The unifying theme of this proposal is control of access of macromolecules to bile. Access of substances to bile is either paracellular or transcellular. Separate sections of this proposal address each. The section on the paracellular pathway deals with mechanisms and consequences of acute alterations in hepatocyte tight junctional (TJ) permeability brought about by 10(-8)M VP. In the isolated perfused rat liver, peptide hormones which mobilize intracellular (Cal') cause an acute reversible but still permselective increase in TJ permeability. This allows leakage from bile of low MW cholephilic molecules, especially cations. When the biliary bile acid is a micelle former (e.g., taurocholate) cations efflux less readily than when the bile acid is not (e.g., taurodehydrocholate) presumably because of cation partitioning into the micelle. These data have been confirmed in vitro using a 3500 MW cutoff dialysis membrane suggesting that the bile-plasma barrier is behaving as a permselective passive dialysis membrane which can change the spectrum of its size selectivity in response to the biologic environment. We now propose to define the transduction pathway of TJ opening by blocking putative steps in this pathway and observing the effects on TJ opening in the perfused liver and on myosin light chain (MLC) phosphorylation in isolated hepatocytes. Agents or interventions will include: hypocalcemia, cytochalasin D, calmodulin inhibitors W-7 and trifluorperazine, the protein kinase C inhibitor H-7 and activators, phorbol esters. MLC phosphorylation is quantified by an immunoprecipitation and autoradiographic technique we have devised. The antimyosin Ab used in immunoprecipitation will also be used to study distribution of myosin about the TJ and to seek morphologic alterations induced by 10(-8) VP. Because immunofluorescence light microscopy will not give the resolution we require, we shall use TEM and immunogold techniques to study TJ ultrastructure before and during 10(-8)M VP stimulation. With regard to the transcellular pathway, we have accumulated much pilot data to suggest it can be regulated. This proposal addresses the mechanism of transcytosis, in particular whether it is terminally a lysosomal event and more particularly whether it is an autolysosomal event. We shall first perform studies to determine the proportion of HRP taken up by parenchymal vs. nonparenchymal cells. We shall then investigate the effect of perturbing transcytosis (e.g., with DBcAMP, elevated biliary pressure, 12mM glymidine) on the ratios of HRP to lysosomal enzymes in bile and in lysosomes. If ratios of biliary HRP to lysosomal enzyme activities change little in these instances, a lysosomal pathway will be supported. We shall study the pathways more closely by using conditions which selectively interrupt the autophagocytic pathway (refeeding and 2/3-hepatectomy) to seek support for our hypothesis, based on pilot data, that HRP transcytosis is via the autophagolysosomal pathway.
| Yamaguchi, Y; Dalle-Molle, E; Hardison, W G (1993) Hepatocyte horseradish peroxidase uptake is saturable and inhibited by mannose-terminal glycoproteins. Am J Physiol 264:G880-5 |
| Poucell, S; Hardison, W G; Miyai, K (1992) Regenerative stimulus increases hepatocyte tight junctional permeability. Hepatology 16:1061-8 |
| Hardison, W G; Heasley, V L; Shellhamer, D F (1991) Specificity of the hepatocyte Na(+)-dependent taurocholate transporter: influence of side chain length and charge. Hepatology 13:68-72 |
| Hardison, W G; Dalle-Molle, E; Gosink, E et al. (1991) Function of rat hepatocyte tight junctions: studies with bile acid infusions. Am J Physiol 260:G167-74 |
| Hardison, W G; Lowe, P J; Shanahan, M (1989) Effect of molecular charge on para- and transcellular access of horseradish peroxidase into rat bile. Hepatology 9:866-71 |
