Evidence for an electrogenic, negatively protein-kinase-A-modulated, Na+-dependent HCO3- transporter in human lymphocytes

Abstract

We studied the effects of external HCO3- on pHi regulation in human lymphocytes after an acid load. Cells were acidified by preincubation with NH4Cl and pHi recovery was measured with the fluorescent dye BCECF. Cells recovering in HCO3--containing medium reached a higher final pHi, the H+ efflux rate was increased and shifted to alkaline pHi compared to that of cells recovering in HCO3--free solution. The resting pHi was higher in a HCO3--containing solution. Experiments carried out in the presence of amiloride, DIDS and in the absence of external Na+ suggest the existence of two major mechanisms acting in the pHi recovery of lymphocytes after an acid load: an amiloride-sensitive Na+/H+ exchanger and a DIDS-sensitive Na+-dependent HCO3- transporter. The last mechanism could be a Na+/HCO3- cotransporter based on membrane potential changes determined with the potential-sensitive fluorescent probe bis-oxonol. Preincubation of cells with forskolin and H-89 showed protein-kinase-A-dependent downregulation of the amiloride-insensitive recovery of pHi in human lymphocytes. In summary, this paper provides functional evidence for the existence of a Na+/HCO3--dependent mechanism involved in pHi recovery in human lymphocytes following an acid load, that is electrogenic and downregulated by PKA.