ISSN:
1432-136X
Keywords:
Key words Fish gills
;
Cultured epithelial cells
;
Intracellular pH
;
Buffer capacity
;
Bicarbonate buffer
Source:
Springer Online Journal Archives 1860-2000
Topics:
Biology
,
Medicine
Notes:
Abstract The influence of a CO2/HCO− 3-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 (pHi) 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/HCO− 3-buffered medium {P CO2=3.71 mmHg, [HCO− 3]= 6.1 mmol l−1, extracellular pH (pHe)=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 [HCO− 3], and was unaffected by most drugs and treatments. However, after transfer to high P CO2=11.15 mmHg, [HCO− 3]= 18.2 mmol l−1 at the same pHe=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-HCO− 3 medium was unaffected by removal of extracellular Cl−. Removal of extracellular Na+ lowered resting pHi and greatly inhibited the rate of pHi 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 pHi, the latter differing from the response in HEPES where amiloride also lowered resting pHi. Similarly 4-acetamido-4′- isothiocyanatostilbene-2,2′-disulfonic acid, sodium salt (0.1 mmol l−1) did not affect resting pHi 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+-(HCO− 3)N co-transport and/or Na+-dependent HCO− 3/Cl− exchange, in addition to Na+/H+ exchange, as mechanisms contributing to “housekeeping” pHi regulation in gill cells in CO2/HCO− 3 media, whereas only Na+/H+ exchange is seen in HEPES. Both Na+-independent Cl−/HCO− 3 exchange and V-type H+-ATPase mechanisms appear to be absent from these cells cultured in isotonic media.
Type of Medium:
Electronic Resource
URL:
http://dx.doi.org/10.1007/s003600050273
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