Preservation of cellular polarity in isolated hepatocytes

Nickola, I.; Frimmer, M.

doi:10.1007/bf00251061

Cited by 17 publications

(1 citation statement)

References 7 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Unlike single cells, hepatocyte couplets retain their apical polarity with tight junctions dividing the canalicular and basolateral domains. The mitochondria, smooth and rough endoplasmic reticulum and Golgi apparatus of these couplets retain normal ultrastructural appearances (Gautam et al, 1987;Nikola and Frimmer, 1986). Functional gap junctions (Reverdin and Weingart, 1988;Spray et al, 1986) as well as tight junctions (Gautum et al, 1987) are maintained between the adjacent cells.…”

Section: Introductionmentioning

confidence: 99%

Periportal- and perivenous-enriched hepatocyte couplets: differences in canalicular activity and in response to oxidative stress

Wilton

Chipman

Lawson

et al. 1993

Biochemical Journal

View full text Add to dashboard Cite

Unlike isolated single hepatocytes, hepatocyte couplets retain their apical polarity, and, during short-term culture form an enclosed canalicular space or vacuole between the two adjacent cells into which biliary secretion is initiated. Hepatocyte couplets were prepared after partial collagenase perfusion of rat liver. Centrifugal elutriation was used to fractionate the preparation into six couplet-containing suspensions. Image analysis was used to determine the size of cultured couplets. The size of the couplets ranged from 34.1 +/- 0.76 microns and 684 +/- 24.1 microns 2 (mean length and area respectively +/- S.E.M.) in Fraction 2, to 43.7 +/- 0.57 microns and 1033 +/- 33.8 microns 2 length and area respectively in Fraction 7. Glutamine synthetase activity was assessed in each freshly eluted fraction and was shown to be predominant in Fractions 6 and 7. Pretreatment of rats with CCl4, which selectively destroys perivenous hepatocytes, decreased the proportion of couplets in these fractions by over 67%, and their glutamine synthetase activity by over 97%. It was concluded that Fractions 2 and 3 contained predominantly couplets of Zone 1 (periportal) origin, Fractions 4 and 5 those from Zone 2, and Fractions 6 and 7 predominantly couplets of Zone 3 (perivenous) origin. The development of canalicular secretory activity was assessed in the couplets after a 15 min incubation with a fluorescent bile acid, cholyl-lysyl-fluorescein (CLF). This was sigmoidal in all fractions, but slower in the periportal couplets, taking 5.1 h for 50% to show secretory activity in Fraction 2, compared with 2.7 h for Fraction 7. Incubation of hepatocyte couplets with 1 or 10 microM taurodehydrocholate, a non-toxic bile acid analogue, did not influence the rate of development of accumulation of CLF by the couplets or the area of the canalicular vacuole in any fraction. However, it did decrease the CLF content of couplets incubated with CLF for 15 min to a greater extent in those of perivenous origin. After subjecting the couplets to oxidative stress by incubation with 20 microM menadione (2-methyl-1,4-naphthoquinone), it was evident that periportal couplets were less able to maintain canalicular secretory activity than perivenous couplets.

show abstract

Section: Introductionmentioning

confidence: 99%

Periportal- and perivenous-enriched hepatocyte couplets: differences in canalicular activity and in response to oxidative stress

Wilton

Chipman

Lawson

et al. 1993

Biochemical Journal

View full text Add to dashboard Cite

show abstract

Kinetics of Taurocholate Efflux From Freshly Isolated Suspensions and Primary Cultures of Rat Hepatocytes

Kukongviriyapan

Stacey

1990

Hepatology

View full text Add to dashboard Cite

Isolated rat hepatocytes have been advocated as a model to study aspects of mechanism of chemical-induced interference with biliary excretory function. Some technical problems do exist in studying efflux, such as the reuptake of the previously excreted substrate. Another concern is the loss of liver-specific functions in hepatocytes with continuing time in culture. It is important to address such technical aspects and to determine whether the process of efflux is compromised in primary cultures of hepatocytes. In the presence of Na+ the apparent efflux of taurocholate from hepatocytes was shown to be significantly confounded by reuptake of substrate. The unidirectional efflux was best demonstrated in buffer where choline replaced Na+. A comparison of efflux kinetics for cultured cells to those in suspension showed that both apparent affinity for transport carriers and transport capacity were greater in the former. The simple diffusion component for efflux increased with the time in culture, but affinity for transport carriers and transport capacity remained unchanged over 6 to 24 hr. However, it was not possible to determine meaningful kinetic constants after 24 hr in culture because the uptake of taurocholate was so low. Primary cultured hepatocytes may therefore be of limited value in the study of efflux of bile salts in the longer term, mainly because of the inability of cells to take up and accumulate a sufficiently high level of bile salts.

show abstract

Cytoskeletal organization in clusters of isolated polarized skate hepatocytes: Structural and functional evidence for microtubule‐dependent transcytosis

et al. 1995

View full text Add to dashboard Cite

Isolated hepatocytes from the marine vertebrate Raja erinacea (the little skate) retain their structural and functional integrity as clusters of cells formed around a single tubular bile canaliculus, and therefore can be used as a model of polarized hepatocytes in situ. In this study we used confocal and conventional epifluorescence microscopy in conjunction with fluorescent markers and immunocytochemistry to examine the structure and function of the cytoskeleton in these cells. Actin filaments in the hepatocyte clusters were found cortically and also concentrated in a pericanalicular array, while microtubules appeared to radiate away from a concentration near the apical membrane of the biliary pole towards the basolateral sinusoidal surfaces. Treatment of clusters with the microtubule disrupting agent, nocodazole, resulted in the microtubules depolymerizing from the basolateral surfaces towards the apical surface, indicating that the microtubules were oriented with their plus ends at the basolateral surface and their minus ends at the apical surface. Nocodazole was also found to disrupt the ability of clusters to transcytose a fluorescent bile salt derivative into their canalicular lumens. We detected cytoplasmic dynein in skate hepatocyte homogenates by Western blotting using an anti-dynein intermediate chain antibody, and immunofluorescent staining of intact hepatocytes revealed a punctate vesicular pattern. The polarized arrangement of microtubules, the presence of cytoplasmic dynein, and the inhibition of bile salt secretion by nocodozole are consistent with the microtubule cytoskeleton playing a fundamental role in the mediation of transcytosis, endocytosis, and bile excretory function in these hepatocytes. These polarized isolated skate hepatocytes represent an excellent experimental model for the in vitro study of hepatic transport, and allow for important comparative studies aimed at elucidating the evolutionarily conserved nature of various hepatocyte structures amongst the vertebrates.

show abstract

Preservation of cellular polarity in isolated hepatocytes

Cited by 17 publications

References 7 publications

Periportal- and perivenous-enriched hepatocyte couplets: differences in canalicular activity and in response to oxidative stress

Periportal- and perivenous-enriched hepatocyte couplets: differences in canalicular activity and in response to oxidative stress

Kinetics of Taurocholate Efflux From Freshly Isolated Suspensions and Primary Cultures of Rat Hepatocytes

Cytoskeletal organization in clusters of isolated polarized skate hepatocytes: Structural and functional evidence for microtubule‐dependent transcytosis

Contact Info

Product

Resources

About