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

Abstract

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.

Recent advances in 2D and 3D in vitro systems using primary hepatocytes, alternative hepatocyte sources and non-parenchymal liver cells and their use in investigating mechanisms of hepatotoxicity, cell signaling and ADME

“…Various techniques were applied to achieve this goal. For instance, hepatocytes from different lobular zones were isolated according to their different size and density by centrifugal elutriation (Wilton et al 1993; Botham et al 1998; Romero et al 1999). The most suitable separation technique leading to consistent results, the so-called digitonin–collagenase perfusion method developed independently by Quistorff (1985) and Lindros and Penttilä (1985), allows isolation of two distinct subpopulations of hepatocytes, one enriched in periportal and the other one enriched in pericentral cells.…”

Canalicular Retention as an in Vitro Assay of Tight Junctional Permeability in Isolated Hepatocyte Couplets: Effects of Protein Kinase Modulation and Cholestatic Agents

“…In line with this view, menadione, which, together with CyA and 17/3-EG, appears to be able to affect only a limited proportion of couplets (see Fig. 5), has been shown to affect differentially periportal from perivenous couplets, as judged by their ability to impair canalicular CLF accumulation (Wilton et al, 1993a). Extension of this study to the analysis of cVR of CLF for this and the other toxicants in subpopulations of periportal and perivenous couplets obtained by centrifugal elutriation (Wilton et ai, 1993a) should contribute to a validation of this observation.…”

Pathobiology and experimental therapeutics in hepatocellular cholestasis: lessons from the hepatocyte couplet model

“…Routine couplet preparations contain cells from all zones of the liver lobule ; by modifying the elutriation procedure, enrichment of ' perivenous ' (small) and ' periportal ' (larger) couplets can be obtained, and these show differences in the rate of bile acid secretion and in sensitivities to compounds affecting bile production [7].…”