Proton Pump Activity of Mitochondria-rich Cells The Interpretation of External Proton-concentration Gradients

We have hypothesized that a major role of the apical H 1 -pump in mitochondria-rich (MR) cells of amphibian skin is to energize active uptake of Cl 2 via an apical Cl 2 /HCO 3 2 -exchanger. The activity of the H 1 pump was studied by monitoring mucosal [H 1 ]-profiles with a pH-sensitive microelectrode. With gluconate as mucosal anion, pH adjacent to the cornified cell layer was 0.98 6 0.07 (mean 6 SEM) pH-units below that of the lightly buffered bulk solution (pH 5 7.40). The average distance at which the pH-gradient is dissipated was 382 6 18 m m, corresponding to an estimated “unstirred layer” thickness of 329 6 29 m m. Mucosal acidification was dependent on serosal p CO 2 , and abolished after depression of cellular energy metabolism, confirming that mucosal acidification results from active transport of H 1 . The [H 1 ] was practically similar adjacent to all cells and independent of whether the microelectrode tip was positioned near an MR-cell or a principal cell. To evaluate [H 1 ]-profiles created by a multitude of MR-cells, a mathematical model is proposed which assumes that the H 1 distribution is governed by steady diffusion from a number of point sources defining a set of particular solutions to Laplace’s equation. Model calculations predicted that with a physiological density of MR cells, the [H 1 ] profile would be governed by so many sources that their individual contributions could not be experimentally resolved. The flux equation was integrated to provide a general mathematical expression for an external standing [H 1 ]–gradient in the unstirred layer. This case was treated as free diffusion of protons and proton-loaded buffer molecules carrying away the protons extruded by the pump into the unstirred layer; the expression derived was used for estimating stationary proton-fluxes. The external [H 1 ]-gradient depended on the mucosal anion such as to indicate that base (HCO ) is excreted in exchange not only for Cl 2 , but also for Br 2 and I 2 , indicating that the active fluxes of these anions can be attributed to mitochondria-rich cells. key words: active Cl 2 transport • rheogenic H 1 pump • unstirred layer • mathematics of diffusion • proton concentration-profiles outside epithelial cells 3 2 i n t r o d u c t i o n In amphibian epidermis, inward fluxes of Na 1 and Cl 2 are coupled to cellular energy metabolism. Thus, these ions can be transported from a relatively dilute solution into the body fluids. This active uptake of Na 1 and Cl 2 compensates for passive losses via the skin and distal renal epithelia. In the skin, as in collecting ducts and urinary bladder, several cell types are involved in the transcellular exchange of ions (reviewed by Steinmetz, 1986; Frömter, 1988; Larsen, 1991; Al-Awqati, 1992; Harvey, 1992; Kaissling and Stanton, 1992; Harvey, 1995; Brown and Stow, 1996; Gluck et. al., 1996). The most abundant cell type, the principal cells, constitutes the major pathway for active uptake of Na 1 accomplished by basolateral Na 1 -K 1 ATPases in series with apical Na 1 channels. The other cell type, mitochondria-rich (MR) 1 or intercalated cells, can be divided into subpopulations serving different body functions. A common feature of MR cells is the expression of proton pumps. The H 1 pump activity and the anion fluxes through a coexpressed Cl 2 /HCO exchanger are coupled to one another via the activity of a cellular carbonic anhydrase. In this way the H 1 -pump can energize the elimination of a body acid-load ( a -phenotype), the elimination of a body base-load ( b -phenotype), or the rheogenic active uptake of chloride ( g -phenotype). The present investigation continues our studies on the active transport of Cl 2 by the skin which seems to be brought about by a proton pump-driven uptake of Cl 2 in exchange for cellular HCO (Larsen et. al., 1992; Larsen et al., 1996). The activity of the apical H 1 -pump can be studied by recording the mucosal [H 1 ]-gradient in the unstirred layer above the isolated epithelium. This method has been employed to localize functionally expressed rheogenic H 1 -pumps to the apical membrane of MR-cells (Harvey, 1992; Larsen et al., 1992). In areas with few MR-cells, we applied the approximation of radial diffusion from point sources. In the present study we have elaborated a more general experimental and theoretical description of [H 1 ]-profiles generated by a large 3 2