Regulation of Na+/Mg2+ antiport in rat erythrocytes

In rat erythrocytes, the regulation of Na+/Mg2+ antiport by protein kinases (PKs), protein phosphatases (PPs), intracellular Mg2+, ATP and Cl- was investigated. In untreated erythrocytes, Na+/Mg2+ antiport was slightly inhibited by the PK inhibitor staurosporine, slightly stimulated by the PP inhibitor calyculin A and strongly stimulated by vanadate. PMA stimulated Na+/Mg2+ antiport. This effect was completely inhibited by staurosporine and partially inhibited by the PKC inhibitors Ro-31-8425 and BIM I. Participation of other PKs such as PKA, the MAPK cascade, PTK, CK I, CK II, CAM II-K, PI 3-K, and MLCK was excluded by use of inhibitors. Na+/Mg2+ antiport in rat erythrocytes can thus be stimulated by PKCalpha. In non-Mg2+ -loaded erythrocytes, ATP depletion reduced Mg2+ efflux and PMA stimulation in NaCl medium. A drastic activation of Na+/Mg2+ antiport was induced by Mg2+ loading which was not further stimulated by PMA. Staurosporine, Ro-31-8425, BIM I and calyculin A did not inhibit Na+/Mg2+ antiport of Mg2+ -loaded cells. Obviously, at high [Mg2+]i Na+/Mg2+ antiport is maximally stimulated. PKCalpha or PPs are not involved in stimulation by intracellular Mg2+. ATP depletion of Mg2+ -loaded erythrocytes reduced Mg2+ efflux and the affinity of Mg2+ binding sites of the Na+/Mg2+ antiporter to Mg2+. In non-Mg2+ -loaded erythrocytes Na+/Mg2+ antiport essentially depends on Cl-. Mg2+ -loaded erythrocytes were less sensitive to the activation of Na+/Mg2+ antiport by [Cl-]i.     

Regulation of Na/Mg antiport in rat erythrocytes

In rat erythrocytes, the regulation of Na⁺/Mg²⁺ antiport by protein kinases (PKs), protein phosphatases (PPs), intracellular Mg²⁺, ATP and Cl⁻ was investigated. In untreated erythrocytes, Na⁺/Mg²⁺ antiport was slightly inhibited by the PK inhibitor staurosporine, slightly stimulated by the PP inhibitor calyculin A and strongly stimulated by vanadate. PMA stimulated Na⁺/Mg²⁺ antiport. This effect was completely inhibited by staurosporine and partially inhibited by the PKC inhibitors Ro-31-8425 and BIM I. Participation of other PKs such as PKA, the MAPK cascade, PTK, CK I, CK II, CAM II-K, PI 3-K, and MLCK was excluded by use of inhibitors. Na⁺/Mg²⁺ antiport in rat erythrocytes can thus be stimulated by PKCα.In non-Mg²⁺-loaded erythrocytes, ATP depletion reduced Mg²⁺ efflux and PMA stimulation in NaCl medium. A drastic activation of Na⁺/Mg²⁺ antiport was induced by Mg²⁺ loading which was not further stimulated by PMA. Staurosporine, Ro-31-8425, BIM I and calyculin A did not inhibit Na⁺/Mg²⁺ antiport of Mg²⁺-loaded cells. Obviously, at high [Mg²⁺]_i Na⁺/Mg²⁺ antiport is maximally stimulated. PKC_α or PPs are not involved in stimulation by intracellular Mg²⁺. ATP depletion of Mg²⁺-loaded erythrocytes reduced Mg²⁺ efflux and the affinity of Mg²⁺ binding sites of the Na⁺/Mg²⁺ antiporter to Mg²⁺. In non-Mg²⁺-loaded erythrocytes Na⁺/Mg²⁺ antiport essentially depends on Cl⁻. Mg²⁺-loaded erythrocytes were less sensitive to the activation of Na⁺/Mg²⁺ antiport by [Cl⁻]_i.     

Effect of Putative Deoxyribonucleic Acid Inhibitors on Macromolecular Synthesis in Saccharomyces cerevisiae

The effects of inhibitors of bacterial deoxyribonucleic acid (DNA) synthesis upon logarithmically growing cultures of Saccharomyces cerevisiae were investigated. Cell division, ribonucleic acid (RNA) synthesis, and DNA synthesis were measured after addition of nalidixic acid, fluorodeoxyuridine, or phenethyl alcohol to cultures of yeast growing in defined and complex media. Both nalidixic acid and fluorodeoxyuridine had only temporary effects on nucleic acid synthesis in cultures growing in defined medium, and little or no observable effect on cultures growing in complex medium. Neither compound inhibited colony formation on complex solid medium, although growth was slow on defined solid medium. Phenethyl alcohol caused complete inhibition of DNA synthesis, RNA synthesis, and cell division in cultures growing in defined medium. In cultures growing in complex medium, RNA synthesis and cell division were inhibited to a lesser extent. A slight increase in DNA was observed in the presence of the inhibitor.     

The staurosporine analog, Ro-31-8220, induces apoptosis independently of its ability to inhibit protein kinase C

A series of bisindolylmaleimide (Bis) compounds were designed as analogs of the natural compound staurosporine (STS), which is a potent inducer of apoptosis. Many of the Bis analogs appear to be highly selective inhibitors of the protein kinase C (PKC) family, including PKC-alpha, -beta, -gamma, -delta, -epsilon, and -zeta, unlike STS, which is an inhibitor of a broad spectrum of protein kinases. In this report we describe the effects of the Bis analogs, Bis-I, Bis-II, Bis-III and Ro-31-8220 on the survival and proliferation of HL-60 cells, which have been widely used as a model cell system for studying the biological roles of PKC. Treatment of HL-60 cells with Bis-I, Bis-II, Bis-III, or Ro-31-8220 blocked phosphorylation of the PKC target protein Raf-1 with equal potency but did not appear to affect the general phosphorylation of proteins by other kinases. However, the biological effects of the Bis compounds were different: Bis-I and Bis-II had no observable effects on either cell survival or proliferation; Bis-III inhibited cell proliferation but not survival, whereas Ro-31-8220 induced apoptosis. These results indicated that the members of the PKC family which could be inhibited by the Bis analogs were required neither for survival nor proliferation of HL-60 cells. Analyses of cells treated with Ro-31-8220 showed that the apoptotic effect of Ro-31-8220 on HL-60 cells was mediated by a well-characterized transduction process of apoptotic signals: i.e., mitochondrial cytochrome c efflux and the activation of caspase-3 in the cytosol. Moreover, the ability of Ro-31-8220 to induce apoptotic activation was completely inhibited by the over-expression of the apoptotic suppressor gene, Bcl-2, in the cells. Interestingly, proliferation of the Bcl-2-over-expressing cells was still sensitive to the presence of Ro-31-8220, suggesting that the inhibitory effects of Ro-31-8220 on viability and cell proliferation were mediated by different mechanisms. In particular, the apoptotic effect of Ro-31-8220 on cells was not altered by the presence of an excess amount of the other Bis analogs, suggesting that this effect is mediated by a factor(s) other than PKC or by a mechanism which was not saturable by the other Bis analogs. Finally, structure-function analyses of compounds related to Ro-31-8220 revealed that a thioamidine prosthetic group in Ro-311-8220 was largely responsible for its apoptotic activity.     

PKCε regulates contraction‐stimulated GLUT4 traffic in skeletal muscle cells

The signaling pathways that stimulate glucose uptake in response to muscle contraction are not well defined. Recently, we showed that carbachol, an acetylcholine analog, stimulates contraction of C2C12 myotube cultures and the rapid arrival of myc‐epitope tagged GLUT4 glucose transporters at the cell surface. Here, we explore a role for protein kinase C (PKC) in regulating GLUT4 traffic. Cell surface carbachol‐induced GLUT4myc levels were partly inhibited by the conventional/novel PKC inhibitors GF‐109203X, Gö6983, and Ro‐31‐8425 but not by the conventional PKC inhibitor Gö6976. C2C12 myotubes expressed several novel isoforms of PKC mRNA with PKCδ and PKCε in greater abundance. Carbachol stimulated phosphorylation of PKC isoforms and translocation of PKCδ and PKCε to membranes within 5 min. However, only a peptidic inhibitor of PKCε translocation (myristoylated‐EAVSLKPT), but not one of PKCδ (myristoylated‐SFNSYELGSL), prevented the GLUT4myc response to carbachol. Significant participation of PKCε in the carbachol‐induced gain of GLUT4myc at the surface of C2C12 myotubes was further supported through siRNA‐mediated PKCε protein knockdown. These findings support a role for novel PKC isoforms, especially PKCε, in contraction‐stimulated GLUT4 traffic in muscle cells. J. Cell. Physiol. 226: 173–180, 2010. © 2010 Wiley‐Liss, Inc.     

Green mould of oranges caused by Penicillium digitatutn Sacc.; effect of additives on spore germination and infection

Water extracts of rind, essential oil and juice from oranges, also citrus pectin and citric acid promoted the formation of lesions when spores of Penicillium digitatum were placed in wounds 1·0 mm deep in flavedo of oranges; fructose, glucose and sucrose had little effect. Rind extracts were less effective in wounds 0·5 mm deep but orange juice and pectin still increased infection. None of the substances allowed the parasite to infect fruit through unwounded surfaces. Germination of spores in water increased as spore concentration decreased but was poor even at low concentrations. Almost all spores germinated in aqueous extracts of flavedo, albedo or whole rind, or in wounds on the surface of fruit. Fructose, glucose, sucrose and xylose were less effective but still caused over two-thirds of spores to germinate but only in the presence of phosphate buffer. Without buffer, germination was little different from that in water. Arabinose and galactose stimulated germination to a lesser extent but with the same phosphate effect. Carboxymethylcellulose and pectin did not affect germination. A variety of substances containing nitrogen increased germination but to different degrees, decreasing in the order, casamino acids, yeast extract, ammonium salts, nitrate. Thiamin and to a lesser extent biotin were also effective. Volatile substances from rind infected with P. digitatum stimulated spore germination and growth of germ tubes. The significance of these results is discussed in relation to infection.     

Hepatocyte resistance to oxidative stress is dependent on protein kinase C-mediated down-regulation of c-Jun/AP-1

The prevention of injury from reactive oxygen species is critical for cellular resistance to many death stimuli. Resistance to death from the superoxide generator menadione in the hepatocyte cell line RALA255-10G is dependent on down-regulation of the c-Jun N-terminal kinase (JNK)/AP-1 signaling pathway by extracellular signal-regulated kinase 1/2 (ERK1/2). Because protein kinase C (PKC) regulates both oxidant stress and JNK signaling, the ability of PKC to modulate hepatocyte death from menadione through effects on AP-1 was examined. PKC inhibition with Ro-31-8425 or bisindolylmaleimide I sensitized this cell line to death from menadione. Menadione treatment led to activation of PKCmicro, or protein kinase D (PKD), but not PKCalpha/beta, PKCzeta/lambda, or PKCdelta/. Menadione induced phosphorylation of PKD at Ser-744/748, but not Ser-916, and translocation of PKD to the nucleus. PKC inhibition blocked menadione-induced phosphorylation of PKD, and expression of a constitutively active PKD prevented death from Ro-31-8425/menadione. PKC inhibition led to a sustained overactivation of JNK and c-Jun in response to menadione as determined by in vitro kinase assay and immunoblotting for the phosphorylated forms of both proteins. Cell death from PKC inhibition and menadione treatment resulted from c-Jun activation, since death was blocked by adenoviral expression of the c-Jun dominant negative TAM67. PKC and ERK1/2 independently down-regulated JNK/c-Jun, since inhibition of either kinase failed to affect activation of the other kinase, and simultaneous inhibition of both pathways caused additive JNK/c-Jun activation and cell death. Resistance to death from superoxide therefore requires both PKC/PKD and ERK1/2 activation in order to down-regulate proapoptotic JNK/c-Jun signaling.     

Study of inhibition of outgrowth in Bacillus cereus T by ethyl picolinate.

The effects of ethyl picolinate on germination, outgrowth, and sporulation of Bacillus cereus T were studied in a synthetic medium containing glucose. Ethyl picolinate specifically inhibited at two stages, outgrowth and sporulation. The initiation of germination and cell division was not affected. The inhibition of outgrowth by ethyl picolinate could be reversed by enrichment of inoculum with aspartic acid, asparagine, lysine, phenylalanine, and tyrosine among the amino acids and by oxalacetate. Nicotinic acid and nicotinamide also possessed this ability. Ethyl picolinate failed to block outgrowth when added to cultures incubated for a short time after inoculation. Enrichment of the medium with lysine plus zinc sulfate stimulated sporulation in the presence of ethyl picolinate to a significance degree.     

Study of inhibition of outgrowth in Bacillus cereus T by ethyl picolinate.

The effects of ethyl picolinate on germination, outgrowth, and sporulation of Bacillus cereus T were studied in a synthetic medium containing glucose. Ethyl picolinate specifically inhibited at two stages, outgrowth and sporulation. The initiation of germination and cell division was not affected. The inhibition of outgrowth by ethyl picolinate could be reversed by enrichment of inoculum with aspartic acid, asparagine, lysine, phenylalanine, and tyrosine among the amino acids and by oxalacetate. Nicotinic acid and nicotinamide also possessed this ability. Ethyl picolinate failed to block outgrowth when added to cultures incubated for a short time after inoculation. Enrichment of the medium with lysine plus zinc sulfate stimulated sporulation in the presence of ethyl picolinate to a significance degree.     

Effect of Initiation Time of Hydrostatic Pressure Shock on Chromosome Set Doubling of Tetraploidization in Turbot <Emphasis Type="Italic">Scophthalmus maximus</Emphasis>

The objective of the study was to clarify the effects of initiation time on chromosome set doubling induced by hydrostatic pressure shock through nuclear phase fluorescent microscopy in turbot Scophthalmus maximus. The ratio of developmentally delayed embryo and chromosome counting was used to assess induction efficiency. For the embryos subjected to a pressure of 67.5 MPa for 6 min at prometaphase (A group), chromosomes recovered to the pre-treatment condition after 11-min recovering. The first nuclear division and cytokinesis proceeded normally. During the second cell cycle, chromosomes did not enter into metaphase after prometaphase, but spread around for about 13 min, then assembled together and formed a large nucleus without anaphase separation; the second nuclear division and cytokinesis was inhibited. The ratio of developmentally delayed embryo showed that the second mitosis of 78% A group embryo was inhibited. The result of chromosome counting showed that the tetraploidization rate of A group was 72%. For the embryos subjected to a pressure of 67.5 MPa for 6 min at anaphase (B group), chromosomes recovered to the pre-treatment condition after about 31-min recovering. Afterwards, one telophase nucleus formed without anaphase separation; the first nuclear division was inhibited. The time of the first cleavage furrow occurrence of B group embryos delayed 27 min compared with that of A group embryos. With the first cytokinesis proceeding normally, 81.3% B group embryos were at two-cell stage around the middle of the second cell cycle after treatment. Those embryos were one of the two blastomeres containing DNA and the other without DNA. The first nuclear division of those embryos was inhibited. During the third cell cycle after treatment, 65.2% of those abovementioned embryos were at four-cell stage, cytokinesis occurred in both blastomeres, and nuclear division only occurred in the blastomere containing DNA. Of those abovementioned embryos, 14.0% were at three-cell stage and cytokinesis only occurred in the blastomere containing DNA. The result of chromosome counting showed that the tetraploidization rate of B group was only 7%. To summarize what had been mentioned above, mechanisms on chromosome set doubling of tetraploid induction would be different with different initiation time of hydrostatic pressure treatment. Chromosome set doubling was mainly due to inhibition of the second mitosis when hydrostatic pressure treatment was performed at prometaphase. Otherwise, chromosome set doubling was mainly due to inhibition of the first nuclear division when hydrostatic pressure treatment was performed at anaphase. Induction efficiency of tetraploidization resulted from inhibition of the second cleavage was higher than which resulted from inhibition of the first nuclear division. This study was the first to reveal biological mechanisms on the two viewpoints of chromosome set doubling through effect of initiation time of hydrostatic pressure treatment on chromosome set doubling in tetraploid induction.     

ET-1 stimulates ERK signaling pathway through sequential activation of PKC and Src in rat myometrial cells

In this study, we analyzed in ratmyometrial cells the signaling pathways involved in the endothelin(ET)-1-induced extracellular signal-regulated kinase (ERK) activationrequired for the induction of DNA synthesis. We found that inhibitionof protein kinase C (PKC) by Ro-31-8220 abolished ERK activation.Inhibition of phospholipase C (PLC) by U-73122 or of phosphoinositide(PI) 3-kinase by wortmannin partially reduced ERK activation. A similarpartial inhibition was observed after treatment with pertussis toxin orPKC downregulation by phorbol ester treatment. The effect of wortmanninwas additive with that produced by PKC downregulation but not with thatdue to pertussis toxin. These results suggest that bothdiacylglycerol-sensitive PKC, activated by PLC products, anddiacylglycerol-insensitive PKC, possibly activated by aG_i-PI 3-kinase-dependent process, are involved inET-1-induced ERK activation. These two pathways were found to beactivated mainly through the ET_A receptor subtype. ET-1 andphorbol ester stimulated Src activity in a PKC-dependent manner, bothresponses being abolished in the presence of Ro-31-8220. Inhibition of Src kinases by PP1 abrogated phorbol ester- and ET-1-induced ERK activation. Finally, ET-1 activated Ras in a PP1-and Ro-31-8220-sensitive manner. Altogether, our results indicatethat ET-1 induces ERK activation in rat myometrial cells through thesequential stimulation of PKC, Src, and Ras.

     

Molecular analysis reveals localization of Saccharomyces cerevisiae protein kinase C to sites of polarized growth and Pkc1p targeting to the nucleus and mitotic spindle

The catalytic activity and intracellular localization of protein kinase C (PKC) are both highly regulated in vivo. This family of kinases contains conserved regulatory motifs, i.e., the C1, C2, and HR1 domains, which target PKC isoforms to specific subcellular compartments and restrict their activity spatially. Saccharomyces cerevisiae contains a single PKC isozyme, Pkc1p, which contains all of the regulatory motifs found in mammalian PKCs. Pkc1p localizes to sites of polarized growth, consistent with its main function in maintaining cell integrity. We dissected the molecular basis of Pkc1p localization by expressing each of its domains individually and in combinations as green fluorescent protein fusions. We find that the Rho1p-binding domains, HR1 and C1, are responsible for targeting Pkc1p to the bud tip and cell periphery, respectively. We demonstrate that Pkc1p activity is required for its normal localization to the bud neck, which also depends on the integrity of the septin ring. In addition, we show for the first time that yeast protein kinase C can accumulate in the nucleus, and we identify a nuclear exit signal as well as nuclear localization signals within the Pkc1p sequence. Thus, we propose that Pkc1p shuttles in and out of the nucleus and consequently has access to nuclear substrates. Surprisingly, we find that deletion of the HR1 domain results in Pkc1p localization to the mitotic spindle and that the C2 domain is responsible for this targeting. This novel nuclear and spindle localization of Pkc1p may provide a molecular explanation for previous observations that suggest a role for Pkc1p in regulating microtubule function.     

Light-dependent translocation of arrestin in rod photoreceptors is signaled through a phospholipase C cascade and requires ATP

Partitioning of cellular components is a critical mechanism by which cells can regulate their activity. In rod photoreceptors, light induces a large-scale translocation of arrestin from the inner segments to the outer segments. The purpose of this project is to elucidate the signaling pathway necessary to initiate arrestin translocation to the outer segments and the mechanism for arrestin translocation.Mouse retinal organotypic cultures and eyes from transgenic Xenopus tadpoles expressing a fusion of GFP and rod arrestin were treated with both activators and inhibitors of proteins in the phosphoinositide pathway. Confocal microscopy was used to image the effects of the pharmacological agents on arrestin translocation in rod photoreceptors. Retinas were also depleted of ATP using potassium cyanide to assess the requirement for ATP in arrestin translocation.In this study, we demonstrate that components of the G-protein-linked phospholipase C (PLC) pathway play a role in initiating arrestin translocation. Our results show that arrestin translocation can be stimulated by activators of PLC and protein kinase C (PKC), and by cholera toxin in the absence of light. Arrestin translocation to the outer segments is significantly reduced by inhibitors of PLC and PKC. Importantly, we find that treatment with potassium cyanide inhibits arrestin translocation in response to light.Collectively, our results suggest that arrestin translocation is initiated by a G-protein-coupled cascade through PLC and PKC signaling. Furthermore, our results demonstrate that at least the initiation of arrestin translocation requires energy input.     

Fermentation of pectin and glucose, and activity of pectin-degrading enzymes in the rabbit caecal bacterium Bacteroides caccae

AIMS: To compare fermentation pattern in cultures of Bacteroides caccae supplied with pectin and glucose, and identify enzymes involved in metabolism of pectin. METHODS AND RESULTS: A strain KWN isolated from the rabbit caecum was used. Fermentation pattern, changes of viscosity and enzyme reactions products were determined. Cultures grown on pectin produced significantly more acetate and less formate, lactate, fumarate and succinate than cultures grown on glucose. Production of cell dry matter and protein per gram of substrate used was the same in pectin- and glucose-grown cultures. The principal enzymes that participated in the metabolism of pectin were extracellular exopectate hydrolase (EC 3.2.1.67), extracellular endopectate lyase (EC 4.2.2.2) and cell-associated 2-keto-3-deoxy-6-phosphogluconate (KDPG) aldolase (EC 4.1.2.14). The latter enzyme is unique to the Entner-Doudoroff pathway. Activities of pectinolytic enzymes in cultures grown on glucose were low. Activity of KDPG aldolase was similar in pectin- and glucose-grown cells. CONCLUSIONS: Metabolites and activities of pectin-degrading enzymes differed in cultures of B. caccae KWN grown on pectin and glucose. Yields of dry matter and protein were the same on both substrates. SIGNIFICANCE AND IMPACT OF THE STUDY: Information on metabolism of pectin in animal strains of Bacteroides is incomplete. This study extends the knowledge on metabolism in bacteria from the rabbit caecum.     

Contrasting effects of ethylene perception and biosynthesis inhibitors on germination and seedling growth of barley (Hordeum vulgare L.)

The effects of the plant growth regulator ethylene, and of ethylene inhibitors, on barley (Hordeum vulgare L.) germination and seedling growth were investigated. Exogenous 1-aminocyclopropane-1-carboxylic acid (ACC) at 100 microM enhanced ethylene production by barley seedlings and stimulated shoot growth, whereas both germination and seedling growth were inhibited by antagonists of ethylene perception (75 microM silver ions, 100 microM 2,5-norbornadiene (NBD)). In contrast, germination was unaffected by, and root and shoot growth of seedlings was strongly stimulated by inhibitors of ethylene biosynthesis (10 microM cobalt chloride, 10 microM aminoethoxyvinylglycine (AVG)). Since the ethylene and polyamine biosynthetic pathways are linked through S:-adenosylmethionine, this prompted further explorations into the role of polyamines in germination and seedling growth. Exogenous polyamines (putrescine, spermidine and spermine) at 1 microM concentration stimulated barley seedling growth in a similar fashion to the ethylene biosynthetic inhibitors. Both polyamines and ethylene biosynthetic inhibitors reversed the inhibitory effects of ethylene perception inhibitors on germination and seedling growth. Blocking endogenous ethylene production with aminoethoxyvinylglycine enhanced the free putrescine and spermidine content of germinating barley grains. Thus endogenous polyamines may play a complementary, growth-promotive, role to ethylene in the normal course of barley germination. Further, experiments that have been carried out using inhibitors of ethylene biosynthesis may have to be re-evaluated to take the possible effect of polyamines into account.     

Productionin vitro of appressoria by the entomopathogenic fungusMetarhizium anisopliae

Germination on complex media induced conidia of the entomopathogenMetarhizium anisopliae to produce infection structures (appressoria and penetration hyphae) when the germ tube contacted a hard surface. The morphology of the infection structures and their rate of formation are very similar to those observed for blowfly cuticle. Differentiation frequencies were greater (more than 70% as compared with less than 40%) on hydrophobic surfaces [Teflon, polyvinyl chloride, polystyrene, polypropylene, polyester (GelBond), aluminum foil] than on hydrophilic surfaces (agarose-coated polyester and cellophane). Differentiation frequencies were similar on both positively and negatively charged surfaces. Differentiationin vitro was stimulated by low levels of complex nitrogenous nutrients. Analysis of one- or multicomponent media suggested that amino acids and the lipid component of epicuticle act in combination with the hydrophobic cuticle surface to stimulate differentiation during pathogenesis. Thigmotropic and chemical stimuli for production of appressoria appear to be translated primarily during the second round of nuclear division because inhibitors of DNA and RNA synthesis do not prevent germination but block differentiation if applied before the second nuclear division. Inhibition of protein synthesis blocked both germination and differentiation.     

Increased contractility of hepatic stellate cells in cirrhosis is mediated by enhanced Ca2+-dependent and Ca2+-sensitization pathways

Activation of hepatic stellate cells (HSCs) results in cirrhosis and portal hypertension due to intrahepatic resistance. Activated HSCs increase their contraction after receptor agonist stimulation; however, the signaling pathways for the regulation of contraction are not fully understood. The aim of this study was to elucidate the change in contractile mechanisms of HSCs after cirrhotic activation. The expression pattern of contractile regulatory proteins was analyzed with quantitative RT-PCR and Western blotting. The phosphorylation levels of myosin light chain (MLC), 17-kDa PKC-potentiated protein phosphatase 1 inhibitor protein (CPI-17), and MLC phosphatase targeting subunit 1 (MYPT1) after endothelin-1 (ET-1) stimulation in culture-activated HSCs were measured using phosphorylation-specific antibodies. In vivo-activated HSCs were isolated from rats subjected to bile duct ligation and repeated dimethylnitrosoamine injections. HSCs showed increased expression of not only α-smooth muscle actin, but also the contractile regulatory proteins MLC kinase (MLCK), Rho kinase 2 (ROCK2), and CPI-17 during HSC activation in vitro. In culture-activated HSCs, ET-1 increased phosphorylation of CPI-17 at Thr18, which was markedly inhibited by the PKC inhibitor Ro-31-8425. ET-1 induced phosphorylation of MYPT1 at Thr853, which was suppressed by the ROCK inhibitor Y-27632. ET-1 induced sustained phosphorylation of MLC at Thr18/Ser19, which was inhibited by both Ro-31-8425 and Y-27632. Consistent with the data obtained from the in vitro study, HSCs isolated from cirrhotic rats showed increased expression of α-smooth muscle actin, MLCK, CPI-17, and ROCK2 compared with HSCs from nontreated rats. Furthermore, MLC phosphorylation in in vivo-activated HSCs was increased, according to enhanced phosphorylation of CPI-17 and MYPT1 in the presence of ET-1. These results suggest that activated HSCs may participate in constriction of hepatic sinusoids in the cirrhotic liver through both Ca(2+)-dependent (MLCK pathway) and Ca(2+)-sensitization mechanism (CPI-17 and MYPT1 pathways).     

The role of PKC isoforms in the inhibition of NF-κB activation by vitamin K2 in human hepatocellular carcinoma cells

Vitamin K (VK) has diverse protective effects against osteoporosis, atherosclerosis and carcinogenesis. We recently reported that menatetrenone, a VK2 analogue, suppressed nuclear factor (NF)-κB activation in human hepatoma cells. Although NF-κB is regulated by isoforms of protein kinase C (PKC), the involvement of PKCs in VK2-mediated NF-κB inhibition remains unknown. Therefore, the effects of VK2 on the activation and the kinase activity of each PKC isoform were investigated. The human hepatoma Huh7 cells were treated with PKC isoform-specific inhibitors and/or siRNAs against each PKC isoform with or without 12-O-tetradecanoylphorbol-13-acetate (TPA). VK2 inhibited the TPA-induced NF-κB activation in Huh7 cells. NF-κB activity was inhibited by the pan-PKC inhibitor Ro-31-8425, but not by the PKCα-specific inhibitor Gö6976. The knockdown of individual PKC isoforms including PKCα, δ and ? showed only marginal effects on the NF-κB activity. However, the knockdown of both PKCδ and PKC?, together with treatment with a PKCα-specific inhibitor, depressed the NF-κB activity. VK2 suppressed the PKCα kinase activity and the phosphorylation of PKC? after TPA treatment, but neither the activation nor the enzyme activity of PKCδ was affected. The knockdown of PKC? abolished the TPA-induced phosphorylation of PKD1, and the effects of PKD1 knockdown on NF-κB activation were similar to those of PKC? knockdown. Collectively, all of the PKCs, including α, δ and ?, and PKD1 are involved in the TPA-mediated activation of NF-κB. VK2 inhibited the NF-κB activation through the inhibition of PKCα and ? kinase activities, as well as subsequent inhibition of PKD1 activation.