Dependent Protein Kinase C Isoforms Research Articles

Receptor-specific regulation of atrial GIRK channel activity by different Ca2+-dependent PKC isoforms

G Protein-activated K+ channels (GIRK) channels are inhibited by depletion of PtdIns(4,5)P2(PIP2), and/or channel phosphorylation by proteinkinase C (PKC). By using FRET-based biosensors, expressed in HEK293 cells or in atrial myocytes, we quantified receptor-specific Gq-coupled receptor (GqPCR) signalling on the level of phospholipase C (PLC) activation by monitoring PIP2-depletion and diacylglycerol (DAG) formation. Simultaneous voltage-clamp experiments on GIRK channel activity were performed as a functional readout for Gq-coupled α1B- and ET-receptor-induced signalling. GqPCR-induced fast inhibition of GIRK channel activity is mediated by depletion of PIP2, whereas phosphorylation of GIRK channels results in delayed, but effective GIRK current inhibition.We demonstrate a receptor-induced inhibitory component on GIRK activity that is independent of PIP2-depletion, but attributed to the activation of Ca2+-dependent PKC isoforms. As a novel finding, we demonstrate receptor-dependent differences in GIRK inhibition according to receptor-specific activation of the Ca2+-dependent PKC isoforms PKCα and PKCβ. Pharmacological inhibition of PKCα, but not of PKCβ, abolishes GIRK inhibition induced by stimulation of α1B-receptors. In contrast, ET-R-induced reduction of GIRK activity is sensitive to pharmacological block of PKCβ, but not of PKCα. Coexpression of α1B-receptors (or ETB-R) and PKCα (or PKCβ) in HEK 293 cells increased homologous receptor desensitization as indicated by a rapid decline of the CKAR FRET signal monitoring receptor activity. These data suggest that receptor-species dependent differences in PKC isoform activation regulate both GIRK channel activity and the strength of the receptor signal via a negative feedback mechanism.

Receptor Species-dependent Desensitization Controls KCNQ1/KCNE1 K+ Channels as Downstream Effectors of Gq Protein-coupled Receptors

Activation of Gq protein-coupled receptors (GqPCRs) might induce divergent cellular responses, related to receptor-specific activation of different branches of the Gq signaling pathway. Receptor-specific desensitization provides a mechanism of effector modulation by restricting the spatiotemporal activation of signaling components downstream of Gq We quantified signaling events downstream of GqPCR activation with FRET-based biosensors in CHO and HEK 293 cells. KCNQ1/KCNE1 channels (IKs) were measured as a functional readout of receptor-specific activation. Activation of muscarinic M1 receptors (M1-Rs) caused robust and reversible inhibition of IKs. In contrast, activation of α1B-adrenergic receptors (α1B-ARs) induced transient inhibition of IKs, which turned into delayed facilitation after agonist withdrawal. As a novel finding, we demonstrate that GqPCR-specific kinetics of IKs modulation are determined by receptor-specific desensitization, evident at the level of Gαq activation, phosphatidylinositol 4,5-bisphosphate (PIP2) depletion, and diacylglycerol production. Sustained IKs inhibition during M1-R stimulation is attributed to robust membrane PIP2 depletion, whereas the rapid desensitization of α1B-AR delimits PIP2 reduction and augments current activation by protein kinase C (PKC). Overexpression of Ca2+-independent PKCδ did not affect the time course of α1B-AR-induced diacylglycerol formation, excluding a contribution of PKCδ to α1B-AR desensitization. Pharmacological inhibition of Ca2+-dependent PKC isoforms abolished fast α1B receptor desensitization and augmented IKs reduction, but did not affect IKs facilitation. These data indicate a contribution of Ca2+-dependent PKCs to α1B-AR desensitization, whereas IKs facilitation is induced by Ca2+-independent PKC isoforms. In contrast, neither inhibition of Ca2+-dependent/Ca2+-independent isoforms nor overexpression of PKCδ induced M1 receptor desensitization, excluding a contribution of PKC to M1-R-induced IKs modulation.

Calcium-Dependent Protein Kinase C Is Not Required for Post-Tetanic Potentiation at the Hippocampal CA3 to CA1 Synapse.

Post-tetanic potentiation (PTP) is a widespread form of short-term synaptic plasticity in which a period of elevated presynaptic activation leads to synaptic enhancement that lasts tens of seconds to minutes. A leading hypothesis for the mechanism of PTP is that tetanic stimulation elevates presynaptic calcium that in turn activates calcium-dependent protein kinase C (PKC) isoforms to phosphorylate targets and enhance neurotransmitter release. Previous pharmacological studies have implicated this mechanism in PTP at hippocampal synapses, but the results are controversial. Here we combine genetic and pharmacological approaches to determine the role of classic PKC isoforms in PTP. We find that PTP is unchanged in PKC triple knock-out (TKO) mice in which all calcium-dependent PKC isoforms have been eliminated (PKCα, PKCβ, and PKCγ). We confirm previous studies and find that in wild-type mice 10 μm of the PKC inhibitor GF109203 eliminates PTP and the PKC activator PDBu enhances neurotransmitter release and occludes PTP. However, we find that the same concentrations of GF109203 and PDBu have similar effects in TKO animals. We also show that 2 μm GF109203 does not abolish PTP even though it inhibits the PDBu-dependent phosphorylation of PKC substrates. We conclude that at the CA3 to CA1 synapse Ca(2+)-dependent PKC isoforms do not serve as calcium sensors to mediate PTP. Neurons dynamically regulate neurotransmitter release through many processes known collectively as synaptic plasticity. Post-tetanic potentiation (PTP) is a widespread form of synaptic plasticity that lasts for tens of seconds that may have important computational roles and contribute to short-term memory. According to a leading mechanism, presynaptic calcium activates protein kinase C (PKC) to increase neurotransmitter release. Pharmacological studies have also implicated this mechanism at hippocampal CA3 to CA1 synapses, but there are concerns about the specificity of PKC activators and inhibitors. We therefore used a molecular genetic approach and found that PTP was unaffected when all calcium-dependent PKC isozymes were eliminated. We conclude that PKC isozymes are not the calcium sensors that mediate PTP at the CA3 to CA1 synapse.

Oxidative stress parameters induced by exposure to either cadmium or 17β-estradiol on Mytilus galloprovincialis hemocytes. The role of signaling molecules

The aim of the present study was to determine and compare the possible effects of exposure to an estrogen, 17β-estradiol and to a metal, cadmium on oxidative parameters of Mytilus galloprovincialis hemocytes and to elucidate the signaling pathways that probably mediate the studied effects exerted by these two chemicals. In addition, it was of interest to investigate if the studied parameters could constitute biomarkers for aquatic pollution monitoring.Our results suggest that micromolar concentrations of either cadmium or 17β-estradiol affected the redox status of mussels by modulating oxidative parameters and antioxidant enzymes gene expression in mussel M. galloprovincialis hemocytes. In particular, our results showed that treatment of hemocytes with either 5μM of cadmium chloride or with 25nM of 17β-estradiol for 30min caused significant increased ROS production; this led to oxidative damage exemplified by significant increased DNA damage, protein carbonylation and lipid peroxidation, as well as increased mRNA levels of the antioxidant enzymes catalase (CAT), superoxide dismoutase (SOD) and glutathione S-transferase (GST). Furthermore, our results suggest that either cadmium or 17β-estradiol signal is mediated either through one of the already known pathways initiated by photatidyl-inositol 3-kinase (PI3K) and reaching Na+/H+ exchanger (NHE) probably through protein kinase C (PKC) or a kinase-mediated signaling pathway that involves in most of the cases NHE, PKC, Ca2+-dependent PKC isoforms, PI3-K, NADPH oxidase, nitric oxide (NO) synthase, c-Jun N-terminal kinase (JNK) and cyclic adenosine-3′-5′-monophosphate (cAMP). Our results also attribute a protective role to cAMP, since pre-elevated intracellular cAMP levels inhibited the signal induced by each exposure. Finally, since aquatic invertebrates have been the most widely used monitoring organisms for pollution impact evaluation in marine environments and taking under consideration the positive correlation obtained between the studied parameters, we can suggest the simultaneous use of these oxidative stress parameters offering an effective early warning system in biomonitoring of aquatic environments.

Protein Kinase C Modulates Aurora-kinase Inhibition Induced by CCT129202 in HMC-1560,816 Cell Line

The human mast cell line HMC-1⁵⁶⁰,⁸¹⁶ carries activating mutations in the proto-oncogene of c-kit that cause autophosphorylation and permanent c-kit receptor activation. The compound CCT129202 is a new and selective inhibitor of Aurora kinase A and B that decreases the viability of a variety of human tumor cell lines. The effect of Aurora kinase inhibition was assessed in the HMC-1⁵⁶⁰,⁸¹⁶ line in order to find a suitable tool for mastocytosis treatment. CCT129202 treatment induces a significant decrease in cell viability in HMC-1⁵⁶⁰,⁸¹⁶ cells after 48 hours of treatment. Moreover, caspase-3 and caspase-8 activation was induced after incubation of HMC-1⁵⁶⁰,⁸¹⁶ cells in the presence of CCT129202. It has been demonstrated that Protein Kinase C (PKC) plays a crucial role in mast cell activation as well as cell migration, adhesion and apoptotic cell death. Co-treatment of Ca²⁺-independent PKCs (δ ε and θ) inhibitor GF109203X with CCT129202, reduces caspase-3 activation which controls cell levels. In contrast, Go6976, an inhibitor of Ca²⁺-dependent PKCs, increases caspase-3 activation. Oppositely, GF109203X does not modify CCT129202-induced apoptosis through the caspase-8 pathway whereas Go6976 treatment abolishes the increase on caspase-8 activity due to CCT129202. This implies that Ca²⁺-independent PKC isoforms seems to be related with CCT129202-induced apoptosis through the caspase- 3 pathway, whereas Ca²⁺-dependent PKC isoforms are related with the CCT129202 effect on the caspase-8 pathway. Interestingly, CCT129202 cytotoxic effect remains even though Ca²⁺-dependent PKCs are inhibited, which shows that the Aurora kinase inhibitor effect is acting through the caspase-3 pathway. On the other hand, Ca²⁺-independent PKCs inhibition does not affect the final apoptotic CCT129202 effect because this seems to be mediated by the caspase-8 pathway. Moreover, CCT129202 does not affect PKCδ and Ca²⁺-dependent PKC translocation, which indicates that PKC translocation pivots on its activation. This demonstrates that Aurora kinase inhibition is not related to this process. Finally, when PKC is silenced in HMC-1⁵⁶⁰,⁸¹⁶ cells, the effect of CCT129202 on the caspase-3 pathway disappears, which indicates that the CCT129202 effect is clearly PKC-dependent.

Circulation Research Thematic Synopsis

<i>Circulation Research</i> Thematic Synopsis

After Damage of Large Bile Ducts by Gamma-Aminobutyric Acid, Small Ducts Replenish the Biliary Tree by Amplification of Calcium-Dependent Signaling and de Novo Acquisition of Large Cholangiocyte Phenotypes

Large cholangiocytes secrete bicarbonate in response to secretin and proliferate after bile duct ligation by activation of cyclic adenosine 3', 5'-monophosphate signaling. The Ca(2+)-dependent adenylyl cyclase 8 (AC8, expressed by large cholangiocytes) regulates secretin-induced choleresis. Ca(2+)-dependent protein kinase C (PKC) regulates small cholangiocyte function. Because gamma-aminobutyric acid (GABA) affects cell functions by activation of both Ca(2+) signaling and inhibition of AC, we sought to develop an in vivo model characterized by large cholangiocyte damage and proliferation of small ducts. Bile duct ligation rats were treated with GABA for one week, and we evaluated: GABA(A), GABA(B), and GABA(C) receptor expression; intrahepatic bile duct mass (IBDM) and the percentage of apoptotic cholangiocytes; secretin-stimulated choleresis; and extracellular signal-regulated kinase1/2 (ERK1/2) phosphorylation and activation of Ca(2+-)dependent PKC isoforms and AC8 expression. We found that both small and large cholangiocytes expressed GABA receptors. GABA: (i) induced apoptosis of large cholangiocytes and reduced large IBDM; (ii) decreased secretin-stimulated choleresis; and (iii) reduced ERK1/2 phosphorylation and AC8 expression in large cholangiocytes. Small cholangiocytes: (i) proliferated leading to increased IBDM; (ii) displayed activation of PKCbetaII; and (iii) de novo expressed secretin receptor, cystic fibrosis transmembrane regulator, Cl(-)/HCO(3)(-) anion exchanger 2 and AC8, and responded to secretin. Therefore, in pathologies of large ducts, small ducts replenish the biliary epithelium by amplification of Ca(2+)-dependent signaling and acquisition of large cholangiocyte phenotypes.

Involvement of Ca 2+-dependent PKCs in the adaptive changes of μ-opioid pathways to sympathetic denervation in the guinea pig colon

In the guinea pig colon, chronic sympathetic denervation entails supersensitivity to inhibitory μ-opioid agents modulating cholinergic neurons. The mechanism underlying such adaptive change has not yet been unravelled, although protein kinase C (PKC) may be involved. A previous study indirectly demonstrated that activation of μ-opioid receptors on myenteric neurons facilitates PKC activity. Such coupling may counteract the inhibitory action of μ-opioid agents on acetylcholine overflow, since PKC, per se, increases this parameter. After chronic sympathetic denervation such restraint abates, representing a possible mechanism for development of supersensitivity to μ-opioid agents. In the present study, this hypothesis was further investigated. After chronic sympathetic denervation, Ca 2+-dependent PKC activity was reduced in colonic myenteric plexus synaptosomes. The μ-opioid agent, DAMGO, increased Ca 2+-dependent PKC activity in synaptosomes obtained from normal, but not from denervated animals. In myenteric synaptosomes obtained from this experimental group, protein levels of Ca 2+-dependent PKC isoforms βI, βII and γ decreased, whereas α levels increased. In whole-mount preparations, the four Ca 2+-dependent PKC isoforms co-localized with μ-opioid receptors on subpopulations of colonic myenteric neurons. The percentage of neurons staining for PKCβII, as well as the number of μ-opioid receptor-positive neurons staining for PKCβII, decreased in denervated preparations. The same parameters related to PKCα, βI or γ remained unchanged. Overall, the present data strengthen the concept that μ-opioid receptors located on myenteric neurons are coupled to Ca 2+-dependent PKCs. After chronic sympathetic denervation, a reduced efficiency of this coupling may predominantly involve PKCβII, although also PKCβI and γ, but not PKCα, may be implicated.

Wnt5a Increases Cardiac Gene Expressions of Cultured Human Circulating Progenitor Cells via a PKC Delta Activation

BackgroundWnt signaling controls the balance between stem cell proliferation and differentiation and body patterning throughout development. Previous data demonstrated that non-canonical Wnts (Wnt5a, Wnt11) increased cardiac gene expression of circulating endothelial progenitor cells (EPC) and bone marrow-derived stem cells cultured in vitro. Since previous studies suggested a contribution of the protein kinase C (PKC) family to the Wnt5a-induced signalling, we investigated which PKC isoforms are activated by non-canonical Wnt5a in human EPC.Methodology/Principal FindingsImmunoblot experiments demonstrated that Wnt5a selectively activated the novel PKC isoform, PKC delta, as evidenced by phosphorylation and translocation. In contrast, the classical Ca2+-dependent PKC isoforms, PKC alpha and beta2, and one of the other novel PKC isoforms, PKC epsilon, were not activated by Wnt5a. The PKC delta inhibitor rottlerin significantly blocked co-culture-induced cardiac differentiation in vitro, whereas inhibitors directed against the classical Ca2+-dependent PKC isoforms or a PKC epsilon-inhibitory peptide did not block cardiac differentiation. In accordance, EPC derived from PKC delta heterozygous mice exhibited a significant reduction of Wnt5a-induced cardiac gene expression compared to wild type mice derived EPC.Conclusions/SignificanceThese data indicate that Wnt5a enhances cardiac gene expressions of EPC via an activation of PKC delta.

Rapid activation of CLOCK by Ca2+‐dependent protein kinase C mediates resetting of the mammalian circadian clock

In mammals, immediate-early transcription of the Period 1 (Per1) gene is crucial for resetting the mammalian circadian clock. Here, we show that CLOCK is a real signalling molecule that mediates the serum-evoked rapid induction of Per1 in fibroblasts through the Ca2+-dependent protein kinase C (PKC) pathway. Stimulation with serum rapidly induced nuclear translocation, heterodimerization and Ser/Thr phosphorylation of CLOCK just before the surge of Per1 transcription. Serum-induced CLOCK phosphorylation was abolished by treatment with PKC inhibitors but not by other kinase inhibitors. Consistently, the interaction between CLOCK and PKC was markedly increased shortly after serum shock, and the Ca2+-dependent PKC isoforms PKCalpha and PKCgamma phosphorylated CLOCK in vitro. Furthermore, phorbol myristic acetate treatment triggered immediate-early transcription of Per1 and also CLOCK phosphorylation, which were blocked by a Ca2+-dependent PKC inhibitor. These findings indicate that CLOCK activation through the Ca2+-dependent PKC pathway might have a substantial role in phase resetting of the circadian clock.

Regulation of Gap Junction Intercellular Communication in Primary Canine Lens Epithelial Cells: Role of Protein Kinase C

Purpose: Gap junction intercellular communication (GJIC) is important in maintaining lens epithelial cell homeostasis and reductions in GJIC may be associated with the development of cataract. Protein kinase C (PKC) activation can disrupt gap junction communication via phosphorylation of connexin 43 (C×43) proteins that compose gap junction channels. This study examined the role of PKC activation in modulating GJIC in a primary canine lens epithelial cell (LEC) line. Methods: TPA (12-O-tetradecanoyl-phorbol-acetate), a potent PKC activator and inhibitor of GJIC, was utilized in the present study. Primary cultures of canine LEC were treated with TPA (0–1000 ng/ml) for 0.5 hr and GJIC was assessed by scrape loading/dye transfer (SL/DT), and immunoblotting to detect phosphorylation of C×43 protein. Inhibition of general and calcium-dependent PKC activity was achieved by pretreatment of cells with GF109203X and Gö6976, respectively. Results: Treatment with TPA (1–1000 ng/ml) significantly decreased GJIC in canine LEC as assessed by SL/DT. Pretreatment with 10 and 100 ng/ml TPA decreased GJIC by 80% as compared to controls and increased C×43 phosphorylation as assessed by immunoblotting. Pretreatment of cells with GF109203X and Gö6976, partially restored TPA-inhibited GJIC by 40% and 60%, respectively, and reduced C×43 phosphorylation. Expression of calcium dependent PKC isoforms was detected in canine whole lens and LEC. Conclusions: Treatment with TPA significantly reduces GJIC in canine LEC. These effects are mediated, in part, by activation of calcium-dependent PKC isoforms. Primary canine LEC are a useful model in the study of the molecular mechanisms involved in GJIC and cataractogenesis.

Angiotensin II inhibits inward rectifier K + channels in rabbit coronary arterial smooth muscle cells through protein kinase Cα

We investigated the effects of the vasoconstrictor angiotensin (Ang) II on the whole cell inward rectifier K + (Kir) current enzymatically isolated from small-diameter (<100 μm) coronary arterial smooth muscle cells (CASMCs). Ang II inhibited the Kir current in a dose-dependent manner (half inhibition value: 154 nM). Pretreatment with phospholipase C inhibitor and protein kinase C (PKC) inhibitors prevented the Ang II-induced inhibition of the Kir current. The PKC activator reduced the Kir currents. The inhibitory effect of Ang II was reduced by intracellular and extracellular Ca 2+ free condition and by Gö6976, which inhibits Ca 2+-dependent PKC isoforms α and β. However, the inhibitory effect of Ang II was unaffected by a peptide that selectively inhibits the translocation of the ε isoform of PKC. Western blot analysis confirmed that PKCα, and not PKCβ, was expressed in small-diameter CASMCs. The Ang II type 1 (AT 1)-receptor antagonist CV-11974 prevented the Ang II-induced inhibition of the Kir current. From these results, we conclude that Ang II inhibits Kir channels through AT 1 receptors by the activation of PKCα.

Protein kinase C-α regulation of gallbladder Na + transport becomes progressively more dysfunctional during gallstone formation

Gallbladder Na+ absorption and biliary Ca2+ are both increased during gallstone formation and may promote cholesterol nucleation. Na+/H+ exchange (NHE) is a major pathway for gallbladder Na+ transport. Ca2+-dependent second messengers, including protein kinase C (PKC), inhibit basal gallbladder Na+ transport. Multiple PKC isoforms with species- and tissue-specific expression have been reported. In this study we sought to characterize Ca2+-dependent PKC isoforms in gallbladder and to examine their roles in Na+ transport during gallstone formation. Gallbladders were harvested from prairie dogs fed either nonlithogenic chow or 1.2% cholesterol-enriched diet for varying periods to induce various stages of gallstone formation. PKC was activated with the use of phorboldibutyrate, and we assessed gallbladder NHE regulation by measuring unidirectional Na+ flux and dimethylamiloride-inhibitable 22Na+ uptake. We measured gallbladder PKC activity with the use of histone III-S phosphorylation and used Gö 6976 to determine PKC-alpha contributions. Gallbladder PKC isoform messenger RNA and protein expression were examined with the use of Northern- and Western-blot analysis, respectively. Prairie dog and human gallbladder expresses PKC-alpha, betaII, and delta isoforms. The PKC activation significantly decreased gallbladder J(Na)(ms) and reduced baseline 22Na+ uptake by inhibiting NHE. PKC-alpha mediated roughly 42% of total PKC activity under basal conditions. PKC-alpha regulates basal gallbladder Na+ transport by way of stimulation of NHE isoform NHE-2 and inhibition of isoform NHE-3. PKC-alpha blockade reversed PKC-induced inhibition of J(Na)(ms) and 22Na+ uptake by about 45% in controls but was progressively less effective during gallstone formation. PKC-alpha contribution to total PKC activity is progressively reduced, whereas expression of PKC-alpha mRNA, and protein increases significantly during gallstone formation. We conclude that PKC-alpha regulation of gallbladder NHE becomes progressively more dysfunctional and may in part account for the increased Na+ absorption observed during gallstone formation.

Protein Kinase C Modulates Agonist-sensitive Release of Ca2+ from Internal Stores in HEK293 Cells Overexpressing the Calcium Sensing Receptor

This study examined the mechanism of Ca2+ entry and the role of protein kinase C (PKC) in Ca2+ signaling induced by activation of the calcium sensing receptor (CaR) in HEK293 cells stably expressing the CaR. We demonstrate that influx of Ca2+ following CaR activation exhibits store-operated characteristics in being associated with Ca2+ store depletion and inhibited by 2-aminoethoxydiphenyl borate. Inhibition of PKC with GF109203X, Go6983, or Go6976 and down-regulation of PKC activity enhanced the release of Ca2+ from internal stores in response to the polyvalent cationic CaR agonist neomycin, whereas activation of PKC with acute 12-O-tetradecanoylphorbol-13-acetate treatment decreased the release. In contrast, overexpression of wild type PKC-alpha or -epsilon augmented the neomycin-induced release of Ca2+ from internal stores, whereas dominant negative PKC-epsilon strongly decreased the release, but dominant negative PKC-alpha had little effect. Prolonged treatment of cells with 12-O-tetradecanoylphorbol-13-acetate effectively down-regulated immunoreactive PKC-alpha but had little effect on the expression of PKC-epsilon. Together these results indicate that diacylglycerol-responsive PKC isoforms differentially influence CaR agonist-induced release of Ca2+ from internal stores. The fundamentally different results obtained when overexpressing or functionally down-regulating specific PKC isoforms as compared with pharmacological manipulation of PKC activity indicate the need for caution when interpreting data obtained with the latter approach.

Histamine H1 receptor antagonist blocks histamine-induced proinflammatory cytokine production through inhibition of Ca 2+-dependent protein kinase C, Raf/MEK/ERK and IKK/IκB/NF-κB signal cascades

Histamine H1 receptor (H1R), a therapeutic target for alleviation of acute allergic reaction, may be also involved in mediating inflammatory responses via effects on cytokine production. However, the mechanisms whereby histamine induces cytokine production are poorly defined. In this study, we comprehensively investigated the signaling pathway involved in cytokine expression caused by histamine, using native human epidermal keratinocytes. We confirmed the expression of functional H1R by reverse transcription-polymerase chain reaction (RT-PCR), Western blotting and histamine-induced Ca 2+ elevation. Histamine induced concentration- and time-dependent production of granulocyte-macrophage-colony stimulating factor (GM-CSF), interleukin (IL)-8 and IL-6, which was completely blocked by olopatadine, an H1 antagonist. Histamine activated the phosphorylation of protein kinase C (PKC), c-Raf, mitogen-activated protein/extracellular signal-regulated kinase kinase (MEK), extracellular signal-regulated kinase (ERK), IκB kinase (IKK), inhibitory κB (IκB)-α and nuclear factor-KB (NF-κB) p65, which was inhibited by Ro-31-8220, a PKC inhibitor. Also, Ro-31-8220 significantly suppressed the expression of these cytokines. BAPTA-AM, an intracellular Ca 2+ chelator, also reduced PKC phosphorylation and cytokine expression. PD98059, a MEK inhibitor, and BAY 11-8702, an IκB-α inhibitor, reduced ERK and NF-κB cascade activation, respectively, with little effect on PKC phosphorylation. PD98059 preferentially inhibited GM-CSF production whereas BAY 11-8702 prevented IL-8 and IL-6 production. Furthermore, in addition to the above cytokines, histamine stimulated the biosynthesis and/or release of numerous keratinocyte-derived mediators, which are probably regulated by the ERK or NF-κB cascades. Our study suggests that histamine activates Ca 2+-dependent PKC isoforms that play crucial roles in the activation of Raf/MEK/ERK and IKK/IκB/NF-κB cascades, leading to up-regulation of cytokine expression. Thus, the anti-inflammatory benefit of H1 antagonists may be in part due to prevention of cytokine production.

Inhibition of Ca2+-dependent PKC isoforms unmasks ERK-dependent hypertrophic growth evoked by phenylephrine in adult ventricular cardiomyocytes.

The duration of extracellular signal-regulated kinase (ERK) activation and the ERK-dependency of hypertrophic growth differ between stimulation of alpha-adrenoceptors or angiotensin II receptors. As both receptor systems activate different protein kinase C (PKC) isoforms, we hypothesized that PKC isoforms contribute to the specific effect of alpha-adrenoceptor stimulation. Isolated adult ventricular cardiomyocytes from rats were used. Different PKC isoforms were inhibited either pharmacologically by six different PKC inhibitors or specifically downregulated by antisense oligonucleotides. ERK activation was determined by phosphorylation relative to total ERK. The rate of protein synthesis was determined by 14C-phenylalanine incorporation. The hypertrophic response of phenylephrine was inhibited in a concentration-dependent fashion by three different inhibitors of Ca2+-independent PKC isoforms (Gö6983, rottlerin, Gö6850), but not by three distinct PKC inhibitors directed preferentially against Ca2+-dependent PKC isoforms (Ro32-0432, HBDDE, Gö6976). Antisense oligonucleotides directed against PKC-alpha, -delta, or -epsilon downregulated their specific isoforms. Their corresponding sense oligonucleotides did not affect PKC isoform expression. The phenylephrine-induced increase in protein synthesis was blocked by antisense oligonucleotides directed against PKC-delta or PKC-epsilon but not PKC-alpha, confirming the pharmacological experiments. Inhibition of Ca2+-dependent PKC isoforms by HBDDE or Gö6976 converted a transient activation of ERK by phenylephrine into a sustained response. Under these conditions, phenylephrine increased protein synthesis in an ERK-dependent way. Inhibition of Ca2+-dependent PKC isoforms converts the ERK-independent effect of phenylephrine on protein synthesis into an ERK-dependent induction of protein synthesis. We conclude that co-activation of Ca2+-dependent PKC isoforms by phenylephrine contributes to the specific effect on adult ventricular cardiomyocytes from rat.

Dehydroepiandrosterone stimulates glucose uptake in human and murine adipocytes by inducing GLUT1 and GLUT4 translocation to the plasma membrane.

Dehydroepiandrosterone (DHEA) has been shown to modulate glucose utilization in humans and animals, but the mechanisms of DHEA action have not been clarified. We show that DHEA induces a dose- and time-dependent increase in glucose transport rates in both 3T3-L1 and human adipocytes with maximal effects at 2 h. Exposure of adipocytes to DHEA does not result in changes of total GLUT4 and GLUT1 protein levels. However, it does result in significant increases of these glucose transporters in the plasma membrane. In 3T3-L1 adipocytes, DHEA increases tyrosine phosphorylation of insulin receptor substrate (IRS)-1 and IRS-2 and stimulates IRS-1- and IRS-2-associated phosphatidylinositol (PI) 3-kinase activity with no effects on either insulin receptor or Akt phosphorylation. In addition, DHEA causes significant increases of cytosolic Ca(2+) concentrations and a parallel activation of protein kinase C (PKC)-beta(2). The effects of DHEA are abrogated by pretreatment of adipocytes with PI 3-kinase and phospholipase C gamma inhibitors, as well as by inhibitors of Ca(2+)-dependent PKC isoforms, including a specific PKC-beta inhibitor. Thus, DHEA increases glucose uptake in both human and 3T3-L1 adipocytes by stimulating GLUT4 and GLUT1 translocation to the plasma membrane. PI 3-kinase, phospholipase C gamma, and the conventional PKC-beta(2) seem to be involved in DHEA effects.

Ca2+-dependent Protein Kinase C Isoforms Induce Cholestasis in Rat Liver

Bile secretion is regulated by different signaling transduction pathways including protein kinase C (PKC). However, the role of different PKC isoforms for bile formation is still controversial. This study investigates the effects of PKC isoform selective activators and inhibitors on PKC translocation, bile secretion, bile acid uptake, and subcellular transporter localization in rat liver, isolated rat hepatocytes and in HepG2 cells. In rat liver activation of Ca(2+)-dependent cPKCalpha and Ca(2+)-independent PKCepsilon by phorbol 12-myristate 13-acetate (PMA, 10nmol/liter) is associated with their translocation to the plasma membrane. PMA also induced translocation of the cloned rat PKCepsilon fused to a yellow fluorescent protein (YFP), which was transfected into HepG2 cells. In the perfused liver, PMA induced marked cholestasis. The PKC inhibitors Gö6850 (1 micromol/liter) and Gö6976 (0.2 micromol/liter), a selective inhibitor of Ca(2+)-dependent PKC isoforms, diminished the PMA effect by 50 and 60%, respectively. Thymeleatoxin (Ttx,) a selective activator of Ca(2+)-dependent cPKCs, did not translocate rat PKCepsilon-YFP transfected in HepG2 cells. However, Ttx (0.5-10 nmol/liter) induced cholestasis similar to PMA and led to a retrieval of Bsep from the canalicular membrane in rat liver while taurocholate-uptake in isolated hepatocytes was not affected. Gö6976 completely blocked the cholestatic effect of Ttx but had no effect on tauroursodeoxycholate-induced choleresis. The data identify Ca(2+)-dependent PKC isoforms as inducers of cholestasis. This is mainly due to inhibition of taurocholate excretion involving transporter retrieval from the canalicular membrane.

Propofol increases pulmonary artery smooth muscle myofilament calcium sensitivity: role of protein kinase C.

Vascular smooth muscle tone is regulated by changes in intracellular free Ca2+ concentration ([Ca2+]i) and myofilament Ca2+ sensitivity. These cellular mechanisms could serve as targets for anesthetic agents that alter vasomotor tone. This study tested the hypothesis that propofol increases myofilament Ca2+ sensitivity in pulmonary artery smooth muscle (PASM) via the protein kinase C (PKC) signaling pathway. Canine PASM strips were denuded of endothelium, loaded with fura-2/AM, and suspended in modified Krebs-Ringer's buffer at 37 degrees C for simultaneous measurement of isometric tension and [Ca2+]i. The KCl (30 mm) induced monotonic increases in [Ca2+]i and tension. Verapamil, an L-type Ca2+ channel blocker, attenuated KCl-induced increases in [Ca2+]i and tension to an equal extent. In contrast, propofol attenuated KCl-induced increases in [Ca2+]i to a greater extent than concomitant changes in tension and caused an upward shift in the peak tension-[Ca2+]i relation. Increasing extracellular Ca2+ in the presence of 30 mM KCl resulted in similar increases in [Ca2+]i in control and propofol-pretreated strips, whereas concomitant increases in tension were greater during propofol administration. The Ca2+ ionophore, ionomycin (0.1 microm), increased [Ca2+]i to approximately 50% of the value induced by 60 mm KCl. Under these conditions, propofol (10, 100 microm) caused increases in tension equivalent to 11 +/- 2 and 28 +/- 3% of the increases in tension in response to 60 mM KCl, whereas [Ca2+]i was slightly decreased. Similar effects were observed in response to the PKC activator, phorbol 12-myristate 13-acetate (PMA, 1 microm). Specific inhibition of PKC with bisindolylmaleimide I before ionomycin administration decreased the propofol- and PMA-induced increases in tension and abolished the propofol- and PMA-induced decreases in [Ca2+]i. Selective inhibition of Ca2+ -dependent PKC isoforms with Gö 6976 also attenuated propofol-induced increases in tension. These results suggest that propofol increases myofilament Ca2+ sensitivity in PASM, and this effect involves the PKC signaling pathway.

Protein kinase C inhibits amyloid β‐peptide neurotoxicity by acting on members of the Wnt pathway

Current evidence supports the notion that the amyloid beta-peptide (Abeta) plays a major role in the neurotoxicity observed in the brain in Alzheimer's disease. However, the signal transduction mechanisms involved still remain unknown. In the present work, we analyzed the effect of protein kinase C (PKC) on some members of the Wnt signaling pathway and its implications for Abeta neurotoxicity. Activation of PKC by phorbol 12-myristate 13-acetate protected rat hippocampal neurons from Abeta toxicity. This effect was accomplished by inhibition of glycogen synthase kinase-3beta (GSK-3beta) activity, which led to the accumulation of cytoplasmic beta-catenin and transcriptional activation via beta-catenin/T-cell factor/lymphoid enhancer factor-1 (TCF/LEF-1) of Wnt target genes, which in the present study were engrailed-1 (en-1) and cyclin D1 (cycD1,). In contrast, inhibition of Ca2+-dependent PKC isoforms activated GSK-3beta and offered no protection from Ab neurotoxicity. Wnt-3a and lithium salts, classical activators of the Wnt pathway, mimicked PKC activation. Our results suggest that regulation of members of the Wnt signaling pathway by Ca2+-dependent PKC isoforms may be important in controlling the neurotoxic process induced by Ab.