Protection by cilostazol against amyloid-β(1-40)-induced suppression of viability and neurite elongation through activation of CK2α in HT22 mouse hippocampal cells

Amyloid-β peptide (Aβ) deposits in the brain are critical in the neurotoxicity induced by Aβ. This study elucidates the underlying signaling pathway by which cilostazol protects HT22 neuronal cells from Aβ(1-40) (3-30 μM)-induced deterioration of cell proliferation, viability, and neurite elongation. Cilostazol rescued HT22 cells from the apoptotic cell death induced by Aβ toxicity through the downregulation of phosphorylated p53 (Ser15), Bax, and caspase-3 and the upregulation of Bcl-2 expression, which improved neuronal cell proliferation and viability. Furthermore, Aβ(1-40) suppressed both phosphorylated CK2α protein expression and CK2 activity in the cytosol; these were concentration dependently recovered by cilostazol (3-30 μM). Cilostazol significantly increased the levels of GSK-3β phosphorylation at Ser9 and β-catenin phosphorylation at Ser675 in the cytosol and nucleus. Cilostazol effects were reversed by KT5720 (1 μM, PKA inhibitor) and TBCA (40 μM, inhibitor of CK2) and CK2α knockdown by siRNA transfection. Likewise, Aβ-stimulated GSK-3β phosphorylation at Tyr 216 was decreased by cilostazol in the control but not in the CK2α siRNA-transfected cells. Furthermore, the Aβ (10 μM)-induced suppression of neurite elongation was recovered by cilostazol; this recovery was attenuated by inhibitors such as KT5720 and TBCA and blocked by CK2α knockdown. In conclusion, increased cAMP-dependent protein kinase-linked CK2α activation underlies the pharmacological effects of cilostazol in downregulating p53 phosphorylation at Ser15 and upregulating GSK-3β phosphorylation at Ser9/β-catenin phosphorylation at Ser675, thereby suppressing Aβ(1-40)-induced neurotoxicity and improving neurite elongation.     

Attenuation of β‐amyloid‐induced tauopathy via activation of CK2α/SIRT1: Targeting for cilostazol

β‐Amyloid (Aβ) deposits and hyperphosphorylated tau aggregates are the chief hallmarks in the Alzheimer's disease (AD) brains, but the strategies for controlling these pathological events remain elusive. We hypothesized that CK2‐coupled SIRT1 activation stimulated by cilostazol suppresses tau acetylation (Ac‐tau) and tau phosphorylation (P‐tau) by inhibiting activation of P300 and GSK3β. Aβ was endogenously overproduced in N2a cells expressing human APP Swedish mutation (N2aSwe) by exposure to medium containing 1% fetal bovine serum for 24 hr. Increased Aβ accumulation was accompanied by increased Ac‐tau and P‐tau levels. Concomitantly, these cells showed increased P300 and GSK3β P‐Tyr216 expression; their expressions were significantly reduced by treatment with cilostazol (3–30 μM) and resveratrol (20 μM). Moreover, decreased expression of SIRT1 and its activity by Aβ were significantly reversed by cilostazol as by resveratrol. In addition, cilostazol strongly stimulated CK2α phosphorylation and its activity, and then stimulated SIRT1 phosphorylation. These effects were confirmed by using the pharmacological inhibitors KT5720 (1 μM, PKA inhibitor), TBCA (20 μM, inhibitor of CK2), and sirtinol (20 μM, SIRT1 inhibitor) as well as by SIRT1 gene silencing and overexpression techniques. In conclusion, increased cAMP‐dependent protein kinase‐linked CK2/SIRT1 expression by cilostazol can be a therapeutic strategy to suppress the tau‐related neurodegeneration in the AD brain. © 2013 Wiley Periodicals, Inc.     

Blockade of phosphodiesterase-III protects against oxygen-glucose deprivation in endothelial cells by upregulation of VE-cadherin

We recently reported that a phosphodiesterase-III inhibitor, cilostazol, prevented the hemorrhagic transformation induced by focal cerebral ischemia in mice treated with tissue plasminogen activator (tPA) and that it reversed tPA-induced cell damage by protecting the neurovascular unit, particularly endothelial cells. However, the mechanisms of cilostazol action are still not clearly defined. The adheren junction (AJ) protein, VE-cadherin, is a known mediator of endothelial barrier sealing and maintenance. Therefore, we tested whether cilostazol might promote expression of adhesion molecules in endothelial cells, thereby preventing deterioration of endothelial barrier functions. Human brain microvascular endothelial cells were exposed to 6-h oxygen-glucose deprivation (OGD). We compared cilostazol with aspirin treatments and examined 2 representative AJ proteins: VE-cadherin and platelet endothelial cell adhesion molecule-1 (PECAM-1). A protein kinase A (PKA) inhibitor, LY294002 (a PI3-K inhibitor), db-cAMP, and RP-cAMPS were used to assess the roles of cAMP, PKA, and PI3-K signaling, respectively, in cilostazol-induced responses. Cilostazol and db-cAMP prevented OGD-stress injury in endothelial cells by promoting VE-cadherin expression, but not PECAM-1. Aspirin did not prevent cell damage. P13-K inhibition by LY294002 had no influence on the effects of cilostazol, but inhibition of cAMP/PKA with PKA inhibitor and Rp-cAMPS suppressed cilostazol-induced inhibition of cell damage and promotion of VE-cadherin expression. In contrast, OGD stress had no detectable effects on VEGF, VEGF receptor, or angiopoietin-1 levels. Cilostazol promotes VE-cadherin expression through cAMP/PKA-dependent pathways in brain endothelial cells; thus, cilostazol effects on adhesion molecule signaling may provide protection against OGD stress in endothelial cells.     

Galpha(s) sensitizes human SH-SY5Y cells to apoptosis independently of the protein kinase A pathway

Disturbances in Galpha(s-L) levels and function have been implicated in the pathophysiology of bipolar disorder, but the role of these changes in the development of the illness is not clear. In view of the critical role of Galpha(s)-mediated cAMP signaling in regulating cell survival, we investigated the potential role of Galpha(s-L) in modulating susceptibility to cellular stressors in human SH-SY5Y neuroblastoma cells. Overexpression of Galpha(s-L) to a level twice that of the vector-transfected cells did not directly affect cell viability but significantly increased the sensitivity to induction of cell death by serum deprivation and other apoptotic stimuli, including staurosporine, H(2)O(2), and tunicamycin. This enhanced sensitivity was associated with increased caspase-3 activation and appearance of fragmented nuclei (Hoechst 33342 staining). The broad-spectrum caspase inhibitor z-VAD-fmk completely suppressed cell death evoked by these apoptotic insults in both vector-transfected and Galpha(s-L)-overexpressing cells. The increased vulnerability conferred by increased Galpha(s-L) expression was neither mimicked by cAMP analogs 8-Br-cAMP, 8-CPT-cAMP, and 8-CPT-2Me-cAMP nor attenuated by PKA inhibitors Rp-cAMPS and KT5720. These data indicate that Galpha(s-L) may modulate apoptotic processes in a caspase-dependent manner through a signaling cascade that is independent of the cAMP/PKA or cAMP/Epac pathway. These results suggest that enhanced Galpha(s-L) expression, as was observed in post-mortem brain of bipolar patients, may impair cellular resilience in response to intracellular stress signals resulting from mitochondrial and/or endoplasmic reticulum dysfunction implicated in this disorder.     

Fencamfamine modulates sodium, potassium-ATPase through cyclic AMP and cyclic AMP-dependent protein kinase in rat striatum

Summary. Dopamine (DA) and fencamfamine (FCF) modulatory action on Na,K-ATPase and Mg-ATPase activity were evaluated in rat striatum. DA and FCF induced a decrease in Na,K-ATPase, without affecting Mg-ATPase activity. The effect of FCF was dose-dependent from 10 to 100 μM, with an IC₅₀ of 4.7 × 10⁻⁵ M. Furthermore, the effect of FCF (100 μM) increasing AMPc levels, but not GMPc, was nonadditive with that of DA (10 μM), which is consistent to a common site of action. The 8-bromo-cyclic AMP also induced a specific reduction in the Na,K-ATPase activity. The reduction of Na,K-ATPase induced by FCF (100 μM) was blocked by either SCH 23390 or sulpiride, which are D1 and D2 receptor antagonists. The decrease in striatal NA,K-ATPase activity induced by FCF was blocked by KT 5720, a selective inhibitor of cyclic AMP-dependent protein kinase (PKA), but not by KT 5823, a selective inhibitor of cyclic GMP-dependent protein kinase (PKG). Otherwise, KT 5720 or KT 5823 did not produce any change in Na,K-ATPase or Mg-ATPase activity. These data suggest that FCF reduces Na,K-ATPase activity through cyclic AMP-dependent changes in protein phosphorylation via a PKA mechanism. Accepted January 20, 1998; received June 2, 1997     

Inhibition of platelet aggregation and the release of P-selectin from platelets by cilostazol

To evaluate the in vitro effects of cilostazol, a phosphodiesterase III inhibitor, on platelet responses, we measured platelet aggregation and the levels of soluble P-selectin, a glycoprotein present on the -granule membrane in resting platelets, and cAMP. Platelet-rich plasma and washed platelets from healthy human volunteers were treated with cilostazol (5, 25 and 50 μM). Platelet-rich plasma was stimulated by ADP (1 and 5 μM) or collagen (5 μg/ml). Washed platelets were stimulated by thrombin (4 U/ml) in the presence or absence of 1 μM forskolin. In vehicle-treated samples, soluble P-selectin levels in response to 1 μM ADP-induced primary aggregation were similar to those of circulating levels of healthy volunteers but the levels in response to 5 μM ADP-induced secondary aggregation and collagen-induced aggregation increased markedly compared to those in response to primary aggregation. This result suggests that P-selectin is released from platelets according to the extent of platelet aggregation. Cilostazol inhibited platelet aggregation as well as P-selectin release in a concentration-dependent manner. Cilostazol inhibited completely thrombin-induced aggregation in the presence of 1 μM forskolin, when cAMP levels were two-fold higher than those in the absence of forskolin. Cilostazol, which increases intracellular cAMP in platelets, may be useful in the treatment of arterial occlusive diseases.     

β-adrenergic enhancement of brain kynurenic acid production mediated via cAMP-related protein kinase A signaling

The central levels of endogenous tryptophan metabolite kynurenic acid (KYNA), an antagonist of N-methyl-d-aspartate (NMDA) and α7-nicotinic receptors, affect glutamatergic and dopaminergic neurotransmission. Here, we demonstrate that selective agonists of β₁-receptors (xamoterol and denopamine), β₂-receptors (formoterol and albuterol), α- and β-receptors (epinephrine), 8pCPT-cAMP and 8-Br-cAMP (analogues of cAMP) increase the production of KYNA in rat brain cortical slices and in mixed glial cultures. Neither betaxolol, β₁-adrenergic antagonist, nor timolol, a non-selective β_1,2-adrenergic antagonist has influenced synthesis of KYNA in both paradigms. In contrast, KT5720, a selective inhibitor of protein kinase A (PKA), strongly reduced KYNA formation in cortical slices (2–10 µM) and in glial cultures (100 nM). β-adrenergic antagonists and KT5720 prevented the β-adrenoceptor agonists-induced increases of KYNA synthesis. In vivo, β-adrenergic agonist clenbuterol (0.1–1.0 mg/kg) increased the cortical endogenous level of KYNA; the effect was blocked with propranolol (10 mg/kg). β-adrenoceptors agonists, cAMP analogues and KT5720 did not affect directly the activity of KAT I or KAT II measured in partially purified cortical homogenate. In contrast, the exposure of intact cultured glial cells to pCPT-cAMP, 8-Br-cAMP and formoterol has lead to an enhanced action of KATs. These findings demonstrate that β-adrenoceptor-mediated enhancement of KYNA production is a cAMP- and PKA-dependent event. PKA activity appears to be an essential signal affecting KYNA formation. Described here novel mechanism regulating KYNA availability may be of a potential importance, considering that various stimuli, among them clinically used drugs, activate cAMP/PKA pathway, and thus could counteract the central deficits of KYNA.     

Cilostazol enhances neovascularization in the mouse hippocampus after transient forebrain ischemia

Cilostazol is known to be a specific type III phosphodiesterase inhibitor, which promotes increased intracellular cAMP levels. We assessed the effect of cilostazol on production of angioneurins and chemokines and recruitment of new endothelial cells for vasculogenesis in a mouse model of transient forebrain ischemia. Pyramidal cell loss was prominently evident 3–28 days postischemia, which was markedly ameliorated by cilostazol treatment. Expression of angioneurins, including endothelial nitric oxide synthase, vascular endothelial growth factor, and brain‐derived neurotrophic factor, was up‐regulated by cilostazol treatment in the postischemic hippocampus. Cilostazol also increased Sca‐1/vascular endothelial growth factor receptor‐2 positive cells in the bone marrow and circulating peripheral blood and the number of stromal cell‐derived factor‐1α‐positive cells in the molecular layer of the hippocampus, which colocalized with CD31. CXCR4 chemokine receptors were up‐regulated by cilostazol in mouse bone marrow‐derived endothelial progenitor cells, suggesting that cilostazol may be important in targeting or homing in of bone marrow‐derived stem cells to areas of injured tissues. CD31‐positive cells were colocalized with almost all bromodeoxyuridine‐positive cells in the molecular layer, indicating stimulation of endothelial cell proliferation by cilostazol. These data suggest that cilostazol markedly enhances neovascularization in the hippocampus CA1 area in a mouse model of transient forebrain ischemia, providing a beneficial interface in which both bone marrow‐derived endothelial progenitor cells and angioneurins influence neurogenesis in injured tissue. © 2010 Wiley‐Liss, Inc.     

Beneficial effect of cilostazol‐mediated neuronal repair following trimethyltin‐induced neuronal loss in the dentate gyrus

Cilostazol acts as an antiplatelet agent and has other pleiotropic effects based on phosphodiesterase‐3‐dependent mechanisms. We evaluated whether cilostazol would have a beneficial effect on neuronal repair following hippocampal neuronal damage by using a mouse model of trimethyltin (TMT)‐induced neuronal loss/self‐repair in the hippocampal dentate gyrus [Ogita et al. (2005) J Neurosci Res 82:609?621]; these mice will hereafter be referred to as impaired animals. A single treatment with cilostazol (10 mg/kg, i.p.) produced no significant change in the number of 5‐bromo‐2′‐deoxyuridine (BrdU)‐incorporating cells in the dentate granule cell layer (GCL) or subgranular zone on day 3 after TMT treatment. However, chronic treatment with cilostazol on days 3–15 posttreatment resulted in an increase in the number of BrdU‐incorporating cells in the dentate GCL of the impaired animals, and these cells were positive for neuronal nuclear antigen or doublecortin. Cilostazol was effective in elevating the level of phosphorylated cyclic adrenosine monophosphate response element‐binding protein (pCREB) in the dentate gyrus of impaired animals. The results of a forced swimming test revealed that the chronic treatment with cilostazol improved the depression‐like behavior seen in the impaired animals. In the cultures of hippocampal neural stem/progenitor cells, exposure to cilostazol produced not only enhancement of proliferation activity but also elevation of pCREB levels. Taken together, our data suggest that cilostazol has a beneficial effect on neuronal repair following neuronal loss in the dentate gyrus through promotion of proliferation and/or neuronal differentiation of neural progenitor cells in the subgranular zone. © 2014 Wiley Periodicals, Inc.     

Multiple treatments with L‐3,4‐dihydroxyphenylalanine modulate dopamine biosynthesis and neurotoxicity through the protein kinase A‐transient extracellular signal‐regulated kinase and exchange protein activation by cyclic AMP‐sustained extracellular signal‐regulated kinase signaling pathways

Multiple treatments with L‐3,4‐dihydroxyphenylalanine (L‐DOPA; 20 µM) induce neurite‐like outgrowth and reduce dopamine biosynthesis in rat adrenal pheochromocytoma (PC) 12 cells. We therefore investigated the effects of multiple treatments with L‐DOPA (MT‐LD) on cell survival and death over a duration of 6 days by using PC12 cells and embryonic rat midbrain primary cell cultures. MT‐LD (10 and 20 µM) decreased cell viability, and both types of cells advanced to the differentiation process at 4–6 days. MT‐LD induced cyclic adenosine monophosphate (cAMP)‐dependent protein kinase A (PKA) phosphorylation and exchange protein activation by cAMP (Epac) expression at 1–3 days, which led to transient extracellular signal‐regulated kinase (ERK1/2) phosphorylation in both cells. In these states, MT‐LD activated cAMP‐response element binding protein (CREB; Ser133) and tyrosine hydroxylase (Ser40) phosphorylation in PC12 cells, which led to an increase in intracellular dopamine levels. In contrast, MT‐LD induced prolonged Epac expression at 4–5 days in both cells, which led to sustained ERK1/2 phosphorylation. In these states, the dopamine levels were decreased in PC12 cells. In addition, MT‐LD induced c‐Jun N‐terminal kinase1/2 phosphorylation and cleaved caspase‐3 expression at 4–6 days in both cells. These results suggest that MT‐LD maintains cell survival via PKA‐transient ERK1/2 activation, which stimulates dopamine biosynthesis. In contrast, at the later time period, MT‐LD induces differentiation via both prolonged Epac and sustained ERK1/2 activation, which subsequently leads to the cell death process. Our data demonstrate that L‐DOPA can cause neurotoxicity by modulating the Epac‐ERK pathways in neuronal and PC12 cells. © 2014 Wiley Periodicals, Inc.     

Cilostazol suppresses β‐amyloid production by activating a disintegrin and metalloproteinase 10 via the upregulation of SIRT1‐coupled retinoic acid receptor‐β

The accumulation of plaques of β‐amyloid (Aβ) peptides, a hallmark of Alzheimer's disease, results from the sequential cleavage of amyloid precursor protein (APP) by activation of β‐ and γ‐secretases. However, the production of Aβ can be avoided by alternate cleavage of APP by α‐and γ‐secretases. We hypothesized that cilostazol attenuates Aβ production by increasing a disintegrin and metalloproteinase 10 (ADAM10)/α‐secretase activity via SIRT1‐coupled retinoic acid receptor‐β (RARβ) activation in N2a cells expressing human APP Swedish mutation (N2aSwe). To evoke endogenous Aβ overproduction, the culture medium was switched from medium containing 10% fetal bovine serum (FBS) to medium containing 1% FBS, and cells were cultured for 3～24 hr. After depletion of FBS in media, N2aSwe cells showed increased accumulations of full‐length APP (FL‐APP) and Aβ in a time‐dependent manner (3–24 hr) in association with decreased ADAM10 protein expression. When pretreated with cilostazol (10–30 μM), FL‐APP and Aβ levels were significantly reduced, and ADAM10 and α‐secretase activities were restored. Furthermore, the effect of cilostazol on ADAM10 expression was antagonized by pretreating Rp‐cAMPS and sirtinol and by SIRT1‐gene silencing. In the N2aSwe cells overexpressing the SIRT1 gene, ADAM10, and sAPPα levels were significantly elevated. In addition, like all‐trans retinoic acid, cilostazol enhanced the protein expressions of RARβ and ADAM10, and the cilostazol‐stimulated ADAM10 elevation was significantly attenuated by LE135 (a RARβ inhibitor), sirtinol, and RARβ‐gene silencing. In conclusion, cilostazol suppresses the accumulations of FL‐APP and Aβ by activating ADAM10 via the upregulation of SIRT1‐coupled RARβ. © 2014 Wiley Periodicals, Inc.     

Second messenger cascade of glial responses evoked by interneuron activity and by a myomodulin peptide in the leech central nervous system

The giant glial cell in the neuropil of segmental ganglia of the leech Hirudo medicinalis responds to the activity of the Leydig interneuron and to a peptide of the myomodulin family, the presumed transmitter mediating the Leydig neuron-to-giant glial cell transmission, with a membrane hyperpolarization due to an increased membrane K+ conductance [Britz et al. (2002) Glia, 38, 215-227]. We have now studied the second messenger cascade initiated by Leydig neuron stimulation and by the endogenous myomodulin (MMHir) in the voltage-clamped giant glial cell. Glial responses to both stimuli are mediated by a G-protein-coupled receptor linked to adenylyl cyclase by the following criteria: (i) injection of GDP-beta-S, but not GDP, resulted in an irreversible decrease of the glial responses to both stimuli; (ii) the responses to both stimuli were reversibly inhibited by the adenylyl cyclase inhibitor SQ22,536; and (3) bath-applied di-butyryl-cyclic AMP, but not di-butyryl-cyclic GMP, elicited an outward current, which reduced the responses elicited by neuronal stimulation or myomodulin. A cocktail of protein kinase (PK) inhibitors (H-8, KT5720), the PKA antagonist Rp-cAMPS, or presumed inhibitors of cyclic nucleotide channels, LY83583 and l-cis-diltiazem, had no effect on the glial responses. Our results suggest that Leydig neuron stimulation and MMHir activate a cAMP-mediated K+ conductance in the glial cell, which appeared neither to be due to the activation of PKA nor of known cyclic nucleotide-gated channels directly.     

Effect of cilostazol on delayed cerebral vasospasm after subarachnoid hemorrhage in rats: evaluation using black blood magnetic resonance imaging

The purpose of this study was to use black blood magnetic resonance imaging (BB-MRI) to assess delayed cerebral vasospasm (DCV) after subarachnoid hemorrhage (SAH) in rats, and evaluate whether delayed treatment with the anti-platelet agent cilostazol was effective on DCV. BA vasospasm was sequentially assessed at 1, 2, and 3 h, and 1-6 days after SAH by BB-MRI. BB-MRI clearly visualized biphasic vasospasm; early vasospasm at 1 h later and the maximal DCV at day 2. Cilostazol was perorally administered twice at day 1 after having confirmed significant DCV using BB-MRI. The effect of cilostazol on DCV was evaluated at day 2. Cilostazol significantly attenuated DCV and suppressed the levels of malondialdehide and 8-isoprostane in CSF after SAH. This study shows that BB-MRI is a useful and less invasive method for the evaluation of DCV, and cilostazol may be effective on DCV.     

Effects of scoparone on dopamine biosynthesis and L‐DOPA‐induced cytotoxicity in PC12 cells

The effects of scoparone on dopamine biosynthesis and L‐DOPA‐induced cytotoxicity in PC12 cells were investigated. PC12 cells treated with scoparone at concentrations of 100–200 μM showed a 128–136% increase in dopamine levels over the course of 24 hr. Scoparone significantly increased the secretion of dopamine into the culture medium. Under the same conditions, the activities of tyrosine hydroxylase (TH) and aromatic L‐amino acid decarboxylase (AADC) were enhanced by treatment with 200 μM scoparone for 6–48 hr, but the activity of TH was regulated for a longer period than that of AADC. The intracellular levels of cyclic AMP and Ca²⁺ were increased by treatment with 200 μM scoparone. The levels of TH mRNA and the phosphorylation of cyclic AMP‐response element‐binding protein (CREB) were also significantly increased by treatment with 200 μM scoparone. In addition, scoparone at a concentration of 200 μM stimulated the activities of cyclic AMP‐dependent protein kinase (PKA), protein kinase C (PKC), and Ca²⁺/calmodulin kinase II (CaMK II). Finally, pretreatment with 200 μM scoparone reduced the cytotoxicity induced by L‐DOPA (20–100 μM) at 24 hr. These results suggest that scoparone enhances dopamine biosynthesis by regulating TH activity and TH gene expression, which is mediated by the PKA, CREB, PKC, and CaMK II pathways, and protects cells from L‐DOPA‐induced cytotoxicity by inducing cyclic AMP‐PKA systems in PC12 cells. © 2009 Wiley‐Liss, Inc.     

Cilostazol attenuates ischemia–reperfusion-induced blood–brain barrier dysfunction enhanced by advanced glycation endproducts via transforming growth factor-β1 signaling

We investigated the effects of cilostazol, a selective inhibitor of phosphodiesterase 3, on blood–brain barrier (BBB) integrity against ischemia–reperfusion injury enhanced by advanced glycation endproducts (AGEs). We used in vitro BBB models with primarily cultured BBB-related cells from rats (brain capillary endothelial cells, astrocytes and pericytes), and subjected cells to either normoxia or 3-h oxygen glucose deprivation (OGD)/24-h reoxygenation with or without AGEs. Treatment of AGEs did not affect the transendothelial electrical resistance (TEER) in the BBB model under normoxia, but there was a significant decrease in TEER under 3-h OGD/24-h reoxygenation conditions with AGEs. Cilostazol inhibited decreases in TEER induced by 3-h OGD/24-h reoxygenation with AGEs. Immunocytochemical and Western blot analyses showed that AGEs reduced the expression of claudin-5, the main functional protein of tight junctions (TJs). In contrast, cilostazol increased the expression of claudin-5 under 3-h OGD/24-h reoxygenation with AGEs. Furthermore, while AGEs increased the production of extracellular transforming growth factor (TGF)-β1, cilostazol inhibited the production of extracellular TGF-β1 and restored the integrity of TJs. Thus, we found that AGEs enhanced ischemia–reperfusion injury, which mainly included decreases in the expression of proteins comprising TJs through the production of TGF-β1. Cilostazol appeared to limit ischemia–reperfusion injury with AGEs by improving the TJ proteins and inhibiting TGF-β1 signaling.     

Protein kinase A modulates retinal ganglion cell growth during development

During development, retinal ganglion cells (RGCs) extend their axons toward their thalamic and mesencephalic targets. Their navigation is largely directed by guidance cues present in their environment. Since cAMP is an important second messenger that mediates the neural response to guidance molecules and its intracellular levels seem to decrease significantly following birth, we tested whether modulation of the cAMP/protein kinase A (PKA) pathway would affect the normal development of RGC axons. At postnatal day 1, hamsters received a unilateral intraocular injection of either 0.9% saline solution, 12 mM of the membrane-permeable cAMP analogue (dibutyryl cAMP; db-cAMP), or 10 µM of the PKA inhibitor KT5720. Intraocular elevation of cAMP significantly accelerated RGC axonal growth while inhibition of PKA activity decreased it. Moreover, when highly purified RGC cultures were treated with forskolin (an activator of adenylate cyclase) or cAMP analogues (db-cAMP and Sp-cAMP), neurite length, growth cone (GC) surface area and GC filopodia number were significantly increased. This indicates that intraocular elevation of cAMP acts directly on RGCs. Since these effects were prevented by PKA inhibitors, it demonstrates that cAMP also exerts its action via the PKA pathway. Taken together, these results suggest that the cAMP/PKA cascade is essential for the normal development of retinothalamic projections.     

Induction of peptidylarginine deiminase 2 and 3 by dibutyryl cAMP via cAMP‐PKA signaling in human astrocytoma U‐251MG cells

Peptidylarginine deiminases (PADs) are posttranslational modification enzymes that citrullinate (deiminate) protein arginine residues in a calcium‐dependent manner, yielding citrulline residues. Enzymatic citrullination abolishes positive charges of native protein molecules, inevitably causing significant alterations in their structure and function. Previously, we reported the abnormal accumulation of citrullinated proteins and an increase of PAD2 content in hippocampi of patients with Alzheimer disease. In this study, we investigated PAD expression by using dibutyryl cAMP (dbcAMP) in human astrocytoma U‐251MG cells. Under normal culture conditions, PAD2 and PAD3 mRNA expression is detectable with quantitative PCR in U‐251MG cells. The addition of dbcAMP in a dose‐dependent manner significantly increased this mRNA expression and protein levels. Moreover, PAD enzyme activity also increased significantly and dose‐dependently. Furthermore, the expression of PAD2 and PAD3 mRNA was inhibited by the cAMP‐dependent PKA inhibitor KT5720, suggesting that such expression of dbcAMP‐induced PAD2 and PAD3 mRNA is mediated by the cAMP‐PKA signaling pathway in U‐251MG cells. This is the first report to document the PAD2 and PAD3 mRNA expression induced by dbcAMP and to attribute the induction of these genes to mediation by the cAMP‐PKA signaling pathway in U‐251MG cells. © 2016 Wiley Periodicals, Inc.     

Sex Differences in Protein Kinase A Signaling of the Latent Postoperative Pain Sensitization That Is Masked by Kappa Opioid Receptors in the Spinal Cord

Latent sensitization (LS) of pain engages pronociceptive signaling pathways in the dorsal horn that include NMDA receptor (NMDAR)→adenylyl cyclase-1 (AC1)→protein kinase A (PKA), and exchange proteins directly activated by cyclic AMP (Epacs). To determine whether these pathways operate similarly between males and females or are under the inhibitory control of spinal κ opioid receptors (KOR), we allowed hyperalgesia to resolve after plantar incision and then blocked KOR with intrathecal administration of LY2456302, which reinstated hyperalgesia and facilitated touch-evoked immunoreactivity of phosphorylated extracellular signal-regulated kinase (pERK) in neurons (NeuN) but not astrocytes (GFAPs) nor microglia (Iba1). LY2456302 reinstated hyperalgesia even when administered 13 months later, indicating that chronic postoperative pain vulnerability persists for over a year in a latent state of remission. In both sexes, intrathecal MK-801 (an NMDAR competitive antagonist) prevented LY2456302-evoked reinstatement of hyperalgesia as did AC1 gene deletion or the AC1 inhibitor NB001. NB001 also prevented stimulus-evoked pERK. In both sexes, the Epac inhibitor ESI-09 prevented reinstatement, whereas the Epac activator 8-CPT reinstated hyperalgesia. By contrast, the PKA inhibitor H89 prevented reinstatement only in male mice, whereas the PKA activator 6-bnz-cAMP itself evoked reinstatement at all doses tested (3–30 nmol, i.t.). In neither sex did incision change gene expression of KOR, GluN1, PKA, or Epac1 in dorsal horn. We conclude that sustained KOR signaling inhibits spinal PKA-dependent mechanisms that drive postoperative LS in a sex-dependent manner. Our findings support the development of AC1, PKA, and Epac inhibitors toward a new pharmacotherapy for chronic postoperative pain.SIGNIFICANCE STATEMENT Because of neural mechanisms that are not well understood, men and women respond differently to treatments for chronic pain. We report that surgical incision recruits a pronociceptive latent pain sensitization that persisted for over a year and was kept in check by the sustained analgesic activity of κ opioid receptors. NMDAR→AC1→cAMP→Epac signaling pathways in the dorsal horn of the spinal cord maintain latent sensitization in both males and females; however, only males recruit a PKA-dependent mechanism. This work presents a novel male-specific mechanism for the promotion of chronic postoperative pain.