Ionic Mechanisms of Cell Volume Regulation in Isolated Rat Hepatocytes

1989 ◽  
Vol 574 (1 Bicarbonate,) ◽  
pp. 385-387 ◽  
Author(s):  
JAMES G. CORASANTI ◽  
DERMOT GLEESON ◽  
JAMES L. BOYER
Keyword(s):  
Cell Volume ◽  
Volume Regulation ◽  
Cell Volume Regulation ◽  
Rat Hepatocytes ◽  
Isolated Rat Hepatocytes ◽  
Ionic Mechanisms ◽  
Isolated Rat

Effects of osmotic stresses on isolated rat hepatocytes. I. Ionic mechanisms of cell volume regulation

1990 ◽  
Vol 258 (2) ◽  
pp. G290-G298
Author(s):  
J. G. Corasanti ◽  
D. Gleeson ◽  
J. L. Boyer
Keyword(s):  
Cell Volume ◽  
Regulatory Volume Decrease ◽  
Cell Volume Regulation ◽  
Rat Hepatocytes ◽  
Relative Volume ◽  
Disulfonic Acid ◽  
Isolated Rat Hepatocytes ◽  
Hypertonic Stress ◽  
Anion Conductance ◽  
Ionic Mechanisms

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.

ClC-2 chloride channels contribute to HTC cell volume homeostasis

2001 ◽  
Vol 280 (3) ◽  
pp. G344-G353 ◽  
Author(s):  
Richard M. Roman ◽  
Roderic L. Smith ◽  
Andrew P. Feranchak ◽  
Gerald H. Clayton ◽  
R. Brian Doctor ◽  
...  
Keyword(s):  
Cell Volume ◽  
Volume Regulation ◽  
Chloride Channels ◽  
Purinergic Receptor ◽  
Cell Volume Regulation ◽  
Rat Hepatocytes ◽  
Hek 293 ◽  
Whole Cell Patch Clamp ◽  
293 Cells ◽  
Htc Cells

Membrane Cl−channels play an important role in cell volume homeostasis and regulation of volume-sensitive cell transport and metabolism. Heterologous expression of ClC-2 channel cDNA leads to the appearance of swelling-activated Cl−currents, consistent with a role in cell volume regulation. Since channel properties in heterologous models are potentially modified by cellular background, we evaluated whether endogenous ClC-2 proteins are functionally important in cell volume regulation. As shown by whole cell patch clamp techniques in rat HTC hepatoma cells, cell volume increases stimulated inwardly rectifying Cl−currents when non-ClC-2 currents were blocked by DIDS (100 μM). A cDNA closely homologous with rat brain ClC-2 was isolated from HTC cells; identical sequence was demonstrated for ClC-2 cDNAs in primary rat hepatocytes and cholangiocytes. ClC-2 mRNA and membrane protein expression was demonstrated by in situ hybridization, immunocytochemistry, and Western blot. Intracellular delivery of antibodies to an essential regulatory domain of ClC-2 decreased ClC-2-dependent currents expressed in HEK-293 cells. In HTC cells, the same antibodies prevented activation of endogenous Cl−currents by cell volume increases or exposure to the purinergic receptor agonist ATP and delayed HTC cell volume recovery from swelling. These studies provide further evidence that mammalian ClC-2 channel proteins are functional and suggest that in HTC cells they contribute to physiological changes in membrane Cl−permeability and cell volume homeostasis.

Effects of osmotic stresses on isolated rat hepatocytes. II. Modulation of intracellular pH

1990 ◽  
Vol 258 (2) ◽  
pp. G299-G307 ◽  
Author(s):  
D. Gleeson ◽  
J. G. Corasanti ◽  
J. L. Boyer
Keyword(s):  
Intracellular Ph ◽  
Volume Regulation ◽  
Regulatory Volume Decrease ◽  
Cell Volume Regulation ◽  
Rat Hepatocytes ◽  
Transport Systems ◽  
Disulfonic Acid ◽  
Isolated Rat Hepatocytes ◽  
Intracellular Buffering Capacity ◽  
Volume Decrease

To assess the roles of acid-base transport systems in cell volume regulation in rat hepatocytes, intracellular pH (pHi) was measured in subconfluent monolayers loaded with 2'-7'-bis(carboxyethyl)-5,6-carboxyfluorescein (BCECF) after exposure to hypotonic and relative hypertonic media, interventions that stimulate regulatory volume decrease (RVD) and increase (RVI), respectively. During RVD, pHi decreased from 6.98 +/- 0.11 to 6.85 +/- 0.08 in the absence of HCO3- and from 7.26 +/- 0.10 to 7.19 +/- 0.06 in its presence. Omission of Na+ or addition of 1 mM amiloride prevented the decline in pHi. Acute withdrawal or replacement of Na+ in hypotonic medium resulted in a slower rate of fall or recovery in pHi, respectively, than when the same maneuvers were carried out in isotonic medium. In contrast, during RVI, pHi increased from 6.86 +/- 0.11 to 7.15 +/- 0.15 in the absence of HCO3-, a rise in pHi that was also completely abolished by Na+ removal or by 1 mM amiloride. In the presence of HCO3-, the rise in pHi was less marked than in its absence, although net acid efflux was greater because of a greater intracellular buffering capacity. Cl- removal in the presence of HCO3- had no effect on the change in pHi during either RVD or RVI. Perfusion with 0.5 mM 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) during RVD lowered pHi further and accentuated the subsequent pHi rise seen after the return to isotonic medium. These data suggest that Na(+)-H+ exchange in rat hepatocytes is downregulated during RVD and activated during RVI. Cl(-)-HCO3- exchange does not appear to be involved in hepatocyte volume regulation.

Role of chloride ions in liver cell volume regulation

1991 ◽  
Vol 261 (2) ◽  
pp. G340-G348 ◽  
Author(s):  
P. Haddad ◽  
J. S. Beck ◽  
J. L. Boyer ◽  
J. Graf
Keyword(s):  
Cell Volume ◽  
Initial Rate ◽  
Regulatory Volume Decrease ◽  
Cell Volume Regulation ◽  
Rat Hepatocytes ◽  
Liver Cells ◽  
Chloride Ions ◽  
Isolated Rat Hepatocytes ◽  
Hypotonic Stress

Hypotonic swelling of liver cells is followed by regulatory volume decrease (RVD), which has been shown to involve facilitated release of K+. In this study, the role of C1- in RVD was examined by videoplanimetric analysis of cell volume and measurement of membrane potential (Vm) and resistance (Rm) in single isolated rat hepatocytes, and by measurement of 36Cl efflux in the isolated perfused liver preloaded with the isotope. Liver cells subjected to hypotonic stress by removal of 50 mM external NaCl (70% of control osmolality) swelled from an initial volume of 6.68 +/- 0.77 to 8.27 +/- 0.88 pl (24.3 +/- 3.4% increase) within 1 min and exhibited RVD at an initial rate of 0.26 +/- 0.01 pl/min. A step decrease in external Cl- accelerated the initial rate of RVD to 0.53 +/- 0.08 pl/min. RVD was abolished in cells that had been depleted of Cl-. Vm and Rm displayed biphasic responses to hypotonic stress. An initial (less than 15 s) hyperpolarization of Vm from -35.4 +/- 2.2 to -38.8 +/- 2.6 mV was followed by a gradual depolarization reaching -30.2 +/- 2.0 mV by 1 min. In parallel, Rm initially (less than 15 s) increased from 101 +/- 13 to 121 +/- 17 M omega (19 +/- 3% increase) and then declined to 55 +/- 4 M omega (59 +/- 4% of initial Rm) within 1 min. These changes were reversible upon return to isotonicity.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals Volume-regulatory K+ efflux during concentrative uptake of alanine in isolated rat hepatocytes

1984 ◽  
Vol 221 (1) ◽  
pp. 265-268 ◽  
Author(s):  
L ∅ Kristensen ◽  
M Folke
Keyword(s):  
Cell Volume ◽  
Rate Constant ◽  
Rat Hepatocytes ◽  
Water Volume ◽  
Bicarbonate Buffer ◽  
Isolated Rat Hepatocytes ◽  
Volume Increase ◽  
Regulatory Response ◽  
Cellular Water ◽  
Isolated Rat

Changes in cell volume and 42K+ efflux associated with concentrative alanine uptake were studied in isolated rat hepatocytes suspended in Krebs-Ringer bicarbonate buffer. After addition of 10 mM-alanine, cellular water volume increased by 15% and the rate constant of 42K+ efflux by 250%. Alanine-induced 42K+ efflux was abolished by quinine and was strongly decreased when the cell-volume increase was counteracted by sucrose. The results suggest that K+ efflux during alanine uptake is implicated in a volume-regulatory response.

scholarly journals Effects of hydration state on the synthesis and secretion of triacylglycerol by isolated rat hepatocytes. Implications for the actions of insulin and glucagon on hepatic secretion

1995 ◽  
Vol 312 (1) ◽  
pp. 57-62 ◽  
Author(s):  
V A Zammit
Keyword(s):  
Water Content ◽  
Cell Volume ◽  
Rat Hepatocytes ◽  
Cell Swelling ◽  
Cell Water ◽  
Cell Shrinkage ◽  
Isolated Rat Hepatocytes ◽  
Acute Changes ◽  
Isolated Rat

The effects of hepatocyte volume on the secretion of triacylglycerol were studied in order to test the suggestion that increases in the portal concentrations of osmolyte amino acids and metal ions during the prandial/early-absorptive phase may be involved in mediating the acute changes in glycerolipid metabolism observed in vivo [Zammit (1995) Biochem Soc. Trans. 23, 506-511]. Incubation of isolated rat hepatocytes with hypo-osmotic medium or in the presence of glutamine (in the presence or absence of leucine), conditions which gave an increase in cell water content of between 8 and 27%, resulted in a decrease in the rate of [14C]triacylglycerol (TAG) secretion when [14C]palmitate was used as substrate. The inhibition was proportional to the increase in cell water content. At low exogenous palmitate concentration (0.05 mM), the inhibition of [14C]TAG secretion was accompanied by a marked shift in the incorporation of label from TAG to phospholipid. In the presence of 0.5 mM palmitate this effect was attenuated, and in the presence of 1 mM palmitate it was abolished. Increased cell volume associated with incubation of hepatocytes with glutamine (in the presence or absence of leucine) also resulted in a decrease in the fraction of newly labelled TAG that was secreted into the medium. Decreased cell volume, achieved by incubation of hepatocytes with hyperosmotic medium (sufficient to decrease cell water content by approx. 9%) decreased overall [14C]TAG secretion, but did not affect the amount of label that was incorporated into phospholipid as a fraction of that incorporated into total glycerolipids. Cell shrinkage, however, diminished the fraction of newly labelled [14C]TAG that was secreted. When intracellular TAG was prelabelled with [3H]glycerol, it was found that cell shrinkage markedly inhibited (preformed) [3H]TAG secretion, whereas cell swelling did not affect this route of TAG secretion. The data are discussed in terms of the possible action of changes in cell hydration at the different loci at which hepatocyte TAG secretion is controlled, with reference to previous observations that both insulin and glucagon are able to inhibit TAG secretion in cultured rat hepatocytes and HepG2 cells.

Sign in / Sign up

Export Citation Format

Share Document