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)-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 Km for Na+ of the intracellular pH recovery mechanism was 8.3 mmol · l-1, and the rate constant at Kmax 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.
Original language | English (US) |
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Pages (from-to) | 37-45 |
Number of pages | 9 |
Journal | Journal of Comparative Physiology B: Biochemical, Systemic, and Environmental Physiology |
Volume | 166 |
Issue number | 1 |
State | Published - Apr 1 1996 |
Keywords
- Epithelial cells
- Fish gills
- Intracellular pH
- Na-H exchange
- Trout, Oncorhynchus
ASJC Scopus subject areas
- Animal Science and Zoology
- Physiology
- Environmental Science(all)
- Physiology (medical)