Protein Kinase A-dependent Mechanism Research Articles

Activation of histamine type 2 receptors enhances intrinsic excitability of medium spiny neurons in the nucleus accumbens.

Histaminergic neurons are exclusively located in the hypothalamic tuberomammillary nucleus, from where they project to many brain areas including the nucleus accumbens (NAc), a brain area that integrates diverse monoaminergic inputs to coordinate motivated behaviours. While the NAc expresses various histamine receptor subtypes, the mechanisms by which histamine modulates NAc activity are still poorly understood. Using whole‐cell patch‐clamp recordings, we found that pharmacological activation of histamine 2 (H2) receptors elevates the excitability of NAc medium spiny neurons (MSNs), while activation of H1 receptors failed to significantly affect MSN excitability. The evoked firing of MSNs increased after seconds of local H2 agonist administration and remained elevated for minutes. H2 receptor (H2R) activation accelerated subthreshold depolarization in response to current injection, reduced the latency to fire, diminished action potential afterhyperpolarization and increased the action potential half‐width. The increased excitability was protein kinase A‐dependent and associated with decreased A‐type K+ currents. In addition, selective pharmacological inhibition of the Kv4.2 channel, the main molecular determinant of A‐type K+ currents in MSNs, mimicked and occluded the increased excitability induced by H2R activation. Our results indicate that histaminergic transmission in the NAc increases MSN intrinsic excitability through H2R‐dependent modulation of Kv4.2 channels. Activation of H2R will significantly alter spike firing in MSNs in vivo, and this effect could be an important mechanism by which these receptors mediate certain aspects of goal‐induced behaviours.Key points Histamine is synthesized and released by hypothalamic neurons of the tuberomammillary nucleus and serves as a general modulator for whole‐brain activity including the nucleus accumbens.Histamine receptors type 2 (HR2), which are expressed in the nucleus accumbens, couple to Gαs/off proteins which elevate cyclic adenosine monophosphate levels and activate protein kinase A.Whole‐cell patch‐clamp recordings revealed that H2R activation increased the evoked firing in medium spiny neurons of the nucleus accumbens via protein kinase A‐dependent mechanisms.HR2 activation accelerated subthreshold depolarization in response to current injection, reduced the latency to fire, diminished action potential medium after‐hyperpolarization and increased the action potential half‐width. HR2 activation also reduced A‐type potassium current.Selective pharmacological inhibition of the Kv4.2 channel mimicked and occluded the increased excitability induced by H2R activation.

Angiopoietin-like peptide 4 regulates insulin secretion and islet morphology

Insulin secretion from pancreatic islet β-cells is primarily regulated by the blood glucose level, and also modulated by a number of biological factors produced inside the islets or released from remote organs. Previous studies have shown that angiopoietin-like protein 4 (Angptl4) controls glucose and lipid metabolism through its actions in the liver, adipose tissue, and skeletal muscles. In this present study, we investigated the possible role of Angptl4 in the regulation of insulin secretion from pancreatic islets. Angptl4 was found to be highly expressed in the α-cells but not β-cells of rodent islets. Moreover, treatment of rodent islets with Angptl4 peptide potentiated glucose-stimulated insulin secretion through a protein kinase A-dependent mechanism. Consistently, Angptl4 knockout mice showed impaired glucose tolerance. In the cultured islets from Angptl4 knockout mice, glucose-stimulated insulin secretion was significantly lower than in islets from wild type mice. Angptl4 peptide replacement partially reversed this reduction. Moreover, Angptl4 knockout mice had dysmorphic islets with abnormally distributed α-cells. In contrast, the β-cell mass and distribution were not significantly altered in these knockout mice. Our current data collectively suggest that Angptl4 may play a critical role in the regulation of insulin secretion and islet morphogenesis.

Tauroursodeoxycholate Protects Rat Hepatocytes from Bile Acid-Induced Apoptosis via β1-Integrin- and Protein Kinase A-Dependent Mechanisms

Background/Aims: Ursodeoxycholic acid, which in vivo is rapidly converted into its taurine conjugate, is frequently used for the treatment of cholestatic liver disease. Apart from its choleretic effects, tauroursodeoxycholate (TUDC) can protect hepatocytes from bile acid-induced apoptosis, but the mechanisms underlying its anti-apoptotic effects are poorly understood. Methods: These mechanisms were investigated in perfused rat liver and isolated rat hepatocytes. Results: It was found that TUDC inhibited the glycochenodeoxycholate (GCDC)-induced activation of the CD95 death receptor at the level of association between CD95 and the epidermal growth factor receptor. This was due to a rapid TUDC-induced β₁-integrin-dependent cyclic AMP (cAMP) signal with induction of the dual specificity mitogen-activated protein (MAP) kinase phosphatase 1 (MKP-1), which prevented GCDC-induced phosphorylation of mitogen-activated protein kinase kinase 4 (MKK4) and c-jun-NH₂-terminal kinase (JNK) activation. Furthermore, TUDC induced a protein kinase A (PKA)-mediated serine/threonine phosphorylation of the CD95, which was recently identified as an internalization signal for CD95. Furthermore, TUDC inhibited GCDC-induced CD95 targeting to the plasma membrane in a β₁-integrin-and PKA-dependent manner. In line with this, the β₁-integrin siRNA knockdown in sodium taurocholate cotransporting polypeptide (Ntcp)-transfected HepG2 cells abolished the protective effect of TUDC against GCDC-induced apoptosis. Conclusion: TUDC exerts its anti-apoptotic effect via a β₁-integrin-mediated formation of cAMP, which prevents CD95 activation by hydrophobic bile acids at the levels of JNK activation and CD95 serine/threonine phosphorylation.

Cathepsin S Causes Inflammatory Pain via Biased Agonism of PAR2 and TRPV4

Serine proteases such as trypsin and mast cell tryptase cleave protease-activated receptor-2 (PAR2) at R(36)↓S(37) and reveal a tethered ligand that excites nociceptors, causing neurogenic inflammation and pain. Whether proteases that cleave PAR2 at distinct sites are biased agonists that also induce inflammation and pain is unexplored. Cathepsin S (Cat-S) is a lysosomal cysteine protease of antigen-presenting cells that is secreted during inflammation and which retains activity at extracellular pH. We observed that Cat-S cleaved PAR2 at E(56)↓T(57), which removed the canonical tethered ligand and prevented trypsin activation. In HEK and KNRK cell lines and in nociceptive neurons of mouse dorsal root ganglia, Cat-S and a decapeptide mimicking the Cat-S-revealed tethered ligand-stimulated PAR2 coupling to Gαs and formation of cAMP. In contrast to trypsin, Cat-S did not mobilize intracellular Ca(2+), activate ERK1/2, recruit β-arrestins, or induce PAR2 endocytosis. Cat-S caused PAR2-dependent activation of transient receptor potential vanilloid 4 (TRPV4) in Xenopus laevis oocytes, HEK cells and nociceptive neurons, and stimulated neuronal hyperexcitability by adenylyl cyclase and protein kinase A-dependent mechanisms. Intraplantar injection of Cat-S caused inflammation and hyperalgesia in mice that was attenuated by PAR2 or TRPV4 deletion and adenylyl cyclase inhibition. Cat-S and PAR2 antagonists suppressed formalin-induced inflammation and pain, which implicates endogenous Cat-S and PAR2 in inflammatory pain. Our results identify Cat-S as a biased agonist of PAR2 that causes PAR2- and TRPV4-dependent inflammation and pain. They expand the role of PAR2 as a mediator of protease-driven inflammatory pain.

Cyclooxygenase-2 Inhibits T Helper Cell Type 9 Differentiation during Allergic Lung Inflammation via Down-regulation of IL-17RB

Helper CD4(+) T cell subsets, including IL-9- and IL-10-producing T helper cell type 9 (Th9) cells, exist under certain inflammatory conditions. Cyclooxygenase (COX)-1 and COX-2 play important roles in allergic lung inflammation and asthma. It is unknown whether COX-derived eicosanoids regulate Th9 cells during allergic lung inflammation. To determine the role of COX metabolites in regulating Th9 cell differentiation and function during allergic lung inflammation. COX-1(-/-), COX-2(-/-), and wild-type (WT) mice were studied in an in vivo model of ovalbumin-induced allergic inflammation and an in vitro model of Th9 differentiation using flow cytometry, cytokine assays, confocal microscopy, real-time PCR, and immunoblotting. In addition, the role of specific eicosanoids and their receptors was examined using synthetic prostaglandins (PGs), selective inhibitors, and siRNA knockdown. Experimental endpoints were not different between COX-1(-/-) and WT mice; however, the percentage of IL-9(+) CD4(+) T cells was increased in lung, bronchoalveolar lavage fluid, lymph nodes, and blood of allergic COX-2(-/-) mice relative to WT. Bronchoalveolar lavage fluid IL-9 and IL-10, serum IL-9, and lung IL-17RB levels were significantly increased in allergic COX-2(-/-) mice or in WT mice treated with COX-2 inhibitors. IL-9, IL-10, and IL-17RB expression in vivo was inhibited by PGD2 and PGE2, which also reduced Th9 cell differentiation of murine and human naive CD4(+) T cells in vitro. Inhibition of protein kinase A significantly increased Th9 cell differentiation of naive CD4(+) T cells isolated from WT mice in vitro. COX-2-derived PGD2 and PGE2 regulate Th9 cell differentiation by suppressing IL-17RB expression via a protein kinase A-dependent mechanism.

Inflammatory Signals Enhance piezo2-Mediated Mechanosensitive Currents

Heightened nociceptor function caused by inflammatory mediators such as bradykinin (BK) contributes to increased pain sensitivity (hyperalgesia) to noxious mechanical and thermal stimuli. Although it is known that sensitization of the heat transducer TRPV1 largely subserves thermal hyperalgesia, the cellular mechanisms underlying mechanical hyperalgesia have been elusive. The role of the mechanically activated (MA) channel piezo2 present in mammalian sensory neurons is unknown. We tested the hypothesis that piezo2 activity is enhanced by BK, an algogenic peptide that induces mechanical hyperalgesia within minutes. We elicited piezo2-mediated currents by poking transiently transfected HEK293T cells with a blunt glass probe. Piezo2 current amplitude increases and inactivation is slowed by activation of exogenous bradykinin receptor B2. The area under the curve was enhanced 6.5-fold by BK compared with vehicle. Inclusion of GDPbetaS in the pipette occluded both effects on amplitude and inactivation. The protein kinase A (PKA) and protein kinase C (PKC) agonists 8-BrcAMP and PMA, respectively, enhanced piezo2 activity. No enhancement of piezo2-mediated currents was observed during exposure to the PLC activator m-3M3FBS (25 microM), suggesting phosphatidylinositol-PLC pathways do not increase piezo2 activity; inhibition of store release by thapsigargin had no effect on BK-induced changes. The PKA inhibitor H-89 and PKC inhibitor BIM I applied together effectively abrogated the BK-induced changes. Finally, piezo2-dependent MA currents in a class of native sensory neurons are enhanced 8-fold by BK via a PKA and PKC dependent mechanism, similarly to that observed in HEK cells. Thus, piezo2 sensitization may contribute to PKA- and PKC-mediated mechanical hyperalgesia. The mechanism described here differs from another model of mechanical hyperalgesia (epinephrine-induced PKCe-mediated) in which the downstream effector of cAMP is EPAC rather than PKA, since the BK effects observed here require PKA activity, and neither PI-PLC nor phospholipase D appears to be involved.

Dual Inhibition of γ-Oryzanol on Cellular Melanogenesis: Inhibition of Tyrosinase Activity and Reduction of Melanogenic Gene Expression by a Protein Kinase A-Dependent Mechanism

The in vitro effects on melanogenesis of γ-oryzanol (1), a rice bran-derived phytosterol, were investigated. The melanin content in B16F1 cells was significantly and dose-dependently reduced (-13% and -28% at 3 and 30 μM, respectively). Tyrosinase enzyme activity was inhibited by 1 both in a cell-free assay and when analyzed based on the measurement of cellular tyrosinase activity. Transcriptome analysis was performed to investigate the biological pathways altered by 1, and it was found that gene expression involving protein kinase A (PKA) signaling was markedly altered. Subsequent analyses revealed that 1 stimulation in B16 cells reduced cytosolic cAMP concentrations, PKA activity (-13% for cAMP levels and -40% for PKA activity), and phosphorylation of the cAMP-response element binding protein (-57%), which, in turn, downregulated the expression of microphthalmia-associated transcription factor (MITF; -59% for mRNA and -64% for protein), a key melanogenic gene transcription factor. Accordingly, tyrosinase-related protein 1 (TRP-1; -69% for mRNA and -82% for protein) and dopachrome tautomerase (-51% for mRNA and -92% for protein) in 1-stimulated B16F1 cells were also downregulated. These results suggest that 1 has dual inhibitory activities for cellular melanogenesis by inhibiting tyrosinase enzyme activity and reducing MITF and target genes in the PKA-dependent pathway.

Cardiotrophin-1 induces sarcoplasmic reticulum Ca2+ leak and arrhythmogenesis in adult rat ventricular myocytes

Plasma levels of cardiotrophin-1 (CT-1) are elevated in several cardiovascular diseases and are correlated with the severity of the pathology. However, the mechanisms by which this inflammatory cytokine participates in the pathology of the heart are not completely understood. It is well established that alterations in intracellular calcium ([Ca(2+)](i)) handling are involved in cardiac dysfunction during heart failure, but it is unknown whether CT-1 modulates [Ca(2+)](i) handling in adult cardiomyocytes. Here we have analyzed for the first time the effects of CT-1 on [Ca(2+)](i) homeostasis in adult rat cardiomyocytes. L-type calcium current (I(CaL)) was recorded using patch-clamp techniques, and [Ca(2+)](i) transients and Ca(2+) sparks were viewed by confocal microscopy. Treatment of cardiomyocytes with 1 nM CT-1 for 20-60 min induced a significant increase in I(CaL) density, [Ca(2+)](i) transients, and cell shortening compared with control cells. Our study reveals that CT-1 increases I(CaL) by a protein kinase A-dependent mechanism, and Ca(2+) sparks by a Ca(2+)/calmodulin kinase II-dependent and protein kinase A-independent mechanism. Cardiomyocytes treated with CT-1 exhibited a higher occurrence of arrhythmogenic behaviour, manifested as spontaneous Ca(2+) waves and aftercontractions. Our findings provide evidence that cardiomyocytes treated with CT-1 present high spontaneous Ca(2+) release during diastole, a mechanism linked to arrhythmogenicity in the pathologic heart.

Glucagon-Like Peptide-1 Receptor Agonist, Exendin-4, Regulates Feeding-Associated Neuropeptides in Hypothalamic Neurons in Vivo and in Vitro

Exendin-4, a long-acting glucagon-like peptide-1 receptor (GLP-1R) agonist, is a potential regulator of feeding behavior through its ability to inhibit gastric emptying, reduce food intake, and induce satiety. GLP-1R activation by exendin-4 induces anorexia; however, the specific populations of neuropeptidergic neurons activated by exendin-4 within the hypothalamus, the central regulator of energy homeostasis, remain unclear. This study determines whether exendin-4 regulates hypothalamic neuropeptide expression and explores the signaling mechanisms involved. The distribution and quantity of exendin-4-induced c-Fos immunoreactivity were evaluated to determine activation of α-melanocyte-stimulating hormone/proopiomelanocortin, neuropeptide Y, neurotensin (NT), and ghrelin neurons in hypothalamic nuclei during exendin-4-induced anorexia in mice. Additionally, exendin-4 action on NT and ghrelin transcript regulation was examined in immortalized hypothalamic neurons. With anorexia induced by intracerebroventricular exendin-4, α-melanocyte-stimulating hormone/proopiomelanocortin and neuropeptide Y neurons were activated in the arcuate nucleus, with simultaneous activation of NT-expressing neurons in the paraventricular nucleus, and ghrelin-expressing neurons in the arcuate nucleus, paraventricular nucleus, and periventricular hypothalamus, suggesting that neurons in one or more of these areas mediate the anorexic action of exendin-4. In the hypothalamic neuronal cell models, exendin-4 increased cAMP, cAMP response element-binding protein/activating transcription factor-1 and c-Fos activation, and via a protein kinase A-dependent mechanism regulated NT and ghrelin mRNA expression, indicating that these neuropeptides may serve as downstream mediators of exendin-4 action. These findings provide a previously unrecognized link between central GLP-1R activation by exendin-4 and the regulation of hypothalamic NT and ghrelin. Further understanding of this central GLP-1R activation may lead to safe and effective therapeutics for the treatment of metabolic disorders.

Activation of TRPV1 reduces vascular lipid accumulation and attenuates atherosclerosis

Activation of transient receptor potential vanilloid type-1 (TRPV1) channels may affect lipid storage and the cellular inflammatory response. Now, we tested the hypothesis that activation of TRPV1 channels attenuates atherosclerosis in apolipoprotein E knockout mice (ApoE(-/-)) but not ApoE(-/-)TRPV1(-/-) double knockout mice on a high-fat diet. Both TRPV1 mRNA and protein expression were identified in vascular smooth muscle cells (VSMC) and in aorta from C57BL/6J mice using RT-PCR, immunoblotting, and immunohistochemistry. In vitro, activation of TRPV1 by the specific agonists capsaicin and resiniferatoxin dose-dependently increased cytosolic calcium and significantly reduced the accumulation of lipids in VSMC from C57BL/6J mice but not from TRPV1(-/-) mice. TRPV1 activation increased ATP-binding cassette transporter A1 (ABCA1) expression and reduced low-density lipoprotein-related protein 1 (LRP1) expression in VSMC by calcium-dependent and calcineurin- and protein kinase A-dependent mechanisms. These results showed increased cellular cholesterol efflux and reduced cholesterol uptake. In vivo, long-term activation of TRPV1 by capsaicin for 24 weeks increased ABCA1 and reduced LRP1 expression in aorta from ApoE(-/-) mice on a high-fat diet. Long-term activation of TRPV1 significantly reduced lipid storage and atherosclerotic lesions in the aortic sinus and in the thoracoabdominal aorta from ApoE(-/-) mice but not from ApoE(-/-)TRPV1(-/-) mice on a high-fat diet. These findings indicated that TRPV1 activation ameliorates high-fat diet-induced atherosclerosis. Activation of TRPV1 may be a novel therapeutic tool to attenuate atherosclerosis caused by a high-fat diet.

Inverse agonism of cannabinoid CB1 receptor blocks the adhesion of encephalitogenic T cells in inflamed brain venules by a protein kinase A-dependent mechanism

It is well known that the cannabinoid system has a significant role in the regulation of the immune responses. Cannabinoid receptors CB1 and CB2 are expressed on T lymphocytes and mediate the immunomodulatory effects of cannabinoids on T cell functions. Here we show that the treatment of proteolipid protein (PLP)139–151-specific T cells with SR141716A, a CB1 inverse agonist and prototype of the diarylpyrazoles series, induced a strong inhibition of firm adhesion in inflamed brain venules in intravital microscopy experiments. In contrast, SR144528, a potent CB2 inverse agonist, had no significant effect on both rolling and arrest of activated T cells. In addition, two analogs of SR141716A and CB1 inverse agonists, AM251 and AM281 inhibited encephalitogenic T cell adhesion suggesting that selective CB1 inverse agonism interfere with lymphocyte trafficking in the CNS. Flow cytometry experiments showed that CB1 inverse agonists have no effect on adhesion molecule expression suggesting that CB1 blockade interferes with signal transduction pathways controlling T cell adhesion in inflamed brain venules. In addition, integrin clustering was not altered after treatment with CB1 inverse agonists suggesting that adhesion blockade is not due to the modulation of integrin valency. Notably, the inhibitory effect exerted by AM251 and AM281 on the adhesive interactions was completely reverted in the presence of protein kinase A (PKA) inhibitor H89, suggesting that cAMP and PKA activation play a key role in the adhesion blockade mediated by CB1 inverse agonists. To further strengthen these results and unveil a previously unknown inhibitory role of cAMP on activated T cell adhesion in vivo in the context of CNS inflammation, we showed that intracellular increase of cAMP induced by treatment with Bt2cAMP, a permeable analog of cAMP, and phosphodiesterase (PDE) inhibitor theophylline efficiently blocked the arrest of encephalitogenic T cells in inflamed brain venules. Our data show that modulation of CB1 function has anti-inflammatory effects and suggests that inverse agonism of CB1 block signal transduction mechanisms controlling encephalitogenic T cells adhesion in inflamed brain venules by a PKA-dependent mechanism.

Multiple Cross Talk between Angiotensin II, Bradykinin, and Insulin Signaling in the Cortical Thick Ascending Limb of Rat Kidney

Cortical thick ascending limb (CTAL) naturally expresses the angiotensin II (AngII) receptor type 1A (AT(1)-R), the bradykinin (BK) receptor type 2 (B(2)-R), and the insulin receptor. This segment is made of a single morphologically distinct cell type. AngII and BK are involved in same transduction pathways but differ markedly in their physiological actions on Na(+) transport. Besides, the insulin signaling intersects with those of AngII and BK at multiple levels and especially by stimulation on Na(+) reabsorption. Thus, the CTAL is a biologically suitable model to study the cross talk between G protein-coupled receptors or G protein-coupled receptors and receptor tyrosine kinase. In this work, the cross talks between AngII, BK, and insulin signaling are studied in rat CTAL by measuring changes in [Ca(2+)](i). We show that BK exerts negative modulatory effects on AngII-induced [Ca(2+)](i) responses dependent on tyrosine kinase and MAPK pathways. Moreover, in the presence of BK, AngII-induced Na(+) transport is suppressed. These effects suggest an interaction between AT(1)-R and B(2)-R. We show a positive interaction between the insulin receptor and the AT(1)-R through a protein kinase A-dependent mechanism that involves MAPK cascade, leading to the stimulation of the Ca(2+) influx induced by AngII. The presence of such interactions brings additional arguments for a complex and fine regulation of CTAL functions and puts forward the potentially beneficial effect of BK across this segment, in case of hyperinsulinemia or insulin resistance, by its negative feedback on AngII actions.

Noradrenaline reduces the ATP-stimulated phosphorylation of p38 MAP kinase via β-adrenergic receptors–cAMP–protein kinase A-dependent mechanism in cultured rat spinal microglia

To elucidate the involvement of the noradrenergic system in the regulation of spinal microglial activity, we examined the effects of noradrenaline (NA) on the phosphorylation of three MAP kinases (extracellular signal-regulated kinase (ERK), p38, or c-Jun N-terminal kinase (JNK)) stimulated by ATP in rat cultured spinal microglia using Western blotting. ATP (100 μM) quickly induced the phosphorylation of three MAP kinases and MKK3/6, which are upstream kinases of p38. Under these conditions, NA inhibited only the ATP-stimulated phosphorylation of p38 in a time (30–60 min)- and dose (10–100 μM)-dependent manner, but did not affect those of ERK, JNK, or MKK3/6. The inhibitory action of NA was completely reversed by pretreatment with propranolol, an antagonist for β-adrenoceptors, or both atenolol and ICI118551, selective antagonists for β1 and β2, respectively. Treatment with dibutyryl cAMP or the selective activator of PKA mimicked the inhibitory effect of NA. Furthermore, treatment with KT5720, an inhibitor of protein kinase A, completely blocked the action of NA. These data suggest that NA could control the activation of p38 through the β1/2-adrenergic pathways, which include the production of cAMP and the activation of PKA. Simultaneously, we found that NA also markedly inhibited the ATP-induced increase in the expression of tumor necrosis factor (TNF)-α mRNA through β-adrenergic pathways. Furthermore, preincubation with either actinomycin D or cyclohexamide, general inhibitors of transcription or protein synthesis, respectively, almost completely blocked the inhibitory action of NA on the ATP-stimulated phosphorylation of p38. These results suggest that de novo synthesis of certain factors by NA through β-adrenoceptors would participate in the modulation of p38 activity. Thus, the inhibitory system via β1/2-adrenergic pathways in spinal microglia appears to have an important role in the modulation of microglial functions through the downregulation of p38 activity.

Rotenone inhibits delayed rectifier K+ current via a protein kinase A-dependent mechanism

Voltage-gated K+ channels (K(V)) regulate cell electrical properties, proliferation, migration, and death. Rotenone is a mitochondrial inhibitor, influencing activity of many channels that potentially participate in cell death processes, but its effect on K(V) channel in neurons remains unclear. This study used whole-cell patch clamp methods and found that rotenone concentration dependently decreased delayed rectifier K+ current (I(DR)) amplitude in cultured ventral mesencephalic neurons, but had no effect on A-type current (I(A)) peak amplitude. Furthermore, the protein kinase A inhibitor H-89 prevented rotenone-induced decrease in I(DR). The inhibition of I(DR) by rotenone was mimicked by extracellular application of forskolin. In summary, our results indicate that rotenone acutely decreased I(DR) currents in cultured mesencephalic neurons via a protein kinase A-dependent mechanism.

Octopamine Regulates Sleep inDrosophilathrough Protein Kinase A-Dependent Mechanisms

Sleep is a fundamental process, but its regulation and function are still not well understood. The Drosophila model for sleep provides a powerful system to address the genetic and molecular mechanisms underlying sleep and wakefulness. Here we show that a Drosophila biogenic amine, octopamine, is a potent wake-promoting signal. Mutations in the octopamine biosynthesis pathway produced a phenotype of increased sleep, which was restored to wild-type levels by pharmacological treatment with octopamine. Moreover, electrical silencing of octopamine-producing cells decreased wakefulness, whereas excitation of these neurons promoted wakefulness. Because protein kinase A (PKA) is a putative target of octopamine signaling and is also implicated in Drosophila sleep, we investigated its role in the effects of octopamine on sleep. We found that decreased PKA activity in neurons rendered flies insensitive to the wake-promoting effects of octopamine. However, this effect of PKA was not exerted in the mushroom bodies, a site previously associated with PKA action on sleep. These studies identify a novel pathway that regulates sleep in Drosophila.

Oleate Reverses Palmitate-induced Insulin Resistance and Inflammation in Skeletal Muscle Cells

Here we report that in skeletal muscle cells the contribution to insulin resistance and inflammation of two common dietary long-chain fatty acids depends on the channeling of these lipids to distinct cellular metabolic fates. Exposure of cells to the saturated fatty acid palmitate led to enhanced diacylglycerol levels and the consequent activation of the protein kinase C/nuclear factor kappaB pathway, finally resulting in enhanced interleukin 6 secretion and down-regulation of the expression of genes involved in the control of the oxidative capacity of skeletal muscle (peroxisome proliferator-activated receptor (PPAR)gamma-coactivator 1alpha) and triglyceride synthesis (acyl-coenzyme A: diacylglycerol acyltransferase 2). In contrast, exposure to the monounsaturated fatty acid oleate did not lead to these changes. Interestingly, co-incubation of cells with palmitate and oleate reversed both inflammation and impairment of insulin signaling by channeling palmitate into triglycerides and by up-regulating the expression of genes involved in mitochondrial beta-oxidation, thus reducing its incorporation into diacylglycerol. Our findings support a model of cellular lipid metabolism in which oleate protects against palmitate-induced inflammation and insulin resistance in skeletal muscle cells by promoting triglyceride accumulation and mitochondrial beta-oxidation through PPARalpha- and protein kinase A-dependent mechanisms.

Role of Epac in cAMP mediated PKA independent mechanism of intestinal Cl‐secretion

cAMP is a major regulator of Cl−secretion. With the recognition that the exchange protein directly activated by cAMP (Epac) transduces cAMP signaling cascades independent of protein kinase A (PKA), we test the hypothesis that both PKA and Epac are involved in forskolin (FSK) stimulated Cl− secretion. FSK is an adenylate cyclase activator. T84 cells were used for short circuit current (Isc) measurement in the Ussing chamber. FSK stimulated Isc was completely inhibited by the additive effects of the PKA inhibitor, H89 (1μM) and the [Ca2+]i chelator, BAPTA‐AM (25μM), suggesting the presence of both PKA dependent and independent mechanisms. Epac1 was found to be expressed in T84 cells. Both FSK and the Epac selective agonist, 8‐pCPT‐2‘‐O‐Me‐cAMP (8‐CPT, 50μM), elevated [Ca2+]i. Activation of Epac1 by 8‐CPT induced Isc in intact T84 cells and in cells permeabilized with nystatin at the basolateral membrane. This effect of 8‐CPT was completely abolished by BAPTA‐AM, but not by H89 (1μM) and the CFTR specific blocker, cftr‐172, suggesting that 8‐CPT stimulated non‐CFTR channel(s) via elevation of [Ca2+]i. FSK stimulated Isc was inhibited by the PLC inhibitor U73122. Both FSK and 8‐CPT increased the amount of activated (GTP‐bound) Rap proteins, which have been shown to activate PLCε to mobilize [Ca2+]i. Our findings in T84 cells were further validated in mouse ileum. 8‐CPT stimulated Isc in mouse ileal sheet and this stimulation was not inhibited by H89. We conclude that PKA and Epac1 contribute to FSK stimulated Cl− secretion in intestine via PKA dependent and Ca2+ dependent pathways, the latter mediated by Epac1‐Rap2‐PLC‐Ca2+ signaling involving non‐CFTR channel(s).

Memantine increases brain production of kynurenic acid via protein kinase A-dependent mechanism

We describe a novel aspect of action of memantine ex vivo, in the brain cortical slices and in vitro, in mixed glial cultures. The drug potently increased the production of kynurenic acid, an endogenous tryptophan metabolite blocking N-methyl-D-aspartate (NMDA) and nicotinic alpha7 receptors. In cortical slices memantine, an open-channel NMDA blocker (100-150 microM), but not the competitive NMDA receptor antagonist, LY235959 increased the production of kynurenic acid. Neither SCH23390, D1 receptor antagonist (50 microM) nor raclopride, D2 receptor antagonist (10 microM) changed the memantine-induced effects. Propranolol (100 microM) has partially reduced its action. Selective cAMP-dependent protein kinase (PKA) inhibitor, KT5720 (1 microM), but not selective protein kinase C (PKC) inhibitor, NPC15437 (30 microM) totally reversed the action of memantine. In mixed glial cultures, 2-24 h incubation with memantine (2-50 microM) enhanced the production of kynurenic acid. Memantine (up to 0.5 mM) has not affected the activity of kynurenic acid biosynthetic enzymes. The obtained data suggest that memantine enhances the production of kynurenic acid in PKA-mediated way. This effect may partially contribute to the therapeutic actions of memantine and be of a potential clinical importance.

A Protein Kinase A-Dependent Mechanism by Which Rotavirus Affects the Distribution and mRNA Level of the Functional Tight Junction-Associated Protein, Occludin, in Human Differentiated Intestinal Caco-2 Cells

We found that at the tight junctions (TJs) of Caco-2 cell monolayers, rhesus monkey rotavirus (RRV) infection induced the disappearance of occludin. Confocal laser scanning microscopy showed the disappearance of occludin from the cell-cell boundaries without modifying the expression of the other TJ-associated proteins, ZO-1 and ZO-3. Western immunoblot analysis of RRV-infected cells showed a significant fall in the levels of the nonphosphorylated form of occludin in both Triton X-100-insoluble and Triton X-100-soluble fractions, without any change in the levels of the phosphorylated form of occludin. Quantitative reverse transcription-PCRs revealed that the level of transcription of the gene that encodes occludin was significantly reduced in RRV-infected cells. Treatment of RRV-infected cells with Rp-cyclic AMP and protein kinase A inhibitors H89 and KT5720 during the time course of the infection restored the distribution of occludin and a normal level of transcription of the gene that encodes occludin.

Anti-apoptotic effect of retinoic acid on retinal progenitor cells mediated by a protein kinase A-dependent mechanism

Retinal progenitor cells (RPCs) are neural stem cells able to differentiate into any normal adult retinal cell type, except for pigment epithelial cells. Retinoic acid (RA) is a powerful growth/differentiation factor that generally causes growth inhibition, differentiation and/or apoptosis. In this study, we demonstrate that RA not only affects mouse RPC differentiation but also improves cell survival by reducing spontaneous apoptotic rate without affecting RPC proliferation. The enhanced cell survival was accompanied by a significant upregulation of the expression of protein kinase A (PKA) and several protein kinase C (PKC) isoforms. Treatment of cells grown in RA-free media with 8-bromoadenosine3',5'-cyclic monophosphate, a known activator of PKA, resulted in an anti-apoptotic effect similar to that caused by RA; whereas the PKA inhibitor N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide dihydrochloride led to a significant ( approximately 32%) increase in apoptosis. In contrast, treatment of RPCs with any of two PKC selective inhibitors, 2,2',3,3',4,4'-hexahydroxy-1,1'-biphenyl-6,6'-dimethanol dimethyl ether and bisindolylmaleimide XI, led to diminished apoptosis; while a PKC activator, phorbol 12-myristate 13-acetate, increased apoptosis. These and other data suggest that the effect of RA on RPC survival is mostly due to the increased anti-apoptotic activity elicited by PKA, which might in turn be antagonized by PKC. Such a mechanism is a new example of tight regulation of important biological processes triggered by RA. Although the detailed mechanisms remain to be elucidated, we provide evidence that the pro-survival effect of RA on RPCs is not mediated by changed expression of p53 or bcl-2, and appears to be independent of beta-amyloid, Fas ligand, TNF-alpha, ganglioside GM1 and ceramide C16-induced apoptotic pathways.