Avi Ring

Ca2+ response of rat mesangial cells to ATP analogues

The aim of this investigation was to characterise the effects of ATP analogues and UTP on the single cell intracellular Ca2+ concentration ([Ca2+]i) in cultured rat mesangial cells. Typically, there were two phases in the Ca2+ response to the agonists, an initial fast transient peak and a subsequent slower decline, or plateau, phase. For the peak amplitude in [Ca2+]i the agonists had about equal effect. But when taking in consideration the percentage of responding cells and the integrated Ca2+ response over 1 min, the order of efficacy of nucleotide agonists (100 microM) was UTP = ATP > ATPgammaS > ADP = 2MeS-ATP (2-methylthio-ATP). Adenosine, AMP and beta,gamma-Me-ATP (100 microM) had no effect. Suramine (100 microM) and reactive blue (50 microM) decreased the number of responding cells. Removing Ca2+ from the bath diminished neither the peak in [Ca2+]i nor the percentage of responding cells, but the average [Ca2+]i increase in 1 min was significantly reduced. The results indicate that P2Y2 receptors are present in rat mesangial cells but it cannot be excluded that there are receptors distinct from P2Y2 which also mediate a rise in [Ca2+]i.

Calcium‐activated chloride fluxes in cultured NCL‐SG3 sweat gland cells

The dependence of chloride permeability of the human sweat gland cell line NCL‐SG3 cell line on cytosolic free calcium ([Ca2+]i) was investigated. X‐ray microanalysis, fura‐2 fluorescence and patch clamp methodology were used. Carbachol and A23187 decreased cellular Cl and K for cells grown on permeable supports, but carbachol had no effect on cells grown on impermeable supports. In perforated patch experiments with impermeable supports, ATP and calcium ionophores increased the inward current (ic) whereas carbachol had no effect. ic was unaffected by cation channel blockers or removal of extracellular Na+ but was blocked by chloride channel blockers. Lowering bath Ca2+ decreased ic. On raising bath Ca2+ ic and [Ca2+]i responded with a transient rise which was not blocked by La3+ or D‐600. La3+, but not D‐600, blocked the entry of Mn2+. K+‐depolarization and Bay‐K‐8644 had little effect on [Ca2+]i. The rise in [Ca2+]i may be mediated primarily via depletion operated Ca2+‐channels. Irrespective of substrate NCL‐SG3 cells have a chloride permeability which depends on [Ca2+]i.

Modulation of gramicidin A open channel lifetime by ion occupancy

The hypothesis that the gramicidin A channel stability depends on the level of ion occupancy of the channel was used to derive a mathematical model relating channel lifetime to channel occupancy. Eyring barrier permeation models were examined for their ability to fit the zero-voltage conductance, current-voltage, as well as lifetime data. The simplest permeation model required to explain the major features of the experimental data consists of three barriers and four sites (3B4S) with a maximum of two ions occupying the channel. The average lifetime of the channel was calculated from the barrier model by assuming the closing rate constant to be proportional to the probability of the internal channel sites being empty. The link between permeation and lifetime has as its single parameter the experimentally determined averaged lifetime of gramicidin A channels in the limit of infinitely dilute solutions and has therefore no adjustable parameters. This simple assumption that one or more ions inside the channel completely stabilize the dimer conformation is successful in explaining the experimental data considering the fact that this model for stabilization is independent of ion species and configurational occupancy. The model is used to examine, by comparison with experimental data, the asymmetrical voltage dependence of the lifetime in asymmetrical solutions, the effects of blockers, and the effects of elevated osmotic pressure.

THE INFLUENCE OF HYPEROSMOTIC STRESS ON THE BLOOD-RETINAL BARRIER EFFECTS ON THE ELECTRORETINOGRAM

Electroretinographic recordings were obtained from cat eyes before and after an intracarotid injection of a hypertonic NaCl‐solution. It was found that after the injection the c‐wave decreased in amplitude or disappeared, but reappeared within a period of four to six hours, suggesting that there is a reversible opening of the tight junctions following osmotic stress.

Measurement of channel lifetime in artificial lipid membranes: dimerization kinetics of gramicidin a

Abstract The autocorrelation function (acf) method has been used to evaluate the lifetime of gramicidin A channels in lipid bilayers. The accuracy of the method was evaluated using computer simulation techniques and experimentally by comparing the acf method with methods to extract the lifetime from single-channel recordings. It was found that a correct evaluation of the lifetime requires registrations for a period of 500-1000 lifetimes and that improving the accuracy of the method by increasing the event activity is not as effective as increasing the total registration time. The effects of noise and filters are examined and a method for constructing the survival-plot, in the case of overlapping channels, is presented. Experimental data for the dependence of lifetime and conductivity on salt activity are presented for CsCl, KCl and LiCl. The general finding is that the lifetime increases with ion concentration and for CsCl and KCl it passes through a maximum at about 1 M. A simple model relating the channel unit conductance and lifetime to the ion occupancy of the channel is described, and the best fit of the model to the data is presented.

Impedance of the frog gastric mucosa, the Teorell-Wersäll effect.

The small signal impedance of frog gastric mucosa has been measured from 1 Hz to 500 kHz. The results obtained in Ringers solution have been compared with the effect of replacing sodium with potassium on the nutrient side of the mucosa. The substitution to potassium reduces the membrane resistance and changes the impedance locus from a flat to a semicircular shape. We have examined this effect, the Teorell-Wersäll effect, in relation to changes in membrane permeabilities. The experimental data are consistent with a model based on the electrical behaviour of transmission cables (the geometric requirement for this may be found in the gastric duct) and the observed electrical properties can be predicted from an increase in conductance of the secretory membrane when replacing sodium with potassium.

Recording and control of ion channel currents in bilayer and patch-clamp experiments

A program was developed for the control and recording of ion-channel activity in lipid bilayers and cell membranes. The software runs on a PC under DOS. Stimulation waveforms may be applied simultaneously with recording at a sample rate of 10 kHz and at 12-bit resolution. Comments may be stored with the recordings, also during timed repetitive pulse applications. The time critical parts and graphics are written in C and assembler whereas the I/O and control is written in Quick-Basic 4.5 (QB). A nested 16-level macro facility allows the use of complex protocols. Array-buffer arithmetic is supported. There is mouse support for cursor measurements and parabolic offset subtractions. DOS applications such as editing macro files may be executed without halting the program. The program runs in the QB interactive environment with the foreign language routines in a library and is open to customization. The library of drawing subroutines support up to 1024 x 768 resolution.