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A rat liver plasma membrane preparation was isolated and characterized both biochemically and morphologically. The isolation procedure was rapid, simple and effective in producing a membrane fraction with the following biochemical characteristics: ~40-fold enrichment in three plasma membrane markers, 5'-nucleotidase, alkaline phosphodiesterase t (both putative bile canalicular membrane enzymes), and the asialo-glycoprotein (ASGP) receptor (a membrane glycoprotein present along the sinusoidal front of hepatocytes); a yield of each of these plasma membrane markers that averaged ~16%; and minimal contamination by lysosomes, nuclei, and mitochondria, but persistent contamination by elements of the endoplasmic reticulum. Morphological analysis of the preparation revealed that all three major domains of the hepatocyte plasma membrane (sinusoidal, lateral, and bile canalicular) were present in substantial amounts. The identification of sinusoidal membrane was further confirmed when ASGP binding site,s were localized predominantly to this membrane in the isolated PM using electron microscope autoradiography. By morphometry, the sinusoidal front membrane accounted for 47% of the total membrane in the preparation, whereas the lateral surface and bile canalicular membrane accounted for 6.8% and 23% respectively. This is the first report of such a large fraction of sinusoidal membrane in a liver plasma membrane preparation.

The goal of this study was to evaluate the permeability characteristics of Calu-3, human bronchial epithelial cells to passive and actively transported drugs and to correlate the data with other in vitro models and rat lung absorption in vivo. Air-interface cultured Calu-3 cells grown on collagen-coated permeable filter supports formed "tight" polarized and well differentiated cell monolayers with apical microvilli and tight-junctional complexes. Within 8-10 days, cell monolayers developed trans-epithelial electrical resistance (TEER) > 1000 ohm cm2 and potential difference about 11-16 mV. Solute permeability was dependent on lipophilicity, and inversely related to molecular size. Calu-3 cells actively transported amino acids, nucleosides and dipeptide analogs, but not organic anions, organic cations or efflux pump substrates. The permeability characteristics of Calu-3 cells correlated well with primary cultured rabbit tracheal epithelial cells in vitro (r2 = 0.91), and the rate of drug absorption from the rat lung in vivo (r2 = 0.94). The absorption predicted from the regression equation correlated well with observed values. In conclusion, in vitro-in vivo correlation studies indicate that the Calu-3 cell culture model is a potentially useful model to predict absorption of inhalation delivery drug candidates.

The human intestinal oligopeptide transporter (PEPT1) facilitates the absorption of dipeptides, tripeptides, and many peptidomimetic drugs. In this study, a large number of peptides were selected to investigate the structural features required for PEPT1 transport. Binding affinity was determined in a Gly-Sar uptake inhibition assay, whereas functional transport was ranked in a membrane depolarization assay. Although most of the peptides tested could bind to PEPT1, not all were substrates. As expected, single amino acids and tetrapeptides could not bind to or be transported by PEPT1. Dipeptide transport was influenced by charge, hydrophobicity, size, and side chain flexibility. The extent of transport was variable, and unexpectedly, some dipeptides were not substrates of PEPT1. These included dipeptides with two positive charges or extreme bulk in either position 1 or 2. Our results identify key features required for PEPT1 transport in contrast to most previously described pharmacophores, which are based on the inhibition of transport of a known substrate.