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.     

Adhesion of rat hepatocytes to collagen

Rat hepatocytes, freshly isolated with a collagenase perfusion technique, were found to attach within 1 h on collagen substrates and on culture dishes coated with cold insoluble globulin (CIG) or asialoceruloplasmin (AC). Spreading was observed on collagen and CIG but not on AC. Both attachment and spreading occurred in a simple balanced salt solution in the absence of serum. In the absence of serum no attachment was observed on plain plastic dishes or on dishes coated with serum albumin or other plasma proteins, unless divalent manganese ions were present. In the presence of manganese the hepatocytes attached to all surfaces tested, but no spreading occurred. Attachment to collagen occurred equally well to collagens type I or type III both in the native, fibrillar state and in the denatured state. Collagen attachment required magnesium ions but did not appear to involve the collagen-linked carbohydrates. Different mechanisms were found to operate in hepatocyte attachment to collagen and to AC; the latter is most likely mediated by the hepatocyte surface receptor involved in recognition and uptake of asialoglycoproteins. The role of CIG in hepatocyte attachment to collagen was investigated. Data are presented suggesting that this glycoprotein, which mediates the adhesion of fibroblasts to collagen, is not required for hepatocyte attachment to collagen.     

Comparison of trout hepatocyte culture on different substrates

Summary Comparisons were made of attachment and viability of rainbow trout (Salmo gairdneri) hepatocytes in short-term (2 days), primary culture on plastic, collagen-coated or extracellular matrix (ECM) coated dishes. Hepatocyte isolation routinely yielded cells with good viability (96%). Cells plated on ECM attached with high efficiency (93%) in contrast to cells cultured on plastic or collagen (∼20%). The cells plated on ECM flattened out and formed monolayers, while the cells on plastic and collagen rounded up and formed multi-cell aggregates in suspension. Viability of cells in all substrates remained high over the 2 day culture period. ECM is the first substrate to support trout-hepatocyte attachment in primary culture. Differentiated liver function was maintained in cells cultured on ECM as evidence by the induction of tyrosine aminotransferase by hydrocortisone (200%). This work was supported in part by research grant R809599010 from the U. S. Environmental Protection Agency. Editor's Statement This paper reports improved methods for culture of trout liver-derived cells that make in vitro investigations of fish metabolism, carcinogenesis and chemical toxicity more feasible than previously applied techniques. Recent interest in fish as models for study and indicators of effects of envionmental and food-related toxins make this work timely, poarticularly since many of the compounds of interest are primarily metabolized by hepatocytes or act on liver as a major target. David W. Barnes     

Viability and differential function of rainbow trout liver cells in primary culture: Coculture with two permanent fish cells

Summary The study investigates the influence of different culture conditions on attachment, viability and functional status of rainbow trout (Oncorhynchus mykiss) liver cells in primary culture. Cells were isolated by a two-step collagenase perfusion and incubated in serum-free, chemically defined minimal essential medium (MEM), (a) as a monolayer on uncoated PRI-MARIA? dishes, (b) as a monolayer on culture dishes coated with calf collagen type 1, and (c) in coculture with the established fish cell lines RTH-149 or RTG-2. Cell attachment was assessed from DNA and protein concentrations per dish, viability was estimated from cellular lactate dehydrogenase release, and the metabolic status was investigated by measuring activities of the phosphoenolpyruvate carboxykinase and biotransformation enzymes as well as the total cytochrome P450 contents. Seeding of hepatocytes on collagen-coated dishes did not alter cell attachment or detachment from the culture substrate, but had a small, but not significant effect on cell viability and metabolic parameters. Coculture of liver cells and RTG-2 cells reduced hepatocyte detachment from the culture substrate, and it was associated with a significant elevation of 7-ethoxyresorufin-O-deethylase activities in the hepatic cells. Cytochrome P450 contents, however, were not altered. The coculture effect on liver cell physiology clearly depended on the type of cell line, because coculture with RTH-149 cells led to similar, but much weaker effects than obtained in cocultures with RTG-2 cells. Electron microscopical observations revealed the existence of gap junctions and possible exocytosis-like transport between cell lines and hepatocytes. The results point to the potential of coculture systems to improve physiological parameters of trout liver cells in primary culture.     

Subculture of proliferating adult rat hepatocytes in medium supplemented with nicotinamide and EGF

Summary To establish parenchymal hepatocyte cell lines, we tried to subculture the primary hepatocytes isolated from adult rats. The hepatocytes were cultured in serum-free modified Dulbecco’s modified Eagle’s medium supplemented with 10 mM nicotinamide and 10 ng/ml epidermal growth factor. When 6×10⁵ cells were plated on 35-mm dishes coated with rat tail collagen, the cells proliferated and reached confluence at Day 6 to Day 8. The first subculture was carried out at Day 8 using 0.005% collagenase and gentle pipettings. Most cells were recovered and plated on the new dishes coated with the collagen (first passage). The attached cells could proliferate and reached near confluence when the cells occupied more than two-thirds of the dish surface. About a week after the first subculture, the second one was conducted. Although the number of the recovered cells was smaller than at the first passage, the cells could attach and proliferate to a certain extent. Thereafter, they were maintained for more than 2 mo. but they never overgrew. Albumin secretion into the culture medium was confirmed in the subcultured cells. Ultrastructurally, these subcultured cells possessed hepatic characteristics such as peroxisomes with a crystalline nucleoid and bile-canaliculus structures. When 10% fetal bovine serum and ascorbic acid 2-phosphate were added to the cells of the second passage, they began to proliferate very slowly. These proliferating cells were mainly mononucleate and had a small cytoplasm. In addition, some of them could differentiate into typical mature hepatocytes by forming a three-dimensional structure interacting with nonparenchymal cells. In this experiment, we showed the successful subculturing of parenchymal hepatocytes isolated from adult rats and provided evidence that the subcultured cells still have the potential to proliferate and to differentiate.     

Substrate ifluences human epidermal melanocyte attachment and sperading in vitro

Summary Previous culture systems for melanocytes have employed serum-supplemented medium and uncoated plastic dishes, prohibiting examination of possible substrate influences on cellular morphology and function. We now report, using a sensitive serum-free system and a quantitative procedure for evaluating cellular morphology, that modification of the plating surface affects human epidermal melanocyte attachment rate and subsequent morphology in vitro. Melanocytes attach and spread more rapidly on surfaces coated with fibronectin or Type I/III collagen or on surfaces previously conditioned by human keratinocytes, dermal fibroblasts, melanocytes, or melanoma cells than do melanocytes on untreated control surfaces. Type IV collagen and laminin, although minimally beneficial for cell attachment, do support a characteristics melanocyte morphology that differs from that seen either on the other coated surfaces or on uncoated plastic controls. Addition of fetal bovine serum at the time of inoculation has no appreciable effect on attachment but markedly improves cell spreading on untreated surfaces, while addition of nerve growth factor with or without serum to this system fails to affect cell attachment or spreading. Our data establish that human epidermal melanocytes are indeed capable of responding morphologically to substrate signals. The ability of several biochemically unrelated surfaces to enhance melanocyte attachment rate and spreading suggests that melanocytes have surface receptors with a variety of specificities. This work is relevant to the development of improved culture systems for melanocytes in vitro and to understanding melanocyte behavior in vivo. This work was supported by the USDA Agricultural Research Service, by a grant from Cheesebrough-Ponds, Inc., and by a Dermatology Foundation Fellowship (Dr. Yaar).     

Switching from differentiation to growth in hepatocytes: control by extracellular matrix.

Studies were carried out to analyze how different extracellular matrix (ECM) molecules regulate hepatocyte growth and differentiation. Freshly isolated rat hepatocytes were cultured on non-adhesive plastic dishes that were pre-coated with defined densities of either laminin, fibronectin, type I collagen, or type IV collagen. Sparse cell plating densities were used to minimize cell-cell contact formation and all studies were carried out in chemically defined medium that contained a saturating amount of soluble growth factors. Dishes coated with a low ECM density (1 ng/cm2) supported hepatocyte attachment, but did not promote cell spreading or growth. Computerized image analysis confirmed that over 80% of cells remained free of contact with other cells under these conditions. Yet, these round cells maintained high levels of albumin gene expression as well as elevated secretion rates for multiple liver-specific proteins (albumin, transferrin, and fibrinogen), regardless of the type of ECM molecule used for cell attachment. When ECM coating densities were raised from 1 to 1,000 ng/cm2, cell spreading, expression of histone mRNA, DNA synthesis, and cell proliferation all increased in parallel. Activation of growth by high ECM densities was also accompanied by a concomitant down-regulation of differentiated functions and again, dishes coated with all four types of ECM molecules produced similar effects. Thus, the ability to switch hepatocytes from differentiation to growth (i.e., between different genetic programs) is not limited to a single ECM molecule, a distinct three dimensional ECM geometry, or due to alteration of cell-cell interactions. Rather, the regulatory signals conveyed by immobilized ECM molecules depend on the density at which they are presented and thus, on their ability to either prohibit or support cell spreading.     

Degradation of extracellular thymidine by cultured hepatocytes from rainbow trout (Salmo gairdneri)

1. Trout hepatocytes cultured as attached monolayers had low rates of [3H]-thymidine ([3H]-TdR) incorporation during replicative or repair synthesis of DNA. 2. Within 2 hr, most [3H]-TdR was metabolized by trout hepatocytes to a major product that eluted in advance of intact [3H]-TdR on Sephacryl S-200 columns. 3. Metabolism of [3H]-TdR by trout hepatocytes rapidly destroyed its ability to label replicating indicator cultures of proliferating rat hepatocytes. 4. These studies demonstrate that [3H]-TdR tracer assays for DNA synthesis cannot be reliably used in cultured trout hepatocytes which catabolize thymidine much more rapidly than do rat hepatocytes.     

Estradiol-17β stimulation of vitellogenin synthesis in primary culture of male rainbow trout hepatocytes

Summary Trout liver was disaggregated by perfusion with collagenase. Highly purified populations of parenchymal cells were obtained, yielding up to 12×10⁸ viable cells per liver. Culture conditions for providing enhanced attachment and long-term cell survival were defined. The hepatocytes, attached to the plastic and cultured in a serum-free medium, were maintained as monolayers for 10 to 12 d without cell division. Cell viability decreased slightly within 1 wk as shown by intracellular lactic dehydrogenase and DNA content. Furthermore, these primary cultures were demonstrated to retain their competence to respond to estrogens by de novo synthesis of vitellogenin allowing the study of gene expression in vitro. This system, therefore, offers many applications for the study of the regulation of trout liver metabolism. This work was supported by CNRS: ATP Bases biologiques de l'Aquaculture 9.82.101 and INSERM.     

The influence of temperature, relative humidity and host factors on the attachment and survival of Boophilus microplus (Canestrini) larvae to skin slices

Attachment of B. microplus larvae was examined using slices (0.5 mm) of bovine skin stretched over a suitable medium. Optimal temperature for attachment lay between 31 and 38°C. At 38°C, 70 to 80% had attached by 4 h but this was followed 6–7 h after the release of the larvae by a significant decrease in the percentage attached. By 8 h, the percentage attached had risen again and remained at 70–80% for the subsequent 16 h. Attachment was not influenced by ambient relative humidity within the range 20–75 % on the time scale studied (16 h). Most larvae denied access to the skin surface at 20 or 45 % R.H. (38°C) died within 24 h. Those allowed to feed survived at 45% but most died at 20% R.H. There was no difference in attachment when bovine or rabbit serum or phosphate buffered saline was used, nor when skin was taken from Zebu × European cattle or Herefords. Attachment was no different when skin was taken from neck, rib or rump, or from cattle with different levels of resistance to B. microplus but there was reduced attachment with mouse skin. Attachment was reduced on skin which had been stored at ? 15°C for 1 day but storage at 10°C for 16 h had no effect. These results are related to problems of tick water balance, host specificity and stimuli for tick attachment.     

Membrane glycoproteins involved in hepatocyte adhesion to collagen type I

Liver membrane glycoproteins with affinity for immobilized collagen type I were subjected to preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by electroelution of the separated proteins. Electroeluted glycoproteins with ability to neutralize the inhibitory effect of anti-CollCAM antibodies on hepatocyte adhesion to collagen were collected from several consecutive runs and used to raise a high titer antiserum, denoted anti-CollCAM II. IgG from this antiserum inhibited the attachment of hepatocytes to dishes coated with collagen type I, but not to fibronectin- or collagen type IV-coated dishes. When the antibodies were immobilized to Sepharose CL-4B they bound three sets of glycoproteins with apparent Mr's of 105,000, 115,000, and 130,000 as analyzed by SDS-PAGE under nonreducing (NR) conditions. Upon reduction (R) the glycoproteins migrated with apparent Mr's of 115,000, 130,000, and 160,000, respectively. The Mr 105,000-115,000 (NR) glycoproteins effectively neutralized the inhibitory effect exerted by both anti-CollCAM and anti-CollCAM II antibodies, on hepatocyte spreading and attachment to collagen type I substrates. Peptide mapping suggested the Mr 160,000 (R) species to be different from the Mr 115,000 (R).     

Down regulation of CYP 1A1 by glucocorticoids in trout hepatocytes in vitro

Summary Short-term culture of rainbow trout (Onchorhynchus mykiss) hepatocytes was used to examine the effect of dexamethasone (DEX) on microsomal CYP 1A1 protein content and 7-ethoxyresorufin-O-deethylase (EROD) activity in vitro. Hepatocytes prepared by controlled collagenase digestion and plated at a density of 0.25 × 10⁶ cells/cm² in plastic culture dishes precoated with trout skin extract (7.6 μg skin protein/cm²) to facilitate cell attachment were maintained at 16° C. Cells were treated with DEX (10⁻⁹ to 10⁻⁷ M) or vehicle (dimethyl sulfoxide, DMSO) at 24 h. Microsomal CYP 1A1 protein content and EROD activities were measured at 72 h. Both CYP 1A1 protein as measured by Western blots using CYP 1A1 specific anti-sera and EROD activity were significantly lower in DEX (10⁻⁸ to 10⁻⁷ M)-treated hepatocytes compared to untreated (control) or DMSO-treated cells. The effect was dose dependent in that a gradual decrease of CYP 1A1 protein and EROD activities were seen with increasing doses of DEX (10⁻⁸ to 10⁻⁷ M). DEX at 10⁻⁹ M was ineffective. Concomitant addition of 10⁻⁶ M RU486, a type II specific glucocorticoid receptor antagonist, to hepatocytes treated with 10⁻⁷ M DEX abolished the DEX effect. RU486 at 10⁻⁸ M was ineffective. Spironolactone (10⁻⁸ to 10⁻⁶ M), a type I specific glucocorticoid receptor antagonist, did not counteract the DEX effect. RU486 or spironolactone (10⁻⁶ M) alone had no effect on CYP 1A1 under similar conditions. DEX thus down regulates CYP 1A1 in fish cultured hepatocytes and this regulation is mediated through the type II glucocorticoid receptor(s).     

Isolation and characterization of biliary epithelial cells from rainbow trout liver

Summary Lectin binding and density gradient centrifugation were explored for isolating epithelial cells from trout liver. Hepatocytes exhibited preferential attachment to coverslips coated withPhaseolus vulgaris erythroagglutinin. Biliary epithelial cells attached with glycine max agglutinin; however, significant attachment of cellular debris limited the use of glycine max agglutinin. Percoll-density gradient centrifugation separated liver cells into two distinct populations with biliary cells and hepatocytes banding at densities of 1.04 and 1.09, respectively. A discontinuous gradient composed of 13% Ficoll (wt/wt) separated biliary cells from hepatocytes. The recovery of highly enriched biliary epithelial cells from trout liver using Ficoll gradients yielded approximately 8 million cells (0.1 ml packed cells) from 10 g liver. Western blot analysis demonstrated that the cytokeratin profile for extracts from biliary epithelial cell-enriched populations differ significantly from those seen with whole liver extracts or with extracts from hepatocyte-enriched populations. Ficoll-gradient purified biliary cells and hepatocytes attached to culture plates coated with trout skin extract and carried out linear incorporation of leucine into protein and thymidine into DNA for 24 h. A mixture of growth hormones (insulin, epidermal growth factor, and dexamethasone) stimulated thymidine incorporation into DNA; however, long-term culture of dividing biliary epithelial cells was not achieved. Chemical analysis of neutral and acidic glycolipids indicated that hepatocytes and biliary cells have similar glycolipid profiles with an exception in the region of GM3 mobility, which is attributable to differences in the ceramide moiety. These studies provide a starting point for further characterization of unique cell types of the trout liver that may be important in their response to toxic and carcinogenic agents.     

Extracellular matrix components influence the survival of adult cardiac myocytes in vitro

Calcium-tolerant myocytes were isolated from adult rat hearts by collagenase perfusion and plated on various substrates in serum-free medium and their adhesion to various extracellular matrix (ECM) components was determined. The myocytes attached readily to dishes coated with collagen type IV (C-IV), laminin (LN), and to fetal bovine serum (FBS) in a manner dependent on the concentration of the components. Substantially fewer myocytes adhered to dishes coated with fibronectin (FN) or to uncoated plastic dishes. Cells adhered equally well to dishes coated with C-IV, LN and FBS within 1-4 h. However, when examined after 2 weeks in culture it was found that only C-IV and LN could support survival of the attached myocytes, and when cultured on C-IV or LN the myocytes were spread and had formed a dense monolayer. The actin filaments had at this time reorganized linearly along the long axis of the cell and the myocytes contracted spontaneously. Rabbit antibodies were raised against myocyte membranes and their ability to inhibit attachment to ECM components was studied. Purified IgG inhibited attachment to C-IV, while having only a minor effect on attachment to LN. These data are compatible with the presence of a specific cell surface component(s) that interacts with ECM substrates and influences cell shape and possibly thereby influences cellular functions.     

Optimization of cryoprotectants for cryopreservation of rat hepatocyte

Rat hepatocytes were cryopreserved in hormonally-defined medium (HDM) containing either fetal bovine serum (FBS), glycerol, dimethyl sulfoxide (DMSO), sucrose or a mixture of these as a cryoprotectant. The best survival was with 10% (v/v) DMSO containing 30% (v/v) FBS using 5 x 10(5) hepatocytes ml(-1) at -70 degrees C for 5 d on type I collagen-coated dishes. After thawing, the cell viability was 81% determined by the MTT-test. The cryopreserved hepatocytes had the capacity of albumin synthesis similar to hepatocytes without cryopreservation. This result shows that cryopreservation of rat hepatocyte can be used for the evaluation of hepatic functions.     

Role of glucose and insulin in regulating glycogen synthase and phosphorylase activities in rainbow trout hepatocytes

This study, using ¹³C nuclear magnetic resonance spectroscopy showed enrichment of glycogen carbon (C1) from ¹³C-labelled (C1) glucose indicating a direct pathway for glycogen synthesis from glucose in rainbow trout (Oncorhynchus mykiss) hepatocytes. There was a direct relationship between hepatocyte glycogen content and total glycogen synthase, total glycogen phosphorylase and glycogen phosphorylase a activities, whereas the relationship was inverse between glycogen content and % glycogen synthase a and glycogen synthase a/glycogen phosphorylase a ratio. Incubation of hepatocytes with glucose (3 or 10 mmol·1^-1) did not modify either glycogen synthase or glycogen phosphorylase activities. Insulin (porcine, 10^-8 mol·1^-1) in the medium significantly decreased total glycogen phosphorylase and glycogen phosphorylase a activities, but had no significant effect on glycogen synthase activities when compared to the controls (absence of insulin). In the presence of 10 mmol·1^-1 glucose, insulin increased % glycogen synthase a and decreased % glycogen phosphorylase a activities in trout hepatocytes. Also, the effect of insulin on the activities of % glycogen synthase a and glycogen synthase a/glycogen phosphorylase a ratio were more pronounced at low than at high hepatocyte glycogen content. The results indicate that in trout hepatocytes both the glycogen synthetic and breakdown pathways are active concurrently in vitro and any subtle alterations in the phosphorylase to synthase ratio may determine the hepatic glycogen content. Insulin plays an important role in the regulation of glycogen metabolism in rainbow trout hepatocytes. The effect of insulin on hepatocyte glycogen content may be under the control of several factors, including plasma glucose concentration and hepatocyte glycogen content.     

Further studies on the effect of diet on serum thyroid hormone concentrations and thyroid histology in rainbow trout,Salmo gairdneri (Pisces,Salmonidae)

Synopsis In a 3 × 2 factorial experiment examining the effects of combinations of ambient temperature (18°, 15°, 9° C) and dietary protein content (35% and 45%) on thyroid activity inSalmo gairdneri, although there was an apparent increase in activity of the thyroid in cold-adapted trout, assessed by histological appearance of the gland, there were no significant changes in serum thyroid hormone titers. In a second experiment examining the effects of combinations of ambient temperature (15°, 12.5°, 10°C) with dietary lipid content (6% and 16%) there was a similar apparent increase in thyroid activity in cold-adapted fish which was accompanied, in fish fed the higher lipid diet, with an increase in serum thyroxine (T4) and triiodothyronine (T3) levels. Trout fed an ascorbic acid-free diet (experiment 3) had lower serum T3 levels than in those given an ascorbic acid supplemented diet (1280 mg·kg^-1). In experiments 2 and 3 serum thyroid hormone concentrations were approximately inversely proportional to ambient temperature and concomitant weight gain, but no such correlation was evident in experiment 1 suggesting that the changes in hormone levels in experiments 2 and 3 were not ipso facto related to differences in either ambient temperature or weight gain but rather to the specific metabolic changes imposed by the dietary lipids or ascorbic acid deficiency.     

The effect of pre-fasting diet and water temperature on liver glycogen and liver weight in rainbow trout, Salmo gairdneri Richardson, during fasting

The effect of feeding an isocaloric and isonitrogenous trout diet that contained different levels of digestible carbohydrate (cerelose) to rainbow trout at either 10 or 15° C on liver glycogen and liver weight was determined in two fasting studies of 12 and 41 days duration. Trout fed diets with increased levels of digestible carbohydrate (HC) had significantly higher liver-body weight ratios (LW) and liver glycogen (LG) than trout reared on low digestible carbohydrate diets (HF). Both LW and LG declined in fasting trout previously fed HC diets but declined little in fasting trout previously fed HF diets. Trout reared at 10° C had higher LW and LG than trout reared at 15° C on either the HC or HF diets. During fasting, the trout reared on HC diets at 10° C required a longer period of time for the LG and LW to decline to the levels of trout reared on the low carbohydrate diets, than did trout reared on the HC diets at 15° C. The results indicate that both pre-fasting diet and water temperature can affect liver glycogen utilization and liver weight in fasting trout. Prolonged elevation of LW and LG in fasting trout could jeopardize the survival rate of stocked trout, particularly at low water temperatures.     

Involvement of plasma membrane dipeptidyl peptidase IV in fibronectin-mediated adhesion of cells on collagen

Dipeptidyl peptidase IV is an exopeptidase found in the serum and in plasma membranes of most animal tissues. The role of this enzyme in cell-matrix interaction of BHK cells and hepatocytes grown on collagen-coated surfaces was investigated by three different approaches. 1) Glass surfaces were derivatized with bovine serum albumin which resulted in a cell-repulsing substratum. When it was further modified with Gly-Pro-Ala tripeptide, which is a substrate for dipeptidyl peptidase IV, BHK fibroblasts spread on it rapidly. The spreading could be inhibited by addition of free Gly-Pro-Ala or other substrates of the enzyme as well as by an inhibitor peptide Val-Pro-Leu. It was not influenced by tripeptides which were neither substrates nor inhibitors of dipeptidyl peptidase IV. 2) The addition of Gly-Pro-Ala to seeded cells slowed down the initial process of cell spreading on denatured collagen in the presence of fibronectin. The presence of both collagen and fibronectin was a necessary precondition for the spreading of cells in a manner sensitive to Gly-Pro-Ala. 3) Antiserum raised against mouse liver dipeptidyl peptidase IV added to the medium delayed the spreading of rat hepatocytes on denatured collagen in the presence of fibronectin in a manner similar to when Gly-Pro-Ala was added to the medium. These observations lead to the conclusion that plasma membrane dipeptidyl peptidase IV may be involved in the initial phase of fibronectin-mediated cell spreading on collagen.