Substrate adhesion of rat hepatocytes: Mechanism of attachment to collagen substrates

Attachment of rat hepatocytes to collagen, which occurs without the aid of fibronectin, was found to be a time-dependent reaction characterized by an initial lag phase of 10–20 min before stable attachment bonds began to form. Increasing the density of molecules in the collagen substrates enhanced the rate of cell attachment. The hepatocytes attached essentially equally well to all the collagen types tested (types I, II, III, IV and V). The initial rate of cell attachment was more rapid to native collagen than to denatured collagen or α1(I) chains, apparently indicating different affinities of the cells for these substrates. However, if cells were incubated for 60 min or more, efficient attachment occurred to the α1(I) chain and to all cyanogen-bromide-treated peptides tested (α1-CB2, α1-CB3, α1-CB4, α1-CB5, α1-CB6A, α1-CB7, α1-CB8, α2-CB2, α2-CB3 and α2-CB4) but not to the aminopropeptide of type I procollagen. A low but significant degree of attachment also took place to substrates made of synthetic peptides with the collagen-like structures (Gly-Ala-Pro)_n, (Gly-Pro-Pro)_n and (Gly-Pro-Hyp)_n, whereas no attachment was observed to polyproline. We suggest that the cell-binding sites in collagen have a simple structure and occur in multiple copies along the collagen molecule. Addition of collagen in solution inhibited intial cell attachment, an effect that persisted longer on substrates made of α1(I) chain than on denatured collagen. The collected data are interpreted in terms of a model for cell-to-collagen adhesion where the formation of stable attachment bonds requires the binding of several low-affinity receptors, clustered at the site of adhesion, to collagen molecules in the substrate.     

Adhesion of human platelets to immobilized trimeric collagen

Human platelets adhere to trimeric Type 1 chick collagen that was covalently linked to plastic slides, providing the basis for a well- defined quantitative assay. The number of platelets that adhere is a function both of platelet concentration and of collagen density on the slides. In contrast with other in vitro assays using collagen that is not covalently linked to the substratum, we found no platelet-platelet aggregation. Adhesion was absolutely dependent on Mg2+, whereas Ca2+ was ineffective. Native trimeric collagen conformation was required for adhesion, since platelets did not bind to slides containing heat- denatured collagen, or isolated alpha 1(1) or or alpha 2(1) chains. Modifications of collagen oligosaccharides had no effect on adhesion. Adhesion was inhibited by cytochalasin D but was not affected by prostaglandin E1, apyrase, acetylsalicylic acid, or theophylline. Because this assay measures platelet-collagen adhesion in the absence of platelet-platelet aggregation, it should facilitate identification of the platelet surface components that directly mediate this adhesion.     

Ultrastructure of ventral membranes of rat hepatocytes spread on type IV collagen

Rat hepatocytes obtained by means of liver perfusion with collagenase were allowed to spread on type IV collagen coated coverslips for 20 h. Interference reflection microscopy revealed a peripheral ring of dark spots. Carbon replicas of ventral membranes left attached to coverslips after lysis and squirting provided high resolution information on the ultrastructure of the protoplasmic surface. Correlative light and electron microscopy of the same ventral membrane showed that the area of the peripheral 'adhesion annulus' was rich in clathrin-coated structures (sheets, pits and vesicles). In vertical thin sections of hepatocyte monolayers numerous small smooth vesicles were observed piled up below the peripheral portion of the cell. These findings suggest high cytotic activity at the cell periphery during spreading. No bundles of microfilaments were observed in cells after squirting or in sections, but a ring of filaments at the cell periphery could be seen in many cells in whole mount preparations after treatment with Triton X-100. The absence of microfilaments associated with the points of adhesion indicates a cytoskeleton independent adhesion mechanism in hepatocytes during the first 20 h of spreading.     

Primary culture of rat hepatocytes using membrane-supported collagen sandwich with hormone-free medium

Summary Primary culture of rat hepatocytes in hormone-free medium using membrane-supported collagen sandwich maintained their cellular morphology and expressed albumin secretion for about 3 weeks in vitro. It was reconfirmed that mimicking the cellular environment in vivo was effective for cellular maintenance.     

Different beta 1-integrin collagen receptors on rat hepatocytes and cardiac fibroblasts

Detergent extracts of primary rat hepatocytes and neonatal cardiac fibroblasts were applied to collagen type I-Sepharose in the presence of 1 mM MnCl2. Elution of bound proteins by 10 mM EDTA yielded one beta 1-integrin heterodimer from hepatocytes with an Mr of 180,000/115,000 under nonreducing conditions. Two beta 1-integrins with Mr's (nonreduced) of 180,000/115,000 and 145,000/115,000 could be isolated from surface-iodinated fibroblasts. A monoclonal antibody, 3A3, directed against the rat homolog of the human integrin VLA-1, precipitated the affinity-purified Mr 180,000/115,000 heterodimer, establishing the relatedness of the Mr 180,000 subunit to the alpha 1-chain of the beta 1-integrin subfamily. Both the alpha 1 beta 1-integrin and the 145,000/beta 1-integrin heterodimers bound specifically to Sepharose beads derivatized with the collagen fragment alpha 1(I) CB3, which lacks RGD sequences. Immunofluorescence staining using the 3A3 monoclonal antibody revealed that the rat alpha 1 beta 1-integrin was present at focal adhesion sites of fibroblasts grown on native collagen type I- but not on fibronectin-coated substrates, although both types of substrates supported the formation of beta 1-integrin containing focal adhesions. Similarly, hepatocytes cultured on substrata coated with collagen type I (but not fibronectin) were stained in a patchy pattern localized to the cell periphery by 3A3 IgG. Furthermore, 3A3 IgG completely inhibited the attachment of hepatocytes to collagen type I, whereas under identical conditions the attachment of fibroblasts to these substrates was inhibited only by approximately 40%. The attachment of both hepatocytes and cardiac fibroblasts to fibronectin was unaffected by the presence of the 3A3 antibody. Collectively these data show that a rat homolog of the human VLA-1 heterodimer both biochemically and functionally fulfills the criteria of a single collagen receptor on rat hepatocytes. In contrast, rat cardiac fibroblasts utilize two different collagen-binding integrins to adhere to collagen, one of which is the rat homolog of the human VLA-1 heterodimer. Furthermore alpha 1(I) CB3 contains cell binding sites for beta 1-integrins.     

Rapid degradation of newly synthesized collagen by primary cultures of adult rat hepatocytes

Greater than 95% of the total radioactive hydroxyproline synthesized by adult rat hepatocytes during the first 24 to 48 hours in monolayer culture is soluble in 5% TCA. During the next 24-hour culture period, the amount of TCA-soluble hydroxyproline is reduced to approximately 40%. Likewise, rat hepatoma cells (HTC) also produce a significant amount of TCA-soluble hydroxyproline (48%). These findings suggest that hepatocytes not only have the capacity to synthesize collagen, but also have the ability to degrade this fibrous protein.     

Attachment of rat hepatocytes in vitro to substrata of serum protein, collagen, or concanavalin A.

Isolated rat hepatocytes attach to, and spread on, the surface of polystyrene tissue culture dishes in the presence of serum. The attachment is essentially complete in 30 min at 37 °C, whereas no attachment occurs at 0 °C. Dead (trypan blue-stainable) cells do not attach; hence the plating efficiency (percentage of cells attaching) is close to the percentage of intact cells in the hepatocyte suspension. Attachment in the presence of serum is relatively independent of pH, but requires divalent cations. Mg²⁺ stimulates attachment more effectively than Ca²⁺, and a combination of both cations gives maximal attachment. Cells do not attach readily to untreated dishes in the absence of serum, but attach to and spread on dishes precoated with adsorbed serum protein, concanavalin A (ConA), or a film of collagen. The attachment-promoting activity in serum is destroyed by acid treatment, by heating to 70 °C, and by protease treatment. It is therefore most probably a protein, which, like collagen and ConA, can bind to receptors on the hepatocyte surface.     

Long-term culture of primary rat hepatocytes with high albumin secretion using membrane-supported collagen sandwich

Primary cultured rat hepatocytes in a membrane-supported collagen sandwich maintained their normal cell morphology and high level of albumin secretion for over 56 days. It was found that the existence of an upper layer of collagen gel is crucial for long-term culture and that the transference of cellular nutrients between the culture media and hepatocytes from both the upper and the lower sides of gel layers promotes albumin secretion. These facts suggest that the membrane-supported collagen sandwich mimics well thein vivo environment of hepatocytes. This method has great potential for the long-term culture of primary cells.     

Isolation of heparan sulfate proteoglycan from beneath the monolayers of rat hepatocytes and its binding to type IV collagen

Primary cultures of rat hepatocytes maintained as monolayer in a serum-free medium synthesise and secrete sulphated proteoglycans. Nearly 5% of the total 35(S)-sulphated material was obtained in a soluble form from beneath the cell layer. A shift in gel filtration pattern on beta-elimination with alkali suggested that it is a sulphated proteoglycan. On ion exchange chromatography over Dowex AG 1 x 2, the major fraction was eluted with 1.25 M NaCl. Further, nearly 80% of the 35(S)-labeled material was susceptible to nitrous acid degradation and more than 90% of the material was resistant to chondroitinase ABC digestion suggesting that it is predominantly a heparan sulphate proteoglycan (HSPG). Since HSPG is a major component of basement membrane, its binding with collagen was studied by a solid phase binding assay. About 75% of the 35(S) HSPG bound to wells coated with type IV collagen whereas only about 20% bound to type I collagen at physiological pH. Binding to collagen IV was reduced by about 50% when free GAG chains were used indicating that the protein core is also involved in interaction with the collagen. These results indicate the possible role of this basal extracellular heparan sulphate proteoglycan in the basal lamina formation.     

Activation of collagen synthesis in primary culture of rat liver parenchymal cells (hepatocytes)

An increase in collagen synthesis by hepatic parenchymal cells (hepatocytes) was observed during 8 days in primary culture by the quantification of total [3H]hydroxyproline as a marker of total collagen synthesis and the ratio of [3H]hydroxyproline in the high-molecular-weight fraction to total [3H]hydroxyproline as a marker of collagen degradation after incubation of the cells with [3H]proline for 24 h. Type analysis of the collagen produced by the cells after 8 days in culture showed the presence of type I and type III collagens in addition to the components corresponding to type IV and type V (alpha A and alpha B) collagens. Only the latter two types were found in the collagens produced by the cells after 2 days in primary culture. The purity of the hepatocytes inoculated was 97%, and the majority of the contaminating small cells were erythrocytes. The rate of serum albumin synthesis, which is a typical function of the hepatocytes, was constant or increased during the culture period. Immuno-electron microscopic observation indicated the production of type I collagen by the hepatocytes after 8 days in primary culture. These results are explained only by the activation of collagen synthesis in the day-8 hepatocytes in primary culture.     

Vasopressin inhibits type-I collagen and albumin gene expression in primary cultures of adult rat hepatocytes

The mechanisms that regulate collagen gene expression in hepatic cells are poorly understood. Accelerated Ca2+ fluxes are associated with inhibiting collagen synthesis selectively in human fibroblasts (Flaherty, M., and Chojkier, M. (1986) J. Biol. Chem. 261, 12060-12065). In suspension cultures of isolated hepatocytes, the Ca2+ agonist vasopressin increases cytosolic levels of free Ca2+ (Thomas, A.P., Marks, J.S., Coll, K.E., and Williamson, J. R. (1983) J. Biol. Chem. 258, 5716-5725). However, whether vasopressin's interactions with plasma membrane V1 receptors attenuate hepatic collagen production is unknown. We investigated this problem by studying vasopressin's effects on collagen synthesis and Ca2+ efflux in long-term primary cultures of differentiated and proliferation-competent adult rat hepatocytes. Twelve-day-old quiescent cultures were exposed to test substances and labeled with [5-3H]proline. Determinations of radioactivity in collagenase-sensitive and collagenase-resistant proteins were used to calculate the relative levels of collagen production. Synthetic [8-arg]vasopressin stimulated 45Ca2+ efflux within 1 min and inhibited hepatocyte collagen production within 3 h by 50%; overall rates of protein synthesis were not affected significantly. In cultures labeled with [35S]methionine, vasopressin also decreased the levels of newly synthesized and secreted albumin, but not fibrinogen, detected in specific immunoprecipitates analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. Northern blot analyses using specific [32P]cDNA probes revealed 70% decreases in hybridizable levels of collagen alpha 1(I) mRNA in hepatocyte cultures treated with either vasopressin or Ca2+ ionophore A23187; hybridizable levels of albumin mRNA also fell approximately 50% following vasopressin treatment. Vasopressin did not affect collagen production in quiescent cultures of mouse Swiss 3T3, human myofibroblast or rat smooth muscle cells; and hepatocyte collagen production was unaffected by treatment with glucagon or dibutyryl cAMP. Thus, accelerated Ca2+ fluxes induced by vasopressin are associated with decreased production of hepatocyte collagen and albumin in primary cultures that simulate quiescent adult rat liver.     

Intracellular cAMP determines the extent of degradation and not the synthesis of collagen by rat hepatocytes

Intracellular collagen degradation in normal rat hepatocytes was exponetially stimulated by db-cAMP (10–100 µM). The effect was manifested as a decrease (p < 0.01) in net collagen production. The extent of degradation directly co-related with the intracellular cAMP levels, only upto a threshold concentration (16.2 ± 1.3 p moles/10⁶ cells) elicited by 100 µM of db-cAMP. Higher concentrations induced no further increment. Forskolin adenylate cyclase activator (10–50 µM), produced similar effects demonstrating cAMP dependence of the phenomenon. Both db-cAMP as well as Forskolin stimulated collagen degradation (p < 0.05) in hepatocytes from rats administered CCL₄. However, the extent of stimulation was significantly (p < 0.01) less compared to that observed in normal hepatocytes. Our data demonstrates that elevated cAMP levels regulate net collagen content by signalling intracellular collagen degradation and not synthesis.Abbreviations cAMP 3,5 cyclic Adenosine Monophosphate - db-cAMP dibutyryl cyclic Adenosine Monophosphate - TCA Trichloroacetic Acid - Coll. Collagen - DMEM Dulbecoo's Minimal Essential Medium     

Sensitization of rat hepatocytes to hyperthermia by calcium

The viability of isolated rat hepatocytes, as assayed by trypan blue exclusion, decreases in a dose-dependent fashion during exposure to hyperthermia (D0 [43 degrees C] = 105 +/- 10 min, D0 [45 degrees C] = 24 +/- 4 min). Hyperthermic sensitivity varies as a function of extracellular Ca2+ concentration in a biphasic manner; optimum survival occurs at 1-5 mM Ca2+, with sensitization in the absence of Ca+ and increasing sensitization at Ca2+ concentrations greater than 10 mM. Ca influx does not correlate well with loss of viability for hepatocytes in 4 mM extracellular Ca2+; influx does not occur until viability decreases to less than 1%. Under sensitizing conditions, Ca2+ influx proceeds loss of viability. Influx begins within 15 min at 45 degrees C in 15 mM Ca2+, and the ionophore A23187 is a potent hyperthermic sensitizer in the presence of extracellular Ca2+. Thus, Ca2+ influx, whether caused by high extracellular Ca2+ or A23187, increases cellular damage caused by supraoptimal temperatures, although some Ca2+ is necessary for maximum resistance, probably because of stabilization of Ca2+ binding proteins against thermal denaturation or possibly to Ca2+-induced decrease in lipid fluidity.     

Microelectrode measurement of cell membrane potential in isolated hepatocytes attached to collagen

Isolated spherical rat hepatocytes attached to collagen-coated cover slips generate a mean membrane potential (Em) of -78 +/- 9 mV as measured with high-resistance microelectrodes. The recordings were biphasic and were stable for upto 20 minutes. The correlation between external potassium concentration and Em was not linear. Several potassium-channel blockers did not effect the membrane potential. Addition of ouabain added to the incubation solution slowly depolarized the cells. The results indicate a high potassium permeability of the isolated spherical hepatocytes attached to collagen.     

Responsiveness of RNA degradation to amino acids in cultured rat hepatocytes: comparison with isolated rat hepatocytes.

The role of amino acids in the regulation of RNA degradation was investigated in cultured hepatocytes from fed rats previously labeled in vivo with [6-14C]orotic acid. Rates of RNA degradation were determined between 42 and 48 h of culture from the release of radioactive cytidine in the presence of 0.5 mM unlabeled cytidine. The fractional rate was about 4.4 +/- 0.4%/h in the absence of amino acids (0x). The catabolism of RNA was decreased to basal level (1.5 +/- 0.3%/h) by the addition of amino acids at 10 times normal plasma concentration (10x). The inhibition of RNA degradation, expressed as percentage of maximal deprivation-induced response (0x minus 10x), averaged 60% at normal plasma levels of amino acids. The degree of responsiveness was greatly improved as compared to freshly isolated hepatocytes (20%) and was similar to the sensitivity previously observed with perfused livers. In cultured hepatocytes, the sensitivity of RNA degradation to amino acids was not affected by varying the volume of medium from 1 to 4 ml per dish. In freshly isolated hepatocytes, the inhibitory effect of amino acids was not modified by changing the cell density from 0.5 to 5 x 10(6) cells per ml. In the range of normal plasma concentration of amino acids, the low sensitivity of RNA degradation in isolated hepatocytes persisted with inhibition ranging from 10 to 20%. These findings suggest that the control of RNA degradation in both cultured and isolated hepatocytes is not affected by the total quantity of amino acids available in the medium, but their concentration is crucial. Electron microscopy observations and the inhibitory effect of 3-methyl-adenine in cultured rat hepatocytes partially confirmed the role of the lysosomal system in the increase of RNA degradation and its regulation by amino acids.     

Primary culture of rat hepatocytes entrapped in cylindrical collagen gels: An in vitro system with application to the bioartificial liver

A static culture model employing cylindrical collagen-hepatocyte gels is reported for large scale testing of conditions relevant to the three compartment hollow fiber bioartificial liver. High density hepatocyte cultivation was achieved by cell entrapment within the collagen-hepatocyte gel. Hepatocyte viability was assessed by vital staining, gel contraction, and insulin utilization. Measures of hepatocyte-specific function included albumin synthesis, ureagenesis, lidocaine biotransformation, and cholate conjugation. Although hepatocyte viability remained stable through the seven day incubation period, hepatocyte functions were not uniformly preserved. Albumin synthesis remained stable, while representative P-450 and conjugation activities decreased with time. This static culture system will facilitate the development of a hollow fiber bioartificial liver which utilizes cylindrical collagen-hepatocyte gels.Abbreviations FDA fluorescein diacetate - EB ethidium bromide - MEGX monoethylglycinexylidide