Selective inhibition of Cl(-) conductance in toad skin by blockers of Cl(-) channels and transporters.

We compared the effects exerted by two classes of Cl(-) transport inhibitors on a Cl(-)-selective, passive anion transport route across the skin of Bufo viridis, the conductance (G(Cl)) of which can be activated by transepithelial voltage perturbation or high cAMP at short circuit. Inhibitors of antiporters (erythrosine, eosin) or cotransporters (furosemide) reduced voltage-activated G(Cl) with IC(50) of 6 +/- 1, 54 +/- 12, and 607 +/- 125 microM, respectively; they had no effect on the cAMP-induced G(Cl). The voltage for half-maximal activation of G(Cl) (V(50)) increased compared with controls, but effects on the maximal G(Cl) at more positive clamp potentials were small. Cl(-) channel blockers from the diphenylamino-2-carboxylic acid (DPC) family [dichloro-DPC, niflumic acid, flufenamic acid, and 5-nitro-2-(3-phenylpropylamino)benzoic acid] reduced the voltage-activated G(Cl) with IC(50) of 8.3 +/- 1.2, 10.5 +/- 0.6, 16.5 +/- 3.4, and 36.5 +/- 11.4 microM, respectively, and also inhibited the cAMP-induced G(Cl), albeit with slightly larger IC(50). V(50) was not significantly changed compared with controls; the maximal G(Cl) was strongly reduced. We conclude that the pathway for Cl(-) is composed of the conductive pore proper, which is blocked by the derivatives of DPC, and a separate, voltage-sensitive regulator, which is influenced by blockers of cotransporters or antiporters. This influence is partly overcome by increasing the clamp potential and removed by high concentrations of cAMP, which renders the pathway insensitive to voltage.

Measurement of pH gradients using an ion-sensitive vibrating probe technique (IP).

The direct measurement and quantification of proton transport in biological structures are below the detection limit of stationary pH-sensitive microelectrodes. We have thus used a more sensitive system to detect and quantify these small pH gradients: a proton-sensitive vibrating ion probe technique. This technique decreases the noise of the system to less than +/- 15 microV, equivalent to pH gradients below 0.0005 pH units, and can be used to measure pH gradients even in the presence of moderate buffer concentrations. At physiological pH the detection limit, analysed with artificial proton sources, is in the range of 5 pmol.s-1.cm-2. Computer simulation indicates that the spatial resolution is sufficient to localize individual proton sources less than 30 microns apart.

Localizing transepithelial conductive pathways using a vibrating voltage probe.

A model simulation of the electrical field distribution for the voltage-activated C1- current across amphibian skin has been carried out for two potential pathways, i.e. transcellularly, through the mitochondria-rich cells (MRCs), or paracellularly, across the tight junctions (TJs) between the outermost living cell layer. The calculations are based on the mean density of MRCs and the typical dimensions of stratum granulosum cells of amphibian skin. It is demonstrated that current flow across MRCs would be detectable by scanning with the vibrating voltage probe in the extracellular space above the epithelium, whereas accurate representation of current flow through the TJs cannot be obtained using a probe of the present design. The experimental data indicate that field patterns corresponding to an MRC origin for the C1- current are never observed. It is concluded that the voltage-activated C1- conductance is localized to ion-selective structures in the paracellular pathway which may be regulated by the TJs.

Mitochondria-rich cells and voltage-activated chloride current in toad skin epithelium: analysis with the scanning vibrating electrode technique.

The pathway for the voltage-activated chloride current across isolated toad skin was analyzed using a scanning 2D-vibrating voltage probe technique, which permits discrimination of local current peaks if their origins are more than 50 microns apart. The epithelium was separated from the corial connective tissue after enzymatic digestion with collagenase. Cl- current was activated by voltage clamping the transepithelial potential to 60-100 mV, serosa positive. Activated inward current was between 85 and 450 microA/cm2. In more than 25 tissue areas of 150 x 100 microns from 10 animals, which were automatically scanned with the vibrating probe, between 0 and 4 peaks of elevated local current (up to 800 microA/cm2) could be identified in individual fields. The density of current peaks, which were generally located at sites of mitochondria-rich (MR) cells, was less than 10% of the density of microscopically identified MR cells. The total current across individual sites of elevated conductance was 3.9 +/- 0.6 nA. Considering the density of peaks, they account for 17 +/- 2.5% of the applied transepithelial clamping current. The time course of current activation over previously identified conductive sites was in most cases unrelated to that of the total transepithelial current. Moreover, initially active sites could spontaneously inactivate. The results indicate that detection of elevated current above some MR cells is not sufficient to verify these cells as the pathway for transepithelial voltage-activated Cl- current. Since the major fraction of activated current is apparently not associated with a route through MR cells, channel-like structures in the tight junctions of the paracellular pathway must be considered as an alternative possibility. Current peaks over MR cells could be due to high density of such sites in tight junctions between MR and surrounding principal cells. Improvement of the spatial resolution of the vibrating probe is required to verify this view.