An inwardly directed electrogenic sodium-bicarbonate co-transport in leech glial cells

1. We have used double-barrelled ion-sensitive microelectrodes to measure the intracellular pH, pHi, the intracellular Na+ activity, aiNa, and the membrane potential in identified glial cells of the central nervous system of the leech Hirudo medicinalis to study the effect of CO2-HCO3-. 2. When a HEPES-buffered saline was exchanged for a saline buffered with 2% CO2 + 11 mM-HCO3-, keeping the pH constant at 7.4, the mean steady-state pHi of the glial cells increased from 6.85 +/- 0.06 to 7.18 +/- 0.13 (mean +/- S.D., n = 25). 3. This CO2-HCO3- -dependent alkalinization was inhibited in the absence of external Na+ (exchanged by N-methyl-D-glucamine), but was unaffected by the inhibitor of Na+-H+ exchange, amiloride (2 mM). 4. The aiNa of the glial cells increased by 2-4 mM from a mean steady state of 7.2 +/- 2 mM (mean +/- S.D., n = 6) upon introduction of CO2-HCO3- -buffered saline. This CO2-HCO3- -dependent rise in aiNa increased to about double when the pHi had been decreased by acid loading the cells (addition and subsequent removal of NH4+). 5. The CO2-HCO3- -dependent increases of pHi and aiNa were inhibited by the stilbene 4,4-diisothiocyanostilbene-2,2'-disulphonic acid (DIDS, 0.5-1.0 mM). 6. Removal of external Cl- and depletion of intracellular Cl- did not inhibit the CO2-HCO3- -dependent alkalinization. 7. The CO2-HCO3- -dependent alkalinization was unaffected by inhibitors of the carbonic anhydrase, acetazolamide (0.2 mM) or ethoxzolamide (2 microM). 8. The membrane potential became more negative by 3-20 mV upon addition of CO2-HCO3-. This hyperpolarization was even further enlarged in the presence of Ba2+ (which reduces the K+ permeability) or at increased external K+ concentration (which depolarizes the membrane and brings the membrane potential to the K+ equilibrium potential). The CO2-HCO3- -induced membrane hyperpolarization was inhibited in Na+-free saline and in the presence of DIDS. Ouabain (0.5 mM) sometimes reduced, but never abolished, the hyperpolarization. 9. The stoichiometry of the co-transport is suggested to be 2 HCO3-:1 Na+ with an equilibrium potential of -90 mV calculated for this coupling ratio in the steady state. 10. It is concluded that in the presence of CO2-HCO3- an inwardly directed electrogenic Na+-HCO3- co-transport is stimulated across the glial membrane, which greatly determines the pHi and thereby affects the intracellular buffering power of the glial cells.