Effect of epsilon toxin-GFP on MDCK cells and renal tubules in vivo.

Epsilon toxin (epsilon-toxin), produced by Clostridium perfringens types B and D, causes fatal enterotoxemia, also known as pulpy kidney disease, in livestock. Recombinant epsilon-toxin-green fluorescence protein (epsilon-toxin-GFP) and epsilon-prototoxin-GFP were successfully expressed in Escherichia coli. MTT assays on MDCK cells confirmed that recombinant epsilon-toxin-GFP retained the cytotoxicity of the native toxin. Direct fluorescence analysis of MDCK cells revealed a homogeneous peripheral pattern that was temperature sensitive and susceptible to detergent. epsilon-Toxin-GFP and epsilon-prototoxin-GFP bound to endothelia in various organs of injected mice, especially the brain. However, fluorescence mainly accumulated in kidneys. Mice injected with epsilon-toxin-GFP showed severe kidney alterations, including hemorrhagic medullae and selective degeneration of distal tubules. Moreover, experiments on kidney cryoslices demonstrated specific binding to distal tubule cells of a range of species. We demonstrate with new recombinant fluorescence tools that epsilon-toxin binds in vivo to endothelial cells and renal tubules, where it has a strong cytotoxic effect. Our binding experiments indicate that an epsilon-toxin receptor is expressed on renal distal tubules of mammalian species, including human.

Gadolinium inhibition of ecto-nucleoside triphosphate diphosphohydrolase activity in Torpedo electric organ.

Ecto-nucleoside triphosphate diphosphohydrolases (E-NTPDases) are widely expressed enzymes implicated in the modulation of nucleotide cell signaling. They dephosphorylate either ATP or ADP in the presence of divalent cations, and efforts have been made to identify efficient inhibitors. E-NTPDase activity has been described in Torpedo electric organ electrocytes. We show here that gadolinium, an established blocker of stretch-activated channels, efficiently inhibits E-NTPDase activity of Torpedo electric organ (Ki = 3 microM for ATPase) as well as apyrase from potato tuber, frequently used in inhibition experiments. To our knowledge, gadolinium is the most potent inhibitor described to date for both membrane-bound and soluble E-NTPDases.

Release of ATP induced by hypertonic solutions in Xenopus oocytes.

ATP mediates intercellular communication. Mechanical stress and changes in cell volume induce ATP release from various cell types, both secretory and non-secretory. In the present study, we stressed Xenopus oocytes with a hypertonic solution enriched in mannitol (300 mM). We measured simultaneously ATP release and ionic currents from a single oocyte. A decrease in cell volume, the activation of an inward current and ATP release were coincident. We found two components of ATP release: the first was associated with granule or vesicle exocytosis, because it was inhibited by tetanus neurotoxin, and the second was related to the inward current. A single exponential described the correlation between ATP release and the hypertonic-activated current. Gadolinium ions, which block mechanically activated ionic channels, inhibited the ATP release and the inward current but did not affect the decrease in volume. Oocytes expressing CFTR (cystic fibrosis transmembrane regulator) released ATP under hypertonic shock, but ATP release was significantly inhibited in the first component: that related to granule exocytosis. Since the ATP measured is the balance between ATP release and ATP degradation by ecto-enzymes, we measured the nucleoside triphosphate diphosphohydrolase (NTPDase) activity of the oocyte surface during osmotic stress, as the calcium-dependent hydrolysis of ATP, which was inhibited by more than 50 % in hypertonic conditions. The best-characterized membrane protein showing NTPDase activity is CD39. Oocytes injected with an antisense oligonucleotide complementary to CD39 mRNA released less ATP and showed a lower amplitude in the inward current than those oocytes injected with water.

Cholinergic currents in Xenopus oocytes transplanted with human brain membranes

Corrientes colinérgicas de cerebro humano fueron registradas en oocitos de Xenopus laevis trasplantados con membranas de cerebro humano procedentes de dos zonas diferentes, la corteza frontal y el hipocampo. Las corrientes registradas fueron activadas por el receptor nicotínico o por el receptor nicotínico o muscarínico de la acetilcolina. Se probaron los efectos de diferentes agonistas nicotínicos como acetilcolina, nicotina y yoduro de 1,1-dimetil-4-fenil-piperazinio (DMPP), y antagonistas del receptor nicotínico como a-bungarotoxina y d-tubocurarina en los oocitos transplantados. Detectamos cuatro clases de cinéticas de corrientes nicotínicas. Las diferencias en la amplitud y en la carga eléctrica total de las corrientes provocadas por varios agonistas en el rango de potencial mantenido no fueron significativas, excepto en el caso del DMPP a un potencial mantenido de -90 mV. Nuestros resultados indican que las formas alfa4beta2, alfa3beta4 y alfa7 son los principales receptores nicotínicos en el cerebro humano

Cholinergic human brain currents were recorded in Xenopus laevis oocytes transplanted with human cerebral membranes from two different zones, the frontal cortex and the hippocampus. The recorded currents were supported by the nicotinic or the muscarinic acetylcholine receptor. We tested the effects of a number of several nicotinic agonists acetylcholine, nicotine and 1,1-dimethyl-4-phenylpiperazinium iodide (DMPP), and the nicotinic receptor antagonists a-bungarotoxin and d-tubocurarine on the transplanted oocytes. We detected four kinds of nicotinic current kinetics. The differences in the amplitude and in the total electric charge of the currents elicited by various agonists at a range of holding potentials were not significant, except in the case of DMPP at a holding potential of -90 mV. Our results indicate that alpha4beta2, alpha3beta4 and alpha7 are the main nicotinic receptors in human brain