Evidence for an electrogenic, negatively protein-kinase-A-modulated, Na+-dependent HCO3- transporter in human lymphocytes.
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.
de la Rosa
L A
LA
Departamentos de Farmacología, Facultad de Veterinaria, E-27002 Lugo, Universidad Santiago de Compostela, Spain.
Cabado
A G
AG
Botana
M A
MA
Vieytes
M R
MR
Vidal
J I
JI
Botana
L M
LM
D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid / Q000494:pharmacology; D000584:Amiloride / Q000494:pharmacology; D001639:Bicarbonates / Q000378:metabolism* / Q000494:pharmacology; D002352:Carrier Proteins / Q000097:blood*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000494:pharmacology*; D005452:Fluoresceins; D005456:Fluorescent Dyes; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008214:Lymphocytes / Q000378:metabolism*; D008564:Membrane Potentials; D012964:Sodium / Q000494:pharmacology*
D001639:Bicarbonates; D002352:Carrier Proteins; D005452:Fluoresceins; D005456:Fluorescent Dyes; D000584:Amiloride; C043829:2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein; D012964:Sodium; D017868:Cyclic AMP-Dependent Protein Kinases; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid