Na/H exchange in cultured epithelial cells from fish gills

Using primary cultures of gill pavement cells from freshwater rainbow trout, a method is described for achieving confluent monolayers of the cells on glass coverslips. A continuous record of intracellular pH was obtained by loading the cells with the pH-sensitive flourescent dye 2′,7′-bis(2-carboxyethyl)-5(6)-carboxy-fluorescein and mounting the coverslips in the flow-through cuvette of a spectrofluorimeter. Experiments were performed in HEPES-buffered media nominally free of HCO₃^-. Resting intracellular pH (7.43 at extracellular pH = 7.70) was insensitive to the removal of Cl^- or the application of 4-acetamido-4′-isothiocyanatostilbene-2,2′-disulfonic acid (0.1 mmol · l^-1 ), but fell by about 0.3 units when Na⁺ was removed or in the presence of amiloride (0.2 mmol · l^-1). Exposure to elevated ammonia ("ammonia prepulse"; 30 mmol · l^-1 as NH₄Cl for 6-9 min) produced an increase in intracellular pH (to about 8.1) followed by a slow decay, and washout of the pulse caused intracellular pH to fall to about 6.5. Intracellular non-HCO₃^- buffer capacity was about 13.4 slykes. Rapid recovery of intracellular pH from intracellular acidosis induced by ammonia prepulse was inhibited more than 80% in Na⁺-free conditions or in the presence of amiloride (0.2 mmol · l^-1). Neither bafilomycin A₁ (3 μmol · l^-1) nor Cl removal altered the intracellular pH recovery rate. The K_m for Na⁺ of the intracellular pH recovery mechanism was 8.3 mmol · l^-1, and the rate constant at K_max was 0.008 · s^-1 (equivalent to 5.60 mmol H⁺ · l^-1 cell water · min^-1), which was achieved at external Na⁺ levels from 25 to 140mmol · l^-1. We conclude that intracellular pH in cultured gill pavement cells in HEPES-buffered, HCO₃^- -free media, both at rest and during acidosis, is regulated by a Na⁺/H⁺ antiport and not by anion-dependent mechanisms or a vacuolar H⁺ -ATPase.