The effect of intracellular acidification on the relationship between cell volume and membrane potential in amphibian skeletal muscle

The relationship between cell volume (V-c) and membrane potential (E-m) in Rana temporaria striated muscle fibres was investigated under different conditions of intracellular acidification. Confocal microscope xz-scanning monitored the changes in V-c, whilst conventional KCl and pH-sensitive microelectrodes measured E-m and intracellular PH (pH(i)), respectively. Applications of Ringer solutions with added NH4Cl induced rapid reductions in V-c that rapidly reversed upon their withdrawal. These could be directly attributed to the related alterations in extracellular tonicity. However: (1) a slower and persistent decrease in V-c followed the NH4Cl withdrawal, leaving Vc up to 10% below its resting value; (2) similar sustained decreases in resting V, were produced by the addition and subsequent withdrawal of extracellular solutions in which NaCl was isosmotically replaced with NH4Cl; (3) the same manoeuvres also produced a marked intracellular acidification, that depended upon the duration of the preceding exposure to NH4Cl, of up to 0.53 +/- 0.10 PH units; and (4) the corresponding reductions in Vc similarly increased with this exposure time. These reductions in V, persisted and became more rapid with Cl- deprivation, thus excluding mechanisms involving either direct or indirect actions of pH(i) upon Cl--dependent membrane transport. However they were abolished by the Na+,K+-ATPase inhibitor ouabain. The E.. changes that accompanied the addition and withdrawal of NH4+ conformed to a Nernst equation modified to include realistic NH4+ permeability terms, and thus the withdrawal of NH4+ restored E-m to close to control values despite a persistent change in V-c. Finally these E-m changes persisted and assumed faster kinetics with Cl- deprivation. The relative changes in Vc, En and pHi were compared to predictions from the recent model of Fraser and Huang published in 2004 that related steady-state values of Vc and E. to the mean charge valency (z(x)) of intracellular membrane-impermeant anions, X-i(-). By assuming accepted values of intracellular buffering capacity (beta(i)), intracellular acidification was shown to produce quantitatively predictable decreases in Vc. These findings thus provide experimental evidence that titration of the anionic z(x) by increased intracellular [H+] causes cellular volume decrease in the presence of normal Na+,K+-ATPase activity, with Cl--dependent membrane fluxes only influencing the kinetics of such changes.