Receptor-Mediated and Fluid-Phase Transcytosis of Horseradish Peroxidase across Rat Hepatocytes

Horseradish peroxidase (HRP) is often used as a fluid-phase marker to characterize endocytic and transcytotic processes. Likewise, it has been applied to investigate the mechanisms of biliary secretion of fluid in rat liver hepatocytes. However, HRP contains mannose residues and thus binds to mannose receptors (MRs) on liver cells, including hepatocytes. To study the role of MR-mediated endocytosis of HRP transport in hepatocytes, we determined the influence of the oligosaccharid mannan on HRP biliary secretion in the isolated perfused rat liver. A 1-minute pulse of HRP was applied followed by marker-free perfusion. HRP appeared in bile with biphasic kinetics: a first peak at 7 minutes and a second peak at 15 minutes after labeling. Perfusion with 0.8 mg/mL HRP in the presence of a twofold excess of mannan reduced the first peak by 41% without effect on the second one. Together with recently published data on MR expression in rat hepatocytes this demonstrates two different mechanisms for HRP transcytosis: a rapid, receptor-mediated transport and a slower fluid-phase transport.

Transport in rat vessel walls. II. Macromolecular leakage and focal spot size growth in rat arteries and veins

Transendothelial lipid transport into and spread in the subendothelial intima of large arteries, and subsequent lipid accumulation, appear to start plaque formation. We experimentally examine transendothelial horseradish peroxidase (HRP) transport in vessels that are usually, e.g., pulmonary artery (PA), or almost always, e.g., inferior vena cava (IVC), atherosclerosis resistant vs. disease prone, e.g., aorta, vessels. In these vessels, HRP traverses the endothelium at isolated, focal spots, rather than uniformly, for short circulation times. For femoral vein HRP introduction, PA spots have 30-s radii [∼53.2 μm (SD10.4); compare aorta: 54.6 μm (SD8.75)] and grow quickly from 30 s to 1 min (40%, P < 0.05) and more slowly afterward ( P > 0.05). This trend resembles the aorta, suggesting the PA has a similarly sparse intima. With carotid artery (CA) HRP introduction, the 30-s spot (132.86 ± 37.32 μm) is far larger than the PAs, grows little (∼28%, P < 0.05) from 30 to 60 s, and is much flatter than the artery curves. Transverse electron microscopic sections after ∼10 min HRP circulation show thin, intense staining immediately beneath both vessels’ endothelia with an almost step change to diffuse staining beyond. This indicates the existence of a sparse, subendothelial intima, even when there is no internal elastic lamina (IVC). This motivates a simple model that translates growth rates into lower bounds for the flow through focal leaks. The model results and our earlier wall and medial hydraulic conductivity data explain these spot growth curves and point to differences in transport patterns that might be relevant in understanding the immunity of IVC to disease initiation.

Role for IL-4 in macromolecular transport across human intestinal epithelium

Increased epithelial permeability is associated with intestinal inflammation, but there is little information on factors that regulate barrier function in the absence of or before inflammation. We examined if interleukin (IL)-4, or serum from atopic individuals, could alter the barrier function of human colonic epithelial (T84) monolayers to antigenic-sized macromolecules. IL-4 and atopic serum significantly decreased T84 monolayer resistance and increased transepithelial horseradish peroxidase (HRP) transport. Bidirectional transport studies demonstrated that IL-4 selectively enhanced apical-to-basal movement of HRP. HRP transport induced by IL-4 was inhibited by cold (4°C) and the tyrosine kinase inhibitor genistein, but not the protein kinase C inhibitor staurosporine. Electron microscopic analysis demonstrated that both transcellular and paracellular pathways were affected. Anti-IL-4 antibodies abolished the increase in HRP transport in response to both IL-4 and serum. We speculate that enhanced production of IL-4 in allergic conditions may be a predisposing factor to inflammation by allowing uptake of luminal antigens that gain access to the mucosal immune system.

Synaptic inputs and morphology of sustained ON-ganglion cells in the mudpuppy retina

1. Sustained ON-ganglion cells from the mudpuppy retina were studied with a combined approach, including intracellular and extracellular recording from the superfused retina-eyecup preparation, pharmacology with bath-applied 2-amino-4-phosphonobutyrate (APB), and retrograde and intracellular staining using horse radish peroxidase (HRP). 2. Bath application of micromolar levels of APB selectively blocks the light response of ON-bipolar cells; APB was used to separate synaptic inputs into those which originate from ON- vs. OFF-bipolar cells. This approach clearly demonstrates that the light response of the vast majority of sustained ON-ganglion cells is primarily the result of sustained excitatory inputs that arise (directly or indirectly) from ON-bipolar cells. 3. APB revealed the presence of transient excitatory OFF-inputs in many sustained ON-ganglion cells that are normally not evident. 4. Five sustained ON-ganglion cells were intracellularly stained with HRP and their morphology was analyzed with the aid of a computer-assisted neuron reconstruction system. The stained cells are anatomically similar, based on quantitative analysis of a number of morphological parameters. The dendritic trees of all five cells are primarily confined to sublamina b of the inner plexiform layer, although some cells have a small number of processes that ramify in sublamina a. These latter processes may relate to the transient excitatory OFF-inputs revealed with APB application. 5. Ganglion cells which are morphologically similar to the stained, intracellularly sustained ON-ganglion cells were found in a collection of Golgi-like cells that were labeled by retrograde HRP transport. This raises the possibility that sustained ON-ganglion cells in the mudpuppy may constitute a morphologically identifiable class of retinal ganglion cells in this species. There is also some suggestion that a morphologically similar class of OFF-cells may be present.

Postnatal development of protein absorption in conventional and germ-free mice

In the jejunal epithelium of adult animals, horseradish peroxidase (HRP) is transported via two functional pathways: a major route involving lysosomal degradation and a minor route allowing transport of the intact protein. The postnatal development of HRP absorption and the influence of conventional microflora were studied in vitro, using jejunal epithelium of conventional and germ-free suckling mice mounted in Ussing chambers. In conventional mice, tritiated peroxidase ([3H]HRP) transport from mucosa to serosa did not change with age except during days 3 and 4, when it increased fivefold. This increase was entirely due to the rise in intact HRP transport and was not related to a decrease in lysosomal cathepsin B and D activities. In germ-free mice, HRP transport developed similarly but decreased by 75% along both pathways. This reduced absorption was associated with lower cathepsin B and D activities. These results strengthen the existence of two transcellular pathways in nonimmunoglobulin protein absorption by the proximal gut with a limiting step situated before the lysosomal system, presumably at the brush-border membrane. Their development is not parallel and does not correlate with gut closure to immunoglobulin transfer.