The contribution of metabolic
bicarbonate to cytosolic pH (pHcyto) regulation was studied on isolated perfused rat liver using
phosphorus-31 NMR spectroscopy. Removal of external HCO3- decreased
proton efflux from 18.6+/-5.0 to 1.64+/-0.29 micromol/min per g liver wet weight (w.w.) and pHcyto from 7.17+/-0.06 to 6.87+/-0.06. In the nominal absence of
bicarbonate, inhibition of
carbonic anhydrase by
acetazolamide induced a further decrease of
proton efflux of 0.69+/-0.26 micromol/min per g liver w.w. reflecting a reduction in metabolic CO2 hydration, and hence a decrease of H+ and HCO3- supplies. Even though 27% of the
proton efflux was
amiloride-sensitive under
bicarbonate-free conditions,
amiloride did not change pHcyto, revealing the contribution of additional regulatory processes. Indeed, pH regulation was affected by the combined use of 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic
acid (
SITS) and
amiloride since pHcyto decreased by 0.16+/-0.05 and
proton efflux by 0.60+/-0.14 micromol/min per g liver w.w. The data suggest that
amiloride-sensitive or
SITS-sensitive transport activities could achieve, by themselves, pHcyto regulation. The involvement of two mechanisms, most likely Na+/H+ antiport and
Na+:HCO3 symport, was confirmed in the whole organ under intracellular and extracellular
acidosis. The evidence of Na-dependent transport of HCO3- in the absence of exogenous
bicarbonate implies that the amount of metabolic
bicarbonate is sufficient to effectively participate to pHcyto regulation.