Existence of phosphoinositide-specific phospholipase C in rat liver nuclei and its change during liver regeneration.

We found phosphoinositide-specific phospholipase C (PtdIns-PLC) activity in nuclei isolated from rat liver. The enzyme hydrolyzed phosphatidylinositol, phosphatidylinositol 4-monophosphate (PIP) and phosphatidylinositol 4,5-bisphosphate in a Ca(2+)-dependent manner, and produced inositol mono-, bis-, and triphosphate, respectively. Neither phosphatidylcholine, phosphatidylethanolamine, nor phosphatidylserine was utilized as a substrate. After partial hepatectomy, the PtdIns-PLC activity in isolated nuclei increased transiently in the S phase (20-22 h post-hepatectomy), to 2.5-fold higher than in the control, when measured with PIP. This result suggests a close relationship between the nuclear PtdIns-PLC, especially its PIP-hydrolyzing activity, and cell proliferation.     

Heterogeneous activation of protein kinase C during rat liver regeneration induced by carbon tetrachloride administration

During rat liver regeneration induced by carbon tetrachloride administration, the protein kinase C alpha subspecies was activated in a heterogeneous fashion, a higher number of hepatocytes expressing the protein kinase C alpha subspecies being detected in the pericentral zone than in the periportal zone. This zonal heterogeneity became maximal at 24 h after the treatment. The distribution of hepatocytes expressing the protein kinase C alpha subspecies was roughly coincident with that of hepatocytes exhibiting DNA synthesis. These results suggest that protein kinase C may play a crucial role in liver regeneration.     

Increased phosphorylation of nuclear substrates for rat brain protein kinase C in regenerating rat liver nuclei.

Protein phosphorylation catalysed by rat brain protein kinase C (PKC) has been studied in nuclei isolated from normal and regenerating rat liver. Histone H1 and a 40,000 molecular weight protein were hyperphosphorylated at all the explored regeneration times, ranging from 3 to 22 h after partial hepatectomy. Phosphorylation of the two substrates was totally dependent on calcium and lipids and was abolished by low concentration of staurosporine. The observed early change of phosphate content of histone H1 and of the 40,000 molecular weight protein on the time scale of liver regeneration suggests that PKC might be involved in the initial nuclear events leading to cell proliferation.     

Immunochemical characterization of protein kinase C in rat liver nuclei and subnuclear fractions

A doublet of immunoreactive bands has been identified in rat liver nuclei, nuclear matrix and lamina by means of a polyclonal antibody against protein kinase C. The two polypeptides show an apparent molecular weight of 77 and 74 kDa on SDS-polyacrylamide gels, and appear to be tightly bound nuclear components, resistant to detergent and high salt extraction. Given the complexity of the genes encoding for protein kinase C, these two forms of the enzyme might be translational products specifically located in the nucleus, involved in the transduction to the genomic apparatus of regulatory signals generated by growth factors and tumor promoters.     

Protease-activated protein kinase C in rat liver

In regenerating rat liver, an elevated protein kinase activity was detected which phosphorylated ribosomal protein S6 and histones. The properties of this enzyme were closely similar with those of protease-activated protein kinase C with Mr 45,000. During the study of the mechanism of proteolytic activation, type III protein kinase C (encoding alpha-sequence) was shown to be subjected to limited proteolysis by trypsin-like protease and converted to protein kinase M in ionic strength- and pH-dependent manner. This reaction was stimulated in the presence of Ca2+ and phospholipid under slightly higher ionic strength condition than physiological level (greater than 140 mM NaCl) and alkaline pH (7.5-8.0). These results suggest that activation of Na+/H+ exchanger in plasma membrane may trigger this type of proteolytic activation of protein kinase C. In addition to protein kinase M, another type of protease-activated kinase with Mr 80,000 was detected when limited proteolysis of protein kinase C was performed on inactive form of this enzyme (in the absence of either Ca2+ or phospholipid or both activators) under lower ionic strength condition. The molecular mass of this active enzyme was slightly smaller (approximately 200) than that of native protein kinase C. However, it is not clear at this time whether this small fragment was released from amino-terminal or carboxy-terminal domain to make protein kinase C partially active in the absence of Ca2+ and phospholipid. Although it has been proposed that proteolytic degradation of protein kinase C is involved in down regulation of this enzyme, the physiological significance of these two types of protease-activated forms of protein kinases in liver has remained obscure.     

Heterogeneity of protein kinase NII from rat liver nuclei

Protein kinase NII from rat liver nuclei was resolved into two fractions, NIIa and NIIb, by DEAE-Sephadex column chromatography. NIIa was eluted at 151 mM (NH₄)₂SO₄ and NIIb at 175 mM. They had an identical molecular size (125,000 daltons, 7.0S) and subunit composition (αα′β₂). However, they showed significantly different Km values and turnover numbers for casein substrate. Furthermore, NIIa was found predominantly as a form bound to the chromatin, while NIIb was in the nucleoplasmic-soluble fraction in addition to the chromatin-bound fraction. These observations suggest that NII consists of a heterogeneous population of at least two molecular species, differing in the activity and functional states in the cell nucleus.     

Relative changes in bile secretion during rat liver regeneration

The influences of partial hepatectomy (66%) on some aspects of rat biliary secretion were studied at different time intervals after surgery (0, 40, 96, 192 and 384 h). Bile salt independent and bile salt dependent fractions were determined. During the first intervals (40 and 96 h) bile salt independent fraction clearly decreased after which it slowly recovered (192 h) until control levels were reached (384 h). These results are interpreted as proof of a faster compensatory hyperplastic regeneration in zone I of the hepatic acinus than in zone III.     

Development of adenylate kinase isoenzymes in rat liver

Total adenylate kinase activity was determined in developing rat liver. The activity was 18 units/g wet weight of tissue in foetal liver; this increased to 41 units/g immediately after birth and continued increasing until adult activities of 150 units/g were reached after two weeks. The adenylate kinase activity was separated into four isoenzymes. Only isoenzymes II and III were observed in foetal rat liver. Isoenzyme II activity was 2 units/g in the foetal liver and increased to 25 units/g in adult liver. Adenylate kinase III activity was 20 units/g in the foetal liver and increased to 118 units/g in adult liver. The possible role that adenylate kinase might have in regulating the energy flow in the developing liver cell is discussed.     

Conformational changes in rat liver chromatin after liver regeneration.

N-Pyrenemaleimide, a fluorescent probe that specifically labels histone H3 of rat liver chromatin in situ, was used to monitor the accessibility of histone H3 in chromatin isolated from rat liver at different times during degeneration. At times of maximum DNA synthesis (18--24 h after hepatectomy), the accessibility of the probe was found to be markedly (40--50%) increased. This increase is abolished, however, by treatment of the chromatin fibres with high salt (2 M-NaCl) or detergent. Tryptophan fluorescence was also enhanced at points of maximum DNA synthesis, suggesting that some non-histone tryptophan-containing protein was being synthesized. The polarization of the labelled histone H3 is not markedly altered, suggesting that fibre aggregation or dissociation does not occur. Mononucleosomes extracted from sham-operated and hepatectomized animals did not exhibit any difference in binding to the probe. Also, analysis of the chromatin protein by electrophoresis on detergent- and acid/urea/ Triton-X-100-containing polyacrylamide gels showed no detectable difference in histone H3 : 1, H3 : 2 or H3 : 3 subclasses.     

Differential changes in protein kinase C associated with regeneration of rat extensor digitorum longus and soleus muscles

We used a model of crush-induced regeneration in rat in order to characterize biochemically and histologically the implication of protein kinase C (PKC) in muscle repair after damage. In this model, slow soleus and fast extensor digitorum longus (EDL) muscle regeneration proceed differently. PKC activity has been assayed in regenerating muscles and their intact contralateral during the first 14 days following crushing. Degeneration (myolysis) occurring shortly after crush was associated with a marked down-regulation of the enzyme in both wound muscles and notable increase in the corresponding contralateral muscles. Muscle fiber reconstruction in EDL was associated with a rise in PKC activity which peaked at day 7 in regenerating muscle where it was twice higher than in intact muscle. At variance, muscle PKC activity in soleus increased slower than that of EDL and reached later intact level. Western blot analysis and immunohistochemical studies of representative members of the three PKC subfamilies were performed. All the isoform tested were much less expressed in regenerating than in control intact muscles suggesting that the overall PKC activity in regenerating muscles was more activable than in controls. We have shown that PKC isoforms were sequentially expressed during regeneration in both muscle types. PKC theta; being present the earliest, then delta, epsilon and alpha and finally zeta, beta and eta. Some isoforms were differentially expressed according muscle type. PKC delta being more expressed in soleus whereas beta and eta appeared earlier in EDL. Histochemical studies have revealed that the isoforms were differently localized in muscle tissue and that fiber regeneration was associated with PKC alpha translocation from sarcoplasma to sarcolemma. Together these data have shown that multiple PKC isoforms are implicated in the regenerative process acting at different in times and location and suggesting that individual isoform may fulfill distinct functions.     

Inactivation of protein kinase C in rat liver during late hypoglycemic phase of sepsis

Changes in protein kinase C (PKC) (calcium- and phospholipid-dependent protein kinase) activity in rat liver during different metabolic phases of sepsis were studied. Sepsis was induced by cecal ligation and puncture (CLP). Experiments were divided into three groups: control, early sepsis, and late sepsis. Early and late sepsis refers to those animals sacrificed at 9 and 18 h, respectively, after CLP. Hepatic PKC was extracted and partially purified by ammonium sulfate fractionation and DEAE-cellulose chromatography. PKC activity was assayed based on the rate of incorporation of ³²p from [-³²P]ATP into histone. The results show that during early sepsis, both membrane-associated and cytosolic PKC activities remained relatively unaltered. During late sepsis, membrane-associated PKC was unaffected while cytosolic PKC activity was decreased by 19.5-34.4%. Kinetic analysis of the data on cytosolic PKC during late phase of sepsis reveals that the V_max values for ATP, histone, Ca²⁺, phosphatidylserine, and diacylglycerol were decreased by 23.4, 22.1, 19.5, 25, and 34.4%, respectively, with no changes in their K_m values. These data indicate that cytosolic PKC activity was inactivated in rat liver during late hypoglycemic phase of sepsis. Since PKC-mediated phosphorylation plays an important role in regulating hepatic glucose metabolism, an inactivation of cytosolic PKC may contribute to the development of hypoglycemia during late phase of sepsis.     

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.     

Protein kinase C located in rat liver nuclei. Partial purification and biochemical and immunochemical characterization

In the rat liver homogenate, maximal protein kinase C activity was found at two calcium concentrations (1.75 and 3.5 mM). Subcellular fractionation of the liver homogenate revealed that the protein kinase C activity requiring 1.75 mM calcium was present only in the cytosolic and particulate subcellular fractions. The protein kinase C activity requiring 3.5 mM calcium concentration was mainly located in the rat liver nuclei preparation. About 19% of the liver homogenate protein kinase C activity requiring 3.5 mM calcium was present in the nuclei. Goat anti-rat brain protein kinase C antibodies revealed a single immunoreactive band at 80-82 kDa in the rat liver nuclear, particulate, or cytosolic fractions. Based on the ratio of plasma membrane marker enzyme activity determined in the nuclear preparation, the purity of the isolated nuclei was ascertained. Rat liver nuclear protein kinase C activity has been partially purified. The purification steps sequentially employed were Triton X-100 extraction of isolated nuclei, DEAE-cellulose chromatography, Phenyl-Superose, and Mono Q (fast protein liquid) chromatography. The final purification step revealed, by silver nitrate staining on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, two protein bands at 80 and 66 kDa, respectively. These findings provide definitive data regarding the nuclear location of protein kinase C. The nuclear location of protein kinase C may lead to an understanding of the molecular pathway involved in signal transduction from the plasma membrane to the nucleus.     

Selective post-transcriptional down-regulation of protein kinase C isoenzymes in leukemic T cells chronically treated with phorbol ester

Binding to, and activation of, protein kinase C (PKC) by phorbol ester (PE) tumor promoters may underlie their tumor-promoting activity. To study the effects of long-term PE treatment on regulation of cellular PKC, we adapted the human leukemic T cell line, Jurkat (JK), to continuous growth in the presence of PE. Such cells (JKPE) displayed loss of CD2 and CD3 cell surface molecules, known to play an important role in agonist-stimulated T cell activation, unresponsiveness to stimuli that induce interleukin 2 (IL2) receptor expression or IL2 production, change in the expression of several cell cycle-regulated genes, and a 6-fold reduction in cellular PKC enzymatic activity. This reduction was accompanied by the disappearance of a major approximately 82-kDa immunoreactive protein in JKPE cytosol when cell extracts were immunoblotted with a polyclonal anti-PKC peptide antibody cross-reactive with the PKC isoenzymes, alpha, beta, and gamma. Analysis with additional anti-peptide antibodies specific for alpha, beta, or gamma PKC indicated that all three types of PKC are expressed in JK cells; however, JKPE cells lost a major approximately 82 kDa immunoreactive cytosolic protein detectable with anti-PKC alpha antibody. In contrast, levels of expression and subcellular distribution of immunoreactive PKC beta and PKC gamma, as well as levels of mRNA specific for the three PKC isoenzymes, were not significantly affected by chronic PE treatment. These results indicate that PE-mediated reduction of PKC in JKPE cells is selective and occurs at the protein, not mRNA, level, and support the notion that distinct isoenzymes encoded by the PKC multigene family may be independently regulated. Moreover, the correlation between phenotypic and functional changes on one hand, and the selective reduction of PKC alpha on the other, raises the possibility that expression of CD2 and/or CD3 and functional activation in JKPE cells are preferentially regulated by PKC alpha.