Next, we tested the hypothesis that increased cellular buffering capacity due to the presence of HCO
3 − and CA activity enhances lactate-H
+ flux in corneal endothelium. To do so, we measured LIA under BF and BR conditions and in the presence and absence of ACTZ, a CA inhibitor.
Figure 5A shows that in the presence of HCO
3 −, there is less LIA relative to that in BF ringer (
Fig. 5B). Treating the cells with 100 μM ACTZ in the presence of HCO
3 − reduced the rate of acidification by 35% but had no effect in BF. To estimate lactate-dependent proton flux under these conditions [J
H + (mM/sec) = β
T dpH
i/dt], the total cellular buffering capacity, β
T, must be known for each condition. β
T is the sum of intrinsic buffering, β
i, and bicarbonate buffering (=2.303 · [HCO
3 −]
i).
23 Endothelial intrinsic buffering (β
i = 10 mM/pH) was previously determined in bovine corneal endothelium.
17 We used the initial rate of pH
i change taken over the first 20 seconds for dpH
i/dt.
Table 2 shows that although the rate of LIA in BR was about half that in BF, because of the increased buffering capacity in BR, lactate-dependent proton flux was three times greater. Moreover, bicarbonate-dependent intracellular buffering was reduced by 41% in the presence of ACTZ. This relative reduction in buffering in the presence of ACTZ was experimentally determined by measuring the change in pH
i during a pulse of 5mM NH
4Cl in the absence and presence of 100 μM ACTZ while being perfused in BR Ringer (data not shown).
Table 2 also shows that ACTZ had no effect on lactate-dependent proton flux in the absence of HCO
3 −; however, in the presence of HCO
3 − proton flux decreased by 56%. These data indicate that lactate/H
+ flux is enhanced by increased buffering capacity as supplied by HCO
3 − and CA activity.