ISSN:
1432-136X
Keywords:
Fish gills
;
Epithelial cells
;
Intracellular pH
;
Na−H exchange
;
Trout, Oncorhynchus
Source:
Springer Online Journal Archives 1860-2000
Topics:
Biology
,
Medicine
Notes:
Abstract 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)-carboxyfluorescein and mounting the coverslips in the flowthrough 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-HCO 3 − 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 V 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 140 mmol·l−1. We conclude that intracellular pH in cultured gill pavement cells in HEPES-buffered, HCO 3 − -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.
Type of Medium:
Electronic Resource
URL:
http://dx.doi.org/10.1007/BF00264637
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