The role of HCO3(-)-dependent mechanisms in pHi regulation during O2 deprivation

We have reported in our previous work that, in the absence of HCO(3)(-), Na(+)/H(+) exchanger is responsible for an anoxia-induced alkalinization in hippocampal CA1 neurons. HCO(3)(-)-dependent mechanisms have been reported to play a key role in pH(i) regulation in nerve cells, but how their function is affected by O(2) deprivation has not been well studied. In this work, pH(i) measurements (obtained from dissociated neurons loaded with carboxy-seminaphthorhodafluor-1 and using confocal microscopy) and whole-cell patch clamp recording techniques were used to investigate the role of HCO(3)(-)-dependent membrane exchangers on CA1 neurons during O(2) deprivation. Anoxia (5 min) induced a small acidification in neurons in the presence of HCO(3)(-) and this acidification was changed to a significant alkalinization when neurons were bathed with Hepes buffer or when 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid was applied in a HCO(3)(-) solution, indicating that HCO(3)(-)-dependent mechanisms were involved. A marked anoxia-induced acidification (0.33+/-0.11 pH unit) was seen when the Na(+)/H(+) exchange was blocked with 3-(methylsulfonyl-4-piperidino-benzoyl)-guanidine methanesulfonate in the presence of HCO(3)(-), but the same anoxia did not cause a significant pH(i) change in a Na(+) free, HCO(3)(-) solution, suggesting that the anoxia-induced acidification in the presence of 3-(methylsulfonyl-4-piperidino-benzoyl)-guanidine methanesulfonate is dependent on both Na(+) and HCO(3)(-). Furthermore, anoxia did not cause a significant pH(i) change when both 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid and 3-(methylsulfonyl-4-piperidino-benzoyl)-guanidine methanesulfonate were present. Current clamp recordings showed a significant membrane depolarization following anoxia in HCO(3)(-) solution but not in Hepes buffer. Our data suggest that, in hippocampal neurons: a) pH(i) regulation during O(2) deprivation is affected not only by metabolism but also by membrane exchangers, and b) besides the activation of Na(+)/H(+) exchange, anoxia activates a 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid-sensitive, Na(+)-dependent acid loader (possibly electrogenic).