TY - JOUR
T1 - Intracellular pH regulation and buffer capacity in CO2/HCO3/--buffered media in cultured epithelial cells from rainbow trout gills
AU - Wood, C. M.
AU - Pärt, P.
N1 - Funding Information:
Acknowledgements We thank Rod Rhem for excellent technical assistance, and Dr. Lisle Gibbs and Dr. Colin Nurse for the loan of equipment. Supported by grants from NSERC (CMW), NSERC Collaborative Program (CMW and PP, Dr. D.G. McDonald, P.I.), the Swedish Environmental Protection Agency (PP), the C.F. Lundstrom Foundation (PP), and an NSERC Foreign Researcher Award (PP). All experimental procedures complied with current animal care regulations in Canada.
Copyright:
Copyright 2018 Elsevier B.V., All rights reserved.
PY - 2000/5
Y1 - 2000/5
N2 - The influence of a CO2/HCO3/--buffered medium on intracellular pH regulation of gill pavement cells from freshwater rainbow trout was examined in monolayers grown in primary culture on glass coverslips; intracellular pH (pH(i)) was monitored by continuous spectrofluorometric recording from cells loaded with 2',7'-bis(2-carboxyethyl)-5(6)-carboxy-fluoroscein. When cells in HEPES-buffered medium at normal pH = 7.70 were transferred to normal CO2/HCO3/--buffered medium {P(CO2) = 3.71 mmHg, [HCO3/-] = 6.1 mmol l-1, extracellular pH (pH(e)) = 7.70}, they exhibited a brief acidosis but subsequently regulated the same pHi (~7.41) as in HEPES. Buffer capacity (β) increased by the expected amount (5.5-8.0 slykes) based on intracellular [HCO3/-], and was unaffected by most drugs and treatments. However, after transfer to high P(CO2) = 11.15 mmHg, [HCO3/-] = 18.2 mmol l-1 at the same pH(e) = 7.70, the final regulated pHi was elevated (~7.53). The rate of correction of alkalosis caused by washout of this high P(CO2), high-HCO3/- medium was unaffected by removal of extracellular Cl-3. Removal of extracellular Na+ lowered resting pH(i) and greatly inhibited the rate of pH(i) recovery from acidosis. Bafilomycin A1 (3 μmol l-1) had no effect on these responses. However amiloride (0.2 mmol l-1) inhibited recovery from acidosis caused by washout of an ammonia prepulse, but did not affect resting pH(i), the latter differing from the response in HEPES where amiloride also lowered resting pH(i). Similarly 4-acetamido-4-isothiocyanatostilbene-2,2'-disulfonic acid, sodium salt (0.1 mmol l-1) did not affect resting pH(i) but slowed the rate of recovery from acidosis, though to a lesser extent than amiloride. Removal of extracellular Cl- also slowed the rate of recovery but greatly increased β by an unknown mechanism; when this was taken into account, H+ extrusion rate was unaffected. These results are consistent with the presence of Na+-(HCO3/-)(N) cotransport and/or Na+-dependent HCO3/-/Cl- exchange, in addition to Na+/H+ exchange, as mechanisms contributing to 'housekeeping' pH(i) regulation in gill cells in CO2/HCO3/- media, whereas only Na+/H+ exchange is seen in HEPES. Both Na+-independent Cl-/HCO3/- exchange and V-type H+-ATPase mechanisms appear to be absent from these cells cultured in isotonic media.
AB - The influence of a CO2/HCO3/--buffered medium on intracellular pH regulation of gill pavement cells from freshwater rainbow trout was examined in monolayers grown in primary culture on glass coverslips; intracellular pH (pH(i)) was monitored by continuous spectrofluorometric recording from cells loaded with 2',7'-bis(2-carboxyethyl)-5(6)-carboxy-fluoroscein. When cells in HEPES-buffered medium at normal pH = 7.70 were transferred to normal CO2/HCO3/--buffered medium {P(CO2) = 3.71 mmHg, [HCO3/-] = 6.1 mmol l-1, extracellular pH (pH(e)) = 7.70}, they exhibited a brief acidosis but subsequently regulated the same pHi (~7.41) as in HEPES. Buffer capacity (β) increased by the expected amount (5.5-8.0 slykes) based on intracellular [HCO3/-], and was unaffected by most drugs and treatments. However, after transfer to high P(CO2) = 11.15 mmHg, [HCO3/-] = 18.2 mmol l-1 at the same pH(e) = 7.70, the final regulated pHi was elevated (~7.53). The rate of correction of alkalosis caused by washout of this high P(CO2), high-HCO3/- medium was unaffected by removal of extracellular Cl-3. Removal of extracellular Na+ lowered resting pH(i) and greatly inhibited the rate of pH(i) recovery from acidosis. Bafilomycin A1 (3 μmol l-1) had no effect on these responses. However amiloride (0.2 mmol l-1) inhibited recovery from acidosis caused by washout of an ammonia prepulse, but did not affect resting pH(i), the latter differing from the response in HEPES where amiloride also lowered resting pH(i). Similarly 4-acetamido-4-isothiocyanatostilbene-2,2'-disulfonic acid, sodium salt (0.1 mmol l-1) did not affect resting pH(i) but slowed the rate of recovery from acidosis, though to a lesser extent than amiloride. Removal of extracellular Cl- also slowed the rate of recovery but greatly increased β by an unknown mechanism; when this was taken into account, H+ extrusion rate was unaffected. These results are consistent with the presence of Na+-(HCO3/-)(N) cotransport and/or Na+-dependent HCO3/-/Cl- exchange, in addition to Na+/H+ exchange, as mechanisms contributing to 'housekeeping' pH(i) regulation in gill cells in CO2/HCO3/- media, whereas only Na+/H+ exchange is seen in HEPES. Both Na+-independent Cl-/HCO3/- exchange and V-type H+-ATPase mechanisms appear to be absent from these cells cultured in isotonic media.
KW - Bicarbonate buffer
KW - Buffer capacity
KW - Cultured epithelial cells
KW - Fish gills
KW - Intracellular pH
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U2 - 10.1007/s003600050273
DO - 10.1007/s003600050273
M3 - Article
C2 - 10841257
AN - SCOPUS:0343962994
VL - 170
SP - 175
EP - 184
JO - Journal of Comparative Physiology B: Biochemical, Systemic, and Environmental Physiology
JF - Journal of Comparative Physiology B: Biochemical, Systemic, and Environmental Physiology
SN - 0174-1578
IS - 3
ER -