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

We studied the effects of external HCO3- on pH(i) regulation in human lymph ocytes after an acid load. Cells were acidified by preincubation with NH4Cl and pH(i) recovery was measured with the fluorescent dye BCECF. Cells reco vering in HCO3--containing medium reached a higher final pH(i), the H+ effl ux rate was increased and shifted to alkaline pH(i) compared to that of cel ls recovering in HCO3--free solution. The resting pH(i) was higher in a HCO 3--containing solution. Experiments carried out in the presence of amilorid e, DIDS and in the absence of external Na+ suggest the existence of two maj or mechanisms acting in the pH(i) recovery of lymphocytes after an acid loa d: an amiloride-sensitive Na+/H+ exchanger and a DIDS-sensitive Na+-depende nt HCO3- transporter. The last mechanism could be a Na+/HCO3- cotransporter based on membrane potential changes determined with the potential-sensitiv e fluorescent probe bis-oxonol. Preincubation of cells with forskolin and H -89 showed protein-kinase-A-dependent downregulation of the amiloride-insen sitive recovery of pH(i) in human lymphocytes. In summary, this paper provi des functional evidence for the existence of a Na+/HCO3--dependent mechanis m involved in pH(i) recovery in human lymphocytes following an acid load, t hat is electrogenic and downregulated by PKA.