Lang, Florian, Gillian L. Busch, Markus Ritter, Harald Volkl, Siegfried Waldegger, Erich Gulbins, and Dieter Haussinger. Functional Significance of Cell Volume Regulatory Mechanisms. Physiol. Rev. ...

Functional Significance of Cell Volume Regulatory Mechanisms

Phenols and their derivatives commonly exist in the environment. These compounds are used as the components of dyes, polymers, drugs and other organic substances. The presence of phenols in the ecosys - tems is also related with production and degradation of numerous pesticides and the generation of industrial and municipal sewages. Some phenols are also formed during natural processes. These compounds may be substituted with chlorine atoms, may be nitrated, methylated or alkylated. both phenols and catechols are harmful ecotoxins. Toxic action of these compounds stems from unspecified toxicity related to hydrophob - ocity and also to the generation of organic radicals and reactive oxygen species. Phenols and catechols re - veal peroxidative capacity, they are hematotoxic and hepatotoxic, provoke mutagenesis and carcinogenesis toward humans and other living organisms.

Phenols - sources and toxicity

Simultaneous in vivo recording of prompt and delayed fluorescence and 820-nm reflection changes during drying and after rehydration of the resurrection plant Haberlea rhodopensis.

Signals transduced by kinases depend on the extent and duration of substrate phosphorylation. We generated genetically encoded fluorescent reporters for PKC activity that reversibly respond to stimuli activating PKC. Specifically, phosphorylation of the reporter expressed in mammalian cells causes changes in fluorescence resonance energy transfer (FRET), allowing real time imaging of phosphorylation resulting from PKC activation. Targeting of the reporter to the plasma membrane, where PKC is activated, reveals oscillatory phosphorylation in HeLa cells in response to histamine. Each oscillation in substrate phosphorylation follows a calcium oscillation with a lag of ∼10 s. Novel FRET-based reporters for PKC translocation, phosphoinositide bisphosphate conversion to IP3, and diacylglycerol show that in HeLa cells the oscillatory phosphorylations correlate with Ca2+-controlled translocation of conventional PKC to the membrane without oscillations of PLC activity or diacylglycerol. However, in MDCK cells stimulated with ATP, PLC and diacylglycerol fluctuate together with Ca2+ and phosphorylation. Thus, specificity of PKC signaling depends on the local second messenger-controlled equilibrium between kinase and phosphatase activities to result in strict calcium-controlled temporal regulation of substrate phosphorylation.

/pdf/a-genetically-encoded-fluorescent-reporter-reveals-4td8o4es40.pdf

A genetically encoded fluorescent reporter reveals oscillatory phosphorylation by protein kinase C

Using chlorophyll (Chl) a fluorescence many aspects of the photosynthetic apparatus can be studied, both in vitro and, noninvasively, in vivo. Complementary techniques can help to interpret changes in the Chl a fluorescence kinetics. Kalaji et al. (Photosynth Res 122:121–158, 2014a) addressed several questions about instruments, methods and applications based on Chl a fluorescence. Here, additional Chl a fluorescence-related topics are discussed again in a question and answer format. Examples are the effect of connectivity on photochemical quenching, the correction of F
 V
 /F
 M
 values for PSI fluorescence, the energy partitioning concept, the interpretation of the complementary area, probing the donor side of PSII, the assignment of bands of 77 K fluorescence emission spectra to fluorescence emitters, the relationship between prompt and delayed fluorescence, potential problems when sampling tree canopies, the use of fluorescence parameters in QTL studies, the use of Chl a fluorescence in biosensor applications and the application of neural network approaches for the analysis of fluorescence measurements. The answers draw on knowledge from different Chl a fluorescence analysis domains, yielding in several cases new insights.

/pdf/frequently-asked-questions-about-chlorophyll-fluorescence-4gyt8jy35u.pdf

Frequently asked questions about chlorophyll fluorescence, the sequel

Single-channel patch-clamp experiments were performed on MDCK cells in order to characterize the ionic channels participating in regulatory volume decrease (RVD). Subconfluent layers of cultured cells were exposed to a hypotonic medium (150 mOsm), and the membrane currents at the single-channel level were measured in cell-attached experiments. The results indicate that MDCK cells respond to a hypotonic swelling by activating several different ionic conductances. In particular, a potassium and a chloride channel appeared in the recordings more frequently than other channels, and this allowed a more detailed study of their properties in the inside-out configuration of the patch-clamp technique. The potassium channel had a linear I/V curve with a unitary conductance of 24±4 pS in symmetrical K+ concentrations (145 mm). It was highly selective for K+ ions vs. Na− ions: PNa/PK<0.04. The time course of its open probability (P0)showed that the cells responded to the hypotonic shock with a rapid activation of this channel. This state of high activity was maintained during the first minute of hypotonicity. The chloride channel participating in RVD was an outward-rectifying channel: outward slope conductance of 63.3+ 4.7 pS and inward slope conductance of 26.1±4.9 pS. It was permeable to both Cl− and NO3−and its maximal activation after the hypotonic shock was reached after several seconds (between 30 and 100 sec). The activity of this anionic channel did not depend on cytoplasmic calcium concentration. Quinine acted as a rapid blocker of both channels when applied to the cytoplasmic side of the membrane. In both cases, 1 mm quinine reversibly reduced single-channel current amplitudes by 20 to 30%. These results indicate that MDCK cells responded to a hypotonic swelling by an early activation of highly selective potassium conductances and a delayed activation of anionic conductances. These data are in good agreement with the changes of membrane potential measured during RVD.

Activation of K+ and Cl− channels in MDCK cells during volume regulation in hypotonic media

When MDCK cells are cultured in MEM, they maintain a high concentration of three amino acids: glutamate (25mm), taurine (19 mm) and glycine (9 mm). With incubation of the cells in hypotonic media, the contents of these amino acids measured by HPLC are reduced in different time courses: taurine decreases most rapidly, followed by glutamate and glycine. All these losses are Na+ independent. To determine the transport mechanism activated by the hypotonic media, increasing external concentrations reaching 60 mm for nine different amino acids in Na+-free media were tested separately. For the five neutral (zwitterionic) amino acids, taurine, glycine, alanine, phenylalanine and tryptophan, cell contents increased linearly with external concentrations in hypotonic media, whereas in isotonic media only a slight rise was observed. The two anionic amino acids, glutamate and aspartate, were also increased linearly with their external concentrations in hypotonic media, but the changes were lower than those found for neutral amino acids. The presence of a negative membrane potential was responsible for this behavior since, using a K+ hypotonic medium which clamps the potential to zero, the glutamate content was found to increase linearly with an amplitude similar to the one observed for neutral amino acid. When external concentrations of two cationic amino acids, arginine and lysine, were increased in hypotonic media, only a small change, similar to that in isotonic media, was observed. These results indicate that a diffusion process for neutral and anionic amino acids is activated by a volume increase and it is suggested that an anion channel is involved.

Activation of amino acid diffusion by a volume increase in cultured kidney (MDCK) cells.

During volume regulation in hypotonic media, glial cells release a large portion of their amino acids These amino acid losses appear to be mediated by a diffusion type of transport and a swelling-activated chloride channel seems to be involved The objective of this project was to provide direct evidence that amino acids could diffuse through a Cl− channel Using a human glial cell line, Cl− currents activated in hypotonic media were measured in whole-cell patch clamp To measure the currents produced by amino acids, it was necessary to increase the pH of external solutions to basic values reaching 96 and 100 to raise the concentration of the anionic form of these amino acids Introducing external hypotonic media containing high concentrations of amino acids, like glycine, taurine, glutamine and glutamate, it was possible to measure their respective current-voltage curves with NMDG-Cl-filled pipettes From the reversal potentials, their permeability ratios with respect to chloride were determined It was found that the low molecular weight amino acids, like glycine, were most permeant, while the larger ones, like glutamine, had a lower permeability with respect to chloride The amino acids with two carboxyl groups, like glutamate, had a much lower permeability ratio The reversal potentials for some metabolites, like lactate and malate were also measured for comparison These results demonstrate that amino acids can diffuse through anion channels and that activation of these channels in pathological conditions could be at least partly responsible for the observed increase in external amino acids

Amino acid current through anion channels in cultured human glial cells.

The membrane potentials and resistances of L cells were measured using a standard electrophysiological technique. The values obtained in physiological media were around--15 mV and 37Momega, respectively. Almost all the large nondividing L cells (giant L cells) showed spontaneous oscillations of the membrane potential between around -15 and -40 mV. Application of an appropriate electrical or mechanical stimulus was also capable of eliciting responses but such were usually induced only once. The total membrane conductance increased significantly and in parallel with such a hyperpolarizing response. Cooling of the cells and application of metabolic inhibitors to the cells completely blocked the spontaneous oscillation despite the fact that the electrically induced hyperpolarizing response remained. Intracellular K+, Na+ and Cl- concentrations were measured by means of a flame photometer and a chloridometer, and the equilibrium potential for each ion was estimated.

Oscillations of membrane potential in L cells. I. Basic characteristics.

Cell-attached and inside-out patch-clamp experiments (O.P. Hamill et al.,Pfluegers Arch.
391: 85–100, 1981) were undertaken in order to characterize the molecular mechanisms responsible for the calcium-dependent potassium permeability observed in HeLa cancer cells. Our result essentially indicate that the HeLa cell external membrane contains potassium channels whose activity can be triggered within an internal calcium concentration range of 0.1 to 1 μm. This particular channel was found to behave as an inward rectifier in symmetrical 200mm KCl with a conductance of 50 and 10 pS at large negative and large positive membrane potentials, respectively.I/V curves were also measured in 10, 20, 75, 200 and 300mm KCl and the data interpreted in terms of a one-site-two-barrier model. The channel activity appeared to be nearly voltage independent within the voltage range −100 to +100mV, an increase ofP
o, the open channel probability, being observed at large negative potentials only. In addition, the results obtained from inside-out experiments on the relationship betweenP
o and the cytoplasmic freecalcium concentration have led to conclude that four calcium ions are probably required in order to open the channel. In this regard it was found that an increase of the internal free-calcium level affects more the number of channel openings per second than the actual channel mean lifetime. Finally, it is concluded following a time interval distribution analysis, that this particular channel has at least three closed states and two open states.

Single-channel analysis of the potassium permeability in HeLa cancer cells: evidence for a calcium-activated potassium channel of small unitary conductance.

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