pH Regulation in tissue-cultured bovine lens epithelial cells |
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Authors: | M R Williams G Duncan P C Croghan R Riach S F Webb |
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Institution: | (1) Biomedical Research Centre, School of Biological Sciences, University of East Anglia, NR4 7TJ Norwich, UK |
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Abstract: | Summary The intracellular pH (pH
i
) of tissue-cultured bovine lens epithelial cells was measured in small groups of 6 to 10 cells using the trapped fluorescent dye 2,7-bis-(2-,carboxyethyl)-5 (and 6)carboxyfluorescein (BCECF). When perifused at 35°C with artificial aqueous humour solution (AAH) containing 16 mM HCO
3
-
and 5% CO2, pH 7.25, pH
i
was 7.19±0.02 (sem, n = 95). On removing HCO
3
-
and CO2 there was an initial transient alkalinization followed by a fall in pH to a steady value of 6.97±0.03 (sem, n = 54). Addition of 0.25 mM 4,4-diisothiocyanatostilbene2, 2-disulfonic acid (DIDS) to AAH containing HCO
3
-
and CO2 led to a rapid and pronounced fall in pH. Exposure to Na+-free AAH again led to a marked fall in pH
i
, but in this case the addition of DIDS did not produce a further fall. Substitution of the impermeant anion gluconate for Cl– in the presence of HCO
3
-
led to a rise in pH
i
, while substitution in the absence of HCO
3
-
led to a fall in pH
i
. The above data indicate a significant role for a sodium-dependent Cl–-HCO
3
-
exchange mechanism in the regulation of pH
i
. Addition of 1 mM amiloride to control AAH in both the presence and absence of HCO
3
-
led to a marked fall in pH
i
, indicating that a Na+/H+ exchange mechanism also has a significant role in the regulation of pH
i
. There is evidence for a lactic acid transport mechanism in bovine lens cells, as addition of lactate to the external medium produced a rapid fall in pH
i
. Larger changes in pH
i
were observed in control compared to HCO
3
-
-free AAH and in the latter case a pronounced alkalinizing overshoot was obtained on removing external lactate. Tissue-cultured bovine lens cells thus possess at least three membrane transport mechanisms that are involved in pH regulation. The buffering capacity of the lens cells was measured by perturbing pH
i
with either NH
4
+
or procaine. The values obtained were similar in both cases and the intrinsic buffering capacity measured in the absence of external HCO
3
-
was 5 mm/pH unit (procaine). However, in the presence of HCO
3
-
and CO2 the buffer capacity increases approximately fourfold, indicating that HCO
3
-
is the principal intracellular buffer.We acknowledge financial support from the Wellcome Trust and the Humane Research Trust for this project. M.R. Williams was in receipt of a Science & Engineering Research Council studentship. |
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Keywords: | Lens pH pH regulation bicarbonate BCECF Cl– -HCO
3
- exchanger |
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