PKCε Translocation Research Articles

Thienoquinolines as Novel Disruptors of the PKCε/RACK2 Protein–Protein Interaction

Ten protein kinase C (PKC) isozymes play divergent roles in signal transduction. Because of sequence similarities, it is particularly difficult to generate isozyme-selective small molecule inhibitors. In order to identify such a selective binder, we derived a pharmacophore model from the peptide EAVSLKPT, a fragment of PKCε that inhibits the interaction of PKCε and receptor for activated C-kinase 2 (RACK2). A database of 330 000 molecules was screened in silico, leading to the discovery of a series of thienoquinolines that disrupt the interaction of PKCε with RACK2 in vitro. The most active molecule, N-(3-acetylphenyl)-9-amino-2,3-dihydro-1,4-dioxino[2,3-g]thieno[2,3-b]quinoline-8-carboxamide (8), inhibited this interaction with a measured IC50 of 5.9 μM and the phosphorylation of downstream target Elk-1 in HeLa cells with an IC50 of 11.2 μM. Compound 8 interfered with MARCKS phosphorylation and TPA-induced translocation of PKCε (but not that of PKCδ) from the cytosol to the membrane. The compound reduced the migration of HeLa cells into a gap, reduced invasion through a reconstituted basement membrane matrix, and inhibited angiogenesis in a chicken egg assay.

Apelin Increases Cardiac Contractility via Protein Kinase Cε- and Extracellular Signal-Regulated Kinase-Dependent Mechanisms

BackgroundApelin, the endogenous ligand for the G protein-coupled apelin receptor, is an important regulator of the cardiovascular homoeostasis. We previously demonstrated that apelin is one of the most potent endogenous stimulators of cardiac contractility; however, its underlying signaling mechanisms remain largely elusive. In this study we characterized the contribution of protein kinase C (PKC), extracellular signal-regulated kinase 1/2 (ERK1/2) and myosin light chain kinase (MLCK) to the positive inotropic effect of apelin.Methods and ResultsIn isolated perfused rat hearts, apelin increased contractility in association with activation of prosurvival kinases PKC and ERK1/2. Apelin induced a transient increase in the translocation of PKCε, but not PKCα, from the cytosol to the particulate fraction, and a sustained increase in the phosphorylation of ERK1/2 in the left ventricle. Suppression of ERK1/2 activation diminished the apelin-induced increase in contractility. Although pharmacological inhibition of PKC attenuated the inotropic response to apelin, it had no effect on ERK1/2 phosphorylation. Moreover, the apelin-induced positive inotropic effect was significantly decreased by inhibition of MLCK, a kinase that increases myofilament Ca2+ sensitivity.ConclusionsApelin increases cardiac contractility through parallel and independent activation of PKCε and ERK1/2 signaling in the adult rat heart. Additionally MLCK activation represents a downstream mechanism in apelin signaling. Our data suggest that, in addition to their role in cytoprotection, modest activation of PKCε and ERK1/2 signaling improve contractile function, therefore these pathways represent attractive possible targets in the treatment of heart failure.

M2 Receptors Exert Analgesic Action on DRG Sensory Neurons by Negatively Modulating VR1 Activity

The peripheral application of the M2 cholinergic agonist arecaidine on sensory nerve endings shows anti-nociceptive properties. In this work, we analyze in vitro, the mechanisms downstream M2 receptor activation causing the analgesic effects, and in vivo the effects produced by M2 agonist arecaidine administration on nociceptive responses in a murine model of nerve growth factor (NGF)-induced pain. Cultured DRG neurons treated with arecaidine showed a decreased level of VR1 and SP transcripts. Conversely, we found an increased expression of VR1 and SP transcripts in DRG from M2/M4(-/-) mice compared to WT and M1(-/-) mice, confirming the inhibitory effect in particular of M2 receptors on SP and VR1 expression. Patch-clamp experiments in the whole-cell configuration showed that arecaidine treatment caused a reduction of the fraction of capsaicin-responsive cells, without altering the mean capsaicin-activated current in responsive cells. We also demonstrated that arecaidine prevents PKCϵ translocation to the plasma membrane after inflammatory agent stimulation, mainly in medium-small sensory neurons. Finally, in mice, we have observed that intraperitoneal injection of arecaidine reduces VR1 expression blocking hyperalgesia and allodynia caused by NGF intraplantar administration. In conclusion, our data demonstrate that in vivo M2 receptor activation induces desensitization to mechanical and heat stimuli by a down-regulation of VR1 expression and by the inhibition of PKCϵ activity hindering its translocation to the plasma membrane, as suggested by in vitro experiments.

HepaCAM inhibits clear cell renal carcinoma 786-0 cell proliferation via blocking PKCε translocation from cytoplasm to plasma membrane

Hepatocyte cell adhesion molecule (HepaCAM) plays a crucial role in tumor progression and has been recognized as a novel tumor suppressor gene. The high protein expression level of protein kinase Cε (PKCε) has been discovered in many tumor types. In the present study, we determined HepaCAM and PKCε protein levels in human clear cell renal cell carcinoma (ccRCC) tissues and analyzed the correlation between them. We observed an inverse relationship in the expression of HepaCAM and PKCε in ccRCC and adjacent normal tissues. In ccRCC tissue, HepaCAM expression was undetectable while PKCε expression was high; the opposite was found in the adjacent normal tissue. Western blot analysis demonstrated that PKCε cytosolic protein levels increased while plasma membrane protein levels decreased without any change in total protein following infection of the ccRCC cell line 786-0 with adenovirus-GFP-HepaCAM (Ad-GFP-HepaCAM). Moreover, the application of Ad-GFP-HepaCAM combined with a PKCε-specific translocation inhibitor (εV1-2) effectively inhibited 786-0 cell growth. Ad-mediated expression of HepaCAM in 786-0 cells reduced the levels of phosphorylated AKT and cyclin D1 and inhibited cell proliferation. In summary, our studies point to interesting connections between HepaCAM and PKCε in tissues and in vitro. HepaCAM may prevent the translocation of PKCε from cytosolic to particulate fractions, resulting in the inhibition of 786-0 cell proliferation. Therapeutic manipulation of these novel protein targets may provide new ways of treating ccRCC.

Mediation of dopamine D2 receptors activation in post-conditioning-attenuated cardiomyocyte apoptosis

The physiological and pathological roles of dopamine D2 receptors (DR2) in the regulation of cardiovacular functions have been recognized. DR2 activation protects hypoxia/reoxygenation (H/R)-induced cardiomyocyte injury and apoptosis, and ischemic post-conditioning (PC) plays a critical role in cardioprotection as well; however the involvement of the DR2 activation in the PC-induced cardioprotection is unknown. In the present study, we found that the H/R increased the expressions of DR2 mRNA and protein in cardiomyocytes, which were significantly enhanced by PC. Bromocriptine (Bro, a DR2 agonist) further increased DR2 expression, but Haloperidol (Hal, a DR2 antagonist) reversed the Bro-induced DR2 expressions. PC protected against H/R-induced apoptosis, the rise of [Ca2+]i, the expressions of cleaved caspase-3 and -9, release of cytochrome c, and mPTP opening. In addition, PC counteracted the reduction of cell viability caused by H/R, increased the phosphorylation of ERK1/2, PI3K, Akt, GSK-3β and mitochondrial membrane potential. PC further increased Bcl-2 expression, promoted PKC-ε translocation to cell membrane, and activated the mitochondrial ATP-sensitive K channels (mKATP). Bro further enhanced the cardioprotective roles of PC, but Hal reversed these effects of Bro. Meanwhile, we found that DR2 was expressed in cell membrane and interacted with PKC-ε in PC. In conclusion, these results suggest that PC attenuates cardiomyocyte apoptosis via inhibition of mPTP opening by DR2-mediated activation of ERK1/2, PI3K–Akt–GSK-3β and PKC-ε–mKATP. These findings provide a novel target for the treatment of ischemic cardiomyopathy.

Effect of mitochondrial ATP-sensitive potassium channel opening on the translocation of protein kinase C epsilon in adult rat ventricular myocytes.

This study aimed to investigate the effects of mitochondrial ATP-sensitive potassium (MitoKATP) channel opening on the translocation of protein kinase C epsilon (PKCε). In addition, we aimed to determine the relationship between PKCε translocation and the production of reactive oxygen species (ROS). PKCε protein expression in cultured adult rat ventricular myocytes was investigated by immunofluorescence and Western blotting. Diazoxide (DZ), a selective MitoKATP channel activator, caused a significant translocation to myofibrillar-like structures in cultured adult rat ventricular myocytes. N-2-Mercaptopropionylglycine, a free radical scavenger, could partially inhibit the translocation of PKCε induced by DZ. By contrast, chelerythrine, a selective PKC inhibitor, could completely block the translocation of PKCε induced by DZ. The opening of MitoKATP channels might activate and cause PKCε to translocate into myofibrillar-like structures. PKCε activation occurred downstream of the MitoKATP channel, possibly as a result of ROS production that occurred after the MitoKATP channels opened.

23 The Role of AMPK and PKCε in the Attenuation of ET-1 Induced Cardiomyocyte Hypertrophy by RIC Human Serum

Rationale We have previously shown that serum from healthy volunteers who have undergone Remote Ischaemic Conditioning (RIC) reduces hypertrophy in rat cardiomyoblasts. Here we investigated the roles of AMPK and PKCe in this process. Methodology Blood was taken from healthy volunteers after 3 cycles of 5 min of upper arm cuff inflation/deflation and the serum applied to H9c2 cardiomyoblasts in culture. Cells were treated with endothelin-1 (ET-1) to stimulate hypertrophy, and cell area determined using immunofluorescence at 48 h. The role of AMPK-signalling in the anti-hypertrophic effect of RIC-serum was probed using the AMPK inhibitor compound-C and AMPK phosphorylation and PKCe translocation were analysed using Western blots. Results ET-1 increased cell surface area by >45% from 1.47 x 104 ± 0.08 μm 2 in control cells to 2.14 x 104 ± 0.09 μm 2 in ET-1 treated cells (n = 8, >1000 cells per treatment, p 2 (p 2 (p 2 ; ns). RIC-serum caused a transient translocation of PKCe after 30 min (76.3%), which dissipated after 48 h to be replaced by a significant increase in cytosolic PKCe (3.4 ± 0.5 fold, p Conclusion Our data suggests that PKCe and AMPK-signalling are involved in the anti-hypertrophic action of RIC-serum.

Interleukin 1ß Modulates the Ventricular L-Type Calcium Current Through ROS Signalling

Introduction & Objective: Several lines of evidence suggest that cytokines are potent mediators of cardiac remodelling including hypertrophy and generation of reactive oxidative species (ROS). Increases in serum cytokines, notably necrosis factor-alpha (TNFα) and interleukin-1β (IL-1β), have been reported in patients suffering from arrhythmias and heart failure. We previously showed that TNFα reduces the repolarizing K+ current in mice; however the effects of cytokines on other cardiac ionic currents and the mechanism by which these effects are mediated remain incompletely understood. Thus, our objective was to investigate the role of TNFα and IL-1β on L-type calcium current (ICaL), a key player in cardiomyocyte excitation-contraction coupling.Methods: Cultured neonatal mouse ventricular myocytes were treated with a pathophysiological concentration (30 pg/mL) of TNFα and IL-1β for 24H. ICaL was recorded using the voltage-clamp technique.Results: The density of ICaL (pA/pF) was decreased by 36% in IL-1β-treated myocytes compared to controls whereas TNFα had no effect. The CaV1.2 mRNA expression was unchanged by IL-1β treatment however there was a significant increase in intracelluar ROS. The antioxidant N-acetyl-L-cystein reduced ROS and restored ICaL density. Furthermore, Western blot experiments reveal that IL-1β increases PKCe membrane translocation. Treatment with a specific PKCe translocation inhibitor or pertussis toxin, the Gαi inhibitor, also reversed the effects of IL-1β.Conclusion: IL-1β significantly decreased ICaL density without affecting the expression of Cav1.2. Our results suggest that IL-1β mediates its effects by ROS signalling implicating Gαi and subsequently PKCe activation. These findings could contribute to explain the role of IL-1β in the development of arrhythmia and heart failure.

Amelioration in Hepatic Insulin Sensitivity by Reduced Hepatic Lipid Accumulation at Short-Term After Roux-en-Y Gastric Bypass Surgery in Type 2 Diabetic Rats

Previous studies showed that early after Roux-en-Y gastric bypass (RYGB), there is a remarkable improvement in type 2 diabetes, which is characterized by insulin resistance. This study aims to gain insight into the underlying mechanisms of this effect. We determined the acute effects of RYGB on hepatic and peripheral insulin sensitivity. A rat model of type 2 diabetes was established using high-fat diet combined with streptozotocin (30 mg/kg, ip). Animals were divided into four groups: diabetic, diabetic RYGB, diabetic RYGB sham, and control rats. Hyperinsulinemic-euglycemic clamps with tracer infusion were completed at 2 weeks postoperatively to assess insulin sensitivity. Triglyceride concentration in liver and muscle tissues was determined. Protein kinase C (PKC) membrane translocation, protein expression of phospho-c-Jun NH2-terminal kinase (JNK), and phospho-IκB kinase β (IKKβ) were assessed with western blot. Malondialdehyde (MDA) and superoxide dismutase (SOD) activities in the liver were also measured. RYGB surgery significantly improved hepatic insulin sensitivity index and decreased hepatic triglyceride concentration (P < 0.05), without an improvement in peripheral insulin sensitivity. Membrane translocation of PKC-ε, PKC-δ, and PKC-θ; the ratio of MDA to SOD; and the expression of p-JNK and p-IKKβ in the liver were lower in the diabetic RYGB group than in the diabetic group. Diabetes remission was induced at short term after RYGB. The improvement of hepatic tissue lipotoxicity decreased the activation of certain PKC isoforms, the activity of JNK and IKK inflammatory signaling pathways, and the degree of oxidative stress. Furthermore, the hepatic insulin sensitivity was ameliorated, which is possibly a mechanism for early diabetes remission.

Hemorrhagic preconditioning improves vascular reactivity after hemorrhagic shock by activation of PKCα and PKCε via the adenosine A1 receptor in rats

Our previous study demonstrated that protein kinase C (PKC) has an important protective role on vascular reactivity and calcium sensitivity after shock. Here, we investigated if hemorrhagic preconditioning could lessen shock-induced vascular hyporeactivity by activating PKC. Using hemorrhagic-shocked rats, the protective effects of different extents of hemorrhagic preconditioning on vascular reactivity and calcium sensitivity; the roles of PKCα, PKCε, and adenosine in this process; as well as hemorrhagic preconditioning-induced systemic effects were observed. Hemorrhage preconditioning (particularly hemorrhage involving 5% of the total estimated blood volume implemented 30 minutes before shock) significantly improved vascular reactivity and calcium sensitivity after shock. Hemorrhage preconditioning enhanced the translocation of PKCα and PKCε from the cytoplasm to the membrane and increased the blood concentration of adenosine after shock. Antagonists of PKCα, PKCε, and the adenosine A1 receptor abolished the hemorrhagic preconditioning-induced protective effects on vascular reactivity and calcium sensitivity. The adenosine A1 receptor antagonist eliminated hemorrhagic preconditioning-induced translocation of PKCα and PKCε. Hemorrhagic preconditioning could significantly increase survival, improve hemodynamic parameters, and increase the blood flow and mitochondrial respiratory function of the liver and kidney in hemorrhagic-shock rats. Hemorrhagic preconditioning could induce the protection of vascular reactivity and calcium sensitivity after hemorrhagic shock through the adenosine-adenosine A1 receptor-PKCα and PKCε signaling pathway and could bring further beneficial systemic effects in hemorrhagic-shock rats.

Involvement of dopamine D2 receptors activation in ischemic post-conditioning-induced cardioprotection through promoting PKC-ε particulate translocation in isolated rat hearts

Dopamine D2 receptors (DR2) are important regulators in many organs, including cardiac system. Protein kinase C (PKC) activation and translocation is associated with cardioprotection against ischemic post-conditioning (PC); however, the regulatory role of DR2 during this process has been unknown. This study hypothesized that the prevention of cardiomyocyte damage by DR2 activation is associated with PKC translocation to the cell membrane. In the present study, we found that the ischemia/reperfusion (I/R) increased the expressions of DR2 mRNA and protein, which were further enhanced by PC. Bromocriptine (DR2 agonist) up-regulated the PC-induced DR2 expressions, and Haloperidol (DR2 antagonist) reversed the increase of DR2 expressions by Bromocriptine. PC reduced I/R-induced cardiomyocytes damage, apoptosis and myocardial infarct size, and improved cardiac function. Compared with PC, Bromocriptine further enhanced the cardioprotective roles of PC, but Haloperidol canceled the protection effect of Bromocriptine. PC up-regulated PKC-ε translocation in the particulate fraction, which was further strengthened by Bromocriptine but canceled by Haloperidol. In the cytosolic fraction, the changes of the PKC-ε translocation were opposite to the particulate fraction. These findings suggest that DR2 activation provides cardioprotection via promoting PC-induced translocation of PKC-ε.

Investigation of signalling cascades induced by neurotrophic synaptolepis factor K7 reveals a critical role for novel PKCε

This study elucidates signalling cascades involved in the neurotrophic effects induced by an active compound of Synaptolepis kirkii, a plant that is used against snakebites and for treatment of epilepsy. The active compound of this plant, synaptolepis factor K7 (K7), is suggested to exert anti-tumoral and neurotrophic actions via modulation of PKC. In SH-SY5Y cells synthesis of the neuronal marker growth-associated protein 43 was increased upon 48h treatment with K7. Immunofluorescent staining of neurites revealed an increased neurite formation by synaptolepis factor K7. Short-term signal transduction events were followed at the level of extracellular-regulated kinase phosphorylation. Extracellular-regulated kinase (ERK) phosphorylation was transiently increased upon stimulation with synaptolepis factor K7 (300nM) with a maximal effect at 30min. Use of the general PKC inhibitor bisindolylmaleimide I blocked the K7-induced ERK phosphorylation suggesting involvement of PKC. Conversely, inhibition of conventional PKCs, α, β and γ by treatment with Go6976 did not inhibit ERK phosphorylation up to 1μM. Use of a specific-PKCε translocation inhibitor peptide or RNAi-mediated knockdown of PKC-epsilon (ε) abolished the K7-induced ERK phosphorylation implicating PKCε in K7 function. This was confirmed by the observed increase in PKCε translocation and autophosphorylation induced by the compound. These data show that synaptolepis factor K7 induces neuronal differentiation of SH-SY5Y cells concomitant with a transient increase in ERK phosphorylation that is mediated by activation of PKCε.

Novel Association between Vasoactive Intestinal Peptide and CRTH2 Receptor in Recruiting Eosinophils: A POSSIBLE BIOCHEMICAL MECHANISM FOR ALLERGIC EOSINOPHILIC INFLAMMATION OF THE AIRWAYS

We explored the relation between vasoactive intestinal peptide (VIP), CRTH2, and eosinophil recruitment. It is shown that CRTH2 expression by eosinophils from allergic rhinitis (AR) patients and eosinophil cell line (Eol-1 cells) was up-regulated by VIP treatment. This was functional and resulted in exaggerated migratory response of cells against PGD2. Nasal challenge of AR patients resulted in a significant increase of VIP contents in nasal secretion (ELISA), and the immunohistochemical studies of allergic nasal tissues showed significant expression of VIP in association with intense eosinophil recruitment. Biochemical assays showed that VIP-induced eosinophil chemotaxis from AR patients and Eol-1 cells was mediated through the CRTH2 receptor. Cell migration against VIP was sensitive to protein kinase C (PKC) and protein kinase A (PKA) inhibition but not to tyrosine kinase or p38 MAPK inhibition or calcium chelation. Western blot demonstrated a novel CRTH2-mediated cytosol-to-membrane translocation of PKC-ε, PKC-δ, and PKA-α, -γ, and -IIαreg in Eol-1 cells upon stimulation with VIP. Confocal images and FACS demonstrated a strong association and co-localization between VIP peptide and CRTH2 molecules. Further, VIP induced PGD2 secretion from eosinophils. Our results demonstrate the first evidence of association between VIP and CRTH2 in recruiting eosinophils.

ARF1-regulated coatomer directs the steady-state localization of protein kinase C epsilon at the Golgi apparatus

Protein kinase C epsilon (PKCε) contributes to multiple signaling pathways affecting human disease. The function of PKCε requires it to undergo changes in subcellular distribution in response to signaling events. While the mechanisms underlying this translocation are incompletely understood, it involves the receptor for activated C kinase protein (RACK2/β′-COP). This receptor also functions as a vesicle coat protein in the secretory pathway where it is regulated by the small GTP-binding protein ADP-ribosylation factor, ARF1. We inhibited ARF1 activation to test the requirement for RACK2/β′-COP in PKCε localization in NIH3T3 fibroblasts. We found that steady-state localization of PKCε at the Golgi complex requires ARF1-regulated RACK2/β′-COP function. By contrast, we did not observe any defects in phorbol ester-induced translocation when ARF1 was inhibited. We also found that PKCε bound to isolated membranes through two distinct mechanisms. One mechanism was dependent upon RACK2/β′-COP while a second was RACK2/β′-COP-independent and stimulated by phorbol esters. Finally, we show that RACK2/β′-COP affects the subcellular distribution of a constitutively active form of PKCε, in a manner similar to what we observed for wild-type PKCε. Together, our data support a role for RACK2/β′-COP in the steady-state localization of PKCε at the Golgi apparatus, which may be independent of its role during PKCε translocation to the cell surface.

Ca++/CaMKII switches nociceptor‐sensitizing stimuli into desensitizing stimuli

Many extracellular factors sensitize nociceptors. Often they act simultaneously and/or sequentially on nociceptive neurons. We investigated if stimulation of the protein kinase C epsilon (PKCε) signaling pathway influences the signaling of a subsequent sensitizing stimulus. Central in activation of PKCs is their transient translocation to cellular membranes. We found in cultured nociceptive neurons that only a first stimulation of the PKCε signaling pathway resulted in PKCε translocation. We identified a novel inhibitory cascade to branch off upstream of PKCε, but downstream of Epac via IP3-induced calcium release. This signaling branch actively inhibited subsequent translocation and even attenuated ongoing translocation. A second 'sensitizing' stimulus was rerouted from the sensitizing to the inhibitory branch of the signaling cascade. Central for the rerouting was cytoplasmic calcium increase and CaMKII activation. Accordingly, in behavioral experiments, activation of calcium stores switched sensitizing substances into desensitizing substances in a CaMKII-dependent manner. This mechanism was also observed by in vivo C-fiber electrophysiology corroborating the peripheral location of the switch. Thus, we conclude that the net effect of signaling in nociceptors is defined by the context of the individual cell's signaling history.

Circulation Research Thematic Synopsis

<i>Circulation Research</i> Thematic Synopsis

Pinacidil Pretreatment Improves Vascular Reactivity After Shock Through PKCα and PKC[Latin Small Letter Open E] in Rats

We investigated the beneficial effect of pinacidil pretreatment on vascular reactivity, calcium sensitivity, and animal survival after hemorrhagic shock, its relationship to protein kinase Cα (PKCα), protein kinase Cε (PKCε), and adenosine. Using hemorrhagic shock rats, the protective effects of different extents of pinacidil pretreatment on vascular reactivity and in which the roles of PKCα, PKCε, and adenosine were observed. Pinacidil pretreatment significantly improved shock-induced decrease of vascular reactivity of superior mesenteric artery, which was antagonized by the PKCα antagonist Gö-6976 (5 × 10 mole/L) and PKCε pseudosubstrate inhibitory peptide (1 × 10 mole/L). Pinacidil pretreatment induced the translocation of PKCα and PKCε from the cytoplasm to the membrane. This translocation of PKCα and PKCε was eliminated by adenosine A1 receptor antagonist DPCPX (1 × 10 mole/L). As compared with simple fluid resuscitation, combination with pinacidil pretreatment significantly improved the vascular reactivity and survival rate of hemorrhagic-shocked rats. These results suggested that pinacidil pretreatment could induce good protective effects on vascular reactivity and calcium sensitivity after hemorrhagic shock mainly through the activation of PKCα and PKCε via adenosine A1 receptor, and this protective effect made an important contribution to the overall outcome of shock therapy.

Crosstalk between adenosine A1 and β1‐adrenergic receptors regulates translocation of PKCε in isolated rat cardiomyocytes

Adenosine A(1) receptor (A(1)R)-induced translocation of PKCε to transverse (t) tubular membranes in isolated rat cardiomyocytes is associated with a reduction in β(1)-adrenergic-stimulated contractile function. The PKCε-mediated activation of protein kinase D (PKD) by endothelin-1 is inhibited by β(1)-adrenergic stimulated protein kinase A (PKA) suggesting a similar mechanism of A(1)R signal transduction modulation by adrenergic agonists may exist in the heart. We have investigated the influence of β(1)-adrenergic stimulation on PKCε translocation elicited by A(1)R. Immunofluorescence imaging and Western blotting with PKCε and β-COP antibodies were used to quantify the co-localization of PKCε and t-tubular structures in isolated rat cardiomyocytes. The A(1)R agonist CCPA increased the co-localization of PKCε and t-tubules as detected by imaging. The β(1)-adrenergic receptor agonist isoproterenol (ISO) inhibited this effect of CCPA. Forskolin, a potent activator of PKA, mimicked, and H89, a pharmacological PKA inhibitor, and PKI, a membrane-permeable PKA peptide PKA inhibitor, attenuated the negative effect of ISO on the A(1)R-mediated PKCε translocation. Western blotting with isolated intact hearts revealed an increase in PKCε/β-COP co-localization induced by A(1)R. This increase was attenuated by the A(1)R antagonist DPCPX and ISO. The ISO-induced attenuation was reversed by H89. It is concluded that adrenergic stimulation inhibits A(1)R-induced PKCε translocation to the PKCε anchor site RACK2 constituent of a coatomer containing β-COP and associated with the t-tubular structures of the heart. In that this translocation has been previously associated with the antiadrenergic property of A(1)R, it is apparent that the interactive effects of adenosine and β(1)-adrenergic agonists on function are complex in the heart.

The G protein Gα11 is essential for hypertrophic signalling in diabetic myocardium

Aims/hypothesisPathological cardiac hypertrophy is an early phenotype in both types 1 and 2 diabetes. The primary stimulus for hypertrophic growth in diabetes is yet unknown and may involve neurohumoral stimulation of Gq-coupled receptors as well as direct glucose-dependent mechanisms. To discriminate between these hypertrophic stimuli we analyzed hypertrophic signalling pathways in wildtype and Gα11-knockout mice. MethodsExperimental diabetes was induced in wildtype and knockout mice by intraperitoneal injection of streptozotocin. 8weeks after induction of diabetes myocardial function and structure was assessed by echocardiography before sacrifice. To identify prohypertrophic signalling pathways expression and translocation of protein kinase C isoforms α, βII, δ, ε and ζ were analyzed by immunohistochemical staining and immunoblot analysis after tissue fractionation. Changes in calcineurin signalling were identified by immunoblot analysis and functional assays. Expression levels of transcription factors GATA4 and NF-κB were quantified by real-time RT-PCR. ResultsDiabetic wildtype mice developed myocardial hypertrophy with preserved cardiac function. Calcineurin signalling was not different between the two groups. However, diabetic wildtype mice showed increased protein levels of PKC-α and PKC-ζ, translocation of PKC-α, -δ and -ε to cellular membranes and higher levels of NF-κB expression. In contrast, diabetic Gα11-knockout mice showed no altered phenotype and no changes in NF-κB or PKC expression, although translocation of PKC-ε occurred as in wildtypes. ConclusionsGα11 is essential for the development of cardiac hypertrophy in type 1-diabetes. Stimulation of hypertrophic signalling through PKC-α, PKC-δ, PKC-ζ, and NF-κB appears to be receptor-dependent, whereas PKC-ε is activated by hyperglycemia, independent of Gα11.

Apolipoprotein E3 (ApoE3) but Not ApoE4 Protects against Synaptic Loss through Increased Expression of Protein Kinase Cϵ

Synaptic loss is the earliest pathological change in Alzheimer disease (AD) and is the pathological change most directly correlated with the degree of dementia. ApoE4 is the major genetic risk factor for the age-dependent form of AD, which accounts for 95% of cases. Here we show that in synaptic networks formed from primary hippocampal neurons in culture, apoE3, but not apoE4, prevents the loss of synaptic networks produced by amyloid β oligomers (amylospheroids). Specific activators of PKCε, such as 8-(2-(2-pentyl-cyclopropylmethyl)-cyclopropyl)-octanoic acid methyl ester and bryostatin 1, protected against synaptic loss by amylospheroids, whereas PKCε inhibitors blocked this synaptic protection and also blocked the protection by apoE3. Blocking LRP1, an apoE receptor on the neuronal membrane, also blocked the protection by apoE. ApoE3, but not apoE4, induced the synthesis of PKCε mRNA and expression of the PKCε protein. Amyloid β specifically blocked the expression of PKCε but had no effect on other isoforms. These results suggest that protection against synaptic loss by apoE is mediated by a novel intracellular PKCε pathway. This apoE pathway may account for much of the protective effect of apoE and reduced risk for the age-dependent form of AD. This finding supports the potential efficacy of newly developed therapeutics for AD.