Role of mitochondria-rich cells for passive chloride transport, with a discussion of Ussing's contribution to our understanding of shunt pathways in epithelia

A non-invasive method is applied for studying ion transport by single isolated epidermal mitochondria-rich (MR) cells. MR cells of toad skin (Bufo bufo) were prepared by trypsin (or pronase) treatment of the isolated epithelium bathed in Ca2+-free Ringer. Glass pipettes were pulled and heat- polished to obtain a tip of 2-4 mm with parallel walls and low tip resistances. The neck of an MR-cell was sucked into the tip of the pipette for being 'clamped mechanically' by the heat-polished glass wall. In this configuration the apical cell membrane faces the pipette solution while the major neck region and the cell body are in the electrically grounded bath. With Ringer in bath and pipette, transcellular voltage clamp currents were composed of an ohmic (I(leak)) and a dynamic (I(dynamic)) component. The dynamic component was studied by stepping the transcellular potential (Vp) from a holding value of +50 mV to the hyperpolarizing region (50 > Vp > or = -100 mV). The steady state I(dynamic)-Vp relationship was strongly outward rectified with I(dynamic) being practically zero for Vp > 0 mV. At Vp = -100 mV, MR cells isolated by trypsin or pronase generated a steady-state I(dynamic) of,-2.72 +/- 0.40 nA/cell (N = 21 MR cells). Continuous superfusion of the MR cell during recording increased the current to -7.99 +/- 1.48 nA/cell (N = 10 MR cells). The time course of the reversible activation of G(dynamic) varied among cells, but was usually sigmoid with T1/2 decreasing with Vp (-25 > or = Vp > or = -100 mV). T1/2 was in the order of 10 sec at Vp = -100 mV. The single-MR-cell currents recorded in this study are fully compatible with Cl- currents estimated by relating density of MR cells to transepithelial ICl or by measurements with the self-referencing ('vibrating') probe technique. In the discussion, Ussing's work on epithelial shunt pathways is considered. His thinking and experiments leading to his theory of isotonic transport in leaky epithelia is emphasized. It is our thesis that the understanding of the physiology of epithelia owes as much to Ussing's studies of shunt pathways as to his studies of the active sodium pathway.