Springer Nature is making SARS-CoV-2 and COVID-19 research free. View research | View latest news | Sign up for updates

Na/H exchange in cultured epithelial cells from fish gills

  • 80 Accesses

  • 15 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)-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.

This is a preview of subscription content, log in to check access.

Abbreviations

BCECF :

2′,7′-bis(2-carboxyethyl)-5(6)-carboxy-fluorescein

BCECF/AM :

2′,7′-bis(2-carboxyethyl)-5(6)-carboxy-fluorescein, acetoxymethylester

Cholin-Cl :

choline chloride

DMSO :

dimethyl sulfoxide

EDTA :

ethylene diamine tetra-acetic acid

FBS :

foetal bovine serum

H + -ATPase :

Proton-dependent adenosine triphosphatase

HEPES :

N-[2-hydroxyethyl]piperazine-N′[2-ethanesulfonic acid]

pH i :

intracellular pH

pH e :

extracellular pH

PBS :

phosphate-buffered saline

SITS :

4-acetamido-4′-isothiocyanatostilbene-2,2′-disulfonic acid

References

  1. Avella M, Bornancin M (1989) A new analysis of ammonia and sodium transport through the gills of freshwater rainbow trout (Salmo gairdneri). J Exp Biol 142:155–175

  2. Boron WF, Weer P de (1976) Intracellular pH transients in squid giant axon caused by CO2, NH3 and metabolic inhibitors. J Gen Physiol 67:91–112

  3. Bowman EJ, Siebers A, Altendorf K (1988) Bafilomycins: a class of inhibitors of membrane ATPase from microorganisms, animal cells, and plant cells. Proc Natl Acad Sci USA 85: 7972–7976

  4. Boyarsky G, Ganz MB, Sterzel RB, Boron WF (1988) pH regulation in single glomerular mesangial cells. I. Acid extrusion in absence and presence of HCO 3 . Am J Physiol 255:C844-C856

  5. Calonge ML, Molina MT, Ilundian A (1992) Chloride-base exchange and cellular pH regulation in enterocytes isolated from chick small intestine. Biochim Biophys Acta 1103: 45–50

  6. Cameron JN, Heisler N (1983) Studies of ammonia in the rainbow trout: physico-chemical parameters, acid-base behaviour and respiratory clearance. N Exp Biol 105:107–125

  7. Chaillet JR, Amsler K, Boron WF (1986) Optical measurements of intracellular pH in single LLC-PK1 cells: demonstration of Cl−HCO3 exchange. Proc Natl Acad Sci USA 83:522–526

  8. Chuard F, Durand J (1992) Coupling between the intracellular pH and the active transport of sodium in an epithelial cell line from Xenopus laevis. Comp Biochem Physiol 102A:7–14

  9. Ehrenfeld J, Garcia-Romeu F, Harvey BJ (1985) Electrogenic active proton pump in Rana esculenta skin and its role in sodium transport. J Physiol (London) 359:331–355

  10. Ehrenfeld J, Lacoste I, Harvey BJ (1989) The key role of the mitochondria-rich cells in Na+ and H+ transport across the frog skin epithelium. Pflügers Arch (Eur J Physiol) 414:59–67

  11. Frelin C, Vigne P, Ladoux A, Lazdunski M (1988) The regulation of intracellular pH in cells from vertebrates. Eur J Biochem 174: 3–14

  12. Goss GG, Perry SF, Wood CM, Laurent PL (1992) Mechanisms of ion and acid-base regulation at the gills of freshwater fish. J Exp Zool 263:143–159

  13. Goss GG, Wood CM (1991) Two-substrate kinetic analysis: a novel approach linking ion and acid-base transport at the gills of freshwater trout, Oncorhynchus mykiss. J Comp Physiol B 161: 635–646

  14. Graber M, Dipola J, Hsiang F, Barry C, Pastoriza E (1991) Intracellular pH in the OK cell: identification of proton conductance and observations on buffering capacity. Am J Physiol 261: C1143-C1153

  15. Harvey BJ (1986) Regulation of intracellular sodium and pH by the electrogenic H+ pump in frog skin. Pflügers Arch (Eur J Physiol) 406:362–366

  16. Ilundain A (1992) Intracellular pH regulation in intestinal and renal epithelial cells. Comp Biochem Physiol 101A:413–424

  17. Justesen NPB, Dall-Larsen T, Klungsöyr L(1993) Proton transport and chloride cells in the gill of rainbow trout (Oncorhynchus mykiss). Can J Fish Aquat Sci 50:988–995

  18. Kirschner LB (1994) Hydromineral metabolism and acid-base regulation in aquatic vertebrates. Handbook of physiology (in press)

  19. Kirschner LB, Greenwald L, Kerstetter TH (1973) Effect of amiloride on sodium transport across body surfaces of freshwater animals. Am J Physiol 224:832–837

  20. Krapf R, Alpern RJ (1993) Cell pH and transepithelial H+/HCO 3 transport in the renal proximal tubule. J Membr Biol 131:1–10

  21. Krogh A (1939) Osmotic Regulation in Aquatic Animals. Cambridge University Press, London

  22. Krayer-Pawlowska D, Helmle-Kolb C, Montrose M, Krapf R, Murer H (1991) Studies on the kinetics of Na+/H+ exchange in OK cells: introduction of a new device for the analysis of polarized transport in cultured epithelia. J Membr Biol. 120:173–183

  23. Lin H, Miller SS (1991) pHi regulation in frog retinal pigment epithelium: two apical membrane mechanisms. Am J Physiol 261:C132-C142

  24. Lin H, Randall DJ (1991) Evidence for the presence of an electrogenic proton pump on the trout gill epithelium. J Exp Biol 161:119–134

  25. Lin H, Randall DJ (1993) H+-ATPase activity in crude homogenates of fish gill tissue: inhibitor sensitivity and environmental and hormonal regulation. J Exp Biol 180:163–174

  26. Lin H, Pfeiffer DC, Vogl AV, Pan J, Randall DJ (1994) Immunolocalization of H+-ATPase in the gill epithelia of rainbow trout. J Exp Biol 195:169–183

  27. Lubman RL, Crandall ED (1992) Regulation of intracellular pH in alveolar epithelial cells. Am J Physiol 262:L1-L14

  28. Madshus IH (1988) Regulation of intracellular pH in eukaryotic cells. Biochem J 250:1–8

  29. Maetz J (1973) Na+/NH 4 + , Na+/H+ exchanges and NH3 movements across the gill of Carassius auratus. J Exp Biol 58:255–275

  30. Maetz J, Garcia-Romeu F (1964) The mechanism of sodium and chloride uptake by gills of a freshwater fish, Carassius auratus: II. Evidence for NH 4 + /Na+ and HCO 3 /Cl-exchanges. J Gen Physiol 47:1209–1227

  31. Marshall WS, Bryson SE (1991) Intracellular pH regulation in trout urinary bladder epithelium: Na+-H+(NH 4 + ) exchange. Am J Physiol 261:R652-R658

  32. Molenaar WH, Tertoolen LGJ, Laat SW de (1984) The regulation of cytoplasmic pH in human fibroblasts. J Biol Chem 259: 7563–7569

  33. Montrose MH, Murer H (1986) Regulation of intracellular pH in LLC-PK1 cells by Na+/H+ exchange. J Membr Biol 93:33–42

  34. Murer M, Hopfer U, Kinne R (1976) Sodium/proton antiport in brush-border-membrane vesicles isolated from rat small intestine and kidney. Biochem J 154:597–604

  35. Nikinmaa M (1992) Membrane transport and control of hemoglobin-oxygen affinity in nucleated erythrocytes. Physiol Rev 72: 301–321

  36. Perry SF, Randall DJ (1981) Effects of amiloride and SITS on branchial ion fluxes in rainbow trout, Salmo gairdneri. J Exp Zool 215:225–228

  37. Potts WTW (1994) Kinetics of sodium uptake in freshwater animals: a comparison of ion-exchange and proton pump hypotheses. Am J Physiol 266:315–320

  38. Pärt P, Norrgren L, Bergström E, Sjöberg P (1993) Primary cultures of epithelial cells from rainbow trout gills. J Exp Biol 175: 219–232

  39. Rink TJ, Tsien RY, Pozzan T (1982) Cytoplasmic pH and free Mg2+ in lymphocytes. J Cell Biol 95:189–196

  40. Rodriguez-Boulan E, Nelson WJ (1989) Morphogenesis of the epithelial cell phenotype. Science 245:718–725

  41. Roos A, Boron WF (1981) Intracellular pH. Physiol Rev 61: 296–434

  42. Vilella S, Guerra L, Helmle KC, Murer H(1992) Characterization of basolateral Na/H exchange (Na/H-1) in MDCK cells. Pflügers Arch (Eur J Physiol) 420:275–281

  43. Walsh PJ (1986) Ionic requirements for intracellular pH regulation in rainbow trout hepatocytes. Am J Physiol 250:R24-R29

  44. Weintraub WH, Machen TE (1989) pH regulation in hepatoma cells: roles for Na−H exchange, Cl−HCO3 exchange and Na−HCO3 cotransport. Am J Physiol 257:G317-G327

  45. Werner G, Hagenmaier H, Drautz H, Baumgartner A, Zähner H (1984) Metabolic products of microorganisms. 224 bafilomycins, a new group of macrolide antibiotics. Production, isolation, chemical structure and biological activity. J Antibiotics 37: 110–117

  46. Williams MR, Duncan G, Croghan PC, Riach R, Webb SF (1992) pH regulation in tissue-cultured bovine lens epithelial cells. J Membr Biol 129:179–187

  47. Wolf K (1963) Physiological salines for fresh-water teleosts. Progr Fish Cult 25:135–140

  48. Wood CM (1991) Branchial ion and acid-base transfer-environmental hyperoxia as a probe. Physiol Zool 64:68–102

  49. Wood CM, LeMoigne J (1991) Intracellular acid-base responses to environmental hyperoxia and normoxic recovery in rainbow trout. Respir Physiol 86:91–113

  50. Wright PA, Wood CM (1985) An analysis of branchial ammonia excretion in the freshwater rainbow trout: effects of environmental pH change and sodium uptake blockade. J Exp Biol 114: 329–353

Download references

Author information

Correspondence to P. Pärt.

Additional information

Communicated by H. Huddart

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Pärt, P., Wood, C.M. Na/H exchange in cultured epithelial cells from fish gills. J Comp Physiol B 166, 37–45 (1996). https://doi.org/10.1007/BF00264637

Download citation

Key words

  • Fish gills
  • Epithelial cells
  • Intracellular pH
  • Na−H exchange
  • Trout, Oncorhynchus