Synthesis and degradation of eicosanoids in primary rat hepatocyte cultures

Arachidonic acid metabolites may play an important role in liver physiology, yet hepatocyte prostaglandin synthesis has not been characterized extensively. We used RIA to study production and clearance of several eicosanoids in confluent primary cultures of rat hepatocytes in serum-free, hormonally-defined medium. Under basal, unstimulated conditions 6-keto-PGF1 alpha (spontaneous breakdown product of prostacyclin) and 13,14-dihydro-15-keto-PGE (DHK-PGE, a metabolite of PGE) accumulated in the culture medium. Hepatocytes cleared 6-keto-PGF1 alpha, thromboxane B2, and DHK-PGE from the medium. Production of eicosanoids by primary cultures appeared resistant to indomethacin and several other cyclooxygenase inhibitors. This apparent resistance to indomethacin was not caused by rapid metabolism of indomethacin, by failure of the drug to enter hepatocytes, or by insensitivity of hepatocyte cyclooxygenase to the drug. Metabolism of PGE to DHK-PGE may be saturated under in vitro conditions. Hepatocytes can synthesize significant amounts of eicosanoids, although they are probably less active in this regard than are non-parenchymal cells.     

Synthesis, transport, and processing of apolipoproteins of high density lipoproteins

Cell biology methods have greatly influenced the elucidation of the biosynthetic pathways of apolipoproteins. In vitro and tissue culture systems allow the study, to a large extent, of the process of synthesis, intracellular processing, secretion, and extracellular processing of the major high density lipoprotein apoproteins apoA-I and A-II and also of a minor component, apoA-IV. Whereas the latter apoprotein is equipped only with a signal sequence, the primary translation products of apoA-I and apoA-II carry N-terminal extensions of preprosequence of 24 amino acids for apoA-I and 23 amino acid residues for apoA-II. The pro-form of apoA-I characterized by a hexapeptide extension is completely stable intracellularly and is secreted as such. The pro-form is further processed by a serum protease specific for an unusual -Gln-Gln-Asp-Glu-sequence site. Pro-apoA-II, a pentapeptide sequence, is partially processed intracellularly to its mature form and secreted together with the residual pro-form. The cleavage site of pro-apoA-II is characterized by two basic amino acid residues Arg-Arg, present also in other known pro-proteins. The biological function of the N-terminal pro-sequences and details of their final processing by the serum protease(s) have yet to be established.     

Synthesis and secretion of albumin and transferrin by foetal rat hepatocyte cultures

Hepatocytes derived from foetal rat liver synthesize and secrete albumin and transferrin when maintained in primary culture. These proteins are produced for at least seven days under the conditions of culture. Studies on hepatocyte cultures derived from 12, 13, 14, 15 and 19-day foetal rats show that the maximal cellular rate of secretion of both proteins increases about 50-fold over this period. The maximal rate of albumin secretion in all cultures is achieved after one day in culture and decreases in hepatocytes from early foetuses after the fourth to sixth day in culture. Transferrin secretion by hepatocytes from 12 to 15 day foetuses increases markedly during the second day of culture and is relatively constant thereafter. In contrast, secretion of transferrin by hepatocytes from 19-day foetuses is constant from the first day of culture. The results show that both albumin and transferrin are synthesized and secreted by the foetal liver as early as the twelfth day of gestation. The increase in the rate of transferrin secretion that occurs during culture of hepatocytes from 12 to 15 day foetuses may reflect the development of a secretory mechanism that is different from that for albumin.     

Biological properties of a hepatocyte growth factor from rat platelets

In an accompanying communication we demonstrated that about half of the potency of rat serum to stimulate DNA synthesis in cultured adult rat hepatocytes resides in a polypeptidelike substance from the platelets. A lysate of rat platelets was able to restore the potency of platelet-poor rat serum, whereas a lysate of human platelets inhibited thymidine incorporation by the hepatocytes. Moreover, addition to these cultures of either highly purified human platelet-derived growth factor (PDGF) or human platelet factor 4 (PF-4) failed to influence DNA synthesis either alone or in the presence of rat or human platelet-poor serum, which is required for expression of PDGF activity. Unlike the human platelet factors, rat platelet lysate (RPL) was moderately active by itself and was augmented equally well by platelet-poor serum from either source. At concentrations below 5%, platelet-poor serum from hypophysectomized rats was as potent as that from normal rats in augmenting RPL activity. This suggests that, unlike PDGF, which is not activated by hypophysectomized rat serum, the hepatotrophic component of RPL does not require the presence of exogenous somatomedins for activity, but interacts instead with other plasma constituents or with somatomedins produced by the hepatocytes in vitro. Rat platelets do, however, appear to contain PDGF or its rat equivalent in addition to the hepatocyte growth factor, since if they are heated to 100 degrees C for 10 min, their ability to stimulate nuclear labeling in confluent BALB/c 3T3 cells is not impaired, while their ability to stimulate DNA synthesis in rat hepatocytes is destroyed. These studies indicate that the hepatocyte growth factor from rat platelets differs from PDGF in its biological as well as physical characteristics, but that rat platelets also contain PDGF or an equivalent substance.     

A comparative study of apolipoproteins in mouse and rat

1. Apolipoproteins isolated from plasma samples of 10 inbred strains of mice and 17 inbred strains of rats were subjected to isoelectric focusing and second-dimension-pore-gradient-SDS-electrophoresis. 2. All major HDL apolipoproteins could be identified by their isoelectric point and mol. wt. 3. In inbred strains of mice polymorphism could be demonstrated for apo A-I and apo A-II. 4. In inbred strains of rats no apolipoprotein polymorphism could be demonstrated.     

Synthesis and properties of a photoaffinity probe for the glucagon receptor in hepatocyte plasma membranes

The reaction of glucagon with 4-fluoro-3-nitrophenylazide has been shown to afford the photosensitive derivative, N^?-4-azido-2-nitrophenyl-glucagon. The structure and properties of this derivative were established by amino acid analysis, absorption and fluorescence spectroscopy, deamination, Edman degradation and photolysis. This photoaffinity derivative of glucagon has been used to label specifically glucagon binding sites on hepatocyte plasma membranes.     

Characterization of the apolipoproteins of rat plasma lipoproteins.

Purified fractions of three major rat high-density lipoproteins (HDL) and one rat very low-density lipoprotein (VLDL) were isolated by Sephadex gel chromatography or preparative sodium dodecyl sulfate gel electrophoresis. These proteins were characterized by amino acid analysis, end-group analysis, molecular-weight determination, polyacrylamide gel electrophoresis, and circular dichroism. One of these rat proteins, of molecular weight 27 000, appears to be homologous with the human A-I protein. However, rat HDL possesses two additional major components not reported in human HDL - an arginine-rich protein of molecular weight 35 000 and a protein of molecular weight 46 000. The arginine-rich protein of the rat is similar in size and amino acid analysis to the arginine-rich protein reported in human VLDL. A major component of rat VLDL of 35 000 molecular weight appears similar or identical to the arginine-rich protein in rat HDL by every criterion employed for their characterization.     

Effect of oxidation on the properties of apolipoproteins A-I and A-II

Purified apolipoprotein A-I has been separated by reversed-phase high performance liquid chromatography (HPLC) into multiple peaks and these peaks have been characterized. One peak, apoA-Ib had a relatively longer retention time on HPLC but its retention time could be shortened by treatment by hydrogen peroxide. CNBr cleavage studies indicated that the differences in apoA-Ib and in its oxidation product, apoA-Ia, were due to the different oxidation states of methionine. This phenomenon was also observed in apoA-II, where methionine oxidation produced two more forms of this apolipoprotein in addition to the native form. These isomers were found to have different secondary structures and affinities for lipid. Model peptide analogs of the amphipathic helix with the same sequence but with methionine and methionine sulfoxide at the nonpolar face of the amphipathic helix were synthesized and studied. It was found that the lipid affinities of these synthetic peptide isomers were very different. They also differed in their secondary structures as studied by circular dichroism (CD). We propose that methionine oxidation introduces hydrophilic residues at the nonpolar face of the amphipathic helical domains of these apolipoproteins and, therefore, alters their secondary structure and lipid affinity.     

Synthesis and properties of a photoaffinity probe for the glucagon receptor in hepatocyte plasma membranes.

The reaction of glucagon with 4-fluoro-3-nitrophenylazide has been shown to afford the photosensitive derivative, Nepsilon-4-azido-2-nitrophenyl-glucagon. The structure and properties of this derivative were established by amino acid analysis, absorption and fluorescence spectroscopy, deamination, Edman degradation and photolysis. This photoaffinity derivative of glucagon has been used to label specifically glucagon binding sites on hepatocyte plasma membranes.     

Scatter factor and hepatocyte growth factor: activities, properties, and mechanism.

Scatter factor (SF) was first identified as a fibroblast-derived protein which disperses (i.e., "scatters") cohesive colonies of epithelium. SF-like proteins were found in human smooth muscle cell conditioned medium, amniotic fluid, and placental tissue. SFs markedly stimulate migration of epithelial, carcinoma, and vascular endothelial cell types at picomolar concentrations. Hepatocyte growth factors (HGFs) were originally described as platelet- and serum-derived proteins which stimulate hepatocyte DNA synthesis. Partial amino acid sequence data for mouse and human SFs indicate significant homology with HGFs. We used biological, biochemical, and immunological assays to evaluate and compare the activities, properties, and mechanisms of action of mouse SF, human SF (fibroblast or placenta derived), and recombinant human HGF (hrHGF). We report the following findings: (a) mouse SF exhibits species-related differences in biological activities relative to the human factors; (b) human SF and hrHGF show significant overlap in biological activities (i.e., hrHGF stimulates motility of multiple normal and carcinoma cell types, whereas human SF stimulates DNA synthesis in several normal cell types); (c) the three factors contain common antigenic determinants; and (d) all three proteins stimulate rapid phosphorylation of tyrosine residues on the c-met protooncogene protein product (the putative receptor for HGF) and on another protein with Mr 110,000. A few biological and immunological differences between human SFs and hrHGF were observed. These may reflect minor variations in amino acid sequence or posttranslational modification related to the sources of the factors. Taken as a whole, our findings suggest that by structural, functional, immunological, and mechanistic criteria, human SF and human HGF are essentially identical.     

Differential effects of insulin, epinephrine, and glucagon on rat hepatocyte mitochondrial activity

Oxidation of [2,3-14C]succinate carbons in the mitochondrial Krebs cycle was used as a probe to investigate the effects of insulin, epinephrine, glucagon, and 2,4-dinitrophenol (2,4-DNP) on isolated rat hepatocytes. Epinephrine, glucagon, and 2,4-DNP had a far greater stimulatory effect on 14CO2 formation from [2,3-14C]succinate than insulin. Unlike insulin, epinephrine and glucagon had no significant effect on the anabolic utilization of succinate carbons for protein synthesis. Our results suggest that although epinephrine, glucagon, and 2,4-DNP enhance the movement of tracer carbons through the Krebs cycle, only insulin is capable of enhancing amphibolite utilization for protein synthesis.     

Quantification of apolipoproteins in rat serum and in cultured rat hepatocytes by enzyme-linked immunosorbent assay

An enzyme-linked immunosorbent assay (ELISA) has been developed to measure apolipoproteins in rat serum. Nondelipidated whole serum was heat-treated at 52 degrees C for 3 h in phosphate-buffered saline containing 0.1% Tween-20 before assay. Monospecific rabbit anti-rat apolipoprotein antibodies were added to 96-well polystyrene microtiter plates which had been coated with purified rat serum apolipoproteins or unknown samples. After incubation and washing, goat anti-rabbit serum antibodies conjugated with horseradish peroxidase were added to the plates and incubated. The bound peroxidase activity was assayed after further washing. Serum apolipoprotein concentrations were calculated by comparison against purified standards that were assayed simultaneously with the unknown samples. The intraassay coefficients of variation for apolipoprotein AI, E, and AIV (Apo AI, E, and AIV) were 2.3, 4.4, and 5.3%, and interassay coefficients of variation were 6.1, 5.5, and 7.9%, respectively. The ELISA assay is sensitive to nanogram quantities of rat serum apolipoproteins and the results agree well with those measured by densitometry. The serum concentrations of Apo AI, E, and AIV of a normal fed rat were found to be 504 +/- 8, 413 +/- 20, and 262 +/- 20 micrograms/ml, respectively. When cultured as monolayers in Waymouth's medium for 1 day, rat hepatocytes secreted Apo AI, E, and AIV at rates of 2.51, 61.8, and 48.9 ng protein/mg cell protein/h.     

Acute modification of biomechanical properties of the bone-ligament insertion to rat limb unweighting

We investigated the acute adaptation of the rat femur-medial collateral ligament-tibia (FMT) complex to 7 days of limb unweighting by means of a hind-limb suspension protocol. Male, young adult, Harlan Sprague-Dawley rats were randomly assigned to either control or suspended groups. Rats deprived of hind limb-to-ground contact forces had a 42% decrease in soleus muscle mass compared with the control group. Medial collateral ligament (MCL) length and cross-sectional area were measured, and each FMT complex was tension tested to failure. All failed at their tibia-MCL insertion. The ultimate load in the FMT and the peak Kirchhoff stress in the MCL (occurring immediately before insertion site failure) were significantly reduced in the suspended group. The suspended MCLs were 9.7% larger in area and 5.7% shorter in length than the controls under the same preload (0.25 N). We found no significant differences between the control and suspended MCLs in Green strain, stretch, or deformation immediately before insertion site failure, nor did we find a significant difference in the MCL tangent modulus. This study indicates that even acute periods of limb unweighting can structurally compromise bone-ligament insertions. Further, this study implies that the adaptations responsible for this structural compromise must involve acute changes in the intrinsic zone (or zones) of the bone-ligament insertion.     

Effect of 2,5-anhydro-D-mannitol on membrane potential in rat hepatocyte couplets and hepatocyte monolayer cultures

The fructose analogue 2,5-anhydro-D-mannitol (2,5-AM), which depletes liver cells of ATP, has been shown to alter liver cell membrane potential (V(m)) in situ and in superfused liver slices. To study this effect of 2,5-AM on hepatocytes in more detail, patch-clamp experiments in the current-clamp mode were performed using two established models, rat hepatocyte couplets and confluent rat hepatocytes in primary culture. 2,5-AM, which has previously been shown to hyperpolarize hepatocytes in superfused liver slices and in vivo, failed to alter V(m) of hepatocyte couplets. Increasing intracellular Ca(2+) by addition of thapsigargin or ionomycin also did not evoke a change of V(m). This is most likely due to a lack of Ca(2+)-dependent K(+) channels in rat hepatocyte couplets. In contrast, 2,5-AM depolarized the cells in confluent hepatocyte monolayers. This depolarization was mimicked after inhibition of Na(+)/K(+) ATPase by ouabain. Ouabain was also able to block 2, 5-AM's effect on monolayer V(m). Thus, 2,5-AM affects the membrane potential of isolated and cultured hepatocytes in a way not comparable with cells integrated in the liver.     

Long-term culture of adult rat hepatocyte spheroids

Hepatocytes from adult rats were cultured on poly-HEMA-coated surface to form spheroids in hormonally defined media as previously shown with newborn rat hepatocytes. Spheroidal aggregates of adult rat hepatocytes were morphologically similar to those of newborn rat hepatocytes and could also form a monolayer of uniform liver parenchyma-like cells when transferred on collagen-coated surfaces even after 2 months of culture. Under these culture conditions, albumin and transferrin secreted in vitro by adult rat hepatocyte spheroids were detectable by immunoprecipitation method at least until 2 months of culture. The production of proteins by hepatocyte spheroids could be regulated in vitro by IL-6: the secretion of alpha 2-macroglobulin was increased and the secretion of albumin was decreased in the presence of this cytokine. In addition, cytochrome P450 IA1 was strongly induced by methylcholanthrene in adult rat hepatocyte spheroids, and the induction remained relatively constant up to 22 days of culture. These cells were also able to metabolize lidocaine to monoethylglycinexylidine when measured up to 14 days of culture, showing the presence of a relatively high level of P450 IIIA2. The UDP-glucuronyltransferase activity, specific for bilirubin conjugation, decreased to 18% of the initial value after 2 weeks of culture. This work showed that adult rat hepatocytes in long-term spheroid culture kept differentiated functions, providing a new model for the in vitro study of hepatocyte functions and complementing that of newborn rat hepatocytes using the same system.     

Distribution of C apolipoproteins between the vascular and lymph compartments of the rat

This study has investigated the kinetics of transfer of C apolipoproteins between the vascular and lymph compartments of the rat. Very-low-density lipoprotein, labeled with [125I]apolipoprotein C, was injected intravenously into lymph duct-cannulated rats and the redistribution of radioactivity between lymph and plasma followed at frequent intervals for 3 h. Equilibration between the two compartments was rapid (10-15 min), and thereafter removal from both compartments continued at similar rates. Specific radioactivity determinations showed that lymph C-III-0, C-III-3, and C-III-2,1 apolipoproteins rapidly reached values identical to those of corresponding plasma C apolipoproteins and the interrelationship between the curves were consistent with precursor-product relationships in which all, or most, of the product (lymph apolipoprotein C-III) was derived from the precursor (plasma). However, the specific radioactivity curves for C-II peptide did not cross; the lower value for lymph C-II apolipoprotein suggests that, unlike C-III apolipoproteins, a substantial proportion (approx. 40%) of lymph C-II peptide is not derived from the plasma compartment. The most likely source of the unlabeled lymph apolipoprotein C-II is synthesis and secretion from the intestine.