Selective Protein Kinase A Inhibitor Research Articles

Influence of Phosphodiesterase Inhibition on CRE- and EGR1-Dependent Transcription in a Mouse Hippocampal Cell Line.

Signaling pathways, depending on the second messenger molecule cAMP, modulate hippocampal cell signaling via influencing transcription factors like cAMP-regulated element-binding protein (CREB) or early growth response 1 EGR1/Krox24/zif268/ZENK (EGR1). Here, we investigated two reporter cell lines derived from an immortalized hippocampal neuronal cell line stably expressing a CRE- or EGR1-luciferase reporter gene (HT22CREluc and HT22EGR1luc, respectively). The cells were subjected to phosphodiesterase inhibitors and other cAMP-modulating agents to investigate dose- and time-dependent phosphodiesterase (PDE)-mediated fine-tuning of cAMP-dependent transcriptional signaling. The non-isoform-specific cyclic nucleotide phosphodiesterase (PDE) inhibitor isobutyl-methyl-xanthine (IBMX), as well as selective inhibitors of PDE3 (milrinone) and PDE4 (rolipram), were tested for their ability to elevate CRE- and EGR1-luciferase activity. Pharmacological parameters like onset of activity, maximum activity, and offset of activity were determined. In summary, phosphodiesterase inhibition appeared similarly potent in comparison to adenylate cyclase stimulation or direct activation of protein kinase A (PKA) via specific cAMP agonists and was at least partly mediated by PKA as shown by the selective PKA inhibitor Rp-8-Br-cAMPS. Moreover, transcriptional activation by PDE inhibition was also influenced by organic anion-exchanger action and interacted with fibroblast growth factor (FGF) receptor-mediated pathways.

Anti-depressant effects of oil from fructus gardeniae via PKA-CREB-BDNF signaling.

The dried ripe fruit of Gardenia jasminoides Ellis was usually applied as an herb medicine in Traditional Chinese Medicine. It was suggested that the Gardenia jasminoides oil extract (oil from Fructus Gardeniae [OFG]) might serve as a potential treatment for depression, whereas its pathogenesis still remained not fully understood. The present research was conducted to evaluate the anti-depressive effect of OFG in mice and explore its potential mechanism. The OFG and ketamine (KET) were intragastrically and intraperitoneally treated, respectively. Thereafter, the animals were subjected to the behavior tests. The expressions of protein kinase A (PKA), brain derived neurotrophic factor (BDNF), cAMP response element-binding protein (CREB) in hippocampus were detected by Western blot. The selective PKA inhibitor H-89 was also applied to confirm the mechanism. As a result, OFG and KET treatment improved the behavior performance. Furthermore, the administrations of OFG effectively enhanced the expressions of PKA, p-CREB, and BDNF. With the application of selective PKA inhibitor H-89, the ameliorated effects caused by OFG were blocked, but not by KET. In conclusion, the presented work indicated that OFG-exerted protective effect on depression through PKA-CREB-BDNF signaling.

Central Role of Protein Kinase A in Promoting Trigeminal Nociception in an In Vivo Model of Temporomandibular Disorders.

To investigate cellular changes in the spinal trigeminal nucleus (STN) and trigeminal ganglion (TG) associated with trigeminal nociception mediated by inflammation in the temporomandibular joint (TMJ). Male Sprague-Dawley rats (n = 86) were utilized to investigate cellular and behavioral responses to prolonged TMJ inflammation caused by bilateral injection of Complete Freund's Adjuvant (CFA) in the TMJ capsules. To investigate the cellular effects of protein kinase A (PKA) in the STN, rats were injected intrathecally with the selective PKA inhibitor KT5720 prior to injection of CFA into both TMJ capsules. Levels of calcitonin gene-related peptide (CGRP), active PKA, and ionized calcium-binding adapter molecule 1 (Iba1) in the STN and expression of phosphorylated extracellular regulated kinases (p-ERK) in the TG were determined with immunohistochemistry (n ≥ 3 experiments per test condition). Nocifensive head withdrawal responses to mechanical stimulation of the cutaneous tissue over the TMJ were monitored following CFA injection in the absence or presence of KT5720 (n = 7). Statistical analysis was performed using parametric analysis of variance (ANOVA) tests. Intrathecal injection of KT5720 significantly inhibited the stimulatory effect of CFA on levels of CGRP, PKA, and Iba1 in the STN. In addition, administration of KT5720 decreased the average number of CFA-induced nocifensive withdrawal responses to mechanical stimulation and the CFA-mediated increase in p-ERK expression in the ganglion. These findings provide evidence that elevated PKA activity in the STN promotes cellular events temporally associated with trigeminal nociception caused by prolonged TMJ inflammation.

Catecholamines facilitate VEGF-dependent angiogenesis via β2-adrenoceptor-induced Epac1 and PKA activation.

Chronic stress has been associated with the progression of cancer and antagonists for β-adrenoceptors (βAR) are regarded as therapeutic option. As they are also used to treat hemangiomas as well as retinopathy of prematurity, a role of endothelial β2AR in angiogenesis can be envisioned. We therefore investigated the role of β2AR-induced cAMP formation by analyzing the role of the cAMP effector molecules exchange factor directly activated by cAMP 1 (Epac1) and protein kinase A (PKA) in endothelial cells (EC). Epac1-deficient mice showed a reduced amount of pre-retinal neovascularizations in the model of oxygen-induced retinopathy, which is predominantly driven by vascular endothelial growth factor (VEGF). siRNA-mediated knockdown of Epac1 in human umbilical vein EC (HUVEC) decreased angiogenic sprouting by lowering the expression of the endothelial VEGF-receptor-2 (VEGFR-2). Conversely, Epac1 activation by β2AR stimulation or the Epac-selective activator cAMP analog 8-p-CPT-2’-O-Me-cAMP (8-pCPT) increased VEGFR-2 levels and VEGF-dependent sprouting. Similar to Epac1 knockdown, depletion of the monomeric GTPase Rac1 decreased VEGFR-2 expression. As Epac1 stimulation induces Rac1 activation, Epac1 might regulate VEGFR-2 expression through Rac1. In addition, we found that PKA was also involved in the regulation of angiogenesis in EC since the adenylyl cyclase (AC) activator forskolin (Fsk), but not 8-pCPT, increased sprouting in Epac1-depleted HUVEC and this increase was sensitive to a selective synthetic peptide PKA inhibitor. In accordance, β2AR- and AC-activation, but not Epac1 stimulation increased VEGF secretion in HUVEC.Our data indicate that high levels of catecholamines, which occur during chronic stress, prime the endothelium for angiogenesis through a β2AR-mediated increase in endothelial VEGFR-2 expression and VEGF secretion.

Insights into the regulation of the MXR response in haemocytes of the Mediterranean mussel (Mytilus galloprovincialis)

This study investigated functional and transcriptional modulation of the Multixenobiotic resistance (MXR) system as a cytoprotective mechanism contributing to the physiological chemoresistance of haemocytes in the Mediterranean mussel. Basal transport activity was assessed using the model substrate rhodamine 123 and specific inhibitors for the MXR-related transporters P-glycoprotein (ABCB mRNA) and Multidrug resistance-related protein (ABCC mRNA). Results showed that MXR activity in mussel haemocytes was mainly supported by the Mrp-mediated efflux. In agreement, ABCC was expressed at higher levels than ABCB. Activation of the cyclic-AMP (cAMP) dependent protein kinase A (PKA) resulted in increased rhodamine efflux, which was counteracted by the selective PKA inhibitor H89. Although serotonin, a physiological modulator of cAMP/PKA signaling and ABCB transcription in haemocytes, did not affect basal MXR transport, the environmental pharmaceuticals fluoxetine, propranolol, and carbamazepine, which interact in different ways with the adrenergic and serotoninergic pathways, were showed to act as modulators and substrates of MXR-related transporters and to affect cell viability. While the increased MXR activity may have lowered the cytotoxic effects of propranolol and carbamazepine, the lack of MXR efflux induction by fluoxetine may play a role in the observed cytotoxicity of the compound.

Spinal 5-HT7 receptors induce phrenic motor facilitation via EPAC-mTORC1 signaling.

Spinal serotonin type 7 (5-HT7) receptors elicit complex effects on motor activity. Whereas 5-HT7 receptor activation gives rise to long-lasting phrenic motor facilitation (pMF), it also constrains 5-HT2 receptor-induced pMF via "cross-talk inhibition." We hypothesized that divergent cAMP-dependent signaling pathways give rise to these distinct 5-HT7 receptor actions. Specifically, we hypothesized that protein kinase A (PKA) mediates cross-talk inhibition of 5-HT2 receptor-induced pMF whereas 5-HT7 receptor-induced pMF results from exchange protein activated by cAMP (EPAC) signaling. Anesthetized, paralyzed, and ventilated rats receiving intrathecal (C4) 5-HT7 receptor agonist (AS-19) injections expressed pMF for >90 min, an effect abolished by pretreatment with a selective EPAC inhibitor (ESI-05) but not a selective PKA inhibitor (KT-5720). Furthermore, intrathecal injections of a selective EPAC activator (8-pCPT-2'-Me-cAMP) were sufficient to elicit pMF. Finally, spinal mammalian target of rapamycin complex-1 (mTORC1) inhibition via intrathecal rapamycin abolished 5-HT7 receptor- and EPAC-induced pMF, demonstrating that spinal 5-HT7 receptors elicit pMF by an EPAC-mTORC1 signaling pathway. Thus 5-HT7 receptors elicit and constrain spinal phrenic motor plasticity via distinct signaling mechanisms that diverge at cAMP (EPAC vs. PKA). Selective manipulation of these molecules may enable refined regulation of serotonin-dependent spinal motor plasticity for therapeutic advantage.

5HT7 Receptor Induced Phrenic Motor Facilitation Requires EPAC and mTORC1, But Not PKA Activity

Serotonin type‐7 (5HT7) receptors in the cervical spinal cord elicit complex effects on respiratory motor activity. For example, intermittent 5HT7 receptor activation gives rise to long‐lasting phrenic motor facilitation (pMF); on the other hand, 5HT7 receptor activation constrains competing serotonin type‐2 induced pMF via protein kinase A (PKA)‐dependent “cross‐talk inhibition.” We hypothesized that divergent signaling pathways give rise to these distinct 5HT7 receptor actions. Specifically, whereas PKA regulates cross‐talk inhibition of 5HT2‐dependent pMF, a different cyclic AMP signaling pathway that requires exchange protein activated by cyclic AMP (EPAC) underlies 5HT7‐dependent pMF. Anesthetized, paralyzed and ventilated rats receiving intermittent 5HT7 receptor agonist injections (3x5uL; AS‐19) into the C4 intrathecal space expressed pMF for at least 90 minutes. This 5HT7‐induced pMF was abolished by pretreatment with a selective EPAC inhibitor (ESI‐05), but not a selective PKA inhibitor (KT‐5720). Further, an intrathecal EPAC activator (8‐pCPT‐2′‐O‐Me‐cAMP) is sufficient to elicit pMF. mTORC1 inhibition (rapamycin) abolished both 5HT7‐ and EPAC‐induced pMF. Thus, spinal 5HT7 receptors elicit pMF by a non‐canonical EPAC‐AKT‐mTORC1 pathway, and constrain alternate pathways to spinal respiratory motor plasticity via PKA signaling.

Coumestrol inhibits carotid sinus baroreceptor activity by cAMP/PKA dependent nitric oxide release in anesthetized male rats

Phytoestrogens could offer multiple beneficial effects on the cardiovascular system. Here, we have examined the effects of coumestrol (CMT) on carotid baroreceptors activity (CBA) and the possible mechanisms in male rats.The functional parameters of carotid baroreceptors were measured by recording sinus nerve afferent discharge in anesthetized male rats with perfused isolated carotid sinus. The levels of protein expression were determined by using ELISA and Western blotting.CMT (1 to 100μmolL−1) inhibited CBA, which shifted the functional curve of the carotid baroreceptor to the right and downward, with a marked decrease in the peak slope and the peak integral value of carotid sinus nerve discharge in a concentration dependent manner. These effects were not blocked by a specific estrogen receptor antagonist ICI 182,780, but were completely abolished by nitric oxide (NO) synthase inhibitor l-NAME (NG-nitro-l-arginine methyl ester). Furthermore, a NO donor, SIN-1(3-morpholion-sydnon-imine), could potentiate these inhibitory effects of CMT. CMT stimulated the phosphorylation of Ser1176-eNOS (endothelial nitric oxide synthase) in a dose-dependent manner in carotid bifurcation tissue over a perfusion period of 15min. The rapid activation of eNOS by CMT was blocked by a highly selective PKA (protein kinase A) inhibitor H89. In addition, inhibition of PI3K (phosphatidylinositol-3-kinase) and ERK (extracellular signal-regulated kinase) pathways had no effect on eNOS activation by CMT.CMT inhibited CBA via eNOS activation and NO synthesis. These effects were mediated by the cAMP/PKA pathway and were unrelated to the estrogenic effect.

Highly expressed protein kinase A inhibitor α and suppression of protein kinase A may potentiate acetaminophen-induced hepatotoxicity

Drug-induced hepatotoxicity is a serious adverse effect with high morbidity and mortality rates but substantial individual to individual variation is observed in its severity. Here we sought to discover factors determining the susceptibility to acetaminophen (APAP)-induced hepatotoxicity by comparing the global gene expression profile (27,342 genes) in pre-dose blood before APAP administration between susceptible and resistant animals (N=5) grouped based on the severity of hepatotoxicity. Forty-one genes were expressed differently (>1.5 fold change and p<0.05) between susceptible and resistant groups. Among them, protein kinase (cAMP-dependent) inhibitor alpha, Pkia, a member of protein kinase A (PKA) inhibitor family, was found to be most significantly and highly expressed in susceptible animals (∼3.5 fold with p<0.01). To investigate the effects of PKA inhibition on APAP-induced hepatotoxicity, we pre-treated H-89, a potent and selective inhibitor of PKA, prior to APAP administration in vivo. As a result, H-89 pretreatment significantly potentiated APAP-induced hepatotoxicity as determined by the increased serum alanine transaminase. These results were further corroborated by the exacerbation of APAP-induced glutathione depletion, suppression of antioxidant enzyme system, superoxide dismutase 1 and glutathione peroxidase 1, and peroxynitrite generation in the liver following H-89 pretreatment, reflecting that PKA may be involved in the protection against, or attenuation of APAP-induced hepatotoxicity, and Pkia can be employed to screen individuals susceptible to APAP-induced hepatotoxicity.

The protective roles of GLP-1R signaling in diabetic nephropathy: possible mechanism and therapeutic potential

Glucagon-like peptide-1 (GLP-1) is a gut incretin hormone that has an antioxidative protective effect on various tissues. Here, we determined whether GLP-1 has a role in the pathogenesis of diabetic nephropathy using nephropathy-resistant C57BL/6-Akita and nephropathy-prone KK/Ta-Akita mice. By in situ hybridization, we found the GLP-1 receptor (GLP-1R) expressed in glomerular capillary and vascular walls, but not in tubuli, in the mouse kidney. Next, we generated C57BL/6-Akita Glp1r knockout mice. These mice exhibited higher urinary albumin levels and more advanced mesangial expansion than wild-type C57BL/6-Akita mice, despite comparable levels of hyperglycemia. Increased glomerular superoxide, upregulated renal NAD(P)H oxidase, and reduced renal cAMP and protein kinase A (PKA) activity were noted in the Glp1r knockout C57BL/6-Akita mice. Treatment with the GLP-1R agonist liraglutide suppressed the progression of nephropathy in KK/Ta-Akita mice, as demonstrated by reduced albuminuria and mesangial expansion, decreased levels of glomerular superoxide and renal NAD(P)H oxidase, and elevated renal cAMP and PKA activity. These effects were abolished by an adenylate cyclase inhibitor SQ22536 and a selective PKA inhibitor H-89. Thus, GLP-1 has a crucial role in protection against increased renal oxidative stress under chronic hyperglycemia, by inhibition of NAD(P)H oxidase, a major source of superoxide, and by cAMP-PKA pathway activation.

NF-κB enhances hypoxia-driven T-cell immunosuppression via upregulation of adenosine A2A receptors

Hypoxia affects inflammation by modulating T-cell activation via the adenosinergic system. We supposed that, in turn, inflammation influences cell hypoxic behavior and that stimulation of T-cells in inflammatory conditions involves the concerted action of the nuclear factor κB (NF-κB) and the related hypoxia-inducible factor 1α (HIF-1α) on the adenosinergic system. We addressed this hypothesis by monitoring both transcription factors and four adenosinergic signaling parameters – namely adenosine, adenosine deaminase (ADA), adenosine A2A receptor (A2AR) and cAMP – in T-cells stimulated using phorbol myristate acetate and phytohemagglutinin and submitted to hypoxic conditions which were mimicked using CoCl2 treatment. We found that cell viability was more altered in stimulated than in resting cells under hypoxia. Detailed analysis showed that: i) NF-κB activation remained at basal level in resting hypoxic cells but greatly increased following stimulation, stimulated hypoxic cells exhibiting the higher level; ii) HIF-1α production induced by hypoxia was boosted via NF-κB activation in stimulated cells whereas hypoxia increased HIF-1α production in resting cells without further activating NF-κB; iii) A2AR expression and cAMP production increased in stimulated hypoxic cells whereas adenosine level remained unchanged due to ADA regulation; and iv) the presence of H2S, an endogenous signaling molecule in inflammation, reversed the effect of stimulation on cell viability by down-regulating the activity of transcription factors and adenosinergic immunosuppression. We also found that: i) the specific A2AR agonist CGS-21680 increased the suppressive effect of hypoxia on stimulated T-cells, the antagonist ZM-241385 exhibiting the opposite effect; and ii) Rolipram, a selective inhibitor of cAMP-specific phosphodiesterase 4, and 8-Br-cAMP, a cAMP analog which preferentially activates cAMP-dependent protein kinase A (PKA), increased T-cell immunosuppression whereas H-89, a potent and selective inhibitor of cAMP-dependent PKA, restored cell viability. Together, these data indicate that inflammation enhances T-cell sensitivity to hypoxia via NF-κB activation. This process upregulates A2AR expression and enhances cAMP production and PKA activation, resulting in adenosinergic T-cell immunosuppression that can be modulated via H2S.

IL-17 Inhibits Chondrogenic Differentiation of Human Mesenchymal Stem Cells

ObjectiveMesenchymal stem cells (MSCs) can differentiate into cells of mesenchymal lineages, such as osteoblasts and chondrocytes. Here we investigated the effects of IL-17, a key cytokine in chronic inflammation, on chondrogenic differentiation of human MSCs.MethodsHuman bone marrow MSCs were pellet cultured in chondrogenic induction medium containing TGF-β3. Chondrogenic differentiation was detected by cartilage matrix accumulation and chondrogenic marker gene expression.ResultsOver-expression of cartilage matrix and chondrogenic marker genes was noted in chondrogenic cultures, but was inhibited by IL-17 in a dose-dependent manner. Expression and phosphorylation of SOX9, the master transcription factor for chondrogenesis, were induced within 2 days and phosphorylated SOX9 was stably maintained until day 21. IL-17 did not alter total SOX9 expression, but significantly suppressed SOX9 phosphorylation in a dose-dependent manner. At day 7, IL-17 also suppressed the activity of cAMP-dependent protein kinase A (PKA), which is known to phosphorylate SOX9. H89, a selective PKA inhibitor, also suppressed SOX9 phosphorylation, expression of chondrogenic markers and cartilage matrix, and also decreased chondrogenesis.ConclusionsIL-17 inhibited chondrogenesis of human MSCs through the suppression of PKA activity and SOX9 phosphorylation. These results suggest that chondrogenic differentiation of MSCs can be inhibited by a mechanism triggered by IL-17 under chronic inflammation.

OP21 Role of phosphodiesterase inhibition and modulation of mitochondrial cAMP levels in the bioenergetic effect of hydrogen sulfide in isolated mitochondria

Introduction Several lines of studies demonstrate the biphasic bioenergetic effect of hydrogen sulfide (H 2 S): at lower concentrations, endogenously produced and exogenously administered H 2 S stimulates mitochondrial electron transport and supports oxidative phosphorylation, whereas at higher concentration, H 2 S inhibits mitochondrial respiration by inhibiting mitochondrial Complex IV (cytochrome C oxidase).In addition, it has recently been demonstrated that in the mitochondrial matrix there is a distinct cAMP pool, which is not related to the cytosolic cAMP pool.Furthermore, a soluble adenylyl cyclase enzyme (sAC) has been localized to the mitochondrial matrix, which is involved in the maintenance of the intra-mitochondrial cAMP levels.Given the recently emerging evidence that (a) H 2 S is an endogenous inhibitor of cAMP and cGMP phosphodiesterases and that (b) intramitochondrial cAMP and PKA act an endogenous stimulators of mitochondrial electron transport and oxidative phosphorylation, the aim of the current study was to explore the potential role of mitochondrial cAMP and its downstream effector, protein kinase A (PKA) in the stimulatory bioenergetic effect of H 2 S in vitro . Methods The presence of PDE2A (a combined cAMP- and cGMP-degrading phosphodiesterase) and of PKA was investigated by western blotting of native isolated rat liver mitochondria, as well as its partially digested forms to yield mitoplasts and post-mitoplasts.In addition, freshly isolated rat liver mitochondria were subjected to H 2 S donor NaHS and analyzed by Extracellular Flux Analysis (Seahorse) in the absence or presence of various modulators of mitochondrial cAMP/PKA, followed by the measurement of oxygen consumption rate in the absence or presence of ADP, as well as a sequential administration of specific modulators (the ATP synthase inhibitor oligomycin, the mitochondrial uncoupler FCCP and the Complex III inhibitor antimycin) of mitochondrial electron transport chain complexes.The effect of the H 2 S donor NaHS on the activity of recombinant PDEA2 (an enzyme involved in the degradation of cAMP) was measured in a cell-free assay. Results Western blot analysis confirmed the presence of PDE2A (a combined cAMP- and cGMP-degrading phosphodiesterase) and of PKA in the mitochondrial preparations.Addition of the membrane-permeable cAMP analog 8-bromo-cAMP or the H 2 S donor NaHS to isolated rat liver mitochondria both increased oxygen consumption rate (OCR) and ATP turnover, as well as the maximal mitochondrial respiration elicited by the uncoupling agent FCCP. The positive bioenergetic effects of 8-bromo-cAMP and of H 2 S were both attenuated by the selective PKA inhibitor Rp-cAMP. H 2 S exerted a concentration-dependent inhibitory effect on the activity of recombinant PDE2A by H 2 S in vitro . Conclusions The current data indicate that H 2 S, in addition to acting as a direct electron donor to the mitochondria, stimulates mitochondrial electron transport and cellular bioenergetic effects by activating a secondary pathway that involves inhibition of phosphodiesterase activity, enhancement of cAMP levels and stimulation of mitochondrial PKA. Based on recent publications, the likely mode of the enhancement of mitochondrial electron transport by PKA may involve the direct phosphorylation of specific electron transport chain components.

Dual Contradictory Effect of H-89 on Neuronal Retraction, Death and Inflammation in Differentiated PC12 Cells Subjected to Oxidative Stress

Interrelation between oxidative stress and neuro-inflammation has been discussed extensively to contribute to neuronal dysfunction in neurodegenerative disorders. In this manner, it seems that there is an intriguing link between protein kinase A (PKA), neuronal apoptosis and inflammation. Rat PC12 pheochromocytoma cell can be induced to differentiate into neuron-like cells possessing elongated neurites by nerve growth factor. In this study, we investigated the effect of H-89, a selective inhibitor of PKA, on the neurite retraction along with evaluation of cell death and inflammatory markers in the differentiated PC12 cells, exposed to H2O2. We found that dose-dependent inhibition of PKA by low and medium concentrations of H-89 (5, 7 and 10 μM) enhanced the parameters of neurite outgrowth and complexity in the cells co-treated with H2O2 as an oxidative stress. Similar concentrations of H-89 significantly inhibited cell death and neurite retraction induced by oxidative stress. Components of TNF-α-NFκB-COX-2 axis, a discussed pathway in neuroinflammation, downregulated dose-dependently by administration of H-89 in H2O2-induced PC12 cells. In this condition, PKA inhibition by the high concentrations of H-89 (15 and 20 μM) led to enhanced cell death and inflammation with decreased neurite outgrowth. These findings indicate that H-89 has a dual contradictory effect on oxidative stress and inflammation that affect neurite outgrowth and complexity in differentiated PC12 cells.

Adenosine pretreatment attenuates angiotensin II-mediated p38 MAPK activation in a protein kinase A dependent manner

AbstractBackground: Adenosine is known as a protective and anti-inflammatory nucleoside. Angiotensin II is the main hormone of the renin-angiotensin system. It is associated with endothelial permeability, recruitment, and activation of the immune cells through induction of inflammatory mediators. Matrix metalloproteinase-9 (MMP-9) plays an important role in inflammatory processes mediated by macrophages. Objectives: Investigate whether adenosine pretreatment modulates angiotensin II-induced MMP-9 expression and activation of signaling molecules. Methods: Human monocytic U-937 cells were treated with either adenosine or angiotensin II alone or angiotensin II following a pretreatment with adenosine. Supernatants were analyzed for MMP-9 activity by zymography method. MMP-9 gene expression was analyzed using real-time PCR. Activation of inflammatory mediators IκB-α, NF-κB, JNK, p38 MAPK, and STAT3 were analyzed by a multi-target ELISA kit. Association of Protein kinase A (PKA) in adenosine effects was studied by pre-incubation with H89, a selective PKA inhibitor. Results: Treatment of the cells with angiotensin II significantly increased MMP-9 production (p &lt;0.05). Adenosine pretreatment did not attenuate this angiotensin II effect. Angiotensin II treatment induced NF-κB, JNK and p38 activation. Pretreatment with adenosine prior to angiotensin II stimulation showed a 40% inhibitory effect on p38 induction (p &lt;0.05). This effect was reversed by PKA inhibition. Conclusion: The present data confirmed that monocytic MMP-9 was a target gene for angiotensin II. Adenosine pretreatment did not inhibit MMP-9 increase in response to angiotensin II. However, it showed a potential inhibitory effect on angiotensin II inflammatory signaling.

CAMP-mediated β-adrenergic signaling negatively regulates Gq-coupled receptor-mediated fetal gene response in cardiomyocytes

The treatment with β-blockers causes an enhancement of the norepinephrine-induced fetal gene response in cultured cardiomyocytes. Here, we tested whether the activation of cAMP-mediated β-adrenergic signaling antagonizes α 1-adrenergic receptor (AR)-mediated fetal gene response. To address this question, the fetal gene program, of which atrial natriuretic peptide (ANP) and the β-isoform of myosin heavy chain are classical members, was induced by phenylephrine (PE), an α 1-AR agonist. In cultured neonatal rat cardiomyocytes, we found that stimulation of β-ARs with isoproterenol, a β-AR agonist, inhibited the fetal gene expression induced by PE. Similar results were also observed when cardiomyocytes were treated with forskolin (FSK), a direct activator of adenylyl cyclase, or 8-CPT-6-Phe-cAMP, a selective activator of protein kinase A (PKA). Conversely, the PE-induced fetal gene expression was further upregulated by H89, a selective PKA inhibitor. To evaluate whether these results could be generalized to Gq-mediated signaling and not specifically to α 1-ARs, cardiomyocytes were treated with prostaglandin F 2α, another Gq-coupled receptor agonist, which is able to promote fetal gene expression. This treatment caused an increase of both ANP mRNA and protein levels, which was almost completely abolished by FSK treatment. The capability of β-adrenergic signaling to regulate the fetal gene expression was also evaluated in vivo conditions by using β1- and β2-AR double knockout mice, in which the predominant cardiac β-AR subtypes are lacking, or by administering isoproterenol (ISO), a β-AR agonist, at a subpressor dose. A significant increase of the fetal gene expression was found in β 1- and β 2-AR gene deficient mice. Conversely, we found that ANP, β-MHC and skACT mRNA levels were significantly decreased in ISO-treated hearts. Collectively, these data indicate that cAMP-mediated β-adrenergic signaling negatively regulates Gq cascade activation-induced fetal gene expression in cultured cardiomyocytes and that this inhibitory regulation is already operative in the mouse heart under physiological conditions.

Activation of the β-Adrenoceptor–Protein Kinase A Signaling Pathway within the Ventral Bed Nucleus of the Stria Terminalis Mediates the Negative Affective Component of Pain in Rats

Pain is an unpleasant sensory and emotional experience. The neural systems underlying the sensory component of pain have been studied extensively, but we are only beginning to understand those underlying its affective component. The bed nucleus of the stria terminalis (BNST) has been implicated in stress responses and negative affective states, such as anxiety, fear, and aversion. Recently, we demonstrated the crucial role of the BNST in the negative affective component of pain using the conditioned place aversion (CPA) test. In the present study, we investigated the involvement of the beta-adrenoceptor-protein kinase A (PKA) signaling pathway within the BNST, in particular, within the ventral part of the BNST (vBNST), in pain-induced aversion in male Sprague Dawley rats. In vivo microdialysis showed that extracellular noradrenaline levels within the vBNST were significantly increased by intraplantar formalin injection. Using the CPA test, we found that intra-vBNST injection of timolol, a beta-adrenoceptor antagonist, dose-dependently attenuated the intraplantar-formalin-induced CPA (F-CPA) without reducing nociceptive behaviors. Experiments with subtype-selective antagonists demonstrated the essential role of beta(2)-adrenoceptors in F-CPA. Intra-vBNST injection of isoproterenol, a beta-adrenoceptor agonist, dose-dependently produced CPA even in the absence of noxious stimulation. This isoproterenol-induced CPA was reversed by the coinjection of Rp-cyclic adenosine monophosphorothioate (Rp-cAMPS), a selective PKA inhibitor. Furthermore, intra-vBNST injection of Rp-cAMPS dose-dependently attenuated the F-CPA. Together, these results suggest that PKA activation within the vBNST via the enhancement of beta-adrenergic transmission is important for the negative affective component of pain.

The involvement of protein kinase A in the immune response of Galleria mellonella larvae to bacteria.

The role of protein kinase A (PKA) in the humoral immune response of the greater wax moth Galleria mellonella larvae to live gram-positive bacteria Micrococcus lysodeikticus and gram-negative bacteria Escherichia coli was investigated. The immune challenge of larvae with both kinds of bacteria caused an increase in fat body PKA activity depending on the injected bacteria. Gram-positive M. lysodeikticus was a much better inducer of the enzyme activity than gram-negative E. coli. The PKA activity was increased about 2.5-fold and 1.5-fold, after M. lysodeikticus and E. coli injection, respectively. The in vivo inhibition of the enzyme activity by a cell permeable selective PKA inhibitor, Rp-8-Br-cAMPS, was correlated with considerable changes of fat body lysozyme content and hemolymph antimicrobial activity in bacteria-challenged insects. The kinetics of changes were different and dependent on the bacteria used for the immune challenge of G. mellonella larvae.

Regulation of transient receptor potential channels of melastatin type 8 (TRPM8): Effect of cAMP, cannabinoid CB 1 receptors and endovanilloids

The transient receptor potential channel of melastatin type 8 (TRPM8), which is gated by low (< 25 °C) temperature and chemical compounds, is regulated by protein kinase C-mediated phosphorylation in a way opposite to that observed with the transient receptor potential channel of vanilloid type 1 (TRPV1), i.e. by being desensitized and not sensitized. As TRPV1 is sensitized also by protein kinase A (PKA)-mediated phosphorylation, we investigated the effect of two activators of the PKA pathway, 8-Br-cAMP and forskolin, on the activity of menthol and icilin at TRPM8 in HEK-293 cells stably overexpressing the channel (TRPM8-HEK-293 cells). We also studied the effect on TRPM8 of: (1) a series of compounds previously shown to activate or antagonize TRPV1, and (2) co-stimulation of transiently co-expressed cannabinoid CB 1 receptors. Both 8-Br-cAMP (100 μM) and forskolin (10 μM) right-shifted the dose–response curves for the TRPM8-mediated effect of icilin and menthol on intracellular Ca 2+. The inhibitory effects of 8-Br-cAMP and forskolin were attenuated by the selective PKA inhibitor Rp-cAMP-S. Stimulation of human CB 1 receptors transiently co-expressed in TRPM8-HEK-293 cells also inhibited TRPM8 response to icilin. Finally, some TRPV1 agonists and antagonists, but not iodinated antagonists, antagonized icilin- and much less so menthol-, induced TRPM8 activation. Importantly, the endovanilloids/endocannabinoids, anandamide and NADA, also antagonized TRPM8 at submicromolar concentrations. Although these findings need to be confirmed by experiments directly measuring TRPM8 activity in natively TRPM8-expressing cells, they support the notion that the same regulatory events have opposing actions on TRPM8 and TRPV1 receptors and identify anandamide and NADA as the first potential endogenous functional antagonists of TRPM8 channels.

The linoleic acid derivative DCP-LA selectively activates PKC-ɛ, possibly binding to the phosphatidylserine binding site

This study examined the effect of 8-[2-(2-pentyl-cyclopropylmethyl)-cyclopropyl]-octanoic acid (DCP-LA), a newly synthesized linoleic acid derivative with cyclopropane rings instead of cis-double bonds, on protein kinase C (PKC) activity. In the in situ PKC assay with reverse-phase high-performance liquid chromatography, DCP-LA significantly activated PKC in PC-12 cells in a concentration-dependent (10 nM-100 microM) manner, with the maximal effect at 100 nM, and the DCP-LA effect was blocked by GF109203X, a PKC inhibitor, or a selective inhibitor peptide of the novel PKC isozyme PKC-epsilon. Furthermore, DCP-LA activated PKC in HEK-293 cells that was inhibited by the small, interfering RNA against PKC-epsilon. In the cell-free PKC assay, of the nine isozymes examined here, DCP-LA most strongly activated PKC-epsilon, with >7-fold potency over other PKC isozymes, in the absence of dioleoyl-phosphatidylserine and 1,2-dioleoyl-sn-glycerol; instead, the DCP-LA action was inhibited by dioleoyl-phosphatidylserine. DCP-LA also activated PKC-gamma, a conventional PKC, but to a much lesser extent compared with that for PKC-epsilon, by a mechanism distinct from PKC-epsilon activation. Thus, DCP-LA serves as a selective activator of PKC-epsilon, possibly by binding to the phosphatidylserine binding site on PKC-epsilon. These results may provide fresh insight into lipid signaling in PKC activation.