REGULATION OF ION CONTENT AND CELL-VOLUME IN ISOLATED RAT RENAL IMCD CELLS UNDER HYPERTONIC CONDITIONS

The adaptive responses of cell volume and intracellular inorganic elec trolytes to hypertonicity were investigated in isolated inner medullar y collecting duct (IMCD) cells in vitro. Intracellular water and eleme nt content were determined simultaneously by a rapid centrifugation te chnique and electron-probe microanalysis, respectively. In the absence of adenosine 3',5'-cyclic monophosphate (cAMP), cells isolated at 600 mosM (268 mM NaCl) shrank in 900 mosM buffer (418 mM NaCl) within 100 s to 79 +/- 2% of the control volume and showed no regulatory volume increase (RVI). K+ content decreased steadily during the first 5 min t o 18% below control. K+ concentration initially increased 25 +/- 2% ab ove control and was normalized after 5 min to 144 +/- 10 mM (n = 6). N a+ and Cl- content increased slowly during the 60-min incubation perio d; at this point intracellular Naf and Cl- concentrations were 97 +/- 6 and 59 +/- 7% (n = 6) above control levels (75 +/- 12 mM Na+ and 143 +/- 29 mM Cl-), respectively. In the presence of 10(-4) M 8-bromo-cAM P (8-BrcAMP; used as a substitute for antidiuretic hormone), IMCD cell s showed RVI within 5 min. K+ content initially dropped 18 +/- 2% belo w control and recovered during the next 4 min but was 12 +/- 4% below control after 60 min. K+ concentration showed similar changes. Na+ and Cl- content increased to a larger extent than in the absence of 8-Brc AMP to 95 +/- 10% (Na+) and 47 +/- 6% (Cl-) above control after 60 min , whereas Na+ and Cl- concentrations were the same as without 8-BrcAMP . 8-BrcAMP-dependent RVI was almost completely prevented by 10(-4) M b umetanide, 10(-3) M amiloride, or 10(-3) M -acetamido-4'-isothiocyanos tilbene-2,2'-disulfonic acid. These results demonstrate that IMCD cell s in vitro in hypertonic solutions undergo RVI only in the presence of 8-BrcAMP. During and after RVI, the gain in total intracellular inorg anic electrolytes is too small to account for the volume increase; thu s additional (possibly organic) osmolytes have to be invoked.