Membrane potentials and intracellular Cl− activity of toad skin epithelium in relation to activation and deactivation of the transepithelial Cl− conductance

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Abstract
The potential dependence of unidirectional36Cl fluxes through toad skin revealed activation of a conductive pathway in the physiological region of transepithelial potentials. Activation of the conductance was dependent on the presence of Cl− or Br− in the external bathing solution, but was independent of whether the external bath was NaCl-Ringer's, NaCl-Ringer's with amiloride, KCl-Ringer's or choline Cl-Ringer's To partition the routes of the conductive Cl− ion flow, we measured in the isolated epithelium with double-barrelled microelectrodes apical membrane potentialV a , and intracellular Cl− activity,a Cl c , of the principal cells indentified by differential interference contrast microscopy. Under short-circuit conditionsI sc=27.0±2.0 μA/cm2, with NaCl-Ringer's bathing both surfaces,V a was −67.9±3.8mV (mean ±se,n=24, six preparations) anda Cl c was 18.0±0.9mM in skins from animals adapted to distilled water. BothV a anda Cl a were found to be positively correlated withI sc (r=0.66 andr=0.70, respectively). In eight epithelia from animals adapted to dry milieu/tap waterV a anda Cl c were measured with KCl Ringer's on the outside during activation and deactivation of the transepithelial Cl− conductance (G Cl) by voltage clamping the transepithelial potential (V) at 40 mV (mucosa positive) and −100 mV. AtV=40 mV; i.e. whenG Cl was deactivated,V a was −70.1±5.0 mV (n=15, eight preparations) anda Cl c was 40.0±3.8mm. The fractional apical membrane resistance (fR a) was 0.69±0.03. Clamping toV=−100 mV led to an instantaneous change ofV a to 31.3±5.6 mV (cell interior positive with respect to the mucosal bath), whereas neithera Cl c norfR a changed significantly within a 2 to 5-min period during whichG Cl increased by 1.19±0.10 mS/cm2. WhenV was stepped back to 40 mV,V a instantaneously shifted to −67.8±3.9 mV whilea Cl c andfR a remained constant during deactivation ofG Cl. Similar results were obtained in epithelia impaled from the serosal side. In 12 skins from animals adapted to either tap water or distilled water the density of mitochondria-rich (D MRC) cells was estimated and correlated with the Cl current (I Cl though the fully activated (V=−100mV) Cl− conductance). A highly significant correlation was revealed (r=−0.96) with a slope of −2.6 nA/m.r. (mitochondria-rich cell and an I-axis intercept not significantly different from zero. In summary, the voltage-dependent Cl currents were not reflected infR a anda Cl a of the principal cells but showed a correlation with the m.r. cell density. We conclude that the pricipal cells do not contribute significantly to the voltage-dependent Cl conductance.