Adenosine A2A receptor (A2AR) is a fine-tune regulator of the collagen1:collagen3 balance

Adenosine is a potent endogenous anti-inflammatory and immunosuppressive metabolite that is a potent modulator of tissue repair. However, the adenosine A_2A receptor (A_2AR)-mediated promotion of collagen synthesis is detrimental in settings such as scarring and scleroderma. The signaling cascade from A_2AR stimulation to increased collagen production is complex and obscure, not least because cAMP and its downstream molecules PKA and Epac1 have been reported to inhibit collagen production. We therefore examined A_2AR-stimulated signaling for collagen production by normal human dermal fibroblasts (NHDF). Collagen1 (Col1) and collagen3 (Col3) content after A_2AR activation by CGS21680 was studied by western blotting. Contribution of PKA and Epac was analyzed by the PKA inhibitor PKI and by knockdowns of the PKA-Cα, -Cβ, -Cγ, Epac1, and Epac2. CGS21680 stimulates Col1 expression at significantly lower concentrations than those required to stimulate Col3 expression. A_2AR stimulates Col1 expression by a PKA-dependent mechanism since PKA inhibition or PKA-Cα and -Cβ knockdown prevents A_2AR-mediated Col1 increase. In contrast, A_2AR represses Col3 via PKA but stimulates both Col1 and Col3 via an Epac2-dependent mechanism. A_2AR stimulation with CGS21680 at 0.1 μM increased Col3 expression only upon PKA blockade. A_2AR activation downstream signaling for Col1 and Col3 expression proceeds via two distinct pathways with varying sensitivity to cAMP activation; more highly cAMP-sensitive PKA activation stimulates Col1 expression, and less cAMP-sensitive Epac activation promotes both Col1 and Col3 expression. These observations may explain the dramatic change in Col1:Col3 ratio in hypertrophic and immature scars, where adenosine is present in higher concentrations than in normal skin.     

EP2 Receptor Signaling Pathways Regulate Classical Activation of Microglia

Activation of EP2 receptors by prostaglandin E2 (PGE₂) promotes brain inflammation in neurodegenerative diseases, but the pathways responsible are unclear. EP2 receptors couple to Gα_s and increase cAMP, which associates with protein kinase A (PKA) and cAMP-regulated guanine nucleotide exchange factors (Epacs). Here, we studied EP2 function and its signaling pathways in rat microglia in their resting state or undergoing classical activation in vitro following treatment with low concentrations of lipopolysaccharide and interferon-γ. Real time PCR showed that PGE₂ had no effect on expression of CXCL10, TGF-β1, and IL-11 and exacerbated the rapid up-regulation of mRNAs encoding cyclooxygenase-2, inducible NOS, IL-6, and IL-1β but blunted the production of mRNAs encoding TNF-α, IL-10, CCL3, and CCL4. These effects were mimicked fully by the EP2 agonist butaprost but only weakly by the EP1/EP3 agonist 17-phenyl trinor PGE₂ or the EP4 agonist CAY10598 and not at all by the EP3/EP1 agonist sulprostone and confirmed by protein measurements of cyclooxygenase-2, IL-6, IL-10, and TNF-α. In resting microglia, butaprost induced cAMP formation and altered the mRNA expression of inflammatory mediators, but protein expression was unchanged. The PKA inhibitor H89 had little or no effect on inflammatory mediators modulated by EP2, whereas the Epac activator 8-(4-chlorophenylthio)-2′-O-methyladenosine 3′,5′-cyclic monophosphate acetoxymethyl ester mimicked all butaprost effects. These results indicate that EP2 activation plays a complex immune regulatory role during classical activation of microglia and that Epac pathways are prominent in this role.     

Anti-inflammatory effects of purine nucleosides, adenosine and inosine, in a mouse model of pleurisy: evidence for the role of adenosine A2 receptors

Adenosine and its metabolite, inosine, have been described as molecules that participate in regulation of inflammatory response. The aim of this study was to investigate the effect of adenosine and inosine in a mouse model of carrageenan-induced pleurisy as well as the participation of adenosine receptors in this response. Injection of carrageenan into the pleural cavity induced an acute inflammatory response characterized by leukocyte migration, pleural exudation, and increased release of interleukin-1β and tumor necrosis factor-α in pleural exudates. The treatment with adenosine (0.3–100 mg/kg, i.p.) and inosine (0.1–300 mg/kg, i.p.) 30 min before carrageenan injection reduced significantly all these parameters analyzed. Our results also demonstrated that A_2A and A_2B receptors seem to mediate the adenosine and inosine effects observed, since pretreatment with selective antagonists of adenosine A_2A (ZM241385) and A_2B (alloxazine) receptors, reverted the inhibitory effects of adenosine and inosine in pleural inflammation. The involvement of A₂ receptors was reinforced with adenosine receptor agonist {"type":"entrez-protein","attrs":{"text":"CGS21680","term_id":"878113053","term_text":"CGS21680"}}CGS21680 treatment, since its anti-inflammatory effects were reversed completely and partially with ZM241385 and alloxazine injection, respectively. Moreover, the combined treatment with subeffective dose of adenosine (0.3 mg/kg) and inosine (1.0 mg/kg) induced a synergistic anti-inflammatory effect. Thus, based on these findings, we propose that inosine contributes with adenosine to exert anti-inflammatory effects in pleural inflammation, reinforcing the notion that endogenous nucleosides play an important role in controlling inflammatory diseases. This effect is likely mediated by the activation of adenosine A₂ subtype receptors and inhibition of production or release of pro-inflammatory cytokines.     

Cross-talk from β-Adrenergic Receptors Modulates α2A-Adrenergic Receptor Endocytosis in Sympathetic Neurons via Protein Kinase A and Spinophilin

Inter-regulation of adrenergic receptors (ARs) via cross-talk is a long appreciated but mechanistically unclear physiological phenomenon. Evidence from the AR literature and our own extensive studies on regulation of α_2AARs by the scaffolding protein spinophilin have illuminated a potential novel mechanism for cross-talk from β to α₂ARs. In the present study, we have characterized a mode of endogenous AR cross-talk in native adrenergic neurons whereby canonical βAR-mediated signaling modulates spinophilin-regulated α_2AAR endocytosis through PKA. Our findings demonstrate that co-activation of β and α_2AARs, either by application of endogenous agonist or by simultaneous stimulation with distinct selective agonists, results in acceleration of endogenous α_2AAR endocytosis in native neurons. We show that receptor-independent PKA activation by forskolin is sufficient to accelerate α_2AAR endocytosis and that α_2AAR stimulation alone drives accelerated endocytosis in spinophilin-null neurons. Endocytic response acceleration by β/α_2AAR co-activation is blocked by PKA inhibition and lost in spinophilin-null neurons, consistent with our previous finding that spinophilin is a substrate for phosphorylation by PKA that disrupts its interaction with α_2AARs. Importantly, we show that α₂AR agonist-mediated α_2AAR/spinophilin interaction is blocked by βAR co-activation in a PKA-dependent fashion. We therefore propose a novel mechanism for cross-talk from β to α₂ARs, whereby canonical βAR-mediated signaling coupled to PKA activation results in phosphorylation of spinophilin, disrupting its interaction with α_2AARs and accelerating α_2AAR endocytic responses. This mechanism of cross-talk has significant implications for endogenous adrenergic physiology and for therapeutic targeting of β and α_2AARs.     

Adenosine A2A receptor activation is determinant for BDNF actions upon GABA and glutamate release from rat hippocampal synaptosomes

Adenosine, through A_2A receptor (A_2AR) activation, can act as a metamodulator, controlling the actions of other modulators, as brain-derived neurotrophic factor (BDNF). Most of the metamodulatory actions of adenosine in the hippocampus have been evaluated in excitatory synapses. However, adenosine and BDNF can also influence GABAergic transmission. We thus evaluated the role of A_2AR on the modulatory effect of BDNF upon glutamate and GABA release from isolated hippocampal nerve terminals (synaptosomes). BDNF (30 ng/ml) enhanced K⁺-evoked [³H]glutamate release and inhibited the K⁺-evoked [³H]GABA release from synaptosomes. The effect of BDNF on both glutamate and GABA release requires tonic activation of adenosine A_2AR since for both neurotransmitters, the BDNF action was blocked by the A_2AR antagonist SCH 58261 (50 nM). In the presence of the A_2AR agonist, {"type":"entrez-protein","attrs":{"text":"CGS21680","term_id":"878113053","term_text":"CGS21680"}}CGS21680 (30 nM), the effect of BDNF on either glutamate or GABA release was, however, not potentiated. It is concluded that both the inhibitory actions of BDNF on GABA release as well as the facilitatory action of the neurotrophin on glutamate release are dependent on the activation of adenosine A_2AR by endogenous adenosine. However, these actions could not be further enhanced by exogenous activation of A_2AR.     

Static magnetic field exposure reproduces cellular effects of the Parkinson's disease drug candidate ZM241385

Background

This study was inspired by coalescing evidence that magnetic therapy may be a viable treatment option for certain diseases. This premise is based on the ability of moderate strength fields (i.e., 0.1 to 1 Tesla) to alter the biophysical properties of lipid bilayers and in turn modulate cellular signaling pathways. In particular, previous results from our laboratory (Wang et al., BMC Genomics, 10, 356 (2009)) established that moderate strength static magnetic field (SMF) exposure altered cellular endpoints associated with neuronal function and differentiation. Building on this background, the current paper investigated SMF by focusing on the adenosine A_2A receptor (A_2AR) in the PC12 rat adrenal pheochromocytoma cell line that displays metabolic features of Parkinson''s disease (PD).

Methodology and Principal Findings

SMF reproduced several responses elicited by ZM241385, a selective A_2AR antagonist, in PC12 cells including altered calcium flux, increased ATP levels, reduced cAMP levels, reduced nitric oxide production, reduced p44/42 MAPK phosphorylation, inhibited proliferation, and reduced iron uptake. SMF also counteracted several PD-relevant endpoints exacerbated by A_2AR agonist {"type":"entrez-protein","attrs":{"text":"CGS21680","term_id":"878113053","term_text":"CGS21680"}}CGS21680 in a manner similar to ZM241385; these include reduction of increased expression of A_2AR, reversal of altered calcium efflux, dampening of increased adenosine production, reduction of enhanced proliferation and associated p44/42 MAPK phosphorylation, and inhibition of neurite outgrowth.

Conclusions and Significance

When measured against multiple endpoints, SMF elicited qualitatively similar responses as ZM241385, a PD drug candidate. Provided that the in vitro results presented in this paper apply in vivo, SMF holds promise as an intriguing non-invasive approach to treat PD and potentially other neurological disorders.     

β-Adrenergic Receptors Activate Exchange Protein Directly Activated by cAMP (Epac), Translocate Munc13-1, and Enhance the Rab3A-RIM1α Interaction to Potentiate Glutamate Release at Cerebrocortical Nerve Terminals

The adenylyl cyclase activator forskolin facilitates synaptic transmission presynaptically via cAMP-dependent protein kinase (PKA). In addition, cAMP also increases glutamate release via PKA-independent mechanisms, although the downstream presynaptic targets remain largely unknown. Here, we describe the isolation of a PKA-independent component of glutamate release in cerebrocortical nerve terminals after blocking Na⁺ channels with tetrodotoxin. We found that 8-pCPT-2′-O-Me-cAMP, a specific activator of the exchange protein directly activated by cAMP (Epac), mimicked and occluded forskolin-induced potentiation of glutamate release. This Epac-mediated increase in glutamate release was dependent on phospholipase C, and it increased the hydrolysis of phosphatidylinositol 4,5-bisphosphate. Moreover, the potentiation of glutamate release by Epac was independent of protein kinase C, although it was attenuated by the diacylglycerol-binding site antagonist calphostin C. Epac activation translocated the active zone protein Munc13-1 from soluble to particulate fractions; it increased the association between Rab3A and RIM1α and redistributed synaptic vesicles closer to the presynaptic membrane. Furthermore, these responses were mimicked by the β-adrenergic receptor (βAR) agonist isoproterenol, consistent with the immunoelectron microscopy and immunocytochemical data demonstrating presynaptic expression of βARs in a subset of glutamatergic synapses in the cerebral cortex. Based on these findings, we conclude that βARs couple to a cAMP/Epac/PLC/Munc13/Rab3/RIM-dependent pathway to enhance glutamate release at cerebrocortical nerve terminals.     

A2A Adenosine Receptors Are Differentially Modulated by Pharmacological Treatments in Rheumatoid Arthritis Patients and Their Stimulation Ameliorates Adjuvant-Induced Arthritis in Rats

A_2A adenosine receptors (ARs) play a key role in the inhibition of the inflammatory process. The purpose of this study was to evaluate the modulation of A_2AARs in rheumatoid arthritis (RA) patients after different pharmacological treatments and to investigate the effect of A_2AAR stimulation in a rat model of arthritis. We investigated A_2AAR density and functionality in RA progression by using a longitudinal study in RA patients before and after methotrexate (MTX), anti-TNFα agents or rituximab treatments. A_2AARs were analyzed by saturation binding assays in lymphocytes from RA patients throughout the 24-month study timeframe. In an adjuvant-induced arthritis model in rats we showed the efficacy of the A_2AAR agonist, CGS 21680 in comparison with standard therapies by means of paw volume assessment, radiographic and ultrasonographic imaging. Arthritic-associated pain was investigated in mechanical allodynia and thermal hyperalgesia tests. IL-10 release following A_2AAR stimulation in lymphocytes from RA patients and in serum from arthritic rats was measured. In lymphocytes obtained from RA patients, the A_2AAR up-regulation was gradually reduced in function of the treatment time and the stimulation of these receptors mediated a significant increase of IL-10 production. In the same cells, CGS 21680 did not affected cell viability and did not produced cytotoxic effects. The A_2AAR agonist CGS 21680 was highly effective, as suggested by the marked reduction of clinical signs, in rat adjuvant-induced arthritis and associated pain. This study highlighted that A_2AAR agonists represent a physiological-like therapeutic alternative for RA treatment as suggested by the anti-inflammatory role of A_2AARs in lymphocytes from RA patients. The effectiveness of A_2AAR stimulation in a rat model of arthritis supported the role of A_2AAR agonists as potential pharmacological treatment for RA.     

Mechanisms involved in the adenosine-induced vasorelaxation to the pig prostatic small arteries

Benign prostatic hypertrophy has been related with glandular ischemia processes and adenosine is a potent vasodilator agent. This study investigates the mechanisms underlying the adenosine-induced vasorelaxation in pig prostatic small arteries. Adenosine receptors expression was determined by Western blot and immunohistochemistry, and rings were mounted in myographs for isometric force recording. A_2A and A₃ receptor expression was observed in the arterial wall and A_2A-immunoreactivity was identified in the adventitia–media junction and endothelium. A₁ and A_2B receptor expression was not obtained. On noradrenaline-precontracted rings, P1 receptor agonists produced concentration-dependent relaxations with the following order of potency: 5′-N-ethylcarboxamidoadenosine (NECA) = {"type":"entrez-protein","attrs":{"text":"CGS21680","term_id":"878113053","term_text":"CGS21680"}}CGS21680 > 2-Cl-IB-MECA = 2-Cl-cyclopentyladenosine = adenosine. Adenosine reuptake inhibition potentiated both NECA and adenosine relaxations. Endothelium removal and ZM241385, an A_2A antagonist, reduced NECA relaxations that were not modified by A₁, A_2B, and A₃ receptor antagonists. Neuronal voltage-gated Ca²⁺ channels and nitric oxide (NO) synthase blockade, and adenylyl cyclase activation enhanced these responses, which were reduced by protein kinase A inhibition and by blockade of the intermediate (IK_Ca)- and small (SK_Ca)-conductance Ca²⁺-activated K⁺ channels. Inhibition of cyclooxygenase (COX), large-conductance Ca²⁺-activated-, ATP-dependent-, and voltage-gated-K⁺ channel failed to modify these responses. These results suggest that adenosine induces endothelium-dependent relaxations in the pig prostatic arteries via A_2A purinoceptors. The adenosine vasorelaxation, which is prejunctionally modulated, is produced via NO- and COX-independent mechanisms that involve activation of IK_Ca and SK_Ca channels and stimulation of adenylyl cyclase. Endothelium-derived NO playing a regulatory role under conditions in which EDHF is non-functional is also suggested. Adenosine-induced vasodilatation could be useful to prevent prostatic ischemia.     

Adenosine A2A receptors inhibit the conductance of NMDA receptor channels in rat neostriatal neurons

Summary Whole-cell patch clamp experiments were carried out in rat striatal brain slices. In a subset of striatal neurons (70–80%), NMDA-induced inward currents were inhibited by the adenosine AZA receptor selective agonist CGS 21680. The non-selective adenosine receptor antagonist 8-(p-sulphophenyl)-theophylline and the AZA receptor selective antagonist 8-(3chlorostyryl) caffeine abolished the inhibitory action of CGS 21680. Intracellular GDP--S, which is known to prevent G protein-mediated reactions, also eliminated the effect of CGS 21680. Extracellular dibutyryl cAMP, a membrane permeable analogue of cAMP, and intracellular Sp-cAMPS, an activator of cAMP-dependent protein kinases (PKA), both abolished the CGS 21680-induced inhibition. By contrast, Rp-cAMPS and PKI 14–24 amide, two inhibitors of PKA had no effect. Intracellular U-73122 (a phospholipase C inhibitor) and heparin (an inositoltriphosphate antagonist) prevented the effect of CGS 21680. Finally, a more efficient buffering of intracellular Ca²⁺ by a substitution of EGTA (11 mM) by BAPTA (5.5 mM) acted like U-73122 or heparin. Hence, AZA receptors appear to negatively modulate NMDA receptor channel conductance via the phospholipase C/inositoltriphosphate/Ca²⁺ pathway rather than the adenylate cyclase/PKA pathway.     

Epac1 mediates protein kinase A–independent mechanism of forskolin-activated intestinal chloride secretion

Intestinal Cl⁻ secretion is stimulated by cyclic AMP (cAMP) and intracellular calcium ([Ca²⁺]_i). Recent studies show that protein kinase A (PKA) and the exchange protein directly activated by cAMP (Epac) are downstream targets of cAMP. Therefore, we tested whether both PKA and Epac are involved in forskolin (FSK)/cAMP-stimulated Cl⁻ secretion. Human intestinal T84 cells and mouse small intestine were used for short circuit current (I_sc) measurement in response to agonist-stimulated Cl⁻ secretion. FSK-stimulated Cl⁻ secretion was completely inhibited by the additive effects of the PKA inhibitor, H89 (1 µM), and the [Ca²⁺]_i chelator, 1,2-bis-(o-aminophenoxy)-ethane-N,N,N’,N’-tetraacetic acid, tetraacetoxymethyl ester (BAPTA-AM; 25 µM). Both FSK and the Epac activator 8-pCPT-2’-O-Me-cAMP (50 µM) elevated [Ca²⁺]_i, activated Ras-related protein 2, and induced Cl⁻ secretion in intact or basolateral membrane–permeabilized T84 cells and mouse ileal sheets. The effects of 8-pCPT-2’-O-Me-cAMP were completely abolished by BAPTA-AM, but not by H89. In contrast, T84 cells with silenced Epac1 had a reduced I_sc response to FSK, and this response was completely inhibited by H89, but not by the phospholipase C inhibitor {"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075","term_text":"U73122"}}U73122 or BAPTA-AM. The stimulatory effect of 8-pCPT-2’-O-Me-cAMP on Cl⁻ secretion was not abolished by cystic fibrosis transmembrane conductance (CFTR) inhibitor 172 or glibenclamide, suggesting that CFTR channels are not involved. This was confirmed by lack of effect of 8-pCPT-2’-O-Me-cAMP on whole cell patch clamp recordings of CFTR currents in Chinese hamster ovary cells transiently expressing the human CFTR channel. Furthermore, biophysical characterization of the Epac1-dependent Cl⁻ conductance of T84 cells mounted in Ussing chambers suggested that this conductance was hyperpolarization activated, inwardly rectifying, and displayed a Cl⁻>Br⁻>I⁻ permeability sequence. These results led us to conclude that the Epac-Rap-PLC-[Ca²⁺]_i signaling pathway is involved in cAMP-stimulated Cl⁻ secretion, which is carried by a novel, previously undescribed Cl⁻ channel.     

Pivotal Role of the α2A-Adrenoceptor in Producing Inflammation and Organ Injury in a Rat Model of Sepsis

Background

Norepinephrine (NE) modulates the responsiveness of macrophages to proinflammatory stimuli through the activation of adrenergic receptors (ARs). Being part of the stress response, early increases of NE in sepsis sustain adverse systemic inflammatory responses. The intestine is an important source of NE release in the early stage of cecal ligation and puncture (CLP)-induced sepsis in rats, which then stimulates TNF-α production in Kupffer cells (KCs) through the activation of the α₂-AR. It is important to know which of the three α₂-AR subtypes (i.e., α_2A, α_2B or α_2C) is responsible for the upregulation of TNF-α production. The aim of this study was to determine the contribution of α_2A-AR in this process.

Methodology/Principal Findings

Adult male rats underwent CLP and KCs were isolated 2 h later. Gene expression of α_2A-AR was determined. In additional experiments, cultured KCs were incubated with NE with or without BRL-44408 maleate, a specific α_2A-AR antagonist, and intraportal infusion of NE for 2 h with or without BRL-44408 maleate was carried out in normal animals. Finally, the impact of α_2A-AR activation by NE was investigated under inflammatory conditions (i.e., endotoxemia and CLP). Gene expression of the α_2A-AR subtype was significantly upregulated after CLP. NE increased the release of TNF-α in cultured KCs, which was specifically inhibited by the α_2A-AR antagonist BRL-44408. Equally, intraportal NE infusion increased TNF-α gene expression in KCs and plasma TNF-α which was also abrogated by co-administration of BRL-44408. NE also potentiated LPS-induced TNF-α release via the α_2A-AR in vitro and in vivo. This potentiation of TNF-α release by NE was mediated through the α_2A-AR coupled Gαi protein and the activation of the p38 MAP kinase. Treatment of septic animals with BRL-44408 suppressed TNF-α, prevented multiple organ injury and significantly improved survival from 45% to 75%.

Conclusions/Significance

Our novel finding is that hyperresponsiveness to α₂-AR stimulation observed in sepsis is primarily due to an increase in α_2A-AR expression in KCs. This appears to be in part responsible for the increased proinflammatory response and ensuing organ injury in sepsis. These findings provide important feasibility information for further developing the α_2A-AR antagonist as a new therapy for sepsis.     

cAMP Mediators of Pulsatile Insulin Secretion from Glucose-stimulated Single β-Cells

Pulsatile insulin release from glucose-stimulated β-cells is driven by oscillations of the Ca²⁺ and cAMP concentrations in the subplasma membrane space ([Ca²⁺]_pm and [cAMP]_pm). To clarify mechanisms by which cAMP regulates insulin secretion, we performed parallel evanescent wave fluorescence imaging of [cAMP]_pm, [Ca²⁺]_pm, and phosphatidylinositol 3,4,5-trisphosphate (PIP₃) in the plasma membrane. This lipid is formed by autocrine insulin receptor activation and was used to monitor insulin release kinetics from single MIN6 β-cells. Elevation of the glucose concentration from 3 to 11 mm induced, after a 2.7-min delay, coordinated oscillations of [Ca²⁺]_pm, [cAMP]_pm, and PIP₃. Inhibitors of protein kinase A (PKA) markedly diminished the PIP₃ response when applied before glucose stimulation, but did not affect already manifested PIP₃ oscillations. The reduced PIP₃ response could be attributed to accelerated depolarization causing early rise of [Ca²⁺]_pm that preceded the elevation of [cAMP]_pm. However, the amplitude of the PIP₃ response after PKA inhibition was restored by a specific agonist to the cAMP-dependent guanine nucleotide exchange factor Epac. Suppression of cAMP formation with adenylyl cyclase inhibitors reduced already established PIP₃ oscillations in glucose-stimulated cells, and this effect was almost completely counteracted by the Epac agonist. In cells treated with small interfering RNA targeting Epac2, the amplitudes of the glucose-induced PIP₃ oscillations were reduced, and the Epac agonist was without effect. The data indicate that temporal coordination of the triggering [Ca²⁺]_pm and amplifying [cAMP]_pm signals is important for glucose-induced pulsatile insulin release. Although both PKA and Epac2 partake in initiating insulin secretion, the cAMP dependence of established pulsatility is mediated by Epac2.     

Adenosine A2A Receptor Deficiency Up-Regulates Cystatin F Expression in White Matter Lesions Induced by Chronic Cerebral Hypoperfusion

In previous studies, we have shown that the inactivation of the adenosine A2A receptor exacerbates chronic cerebral hypoperfusion-induced white matter lesions (WMLs) by enhancing neuroinflammatory responses. However, the molecular mechanism underlying the effect of the adenosine A2A receptor remains unknown. Recent studies have demonstrated that cystatin F, a potent endogenous cysteine protease inhibitor, is selectively expressed in immune cells in association with inflammatory demyelination in central nervous system diseases. To understand the expression of cystatin F and its potential role in the effect of A2A receptor on WMLs induced through chronic cerebral hypoperfusion, we investigated cystatin F expression in the WMLs of A2A receptor gene knockout mice, the littermate wild-type mice and wild-type mice treated daily with the A2A receptor agonist {"type":"entrez-protein","attrs":{"text":"CGS21680","term_id":"878113053","term_text":"CGS21680"}}CGS21680 or both {"type":"entrez-protein","attrs":{"text":"CGS21680","term_id":"878113053","term_text":"CGS21680"}}CGS21680 and A2A receptor antagonist {"type":"entrez-protein","attrs":{"text":"SCH58261","term_id":"1052882304","term_text":"SCH58261"}}SCH58261 after chronic cerebral hypoperfusion. The results of quantitative-PCR and western blot analysis revealed that cystatin F mRNA and protein expression were significantly up-regulated in the WMLs after chronic cerebral hypoperfusion. In addition, cystatin F expression in the corpus callosum was significantly increased in A2A receptor gene knockout mice and markedly decreased in mice treated with {"type":"entrez-protein","attrs":{"text":"CGS21680","term_id":"878113053","term_text":"CGS21680"}}CGS21680 on both the mRNA and protein levels. Additionally, {"type":"entrez-protein","attrs":{"text":"SCH58261","term_id":"1052882304","term_text":"SCH58261"}}SCH58261 counteracted the attenuation of cystatin F expression produced by {"type":"entrez-protein","attrs":{"text":"CGS21680","term_id":"878113053","term_text":"CGS21680"}}CGS21680 after chronic cerebral hypoperfusion. Moreover, double immunofluorescence staining revealed that cystatin F was co-localized with the activated microglia marker CD11b. In conclusion, the cystatin F expression in the activated microglia is closely associated with the effect of the A2A receptors, which may be related to the neuroinflammatory responses occurring during the pathological process.     

Hematopoietic Protein Tyrosine Phosphatase Mediates β2-Adrenergic Receptor-Induced Regulation of p38 Mitogen-Activated Protein Kinase in B Lymphocytes

Stimulation of the β₂-adrenergic receptor (β₂AR) on a CD40L/interleukin-4-activated B lymphocyte increases the level of immunoglobulin E (IgE) in a protein kinase A (PKA)- and p38 mitogen-activated protein kinase (MAPK)-dependent manner. However, the mechanism by which β₂AR stimulation mediates the increase in the level of p38 MAPK activation has remained unclear. Here we show that the β₂AR-induced increase in p38 MAPK activation occurred via a hematopoietic protein tyrosine phosphatase (HePTP)-mediated cross talk between PKA and p38 MAPK. β₂AR agonists, cAMP-elevating agents, and PKA inhibitors were used to show that β₂AR stimulation resulted in a PKA-dependent increase in p38 MAPK phosphorylation. Pharmacological agents and gene-deficient mice revealed that p38 MAPK phosphorylation was regulated by the G-stimulatory (Gs)/cAMP/PKA pathway independently of the G-inhibitory or β-arrestin-2 pathways. Coimmunoprecipitation and Western blot analysis showed that HePTP was phosphorylated in a PKA-dependent manner, which inactivated HePTP and allowed for increased free p38 MAPK to be phosphorylated by the MAPK cascade that was activated by CD40L. HePTP short hairpin RNA confirmed that HePTP played a role in regulating the level of p38 MAPK phosphorylation in a B cell. Thus, β₂AR stimulation on a B cell phosphorylates and inactivates HePTP in a Gs/cAMP/PKA-dependent manner to release bound p38 MAPK, making more available for phosphorylation and subsequent IgE regulation.     

β-Agonist-mediated Relaxation of Airway Smooth Muscle Is Protein Kinase A-dependent

Inhaled β-agonists are effective at reversing bronchoconstriction in asthma, but the mechanism by which they exert this effect is unclear and controversial. PKA is the historically accepted effector, although this assumption is made on the basis of associative and not direct evidence. Recent studies have asserted that exchange protein activated by cAMP (Epac), not PKA, mediates the relaxation of airway smooth muscle (ASM) observed with β-agonist treatment. This study aims to clarify the role of PKA in the prorelaxant effects of β-agonists on ASM. Inhibition of PKA activity via expression of the PKI and RevAB peptides results in increased β-agonist-mediated cAMP release, abolishes the inhibitory effect of isoproterenol on histamine-induced intracellular calcium flux, and significantly attenuates histamine-stimulated MLC-20 phosphorylation. Analyses of ASM cell and tissue contraction demonstrate that PKA inhibition eliminates most, if not all, β-agonist-mediated relaxation of contracted smooth muscle. Conversely, Epac knockdown had no effect on the regulation of contraction or procontractile signaling by isoproterenol. These findings suggest that PKA, not Epac, is the predominant and physiologically relevant effector through which β-agonists exert their relaxant effects.     

S100-β aggravates spinal cord injury via activation of M1 macrophage phenotype

Objectives:S100-β has been identified as a sensitive biomarker in central nervous system injuries. However, the functions and mechanisms of S100-β are unknown in spinal cord injury.Methods:Spinal cord injury (SCI) mouse model was generated by surgical operation, microglia activation model was established by inducing BV-2 cells with LPS. The SCI model was evaluated by Basso-Beattie-Bresnahan (BBB) behavioral score, HE staining, and Nissl staining. The expression level of S100-β was detected by qRT-PCR, western blot, and immunofluorescence. qRT-PCR and western blot were used to detect the expression of iNOS and CD16. Pro-inflammatory cytokines TNF-α and IL-1β levels were detected by qRT-PCR and ELISA.Results:The expression of IL-1β, TNF-α, iNOS, and CD16 increased at 3^rd day after SCI. In BV2 microglia, LPS treatment promoted the expression of S100-β, IL-1β, TNF-α, iNOS, and CD16. Knockdown of S100-β reduced the expression of iNOS stimulated by LPS. Over-expression of S100-β increased IL-1β and TNF-α, and S100-β inhibition suppressed IL-1β and TNF-α. In SCI mice, knockdown of S100-β attenuated the spinal cord injury and inhibited the expression of iNOS, IL-1β, and TNF-α.Conclusions:Down-regulation of S100-β could inhibit the pathogenesis of SCI and inhibit the activation of M1 macrophages. S100-β may be a useful diagnostic biomarker or therapeutic target for SCI.     

Lactosylceramide Interacts with and Activates Cytosolic Phospholipase A2α

Lactosylceramide (LacCer) is a member of the glycosphingolipid family and is known to be a bioactive lipid in various cell physiological processes. However, the direct targets of LacCer and cellular events mediated by LacCer are largely unknown. In this study, we examined the effect of LacCer on the release of arachidonic acid (AA) and the activity of cytosolic phospholipase A₂α (cPLA₂α). In CHO-W11A cells, treatment with 1-phenyl-2-palmitoylamino-3-morpholino-1-propanol (PPMP), an inhibitor of glucosylceramide synthase, reduced the glycosphingolipid level, and the release of AA induced by {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187 or platelet-activating factor was inhibited. The addition of LacCer reversed the PPMP effect on the stimulus-induced AA release. Exogenous LacCer stimulated the release of AA, which was decreased by treatment with an inhibitor of cPLA₂α or silencing of the enzyme. Treatment of CHO-W11A cells with LacCer induced the translocation of full-length cPLA₂α and its C2 domain from the cytosol to the Golgi apparatus. LacCer also induced the translocation of the D43N mutant of cPLA₂α. Treatment of L929 cells with TNF-α induced LacCer generation and mediated the translocation of cPLA₂α and AA release, which was attenuated by treatment with PPMP. In vitro studies were then conducted to test whether LacCer interacts directly with cPLA₂α. Phosphatidylcholine vesicles containing LacCer increased cPLA₂α activity. LacCer bound to cPLA₂α and its C2 domain in a Ca²⁺-independent manner. Thus, we propose that LacCer is a direct activator of cPLA₂α.