Epidermal growth factor receptor activity upregulates lactate dehydrogenase A expression, lactate dehydrogenase activity, and lactate secretion in cultured IB3-1 cystic fibrosis lung epithelial cells.

Cystic fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. It has been postulated that reduced HCO3- transport through CFTR may lead to a decreased airway surface liquid pH. In contrast, others have reported no changes in the extracellular pH (pHe). We have recently reported that in carcinoma Caco-2/pRS26 cells (transfected with short hairpin RNA for CFTR) or CF lung epithelial IB3-1 cells, the mutation in CFTR decreased mitochondrial complex I activity and increased lactic acid production, owing to an autocrine IL-1ß loop. The secreted lactate accounted for the reduced pHe, because oxamate fully restored the pHe. These effects were attributed to the IL-1ß autocrine loop and the downstream signaling kinases c-Src and JNK. Here we show that the pHe of IB3-1 cells can be restored to normal values (∼7.4) by incubation with the epidermal growth factor receptor (EGFR, HER1, ErbB1) inhibitors AG1478 and PD168393. PD168393 fully restored the pHe values of IB3-1 cells, suggesting that the reduced pHe is mainly due to increased EGFR activity and lactate. Also, in IB3-1 cells, lactate dehydrogenase A mRNA, protein expression, and activity are downregulated when EGFR is inhibited. Thus, a constitutive EGFR activation seems to be responsible for the reduced pHe in IB3-1 cells.

Physiological and Proteomic Changes in the Apoplast Accompany Leaf Senescence in Arabidopsis.

The apoplast, i.e. the cellular compartment external to the plasma membrane, undergoes important changes during senescence. Apoplastic fluid volume increases quite significantly in senescing leaves, thereby diluting its contents. Its pH elevates by about 0.8 units, similar to the apoplast alkalization in response to abiotic stresses. The levels of 159 proteins decrease, whereas 24 proteins increase in relative abundance in the apoplast of senescing leaves. Around half of the apoplastic proteins of non-senescent leaves contain a N-terminal signal peptide for secretion, while all the identified senescence-associated apoplastic proteins contain the signal peptide. Several of the apoplastic proteins that accumulate during senescence also accumulate in stress responses, suggesting that the apoplast may constitute a compartment where developmental and stress-related programs overlap. Other senescence-related apoplastic proteins are involved in cell wall modifications, proteolysis, carbohydrate, ROS and amino acid metabolism, signaling, lipid transport, etc. The most abundant senescence-associated apoplastic proteins, PR2 and PR5 (e.g. pathogenesis related proteins PR2 and PR5) are related to leaf aging rather than to the chloroplast degradation program, as their levels increase only in leaves undergoing developmental senescence, but not in dark-induced senescent leaves. Changes in the apoplastic space may be relevant for signaling and molecular trafficking underlying senescence.

Extracellular pH and lung infections in cystic fibrosis.

Cystic fibrosis (CF) is an autosomal recessive disease caused by CFTR mutations. It is characterized by high NaCl concentration in sweat and the production of a thick and sticky mucus, occluding secretory ducts, intestine and airways, accompanied by chronic inflammation and infections of the lungs. This causes a progressive and lethal decline in lung function. Therefore, finding the mechanisms driving the high susceptibility to lung infections has been a key issue. For decades the prevalent hypothesis was that a reduced airway surface liquid (ASL) volume and composition, and the consequent increased mucus concentration (dehydration), create an environment favoring infections. However, a few years ago, in a pig model of CF, the Na+/K+ concentrations and the ASL volume were found intact. Immediately a different hypothesis arose, postulating a reduced ASL pH as the cause for the increased susceptibility to infections, due to a diminished bicarbonate secretion through CFTR. Noteworthy, a recent report found normal ASL pH values in CF children and in cultured primary airway cells, challenging the ASL pH hypothesis. On the other hand, recent evidences revitalized the hypothesis of a reduced ASL secretion. Thus, the role of the ASL pH in the CF is still a controversial matter. In this review we discuss the basis that sustain the role of CFTR in modulating the extracellular pH, and the recent results sustaining the different points of view. Finding the mechanisms of CFTR signaling that determine the susceptibility to infections is crucial to understand the pathophysiology of CF and related lung diseases.

Programmed cell death in yeast by thionin-like peptide from Capsicum annuum fruits involving activation of caspases and extracellular H+ flux.

CaThi is a thionin-like peptide isolated from fruits of Capsicum annuum, which has strong antimicrobial activity against bacteria, yeasts and filamentous fungi, and induced reactive oxygen species (ROS) in fungi. ROS are molecules that appear in the early stages of programmed cell death or apoptosis in fungi. Due to this fact, in this work we analyzed some events that may be related to process of apoptosis on yeast induced by CaThi. To investigate this possibility, we evaluated phosphatidylserine (PS) externalization, presence of active caspases and the ability of CaThi to bind to DNA in Candida tropicalis cells. Additionally, we investigated mitochondrial membrane potential, cell surface pH, and extracellular H+ fluxes in C. tropicalis cells after treatment with CaThi. Our results showed that CaThi induced PS externalization in the outer leaflet of the cell membrane, activation of caspases, and it had the ability for DNA binding and to dissipate mitochondrial membrane potential. In addition, the cell surface pH increased significantly when the C. tropicalis cells were exposed to CaThi which corroborates with ~96% inhibition on extracellular H+ efflux. Taking together, these data suggest that this peptide is capable of promoting an imbalance in pH homeostasis during yeast cell death playing a modulatory role in the H+ transport systems. In conclusion, our results strongly indicated that CaThi triggers apoptosis in C. tropicalis cells, involving a pH signaling mechanism.

El plomo inhibe la corriente activada por protones (ASIC) en las neuronas de los ganglios dorsales / Lead inhibits proton gated currents (ASIC) in dorsal root ganglion neurons

Antecedentes. Los canales iónicos ASIC (del inglés Acid Sensing Ion Channel) son canales iónicos activados por reducciones transitorias en el pH extracelular. Pese a no conocerse con exactitud su mecanismo, la activación ocurre por medio de la unión de protones al dominio extracelular del canal y es modulada por iones calcio y zinc. Objetivo. El hecho de que los cationes divalentes modifiquen el funcionamiento del canal nos llevó a preguntar si el plomo, otro catión divalente, sería capaz de alterar el funcionamiento de los ASIC. Métodos y resultados. Mediante el uso de la técnica de fijación de voltaje en configuración de célula completa en las neuronas de los ganglios de la raíz dorsal de la rata, encontramos que el plomo inhibe la corriente ASIC en forma dependiente de la concentración. Conclusiones. Estos resultados contribuyen a definir los mecanismos de activación de los canales ASIC y a explicar algunos de los mecanismos tóxicos del plomo en el organismo.

BACKGROUND: Acid sensing ion channels (ASIC) are ionic channels activated by transient pH reductions in the ext raceilularenvi ronment. Although the activation mechanism is not fully elucidated, it is clear that the channel is activated by proton binding to its extraceilular domain, a process that is modulated by calcium and zinc. OBJECTIVE: The fact that divalent cations are able to modify ASIC operation, lead us to consider if lead, anotherdivalent cation and widely distributed neurotoxicant, is also capable to affect ASIC function. METHODS: For this purpose, we recordedASiC currents in rat dorsal root ganglion neurons using the whole cell patch-clamp technique. RESULTS: The results indicated that lead inhibits ASIC currents in a concentration -dependent fashion. CONCLUSIONS: These results contribute to the understanding of the activation mechanism of ASIC and to explain some of the toxic mechanisms of lead in the organism.