Na⁺-independent proton secretion in MDCK–C11 cells

Abstract.

In this work we studied the proton secretion mechanisms in recently cloned MDCK–C11 cells. We measured intracellular pH (pHi) in monolayers grown on permeable filters, using the pH-sensitive probe BCECF and an inverted epifluorescence microscope. The cells have a basal pHi of 7.20±0.01 (n=136) and after an acid-releasing NH₄Cl pulse pHi recovered at a rate (dpHi/dt) of 0.167±0.006 pH units/ per minute (n=20). This rate decreased significantly when Na⁺ was removed from both cell surfaces, and was further reduced when they were both perfused with a solution containing no Na⁺ and K⁺. pHi recovery fell again in the presence of concanamycin (at a concentration of 4.6×10^–8 M; a specific inhibitor of the vacuolar H⁺-ATPase). When Na⁺ was removed from the apical or the basolateral side, pHi recovery (in pH units per minute) was significantly reduced to 0.099±0.008 (n=11) and 0.086±0.01 (n=10), respectively. The Na⁺-independent mechanism of pHi recovery was significantly inhibited by the presence of 5×10^–5 M Schering 28080 (an inhibitor of the H⁺-K⁺-ATPase) at the apical side (0.065±0.01 versus 0.099±0.008 pH units per minute, P<0.05), but not at the basolateral side (0.072±0.01 versus 0.086±0.01 pH units per minute). On the other hand, concanamycin inhibited the Na⁺-independent pHi recovery when applied apically (0.0304±0.005 pH units per minute, n=7) and basolaterally (0.025±0.004 pH units per minute, n=7). From these results we conclude that monolayers of MDCK–C11 cells have a Na⁺/H⁺ exchanger and a concanamycin-sensitive H⁺-ATPase on their apical and basolateral membranes; and a K⁺-dependent, Schering 28080-sensitive H⁺-K⁺-ATPase on their apical side.