Protein Kinase C Isoforms Epsilon Research Articles

Protein kinase Cɛ activity regulates mGluR5 surface expression in the rat nucleus accumbens.

Type 5 metabotropic glutamate receptors (mGluR5) activate protein kinase C (PKC) via coupling to Gαq/11 protein signaling. We have previously demonstrated that the epsilon isoform of PKC (PKCɛ) is a critical downstream target of mGluR5 in regulating behavioral and biochemical responses to alcohol. Recent evidence suggests that PKC-mediated phosphorylation of mGluR5 can lead to receptor desensitization and internalization. We therefore sought to examine the specific involvement of PKCɛ in the regulation of mGluR5 surface expression in the nucleus accumbens (NAc), a key regulator of alcohol-associated behaviors. Coronal brain sections from male Wistar rats were analyzed for either colocalization of mGluR5 and PKCɛ via immunohistochemistry or changes in mGluR5 surface expression and PKCɛ phosphorylation following local application of PKCɛ translocation activator or inhibitor peptides and/or an orthosteric mGluR5 agonist. We observed colocalization of mGluR5 and PKCɛ in the NAc. We also showed that intra-NAc infusion of the PKCɛ translocation inhibitor ɛV1-2 increased mGluR5 surface expression under baseline conditions. Stimulation of mGluR5 with an orthosteric agonist DHPG, dose dependently increased ERK1/2 and PKCɛ phosphorylation as well as mGluR5 internalization in acute NAc slices. Finally, we observed that activation of PKCɛ translocation with Tat-ΨɛRACK peptide mediates agonist-independent mGluR5 internalization, whereas PKCɛ translocation inhibitor ɛV1-2 prevents agonist-dependent internalization of mGluR5 in NAc slice preparations. These findings suggest that the subcellular localization of mGluR5 in the NAc is regulated by PKCɛ under basal and stimulation conditions, which may influence the role of mGluR5-PKCɛ signaling in alcohol-related behaviors. © 2016 Wiley Periodicals, Inc.

Abstract 940: The role of protein kinase C isoforms in telomerase activity and alphafetoprotein secretion by the hepatocellular carcinoma cell lines

Abstract Background: Hepatocellular carcinoma (HCC) is the third largest contributor to cancer mortality in the world. Alphafetoprotein (AFP) is synthetized and secreted by the majority of HCCs. Despite its controversial value as classical HCC diagnostic and follow-up marker, the AFP was lately shown to correlate with the volume of liver cancer. It is also useful as a predictive marker for radiotherapy treatment response and patients prognosis. On the molecular level, AFP has a proliferative role in HCC since it acts on its own receptor on the surface of hepatocytes; however, its mechanism of action remains unclear. Our laboratory have previously demonstrated that telomerase and protein kinase C (PKC) modulate AFP secretion in HCC cell lines and this effect is additive. The aim of this study is to elucidate the PKC isoforms involved in AFP secretion modulation and telomerase expression. Methods: Two AFP secretory cell lines, HepG2/C3a and PLC/PRF/5, and one non-secretory AFP cell line, SNU-387, were cultured in DMEM (RPMI-1640 for SNU-387) media with 10% FBS and 1% Penicillin/Streptomycin and incubated in humid 5% CO2 incubator. The RNA was extracted; the RT-PCR was performed to characterize the PKC isoforms and the modulation of hTERT by these isoforms. The role of each isoform on AFP secretion was elucidated using different PKC isoform modulators; the AFP concentration was measured using ELISA technique. The viability and toxicity of cells were assessed using WST-1. Results: The three cell lines express the PKC alpha, delta and epsilon isoforms. Whereas the other isoforms Beta 1 and 2 are slightly expressed in C3a, the gamma and beta isoforms are slightly expressed in PLC and SNU respectively. Dose-dependent inhibition of PKC alpha and beta by GO6976 decreased AFP secretion by 40% at 10 uM by the HepG2/C3a cell line. The inhibition of PKC delta by Rottlerin dose-dependently decreased AFP secretion by 30% at 5 uM. The PKC epsilon modulator FR236924 dose-dependently inhibited AFP secretion by 50% at 1 uM. Pan PKC inhibitor, GO6983, decreased AFP secretion by 45% at 5 uM. The same profile was obtained for PLC/PRF/5 with a maximum 30% of AFP down-regulation except for Rottlerin which showed a 75% of AFP inhibition. The catalytic subunit expression of telomerase, hTERT, was only inhibited by Rottlerin at 5 uM. Our experiments did not show any cell death nor cell proliferation modulation when incubating the three cell lines with PKC inhibitors. Conclusion: Combining the above results with our previous ones, we suggest for the first time, that PKC delta modulates AFP secretion through telomerase however the mechanism by which PKC alpha and epsilon modulate AFP secretion is to be elucidated. However, further experiments are needed to support this statement. Citation Format: Raia Doumit, Roula Tahtouh, Rita Ammoury, Riad Sarkis, Nada Alaaddine, George Hilal. The role of protein kinase C isoforms in telomerase activity and alphafetoprotein secretion by the hepatocellular carcinoma cell lines. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 940.

The effects of acute ethanol challenge on amygdala protein and gene expression of mglu5 and pkc epsilon during ethanol withdrawal induced anxiety in rats

Event Abstract Back to Event The effects of acute ethanol challenge on amygdala protein and gene expression of mglu5 and pkc epsilon during ethanol withdrawal induced anxiety in rats Jaya Kumar1, 2*, Hermizi Hapidin3, Yvonne Tee Get Bee3 and Zalina Ismail2 1 Universiti Kebangsaan Malaysia, Department of Physiology, UKM Medical Centre, Malaysia 2 Universiti Sains Malaysia, BRAINetwork Centre for Neurocognitive Sciences, Malaysia 3 Universiti Sains Malaysia, School of Health Sciences, Malaysia Cessation from prolonged and excessive ethanol intake can lead to hyperexcitability of glutamatergic neurotransmission in the amygdala, which induces an anxiety-like syndrome. Most alcoholics that suffer from such symptoms frequently depend on habitual drinking as self-medication to alleviate their symptoms. Metabotropic glutamate receptors 5 (mGlu5) and Protein Kinase C (PKC) epsilon have been related to ethanol consumption and anxiety-related behaviours. mGlu5 signalling has been associated with PKC activities and the epsilon isoform of PKC has been suggested as a crucial signalling target for modulation of ethanol consumption by mGlu5. This study explores the changes in mGlu5 and PKC epsilon at protein and gene-levels in the amygdala following acute ethanol challenge during EW induced anxiety in rats. Male Wistar rats were fed a modified liquid diet containing low fat cow milk, sucrose, and maltodextrin, with a gradual introduction of 2.4%, 4.8% and 7.2% ethanol for 20 days. Six hours into EW, the rats were intraperitoneally injected with normal saline and ethanol (2.5?g/kg, 20% v/v), and exposed to open field and elevated plus maze tests. Then, amygdala tissue was dissected from the rat brain for Western blot and gene expression studies. EW-induced anxiety was accompanied by a significant increment of mGlu5, total PKC epsilon and phosphorylated PKC epsilon protein levels and also of mRNA of mGlu5 (GRM5) in the amygdala. Acute administration of ethanol significantly attenuated EW-induced anxiety as well as an EW-induced increase in GRM5. The acute challenge of ethanol to EW rats had little effect on the phosphorylated and total protein levels of PKC epsilon in the amygdala. Our results show that amygdala PKC epsilon may not be directly involved in the development of anxiety following EW. Keywords: Amygdala, hyperexcitability, PKC, mGlu5, ethanol challenge Conference: 14th Meeting of the Asian-Pacific Society for Neurochemistry, Kuala Lumpur, Malaysia, 27 Aug - 30 Aug, 2016. Presentation Type: YIC05: Young Investigator Colloquium 5 Topic: 14th Meeting of the Asian-Pacific Society for Neurochemistry Citation: Kumar J, Hapidin H, Tee Get Bee Y and Ismail Z (2016). The effects of acute ethanol challenge on amygdala protein and gene expression of mglu5 and pkc epsilon during ethanol withdrawal induced anxiety in rats. Conference Abstract: 14th Meeting of the Asian-Pacific Society for Neurochemistry. doi: 10.3389/conf.fncel.2016.36.00069 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 04 Aug 2016; Published Online: 11 Aug 2016. * Correspondence: Dr. Jaya Kumar, Universiti Kebangsaan Malaysia, Department of Physiology, UKM Medical Centre, Cheras, Kuala Lumpur, Malaysia, jayakumar@ukm.edu.my Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Jaya Kumar Hermizi Hapidin Yvonne Tee Get Bee Zalina Ismail Google Jaya Kumar Hermizi Hapidin Yvonne Tee Get Bee Zalina Ismail Google Scholar Jaya Kumar Hermizi Hapidin Yvonne Tee Get Bee Zalina Ismail PubMed Jaya Kumar Hermizi Hapidin Yvonne Tee Get Bee Zalina Ismail Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Nociceptor beta II, delta, and epsilon isoforms of PKC differentially mediate paclitaxel-induced spontaneous and evoked pain.

As one of the most effective and frequently used chemotherapeutic agents, paclitaxel produces peripheral neuropathy (paclitaxel-induced peripheral neuropathy or PIPN) that negatively affects chemotherapy and persists after cancer therapy. The mechanisms underlying this dose-limiting side effect remain to be fully elucidated. This study aimed to investigate the role of nociceptor protein kinase C (PKC) isoforms in PIPN. Employing multiple complementary approaches, we have identified a subset of PKC isoforms, namely βII, δ, and ϵ, were activated by paclitaxel in the isolated primary afferent sensory neurons. Persistent activation of PKCβII, PKCδ, and PKCϵ was also observed in the dorsal root ganglion neurons after chronic treatment with paclitaxel in a mouse model of PIPN. Isoform-selective inhibitors of PKCβII, PKCδ, and PKCϵ given intrathecally dose-dependently attenuated paclitaxel-induced mechanical allodynia and heat hyperalgesia. Surprisingly, spinal inhibition of PKCβII and PKCδ, but not PKCϵ, blocked the spontaneous pain induced by paclitaxel. These data suggest that a subset of nociceptor PKC isoforms differentially contribute to spontaneous and evoked pain in PIPN, although it is not clear whether PKCϵ in other regions regulates spontaneous pain in PIPN. The findings can potentially offer new selective targets for pharmacological intervention of PIPN.

MTOR complex 2 mediates Akt phosphorylation that requires PKCε in adult cardiac muscle cells

Our earlier work showed that mammalian target of rapamycin (mTOR) is essential to the development of various hypertrophic responses, including cardiomyocyte survival. mTOR forms two independent complexes, mTORC1 and mTORC2, by associating with common and distinct cellular proteins. Both complexes are sensitive to a pharmacological inhibitor, torin1, although only mTORC1 is inhibited by rapamycin. Since mTORC2 is known to mediate the activation of a prosurvival kinase, Akt, we analyzed whether mTORC2 directly mediates Akt activation or whether it requires the participation of another prosurvival kinase, PKCε (epsilon isoform of protein kinase-C). Our studies reveal that treatment of adult feline cardiomyocytes in vitro with insulin results in Akt phosphorylation at S473 for its activation which could be augmented with rapamycin but blocked by torin1. Silencing the expression of Rictor (rapamycin-insensitive companion of mTOR), an mTORC2 component, with a sh-RNA in cardiomyocytes lowers both insulin-stimulated Akt and PKCε phosphorylation. Furthermore, phosphorylation of PKCε and Akt at the critical S729 and S473 sites respectively was blocked by torin1 or Rictor knockdown but not by rapamycin, indicating that the phosphorylation at these specific sites occurs downstream of mTORC2. Additionally, expression of DN-PKCε significantly lowered the insulin-stimulated Akt S473 phosphorylation, indicating an upstream role for PKCε in the Akt activation. Biochemical analyses also revealed that PKCε was part of Rictor but not Raptor (a binding partner and component of mTORC1). Together, these studies demonstrate that mTORC2 mediates prosurvival signaling in adult cardiomyocytes where PKCε functions downstream of mTORC2 leading to Akt activation.

Activation of neuromedin U type 1 receptor inhibits L-type Ca 2+ channel currents via phosphatidylinositol 3-kinase-dependent protein kinase C epsilon pathway in mouse hippocampal neurons

Neuromedin U (NMU) plays very important roles in the central nervous system. However, to date, any role of NMU in hippocampal neurons and the relevant mechanisms still remain unknown. In the present study, we report that NMU selectively inhibits L-type high-voltage-gated Ca 2+ channels (HVGCC) in mouse hippocampal neurons, in which NMU type 1 receptor (NMUR1), but not NMUR2, is endogenously expressed. In wild type mice, NMU (0.1 μM) reversibly inhibited HVGCC barium currents (I Ba) by ∼ 28%, while in NMUR1 −/− mice NMU had no significant effects. Intracellular infusion of GDP-β-S or a selective antibody raised against the G oα, as well as pretreatment of the neurons with pertussis toxin, blocked the inhibitory effects of NMU, indicating the involvement of G o-protein. This NMUR1-mediated effect did not display the characteristics of a direct interaction between G-protein βγ subunit (G βγ) and L-type HVGCC, but was abolished by dialyzing cells with QEHA peptide or an antibody to the G β. The classical and novel protein kinase C (PKC) antagonist calphostin C, as well as phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002, abolished NMU responses, whereas the classical PKC antagonist Gö6976 had no such effects. Cells dialyzed with a PKC epsilon isoform (PKCε) specific inhibitor peptide, GAVSLLPT, abolished NMU responses. In contrast, in cells dialyzed with an inactive PKCε control scramble peptide, LSGTLPAV, no significant effects were observed. In summary, these results suggest that NMU inhibits L-type HVGCC via activation of NMUR1 and downstream G βγ, PI3K, and a novel PKCε signaling pathway.

Cardiopulmonary Bypass with Cardioplegic Arrest Activates Protein Kinase C in the Human Myocardium

The protein kinase C (PKC) family consists of 12 isoforms, 6 of which have been found in human myocardium (PKC alpha, beta I/beta II, delta, epsilon, eta, and lambda/iota). These kinases function in regulation of contractility, ion channels, and in cellular protection or damage during ischemia-reperfusion injury. This study investigated the effects of controlled ischemia-reperfusion injury through cardioplegic arrest on PKC activity in patients undergoing cardiac surgery. Myocardium and skeletal muscle were harvested from patients before and after cardiopulmonary bypass and cardioplegic arrest. Total PKC (n=12) was isolated and specifically, the PKC delta (n=8) and PKC epsilon (n=8) isoforms were immunoprecipitated for use in functional kinase assays. Cellular fractions (n=4) were separated by differential ultracentrifugation and analyzed by Western blotting for membrane translocation (an indirect indicator of increased activity). Immunofluorescent staining for PKC delta and PKC epsilon was performed on myocardial sections. An in vitro assay of hypoxic cardioplegic arrest followed by reoxygenation was performed using isolated cardiomyocytes, and apoptosis was assessed. Cardioplegic arrest was associated with a 24.4% +/- 4.6% increase (p < 0.05) in total PKC activity, a 26.7% +/- 4.9% increase (p < 0.05) in PKC delta activity, and a 35.3% +/- 14% increase (p < 0.05) in PKC epsilon activity in myocardium. Cardioplegic arrest induced migration of PKC delta and epsilon to the z-line of the cardiomyocyte. Inhibition of PKC delta in the in vitro studies demonstrated a considerable reduction in apoptotic cells. PKC delta and epsilon have previously been shown to mediate and protect, respectively, from ischemia-reperfusion injury after myocardial ischemia. Demonstration of increases in their activity after cardioplegic arrest provides support for their possible role in myocardial function after cardiac surgery. Isoform-specific modulators may be of potential therapeutic value in treating postoperative myocardial dysfunction.

Tamoxifen effect on L-DOPA induced response complications in parkinsonian rats and primates

The contribution of striatal protein kinase C (PKC) isoform changes in levodopa (L-DOPA) induced motor response complications in parkinsonian rats was investigated and the ability of tamoxifen, an antiestrogen with a partial PKC antagonist property, to prevent these response alterations in 6-hydroxydopamine (6-OHDA) lesioned rats as well as in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) treated cynomologous monkeys was studied. Following treatment of adult male rats with L-DOPA twice daily for 3 weeks, protein levels of left (lesioned) and right (intact) striatal PKC isoforms were measured. Western blot analysis showed increased protein expression of both the novel PKC epsilon isoform and the atypical PKC lambda isoform ipsilateral to the lesion (174 ± 17% for epsilon, 140 ± 9% for lambda, of intact striatum in 6-OHDA lesioned plus chronic L-DOPA treated animals) in acute L-DOPA treated rats. No enhancement was observed in PKC immunoreactivity for other isoforms. Tamoxifen (5.0 mg/kg p.o.) significantly attenuated the L-DOPA induced augmentation of protein expression of PKC epsilon and PKC lambda, but had no effect on immunoreactivity for other PKC isoforms. In chronic L-DOPA treated parkinsonian rats, tamoxifen prevented (5.0 mg/kg p.o.) as well as ameliorated (5.0 mg/kg p.o.) the characteristic shortening in duration of motor response to L-DOPA challenge. In MPTP lesioned primates, similar to the ameliorative effect seen in rats, tamoxifen (1 and 3 mg/kg p.o) reduced the appearance of L-DOPA induced dyskinesia by 61% and 55% respectively ( p < 0.05). These results suggest that changes in specific striatal PKC isoforms contribute to the pathogenesis of L-DOPA induced motor complications and further that drugs able to selectively inhibit these signaling kinases might provide adjunctive benefit in the treatment of Parkinson's disease.

GABAA receptor regulation of voluntary ethanol drinking requires PKCepsilon.

Protein kinase C (PKC) regulates a variety of neural functions, including ion channel activity, neurotransmitter release, receptor desensitization and differentiation. We have shown previously that mice lacking the epsilon-isoform of PKC (PKCepsilon) self-administer 75% less ethanol and exhibit supersensitivity to acute ethanol and allosteric positive modulators of GABA(A) receptors when compared with wild-type controls. The purpose of the present study was to examine involvement of PKCepsilon in GABA(A) receptor regulation of voluntary ethanol drinking. To address this question, PKCepsilon null-mutant and wild-type control mice were allowed to drink ethanol (10% v/v) vs. water on a two-bottle continuous access protocol. The effects of diazepam (nonselective GABA(A) BZ positive modulator), zolpidem (GABA(A) alpha1 agonist), L-655,708 (BZ-sensitive GABA(A) alpha5 inverse agonist), and flumazenil (BZ antagonist) were then tested on ethanol drinking. Ethanol intake (grams/kg/day) by wild-type mice decreased significantly after diazepam or zolpidem but increased after L-655,708 administration. Flumazenil antagonized diazepam-induced reductions in ethanol drinking in wild-type mice. However, ethanol intake by PKCepsilon null mice was not altered by any of the GABAergic compounds even though effects were seen on water drinking in these mice. Increased acute sensitivity to ethanol and diazepam, which was previously reported, was confirmed in PKCepsilon null mice. Thus, results of the present study show that PKCepsilon null mice do not respond to doses of GABA(A) BZ receptor ligands that regulate ethanol drinking by wild-type control mice. This suggests that PKCepsilon may be required for GABA(A) receptor regulation of chronic ethanol drinking.

Protein Kinase C Involvement in Deoxycholate-Induced Apoptosis in Human Gastric Cells

Bile acids, such as deoxycholic acid (DC), are known to mediate some of their actions by differentially activating various protein kinase C (PKC) isoforms. This study confirms that DC induces apoptosis in gastric epithelial cells through PARP and caspase cascade activation, and examined the role of PKC in DC-induced apoptosis. We found increased activation of PKC in membrane fractions in response to DC that was concentration and time related. The PKC (beta(I)) isoform expression increased with translocation into the cell membrane fraction after DC (300 microM) stimulation. In contrast, PKCepsilon expression markedly decreased in response to DC treatment in a time- and concentration-dependent manner. In addition, this process was regulated by caspases, since the pan-caspase inhibitor z-VAD-fmk and caspase-3-, -6-, and -9-specific inhibitors prevented PKC (beta(I)) and (epsilon epsilon processing induced by DC. Treatment with the caspase-8-specific inhibitor, however, did not affect expression of either PKC isoform. No significant differences in the apoptotic response were observed when PKC (epsilon) overexpressed cells were exposed to DC in the presence of calcium-dependent conventional PKC inhibitors (Gö 6850 or Gö 6976). Our findings demonstrate that PKC is activated in gastric epithelial cells treated with DC with the PKC (beta(I)) and PKC (epsilon) isoforms being particularly involved in this process. The processing of PKC (beta(I) and epsilon) was shown to be closely regulated by caspases; however, modulations in PKC isoform concentrations by themselves have no effect on the apoptotic death of gastric mucosal cells induced by DC.

Serotonin via 5‐HT7 receptors activates p38 mitogen‐activated protein kinase and protein kinase C ɛ resulting in interleukin‐6 synthesis in human U373 MG astrocytoma cells

Serotonin [5-hydroxytryptamine (5-HT)] is a widely distributed neurotransmitter which is involved in neuroimmunomodulatory processes. Previously, it has been demonstrated that 5-HT may induce interleukin (IL)-6 expression in primary rat hippocampal astrocytes. The present study was undertaken to investigate the molecular pathways underlying this induction of IL-6 synthesis. As a model system, we used the human astrocytoma cell line U373 MG, which synthesizes IL-6 upon stimulation with various inducers. 5-HT dose- and time-dependently induced IL-6 protein synthesis. We identified several 5-HT receptors to be expressed on U373 MG cells, including the 5-HT1D, 5-HT2A, 5-HT3 and 5-HT7 receptors. In this report, we show that the 5-HT-induced IL-6 release is mediated by the 5-HT7 receptor based on several agonist/antagonists that were used. 5-HT-induced IL-6 synthesis is inhibited by the partially selective 5-HT7 receptor antagonist, pimozide, and the selective antagonist SB269970. Furthermore, IL-6 synthesis was induced by the 5-HT7 receptor agonist carboxamidotryptamin. In addition, we found p38 MAPKs and protein kinase C (PKC) epsilon to be involved in 5-HT-induced IL-6 synthesis as specific inhibitors of these enzymes (SB202190 and RO-31-8425, respectively) blocked 5-HT-induced IL-6 synthesis. Furthermore, 5-HT mediated the phosphorylation of both p38 MAPK as well as the PKC epsilon isoform. The p42/44 MAPKs, however, were not involved in 5-HT-induced IL-6 synthesis. This study shows, for the first time, a central role of 5-HT7 receptor linked to p38 MAPK and PKC epsilon for the induction of cytokine synthesis in astrocytic cells.

IL-6 expression induced by adenosine A 2b receptor stimulation in U373 MG cells depends on p38 mitogen activated kinase and protein kinase C

Adenosine binds to a class of G-protein coupled receptors, which are further distinguished as A 1, A 2a, A 2b and A 3 adenosine receptors. As we have shown earlier, the stable adenosine analogue NECA ( N6-( R)-phenylisopropyladenosine) stimulates IL-6 expression in the human astrocytoma cell line U373 MG via the A 2b receptor. The mechanism by which NECA promotes astrocytic IL-6 expression has not been identified. By using various inhibitors of signal transduction, we found that p38 mitogen-activated protein kinases (MAPK) activation (inhibitor SB202190), but not extracellular signal-regulated kinase (ERK) (PD98059) and c-jun N-terminal kinase (JNK)(SP600125), is essential in the NECA-induced signalling cascade that leads to the increase in IL-6 synthesis in U373 MG cells. Results obtained with protein kinase C (PKC) inhibitors that have different substrate specificities, indicated that the PKC delta and epsilon isoforms are also involved in adenosine receptor A 2b dependent upregulation of IL-6 expression. This is supported by the fact that NECA induced the activation of PKC delta and epsilon in U373 MG cells.

Myofilament anchoring of protein kinase C-epsilon in cardiac myocytes.

Regulatory proteins on muscle filaments are substrates for protein kinase C (PKC) but mechanisms underlying activation and translocation of PKC to this non-membrane compartment are poorly understood. Here we demonstrate that the epsilon isoform of PKC (epsilon-PKC) activated by arachidonic acid (AA) binds reversibly to cardiac myofibrils with an EC(50) of 86 nM. Binding occurred near the Z-lines giving rise to a striated staining pattern. The delta isoform of PKC (delta-PKC) did not bind to cardiac myofibrils regardless of the activator used, and the alpha isoform (alpha-PKC) bound only under strong activating conditions. Three established PKC anchoring proteins, filamentous actin (F-actin), the LIM domain protein Cypher-1, and the coatamer protein beta'-COP were each tested for their involvement in cytoskeletal anchoring. F-actin bound epsilon-PKC selectively over delta-PKC and alpha-PKC, but this interaction was readily distinguishable from cardiac myofilament binding in two ways. First, the F-actin/epsilon-PKC interaction was independent of PKC activation, and second, the synthetic hexapeptide LKKQET derived from the C1 region of epsilon-PKC effectively blocked epsilon-PKC binding to F-actin, but was without effect on its binding to cardiac myofilaments. Involvement of Cypher-1 was ruled out on the basis of its absence from detergent-skinned myofibrils that bound epsilon-PKC, despite its presence in intact cardiac myocytes. The epsilon-PKC translocation inhibitor peptide EAVSLKPT reduced activated epsilon-PKC binding to cardiac myofibrils in a concentration dependent manner, suggesting that a RACK2 or a similar protein plays a role in epsilon-PKC anchoring in cardiac myofilaments.

Protein kinase C translocation and Src protein tyrosine kinase activation mediate isoflurane-induced preconditioning in vivo: potential downstream targets of mitochondrial adenosine triphosphate-sensitive potassium channels and reactive oxygen species.

The authors tested the hypotheses that protein kinase C (PKC)-specific isoform translocation and Src protein tyrosine kinase (PTK) activation play important roles in isoflurane-induced preconditioning in vivo. Rats (n = 125) instrumented for measurement of hemodynamics underwent 30 min of coronary artery occlusion followed by 2 h of reperfusion and received 0.9% saline (control); PKC inhibitors chelerythrine (5 mg/kg), rottlerin (0.3 mg/kg), or PKC-epsilonV1-2 peptide (1 mg/kg); PTK inhibitors lavendustin A (1 mg/kg) or 4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP1; 1 mg/kg); mitochondrial adenosine triphosphate-sensitive potassium channel antagonist 5-hydroxydecanote (10 mg/kg); or reactive oxygen species scavenger N-acetylcysteine (150 mg/kg) in the absence and presence of a 30-min exposure to isoflurane (1.0 minimum alveolar concentration) in separate groups. Isoflurane was discontinued 15 min before coronary occlusion (memory period). Infarct size was determined using triphenyltetrazolium staining. Immunohistochemistry and confocal microscopic imaging were performed to examine PKC translocation in separate groups of rats. Isoflurane significantly (P < 0.05) reduced infarct size (40 +/- 3% [n = 13]) as compared with control experiments (58 +/- 2% [n = 12]). Chelerythrine, rottlerin, PKC-epsilonV1-2 peptide, lavendustin A, PP1, 5-hydroxydecanote, and N-acetylcysteine abolished the anti-ischemic actions of isoflurane (58 +/- 2% [n = 8], 50 +/- 3% [n = 9], 53 +/- 2% [n = 9], 59 +/- 3% [n = 6], 57 +/- 3% [n = 7], 60 +/- 3% [n = 7], and 53 +/- 3% [n = 6], respectively). Isoflurane stimulated translocation of the delta and epsilon isoforms of PKC to sarcolemmal and mitochondrial membranes, respectively. Protein kinase C-delta, PKC-epsilon, and Src PTK mediate isoflurane-induced preconditioning in the intact rat heart. Opening of mitochondrial adenosine triphosphate-sensitive potassium channels and generation of reactive oxygen species are upstream events of PKC activation in this signal transduction process.

Effects of nifedipine-induced pulmonary vasodilatation on cardiac receptors and protein kinase C isoforms in the chronically hypoxic rat.

In chronic hypoxia, pulmonary hypertension induces a right ventricular (RV) hypertrophy (RVH) and the catecholamine-activated adrenergic system modulates cardiovascular responses through alpha- and beta-adrenergic pathways. The alpha(1)-adrenergic receptor (alpha(1)-AR) and protein kinase C (PKC) may play an important role in the signaling pathway leading to RVH. The aim of this study was to examine the relationship between nifedipine-induced pulmonary vasodilatation, the blunting of RVH and the modifications in the density of alpha(1)-AR, PKC activity and expression of PKC isoforms. In rats exposed to 15 days of hypoxia (380 torr, 50.66 kPa), RV pressure increased and RVH developed. Nifedipine, a calcium antagonist, given through gastric administration, partially decreased RV pressure and RVH. In both ventricles, hypoxia decreased alpha(1)-AR and beta-AR density and increased muscarinic acetylcholine receptor density. Nifedipine decreased alpha(1)-AR density only in normoxia. Expression of epsilon, delta and zeta PKC isoforms increased with RVH and normalized with nifedipine treatment. In conclusion, in this in vivo model of hypoxic rat, no relation was found between a RVH decrease and cardiac receptor densities. However, the development and regression of pulmonary hypertension and RVH were related to the expression of some PKC isoforms suggesting that pathways other than alpha(1)-AR might be involved in hypoxia-induced ventricular hypertrophy.

Critical role of protein kinase C ε for lipopolysaccharide-induced IL-12 synthesis in monocyte-derived dendritic cells

In the present study we have investigated the potential involvement of protein kinase C (PKC) in the maturation of human dendritic cells (DC) by bacterial lipopolysaccharide (LPS). LPS stimulation of DC derived from human monocytes resulted in PKC phosphorylation. Inhibition of PKC activation using bisindolylmaleimide (Bis), a pan-PKC inhibitor, was associated with a dose-dependent decrease of LPS-induced IL-12 production. In contrast, up-regulation of MHC class II, CD80 and CD86 was not altered. Consistent with the diminished IL-12 synthesis, DC stimulated with LPS in presence of Bis were deficient in the induction of IFN-gamma production by allogeneic CD4+ T cells. Furthermore, we found that PKC inhibition impaired LPS-induced I kappa B-alpha degradation and subsequent nuclear factor (NF)-kappa B activation in DC. LPS resulted in the phosphorylation of conventional alpha/beta and novel epsilon PKC isoforms in DC. Inhibition of LPS-induced PKC activity using pseudosubstrate peptides specific for PKC isoforms established that PKC epsilon but not PKC alpha/beta was involved in the production of IL-12 and TNF-alpha. Overall, these data provide evidence that PKC inhibition impairs LPS signaling in DC and identify PKC epsilon as a potential target for the inhibition of Toll-like receptor-4-mediated, IL-12-dependent Th1 type responses.

Docosahexaenoic acid modulates phorbol ester-induced activation of extracellular signal-regulated kinases 1 and 2 in NIH/3T3 cells.

Phosphorylation of extracellular signal-regulated kinases (ERK1/ERK2) has been implicated in cell proliferation of mammalian cells. In the present study, we investigated the role of docosahexaenoic acid (DHA) in the modulation of ERK1/ERK2 phosphorylation, stimulated either with phorbol 12-myristate 13-acetate (PMA) or transforming growth factor-alpha (TGFalpha) in NIH/3T3 cells. We observed that both PMA and TGFalpha induced ERK1/ERK2 phosphorylation within 5 min of stimulation. PMA acts upstream of MEK and via activation of protein kinase C (PKC), as GF109203X, a potent PKC inhibitor, and U0126, a MEK inhibitor, abolished its actions on ERK1/ERK2 phosphorylation. TGFalpha did not act via PKC because GF109203X failed to curtail the degree of ERK1/ERK2 phosphorylation in these cells. DHA alone failed to induce the phosphorylation of these mitogen-activated protein (MAP) kinases; however, this fatty acid significantly curtailed the PMA- but not TGFalpha-induced MAP kinase enzyme activity and phosphorylation in NIH/3T3 cells. Furthermore, we observed that DHA significantly inhibited PMA-induced translocation of two PKC isoforms, PKC alpha and PKC epsilon, from cytosol to plasma membrane. Interestingly, DHA failed to inhibit the PMA-induced translocation PKC delta isoform in these cells. Furthermore, DHA decreased PMA-induced proliferation of NIH/3T3 cells. In this study, we show for the first time that DHA inhibits MAP kinase ERK1/ERK2) activation and proliferation of NIH/3T3 cells via its inhibitory action on PKC alpha and epsilon isoforms.

Preconditioning attenuates apoptosis and necrosis: role of protein kinase C epsilon and -delta isoforms.

Preconditioning reduces cardiomyocyte necrosis in vivo and in vitro, but it is unknown whether preconditioning blocks apoptosis. We wanted to compare the effects of preconditioning on necrosis and apoptosis in cardiomyocytes. Necrosis was detected with propidium iodide, and apoptosis was quantified by three complementary techniques: flow cytometry, TdT-mediated dUTP nick-end labeling assay, and DNA-laddering electrophoresis. Apoptosis increased with simulated ischemia time (6 h, 19 +/- 1%; 12 h, 27 +/- 2%; 18 h, 40 +/- 4%; 24 h, 54 +/- 4%; and 36 h, 83 +/- 4%; n = 6 for each group). Simulated ischemia and reoxygenation contributed equally to apoptosis (12-h ischemia, 27 +/- 2%, n = 6; 12-h ischemia and 12-h reoxygenation, 51 +/- 4%, n = 6; and 24-h ischemia, 54 +/- 5%, n = 8). Necrosis occurred primarily during reoxygenation; none was detected during simulated ischemia. Preconditioning with 10 min of simulated ischemia reduced necrosis (18 +/- 6%, n = 8) but had no effect on apoptosis. However, three 1-min cycles of simulated ischemia separated by 5 min of reoxygenation reduced necrosis and apoptosis similarly. The protein kinase C (PKC) inhibitors Go6976 (0.1 microM) or chelerythrene (4 microM) abolished the effect of preconditioning. Preconditioning selectively activated PKC epsilon but had no effect on PKC delta and on total PKC enzyme activity. Preconditioning protected against necrosis and apoptosis, but the preconditioning ischemia required for blocking apoptosis was less than that for reducing necrosis. Activation of PKC epsilon isoform is important in mediating the protection.

Na+/K+ATPase activity inhibition and isoform-specific translocation of protein kinase C following angiotensin II administration in isolated eel enterocytes.

In the eel, angiotensin II (Ang II) has a role at the level of both gill chloride and kidney tubular cells, regulating sodium balance and therefore osmoregulation. The present study extends these findings to another important osmoregulatory organ - the intestine. Enterocytes were obtained from sea-water (SW)-acclimated eels to investigate the role of Ang II on the intestinal Na+/K+ATPase activity, because in SW-acclimated animals the intestine represents an important site of water and NaCl transport from the mucosal to the serosal side. This paper demonstrates that isolated enterocytes stimulated with increasing Ang II concentrations (0.01-100 nM) showed a dose-dependent inhibition of the Na+/K+ATPase activity. The threshold decrease was at 0.01 nM Ang II; it reached a maximum at 10 nM (81.5% inhibition) and did not decrease further with the use of higher hormone doses. These hormonal effects were blocked by a specific competitive antagonist of the AT1 receptor subtype, DuP-753 (100% inhibition at 10 microM), indicating that these effects are mediated by an AT1-like receptor. Isolated enterocytes stimulated with 10 nM Ang II showed a transient increase in intracellular calcium ([Ca2+]i), followed by a lower sustained phase. Removal of extracellular Ca2+ did not reduce the initial transient response and completely abolished the plateau phase. The inhibition of the Na+/K+ATPase activity was dependent on protein kinase C (PKC) activation since PKC antagonists (calphostin C and staurosporine) abolished the inhibitory effect of Ang II, and the PKC activator phorbol 12-myristate 13-acetate reduced transporter activity. Western blot analysis with antibodies to PKC alpha, beta I, beta II, gamma, delta, epsilon, iota, eta and zeta isoforms showed that eel enterocytes expressed the conventional isoforms (alpha and beta I), the novel isoforms (delta and eta) and the atypical isoforms (zeta and iota). Ang II stimulated the translocation from the cytosol to the plasma membrane of PKC alpha, PKC delta and PKC eta isoforms. In conclusion, our results suggest that Ang II modulates intestinal Na+/K+ATPase in SW-acclimated eels via calcium mobilization and PKC activation.

Increased neuronal and glial expression of protein kinase C isoforms in neocortex of transgenic Tg2576 mice with amyloid pathology

We investigated the influence of five- to sevenfold neuronal overexpression of the Swedish mutation of human APP695 (APPsw) in the transgenic mouse strain Tg2576 on neocortical protein kinase C (PKC) expression and subcellular distribution. Using specific antibodies to PKC alpha, PKC beta, PKC gamma, PKC epsilon and PKC zeta isoforms for Western blot analysis, we observed increased immunoreactivity for PKC alpha and PKC gamma isoforms in crude tissue homogenates from the neocortex of 16-month-old APPsw mice as compared with nontransgenic littermates, which was not present in 6 month-old Tg2576 mice. We also observed elevated levels of PKC alpha, PKC beta, PKC gamma and PKC zeta in membrane fractions and reduced concentrations of PKC alpha and PKC gamma in cytosolic fractions of aged Tg2576 mice, indicating that these PKC isoforms are in their activated state. In young, 6-month-old Tg2576 mice, however, the increase in membrane-bound PKC isoforms and concomitant decrease in cytosolic PKC isoforms was much less pronounced, demonstrating the age-dependent nature of alterations in PKC isoforms. Immunocytochemistry of brain sections supported these findings and revealed increased neuronal labelling for PKC alpha, PKC gamma and PKC lambda isoforms in neocortex of 16-month-old APPsw mice compared with nontransgenic littermates, with the increase being strongest for PKC gamma and PKC lambda isoforms. Additionally, PKC gamma and to a lesser extent PKC lambda isoforms were induced in reactive astrocytes in proximity to amyloid plaques. Our data indicate that neuronal overexpression of APPsw causes a dynamic change in neuronal expression and activation of multiple PKC isoforms known to be regulators of proteolytic amyloid precursor protein (APP) processing (PKC alpha) and of neuronal survival (PKC lambda and PKC zeta). The induction of the PKC gamma and PKC lambda isoforms in reactive astrocytes surrounding amyloid plaques might be required for astrocyte activation and astrocytic cytokine expression in response to amyloid plaque formation.