title: Control by signaling modulators of the sorting of canalicular transporters in rat hepatocyte couplets: role of the cytoskeleton.
abstract: Hormonal control of the restoration of hepatocanalicular polarity in short-term cultured hepatocyte couplets was analyzed. One hour following isolation, couplets were unable to accumulate the fluorescent bile acid analogue, cholyl-lysyl-fluorescein (CLF), and showed a nonpolarized distribution of F-actin and mrp2 over the cell body. A progressive, time-dependent restoration of couplet-polarized function and morphology was reached after 4 hours of culture. Both dibutyryl cyclic adenosine monophosphate (DBcAMP) and the Ca(2+)-elevating compound, thapsigargin, accelerated restoration of normal couplet morphology and function. The DBcAMP-mediated stimulus was inhibited by the Ca(2+) chelator, 1, 2-bis-(o-aminophenoxy)-ethene-N,N,N',N'-tetra-acetate tetra-(acetomethyl)ester (BAPTA/AM), but not by the protein kinase A (PKA) inhibitors, KT5720 or H89, suggesting that Ca(2+) elevation rather than PKA activation is involved. N-(6-aminohexyl-5-chloro-1-napththalenesulfonamide (W-7), a calmodulin inhibitor, and the protein kinase C (PKC) activator, phorbol dibutyrate, inhibited both the basal and the DBcAMP-stimulated recovery of functional polarity, whereas staurosporine and Gö 6976, 2 PKC inhibitors, accelerated the basal recovery of polarized function. Disruption of the microtubule cytoskeleton by colchicine induced only minor changes under basal, but not under DBcAMP-stimulated, conditions. The Golgi complex disruptor, brefeldin A, significantly delayed, and the microfilament-disrupting agent, cytochalasin D, fully blocked, both processes. However, DBcAMP stimulated trafficking of vesicles containing CLF to the pericanalicular region under the last condition. Our results indicate that restoration of couplet polarity following isolation occurs via a Ca(2+)-calmodulin-mediated mechanism, which depends on microfilament, but not on microtubule integrity. A second pathway is activated by DBcAMP activation via Ca(2+)-calmodulin formation, whose requirements with respect to cytoskeletal components are opposite. PKC has a negative regulatory role in both pathways.
authors.affiliation: Institute of Experimental Physiology, CONICET-University of Rosario, Argentina. L.Klomp@wkz.azu.nl
authors.forename: M G
authors.initials: MG
authors.lastname: Roma
authors.forename: P
authors.initials: P
authors.lastname: Milkiewicz
authors.forename: E
authors.initials: E
authors.lastname: Elias
authors.forename: R
authors.initials: R
authors.lastname: Coleman
mesh_terms: D000199:Actins / Q000502:physiology; D000818:Animals; D027321:Anion Transport Proteins; D001648:Bile Canaliculi / Q000378:metabolism* / Q000502:physiology; D002352:Carrier Proteins / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D016922:Cellular Senescence / Q000502:physiology; D002793:Cholic Acids / Q000493:pharmacokinetics; D003593:Cytoplasm / Q000378:metabolism; D003599:Cytoskeleton / Q000502:physiology*; D005452:Fluoresceins / Q000493:pharmacokinetics; D005456:Fluorescent Dyes / Q000493:pharmacokinetics; D022781:Hepatocytes / Q000378:metabolism*; D006728:Hormones / Q000502:physiology; D008297:Male; D051381:Rats; D017208:Rats, Wistar; D015398:Signal Transduction / Q000502:physiology*; D014617:Vacuoles / Q000502:physiology
chemical_list: D000199:Actins; D027321:Anion Transport Proteins; D002352:Carrier Proteins; D002793:Cholic Acids; D005452:Fluoresceins; D005456:Fluorescent Dyes; D006728:Hormones; C072313:cholyl-lysylfluorescein