Previously, we demonstrated that bicarbonate and carbonic anhydrase enhanced lactate-H
+ cotransport in cultured bovine corneal endothelial cells (CEC).
24 We attributed the facilitated transport to increased intracellular buffering capacity. To determine if this mechanism was also significant in rabbit endothelium, we first examined the effect of bicarbonate on lactate-induced acidification. In the absence of bicarbonate, there was significant acidification when 20 mM lactate was perfused for 1 minute on either the apical or basolateral surface (
Fig. 3A). However, in the presence of bicarbonate, the amount of acidification was significantly less on the basolateral surface and unmeasurable on the apical surface (
Fig. 3A). Next, we decreased intracellular buffering by incubating the tissue in 100 μM acetazolamide, a carbonic anhydrase inhibitor. Acetazolamide did not change the rate of LIA in tissues perfused without bicarbonate (0.00167–0.00163 pH units/sec,
n = 5,
P = 0.57) (
Fig. 3B). However, in the presence of bicarbonate, acetazolamide produced a small, but significant increase in the rate of LIA on the BL surface (from 0.0006 to 0.0008 pH units/sec,
n = 8,
P = 0.03) (
Fig. 3B). To determine if lactate-dependent proton flux (J
H +[mM/sec] = β
T*dpH
i/dt) was affected by HCO
3 − or ACTZ, total buffering capacity (β
T = β
i + β
HCO3) was determined under each condition as previously described.
12,24 The intrinsic buffering capacity (β
i = 20 mM/pH) of the rabbit endothelium was determined using dpH
i/dt of the first 20 seconds following exposure to NH
4Cl.
12 We found that β
HCO3 (= 2.303*[HCO
3 −]
i)
28 was reduced by 43% in the presence of ACTZ. This relative reduction in buffering was experimentally determined by measuring the change in pH
i during a pulse of 2 mM NH
4Cl in the absence and presence of 100 μM ACTZ in BR Ringer's solution (data not shown). Although the rate of LIA was significantly smaller in BR Ringer's solution, with the increased buffering supplied by bicarbonate, lactate-dependent proton flux was 13% greater; however, this was not significant (
P > 0.05;
Table 1).
Table 1 also shows that ACTZ had no effect on lactate-dependent proton flux in the absence of HCO
3 − as expected; however, in the presence of HCO
3 − proton flux decreased by 6%, but again not significant (
P > 0.05). So, unlike the cultured bovine corneal endothelium,
24 we could not demonstrate significant HCO
3 −/CA–facilitated lactate-dependent H
+ fluxes in vitro in the rabbit endothelium. A major difference is that the rate of LIA in the rabbit endothelium is more than 10-fold smaller (e.g., dpH/sec = 0.011 in bovine cells and 0.0008 in rabbit in BR + ACTZ), which significantly limits the ability to resolve differences. Therefore, we next explored disruption of HCO
3 − buffering using an in vivo approach.