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.