Figure 1. NHE1 expression and buffer capacities in MDCK-F cells.

A, the presence of NHE activity in NHE-deficient (NHE⁻, black curve) and parent MDCK-F cells (NHE⁺, grey curve) is tested with the NH₄⁺-prepulse technique. After superfusing cells with Hepes-buffered Ringer solution (con), cells are alkalinized by a brief pulse of 20 mm NH₄Cl (NH₄⁺). Removal of NH₄Cl in the absence of extracellular Na⁺ (Na⁺-free) elicits a marked intracellular acidosis. Intracellular pH (pH_i) recovers in parent MDCK-F cells upon the re-addition of extracellular Na⁺ (con). Na⁺-dependent recovery of pH_i is virtually absent in NHE-deficient MDCK-F cells, when they are superfused with Hepes-buffered Ringer solution. B, immunoblot of a crude membrane protein preparation of normal (NHE⁺) and NHE-deficient (NHE⁻) MDCK-F cells. Equal amount of proteins were loaded. The NHE1 antibody detects the mature NHE1 protein in both cell types (arrow). However, it is approximately three times more abundant in normal than in NHE-deficient MDCK-F cells. C, original recordings of intracellular pH for the determination of the intrinsic buffering capacity. NHE-deficient MDCK-F cells (NHE⁻; black curve); parental MDCK-F cells (NHE⁺; grey curve). D, buffering capacity of NHE-deficient (NHE⁻, black curve) and normal MDCK-F cells (NHE⁺, grey curve).