence of 1 mM DIDS (stilbene-disulfonic acid) in the renal tubule system, was evaluated. Urine fractions were collected after two-minutes stop-flow. Changes in the urine fraction (Fr) related to those in free-flow urine samples (Ff) were related to the respective polyfructosan (Inutest) ratio. Renal kallikrein activity (Fr:Ff kallikrein/Fr:Ff polyfructosan) increased significantly in the first 120 microliters urine fraction collected after glandular 1 microgram/ml kallikrein, P < 0.05, (first stop-flow) and after glandular 1 microgram/ml kallikrein plus 1 mM. DIDS P < 0.05 (second stop flow). Bicarbonate secretion rate (Fr:Ff HCO3-/Fr:Ff polyfructosan) of collecting ducts was significantly reduced in the first 120 microliters urine fraction collected, related to control, during the first and second stop-flow periods. No difference was shown in bicarbonate excretion between the first 120 microliters urine fractions collected after administration of glandular kallikrein and glandular kallikrein plus DIDS. To measure H(+)-ATPase activity, rat microdissected cortical collector tubules (CCD) were incubated in the presence of increasing glandular kallikrein doses (A: 93, B: 187 and C: 375 mU/200 microL) in the presence of ouabain (4 microM) and omeprazole (100 microM) to inhibit Na(+)-K(+)-ATPase and H(+)-K(+)-ATPase, respectively. In CCD, bafilomycin-sensitive H(+)-ATPase activity (pmol/mm/min) after increasing kallikrein doses did not differ significantly from control...

Photoaffinity labeling and photoaffinity crosslinking of enzymes

Photoaffinity labeling is a special type of chemical modification, where the label is activated by the action of light. This article presents the general principles and limitations of this technique, its application to the study of Micrococcus luteus ATPase and the use of photoaffinity crosslinking to probe the structure of this enzyme

The H+ -ATP synthase: a biochemical challenge

ATP is a high energy compound that living cells utilize for driving most of their endergonic reactions. Directly or indirectly, ATP yields energy through the splitting of its terminal pyrophosphate bond. In cells, the ATP synthase of energy transducing membranes is responsible for forming from ADP and phosphate most of the ATP that cells need for survival and reproduction. The question of how the enzyme catalyzes ATP synthesis has been addressed by numerous workers for over thirty years. A fundamental discovery was that the enzyme is localized in membranes, and that the energy for ATP formation derives from electrochemical gradients built up by enzymes that catalyze electron transfer and that are localized in those membranes. However, the molecular events that take place in the H+ -ATP synthase during the transformation of the energy of electrochemical gradients into the chemical energy of ATP have not been entirely unveiled. Studies of its structure have shown that the H+ -ATP synthase is one of the most complex enzymes discovered. It has a H+ conducting multisubunit pathway and a multisubunit complex where the catalytic events in ATP synthesis take place. Moreover, it is an enzyme that is regulated by numerous and different factors, i.e., adenine nucleotides, electrochemical H+ gradients and protein-protein interactions. Studies on the mechanisms of energy transduction have shown that synthesis of ATP at the catalytic site of the enzyme is a spontaneous process; this indicates that depending on the environment ATP may be a high or a low energy compound. Thus, even though the enzyme presents many unknowns, it continues to be a source of fundamental and unsuspected aspects of basic biochemistry.

The role of the distal nephron in the regulation of acid-base equilibrium by the kidney

The present paper reviews mechanisms by which the kidney controls systemic acid-base balance, with emphasis on the role of the distal nephron, and particularly of the cortical distal tubule. These mechanisms are essentially based on H-ion transport along the whole nephron. In proximal tubule cells, approximately 80 of H-ion secretion is mediated by Na+/H+ exchange, and 20 by H(+)-ATPase. In the distal nephron, acid-base transport mechanisms are located mainly in intercalated cells. H-ion secretion is effected by vacuolar H(+)-ATPase in alpha-intercalated cells and, in K-depleted animals, also by the gastric type H/K ATPase. In animals in alkalosis, beta-intercalated cells secrete bicarbonate by an apical Cl-/HCO3- exchanger, while a basolateral H-ATPase transfers H-ions into the interstitium. In cortical distal tubule, these mechanisms have been shown to be present in the intercalated cells of the connecting segment and of the initial collecting duct (the late distal tubule of micropuncture experiments). In the convoluted distal tubule (early distal tubule), most H-ion secretion occurs by means of the Na+/H+ exchanger. These data show that the distal nephron, including the cortical distal tubule, is a nephron segment responsible for a sizeable portion of bicarbonate reabsorption and titratable acid generation, as well as for bicarbonate secretion under appropriate metabolic conditions, being therefore the site of fine regulation of renal mechanisms that maintain acid-base homeostasis.

Effect of bafilomycin on proximal bicarbonate absorption in the rat

To evaluate the relative importance of the V-type H(+)-ATPase in proximal bicarbonate reabsorption in vivo, proximal tubules of male and female Wistar rats (180 to 260 g) were perfused with bicarbonate-Ringer solution with and without the addition of 2 microM bafilomycin A1. Bafilomycin significantly increased stationary pH from 6.75 +/- 0.05 (N = 39) to 6.86 +/- 0.03 (N = 82), the stationary concentration of bicarbonate from 5.24 +/- 0.62 to 6.33 +/- 0.46 mM and the half-time of acidification from 3.72 +/- 0.22 to 4.65 +/- 0.25 s, and significantly decreased net bicarbonate reabsorption from 3.17 +/- 0.21 to 2.55 +/- 0.15 nmol s-1 cm-2, that is, by 20 per cent . Since bafilomycin is considered to be a specific inhibitor for V-type H(+)-ATPase, these data establish 1) the existence of this type of transport in the rat proximal tubule and 2) that approximately a fifth of the total proximal bicarbonate reabsorption is due to this mechanism of transport

Papel de las H+ ATPasas en las membranas plasmáticas del musculo liso de las vías aéreas / Role of H+ ATPases in plasma membranes from airway smooth muscle

Los protones generadores en el interior de las células debido a la actividad, deben ser removidos mediante mecanismos activos del interior celular hacia el exterior. Uno de los sistemas encargados de transportar protones son las V-ATPasas que son complejos oligométricos presentes en la membranas de varias organelas subcelulares donde participan creando un gradiente de protones y un potencial que energiza dichas membranas. En las membranas plasmáticas del músculo liso de las vías aéreas describimos una V-ATPasas, componente de membranas, que expresa una actividad de Mg2+ ATPasas estimulada por el anión cloruro (Cl-). El efecto activador de cloruro no es específico de este anión sino que otros halógenos como son ioduro (I-) y el bromuro (Br-), la estimulan, con la exepción del fluoruro (F-). Esta ATPasas activada por Cl- es una nucleótido y no es capaz de hidrolizar nucleótidos difosfatos o monofosfatos. Los cationes divalentes activan en orden decreciente (Mg2+ > Mn2+ > Ca2+) a la Mg2+ - ATPasas activada por Cl-. Esta Mg2+ - ATPasas estimulada por Cl-, es insensible a la ouabaína, el vanadato, la azida de sodio y la rutaminina. La NEM (N-etilmaleimida) inhibió parcialmente la actividad de esta ATPsas pero el p-CMB (p-cloromercuribenzoato) produjo una fuerte inhibición de la actividad enzimática. La existencia de una H+ -ATPasas se demostró al estudiar el comportamiento de inhibidores específicos del transporte de protones. Así, la fuerte inhibición producida por la DCCD (diciclohexilcarbodiimida) indica la presencia de subunidad hidrofóbicas de un canal de protones. Adicionalmente, los protonóforos (1799 y FCCP), inducen un aumento en la actividad de Mg2+ - ATPasas estimulada por el cloruro, lo cual indica la presencia de una H+ ATPsas en estas membranas plasmáticas. El requerimiento del Cl- puede ser explicado por la existencia de un conductor de cloruro que funciona en paralelo con la bomba de protones (H+ - ATPasas tipo V), lo cual fue demostrado en este sistema debido al efecto inhibitorio de la duramicina. Las células del músculo liso de las vías aéreas no estan especializadas en la secreción de protones, pero dicha actividad al funcionar como una bomba primaria de protones, energizaría diversos movimientos de iones a través de la membrana plasmática como se ilustra en el modelo propuesto. Estos movimientos ionicos a su vez estan vínculados con fenómenos osmóticos relacionados con los procesos de la contracción del músculo liso de las aéreas