Proceedings of The Physiological Society

University of York (2002) J Physiol 539P, S022


Measurement of intracapillary pH to investigate the role of bicarbonate in H+ transport across the blood-brain barrier in the anaesthetized rat

Dola Akanmu and P.A. Fraser

Centre for Cardiovascular Biology & Medicine, King's College London, Guy's Campus, London SE1 1UL, UK

  • Figure 1. Record of the effect of DIDS on luminal pH from a single pial venular capillary (15 µm diameter) during superfusion with a Hepes buffer.

H+ formed during normal cerebral function must be removed, and the aim of this study is to examine the mechanisms by which this occurs. Experiments accorded with UK legislation. Single venular capillaries of rats (anaesthetized with pentobarbitone: 60 mg kg-1 I.P., an overdose of which was administered to kill humanely) were loaded 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 (mixed with blood) was trapped in the lumen of a venular capillary 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 was proportional to the pH of test solutions between pH 6.8 and 7.6.

During a 120 s occlusion the intracapillary pH fell at a rate of 0.87 ± 0.20 milli-pH units s-1 (mpH; mean ± S.E.M., n = 18) while the vessel was superfused with a bicarbonate-CO2 buffer at pH 7.32. The rate of acidification was no different when a bicarbonate-free Hepes-based buffer was applied (0.79 ± 0.22, n = 38). When the carbonic anhydrase inhibitor, acetazolamide (0.2 mM), was applied 40 s after the beginning of an occlusion, luminal pH increased to a new steady-state value of 105 ± 5.1 mpH units in the presence of bicarbonate (n = 5), but only by 42 ± 10 (n = 9) in the presence of the Hepes buffer. These increases reversed within 10 s when acetazolamide was removed. Inhibition of bicarbonate transport by 4,4fi-diisothiocyanostilbene-2,2fi-disulfonic acid (DIDS, 0.1 mM) added to the superfusate, resulted in a pH increase of 164 ± 30 mpH units (n = 7) with bicarbonate buffer and 115 ± 20 mpH units (n = 21) with Hepes.

Where applicable, experiments conform with Society ethical requirements