Effects of osmotic stresses on isolated rat hepatocytes. I. Ionic mechanisms of cell volume regulation.
Isolated hepatocyte suspensions were exposed to hypotonic and hypertonic stresses and serial cell volume measurements were made with an electronic particle size analyzer. With the exposure to hypotonic (160 mosM) buffer, hepatocytes swelled within 30-60 s as osomometers [relative volume (RV) = 1.44 +/- 0.08] and subsequently underwent regulatory volume decrease (RVD) back toward the resting (isotonic) level (1.16 +/- 0.05). This volume recovery was blocked by 65 mM extracellular K+ concentration and inhibited by barium (1 mM) and quinine (0.5 mM) but not by bumetanide (0.1 mM). Chloride depletion inhibited RVD by approximately 40% while 0.5 mM 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) blocked the recovery by almost 90%. Calcium deprivation had no effect on RVD, nor did ouabain, amiloride, or sodium replacement. When exposed to buffer made hypertonic by addition of 200 mM sucrose, cells shrunk as osmometers (RV = 0.74 +/- 0.02) but did not exhibit regulatory volume increase (RVI). However, when cells that had first undergone RVD were reexposed to isotonic medium (relative hypertonic stress) RVI could be demonstrated from RV 0.77 +/- 0.17 to 0.91 +/- 0.20. This response was dependent on sodium, partially dependent on bicarbonate and chloride, and inhibited by the Na(+)-H+ exchange inhibitor amiloride (1 mM) but not by DIDS. Our findings suggest that RVD in rat hepatocytes is mediated by quinine- and barium-sensitive K+ conductance and DIDS-sensitive anion conductance, which is partly accounted for by Cl-; RVI is mediated by activation of Na(+)-H+ exchange coupled with a bicarbonate- and chloride-dependent but DIDS-insensitive process.
Corasanti
J G
JG
Department of Medicine and Liver Center, Yale University School of Medicine, New Haven, Connecticut 06510.
Gleeson
D
D
Boyer
J L
JL
D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D000584:Amiloride / Q000494:pharmacology; D000818:Animals; D001464:Barium / Q000494:pharmacology; D001639:Bicarbonates / Q000494:pharmacology; D002034:Bumetanide / Q000494:pharmacology; D002478:Cells, Cultured; D002712:Chlorides / Q000494:pharmacology; D006982:Hypertonic Solutions; D007038:Hypotonic Solutions; D007700:Kinetics; D008099:Liver / Q000166:cytology* / Q000187:drug effects / Q000502:physiology; D008297:Male; D009994:Osmolar Concentration; D011189:Potassium Chloride / Q000494:pharmacology; D011803:Quinine / Q000494:pharmacology; D051381:Rats; D011919:Rats, Inbred Strains; D012965:Sodium Chloride / Q000494:pharmacology
D001639:Bicarbonates; D002712:Chlorides; D006982:Hypertonic Solutions; D007038:Hypotonic Solutions; D002034:Bumetanide; D001464:Barium; D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; D012965:Sodium Chloride; D011189:Potassium Chloride; D000584:Amiloride; D011803:Quinine; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid