The rapid removal of H⁺ from the brain is important for homeostasis, but the cerebral endothelium forms a barrier that prevents the free diffusion of ions. We are using the single pial microvessel preparation to study the mechanisms by which this H⁺ transport occurs. Single venular capillaries of rats (anaesthetized with pentobarbitone: 60 mg kg^-1 I.P., an overdose of which was administered to kill humanely; experiments accorded with UK legislation), were filled with the fluorescent ratiometric pH indicator dye, 8-hydroxypyrene-1,3,6-trisulphonic acid (HPTS, 0.5 mM) by a bolus intracarotid injection. The dye-blood mixture was trapped in a selected vessel by lowering two probes positioned at least 100 µm apart. The ratio of the emitted light at wavelength greater than 510 nm for 440 nm and 380 nm excitation is found to be proportional to pH between 6.8 and 7.6.

During an occlusion the luminal pH fell by 0.039 ± 0.011 pH units (mean ± S.E.M., n = 21) when the brain surface was superfused with a bicarbonate-free Hepes-based buffer at pH 7.32, the steady state being reached between 40 and 60 s. Lowering the superfusing pH to 6.8 resulted in luminal acidification by 0.21 ± 0.08 (n = 4). The Na⁺/H⁺ exchange inhibitor EIPA (ethyl isopropyl amiloride; 50 µM) prevented this fall (0.03 ± 0.05; n = 4). When superfusate Na⁺ was substituted by choline (at pH 7.32) the luminal pH fell by 0.09 ± 003 (n = 8). The possibility that this was due to the Na⁺ gradient driving an influx of H⁺ was tested by applying EIPA, and this resulted in an alkalization to 0.06 ± 0.01 (n = 12). EIPA, however, when applied in the presence of Na⁺ and with superfusate pH 7.32, resulted in a similar alkalization by 0.15 ± 0.03 (n = 4). The Na⁺/H⁺ exchanger cannot be the sole mechanism by which H⁺ move from brain to blood.