PKA Phosphorylation Research Articles

Apigenin Improves Hypertension and Cardiac Hypertrophy Through Modulating NADPH Oxidase-Dependent ROS Generation and Cytokines in Hypothalamic Paraventricular Nucleus.

Apigenin, identified as 4', 5, 7-trihydroxyflavone, is a natural flavonoid compound that has many interesting pharmacological activities and nutraceutical potential including anti-inflammatory and antioxidant functions. Chronic, low-grade inflammation and oxidative stress are involved in both the initiation and progression of hypertension and hypertension-induced cardiac hypertrophy. However, whether or not apigenin improves hypertension and cardiac hypertrophy through modulating NADPH oxidase-dependent reactive oxygen species (ROS) generation and inflammation in hypothalamic paraventricular nucleus (PVN) has not been reported. This study aimed to investigate the effects of apigenin on hypertension in spontaneously hypertensive rats (SHRs) and its possible central mechanism of action. SHRs and Wistar-Kyoto (WKY) rats were randomly assigned and treated with bilateral PVN infusion of apigenin or vehicle (artificial cerebrospinal fluid) via osmotic minipumps (20μg/h) for 4weeks. The results showed that after PVN infusion of apigenin, the mean arterial pressure (MAP), heart rate, plasma norepinephrine (NE), Beta 1 receptor in kidneys, level of phosphorylation of PKA in the ventricular tissue and cardiac hypertrophy, perivascular fibrosis, heart level of oxidative stress, PVN levels of oxidative stress, interleukin 1β (IL-1β), interleukin 6 (IL-6), iNOS, monocyte chemotactic protein 1 (MCP-1), tyrosine hydroxylase (TH), NOX2 and NOX4 were attenuated and PVN levels of interleukin 10 (IL-10), superoxide dismutase 1 (Cu/Zn-SOD) and the 67-kDa isoform of glutamate decarboxylase (GAD67) were increased. These results revealed that apigenin improves hypertension and cardiac hypertrophy in SHRs which are associated with the down-regulation of NADPH oxidase-dependent ROS generation and inflammation in the PVN.

197-LB: The Regulation of HSL by Macrophage Migration Inhibitory Factor (MIF) Contributes to Adipocyte Hypertrophy and Development of Obesity

Inflammation is associated with lipolysis in adipose tissue. HSL is a key enzyme in mediating lipolysis through augmenting hydrolysis of triglycerides and diacylglycerides. Our study found that the procytokine MIF has both transcriptional and non-transcriptional effects on mediating HSL through activating AMPK signaling pathway. The activated AMPK downregulates HSL gene expression in adipocytes. It also upregulates a phosphorylation of HSL Ser565, an inhibitory phosphorylation site, leading to the reduction of HSL active site, Ser563 phosphorylation. These mechanisms are operative in either transgenic overexpressed MIF mouse models or WT mice with high circulating MIF levels following a high caloric diet feeding (HFD). The MIF/AMPK/HSL axis regulates the development of adipocyte hypertrophy and obesity. We also confirm that high plasma MIF levels are associated with decreased adipose HSL gene expression in human obesity. Interestingly, in Mif-/-mice, HSL gene expression is also inhibited, suggesting that MIF genotype is important to maintain adipose HSL expression. Following HFD feeding, Mif-/-mice demonstrates higher Akt and lower PKA phosphorylation, which further decrease HSL activation and exacerbate obesity compared to WT. Thus, HSL regulation by MIF may be an important molecular mechanism in the development of obesity.View largeDownload slideView largeDownload slide DisclosureL. Chen: None. Y. Huang: None. L. Li: None. H. Zabihi: None. Y. Qi: None. L. Leng: None. G. Sun: None. R. Bucala: None. D. Qi: None.FundingNational Sciences and Engineering Research Council of Canada (RGPIN-2017-04542); Canadian Institutes of Health Research (PJT-156116)

Maternal omega-3 intake differentially affects the endocannabinoid system in the progeny`s neocortex and hippocampus: Impact on synaptic markers

Omega-3 (n-3) polyunsaturated fatty acids (PUFA) and the endocannabinoid system (ECS) modulate several functions through neurodevelopment including synaptic plasticity mechanisms. The interplay between n-3PUFA and the ECS during the early stages of development, however, is not fully understood. This study investigated the effects of maternal n-3PUFA supplementation (n-3Sup) or deficiency (n-3Def) on ECS and synaptic markers in postnatal offspring. Female rats were fed with a control, n-3Def, or n-3Sup diet from 15 days before mating and during pregnancy. The cerebral cortex and hippocampus of mothers and postnatal 1-2 days offspring were analyzed. In the mothers, a n-3 deficiency reduced CB1 receptor (CB1R) protein levels in the cortex and increased CB2 receptor (CB2R) in both cortex and hippocampus. In neonates, a maternal n-3 deficiency reduced the hippocampal CB1R amount while it increased CB2R. Additionally, total GFAP isoform expression was increased in both cortex and hippocampus in neonates of the n-3Def group. Otherwise, maternal n-3 supplementation increased the levels of n-3-derived endocannabinoids, DHEA and EPEA, in the cortex and hippocampus and reduced 2-arachidonoyl-glycerol (2-AG) concentrations in the cortex of the offspring. Furthermore, maternal n-3 supplementation also increased PKA phosphorylation in the cortex and ERK phosphorylation in the hippocampus. Synaptophysin immunocontent in both regions was also increased. In vitro assays showed that the increase of synaptophysin in the n-3Sup group was independent of CB1R activation. The findings show that variations in maternal dietary omega-3 PUFA levels may impact differently on the ECS and molecular markers in the cerebral cortex and hippocampus of the progeny.

Elevated MAOA impairs intracellular β 1 ‐adrenegic signaling and myocardial contractility in heart failure

Rationale: We recently identified a pool of β1ARs at the sarcoplasmic reticulum (SR) senses cytosolic catecholamines and promotes cardiac contractility. Monoamine oxidase A (MAOA), the major enzyme metabolizing myocardial catecholamines is upregulated in heart failure (HF) together with blunted β1AR signaling. However, the regulation of intracellular β1ARs signaling in HF remains unknown. Objective To uncover the role of MAOA in the spatiotemporal regulation of intracellular β1AR signaling and cardiac contractility. Methods and Results Echocardiogram analysis showed that cardiac-specific deletion (cKO) or pharmacological inhibition of MAOA did not affect the baseline cardiac output but significantly amplified the inotropy of catecholamine (epinephrine) in vivo. Enzyme immunoassay revealed elevated catecholamine concentrations and cAMP/PKA activities in MAOA-cKO mice hearts compared with their flox/flox (FF) littermates. We measured the subcellular-localized β1AR in living cardiomyocytes using PKA biosensors targeted at SR or plasma membrane (PM). Compared with FF cardiomyocytes, MAOA-cKO exhibited an increased PKA activity at SR, but not PM upon epinephrine stimulation. Consistently, phosphorylation of phospholamban (PLB), a major target of SR-localized β1AR signals, was enhanced by MAOA-cKO. More importantly, deleting MAOA increased the response of calcium transients and contractility to epinephrine. Blocking the entry of catecholamines by organic cation transporter 3 (OCT3) inhibitor abolished the differential effects in MAOA-cKO vs FF. Similarly, the effect of MAOA-cKO on intracellular β1AR/PKA/PLB signaling and cardiac excitation-contraction coupling (EC-coupling) was recapitulated by pharmacological inhibition of MAOA. In contrast, overexpression of MAOA in cardiomyocytes suppressed epinephrine-induced PKA activation at the SR, PLB phosphorylation and contractility. Furthermore, the increased expression of MAOA in failing cardiomyocytes suppressed activation of intracellular β1AR and attenuated PKA phosphorylation of PLB and inotropic responses. Inhibition of MAOA restored intracellular β1AR signaling and EC coupling in failing cardiomyocytes, and rescued the inotropy response in HF mice. Conclusion MAOA modulates catecholamine-promoted EC coupling via tuning the intracellular β1AR signaling and PKA phosphorylation of PLB. Inhibition of MAOA restores β1AR/PKA signaling at the SR and cardiac contractility in HF.

Cardiovascular and Renal Actions of the PDZ protein SAP97

Synapse Associated Protein-97 (SAP97) is a PDZ protein that is expressed in the CNS as well as in peripheral organs. SAP97 is a major compartmentalizing protein involved in organizing signaling complexes. We investigated the role of SAP97 in anterograde translocation of beta1-adrenergic receptors (beta1-AR) in adult rat ventricular myocytes (ARVM) and in translocation of aquaporin-2 forming channels in kidney cells. Interactions between SAP97 and other proteins are mediated by binding of one of its three PDZ domains with type-1 PDZ binding motifs in target proteins. We determined that SAP97 forms a ternary complex with the beta1-AR and with A-kinase anchoring protein 79/150 (AKAP5)-PKA, which targets the beta1-AR to AKAP-PKA. Activation of the beta1-AR by agonists promotes induces PKA phosphorylation of the beta1-AR at serine312 in the third intracellular loop and its internalization to endosomes. This phospho-serine along with the type- PDZ binding motif in the C-tail of the beta1-AR serve as trafficking barcodes which promote the recycling of the agonist-internalized beta1-AR back to the membrane. Similarly, SAP97 binds to AQP2 via its type1-PDZ binding motif and to an unidentified AKAP-PKA complex. Exposing renal collecting cells to vasopressin, promotes the phosphorylation of AQP2 on serine256 in the C-tail, which is a primary signal for translocating cytoplasmic AQP2 storage vesicles to the apical membrane. These two pathways were dependent on targeting these trafficking proteins to an AKAP-PKA complex via SAP97. In this regard we verified that anterograde translocation of beta1-AR and AQP2 was dependent on their type-1 PDZs, and on the SAP97-anchored complex to faithfully phosphorylate a specific trafficking serine. In addition, we determined that SAP97 co-localized with beta1-AR in ARVM and with AQP2 in the kidneys, and knockdown of SAP97 inhibited anterograde translocation of these proteins, without affecting their retrograde internalization. Here in we describe these pathways and will show that SAP97 provides a novel paradigm to decipher trafficking events localized around cis-acting barcodes and their trans-acting proteins that might ultimately be used for therapeutic advantage.

Cardiac MyBP-C phosphorylation regulates the Frank-Starling relationship in murine hearts.

The Frank-Starling relationship establishes that elevated end-diastolic volume progressively increases ventricular pressure and stroke volume in healthy hearts. The relationship is modulated by a number of physiological inputs and is often depressed in human heart failure. Emerging evidence suggests that cardiac myosin-binding protein-C (cMyBP-C) contributes to the Frank-Starling relationship. We measured contractile properties at multiple levels of structural organization to determine the role of cMyBP-C and its phosphorylation in regulating (1) the sarcomere length dependence of power in cardiac myofilaments and (2) the Frank-Starling relationship in vivo. We compared transgenic mice expressing wild-type cMyBP-C on the null background, which have ∼50% phosphorylated cMyBP-C (Controls), to transgenic mice lacking cMyBP-C (KO) and to mice expressing cMyBP-C that have serine-273, -282, and -302 mutated to aspartate (cMyBP-C t3SD) or alanine (cMyBP-C t3SA) on the null background to mimic either constitutive PKA phosphorylation or nonphosphorylated cMyBP-C, respectively. We observed a continuum of length dependence of power output in myocyte preparations. Sarcomere length dependence of power progressively increased with a rank ordering of cMyBP-C KO = cMyBP-C t3SA < Control < cMyBP-C t3SD. Length dependence of myofilament power translated, at least in part, to hearts, whereby Frank-Starling relationships were steepest in cMyBP-C t3SD mice. The results support the hypothesis that cMyBP-C and its phosphorylation state tune sarcomere length dependence of myofibrillar power, and these regulatory processes translate across spatial levels of myocardial organization to control beat-to-beat ventricular performance.

A high-throughput fluorescence lifetime-based assay to detect binding of myosin-binding protein C to F-actin.

Binding properties of actin-binding proteins are typically evaluated by cosedimentation assays. However, this method is time-consuming, involves multiple steps, and has a limited throughput. These shortcomings preclude its use in screening for drugs that modulate actin-binding proteins relevant to human disease. To develop a simple, quantitative, and scalable F-actin-binding assay, we attached fluorescent probes to actin's Cys-374 and assessed changes in fluorescence lifetime upon binding to the N-terminal region (domains C0-C2) of human cardiac myosin-binding protein C (cMyBP-C). The lifetime of all five probes tested decreased upon incubation with cMyBP-C C0-C2, as measured by time-resolved fluorescence (TR-F), with IAEDANS being the most sensitive probe that yielded the smallest errors. The TR-F assay was compared with cosedimentation to evaluate in vitro changes in binding to actin and actin-tropomyosin arising from cMyBP-C mutations associated with hypertrophic cardiomyopathy (HCM) and tropomyosin binding. Lifetime changes of labeled actin with added C0-C2 were consistent with cosedimentation results. The HCM mutation L352P was confirmed to enhance actin binding, whereas PKA phosphorylation reduced binding. The HCM mutation R282W, predicted to disrupt a PKA recognition sequence, led to deficits in C0-C2 phosphorylation and altered binding. Lastly, C0-C2 binding was found to be enhanced by tropomyosin and binding capacity to be altered by mutations in a tropomyosin-binding region. These findings suggest that the TR-F assay is suitable for rapidly and accurately determining quantitative binding and for screening physiological conditions and compounds that affect cMyBP-C binding to F-actin for therapeutic discovery.

Cardiac Troponin I N-Terminal Pseudo-Phosphorylation Regulates Power Output and Stretch Activation

Beat-to-beat heart function is modulated by β-adrenergic stimulation, which elicits PKA phosphorylation of several cardiac myocyte proteins including cardiac troponin I (cTnI). PKA phosphorylation of the cTnI N-terminal region is known to regulate myofilament Ca2+ sensitivity of force. Our lab has also observed PKA phosphorylation increases cardiac myofilament power and augments transient force overshoot. However, it is unknown if cTnI N-terminal phosphorylation per se affects myofilament power and stretch activation. We tested the hypothesis that cTnI N-terminal pseudo-phosphorylation is sufficient to increase power and stretch activation. Molecular sufficiency was investigated by measuring power and stretch activation after cTn exchange into rat permeabilized slow-twitch skeletal muscle fibers, since slow-skeletal (ss)TnI lacks N-terminal phosphorylation sites. Four different human cTnI N-terminal pseudo-phosphorylation states were tested: (i) non-phosphorylated WT, (ii) the canonical S22/23D PKA sites, (iii) the tyrosine kinase Y26E site, also known to modulate myofilament Ca2+ sensitivity, and (iv) the combinatorial S22/23D + Y26E cTnI. Following S22/23D cTnI exchange, fiber power increased ∼18% compared to WT cTn exchange where power decreased ∼3% (p < 0.05). Following Y26E cTnI exchange, power also increased (∼6%), albeit to a less degree than S22/23D. Exchange of combined S22/23D + Y26E cTnI resulted in a similar power increase (∼17%) as S22/23D alone, indicating S22/23D cTnI pseudo-phosphorylation is sufficient to regulate myofilament power. Exchange with S22/23D, Y26E, and S22/23D + Y26E cTnI all augmented stretch activation to a similar amount, implicating redundant site-specific stretch regulation. Finally, only the combinatorial S22/23D + Y26E cTnI increased stretch-activated rates of force development, implicating synergistic regulation of stretch-activated force kinetics. Overall, cTnI N-terminal pseudo-phosphorylation regulates myofilament power and stretch activation by a combination of dominant, redundant and synergistic biophysical mechanisms, which may complement each other to tune beat-to-beat myocardial output demand.

Emergence of the N-Terminal Regulatory Domain of Cardiac Troponin I for Living on Land and the Function of Adult Heart of Higher Vertebrates

The troponin complex plays a central role in regulating the contraction and relaxation of striated muscles. Troponin-based thin filament regulation of muscle contraction emerged approximately 700 million years ago with largely conserved functions. Troponin I (TnI) is the inhibitory subunit of troponin and has evolved into three muscle type-specific isoforms in vertebrates. Cardiac TnI has an N-terminal extension that is specifically expressed in the adult heart, implicating a unique selection value. Cardiac TnI of higher vertebrate species contains beta-adrenergic regulated PKA phosphorylation sites in the N-terminal extension, which is a mechanism to enhance cardiac muscle relaxation and facilitate ventricular filling. Our data mining showed that the N-terminal extension of cardiac TnI first emerged in the genomes of early tetrapods as well as primordial lobe-finned fishes such as the coelacanth whereas it is absent in ray-finned fish. An intriguing finding is the lack of a transitional species that has an N-terminal extension in cardiac TnI without the PKA phosphorylation sites. This apparently rapid evolution of the beta-adrenergic regulation of cardiac function suggests a high selection value in the cardiac function of vertebrate animals on land. To understand the evolution and original function of the N-terminal extension in modulating cardiac muscle contractility, our study comparing the cardiac muscle of primitive lobe-finned fish with that of mammals and ray-finned fish investigates its role in cardiac adaptation to the increased energetic demands of life on land and significance in human health and the treatment of heart failure.

Cynandione A Alleviates Neuropathic Pain Through α7-nAChR-Dependent IL-10/β-Endorphin Signaling Complexes.

Cynandione A, an acetophenone isolated from Cynanchum Wilfordii Radix, exhibits antineuropathic pain effect. This study further explored the target molecule and signaling mechanisms underlying cynandione-A-induced antineuropathic pain. Intrathecal injection of cynandione A significantly attenuated mechanical allodynia in neuropathic rats and substantially increased spinal expression of IL-10 and β-endorphin but not dynorphin A. Cynandione A treatment also enhanced expression of IL-10 and β-endorphin but not α7 nicotinic acetylcholine receptors (nAChRs) in cultured microglia. The IL-10 antibody attenuated cynandione-A-induced spinal or microglial gene expression of β-endorphin and mechanical allodynia, whereas the β-endorphin antiserum blocked cynandione-A-induced mechanical antiallodynia but not spinal or microglial IL-10 gene expression. The α7 nAChR antagonist methyllycaconitine significantly reduced cynandione-A-induced mechanical antiallodynia and spinal or microglial expression of IL-10 and β-endorphin. Furthermore, cynandione A stimulated microglial phosphorylation of PKA, p38, and CREB in an α7-nAChR-dependent manner, and treatment with their inhibitors attenuated cynandione-A-induced mechanical antiallodynia and spinal or microglial expression of IL-10 and β-endorphin. In addition, cynandione A stimulated spinal phosphorylation of the transcription factor STAT3, which was inhibited by methyllycaconitine, the PKA activation inhibitor or IL-10 antibody. The STAT3 inhibitor NSC74859 also abolished cynandione-A-induced mechanical antiallodynia and spinal expression of β-endorphin. These findings suggest that cynandione A suppresses neuropathic pain through α7-nAChR-dependent IL-10/β-endorphin signaling pathway in spinal microglia.

Inducibility, but not stability, of atrial fibrillation is increased by NOX2 overexpression in mice.

AimsGp91-containing NADPH oxidases (NOX2) are a significant source of myocardial superoxide production. An increase in NOX2 activity accompanies atrial fibrillation (AF) induction and electrical remodelling in animal models and predicts incident AF in humans; however, a direct causal role for NOX2 in AF has not been demonstrated. Accordingly, we investigated whether myocardial NOX2 overexpression in mice (NOX2-Tg) is sufficient to generate a favourable substrate for AF and further assessed the effects of atorvastatin, an inhibitor of NOX2, on atrial superoxide production and AF susceptibility.Methods and resultsNOX2-Tg mice showed a 2- to 2.5-fold higher atrial protein content of NOX2 compared with wild-type (WT) controls, which was associated with a significant (twofold) increase in NADPH-stimulated superoxide production (2-hydroxyethidium by HPLC) in left and right atrial tissue homogenates (P = 0.004 and P = 0.019, respectively). AF susceptibility assessed in vivo by transoesophageal atrial burst stimulation was modestly increased in NOX2-Tg compared with WT (probability of AF induction: 88% vs. 69%, respectively; P = 0.037), in the absence of significant alterations in AF duration, surface ECG parameters, and LV mass or function. Mechanistic studies did not support a role for NOX2 in promoting electrical or structural remodelling, as high-resolution optical mapping of atrial tissues showed no differences in action potential duration and conduction velocity between genotypes. In addition, we did not observe any genotype difference in markers of fibrosis and inflammation, including atrial collagen content and Col1a1, Il-1β, Il-6, and Mcp-1 mRNA. Similarly, NOX2 overexpression did not have consistent effects on RyR2 Ca2+ leak nor did it affect PKA or CaMKII-mediated RyR2 phosphorylation. Finally, treatment with atorvastatin significantly inhibited atrial superoxide production in NOX2-Tg but had no effect on AF induction in either genotype.ConclusionTogether, these data indicate that while atrial NOX2 overexpression may contribute to atrial arrhythmogenesis, NOX2-derived superoxide production does not affect the electrical and structural properties of the atrial myocardium.

Neuroprotective effects of Ginkgolide B in focal cerebral ischemia through selective activation of prostaglandin E2 receptor EP4 and the downstream transactivation of epidermal growth factor receptor.

The present study was undertaken to identify whether prostaglandin E2 receptor is the potential receptor/binding site for Ginkgolide A, Ginkgolide B, Ginkgolide K, and Bilobalide, the four main ingredients of the Ginkgo biloba L., leaves. Using functional assays, we identified EP4, coupled with Gs protein, as a target of Ginkgolide B. In human neuroblastoma SH-SY5Y cells suffered from oxygen-glucose deprivation/reperfusion, Ginkgolide B-activated PKA, Akt, and ERK1/2 as well as Src-mediated transactivation of epidermal growth factor receptor. These resulted in downstream signaling pathways, which enhanced cell survival and inhibited apoptosis. Knockdown of EP4 prevented Ginkgolide B-mediated Src, epidermal growth factor receptor (EGFR), Akt, and ERK1/2 phosphorylation and neuroprotective effects. Moreover, Src inhibitor prevented Ginkgolide B-mediated EGFR transactivation and the downstream Akt and ERK1/2 activation, while the phosphorylation of PKA induced by Ginkgolide B was not affected, indicating Ginkgolide B might transactivate EGFR in a ligand-independent manner. EP4 knockdown in a rat middle cerebral artery occlusion (MCAO) model prevented Ginkgolide B-mediated infarct size reduction and neurological assessment improvement. At the same time, the increased expressions of p-Akt, p-ERK1/2, p-PKA, p-Src, and p-EGFR and the deceased expression of cleaved capases-3 induced by Ginkgolide B in cerebral cortex were blocked due to EP4 knockdown. In conclusion, Ginkgolide B exerts neuroprotective effects in rat MCAO model through the activation of EP4 and the downstream transactivation of EGFR.

Epigenomic, Transcriptomic, and Protective Effect of Carotenoid Fucoxanthin in High Glucose-Induced Oxidative Stress in Mes13 Kidney Mesangial Cells

Diabetic nephropathy (DN) is the major cause of kidney related diseases in patients induced by high glucose (HG) affecting around 40% of type 1 and 2 diabetic patients. It is characterized by excessive inflammation inducing factors, reactive oxygen species (ROS) overproduction, and potential epigenomic related changes. Fucoxanthin (FX), a carotenoid found in brown seaweed, has a structure which includes an allenic bond and a 5,6-monoepoxide in the molecule, with strong antioxidant and anti-inflammatory activity. However, understanding of the impact of FX on DN was lacking. In this study we tested the early effects of high glucose (HG) on mouse mesangial kidney Mes13 cells, a potential in vitro cell culture model of DN. Our results show that HG induced oxidative stress on kidney mesangial Mes13 cells, while FX treatment attenuates the oxidative stress by decreasing the ROS, demonstrated by flow cytometry. Next, we utilized next-generation sequencing (NGS) to profile the HG-induced early epigenomic and transcriptomic changes in this in vitro DN model and the protective effects of FX. Differentially expressed genes (DEGs) and differentially methylated regions (DMRs) were analyzed using R software in HG and FX treated groups. Differential regulation of signaling pathways was studied using Reactome Pathway Analysis in the comparison. DEG analysis shows that novel biomarkers with specific pathways, including interleukin regulation, Toll-like receptor pathway, and PKA phosphorylation pathways, were found to be modulated by the FX treatment. TGF β 1i1 (TGFB 1i1), MAP-3-kinase-13(MAP3K13) involved in crucial cellular processes including glucose metabolism, phosphodiesterase regulation was methylated in HG, which was demethylated with FX treatment. Integrated transcriptomic and CpG methylome analysis of DEGs and DMRs revealed that genes like adenylate cyclase (Adcy7), calponin 1 (CNN1), potassium voltage-gated channel interacting protein 2 (KCNIP2), phosphatidylinositol-4-phosphate 5-kinase type 1 β (PIP5K1B), and transmembrane protein with EGF-like and two follistatin-like domains 1 (TMEFF1), which were modulated by FX in HG-exposed Mes13 cells, potentially modulate ion channel transport and glucose metabolism. In summary, our current study shows that novel early epigenomic and transcriptomic biomarkers were altered during the disease progression of HG-induced DN and that FX modified these alterations potentially contributing to the protective effects of mesangial cells from the HG-induced oxidative stress and damage.

Stretch-Induced Activation of Pannexin 1 Channels Can Be Prevented by PKA-Dependent Phosphorylation.

Pannexin 1 channels located in the cell membrane are permeable to ions, metabolites, and signaling molecules. While the activity of these channels is known to be modulated by phosphorylation on T198, T308, and S206, the possible involvement of other putative phosphorylation sites remains unknown. Here, we describe that the activity of Panx1 channels induced by mechanical stretch is reduced by adenosine via a PKA-dependent pathway. The mechanical stretch-induced activity—measured by changes in DAPI uptake—of Panx1 channels expressed in HeLa cell transfectants was inhibited by adenosine or cAMP analogs that permeate the cell membrane. Moreover, inhibition of PKA but not PKC, p38 MAPK, Akt, or PKG prevented the effects of cAMP analogs, suggesting the involvement of Panx1 phosphorylation by PKA. Accordingly, alanine substitution of T302 or S328, two putative PKA phosphorylation sites, prevented the inhibitory effect of cAMP analogs. Moreover, phosphomimetic mutation of either T302 or S328 to aspartate prevented the mechanical stretch-induced activation of Panx1 channels. A molecular dynamics simulation revealed that T302 and S328 are located in the water–lipid interphase near the lateral tunnel of the intracellular region, suggesting that their phosphorylation could promote conformational changes in lateral tunnels. Thus, Panx1 phosphorylation via PKA could be modulated by G protein-coupled receptors associated with the Gs subunit.

Intrathecal administration of neuronostatin induces an antinociceptive effect in a mouse visceral pain model

Neuronostatin (NST) is a peptide encoded by the somatostatin gene that serves important physiological functions in diverse tissues. Previous studies have shown that intracerebroventricular administration of NST induces antinociceptive effects and hyperalgesic effects as determined by the tail immersion assay and formalin test, respectively. In the present study, we aimed to evaluate the effects of intrathecal (i.t.) injection of NST on nociception in a model of visceral pain, and determine possible mechanisms of action in mice. NST (1, 3, 6, or 12 nmol) was administered to mice, leading to a dose‐dependent antinociceptive effect as determined by the acetic acid‐induced writhing test in mice. NST (1 nmol) also enhanced the antinociceptive effect of morphine (2.5 and 5 μg/kg) in the spine. Naloxone and β‐funaltrexamine hydrochloride significantly antagonized the antinociceptive effect of NST. The expression of G‐protein‐coupled receptor 107 (GPR107) protein and the phosphorylation of PKA at Thr197 were increased after i.t. administration of NST, suggesting that the μ‐opioid receptor and GPR107/PKA signaling pathway are involved in the analgesic response. In conclusion, i.t. injection of NST may potentially be used as a new approach in the mediation of visceral pain.

The RhoA regulators Myo9b and GEF‐H1 are targets of cyclic nucleotide‐dependent kinases in platelets

BackgroundCirculating platelets are maintained in an inactive state by the endothelial lining of the vasculature. Endothelium‐derived prostacyclin and nitric oxide stimulate cAMP‐ and cGMP‐dependent kinases, PKA and PKG, to inhibit platelets. PKA and PKG effects include the inhibition of the GTPase RhoA, which has been suggested to involve the direct phosphorylation of RhoA on serine 188. ObjectivesWe wanted to confirm RhoA S188 phosphorylation by cyclic nucleotide‐dependent kinases and to identify possible alternative mechanisms of RhoA regulation in platelets. MethodsPhosphoproteomics data of human platelets were used to identify candidate PKA and PKG substrates. Phosphorylation of individual proteins was studied by Western blotting and Phos‐tag gel electrophoresis in human platelets and transfected HEK293T cells. Pull‐down assays were performed to analyze protein interaction and function. ResultsOur data indicate that RhoA is not phosphorylated by PKA in platelets. Instead, we provide evidence that cyclic nucleotide effects are mediated through the phosphorylation of the RhoA‐specific GTPase‐activating protein Myo9b and the guanine nucleotide exchange factor GEF‐H1. We identify Myo9b S1354 and guanine nucleotide exchange factor‐H1 (GEF‐H1) S886 as PKA and PKG phosphorylation sites. Myo9b S1354 phosphorylation enhances its GTPase activating protein function leading to reduced RhoA‐GTP levels. GEF‐H1 S886 phosphorylation stimulates binding of 14‐3‐3β and has been shown to inhibit GEF function by facilitating binding of GEF‐H1 to microtubules. Microtubule disruption increases RhoA‐GTP levels confirming the importance of GEF‐H1 in platelets. ConclusionPhosphorylation of RhoA regulatory proteins Myo9b and GEF‐H1, but not RhoA itself, is involved in cyclic nucleotide‐mediated control of RhoA in human platelets.

Phosphodiesterase PDE4D Is Decreased in Frontal Cortex of Aged Rats and Positively Correlated With Working Memory Performance and Inversely Correlated With PKA Phosphorylation of Tau.

Age is the largest risk factor for Alzheimer’s disease (AD) and contributes to cognitive impairment in otherwise healthy individuals. Thus, it is critical that we better understand the risk aging presents to vulnerable regions of the brain and carefully design therapeutics to address those effects. In this study we examined age-related changes in cAMP-regulatory protein, phosphodiesterase 4D (PDE4D). Inhibition of PDE4D is currently under investigation as a therapeutic target for AD based on memory-enhancing effects in rodent hippocampus. Therefore, it is important to understand the role of PDE4D in brain regions particularly vulnerable to disease such as the frontal association cortex (FC), where cAMP signaling can impair working memory via opening of potassium channels. We found that PDE4D protein level was decreased in the FC of both moderately and extremely aged rats, and that PDE4D level was correlated with performance on a FC-dependent working memory task. In extremely aged rats, PDE4D was also inversely correlated with levels of phosphorylated tau at serine 214 (S214), a site phosphorylated by protein kinase A. In vitro studies of the PDE4D inhibitor, GEBR-7b, further illustrated that inhibition of PDE4D activity enhanced phosphorylation of tau. pS214-tau phosphorylation is associated with early AD tau pathology, promotes tau dissociation from microtubules and primes subsequent tau hyperphosphorylation at other critical AD-related sites. Age-related loss of PDE4D may thus contribute to the specific vulnerability of the FC to degeneration in AD, and play a critical role in normal cAMP regulation, cautioning against the use of pan-PDE4D inhibitors as therapeutics.

PKC- and PKA-dependent phosphorylation modulates TREK-1 function in naïve and neuropathic rats.

PKC and PKA phosphorylation inhibit TREK-1 channels downstream of Gs protein-coupled receptor activation in vitro. However, the role of phosphorylation of TREK-1 in neuropathic pain is unknown. The purpose of this study was to investigate whether altered TREK-1 channel function by PKA and PKC modulators contributes to antiallodynia in neuropathic rats. Furthermore, we investigated if the in vitro described sites for PKC and PKA phosphorylation (S300 and S333, respectively) participate in the modulation of TREK-1 in naïve and neuropathic rats. L5/L6 spinal nerve ligation (SNL) induced tactile allodynia. Intrathecal injection of BL-1249 (TREK-1 activator) reversed nerve injury-induced tactile allodynia, whereas spadin (TREK-1 blocker) produced tactile allodynia in naïve rats and reversed the antiallodynic effect induced by BL-1249 in neuropathic rats. Intrathecal administration of rottlerin or Rp-cAMPs (PKC and PKA inhibitors, respectively) enhanced the antiallodynia observed with BL-1249 in neuropathic rats. In contrast, pretreatment with PdBu or forskolin (PKC and PKA activators, respectively) reduced the BL-1249-induced antiallodynia. Intrathecal injection of two high-activity TREK-1 recombinant channels, using a in vivo transfection method with lipofectamine, with mutations at PKC/PKA phosphosites (S300A and S333A) reversed tactile allodynia in neuropathic rats, with no effect in naïve rats. In contrast, transfection of two low-activity TREK-1 recombinant channels with phosphomimetic mutations at those sites (S300D and S333D) produced tactile allodynia in naïve rats and interfered with antiallodynic effects of rottlerin/BL-1249 or Rp-cAMPs/BL-1249. Data suggest that TREK-1 channel activity can be dynamically tuned in vivo by PKC/PKA to provoke allodynia and modulate its antiallodynic role in neuropathic pain.

Regulation of E2F1 activity via PKA-mediated phosphorylations.

E2F1 becomes activated during the G1 phase of the cell cycle, and posttranslational modifications modulate its activity. Activation of G-protein coupled receptors (GPCR) by many ligands induces the activation of adenylate cyclases and the production of cAMP, which activates the PKA enzyme. Activated PKA elicits its biological effect by phosphorylating the target proteins containing serine or threonine amino acids in the RxxS/T motif. Since PKA activation negatively regulates cell proliferation, we thought that activated PKA would negatively affect the activity of E2F1. In line with this, when we analyzed the amino acid sequence of E2F1, we found 3 hypothetical consensus PKA phosphorylation sites located at 127-130, 232-235, and 361-364 positions and RYET, RLLS, and RMGS sequences. After showing the binding and phosphorylation of E2F1 by PKA, we converted the codons of Threonine-130, Serine-235, and Serine-364 to Alanine and Glutamic acid codons on the eukaryotic E2F1 expression vector we had previously created. We confirmed the phosphorylation of T130, S235, and S364 by developing monoclonal antibodies against phospho-specific forms of these sites and showed that their phosphorylation is cell cycle-dependent. According to our results, PKA-mediated phosphorylation of E2F1 by PKA inhibits proliferation and glucose uptake and induces caspase-3 activation and senescence.

Attenuation effect of polydeoxyribonucleotide on inflammatory cytokines and apoptotic factors induced by particulate matter (PM10) damage in human bronchial cells.

Particulate matter (PM) of 10-μm-sized fine dust in the air penetrates the respiratory tract and contributes to the increasing incidence of various lung diseases, but its definite mechanism is not known. Recently, polydeoxyribonucleotide (PDRN) has been shown to have anti-inflammatory and regenerative effects in various tissues. However, the bronchial-related mechanism is not well-understood. Hence, this experiment is intended to demonstrate the beneficial effect of PDRN administration on PM10-induced injury in human bronchial-derived NCI-H358 cells. To confirm the protective effect of PDRN, PM10 was applied after PDRN pretreatment to confirm changes in NCI-H358 cells. Experiments were conducted to measure cell survival, cytotoxicity, inflammation, and apoptotic factor changes. WST-8 assay was used to confirm cell viability, and lactate dehydrogenase assay was used to obtain cytotoxicity. In addition, changes in inflammatory cytokines and apoptotic factors were confirmed by enzyme-linked immunosorbent assay and Western blot. Decreased cell viability and increased cytotoxicity, inflammatory cytokines, and apoptotic factors were observed after exposure to PM10. However, pretreatment with PDRN enhanced cell viability and reduced cytotoxicity. In addition, the expression of inflammatory cytokines such as tumor necrosis factor-α, interleukin-6 (IL-6), and IL-1β, and cell death factors such as Apaf-1, cyt c, caspase-3, caspase-9, Bid, and Bax/Bcl-2 ratio were decreased by PDRN administration in PM10-exposed NCI-H358 cells. PDRN, an A2AR agonist, affects cAMP activation and regulation of phosphorylation of PKA and CREB. In addition, treatment with A2AR antagonist 3,7-dimethyl-1-propargylxanthine significantly blocked PDRN's effect. These anti-cytotoxicity, anti-inflammation, and anti-apoptosis effects of PDRN can be attributed to the adenosine A2AR enhancing effect on PM10-exposed bronchial cells.