Volume regulation determinants in isolated proximal tubules in hypertonic medium

Isolated nonperfused proximal tubules maintained their cell volume at a constant level [isovolumetric regulation (IVR)] when extracellular osmolality was gradually increased above 295 mosmol at 1.5 mosmol/min. Cell volume remained constant between 295 and 361 ± 7 mosmol in medium containing HCO₃^--CO₂ as the major buffer. In N-2-hydroxyethyl piperazine-N'-2-ethanesulfonic acid-buffered medium, IVR maintained cell size constant between 295 and 370 ± 11 mosmol; thus, the volume regulatory process did not require the presence of external HCO₃^--CO₂. Omission of medium phosphate and sulfate from defined bathing medium had no effect on the volume regulatory process, whereas acetate (5 mM) was required to maintain cell volume constant. Hypertonic IVR was inhibited by acetazolamide (10^-6 M), 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (2.5 x 10^-5 M), and, in medium in which choline replaced all but 25 mM of Na⁺, amiloride (1 mM). These results suggest the involvement of carbonic anhydrase, Cl^-, and HCO₃^- transport, and Na-H exchange in IVR; and provide indirect support for the view that IVR in hyperosmotic medium depends on the intracellular accumulation of electrolytes resulting from altered rates of membrane ionic exchange mechanisms.