Figure 9.
Block of the negative Egly shift by HCO3−/CO2 removal. A, The effect on Egly and pHi of removing HCO3−/CO2 from perfusion (HEPES/O2) (i) and then adding AZA (50 μm) (ii) in one cell. Superimposed control–treatment pairs of Egly/pHi series were chosen on the basis of similar numbers of evoked Ca2+ spikes during the 8 s depolarization. The periods of 8 s Ca2+ spiking are marked with thick bars. Ai, The injected current and number of evoked Ca2+ spikes were 180 pA/16 and 120 pA/18 Ca2+ spikes for the control and HEPES/O2, respectively. Aii, The injected current and number of evoked Ca2+ spikes were 130 pA/30, 110 pA/31, and 110 pA/15 Ca2+ spikes for HEPES/O2, after AZA addition (+AZA) and 17 min after removing AZA (−AZA), respectively. The effects of AZA were fully reversible if it had been applied for less than ∼15 min. B, Another example showing the slowed pHi recovery and positive Egly shift after addition of AZA in HEPES/O2 after Ca2+ spiking. Time of peak acidification in AZA is indicated with an arrow, and the period of Ca2+ spiking is shaded. The injected current and number of evoked Ca2+ spikes were 170 pA/23 and 160 pA/22 Ca2+ spikes for HEPES/O2 and +AZA, respectively. C, Plot of peak acidification (i) and half-recovery time (ii) against the peak negative Egly shift and the peak positive Egly shift for HEPES/O2 and +AZA for six cases (from different cells) in which the numbers of evoked Ca2+ spikes were similar between the two conditions. The number of evoked Ca2+ spikes in +AZA condition ranged from 20 to 47 in the six cases. The difference in peak acidification was not significant, but the half-recovery time was significantly lengthened in AZA added to HEPES/O2. D, Chloride transporters potentially affecting neuronal [Cl−]i (i), and the proposed mechanism of activity-dependent negative shift in Egly in CWCs (ii). KCC, K+-Cl− cotransporters; NKCC, Na+-K+-Cl− cotransporters; AE, Na+-independent anion (Cl−-HCO3−) exchanger; NDCBE, Na+-driven Cl−-HCO3− exchanger, also known as NDAE.