Role of proton pump of mitochondria-rich cells for active transport of chloride ions in toad skin epithelium

Abstract

1. Active Cl- currents were studied in short-circuited toad skin epithelium in which the passive voltage-activated Cl- current is zero. Under visual control double-barrelled microelectrodes were used for impaling principal cells from the serosal side, or for measuring the pH profile in the solution bathing the apical border. 2. The net inward (active) 36Cl- flux of 27 +/- 8 pmol s-1 cm-2 (16) (mean +/- S.E.M (number of observation)) was abolished by 2 mM-CN- (6.3 +/- 3.5 pmol s-1 cm-2 (8)). The active flux was maintained in the absence of active Na+ transport when the latter was eliminated by either 100 microM-mucosal amiloride, replacement of mucosal Na+ with K+, or by 3 mM-serosal ouabain. 3. In Ringer solution buffered by 24 mM-HCO3- -5% CO2 mucosal amiloride reversed the short circuit current (ISC). The outward ISC was maintained when gluconate replaced mucosal Cl-, and it was reversibly reduced in CO2-free 5 mM-Tris-buffered Ringer solution (pH = 7.40) or by the proton pump inhibitor oligomycin. These observations indicate that the source of the outward ISC is an apical proton pump. 4. Amiloride caused principal cells to hyperpolarize from a basolateral membrane potential, Vb, of -73 +/- 3 (22) to -93 +/- 1 mV (26), and superfusion with CO2-free Tris-buffered Ringer solution induced a further hyperpolarization (Vb = -101 +/- 1 mV (26)) which could be blocked by Ba2+. The CO2-sensitive current changes were null at Vb = EK (potassium reversal potential, -106 +/- 2 mV (55)) implying that they are carried by K+ channels in the basolateral membrane. Such a response cannot account for the inhibition of the outward ISC which by default seems to be located to mitochondria-rich (MR) cells. 5. In the absence of mucosal Cl- a pH gradient was built up above MR cells with pH = 7.02 +/- 0.04 (42) and pH increasing to 7.37 +/- 0.02 (10) above principal cells (pH = 7.40 in bulk solution buffered by 0.1 mM-Tris). This observation localizes a proton pump to the apical membrane of MR cells. Using the integrated diffusion equation it was shown that the measured external pH gradient would account within an order of magnitude for measured currents. 6. Standing gradients of protons were eliminated in the presence of mucosal Cl- suggesting that active uptake of Cl- is associated with the exit of base equivalents across the apical membrane of MR cells.(ABSTRACT TRUNCATED AT 400 WORDS)