Isozymic forms of protein kinase C in regenerating rat liver

The expression of multiple forms of protein kinase C (PK-C) was studied in regenerating rat liver using hydroxyapatite column chromatography. Two forms of the enzyme were found in the cytosolic as well as membrane fraction of livers from partially hepatectomized rats. The kinetic variation in the activation of these two liver isozymes by fatty acids, phosphatidylserine and diacylglycerol was similar to that reported for the PK-C subspecies from rat brain, designated types II and III. Intracellular redistribution of PK-C caused by phorbol 12-myristate 13-acetate (PMA) was concentration-dependent and was due to translocation of isozyme III, because type II was insensitive to 5 x 10(-8) M PMA. The activity ratio of the two isozymes in either the particulate or cytosolic fraction was the same at 22 h as compared to 4 h after partial hepatectomy.     

Agonist-induced desensitization of cholinergically stimulated phosphoinositide breakdown is independent of endogenously activated protein kinase C in HT-29 human colon carcinoma cells.

Activation of M3 muscarinic receptors in HT-29 cells by carbachol rapidly increases polyphosphoinositide breakdown. Pretreatment of these cells with carbachol (0.1 mM) for 5 h completely inhibits the subsequent ability of carbachol to increase [3H]inositol monophosphate ([3H]InsP) accumulation, paralleled by a total loss of muscarinic binding sites. In contrast, protein kinase C (PK-C)-mediated desensitization by incubation with phorbol esters [PMA (phorbol 12-myristate 13-acetate)], leading to a time- and dose-dependent inhibition of cholinergically stimulated InsP release (95% inhibition after 4 h with 0.1 microM-PMA), is accompanied by only a 40% decrease in muscarinic receptor binding, which suggests an additional mechanism of negative-feedback control. Neither carbachol nor PMA pretreatment had any effect on receptor affinity. Incubation with carbachol for 15 min caused a small increase of membrane-associated PK-C activity (15% increase, P less than 0.05) as compared with the potency of phorbol esters (PMA) (3-4-fold increase, P less than 0.01). Long-term incubation (4-24 h) with PMA resulted in a complete down-regulation of cytosolic and particulate PK-C activity. Stimulation of InsP release by NaF (20 mM) was not affected after a pretreatment with phorbol esters or carbachol, demonstrating an intact function of G-protein and phospholipase-C (PL-C) at the effector side. Determination of PL-C activity in a liposomal system with [3H]PtdInsP2 as substrate, showed no change in PL-C activity after carbachol (13 h) and short-term PMA (2.5 h) pretreatment, whereas long-term preincubation with phorbol esters (13 h) caused a small but significant decrease in PL-C activity (19%, P less than 0.05). Our results indicate that agonist-induced desensitization of phosphoinositide turnover occurs predominantly at the receptor level, with a rapid loss of muscarinic receptors. Exogenous activation of PK-C by phorbol esters seems to dissociate the interaction between receptor and G-protein/PL-C, without major effects on total cellular PL-C activity.     

Cyclosporin A inhibits proteolytic cleavage and degradation of membrane-bound protein kinase C in hepatocytes after stimulation by phorbol ester

Stimulation of hepatocytes by the tumor promoter phorbol 12-myristate 13-acetate (PMA) caused translocation of cytosolic Ca2+/phospholipid-dependent protein kinase C (PK-C). The major part of PK-C activity (greater than 80%) was associated with the membrane fraction after 30 min. During the following 6 h protein kinase C activity decreased to less than 10%. Minor amounts of Ca2+/phospholipid-independent PK-C activity were found in the cytosol fraction at all times; they temporarily increased 2.5-fold with PMA and decreased after 1 h. Cyclosporin A did not affect the translocation of PK-C from the cytoplasm to the membrane fraction, but the decrease of PK-C activity following translocation was blocked. No marked increase of Ca2+/phospholipid-independent PK-C activity was observed in the cytosol in the presence of cyclosporin A. Leupeptin, which is known to inhibit Ca2+-requiring non-lysosomal proteinases (e.g. calpain), showed an effect similar to cyclosporin A. Both agents reduced proteolytic degradation of cellular proteins observed in isolated hepatocytes after PMA treatment. Ca2+-ionophore A23187 in high doses (greater than 10(5) M) partly reversed cyclosporin A and leupeptin action.     

Inhibiting effect of plasma from normal and tumour bearing mice on the mitotic rate of regenerating liver

Plasma from normal mice and from mice bearing the ES2 transplantable malignant tumour was injected intraperitoneally at a dose of 0.01 ml/g body weight in partially hepatectomized mice. Control animals were injected with a solution of sodium citrate in saline. The recipients were killed at the first (14:00 hours/48 h). These times are the time of day and the number of h after partial hepatectomy and second (14:00 hours/72 h) peak times after partial hepatectomy. The number of colchicine metaphases per 1000 nuclei was determined for hepatocytes and litoral cells. A different effect was obtained with plasma from tumour-bearing compared with normal mice. Plasma from both sources when injected 26 h after partial hepatectomy (16:00 hours/26 h) inhibited the mitotic activity of hepatocytes at the next peak of regenerative activity (14:00 hours/48 h). The plasma from tumour-bearing mice also inhibited the peak on the following day (14:00 hours/72 h), whereas plasma from normal mice had no inhibitory effect and, indeed, a compensatory wave was observed at this time. Furthermore, plasma from tumour-bearing mice also showed an inhibitory effect at the first peak (14:00 hours/48 h) when injected at the time of partial hepatectomy (14:00 hours/00 h) or at 22 h before partial hepatectomy (16:00 hours/-22 h) whereas the injection of plasma from normal mice at these times had no inhibitory effect. In the litoral cells the injection of plasma from tumour-bearing mice made 22 h before hepatectomy (16:00 hours/-22 h) led to a stimulation of mitotic activity which was controlled at 14:00 hours/48 h.(ABSTRACT TRUNCATED AT 250 WORDS)     

Phorbol ester induces desensitization of PTH-stimulated cyclic AMP production by decreasing the PTH receptor binding in UMR-106 cells

Pretreatment of UMR-106 cells (rat osteoblast like osteosarcoma cell line) with the protein kinase C(PK-C) activating phorbol ester, phorbol 12-myristate 13-acetate (PMA) results in a time dependent (1-12h) desensitization of PTH-stimulated cAMP production. Compared to controls, PMA-treated cells showed 50% decrease of PTH-stimulated cAMP production. PK-C inhibitor, H-7 significantly blocked this PMA-induced desensitization. PTH receptor binding, assessed with 125I-[Nle8,Nle18,Tyr34]PTH-(1-34) as radioligand, was decreased by about 20% in PMA-treated cells. H-7 treatment completely restored receptor binding in PMA-treated cells. These data suggest that PK-C might act directly on PTH receptor which is coupling to adenylate cyclase, and induce desensitization.     

The Mitogen-Activated Protein Kinase Kinase/Extracellular Signal-Regulated Kinase Cascade Activation Is a Key Signalling Pathway Involved in the Regulation of G1 Phase Progression in Proliferating Hepatocytes

In this study, activation of the mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) signalling pathway was analyzed in proliferating rat hepatocytes both in vivo after partial hepatectomy and in vitro following epidermal growth factor (EGF)-pyruvate stimulation. First, a biphasic MEK/ERK activation was evidenced in G(1) phase of hepatocytes from regenerating liver but not from sham-operated control animals. One occurred in early G(1) (30 min to 4 h), and the other occurred in mid-late G(1), peaking at around 10.5 h. Interestingly, the mid-late G(1) activation peak was located just before cyclin D1 induction in both in vivo and in vitro models. Second, the biological role of the MEK/ERK cascade activation in hepatocyte progression through the G(1)/S transition was assessed by adding a MEK inhibitor (PD 98059) to EGF-pyruvate-stimulated hepatocytes in primary culture. In the presence of MEK inhibitor, cyclin D1 mRNA accumulation was inhibited, DNA replication was totally abolished, and the MEK1 isoform was preferentially targeted by this inhibition. This effect was dose dependent and completely reversed by removing the MEK inhibitor. Furthermore, transient transfection of hepatocytes with activated MEK1 construct resulted in increased cyclin D1 mRNA accumulation. Third, a correlation between the mid-late G(1) MEK/ERK activation in hepatocytes in vivo after partial hepatectomy and the mitogen-independent proliferation capacity of these cells in vitro was established. Among hepatocytes isolated either 5, 7, 9, 12 or 15 h after partial hepatectomy, only those isolated from 12- and 15-h regenerating livers were able to replicate DNA without additional growth stimulation in vitro. In addition, PD 98059 intravenous administration in vivo, before MEK activation, was able to inhibit DNA replication in hepatocytes from regenerating livers. Taken together, these results show that (i) early induction of the MEK/ERK cascade is restricted to hepatocytes from hepatectomized animals, allowing an early distinction of primed hepatocytes from those returning to quiescence, and (ii) mid-late G(1) MEK/ERK activation is mainly associated with cyclin D1 accumulation which leads to mitogen-independent progression of hepatocytes to S phase. These results allow us to point to a growth factor dependency in mid-late G(1) phase of proliferating hepatocytes in vivo as observed in vitro in proliferating hepatocytes and argue for a crucial role of the MEK/ERK cascade signalling pathway.     

Role of cell-surface modulator of DNA synthesis in liver regeneration

A cell-surface modulator of DNA synthesis by cultured rat hepatocytes was studied in relation to the liver regeneration process. When rat hepatocytes isolated 24 h after partial hepatectomy were cultured, the first burst of DNA synthesis peaked at 5-8 h and declined until 24 h, followed by the second burst. Rat liver plasma membranes added 2 h after plating inhibited only the second burst, while in the case of the normal hepatocytes where the DNA synthesis began to increase after 5 h, this inhibition was observed at 16 h but not at 8 h. The inhibition did not differ when the membranes obtained from regenerating livers 12 h after partial hepatectomy were used. Epidermal growth factor binding to the cultured hepatocytes was not hindered by the membranes. These results suggest that the modulator inhibits hepatocyte proliferation at the early G1-phase of the cell cycle and that its action might be controlled by other factors in the process of liver regeneration.     

Intracellular signalling of epinephrine in rat hepatocytes during fetal development and hepatic regeneration

The changes in intracellular calcium concentration and IP₃ production after the addition of epinephrine were analysed in adult, fetal (20th–22nd day of intrauterine life), and regenerating rat hepatocytes (4 h–24 h after partial hepatectomy) to determine whether the signal transduction is the same in quiescent proliferating and differentiating cells.The epinephrine treatment causes a significative cytosolic calcium transient in hepatocytes isolated in the last day of fetal life (22-day old) and in the early stage of regeneration (4 h). This effect is not significant in the previous stage of fetal life (20-day old) and at the onset of M phase of cell cycle after partial hepatectomy (24 h).[³H]myo inositol incorporation into IP₃ and IP₄ is higher in 20 day fetal and regenerating hepatocytes with respect to the control. In these cells the epinephrine does not affect basal level of IP₃ and IP₄, while it causes a substantial increase of these inositol phosphates in adult hepatocytes.[³H]myo inositol incorporation into PIP₂ is very low at the 20th day of fetal life. Epinephrine has no effect on this parameter in fetal and regenerating hepatocytes.Our results show that the epinephrine signal is mediated differently in proliferating and in quiescent hepatocytes.     

Preferential suppression of insulin-stimulated proliferation of cultured hepatocytes by somatostatin: Evidence for receptor-mediated growth regulation

The role of somatostatin (SS-14) in the regulation of rat liver regeneration was examined by using thymidine incorporation into hepatocyte DNA labeled with tritiated thymidine, a nuclear-labeling index, and the binding of ¹²⁵I-tyr¹¹-SS-14 to hepatocytes isolated at various times after partial hepatectomy. The data demonstrated no suppressive effect of SS-14 on insulin and glucagon-stimulated thymidine incorporation into hepatocyte DNA as early as 2 h after partial hepatectomy. These data were substantiated by a nuclear labeling index studies. At 2 h, ¹²⁵I-tyr¹¹-SS-14 binding to its specific sites on isolated hepatocytes was undetectable. There was a time-dependent increase in binding of ¹²⁵I-tyr¹¹-SS-14 to hepatocytes obtained at various times after partial hepatectomy. There was a significant decrease in the number of binding sites after partial hepatectomy as determined by Scatchard analysis. The data were supported by autoradiography analysis of affinity labeled ¹²⁵I-tyr¹¹-SS-14-binding protein complex followed by SDS-PAGE. SS-14 also inhibited intracellular cAMP in hepatocytes obtained at 18 h after hepatectomy. The data are consistent with the hypothesis that SS-14 participates via its own receptor in the regulation of the liver regeneration. © 1995 Wiley-Liss, Inc.     

Synergistic effect of interferon-γ and phorbol myristate acetate on superoxide production by human monocytes.

This study demonstrates a synergistic effect of IFNγ and PMA on superoxide generation by human monocytes. A strict correlation was observed between the induction of superoxide production and PK-C activation by PMA alone. No such correlation was evident for IFNγ. However, exposure of the cells to IFNγ for 10 to 15 hr prior to PMA treatment enhanced both superoxide production and PK-C activation. Using protein kinase inhibitors, we noticed that while PMA exerted its effect by activating PK-C, IFNγ operated via activation of calcium/calmodulin-dependent or some other calcium-dependent protein kinases. These kinases appeared to be involved in the effect of IFNγ on superoxide production, as well as in its potentiation of PMA activity.     

Comparison of subcellular activation of the human neutrophil NADPH-oxidase by arachidonic acid, sodium dodecyl sulfate (SDS), and phorbol myristate acetate (PMA)

The phorbol myristate acetate (PMA) stimulation of the human neutrophil NADPH-oxidase has been demonstrated through the activation of protein kinase C (PK-C), using light membrane fractions from nitrogen-cavitated cells. Both arachidonic acid (AA) and sodium dodecyl sulfate (SDS) can also generate an active oxidase in cellfree systems. That the source of O2- with AA and SDS activation is the same NADPH-oxidase as previously studied was confirmed by the similar pH optima and Km values for NADPH as those previously described for the O2- -generating activity harvested from pre-stimulated human neutrophils. In contrast to the stimulation by PMA, however, the stimulation of the NADPH-oxidase by AA and SDS does not appear to require protein kinase C activation: the action of AA and SDS is independent of the addition of PK-C cofactors to the system, and the inhibitor of PK-C activity, H-7, had no effect on the stimulation by AA or SDS. AA and SDS activation are comparable, but the level of NADPH-oxidase expression is sixfold greater with each of these agents than that obtained with a reconstituted PK-C system. The basis of this difference in oxidase expression is unclear, but these findings suggest strongly that although activated PK-C is capable of stimulating a dormant NADPH-oxidase in a cellfree system, this is not the sole pathway for oxidase activation.     

Autoradiographic study of RNA synthesis and secretion into the cytoplasm during the early stages of liver regeneration

Autoradiographic experiments with regeneration of rat liver showed that RNA transport from nucleus to cytoplasm occurs more quickly in 3h following partial hepatectomy while the total RNA synthesis increases insignificantly. Nucleolar RNA synthesis at this time grows in some hepatocytes and decreases in others though it does not differ on the average from the control one. By the 6th h after partial hepatectomy total RNA synthesis significantly increases simultaneously with the acceleration of RNA transport to cytoplasm. Nucleolar RNA synthesis intensifies in all hepatocytes.     

Ultrastructural zonal heterogeneity of hepatocytes and mitochondria within the hepatic acinus during liver regeneration after partial hepatectomy

BACKGROUND INFORMATION: Partial hepatectomy (70%) induces cell proliferation until the original mass of the liver is restored. In the first 24 h after partial hepatectomy, drastic changes in the metabolism of the remaining liver have been shown to occur. To evaluate changes in hepatocyte ultrastructure within the hepatic acinus during the liver regenerative process, we investigated, by light and electron microscopy observations on specimens taken 0 h, 24 h and 96 h after partial hepatectomy, the hepatocyte structure and ultrastructure in the periportal and pericentral area of the hepatic acinus, with a particular emphasis on mitochondria ultrastructure. Moreover, some biochemical events that could affect the mitochondria ultrastructure and function were investigated. RESULTS: We found that, 24 h after partial hepatectomy, mitochondria with altered ultrastructure were preferentially localized in the periportal area. Periportal hepatocytes showed also an increase in the number of peroxisomes, free ribosomes, lysosomes and autophagosomes. Altered mitochondria showed swelling, an ultrastructural index of increased membrane permeability, a reduction in the number of cristae, and a rarefied, often vacuoled, matrix. Consistently, an increase in the mitochondrial oxidized/reduced glutathione ratio was found as well as calcium release from mitochondria in a manner inhibited by cyclosporin A. Interestingly, light and electron microscopy analysis showed that the hepatocytes in the periportal area were the cells with the major structural attributes to proliferate. At 96 h after partial hepatectomy, the preferential zonation of altered mitochondria was lost and the normal mitochondrial membrane permeability properties were restored. CONCLUSIONS: We suggest that 24 h after partial hepatectomy, a preferential zonation of altered mitochondria in the periportal hepatocytes could be involved in the changes of metabolic and functional heterogeneity of the hepatocytes within the hepatic acinus during the regenerative process.     

Immunocytochemical determination of ploidy class-dependent bromodeoxyuridine incorporation in rat liver parenchymal cells after partial hepatectomy

Summary Immunocytochemistry of bromodeoxyuridine (BrdU) incorporated in DNA was performed on cryostat sections of rat liver and on isolated hepatocytes after partial hepatectomy using a two-step labeling technique. The method enabled the detection of S-phase nuclei in both tissue preparations. Quantification of the number of labeled nuclei in sections showed that the number of nuclei in S-phase increased from 0.3% in control liver to about 36% at 24 h after partial hepatectomy. The detection of BrdU in isolated hepatocytes showed the same labeling index of binuclear diploid, mononuclear tetraploid and binuclear tetraploid cells. A special role for mononuclear diploid cells in proliferation did not seem to occur.     

Signal transduction during liver regeneration: role of insulin and vasopressin.

The relationship between cell proliferation and inositol lipid turnover has been studied by comparing the steady state of inositol derivative metabolism in quiescent and regenerating rat hepatocytes isolated at 4 h (G1 phase of first cell cycle) and 24 h (onset of M phase) after partial hepatectomy. The effect of two hormones able to regulate hepatic regeneration, insulin and vasopressin, has been considered, and the results can be summarized as follows: (i) at 4 h after partial hepatectomy, the precursor incorporation into inositol polyphosphates and the particulate phospholipase C activity increase with respect to quiescent hepatocytes, whereas the content of 11, 4, 5P3 does not change, suggesting an increased turnover of this molecule in this step of cell cycle priming; (ii) 24 h after partial hepatectomy, the radioactivity linked to IP3 and IP4, as well as soluble and particulate phospholipase C activity, and IP3 content increase, suggesting the presence, at the onset of M phase, of second messenger accumulation; (iii) only 24 h after partial hepatectomy, the inositol derivative metabolism is affected by vasopressin; and (iv) insulin exerts a modulatory role on inositol polyphosphate production without involving membrane-bound PLC activity or phosphoinositide hydrolysis. These data suggest that inositol-derived signal molecules are associated with hepatic regeneration; moreover, the metabolic pathway of such compounds seems to be regulated so that only specific inositol phosphates are present in each step of the cell cycle.     

The control of liver regeneration by parathyroid hormone and Calcium

Following partial hepatectomy in rats, there were two bursts of hepatocyte DNA-synthetic and mitotic activity which were produced by two subpopulations having different rates of (nearly synchronous) proliferative development. Only about 50% of the cells in both subpopulations could initiate DNA synthesis and enter mitosis when exposed to the hypocalcemic conditions in the parathyroprivic rat for 24 hours before partial hepatectomy. The proliferatively incompetent hepatocytes in these hypocalcemic rats could be induced to initiate their DNA synthetic and mitotic activity by an intraperitoneal injection of the calcium-mobilizing parathyroid hormone (50 USP units/100 g) as late as 12 hours after partial hepatectomy. Single intraperitoneal injections of calcium (0.25 mg/100 g) could also restore the proliferative competence of these hepatocytes, but only when injected at specific periods following partial hepatectomy. The injection of calcium 12 to 15 hours after partial hepatectomy induced hepatocytes in the first subpopulation to finish their development and enter mitosis, but did not affect the second, more slowly developing, subpopulation. Calcium had to be injected 25 hours after partial hepatectomy to stimulate proliferation in this second subpopulation. These data suggest that the hepatocytes which became proliferatively incompetent by prolonged exposure to a hypocalcemic environment are proliferatively activated by partial hepatectomy, but their proliferative development stops at a calcium-dependent stage near the end of the pre-replicative phase of development.     

The control of liver regeneration by parathyroid hormone and calcium.

Following partial hepatectomy in rats, there were two bursts of hepatocyte DNA-synthetic and mitotic activity which were produced by two subpopulations having different rates of (nearly synchronous) proliferative development. Only about 50% of the cells in both subpopulations could initiate DNA synthesis and enter mitosis when exposed to the hypocalcemic conditions in the parathyroprivic rat for 24 hours before partial hepatectomy. The proliferatively incompetent hepatocytes in these hypocalcemic rats could be induced to initiate their DNA synthetic and mitotic activity by an intraperitoneal injection of the calcium-mobilizing parathyroid hormone (50 USP units/100 g) as late as 12 hours after partial hepatectomy. Single intraperitoneal injections of calcium (0.25 mg/100 g) could also restore the proliferative competence of these hepatocytes, but only when injected at specific periods following partial hepatectomy. The injection of calcium 12 to 15 hours after partial hepatectomy induced hepatocytes in the first subpopulation to finish their development and enter mitosis, but did not affect the second, more slowly developing, subpopulation. Calcium had to be injected 25 hours after partial hepatectomy to stimulate proliferation in this second subpopulation. These data suggest that the hepatocytes which became proliferatively incompetent by prolonged exposure to a hypocalcemic environment are proliferatively activated by partial hepatectomy, but their proliferative development stops at a calcium-dependent stage near the end of the pre-replicative phase of development.     

Inhibiting effect of a hepatoma extract on the mitotic rate of regenerating liver

Aqueous tumor extracts were prepared by the homogenization of a fast-growing, undifferentiated, transplantable malignant murine hepatoma in distilled water. After centrifugation, an aliquot of 0.01 ml of the supernatant g body weight was injected intraperitoneally into partially hepatectomized mice. Control animals were injected with saline. Groups of mice were killed at various times in relation to the hepatectomy. Four h before killing the animals were given Colcemid (1 microgram/g body weight). The number of Colcemid-arrested mitoses in the hepatocytes and in the littoral cells, respectively, were counted in 140 microscopic fields. The extract significantly inhibited the mitotic rate in hepatocytes when the injection was given between 22 h before, and up to 26 h after hepatectomy. In the littoral cells, a slight initial stimulation was followed by a slight but significant inhibition which occurred when the injection was given at hepatectomy or until 18 h after hepatectomy. The effect was not modified by exposing the extracts to temperatures of 47 degrees C for 30 min or 22 degrees C for 24 h, but 10 min of boiling destroyed their inhibitory effect. Lyophilization and storing at -18 degrees C for up to 4 weeks did not modify the effect. The mitosis-inhibiting effect was also measurable when the extract was injected subcutaneously. There was an almost linear dose-response curve. The results are discussed in relation to circadian rhythms, the pattern of liver cell proliferation after hepatectomy, and recent similar reports from the literature. The conclusion is drawn that extracts of a hepatoma contain one or more growth-inhibitory factors significantly active on regenerating liver cells, and less significantly on littoral cells.