Assessment of evidence for K+-H+ exchange in isolated type-1 cells of neonatal rat carotid body.
Intracellular pH (pHi) was measured in enzymically isolated, neonatal rat carotid body type-1 cells, using the fluorophore carboxy-SNARF-1 (AM-loaded), and using the nigericin technique for in situ fluorescence calibration (nigericin is a membrane-soluble K+-H+ exchanger). In CO2/HCO3--free media, inhibiting Na+-H+ exchange produced a prompt fall of pHi (background acid-loading), the rate of which was reduced by raising the extracellular K+ concentration, [K+]o. pHi recovery from an intracellular acid or alkali load was also sensitive to changes of [K+]o. These results are similar to those of Wilding et al. (J Gen Physiol 100:593-608, 1992), who proposed the existence of an acid-loading, K+-H+ exchanger (KHE) in the type-1 cell. However, when nigericin was not used for post-experimental calibration, and the superfusion system was flushed exhaustively with strong detergent, alcohol and distilled water, then background acid-loading was attenuated, and the K+o sensitivity of pHi insignificant. Background loading was increased again, and K+o sensitivity restored, when cells were monitored in a superfusion system which had previously been exposed to a single nigericin-calibration protocol (followed by a short system wash with strong detergent and distilled water). We conclude that the previously reported expression of KHE in carotid body type-1 cells is an artefact caused by nigericin contamination. We have therefore quantified the pHi dependence of background loading in uncontaminated type-1 cells. We consider the possible implications of our work for reports of KHE in other cell types.
Richmond
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PH
University Laboratory of Physiology, Parks Road, Oxford OX1 3PT, UK.
Vaughan-Jones
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RD
D000143:Acids / Q000494:pharmacology; D000818:Animals; D000831:Animals, Newborn / Q000378:metabolism*; D017920:Antiporters / Q000378:metabolism*; D016477:Artifacts; D002344:Carotid Body / Q000166:cytology / Q000378:metabolism*; D002469:Cell Separation; D006863:Hydrogen-Ion Concentration; D009550:Nigericin / Q000494:pharmacology; D009994:Osmolar Concentration; D011188:Potassium / Q000378:metabolism; D027961:Potassium-Hydrogen Antiporters; D051381:Rats
D000143:Acids; D017920:Antiporters; D027961:Potassium-Hydrogen Antiporters; D009550:Nigericin; D011188:Potassium