- Email: [email protected]
A view of exocytotic vesicle fusion in living secretory cells
HEPATOLOGY Vol. 26, No. 1, 1997
HEPATOLOGY Elsewhere
disease course among various PBC cohorts. However, until more information is available, caution should be exercised in applying observations made in the Metcalf cohort to the usual PBC patient that presents with cholestatic liver biochemistries, a positive AMA, and a compatible liver biopsy. KEITH D. LINDOR, M.D. Division of Gastroenterology Mayo Clinic Foundation Rochester, MN REFERENCES 1. Mitchison HC, Massendine MF, Hendrick A, Bennett MK, Bird G, Watson AJ, James OFW. Positive antimitochondrial antibody but normal alkaline phosphatase: is this primary biliary cirrhosis? HEPATOLOGY 1986;6:1279-1284. 2. Metcalf JV, Mitchison HC, Palmer JM, Jones DE, Bassendine MF, James OFW. Natural history of early primary biliary cirrhosis. Lancet 1996; 348:1399-1402. 3. Balasubramaniam K, Grambsch PM, Wiesner RH, Lindor KD, Dickson ER. Diminished survival in asymptomatic primary biliary cirrhosis. A prospective study. Gastroenterology 1990;98:1567-1571. 4. Heathcote EJ, Cauch-Dudek K, Walker V, Bailey RJ, Blendis LM, Ghent CN, Michieletti P, et al. The Canadian multicenter double-blind randomized controlled trial of ursodeoxycholic acid in primary biliary cirrhosis. HEPATOLOGY 1994;19:1149-1156. 5. Lindor KD, Dickson ER, Baldus WP, Jorgensen RA, Ludwig J, Murtaugh PA, Harrison JM, et al. Ursodeoxycholic acid in the treatment of primary biliary cirrhosis. Gastroenterology 1994;106:1284-1290. 6. Locke GR, Therneau TM, Ludwig J, Dickson ER, Lindor KD. Time course of histological progression in primary biliary cirrhosis. HEPATOLOGY 1996;23:52-56. 7. Heseltine L, Turner IB, Fussey SPM, Kelly PJ, James OFW, Yeaman SJ, Bassendine MF. Primary biliary cirrhosis. Quantitation of autoantibodies to purified mitochondrial enzymes and correlation with disease progression. Gastroenterology 1990;99:1786-1792. 8. VanNorstrand MD, Malinchoc M, Lindor KD, Therneau TM, Gershwin ME, Leung PSC, Dickson ER, et al. Quantitative measurement of autoantibodies to recombinant mitochondrial antigens in patients with primary biliary cirrhosis: relationship of levels of autoantibodies to disease progression. Hepatology 1997;25:6-11. 9. Portmann B, Popper H, Neuberger J, Williams R. Sequential and diagnostic features in primary biliary cirrhosis based on serial histologic study in 209 patients. Gastroenterology 1985;88:1777-1790. 10. Lindor KD, Hoofnagle J, Maddrey WC, Mackay IR, Dickson ER. Primary biliary cirrhosis clinical research single-topic conference. HEPATOLOGY 1996;23:639-644.
A VIEW OF EXOCYTOTIC VESICLE FUSION IN LIVING SECRETORY CELLS
Schneider SW, Sritharan KC, Geibel JP, Oberleithner H, Jena BP. Surface dynamics in living acinar cells imaged by atomic force microscopy: identification of plasma membrane structures involved in exocytosis. Reprinted with permission. Proc Natl Acad Sci U S A 1997;94:316-321. ABSTRACT
The dynamics at the plasma membrane resulting from secretory vesicle docking and fusion and compensatory endocytosis has been difficult to observe in living cells primarily because of limited resolution at the light microscopic level. Using the atomic force microscope, we have been able to image and record changes in plasma membrane structure at ultrahigh resolution after stimulation of secretion from isolated pancreatic acinar cells. ‘‘Pits’’ measuring 500 to 2,000
AID
Hepa 0057
/
5p22$$1521
06-12-97 11:13:41
241
nm and containing 3 to 20 depressions measuring 100 to 180 nm in diameter were observed only at the apical region of acinar cells. The time course of an increase and decrease in ‘‘depression size’’ correlated with an increase and decrease of amylase secretion from live acinar cells. Depression dynamics and amylase release were found to be regulated in part by actin. No structural changes were identified at the basolateral region of these cells. Our results suggest depressions to be fusion pores identified earlier in mast cells by freeze-fracture electron microscopy and by electrophysiological measurements. The atomic force microscope has enabled us to observe plasma membrane dynamics of the exocytic process in living cells in real time. COMMENTS
Exocytotic vesicle fusion represents the final step of vesicle-mediated transport pathways in a wide variety of secretory cells. During the resting state, ‘‘constitutive’’ exocytosis can be observed in secretory cells that occur independently of any regulatory signal and allow a continuous supply of membrane material to be inserted into the plasma membranes. In contrast, ‘‘regulated’’ exocytosis requires stimulation of the cell by a secretagogue that induces a Ca//-dependent fusion of vesicles with their target membrane. Molecular mechanisms leading to regulated exocytosis have been summarized in the SNARE hypothesis (synaptosomal-associated protein receptor).1 The following four steps can be distinguished: 1) Docking of the vesicle to the plasma membrane. SNAREs have been identified both on the vesicle membrane (v-SNAREs) and on the target membrane (t-SNAREs) in synaptic nerve terminals. The docking site is assumed to be formed by the tight binding of one v-SNARE, synaptobrevin or vesicle-associated membrane protein (VAMP), with two t-SNAREs, syntaxin and synaptosome-associated protein (SNAP-25), resulting in a stable trimeric core complex. (Synaptobrevin, SNAP-25, and syntaxin are targets of the proteolytic activities of botulinum and tetanus toxins that inhibit formation of the trimeric core complex and prevent exocytosis.) 2) Priming of the exocytotic machinery. After docking, vesicles are not immediately competent to fuse with their target membrane. The trimeric core complex described above serves as a binding site for N-ethylmaleimide-sensitive fusion protein (NSF; a trimeric Mg-ATPase) via one of a group of NSF-attachment proteins (a-, b-, and g-SNAPs). After binding, NSF crosslinks multiple core complexes and disrupts these adenosine triphosphate-dependently (possibly supported by a second Mg-ATPase, phosphatidylinositol-4phosphate-5-kinase), leading to hemifusion of vesicle and target membrane.1-3 3) Triggering of exocytosis by [Ca//]i . Synaptotagmins are v-SNAREs that are assumed to be part of a clamping apparatus that prevents spontaneous fusion of vesicles with their target membrane. Synaptotagmins might serve as Ca// sensors and might, after a subplasmalemmal rise in [Ca//]i , interact with syntaxins (t-SNAREs) as well as membrane phospholipids and other yet unidentified target proteins to allow, 4) fusion of the vesicular membrane with the plasmalemma.1-3 The molecular mechanisms involved in the process of exocytotic vesicle fusion and its regulation have been studied most intensely in nerve tissue.1,2 However, many aspects of the synaptic exocytotic machinery seem to be applicable to
hepas
WBS: Hepatology
242 HEPATOLOGY Elsewhere
HEPATOLOGY July 1997
other types of secretory cells3 because a remarkably high degree of conservation of the molecular machinery for secretion has been observed from yeast to mammalian neurons.4 Pancreatic acinar cells represent an exemplary model for the study of regulated exocytosis by nonexcitable secretory cells. Their subplasmalemmal actin network is regarded as a barrier against constitutive exocytosis. Recent experimental evidence indicates that limited actin filament disassembly is necessary to allow regulated exocytosis although a minimal actin structure is required for regulated exocytosis to occur.5 The exact role of small guanosine triphosphate-binding proteins of the rab family in controlling regulated exocytosis is as yet unclear. Indirect evidence, however, indicates that rab3-like proteins control an important step in regulated amylase secretion by pancreatic acinar cells.6 The present knowledge on exocytotic vesicle fusion is based mainly on biochemical and electrophysiological studies in living cells as well as on light and electron microscopical investigations in chemically-fixed or frozen tissues. Schneider and coworkers7 used an atomic force microscope to image the apical region of living isolated pancreatic acinar cells in the resting state and during regulated exocytosis. They observed pits of 500 to 2,000 nm in diameter containing 3 to 20 depressions of about 150 nm in diameter. After stimulation with mastoparan, a tetradecapeptide from wasp venom, which has been shown to stimulate heterotrimeric Gi and Go proteins in pancreatic acinar cells,7 the depressions significantly widened and later narrowed again in a time-dependent fashion. In parallel, the rate of amylase secretion increased in these cells. However, when cells were exposed to cytochalasin B, a fungal toxin that inhibits actin polymerization, a significant decrease in depression size was observed, suggesting an important role of actin in maintenance of the depression structure during exocytosis. In summary, the depressions observed in the apical membrane were highly suggestive of exocytotic fusion pores during regulated exocytosis. Thus, Schneider et al. may be the first to have succeeded in imaging exocytosis in living cells. The atomic force microscope (AFM) allows three-dimensional imaging of biological samples ranging from living cells to macromolecular structures.8 A sharp tip attached to a soft cantilever is moved over the surface of the sample under study. Deflections of the tip are recorded, and a three-dimensional topographic image of the sample is created. The precision of the AFM depends on the elasticity of the sample, however, and may be reduced when applied to soft samples such as living cells. Thus, the images obtained in this study7 may be a complex function of both the surface and the elasticity of the apical membrane of acinar cells rather than a simple recording of the membrane structure. It remains to be proven that the depressions observed represent the fusion pores of acinar cells during exocytosis. However, these exciting studies show that the progress in the understanding of cellular physiology and pathophysiology is critically dependent on the progress in the development of new technologies. How does all this apply to the field of hepatology? The liver cell (as well as the cholangiocyte) shares a number of features with the pancreatic acinar cell. Both are highly polarized nonexcitable cells, their apical pole representing the major excretory site. Vesicular transport across the cell occurs within minutes rather than seconds when compared with neuroendocrine cells. The mechanism of exocytosis has
AID
Hepa 0057
/
5p22$$1101
06-12-97 11:13:41
been less intensely studied in the liver as compared with neural tissue. This may be partly because of the experimental difficulty in providing a polarized liver cell system in vitro for functional studies at the cellular level. However, the use of hepatocyte couplets together with studies in the isolated perfused liver has led to an advanced understanding of the regulation of exocytosis in liver cells.9,10 Although exocytosis has been estimated to account for 1.5% to 8% of net bile flow under basal conditions,9,10 the exocytotic insertion of transport proteins into the canalicular membrane may be of greater functional importance for the formation of bile.11 This process is highly regulated in many secretory cells12 including the hepatocyte.9 Exocytotic vesicle fusion and insertion of membrane proteins into the canalicular membrane are impaired in experimental cholestasis.13,14 The hydrophilic bile acid tauroursodeoxycholic acid, which improves cholestatic features in man and experimental animals has been shown to stimulate hepatocellular exocytosis by Ca//- and putatively by protein kinase C-dependent mechanisms.15,16 Thus, the idea that the anticholestatic effect of tauroursodeoxycholic acid could at least partly be explained by the stimulation of regulated hepatocellular exocytosis and reactivation of impaired exocytotic insertion of transport proteins into the canalicular membrane15,17 remains attractive. However, further studies, perhaps employing techniques such as AFM, are needed to prove this hypothesis. The potential of AFM to study regulation of exocytotic vesicle fusion in real time in a model living secretory cell as shown in this study7 is intriguing and will certainly stimulate similar studies in the field of hepatology. Putative tools for these in vitro studies are now available: the hepatocyte-derived WIF-B cell line18 is highly polarized and its apical pole faces at least for a certain time period upwards (towards the AFM tip). Therefore, further insights into the regulation of hepatocellular exocytotic vesicle fusion can be expected in the near future. ULRICH BEUERS, M.D. Department of Internal Medicine II Klinikum Grosshadern, University of Munich Munich, Germany REFERENCES 1. Rothmann JE. Mechanisms of intracellular protein transport. Nature 1994;372:55-63. 2. Calakos N, Scheller RH. Synaptic vesicle biogenesis, docking and fusion: a molecular description. Physiol Rev 1996;76:1-29. 3. Parsons TD, Coorssen JR, Horstmann H, Lee AK, Tse FW, Almers W. The last seconds in the life of a secretory vesicle. Cold Spring Harb Symp Quant Biol 1995;60:389-396. 4. Bennett MK, Scheller RH. The molecular machinery for secretion is conserved from yeast to neurons. Proc Natl Acad Sci U S A 1993;90: 2559-2563. 5. Muallem S, Kwiatkowska K, Xu X, Yin HL. Actin filament disassembly is a sufficient final trigger for exocytosis in nonexcitable cells. J Cell Biol 1995;128:589-598. 6. Padfield PJ, Balch WE, Jamieson JD. A synthetic peptide of the rab3a effector domain stimulates amylase release from permeabilized pancreatic acini. Proc Natl Acad Sci U S A 1992;89:1656-1660. 7. Schneider SW, Sritharan KC, Geibel JP, Oberleithner H, Jena BP. Surface dynamics in living acinar cells imaged by atomic force microscopy: identification of plasma membrane structures involved in exocytosis. Proc Natl Acad Sci U S A 1997;94:316-321. 8. Lal R, John SA. Biological applications of atomic force microscopy. Am J Physiol 1994;266:C1-C21.
hepas
WBS: Hepatology
HEPATOLOGY Vol. 26, No. 1, 1997
HEPATOLOGY Elsewhere
9. Nathanson MH, Boyer JL. Vesicular trafficking in the hepatocyte. In: Arias IM, Boyer JL, Fausto N, Jakoby WB, Schachter DA, Shafritz DA, eds. The Liver: Biology and Pathobiology. New York: Raven, 1994:655664. 10. Crawford JM. Role of vesicle-mediated transport pathways in hepatocellular bile secretion. Semin Liver Dis 1996;16:169-189. 11. Boyer JL, Soroka CJ. Vesicle targeting to the apical domain regulates bile excretory function in isolated rat hepatocyte couplets. Gastroenterology 1995;109:1600-1611. 12. Forte JG. Regulation of secretion and absorption by recruitment and recycling of primary transport proteins. Gastroenterology 1995;109: 1706-1710. 13. Larkin JM, Palade GE. Transcytotic vesicular carriers for polymeric IgA receptors accumulate in rat hepatocyte after bile duct ligation. J Cell Sci 1991;98:205-216. 14. Barr V, Hubbard AL. Newly synthesized plasma membrane proteins are
AID
Hepa 0057
/
5p22$$1101
06-12-97 11:13:41
15.
16.
17. 18.
243
transported in transcytotic vesicles in the bile duct-ligated rat. Gastroenterology 1993;105:554-571. Beuers U, Nathanson MH, Isales CM, Boyer JL. Tauroursodeoxycholic acid stimulates hepatocellular exocytosis and mobilizes extracellular Ca// mechanisms defective in cholestasis. J Clin Invest 1993;92:29842993. Beuers U, Throckmorton DO, Anderson MH, Isales CM, Thasler W, Kullak-Ublick GA, Sauter G, et al. Tauroursodeoxycholic acid stimulates protein kinase C in isolated rat hepatocytes. Gastroenterology 1996; 110:1553-1563. Ha¨ussinger D, Saha N, Hallbrucker C, Lang F, Gerok W. Involvement of microtubules in the swelling-induced stimulation of transcellular taurocholate transport in perfused rat liver. Biochem J 1993;291:355-360. Shanks MR, Cassio D, Lecoq O, Hubbard AL. An improved polarized rat hepatoma hybrid cell line. Generation and comparison with its hepatoma relatives and hepatocytes in vivo. J Cell Sci 1994;107:813-825.
hepas
WBS: Hepatology
HEPATOLOGY Elsewhere
disease course among various PBC cohorts. However, until more information is available, caution should be exercised in applying observations made in the Metcalf cohort to the usual PBC patient that presents with cholestatic liver biochemistries, a positive AMA, and a compatible liver biopsy. KEITH D. LINDOR, M.D. Division of Gastroenterology Mayo Clinic Foundation Rochester, MN REFERENCES 1. Mitchison HC, Massendine MF, Hendrick A, Bennett MK, Bird G, Watson AJ, James OFW. Positive antimitochondrial antibody but normal alkaline phosphatase: is this primary biliary cirrhosis? HEPATOLOGY 1986;6:1279-1284. 2. Metcalf JV, Mitchison HC, Palmer JM, Jones DE, Bassendine MF, James OFW. Natural history of early primary biliary cirrhosis. Lancet 1996; 348:1399-1402. 3. Balasubramaniam K, Grambsch PM, Wiesner RH, Lindor KD, Dickson ER. Diminished survival in asymptomatic primary biliary cirrhosis. A prospective study. Gastroenterology 1990;98:1567-1571. 4. Heathcote EJ, Cauch-Dudek K, Walker V, Bailey RJ, Blendis LM, Ghent CN, Michieletti P, et al. The Canadian multicenter double-blind randomized controlled trial of ursodeoxycholic acid in primary biliary cirrhosis. HEPATOLOGY 1994;19:1149-1156. 5. Lindor KD, Dickson ER, Baldus WP, Jorgensen RA, Ludwig J, Murtaugh PA, Harrison JM, et al. Ursodeoxycholic acid in the treatment of primary biliary cirrhosis. Gastroenterology 1994;106:1284-1290. 6. Locke GR, Therneau TM, Ludwig J, Dickson ER, Lindor KD. Time course of histological progression in primary biliary cirrhosis. HEPATOLOGY 1996;23:52-56. 7. Heseltine L, Turner IB, Fussey SPM, Kelly PJ, James OFW, Yeaman SJ, Bassendine MF. Primary biliary cirrhosis. Quantitation of autoantibodies to purified mitochondrial enzymes and correlation with disease progression. Gastroenterology 1990;99:1786-1792. 8. VanNorstrand MD, Malinchoc M, Lindor KD, Therneau TM, Gershwin ME, Leung PSC, Dickson ER, et al. Quantitative measurement of autoantibodies to recombinant mitochondrial antigens in patients with primary biliary cirrhosis: relationship of levels of autoantibodies to disease progression. Hepatology 1997;25:6-11. 9. Portmann B, Popper H, Neuberger J, Williams R. Sequential and diagnostic features in primary biliary cirrhosis based on serial histologic study in 209 patients. Gastroenterology 1985;88:1777-1790. 10. Lindor KD, Hoofnagle J, Maddrey WC, Mackay IR, Dickson ER. Primary biliary cirrhosis clinical research single-topic conference. HEPATOLOGY 1996;23:639-644.
A VIEW OF EXOCYTOTIC VESICLE FUSION IN LIVING SECRETORY CELLS
Schneider SW, Sritharan KC, Geibel JP, Oberleithner H, Jena BP. Surface dynamics in living acinar cells imaged by atomic force microscopy: identification of plasma membrane structures involved in exocytosis. Reprinted with permission. Proc Natl Acad Sci U S A 1997;94:316-321. ABSTRACT
The dynamics at the plasma membrane resulting from secretory vesicle docking and fusion and compensatory endocytosis has been difficult to observe in living cells primarily because of limited resolution at the light microscopic level. Using the atomic force microscope, we have been able to image and record changes in plasma membrane structure at ultrahigh resolution after stimulation of secretion from isolated pancreatic acinar cells. ‘‘Pits’’ measuring 500 to 2,000
AID
Hepa 0057
/
5p22$$1521
06-12-97 11:13:41
241
nm and containing 3 to 20 depressions measuring 100 to 180 nm in diameter were observed only at the apical region of acinar cells. The time course of an increase and decrease in ‘‘depression size’’ correlated with an increase and decrease of amylase secretion from live acinar cells. Depression dynamics and amylase release were found to be regulated in part by actin. No structural changes were identified at the basolateral region of these cells. Our results suggest depressions to be fusion pores identified earlier in mast cells by freeze-fracture electron microscopy and by electrophysiological measurements. The atomic force microscope has enabled us to observe plasma membrane dynamics of the exocytic process in living cells in real time. COMMENTS
Exocytotic vesicle fusion represents the final step of vesicle-mediated transport pathways in a wide variety of secretory cells. During the resting state, ‘‘constitutive’’ exocytosis can be observed in secretory cells that occur independently of any regulatory signal and allow a continuous supply of membrane material to be inserted into the plasma membranes. In contrast, ‘‘regulated’’ exocytosis requires stimulation of the cell by a secretagogue that induces a Ca//-dependent fusion of vesicles with their target membrane. Molecular mechanisms leading to regulated exocytosis have been summarized in the SNARE hypothesis (synaptosomal-associated protein receptor).1 The following four steps can be distinguished: 1) Docking of the vesicle to the plasma membrane. SNAREs have been identified both on the vesicle membrane (v-SNAREs) and on the target membrane (t-SNAREs) in synaptic nerve terminals. The docking site is assumed to be formed by the tight binding of one v-SNARE, synaptobrevin or vesicle-associated membrane protein (VAMP), with two t-SNAREs, syntaxin and synaptosome-associated protein (SNAP-25), resulting in a stable trimeric core complex. (Synaptobrevin, SNAP-25, and syntaxin are targets of the proteolytic activities of botulinum and tetanus toxins that inhibit formation of the trimeric core complex and prevent exocytosis.) 2) Priming of the exocytotic machinery. After docking, vesicles are not immediately competent to fuse with their target membrane. The trimeric core complex described above serves as a binding site for N-ethylmaleimide-sensitive fusion protein (NSF; a trimeric Mg-ATPase) via one of a group of NSF-attachment proteins (a-, b-, and g-SNAPs). After binding, NSF crosslinks multiple core complexes and disrupts these adenosine triphosphate-dependently (possibly supported by a second Mg-ATPase, phosphatidylinositol-4phosphate-5-kinase), leading to hemifusion of vesicle and target membrane.1-3 3) Triggering of exocytosis by [Ca//]i . Synaptotagmins are v-SNAREs that are assumed to be part of a clamping apparatus that prevents spontaneous fusion of vesicles with their target membrane. Synaptotagmins might serve as Ca// sensors and might, after a subplasmalemmal rise in [Ca//]i , interact with syntaxins (t-SNAREs) as well as membrane phospholipids and other yet unidentified target proteins to allow, 4) fusion of the vesicular membrane with the plasmalemma.1-3 The molecular mechanisms involved in the process of exocytotic vesicle fusion and its regulation have been studied most intensely in nerve tissue.1,2 However, many aspects of the synaptic exocytotic machinery seem to be applicable to
hepas
WBS: Hepatology
242 HEPATOLOGY Elsewhere
HEPATOLOGY July 1997
other types of secretory cells3 because a remarkably high degree of conservation of the molecular machinery for secretion has been observed from yeast to mammalian neurons.4 Pancreatic acinar cells represent an exemplary model for the study of regulated exocytosis by nonexcitable secretory cells. Their subplasmalemmal actin network is regarded as a barrier against constitutive exocytosis. Recent experimental evidence indicates that limited actin filament disassembly is necessary to allow regulated exocytosis although a minimal actin structure is required for regulated exocytosis to occur.5 The exact role of small guanosine triphosphate-binding proteins of the rab family in controlling regulated exocytosis is as yet unclear. Indirect evidence, however, indicates that rab3-like proteins control an important step in regulated amylase secretion by pancreatic acinar cells.6 The present knowledge on exocytotic vesicle fusion is based mainly on biochemical and electrophysiological studies in living cells as well as on light and electron microscopical investigations in chemically-fixed or frozen tissues. Schneider and coworkers7 used an atomic force microscope to image the apical region of living isolated pancreatic acinar cells in the resting state and during regulated exocytosis. They observed pits of 500 to 2,000 nm in diameter containing 3 to 20 depressions of about 150 nm in diameter. After stimulation with mastoparan, a tetradecapeptide from wasp venom, which has been shown to stimulate heterotrimeric Gi and Go proteins in pancreatic acinar cells,7 the depressions significantly widened and later narrowed again in a time-dependent fashion. In parallel, the rate of amylase secretion increased in these cells. However, when cells were exposed to cytochalasin B, a fungal toxin that inhibits actin polymerization, a significant decrease in depression size was observed, suggesting an important role of actin in maintenance of the depression structure during exocytosis. In summary, the depressions observed in the apical membrane were highly suggestive of exocytotic fusion pores during regulated exocytosis. Thus, Schneider et al. may be the first to have succeeded in imaging exocytosis in living cells. The atomic force microscope (AFM) allows three-dimensional imaging of biological samples ranging from living cells to macromolecular structures.8 A sharp tip attached to a soft cantilever is moved over the surface of the sample under study. Deflections of the tip are recorded, and a three-dimensional topographic image of the sample is created. The precision of the AFM depends on the elasticity of the sample, however, and may be reduced when applied to soft samples such as living cells. Thus, the images obtained in this study7 may be a complex function of both the surface and the elasticity of the apical membrane of acinar cells rather than a simple recording of the membrane structure. It remains to be proven that the depressions observed represent the fusion pores of acinar cells during exocytosis. However, these exciting studies show that the progress in the understanding of cellular physiology and pathophysiology is critically dependent on the progress in the development of new technologies. How does all this apply to the field of hepatology? The liver cell (as well as the cholangiocyte) shares a number of features with the pancreatic acinar cell. Both are highly polarized nonexcitable cells, their apical pole representing the major excretory site. Vesicular transport across the cell occurs within minutes rather than seconds when compared with neuroendocrine cells. The mechanism of exocytosis has
AID
Hepa 0057
/
5p22$$1101
06-12-97 11:13:41
been less intensely studied in the liver as compared with neural tissue. This may be partly because of the experimental difficulty in providing a polarized liver cell system in vitro for functional studies at the cellular level. However, the use of hepatocyte couplets together with studies in the isolated perfused liver has led to an advanced understanding of the regulation of exocytosis in liver cells.9,10 Although exocytosis has been estimated to account for 1.5% to 8% of net bile flow under basal conditions,9,10 the exocytotic insertion of transport proteins into the canalicular membrane may be of greater functional importance for the formation of bile.11 This process is highly regulated in many secretory cells12 including the hepatocyte.9 Exocytotic vesicle fusion and insertion of membrane proteins into the canalicular membrane are impaired in experimental cholestasis.13,14 The hydrophilic bile acid tauroursodeoxycholic acid, which improves cholestatic features in man and experimental animals has been shown to stimulate hepatocellular exocytosis by Ca//- and putatively by protein kinase C-dependent mechanisms.15,16 Thus, the idea that the anticholestatic effect of tauroursodeoxycholic acid could at least partly be explained by the stimulation of regulated hepatocellular exocytosis and reactivation of impaired exocytotic insertion of transport proteins into the canalicular membrane15,17 remains attractive. However, further studies, perhaps employing techniques such as AFM, are needed to prove this hypothesis. The potential of AFM to study regulation of exocytotic vesicle fusion in real time in a model living secretory cell as shown in this study7 is intriguing and will certainly stimulate similar studies in the field of hepatology. Putative tools for these in vitro studies are now available: the hepatocyte-derived WIF-B cell line18 is highly polarized and its apical pole faces at least for a certain time period upwards (towards the AFM tip). Therefore, further insights into the regulation of hepatocellular exocytotic vesicle fusion can be expected in the near future. ULRICH BEUERS, M.D. Department of Internal Medicine II Klinikum Grosshadern, University of Munich Munich, Germany REFERENCES 1. Rothmann JE. Mechanisms of intracellular protein transport. Nature 1994;372:55-63. 2. Calakos N, Scheller RH. Synaptic vesicle biogenesis, docking and fusion: a molecular description. Physiol Rev 1996;76:1-29. 3. Parsons TD, Coorssen JR, Horstmann H, Lee AK, Tse FW, Almers W. The last seconds in the life of a secretory vesicle. Cold Spring Harb Symp Quant Biol 1995;60:389-396. 4. Bennett MK, Scheller RH. The molecular machinery for secretion is conserved from yeast to neurons. Proc Natl Acad Sci U S A 1993;90: 2559-2563. 5. Muallem S, Kwiatkowska K, Xu X, Yin HL. Actin filament disassembly is a sufficient final trigger for exocytosis in nonexcitable cells. J Cell Biol 1995;128:589-598. 6. Padfield PJ, Balch WE, Jamieson JD. A synthetic peptide of the rab3a effector domain stimulates amylase release from permeabilized pancreatic acini. Proc Natl Acad Sci U S A 1992;89:1656-1660. 7. Schneider SW, Sritharan KC, Geibel JP, Oberleithner H, Jena BP. Surface dynamics in living acinar cells imaged by atomic force microscopy: identification of plasma membrane structures involved in exocytosis. Proc Natl Acad Sci U S A 1997;94:316-321. 8. Lal R, John SA. Biological applications of atomic force microscopy. Am J Physiol 1994;266:C1-C21.
hepas
WBS: Hepatology
HEPATOLOGY Vol. 26, No. 1, 1997
HEPATOLOGY Elsewhere
9. Nathanson MH, Boyer JL. Vesicular trafficking in the hepatocyte. In: Arias IM, Boyer JL, Fausto N, Jakoby WB, Schachter DA, Shafritz DA, eds. The Liver: Biology and Pathobiology. New York: Raven, 1994:655664. 10. Crawford JM. Role of vesicle-mediated transport pathways in hepatocellular bile secretion. Semin Liver Dis 1996;16:169-189. 11. Boyer JL, Soroka CJ. Vesicle targeting to the apical domain regulates bile excretory function in isolated rat hepatocyte couplets. Gastroenterology 1995;109:1600-1611. 12. Forte JG. Regulation of secretion and absorption by recruitment and recycling of primary transport proteins. Gastroenterology 1995;109: 1706-1710. 13. Larkin JM, Palade GE. Transcytotic vesicular carriers for polymeric IgA receptors accumulate in rat hepatocyte after bile duct ligation. J Cell Sci 1991;98:205-216. 14. Barr V, Hubbard AL. Newly synthesized plasma membrane proteins are
AID
Hepa 0057
/
5p22$$1101
06-12-97 11:13:41
15.
16.
17. 18.
243
transported in transcytotic vesicles in the bile duct-ligated rat. Gastroenterology 1993;105:554-571. Beuers U, Nathanson MH, Isales CM, Boyer JL. Tauroursodeoxycholic acid stimulates hepatocellular exocytosis and mobilizes extracellular Ca// mechanisms defective in cholestasis. J Clin Invest 1993;92:29842993. Beuers U, Throckmorton DO, Anderson MH, Isales CM, Thasler W, Kullak-Ublick GA, Sauter G, et al. Tauroursodeoxycholic acid stimulates protein kinase C in isolated rat hepatocytes. Gastroenterology 1996; 110:1553-1563. Ha¨ussinger D, Saha N, Hallbrucker C, Lang F, Gerok W. Involvement of microtubules in the swelling-induced stimulation of transcellular taurocholate transport in perfused rat liver. Biochem J 1993;291:355-360. Shanks MR, Cassio D, Lecoq O, Hubbard AL. An improved polarized rat hepatoma hybrid cell line. Generation and comparison with its hepatoma relatives and hepatocytes in vivo. J Cell Sci 1994;107:813-825.
hepas
WBS: Hepatology