- Email: [email protected]
cAMP and volume activated chloride conductance in rat hepatocytes
308A 805
807
AASLD
ABSTRACTS
HEPATOLOGY O c t o b e r 1995
cAMP AND VOLUME ACTIVATED CHLORIDE CONDUCTANCE IN RAT HEPATOCYTES. X-J Mere, and SA Weinman. University of Texas Medical Branch, Galveston, TX 77555 Chloride channels account for a major fraction of sinusoidal membrane conductance in hepatocytes and play a role in volume regulation. Our aim was to compare the effects of cAMP and cell swelling on hepatoeyte membrane condactances since both stimuli are known to activate chloride channels in many cell types. METHODS: Primary isolated hepatoeytes were prepared from rat liver using the two-step coIlagenase perfusion procedure. Whole-cell currents were measured with the patch-clamp technique. Cell volume was estimated from eross-seetional images. RESULTS: Cells were patched with cAMP (100 ~tM) included in the pipet. Initial conductance was linear (3. l+1.5 nS) but within 3 rain a large outwardly-rectifying conductance appeared (23.8+_3.0 nS, n=20). This conductance was highly anion selective with a CI- : K+ permeability ratio of 12.1. It was abolished by Ca2* chelation in the pipet and removal from the bath, completely inhibited by the anion channel blocker NPPB (200 laM, n=6), and partially inhibited by the stilbene derivative D1DS (150 ~tM, n=7). Halide selectivitywas I'>Br>CI-. Reduction of bath esmolality from 300 to 250 mOsm/kg in the absence of cAMP activated a similar outwardly-rectifying CI" conductance (ORCC) of 20.5+_2.2 nS (n=4). The volume-activated ORCC had identical ion-selectivity and blocker sensitivity as the cAMP-activated conductance. 8-Br-cAMP (500 I.tM) in the bath also activated ORCC but did not cause cell swelling. However, cell shrinkage with hyperosmotie bath (350 mOsm/kg), either before or after exposure to cAMP, inhibited the cAMP-activated ORCC. The two methods of activation of the ORCC, cAMP and cell swelling, were not additive. CONCLUSION: Cyclic AMP and cell swelling activate the same outward-rectifying CI- conductance in rat hepatocytes. In both eases the conductance is blocked by cell shrinkage. It has the properties of the volumeactivated ORCC present in many cell types and is functionally distinct from CFTR-assoeiated chloride conductance. Cyclic AMP may activate by altering the volume set-point of the channel. (Supported by NIH DK42917)
806 PROTEIN PHOSPHATASE 2A-MEDIATED EFFECTS ON
MODULATION OF KUPFFER CELL FUNCTION BY ATRIAL NATRIURETIC PEPTIDE (ANP). M Bilzer, G Paumgartner, AL Gerbes. Department of Medicine II, Klinikum Gro~hadem, Ludwig-MaximiliansUniversity of Munich, Munich, Germany
808 CHRONIC ETHANOL CONSUMPTION IMPAIRS RECEPTOR
Recently we have shown that ANP protects the liver against cell damage induced by activated Kupffer cells (KC). This effect may be due to a protective effect on the hepatocyte or to modulation of Kupffer cell function. Therefore, we evaluated the influence of ANP on KC function determined by particle phagocytosis in the perfused rat liver. Methods: Livers of male Sprague-Dawley rats were perfused with Krebs-Henseleit buffer (pH 7.4, 37°C) in a nonreciroulating fashion. Zymosan (150 lug/ml), cell wall particles from yeast which are selectively taken up by KC, were infused from 40-46 min (n=6). ANP (2 and 200 nM) or 8-Br-cGMP (50 pM), an analogue of cGMP, were infused from 30-50 min after starting perfusion (n=4 each). Uptake of zymosan was assessed by the difference in absorbance (540 nm) of the inflow and outflow perfusion buffer.
Results: (x.±SD). Infusion of zymosan into the portal vein resulted in zymosan extraction of 77+14%, 64+11% and 68+11% at 42, 44 and 46 min of perfusion time. When livers were perfused with 200 nM ANP, zymosan uptake decreased to 46±12%, 41±11% (p<0.05) and 56±9%. Similar results were obtained with 2 nM ANP which reduced zymosan uptake to 47+12%, 35+11% (p<0.05) and 51+7%. Infusion of 50 luM 8Br-cGMP, an analogue of cGMP, the second messenger of ANP action, resulted in a comparable reduction (p<0.05) of zymosan uptake at 44 min (40±11%) and 46 min (47+11%). Conclusions: ANP inhibits zymosan phagocytosis by KC in the perfused rat liver. This effect was mimicked by 8-Br-cGMP supporting an ANP-receptor-guanylate cyclase-cGMP mediated mechanism. Therefore, protective effects eadier described with ANP and 8-Br-cGMP could be related to a direct modulation of KC-function.
MICROTUBULE-DEPENDENT VESICLE TRANSPORT AND RECEPTOR-MEDIATED ENDOCYTOSIS IN HEPATOCYTES. SF Hamm-Alvarez. X-H Wei. N Berndt. and M Runneaar, Center for Liver Diseases and Departments of Pharmaceutical Sciences, Pathology and Medicine, University of Southern California, Los Angeles CA Although microtubule (MT)-dependent transport has been implicated in membrane trafficking in hepatocytes, little is known about how regulation of the components of MT-based transport impacts on membrane recycling. We have explored the role of protein phosphorylation in the regulation of MTdependent vesicle transport and recaptor-mediated endocytosis, specifically the contributions of protein phosphatases (PP), PP1 and PP2A. We compared the effects of two PP inhibitors with different potency, microcystin (MCYST, inhibits PP1 and PP2A equally) and okadaic acid (OKA, inhibits only PP2A at the doses used hera with phosphorylase as substrate). By video microscopy, we have identified a population of vesicles that move on MTs using nocodazole inhibition. The frequency velocity, and run length of these vesicles were significantly decreased in a dose-dependent manner from 50-500 nM of each PP inhibitor. To understand how changes in MT-based transport impacted on receptor-mediated endocytosis, we measured the accumulation of transferrin (Tf)-Tf receptor under steady state conditions (90 min of incubation with 1251-Tf). Two conditions (125 nM OKA and 500 nM MCYST, 60 min) which inhibited MT-dependent transport (to 30-+1% and 54.7+0.3% of control for MCYST and OKA respectively, n=3-4 cell preparations) while not causing major changes in MT organization were chosen. In both cases inhibition of MTd e p e n d e n t transport was correlated with reduced accumulation of Tf receptor to 71-+14% for OKA (n=4) and 65-+5% for MCYST (n=5). The similar spectrum of inhibition of MT-dependent transport and Tf receptor accumu-lation by these PP inhibitors suggests that PP2A (but not PP1) mediates these processes. Our data also show that regulation of MTdependent transport can alter hepatocyte function through changes in membrane recycling.
MEDIATED ENDOCYTOSlS BY ISOLATED LIVER ENDOTHELIAL CELLS. GM Thiele, JA Miller, LW Klassen, CC Miller, MF Sorrell and DJ Tuma. Experimental Immunology Laboratory and Liver Study Unit, VA Medical Center and University of Nebraska Medical Center, Omaha, NE. Chronic ethanol ingestion is known to have a deleterious effect on protein trafficking pathways including receptor-mediated endocytosis (RME) in hepatocytes. Recent in situ liver perfusion studies have indicated that RME in liver endothelial cells (LECs) may be similarly altered, but the mechanism of this impairment is currently unknown. Therefore, it was the purpose of these studies to investigate whether chronic ethanol ingestion has similar effects on RME in isolated LECs as compared to studies using in situ perfused rat livers. Male, Wistar rats were chow-fed, fed either an ethanolcontaining liquid diet or an isocaloric amount of a control liquid diet for 4 weeks. Livers were perfusad in situ, cyclically perfused with 2 mg of 12%Albumin modified with 20% formaldehyde (f-AIb), and at indicated times aliquots were taken to assess the degradation of the f-AIb ligand by LECs. Alternatively, the livers were perfused with collaganase, the LECs isolated, and placed in culture at 2 X 106 cells/ml. The f-AIb was added to the cultures at 25 pg/ml of culture fluid, incubated for up to 3 hours, and at selected times during the incubation samples were taken and assessed for the amount of f-AIb degraded. By the end of 3 hours of in situ perfusion, 400 + 42 (chow- fed) and 447 ± 57 (pair-fed) lug/total liver of f-AIb was degraded. In contrast, ethanol-fed rats demonstrated approximately a 40% (252 + 22 pg/total liver) decrease in the degradation of the f-AIb ligand. Isolated LECs demonstrated that 2.7 ± 0.7 (chow-fed) and 2.8 ± 0.5 (pair-fed) lug of f-AIb/1 X 106 cells was degraded. Similar to in situ perfusion studies, LECs isolated from ethanol- fed rats showed a decrease in the degradation of f-AIb (1.0 ± 0.12 lug/1 X 106 cells; or 63%). These data demonstrate that impaired RME, observed in perfused livers of ethanol-fed rats, is also observed in isolated LECs. Thus, it appears that the altered RME of modified proteins via the scavenger receptor in the liver occurs at the level of the LEC.
807
AASLD
ABSTRACTS
HEPATOLOGY O c t o b e r 1995
cAMP AND VOLUME ACTIVATED CHLORIDE CONDUCTANCE IN RAT HEPATOCYTES. X-J Mere, and SA Weinman. University of Texas Medical Branch, Galveston, TX 77555 Chloride channels account for a major fraction of sinusoidal membrane conductance in hepatocytes and play a role in volume regulation. Our aim was to compare the effects of cAMP and cell swelling on hepatoeyte membrane condactances since both stimuli are known to activate chloride channels in many cell types. METHODS: Primary isolated hepatoeytes were prepared from rat liver using the two-step coIlagenase perfusion procedure. Whole-cell currents were measured with the patch-clamp technique. Cell volume was estimated from eross-seetional images. RESULTS: Cells were patched with cAMP (100 ~tM) included in the pipet. Initial conductance was linear (3. l+1.5 nS) but within 3 rain a large outwardly-rectifying conductance appeared (23.8+_3.0 nS, n=20). This conductance was highly anion selective with a CI- : K+ permeability ratio of 12.1. It was abolished by Ca2* chelation in the pipet and removal from the bath, completely inhibited by the anion channel blocker NPPB (200 laM, n=6), and partially inhibited by the stilbene derivative D1DS (150 ~tM, n=7). Halide selectivitywas I'>Br>CI-. Reduction of bath esmolality from 300 to 250 mOsm/kg in the absence of cAMP activated a similar outwardly-rectifying CI" conductance (ORCC) of 20.5+_2.2 nS (n=4). The volume-activated ORCC had identical ion-selectivity and blocker sensitivity as the cAMP-activated conductance. 8-Br-cAMP (500 I.tM) in the bath also activated ORCC but did not cause cell swelling. However, cell shrinkage with hyperosmotie bath (350 mOsm/kg), either before or after exposure to cAMP, inhibited the cAMP-activated ORCC. The two methods of activation of the ORCC, cAMP and cell swelling, were not additive. CONCLUSION: Cyclic AMP and cell swelling activate the same outward-rectifying CI- conductance in rat hepatocytes. In both eases the conductance is blocked by cell shrinkage. It has the properties of the volumeactivated ORCC present in many cell types and is functionally distinct from CFTR-assoeiated chloride conductance. Cyclic AMP may activate by altering the volume set-point of the channel. (Supported by NIH DK42917)
806 PROTEIN PHOSPHATASE 2A-MEDIATED EFFECTS ON
MODULATION OF KUPFFER CELL FUNCTION BY ATRIAL NATRIURETIC PEPTIDE (ANP). M Bilzer, G Paumgartner, AL Gerbes. Department of Medicine II, Klinikum Gro~hadem, Ludwig-MaximiliansUniversity of Munich, Munich, Germany
808 CHRONIC ETHANOL CONSUMPTION IMPAIRS RECEPTOR
Recently we have shown that ANP protects the liver against cell damage induced by activated Kupffer cells (KC). This effect may be due to a protective effect on the hepatocyte or to modulation of Kupffer cell function. Therefore, we evaluated the influence of ANP on KC function determined by particle phagocytosis in the perfused rat liver. Methods: Livers of male Sprague-Dawley rats were perfused with Krebs-Henseleit buffer (pH 7.4, 37°C) in a nonreciroulating fashion. Zymosan (150 lug/ml), cell wall particles from yeast which are selectively taken up by KC, were infused from 40-46 min (n=6). ANP (2 and 200 nM) or 8-Br-cGMP (50 pM), an analogue of cGMP, were infused from 30-50 min after starting perfusion (n=4 each). Uptake of zymosan was assessed by the difference in absorbance (540 nm) of the inflow and outflow perfusion buffer.
Results: (x.±SD). Infusion of zymosan into the portal vein resulted in zymosan extraction of 77+14%, 64+11% and 68+11% at 42, 44 and 46 min of perfusion time. When livers were perfused with 200 nM ANP, zymosan uptake decreased to 46±12%, 41±11% (p<0.05) and 56±9%. Similar results were obtained with 2 nM ANP which reduced zymosan uptake to 47+12%, 35+11% (p<0.05) and 51+7%. Infusion of 50 luM 8Br-cGMP, an analogue of cGMP, the second messenger of ANP action, resulted in a comparable reduction (p<0.05) of zymosan uptake at 44 min (40±11%) and 46 min (47+11%). Conclusions: ANP inhibits zymosan phagocytosis by KC in the perfused rat liver. This effect was mimicked by 8-Br-cGMP supporting an ANP-receptor-guanylate cyclase-cGMP mediated mechanism. Therefore, protective effects eadier described with ANP and 8-Br-cGMP could be related to a direct modulation of KC-function.
MICROTUBULE-DEPENDENT VESICLE TRANSPORT AND RECEPTOR-MEDIATED ENDOCYTOSIS IN HEPATOCYTES. SF Hamm-Alvarez. X-H Wei. N Berndt. and M Runneaar, Center for Liver Diseases and Departments of Pharmaceutical Sciences, Pathology and Medicine, University of Southern California, Los Angeles CA Although microtubule (MT)-dependent transport has been implicated in membrane trafficking in hepatocytes, little is known about how regulation of the components of MT-based transport impacts on membrane recycling. We have explored the role of protein phosphorylation in the regulation of MTdependent vesicle transport and recaptor-mediated endocytosis, specifically the contributions of protein phosphatases (PP), PP1 and PP2A. We compared the effects of two PP inhibitors with different potency, microcystin (MCYST, inhibits PP1 and PP2A equally) and okadaic acid (OKA, inhibits only PP2A at the doses used hera with phosphorylase as substrate). By video microscopy, we have identified a population of vesicles that move on MTs using nocodazole inhibition. The frequency velocity, and run length of these vesicles were significantly decreased in a dose-dependent manner from 50-500 nM of each PP inhibitor. To understand how changes in MT-based transport impacted on receptor-mediated endocytosis, we measured the accumulation of transferrin (Tf)-Tf receptor under steady state conditions (90 min of incubation with 1251-Tf). Two conditions (125 nM OKA and 500 nM MCYST, 60 min) which inhibited MT-dependent transport (to 30-+1% and 54.7+0.3% of control for MCYST and OKA respectively, n=3-4 cell preparations) while not causing major changes in MT organization were chosen. In both cases inhibition of MTd e p e n d e n t transport was correlated with reduced accumulation of Tf receptor to 71-+14% for OKA (n=4) and 65-+5% for MCYST (n=5). The similar spectrum of inhibition of MT-dependent transport and Tf receptor accumu-lation by these PP inhibitors suggests that PP2A (but not PP1) mediates these processes. Our data also show that regulation of MTdependent transport can alter hepatocyte function through changes in membrane recycling.
MEDIATED ENDOCYTOSlS BY ISOLATED LIVER ENDOTHELIAL CELLS. GM Thiele, JA Miller, LW Klassen, CC Miller, MF Sorrell and DJ Tuma. Experimental Immunology Laboratory and Liver Study Unit, VA Medical Center and University of Nebraska Medical Center, Omaha, NE. Chronic ethanol ingestion is known to have a deleterious effect on protein trafficking pathways including receptor-mediated endocytosis (RME) in hepatocytes. Recent in situ liver perfusion studies have indicated that RME in liver endothelial cells (LECs) may be similarly altered, but the mechanism of this impairment is currently unknown. Therefore, it was the purpose of these studies to investigate whether chronic ethanol ingestion has similar effects on RME in isolated LECs as compared to studies using in situ perfused rat livers. Male, Wistar rats were chow-fed, fed either an ethanolcontaining liquid diet or an isocaloric amount of a control liquid diet for 4 weeks. Livers were perfusad in situ, cyclically perfused with 2 mg of 12%Albumin modified with 20% formaldehyde (f-AIb), and at indicated times aliquots were taken to assess the degradation of the f-AIb ligand by LECs. Alternatively, the livers were perfused with collaganase, the LECs isolated, and placed in culture at 2 X 106 cells/ml. The f-AIb was added to the cultures at 25 pg/ml of culture fluid, incubated for up to 3 hours, and at selected times during the incubation samples were taken and assessed for the amount of f-AIb degraded. By the end of 3 hours of in situ perfusion, 400 + 42 (chow- fed) and 447 ± 57 (pair-fed) lug/total liver of f-AIb was degraded. In contrast, ethanol-fed rats demonstrated approximately a 40% (252 + 22 pg/total liver) decrease in the degradation of the f-AIb ligand. Isolated LECs demonstrated that 2.7 ± 0.7 (chow-fed) and 2.8 ± 0.5 (pair-fed) lug of f-AIb/1 X 106 cells was degraded. Similar to in situ perfusion studies, LECs isolated from ethanol- fed rats showed a decrease in the degradation of f-AIb (1.0 ± 0.12 lug/1 X 106 cells; or 63%). These data demonstrate that impaired RME, observed in perfused livers of ethanol-fed rats, is also observed in isolated LECs. Thus, it appears that the altered RME of modified proteins via the scavenger receptor in the liver occurs at the level of the LEC.