Rab3D, a small GTP-binding protein implicated in regulated secretion, is associated with the transcytotic pathway in rat hepatocytes

Rab3 isotypes are expressed in regulated secretory cells. Here, we report t hat rab3D is also expressed in rat hepatocytes, classic models for constitu tive secretion. Using reverse transcriptase polymerase chain reaction (RT-P CR) with primers specific for rat rab3D, we amplified a 151 base pair rab3D fragment from total RNA extracted from primary cultures of rat hepatocytes . Immunoblot analysis using polyclonal antibodies to peptides representing the N- and C-terminal hypervariable regions of murine rab3D recognized a pr otein of similar to 25 kd in hepatocyte lysates, hepatic subcellular fracti ons, and tissue extracts. The distribution of rab3D was primarily cytosolic ; however, only membrane-associated rab3D significantly bound guanosine tri phosphate (GTP) in overlay assays. Several lines of investigation indicate that rab3D is associated with the transcytotic pathway. First, rab3D was en riched in a crude vesicle carrier fraction (CVCF), which includes transcyto tic carriers. Vesicular compartments immunoisolated from the CVCF on magnet ic beads coated with anti-rab3D antibody were enriched in the transcytosed form of the polymeric IgA receptor (pIgA-R), but lacked not only the pIgA-R precursor form associated with the secretory pathway, but also a Golgi mar ker protein. Second, indirect immunofluorescence on frozen liver sections a nd in polarized cultured hepatocytes localized rab3D-positive sites at or n ear the apical plasma membrane and to the pericanalicular cytoplasm. Finall y, cholestasis induced by bile duct ligation (BDL), a manipulation known to slow transcytosis, caused rab3D to accumulate in the pericanalicular cytop lasm of cholestatic hepatocytes. Our results indicate that rab3D plays a ro le in the regulation of apically directed transcytosis in rat hepatocytes.