Intracellular pH regulation in the embryonic chicken lens epithelium.
1. The intracellular pH (pHi) of embryonic lens epithelia was measured by the emission ratio technique using the fluorescent pH probe carboxy-seminaphthorhodafluor-1 (Snarf-1). 2. In artificial aqueous humour solutions (AAH) containing HCO3-, pHi was 7.45, a value more alkaline than that of the bathing medium (pH = 7.3). In HCO3- -free AAH, pHi was 7.29. 3. Acetazolamide, an inhibitor of carbonic anhydrase, had no effect on resting pHi. 4. The pHi could be manipulated experimentally by changing the external pH (pHo) of HEPES-buffered AAH, the addition or withdrawal of CO2-HCO3-, or by perfusion with the weak bases NH4Cl and procaine. 5. The pHi change induced by withdrawal of 5 mM-procaine was used to calculate a value for the intrinsic cytoplasmic buffering capacity (beta i) of 16.5 mM. 6. The addition of amiloride (1 mM) or treatment with low-Na+ AAH solutions led to a decrease in pHi of 0.23 over the 10 min exposure. In addition, these treatments inhibited pHi recovery from NH4(+)-induced acidosis. These observations are consistent with the presence of amiloride-sensitive N(+)-H+ antiport. 7. Addition of exogenous antiport activity in the form of 50 microM-monensin caused an increase in pHi of 0.24. 8. In HCO3(-)-containing media, replacing Cl- by gluconate or isothionate led to an immediate, reversible increase in pHi which could be completely inhibited by 2 mM-4-acetamido-4'-isothiocyanato-stillbene-2,2'-disulphonic acid (SITS). This indicates the presence of Cl(-)-HCO3- exchange in this tissue. 9. Under HCO3(-)-free conditions, replacement of Cl- by gluconate or isothionate caused a small transient acidification followed, 5 min later, by a large sustained alkalinization. The delayed increase in pHi could be completely blocked by 1 mM-amiloride and may reflect volume-sensitive stimulation of the Na(+)-H+ antiporter as cell volume (estimated by cell height measurements) was shown to decrease significantly during this period.
Bassnett
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Department of Anatomy, Uniformed Services University of the Health Sciences, Bethesda, MD 20814.
D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid / Q000494:pharmacology; D000086:Acetazolamide / Q000494:pharmacology; D000136:Acid-Base Equilibrium / Q000502:physiology; D000584:Amiloride / Q000494:pharmacology; D000643:Ammonium Chloride / Q000494:pharmacology; D000818:Animals; D001578:Benzopyrans; D001639:Bicarbonates / Q000494:pharmacology; D002245:Carbon Dioxide / Q000494:pharmacology; D002642:Chick Embryo; D004848:Epithelium / Q000502:physiology; D005942:Gluconates / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration / Q000187:drug effects; D066298:In Vitro Techniques; D007908:Lens, Crystalline / Q000196:embryology / Q000502:physiology*; D008985:Monensin / Q000494:pharmacology; D009284:Naphthols; D011343:Procaine / Q000494:pharmacology; D012235:Rhodamines
D001578:Benzopyrans; D001639:Bicarbonates; D005942:Gluconates; D009284:Naphthols; D012235:Rhodamines; C068301:carboxy-seminaphthorhodaminefluoride; D000643:Ammonium Chloride; D002245:Carbon Dioxide; D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; D011343:Procaine; D000584:Amiloride; D008985:Monensin; D000086:Acetazolamide