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University Laboratory of Physiology, Oxford.
1. Intrinsic, intracellular H+ buffering power (beta) was estimated in the isolated sheep cardiac Purkinje fibre at various values of intracellular pH (pHi) in the range 6.2-7.5 and for various values of extracellular pH (pHo) in the range 6.5-8.5. Buffering power was calculated from the fall of pHi (recorded with an intracellular pH-selective microelectrode) induced by addition and removal of extracellular, permeant weak acids and bases (NH4Cl, trimethylamine chloride, sodium propionate). Experiments were performed under conditions nominally free of CO2-HCO3. 2. beta was estimated firstly following acid loads induced by NH4Cl removal (10-20 mM) under conditions where Na(+)-H+ exchange was operational (i.e. in Na(+)-containing Tyrode solution). At constant pHi, the value of beta appeared to double (from a control level of 39.7 mM) as pHo was increased from 7.5 to 8.5. Notably, raising pHo in this range greatly accelerated pHi recovery from an intracellular acid load, indicating stimulation of acid extrusion. It is likely that this stimulation results in an overestimation of beta because it blunts the intracellular acid load. The apparent elevation of beta at high pHo may therefore be an artifact. 3. Estimates of beta were compared (NH4Cl removal) before and after inhibiting Na(+)-H+ exchange in Na(+)-free solution or with amiloride (1 mM). The acid load was larger and in many (but not all) cases the apparent value of beta decreased after inhibition of acid extrusion. This indicates that, if Na(+)-H+ exchange is operational, it can result in an overestimate of beta. In amiloride, beta was 26.6 +/- 1.4 mM (n = 8) at a mean pHi of 6.84 +/- 0.03. 4. Small stepwise reductions of external NH4Cl (from 40 to 0 mM), in the presence of Na(+)-free solution plus 5 mM-BaCl2 at constant pHo, resulted in small stepwise reductions of pHi (approximately 0.1 units). When these were used to calculate beta, we observed that beta increased roughly linearly as pHi became more acid. For a pHi of 7.2, beta approximately 20 mM. 5. An almost identical relationship between beta and pHi was found when using the method of sodium propionate addition (10-50 mM): amiloride (1 mM) was present and pHi was manipulated to various test levels by changing pHo. This confirms that beta varies inversely with pHi and also that it is independent of pHo. We conclude that the apparent variation of beta with pHo observed earlier was indeed an artifact.(ABSTRACT TRUNCATED AT 400 WORDS)
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