H+ ATPase and Cl− Interaction in Regulation of MDCK Cell pH
MDCK cells display several acid-base transport systems found in intercalated cells, such as Na+-H+ exchange, H+–K+ ATPase and Cl−/HCO− 3 exchange. In this work we studied the functional activity of a vacuolar H+-ATPase in MDCK cells and its chloride dependence. We measured intracellular pH (pHi) in monolayers grown on glass cover slips utilizing the pH sensitive probe BCECF. To analyze the functional activity of the H+ transporters we observed the intracellular alkalinization in response to an acute acid load due to a 20 mm NH+ 4 pulse, and calculated the initial rate of pHi recovery (dpHi/dt). The cells have a basal pHi of 7.17 ± 0.01 (n= 23) and control dpHi/dt of 0.121 ± 0.006 (n= 23) pHi units/min. This pHi recovery rate is markedly decreased when Na+ was removed, to 0.069 ± 0.004 (n= 16). It was further reduced to 0.042 ± 0.005 (n= 12) when concanamycin 4.6 × 10−8 m (a specific inhibitor of the vacuolar H+-ATPase) was added to the zero Na+ solution. When using a solution with zero Na+, low K+ (0.5 mm) plus concanamycin, pHi recovery fell again, significantly, to 0.023 ± 0.006 (n= 14) as expected in the presence of a H+–K+-ATPase. This result was confirmed by the use of 5 × 10−5 m Schering 28080. The Na+ independent pHi recovery was significantly reduced from 0.069 ± 0.004 to 0.042 ± 0.004 (n= 12) when NPPB 10−5 m (a specific blocker of Cl− channels in renal tubules) was utilized. When the cells were preincubated in 0 Cl−/normal Na+ solution for 8 min. before the ammonium pulse, the pHi recovery fell from 0.069 ± 0.004 to 0.041 ± 0.007 (n= 12) in a Na+ and Cl− free solution. From these results we conclude that: (i) MDCK cells have two Na+-independent mechanisms of pHi recovery, a concanamycin sensitive H+-ATPase and a K+ dependent, Schering 28080 sensitive H+–K+ ATPase; and, (ii) pHi recovery in Na+-free medium depends on the presence of a chloride current which can be blocked by NPPB and impaired by preincubation in Cl−–free medium. This finding supports a role for chloride in the function of the H+ ATPase, which might be electrical shunting or a biochemical interaction.
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