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Altered cytoskeletal protein function and gene expression during cell volume changes in rat hepatocytes
S19 WORKING PARTY 3: Liver cell volume and liver cell function
ALTERED CYTOSKELETAL PROTEIN FUNCTION A N D OENE EXPRESSION DURINO CELL VOLUME CHANOES IN RAT HEPATOCYTES
C Stournaras*. P.A.Theodoropoulos.* B. StolL D. H~iussingeL *Dept. Biochemistry, School of Medicine, University of Crete, Heraklion, Greece and Medizinische Universit~itsklinik~ Freiburg, Germany. Alterations of hepatocellular hydration, reflecting changes of liver cell volume, occur within minutes under the influence of hormones, amino acids, or oxidative stress. These volume changes were recently recognized as another second messenger of hormone and amino acid action modifying hepatic metabolism. Here we report on the relationship between cell volume changes and alterations of cytoskeletal protein function and gene expression. Exposure of isolated rat hepatocytes to conditions inducing cell swelling were followed by a rapid actin depolymerization, as indicated by lowered intracellular G-actin content without affecting the total actin level. Thus hypotonic incubation of rat hepatocytes reduced the cellular G/total actin ratio by 15.5+1,4% (n--'7). Similarly, following isotonic cell swelling by addition of glutamine (10mM), or insulin (100nM) the G/total actin ratio was decreased by 13,51:2.1% (n=5) and 14,1-t:1,1% (n=ll) respectively. These effects on the G/total actin ratio occurred within 1 rain. and persisted for at least 2 hours. Morover, after 120 min exposure of rat hepatocytes to hypotonic media, glutamine, or insulin, the actin mRNA levels were increased 2,4-, 2,0- and 3,6-fold respectively. Tubulin mRNA levels increased as well 1,9-, 2,7- and 2,1fold in isolated rat hepatocytes exposed for 120 rain to hypotonic medium, glutamine, or insulin respectively. Finally, as shown by immunofluorescence microscopy experiments in the absence or presence of antimicrotubular agents (triethyllead, colchicine), an apparent stabilization of the microtubule network became evident in hepatocytes exposed to cell swelling conditions. From these results we suggest that cytoskeletal proteins and structures are involved in transducing the effects of cell swelling on cellular function.
E L E C T R O P H Y S I O L O G Y OF CELL VOLUME REGULATION IN HEPATOCYTES AND VASCULAR SMOOTH MUSCLE CELLS. F. Lallg. J. Ditlevsen. A. Busch. H. Aofel. S. Breit. P. Kritme{t-Drews. J. Fingerle. S. Meierko-rd. H. Heinle. D. Htlussinger*. A. Kolb. Institute for Physiology, University of Ttibingen, and * University Hospital Freiburg, Germany. Exposure of perfused liver to hypotonic extracellular fluid leads to a cell volume regulatory K + release, paralleled by a variable transient increase of perfusion resistance. Electrophysiological experiments have been performed to elucidate cellular mechanisms involved. The potential difference across the cell membrane (PD) of hepatocytes was -44 + 5 mV (n -- 10). A decrease of extracellular osmolarity by 60 mOsm led to a transient increase of PD by -11 + 3 mV, followed by partial decline of the PD by +7 + 4 inV. Readdition of isotonic extracellular fluid resulted in a transient decrease of PD by +16 + 3 mV. In vascular smooth muscle cells PD was -51 + 5 mV. Exposure to hypotonic extracellular fluid decreased PD by +13 + 1 mV and increased CI- selectivity of the cell membrane from 16 + 8% to 32 + 4 %. Furthermore, it led to contraction of vascular strips, which was inhibited by Ca ++ channel block. In conclusion, the K + release from perfused liver is at least partially due to activation of K + channels leading to hyperpolarization, while cell shrinkage inhibits the K + channels, leading to depolarization. The vasoconstriction following exposure to hypotonic perfusate is due to activation of anion channels, leading to depolarization,and subsequent activation of voltage sensitive Ca++ channels.
CELL SWELLING AND THE CONTROL OF GLYCOGEN METABOLISM IN RAT HEPATOCYTES
ACTIVATION OF MAP KINASES BY INSULIN IN HEPATOCYTES IS DEPENDENT ON Na+UPTAKE
L.A. Oustafson, A.J. Meijer. Academic Medical Center, University of Amsterdam, E.C. Slater Institute, P.O. Box 22700, 1100 DE Amsterdam, Amsterdam, The Netherlands.
M. Peak, S,J. Yeamnan, L. A_gLu.~ Dept. Medicine, Univeristy of Newcastle upon Tyne Newcastle upon Tyne, NE2 4HH, U.K.
Cell swelling is known to act as an anabolic signal. An increase in hepatocyte volume by either an increase in intraceUular osmolarity, due to Na÷ dependent influx of amino acids, or by hypo-osmolarity of the extracellular fluid, strongly activates glycogen synthesis from glucose. The mechanism of activation of glycogen synthesis by cell swelling involves activation of glycogen synthase phosphatase by the fall in intracellular chloride, an inhibitor of the enzyme, combined with a rise in intracellular glutamate and aspartate which are activators of glycogen synthase phosphatase (Meijer et al. (1992) J. Biol. Chem. 267, 58235828). In hepatocytes from fasted rats, under glycogen synthesizing conditions, active glycogen phosphorylase, in addition to active glycogen synthase, was associated with newly synthesized glycogen. Experiments with [14C]glucose indicated that glycogen cycling was significant and increased with increasing size of the intracellular glycogen pool. Cell swelling increased net glycogen synthesis by activating flux through glycogen synthase with little effect on phosphorylase flux. Proline stimulated net glycogen synthesis by activation of flux through glycogen synthase and inhibiting that through phosphorylase. In hepatocytes from fed rats, with a large glycogen pool, the cycling of glycogen became extensive due to the increase in glycogen associated phosphorylase activity.
A c t i v a t i o n of m i t o g e n - a c t i v a t e d protein (MAP) k i n a s e s by i n s u l i n h a s b e e n i m p l i c a t e d in the m e c h a n i s m by which insulin s t i m u l a t e s g l y c o g e n synthesis. In hepatocytes the stimulation ot glycogen s y n t h e s i s b y i n s u l i n is d e p e n d e n t on u p t a k e o f extracellular'Na + and an increase in cell volume. We investigated the role of Na+-mediated cell swellling and of the microfilament cytoskeleton in the activation of MAP-kinases by insulin. MAP kinase activity was d e t e r m i n e d in triton-X 100 e x t r a c t s of h e p a t o c y t e s fractionated by chromatography on Mono-Q. Insulin (100nM, 5min) stimulated 4 p e a k s of M A P - k i n a s e activity in a balanced-salts medium and this effect of insulin was counteracted by glucagon. The activation o f M A P - k i n a s e s by i n s u l i n w a s a b o l i s h e d in an isoosmolar Na+-free medium. However, hypoosmotic swelling activated MAP-kinases in a Na+-free medium indicating that activation of MAP-kinases is dependent on cell s w e l l i n g but d o e s not h a v e an a b s o l u t e requirement for lqa +. Cytochalasin-D which disrupts the microfilament cytoskeleton abolished the activataon of MAP-kinase by insulin suggesting that cell swelling as well as an intact cytoskeleton are essential for the activation of MAP kinase by insulin.
ALTERED CYTOSKELETAL PROTEIN FUNCTION A N D OENE EXPRESSION DURINO CELL VOLUME CHANOES IN RAT HEPATOCYTES
C Stournaras*. P.A.Theodoropoulos.* B. StolL D. H~iussingeL *Dept. Biochemistry, School of Medicine, University of Crete, Heraklion, Greece and Medizinische Universit~itsklinik~ Freiburg, Germany. Alterations of hepatocellular hydration, reflecting changes of liver cell volume, occur within minutes under the influence of hormones, amino acids, or oxidative stress. These volume changes were recently recognized as another second messenger of hormone and amino acid action modifying hepatic metabolism. Here we report on the relationship between cell volume changes and alterations of cytoskeletal protein function and gene expression. Exposure of isolated rat hepatocytes to conditions inducing cell swelling were followed by a rapid actin depolymerization, as indicated by lowered intracellular G-actin content without affecting the total actin level. Thus hypotonic incubation of rat hepatocytes reduced the cellular G/total actin ratio by 15.5+1,4% (n--'7). Similarly, following isotonic cell swelling by addition of glutamine (10mM), or insulin (100nM) the G/total actin ratio was decreased by 13,51:2.1% (n=5) and 14,1-t:1,1% (n=ll) respectively. These effects on the G/total actin ratio occurred within 1 rain. and persisted for at least 2 hours. Morover, after 120 min exposure of rat hepatocytes to hypotonic media, glutamine, or insulin, the actin mRNA levels were increased 2,4-, 2,0- and 3,6-fold respectively. Tubulin mRNA levels increased as well 1,9-, 2,7- and 2,1fold in isolated rat hepatocytes exposed for 120 rain to hypotonic medium, glutamine, or insulin respectively. Finally, as shown by immunofluorescence microscopy experiments in the absence or presence of antimicrotubular agents (triethyllead, colchicine), an apparent stabilization of the microtubule network became evident in hepatocytes exposed to cell swelling conditions. From these results we suggest that cytoskeletal proteins and structures are involved in transducing the effects of cell swelling on cellular function.
E L E C T R O P H Y S I O L O G Y OF CELL VOLUME REGULATION IN HEPATOCYTES AND VASCULAR SMOOTH MUSCLE CELLS. F. Lallg. J. Ditlevsen. A. Busch. H. Aofel. S. Breit. P. Kritme{t-Drews. J. Fingerle. S. Meierko-rd. H. Heinle. D. Htlussinger*. A. Kolb. Institute for Physiology, University of Ttibingen, and * University Hospital Freiburg, Germany. Exposure of perfused liver to hypotonic extracellular fluid leads to a cell volume regulatory K + release, paralleled by a variable transient increase of perfusion resistance. Electrophysiological experiments have been performed to elucidate cellular mechanisms involved. The potential difference across the cell membrane (PD) of hepatocytes was -44 + 5 mV (n -- 10). A decrease of extracellular osmolarity by 60 mOsm led to a transient increase of PD by -11 + 3 mV, followed by partial decline of the PD by +7 + 4 inV. Readdition of isotonic extracellular fluid resulted in a transient decrease of PD by +16 + 3 mV. In vascular smooth muscle cells PD was -51 + 5 mV. Exposure to hypotonic extracellular fluid decreased PD by +13 + 1 mV and increased CI- selectivity of the cell membrane from 16 + 8% to 32 + 4 %. Furthermore, it led to contraction of vascular strips, which was inhibited by Ca ++ channel block. In conclusion, the K + release from perfused liver is at least partially due to activation of K + channels leading to hyperpolarization, while cell shrinkage inhibits the K + channels, leading to depolarization. The vasoconstriction following exposure to hypotonic perfusate is due to activation of anion channels, leading to depolarization,and subsequent activation of voltage sensitive Ca++ channels.
CELL SWELLING AND THE CONTROL OF GLYCOGEN METABOLISM IN RAT HEPATOCYTES
ACTIVATION OF MAP KINASES BY INSULIN IN HEPATOCYTES IS DEPENDENT ON Na+UPTAKE
L.A. Oustafson, A.J. Meijer. Academic Medical Center, University of Amsterdam, E.C. Slater Institute, P.O. Box 22700, 1100 DE Amsterdam, Amsterdam, The Netherlands.
M. Peak, S,J. Yeamnan, L. A_gLu.~ Dept. Medicine, Univeristy of Newcastle upon Tyne Newcastle upon Tyne, NE2 4HH, U.K.
Cell swelling is known to act as an anabolic signal. An increase in hepatocyte volume by either an increase in intraceUular osmolarity, due to Na÷ dependent influx of amino acids, or by hypo-osmolarity of the extracellular fluid, strongly activates glycogen synthesis from glucose. The mechanism of activation of glycogen synthesis by cell swelling involves activation of glycogen synthase phosphatase by the fall in intracellular chloride, an inhibitor of the enzyme, combined with a rise in intracellular glutamate and aspartate which are activators of glycogen synthase phosphatase (Meijer et al. (1992) J. Biol. Chem. 267, 58235828). In hepatocytes from fasted rats, under glycogen synthesizing conditions, active glycogen phosphorylase, in addition to active glycogen synthase, was associated with newly synthesized glycogen. Experiments with [14C]glucose indicated that glycogen cycling was significant and increased with increasing size of the intracellular glycogen pool. Cell swelling increased net glycogen synthesis by activating flux through glycogen synthase with little effect on phosphorylase flux. Proline stimulated net glycogen synthesis by activation of flux through glycogen synthase and inhibiting that through phosphorylase. In hepatocytes from fed rats, with a large glycogen pool, the cycling of glycogen became extensive due to the increase in glycogen associated phosphorylase activity.
A c t i v a t i o n of m i t o g e n - a c t i v a t e d protein (MAP) k i n a s e s by i n s u l i n h a s b e e n i m p l i c a t e d in the m e c h a n i s m by which insulin s t i m u l a t e s g l y c o g e n synthesis. In hepatocytes the stimulation ot glycogen s y n t h e s i s b y i n s u l i n is d e p e n d e n t on u p t a k e o f extracellular'Na + and an increase in cell volume. We investigated the role of Na+-mediated cell swellling and of the microfilament cytoskeleton in the activation of MAP-kinases by insulin. MAP kinase activity was d e t e r m i n e d in triton-X 100 e x t r a c t s of h e p a t o c y t e s fractionated by chromatography on Mono-Q. Insulin (100nM, 5min) stimulated 4 p e a k s of M A P - k i n a s e activity in a balanced-salts medium and this effect of insulin was counteracted by glucagon. The activation o f M A P - k i n a s e s by i n s u l i n w a s a b o l i s h e d in an isoosmolar Na+-free medium. However, hypoosmotic swelling activated MAP-kinases in a Na+-free medium indicating that activation of MAP-kinases is dependent on cell s w e l l i n g but d o e s not h a v e an a b s o l u t e requirement for lqa +. Cytochalasin-D which disrupts the microfilament cytoskeleton abolished the activataon of MAP-kinase by insulin suggesting that cell swelling as well as an intact cytoskeleton are essential for the activation of MAP kinase by insulin.