Regulation of intracellular pH in reticulospinal neurones of the lamprey, Petromyzon marinus

Abstract

1. The regulation of intracellular pH (pHi) in lamprey reticulospinal neurones was investigated with pH-sensitive micro-electrodes based on a neutral carrier liquid membrane. Experiments were performed using an in vitro brain-stem preparation. 2. In HEPES-buffered solutions, extracellular pH (pHo) was consistently more acidic than the pH of the bathing solution (pHb). In HCO3(-)-buffered solutions, the brain was also relatively acidic, but the brain pH gradient was smaller. 3. In HEPES- and HCO3(-)-buffered solutions, mean pHi was 7.40-7.50. This range was too high to be explained by a passive distribution of H+, OH- or HCO3-. 4. In nominally HCO3(-)-free, HEPES-buffered solution, cells were acid loaded by addition and subsequent withdrawal of NH4+ from the superfusate. pHi recovered from acid loading by an energy-dependent process in 10-20 min. Recovery from acid loading in HEPES-buffered solutions was blocked by exposure to amiloride. 5. Removal of extracellular Na+ caused a slow, accelerating fall of pHi. Return of Na+ to the bath caused an immediate reversal of this acidification, followed by a slow recovery of pHi. Measurement with Na+-sensitive micro-electrodes during acid loading showed a rapid rise in the intracellular Na+ activity [( Na+]i). 6. Following acid loading, transition from HEPES- to HCO3(-)-buffered solutions caused an increase in the acid extrusion rate of at least 48%. The effect of these solution changes was dependent on pHo. After blocking pHi recovery with amiloride, transition from HEPES- to HCO3(-)-buffered Ringer plus amiloride produced a slow recovery of pHi. 7. Recovery from acid loading in HCO3(-)-buffered solutions was inhibited 65% by the anion transport blocker DIDS (4,4'-diisothiocyanostilbene-2,2'-disulphonic acid). Recovery from acid loading after incubation in Cl(-)-free solution was slower than recovery after replenishment of Cl-. 8. It is concluded that in HCO3(-)-free solutions, pHi regulation is accomplished by a Na-H exchange mechanism. In the presence of extracellular HCO3- an additional mechanism can operate to extrude intracellular acid.