Na/H exchange in cultured epithelial cells from fish gills

P. Pärt, C. M. Wood

Research output: Contribution to journalArticlepeer-review

34 Scopus citations

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 languageEnglish (US)
Pages (from-to)37-45
Number of pages9
JournalJournal of Comparative Physiology B: Biochemical, Systemic, and Environmental Physiology
Volume166
Issue number1
StatePublished - 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)

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