Maxi K+ channels in the basolateral membrane of the exocrine frog skin gland regulated by intracellular calcium and pH

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A primary culture of mouse proximal tubular cells, established on collagen-coated membranes.

A simple method is described to establish primary cultures of kidney proximal tubule cells (PTC) on membranes. The permeable membranes represent a unique culture surface, allowing a high degree of differentiation since both apical and basolateral membranes are accessible for medium. Proximal tubule (PT) segments from collagenase-digested mouse renal cortices were grown for 7 days, by which time cells were organized as a confluent monolayer. Electron microscopic evaluation revealed structurally polarized epithelial cells with numerous microvilli, basolateral invaginations, and apical tight junctions. Immunoblotting for markers of distinct parts of the nephron demonstrated that these primary cultures only expressed PT-specific proteins. Moreover immunodetection of distinct components of the receptor-mediated endocytic pathway and uptake of FITC-albumin indicated that these cells expressed a functional endocytotic apparatus. In addition, primary cultures possessed the PT brush-border enzymes, alkaline phosphatase, and gamma-glutamyl-transferase, and a phloridzin-sensitive sodium-dependent glucose transport at their apical side. Electrophysiological measurements show that the primary cultured cells have a low transepithelial resistance and high short-circuit current that was completely carried by Na(+) similar to a leaky epithelium like proximal tubule cells. This novel method established well-differentiated PTC cultures

A highly reliable and budget-friendly Peltier-cooled camera for biological fluorescence imaging microscopy

The SAC8.5, a low-cost Peltier-cooled black and white 8-bit CCD camera for astronomy, was evaluated for its use in imaging microscopy. Two camera-microscope configurations were used: an epifluorescence microscope (Nikon Eclipse TE2000-U) and a bottom port laser scanning confocal microscope system (Zeiss LSCM 510 META). Main advantages of the CCD camera over the currently used photomultiplier detection in the scanning setup are fast image capturing, stable background, an improved signal-to-noise ratio and good linearity. Based on DAPI-labelled Chinese Hamster Ovarian cells, the signal-to-noise ratio was estimated to be 4 times higher with respect to the currently used confocal photomultiplier detector. A linear relationship between the fluorescence signal and the FITC-inulin concentrations ranging from 0.05 to 1.8 mg mL(-1) could be established. With the SAC8.5 CCD camera and using DAPI, calcein-AM and propidium iodide we could also distinguish between viable, apoptotic and necrotic cells: exposure to CdCl2 caused necrosis in A6 cells. Additional examples include the observation of wire-like mitochondrial networks in Mito Tracker Green-loaded Madin-Darby canine kidney cells. Furthermore, it is straightforward to interface the SAC8.5 with automated shutters to prevent rapid fluorophore photobleaching via easy to use ASTROVIDEO software. In this study, we demonstrate that the SAC8.5 black and white CCD camera is an easy-to-implement and cost-conscious addition to quantitative fluorescence microfluorimetry on living tissues and is suitable for teaching laboratories

Cadmium-Induced Pathologies: Where Is the Oxidative Balance Lost (or Not)?

Over the years, anthropogenic factors have led to cadmium (Cd) accumulation in the environment causing various health problems in humans. Although Cd is not a Fenton-like metal, it induces oxidative stress in various animal models via indirect mechanisms. The degree of Cd-induced oxidative stress depends on the dose, duration and frequency of Cd exposure. Also the presence or absence of serum in experimental conditions, type of cells and their antioxidant capacity, as well as the speciation of Cd are important determinants. At the cellular level, the Cd-induced oxidative stress either leads to oxidative damage or activates signal transduction pathways to initiate defence responses. This balance is important on how different organ systems respond to Cd stress and ultimately define the pathological outcome. In this review, we highlight the Cd-induced oxidant/antioxidant status as well as the damage versus signalling scenario in relation to Cd toxicity. Emphasis is addressed to Cd-induced pathologies of major target organs, including a section on cell proliferation and carcinogenesis. Furthermore, attention is paid to Cd-induced oxidative stress in undifferentiated stem cells, which can provide information for future therapies in preventing Cd-induced pathologies

Dorsal unpaired median neurons of Locusta migratoria express ivermectin- and fipronil-sensitive glutamate-gated chloride channels

Together with type A GABA and strychnine-sensitive glycine receptors, glutamate-gated chloride channels ( GluCl) are members of the Cys-loop family of ionotropic receptors, which mediate fast inhibitory neurotransmission. To date, GluCls are found in invertebrates only and therefore represent potential specific targets for insecticides, such as ivermectin and fipronil. In this study, we identified the functional expression of GluCls in dorsal unpaired median ( DUM) neurons of the metathoracic ganglion of Locusta migratoria using electrophysiological and molecular biological techniques. In whole cell patch-clamped DUM neurons, glutamate-induced changes in both their membrane potentials (current-clamp) and currents (voltage-clamp) were dependent on the chloride equilibrium potential. On continuous application of glutamate, the glutamate-elicited current response became rapidly and completely desensitized. Application of glutamate in the presence of 10 mu M fipronil or 100 mu M picrotoxin reversibly decreased GluCl-mediated currents by 87 and 39%, respectively. Furthermore, 1 mu M ivermectin induced a persistent chloride current, suggesting the expression of ivermectin-sensitive GluCl alpha subunits. A degenerate PCR/RACE strategy was used to clone the full-length L. migratoria LmGlC1 alpha subunit. Finally, RT-PCR experiments demonstrated the presence of LmGluC1 alpha transcripts in locust DUM neurons. Our results provide the first direct evidence of a functional ivermectin-sensitive GluCl channel on the cell surface of DUM neurons of L. migratoria