Robert M. Ezzell

Culture matrix configuration and composition in the maintenance of hepatocyte polarity and function

Several extracellular matrix (ECM) configurations involving type I collagen and Matrigel were examined for their ability to support differentiated function and polarity of cultured adult rat hepatocytes. Collagen sandwich- and Matrigel-based cultures yielded superior and comparable albumin secretion for at least 2 weeks. In collagen sandwich, hepatocytes were polygonal, and formed multicellular arrays. Collagen sandwich was also found to promote in vivo-like polarization of F-actin, cell adhesion molecules (E-cadherin), and lateral (Na+, K(+)-ATPase, glucose transporter) and apical (dipeptidyl peptidase, aminopeptidase) membrane polarity markers, but not the expression of the gap junction protein connexin 32 and the epidermal growth factor (EGF) receptor. In contrast, hepatocytes cultured in or on Matrigel were more rounded and formed aggregates. Matrigel-based cultures also elicited detectable levels of connexin and EGF receptor and an altered distribution of F-actin, E-cadherin, and apical and lateral membrane proteins. Composite sandwich configurations containing collagen I and Matrigel restored markers lacking in the collagen sandwich, and showed a variable morphology and membrane polarity. Hepatocyte polarity could thus be manipulated by the overall ECM composition. Furthermore, in composite sandwich cultures, these manipulations can be effected largely independent of changes in hepatocyte morphology and albumin secretion.

Dynamics of cell membrane permeability changes at supraphysiological temperatures.

A quantitative fluorescent microscopy system was developed to characterize, in real time, the effects of supraphysiological temperatures between 37 degrees and 70 degrees C on the plasma membrane of mouse 3T3 fibroblasts and isolated rat skeletal muscle cells. Membrane permeability was assessed by monitoring the leakage as a function of time of the fluorescent membrane integrity probe calcein. The kinetics of dye leakage increased with increasing temperature in both the 3T3 fibroblasts and the skeletal muscle cells. Analytical solutions derived from a two-compartment transport model showed that, for both cell types, a time-dependent permeability assumption provided a statistically better fit of the model predictions to the data than a constant permeability assumption. This finding suggests that the plasma membrane integrity is continuously being compromised while cells are subjected to supraphysiological temperatures.

Relationship of Tissue and Cellular Interleukin-1 and Lipopolysaccharide after Endotoxemia and Bacteremia

Distributions of immunoreactive interleukin-1 (IL-1) and lipopolysaccharide (LPS) were studied in the tissues of rats after intravenous injection of purified LPS or live Escherichia coli bacteria. IL-1 staining in the spleen peaked at 4-8 h, colocalized with LPS in marginal zone macrophages, and was undetectable 24 h after injection, whereas LPS staining peaked at 24 h and was detectable for 4 weeks. The tissue IL-1 response was similar for LPS and live bacteria. Thus, tissue IL-1 is down-regulated within hours despite maintenance of LPS in the same cells for weeks. Macrophages in liver and lung had only slight IL-1 staining despite intense staining for LPS. Tissue IL-1 production appears to be differentially regulated after gram-negative bacteremia; LPS cleared by liver and lung macrophages elicit minimal IL-1, whereas there is high local IL-1 production in the marginal zone of the spleen that may increase immune responses to bacterial wall antigens.

Differences in F9 and 5.51 cell elasticity determined by cell poking and atomic force microscopy

We studied the elasticity of both a wild type (F9) mouse embryonic carcinoma and a vinculin‐deficient (5.51) cell line, which was produced by chemical mutagenesis. Using cell poking, we measured the effects of loss of vinculin on the elastic properties of these cells. F9 cells were about 20% more resistant to indentation by the cell poker (a glass stylus) than were 5.51 cells. Using the atomic force microscope to map the elasticity of wild type and vinculin‐deficient cells by 128×128 force scans, we observed a correlation of elasticity with cell poking elastometric measurements. These findings, as well as previous atomic force, rheologic, and magnetometric measurements [Goldmann and Ezzell, Exp. Cell Res. 226 (1996) 234–237; Ezzell et al., Exp. Cell Res. 231 (1997) 14–26], indicate that vinculin is an integral part of the cytoskeletal network.

Vinculin, talin and focal adhesions

One of the most complicated connections between actin and the plasma membrane is the focal adhesion, a complex of proteins and lipids that forms at sites where cells attach to the extracellular matrix. A major protein component of focal adhesions is vinculin. As with other components of the focal adhesion complex, vinculin illustrates the apparent redundancy of proteins that mediate the connection of actin to the plasma membrane. During the past 5 years there have been several studies examining the role of vinculin in cell function. The purpose of this article is to discuss these findings and present an integrated model of vinculin’s role in the cell. Vinculin associates with talin and alpha-actinin via its N-terminal region (Burridge & Mangeat, 1984; Wachsstock et al., 1987). It also self-associates to form head-to-tail dimers (Molony & Burridge, 1985; Johnson & Craig, 1994). There is further convincing evidence that vinculin contains an actin-binding domain (Menkel et al., 1994; Johnson & Craig, 1995). Vinculin is also a ligand for the focal adhesion complex protein paxillin (Turner et al, 1990). Furthermore, vinculin binds phospholipid bilayers non-covalently with an apparent two-step mechanism involving both electrostatic interactions with acidic head groups and insertion into the hydrophobic domain of lipid bilayers (Niggli & Burger, 1987). It has also been shown in in vitro lipid photolabelling studies that vinculin as well as talin directly inserts into the hydrophobic region of lipid bilayers (Goldmann et al., 1992; Niggli et al., 1994). In addition, vinculin interacts with phosphatidylinositol-4,5-bisphosphate (Fukami et al, 1994). Therefore, vinculin binds the lipid bilayer directly through one of several mechanisms and also binds actin directly.

Localization of Endotoxin in the Rat Intestinal Epithelium

High levels of circulating lipopolysaccharide (LPS) cause intestinal inflammation and increased permeability to bacteria and toxins. To better understand the effects of LPS on the gut, confocal microscopy and immunofluorescence staining were used to examine the distribution of LPS in the rat intestine after intravenous or enteral administration. LPS was localized in macrophages in the lamina propria from 1 h to >28 days after intravenous injection. LPS was also detected in the epithelial cells from 8 h to 7 days after injection. In contrast, LPS administered enterally was found in the gut lumen in close proximity to the mucosa but was not detected in enterocytes at any time. The concentration of LPS in enterocytes near the villus tip provides a mechanism for the clearance of endotoxin, by the turnover and shedding of LPS-containing enterocytes into the gut lumen, that has not been previously described.

Effects of dimethyl sulfoxide on cultured rat hepatocytes in sandwich configuration

A recently developed sandwich culture system, in which hepatocytes are sandwiched between two layers of collagen, has been shown to be capable of maintaining long-term expression of hepatocellular function (J. C. Y. Dunn et al., Biotechnol. Prog. 7, 237-245, 1991). The development of an adequate technique for the cryopreservation of hepatocytes in such a stable culture configuration would ensure a ready supply of hepatocytes for use in bioreactors or bioartificial liver support devices. This report describes the effects of exposing hepatocytes in sandwich culture to different concentrations of the cryoprotectant dimethyl sulfoxide (Me2SO) at 22 degrees C on Day 7 of culture. Cell function, morphology, and cytoskeletal organization were followed for 14 days after exposure. Hepatocellular morphology and albumin secretion remained normal when cultures were exposed for up to 120 min to predicted final Me2SO concentrations up to 1.33 M. Exposure for less than 60 min to equilibrium concentrations of up to 3.33 M Me2SO did not adversely affect cell morphology or albumin secretion rate, but at the highest concentration (3.33 M), increase of the exposure time to 60 or 120 min resulted in dramatic, irreversible cell damage and loss of function. Actin filament organization was shown to be undisturbed when the cells were exposed to 1.33 M Me2SO for 60 min, but was irreversibly disrupted by exposure to 3.33 M for 120 min. Based on these results, a simple and safe procedure is suggested for the addition of Me2SO to hepatocytes in a sandwich culture configuration and its subsequent removal, which will be valuable for studies on hepatocyte cryopreservation.

Transport of fluorescent dextrans across the rat ileum after cutaneous thermal injury.

ObjectiveTo determine the time course and spatial distribution of uptake of macromole-cules in the small intestine of rats subjected to cutaneous thermal injury. DesignProspective, controlled animal study. SubjectsFifty-five female Sprague-Dawley (CD) rats subjected to scald burn injury covering 20% (small injury; n = 29) and 40% (large injury; n = 6) of the total body surface area between 3 and 72 hrs after injury. Animals subjected to sham injury (n = 20) were used as controls. InterventionsThe intestine was cannulated near the distal ileum and incised 7 cm upstream. After perfusion with physiologic buffer, this intestinal loop was filled with the same buffer containing fluorescent-labeled dextrans (3 and 70 kilodaltons molecular weight) and ligated 4 cm from the injection point. After a 2-hr incubation period, the tissues were fixed with paraformaldehyde and cryosections were examined by laser confocal microscopy. The mesentery was also observed by laser confocal microscopy during incubation with the permeability probes. The disappearance of fluorescence was studied after washing the dextran probes from the gut lumen. Measurements and Main ResultsIn small injuries, there was a transient uptake of the 3-kilodalton dextran by the epithelium in focal regions of the ileum with the effects seen between 7 and 21 hrs after injury. In large injuries, epithelial staining was visible within 3 hrs, and the marker was seen to translocate both to the lymphatics and the blood vessels of the mesentery. In comparison, the 70-kilodalton dextran was visible within the intercellular spaces. Little or no epithelial staining was seen in sham-injured animals. ConclusionsThese results suggest that a transcellular pathway for the translocation of small macromolecules from the lumen to the mesentery can be activated after burn injury. The novel techniques described here will be useful to examine intestinal transport in various pathologic situations. (Crit Care Med 1994; 22:455–464)

The Study of Fast Reactions by the Stopped Flow Method

The mechanisms involved in protein reactions may be elucidated by investigating the reaction kinetics. There are two investigative approaches in ascertaining the combination of elementary steps which constitute these mechanisms:(1) steady state kinetics permits analysis of the overall reaction in which protein substrates are converted into products without examining the protein molecule itself; and (2) transient kinetics allows the direct measurement of each component in the overall reaction. In this latter case, attention is focused on changes occurring in the molecule upon binding to another protein whereas the study of the equilibrium (steady state) reaction does not examine the protein molecule directly. Equilibrium studies have wider applicability since they usually require only a small amount of protein and do not involve the use of special equipment. Necessarily, the information obtained is indirect and often ambiguous. Although transient kinetics requires special techniques for measuring the rates of fast reactions in solutions, it provides information which is far more direct and useful for elucidating complicated mechanisms of reactions. Thus, the two approaches are complementary and both are indispensable for the study of protein reactions.