Amphiuma red cells were incubated for several hours in hypotonic or hypertonic media. They regulate their volume in both media by using ouabain-insensitive salt transport mechanisms . After initially enlarging osmotically, cells in hypotonic media return toward their original size by losing K, Cl, and H2O. During this volume-regulatory decrease (VRD) response, K loss results from a >10-fold increase in K efflux . Cells in hypertonic media initially shrink osmotically, but then return toward their original volume by gaining Na, Cl, and H2O. The volume-regulatory increase (VRI) response involves a large (>100-fold) increase in Na uptake that is entirely blocked by the diuretic amiloride (10-g M). Na transport in the VRI response shares many of the characteristics of amiloride-sensitive transport in epithelia: (a) amiloride inhibition is reversible ; (b) removal of amiloride from cells pretreated with amiloride enhances Na uptake relative to untreated controls ; (c) amiloride appears to act as a competitive inhibitor (K i = 1-3 AM) of Na uptake; (d) Na uptake is a saturable function of external Na (K m -29 mM); (e) Li can substitute for Na but K cannot . Anomalous Na/K pump behavior is observed in both the VRD and the VRI responses. In the VRD response, pump activity increases 3-fold despite a decrease in intracellular Na concentration, while in the VRI response, a 10-fold increase in pump activity is observed when only a doubling is predicted from increases in intracellular Na .

When Amphiuma red cells are shrunken in hypertonic media, they return toward their original volume by gaining Na through an amiloridesensitive pathway . As cells recover their volume during this volume-regulatory increase (VRI) response, acid is extruded into the medium . Medium acidification is correlated with cell Na uptake . Both medium acidification and cell Na uptake are blocked by 10-s M amiloride or by replacing medium Na with K or choline . Perturbations that increase cell Na uptake (such as increasing medium osmolality) also increase medium acidification . As the medium becomes more acidic, the cells become more alkaline . These changes in cell and medium pH are increased if pH equilibration across the cell membrane is prevented by inhibiting the anion exchanger with SITS (4-acetamido-4'-isothiocyano-2,2'-stilbene disulfonic acid) . The quantity of acid extruded by SITS-treated cells is the same as the quantity of Na gained, which strongly suggests 1 :1 exchange of Na for H . Cell enlargement in SITS-treated cells results from the exchange of osmotically active Na ions for H ions that are not osmotically active when combined with cellular buffers . Previous evidence indicates that the normal VRI response involves an increase in the cellular content of Cl as well as Na .We show that SITS completely blocks net Cl uptake, which suggests that Cl enters via the anion exchanger. SITS also slows Na entry, presumably as a result of the above-mentioned increase in cell pH caused by SITS . We suggest that the initial event in the VRI response is net Na uptake via a Na/H exchanger, and that net Cl uptake results from secondary Cl/HCO 3 exchange via the anion exchanger .

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