Redistribution of canalicular organic anion transport activity in isolated and cultured rat hepatocytes

Abstract

The hepatocanalicular transport of a large number of organic anions, such as bilirubin glucuronides and glutathione conjugates in the rat, is mediated by an adenosine triphosphate (ATP)-dependent transport system, which is termed canalicular multispecific organic anion transporter (cMOAT). This system is mainly defined by its deficiency in mutant TR rats. We have previously reported that in cultured hepatocytes the fluorescent organic anion glutathione-bimane (GS-B) accumulates in intracellular vesicles and that this transport is mediated by cMOAT. We now show that this intracellular accumulation of fluorescent organic anion is largely absent in freshly isolated hepatocytes but appears when cells are incubated in suspension at 37 degrees C or cultured for periods of 1 to 24 hours. The appearance of intracellular cMOAT activity coincides with the disappearance of 70% of cMOAT activity from the plasma membrane as measured by the transport activity of the cells for the organic anion dinitrophenyl-glutathione (GS-DNP). Both the appearance of intracellular cMOAT and the disappearance of transport activity from the plasma membrane were completely inhibited at temperatures below 20 degrees C. Residual cMOAT activity in 24-hour cultured hepatocytes could be further diminished by incubation of the cells with 1 mumol/L monensin or 10 mmol/L methylamine. We conclude that after disruption of the cell polarity by collagenase isolation of the hepatocytes, remnants of apical membrane containing cMOAT are rapidly endocytosed when the cells are kept at 37 degrees C. Evidence suggests that at least part of the transporters may recycle back to the plasma membrane after endocytosis. These observations may be relevant for the understanding of regulation of canalicular transport.

Redistribution of canalicular cell membrane enzyme in hepatocytes following their isolation and during early cell culture

“…brane marker enzyme, dipeptidylpeptidase IV (DPP-IV) (antibody kindly provided by A. Quaroni), we see initially the canalicular membrane as a continuous belt around isolated cells that becomes fragmented within the first hour of cell culture at 37ЊC. Over time, DPP-IV appears in cytoplasmic These observations on apical cell membrane endocytosis vesicles that are preferentially localized around the nucleus are consistent with the observations made by Roelofsen et al 1 and toward the zone of contact to the neighbor cell (Fig. 1B).…”

“…MemWe have read with great interest the article by Roelofsen et brane retrieval and insertion are considered to be mechaal. 1 describing the redistribution of canalicular organic anion nisms that regulate the surface density of canalicular memtransport activity in rat hepatocytes during cell culture, fol-brane transporters. Either of these mechanisms may prevail lowing their isolation from the intact organ with collagenase.…”

Evaluation of Time-Dependent Inactivation of Cyp3a in Cryopreserved Human Hepatocytes

“…The disruption of polarity upon collagenase isolation of hepatocytes may lead to the loss of P-glycoprotein. Roelofsen et al (1995) have demonstrated the endocytosis of canalicular multispecific organic anion transporter after isolation of rat hepatocytes, which was confirmed by the disappearance of transport activity from the cell surface and the appearance of intracellular vesicles that accumulate the substrate. Assuming P-glycoprotein undergoes similar changes, one would expect the decreased availability of a P-glycoprotein substrate to cytoplasmic components (such as CYP3A) due to efflux by the remaining cell membrane P-glycoprotein activity and/or accumulation into the intracellular vesicles by P-glycoprotein.…”

Role of Glycosylation in Trafficking of Mrp2 in Sandwich-Cultured Rat Hepatocytes

“…After disruption of cell polarity by collagenase treatment during hepatocyte isolation, the apical membrane containing Mrp2 is rapidly endocytosed (Roelofsen et al, 1995). Mrp2 in fresh liver and day 0 sandwichcultured rat hepatocytes was partially glycosylated and was glycosylated further over days in culture, resulting in an increase in the molecular mass of Mrp2 by ϳ10 kDa.…”