Volume regulation by Amphiuma red blood cells. The membrane potential and its implications regarding the nature of the ion-flux pathways.

Abstract

After osmotic perturbation, the red blood cells of Amphiuma exhibited a volume-regulatory response that returned cell volume back to or toward control values. After osmotic swelling, cell-volume regulation (regulatory volume decrease; RVD) resulted from net cellular loss of K, CI, and osmotically obliged H20. In contrast, the volume-regulatory response to osmotic shrinkage (regulatory volume increase; RVI) was characterized by net cellular uptake of Na, CI, and H20. The net K and Na fluxes characteristic of RVD and RVI are increased by 1-2 orders of magnitude above those observed in studies of volumestatic control cells. The cell membrane potential of volume-regulating and volume-static cells was measured by impalement with glass microelectrodes. The information gained from the electrical and ion-flux studies led to the conclusion that the ion fluxes responsible for cell-volume regulation proceed via electrically silent pathways. Furthermore, it was observed that Na fluxes during RVI were profoundly sensitive to medium [HCO3] and that during RVI the medium becomes more acid, whereas alkaline shifts in the suspension medium accompany RVD. The experimental observations are explained by a model featuring obligatorily coupled alkali metal-H and CI-HCO3 exchangers. The anion-and cation-exchange pathways are separate and distinct yet functionally coupled via the net flux of H. As a result of the operation of such pathways, net alkali metal, CI, and H20 fluxes proceed in the same direction, whereas H and HCOa fluxes are cyclic. Data also are presented that suggest that the ion-flux pathways responsible for cell-volume regulation are not activated by changes in cell volume per se but by some event associated with osmotic perturbation, such as changes in intracellular pH.

Volume-regulatory responses of Amphiuma red cells in anisotonic media. The effect of amiloride.

“…Our Vm values estimated using Eq. 5 are in good agreement with the microelectrode measurements of Cala (1980), but suggest that minor gradual changes in Vm occur during volume recovery that were not detected in the microelectrode study (Cala, 1980) .…”

“…Possible explanations for the non-Nernstian slope include influences of ions other than Cl on the 53 2 THE JOURNAL OF GENERAL PHYSIOLOGY " VOLUME 86 -1985 membrane potential, exclusion of Cl from the nucleus, and differences in activity coefficients for Cl between the cell and the medium (Stoner and Kregenow, 1980) . Microelectrode studies of Vm in Amphiuma red cells have also been performed on cells recovering their volume under anisotonic conditions similar to those of the present study (Cala, 1980). Cala (1980) found that there were no detectable changes in Vm during volume recovery, which suggests that the volume-regulatory mechanisms are electroneutral .…”

“…Microelectrode studies of Vm in Amphiuma red cells have also been performed on cells recovering their volume under anisotonic conditions similar to those of the present study (Cala, 1980). Cala (1980) found that there were no detectable changes in Vm during volume recovery, which suggests that the volume-regulatory mechanisms are electroneutral . It must be emphasized that if volume-regulatory Na and K movements were through conductive pathways, rCl would not be an accurate indicator of the membrane potential (Lassen, 1977 ;Wieth and Brahm, 1980) .…”

“…Importantly, several characteristics of Na transport that are shared by amiloride-sensitive epithelia (Cuthbert and Fanelli, 1978 ;Benos, 1982) also characterize the Na movements of the volumeregulatory increase (VRI) response . The findings of this study have been presented in preliminary form (Siebens and Kregenow, 1978, 1980, 1982 and reviewed (Kregenow, 1981 ;Benos, 1982 ;Cala, 1983 ;Siebens, 1985) . Other investigators using both Amphiuma (Gala, 1980) and frog (Palfrey and Greengard, 1981) red cells have confirmed and expanded on some of these findings .…”

“…This Na gain increased the cell Na content to about five times its normal value and increased the Na + K content by^-40% . The Na gain was regulated in that the cells did not continue to enlarge beyond their isotonic control volume, even though both the chemical gradient for Na ([Na]o/[Na]i = -160 mM/^"60 mM) and the membrane potential (^-20 mV, inside-negative; Cala, 1980; Table II) favored continued Na entry. Experiments in which volume recovery was prevented by replacing medium Na with choline (data not shown) further support the concept that changes in cell Na content bring about the observed volume changes.…”