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 electrolytes to hypertonicity were investigated in isolated inner medullary collecting duct (IMCD) cells in vitro. Intracellular water and element content were determined simultaneously by a rapid centrifugation technique 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 to 18% below control. K+ concentration initially increased 25 +/- 2% above control and was normalized after 5 min to 144 +/- 10 mM (n = 6). Na+ and Cl- content increased slowly during the 60-min incubation period; 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-cAMP (8-BrcAMP; used as a substitute for antidiuretic hormone), IMCD cells showed RVI within 5 min. K+ content initially dropped 18 +/- 2% below 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-BrcAMP 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 bumetanide, 10(-3) M amiloride, or 10(-3) M 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid. These results demonstrate that IMCD cells in vitro in hypertonic solutions undergo RVI only in the presence of 8-BrcAMP. During and after RVI, the gain in total intracellular inorganic electrolytes is too small to account for the volume increase; thus additional (possibly organic) osmolytes have to be invoked.