RESUMO
Resveratrol, a polyphenol in red wine, has been reported as a calorie restriction mimetic with potential antiaging and antidiabetogenic properties. It is widely consumed as a nutritional supplement, but its mechanism of action remains a mystery. Here, we report that the metabolic effects of resveratrol result from competitive inhibition of cAMP-degrading phosphodiesterases, leading to elevated cAMP levels. The resulting activation of Epac1, a cAMP effector protein, increases intracellular Ca(2+) levels and activates the CamKKß-AMPK pathway via phospholipase C and the ryanodine receptor Ca(2+)-release channel. As a consequence, resveratrol increases NAD(+) and the activity of Sirt1. Inhibiting PDE4 with rolipram reproduces all of the metabolic benefits of resveratrol, including prevention of diet-induced obesity and an increase in mitochondrial function, physical stamina, and glucose tolerance in mice. Therefore, administration of PDE4 inhibitors may also protect against and ameliorate the symptoms of metabolic diseases associated with aging.
Assuntos
3',5'-AMP Cíclico Fosfodiesterases/antagonistas & inibidores , Envelhecimento/metabolismo , Restrição Calórica , Transdução de Sinais , Estilbenos/administração & dosagem , 3',5'-AMP Cíclico Fosfodiesterases/química , 3',5'-AMP Cíclico Fosfodiesterases/metabolismo , Quinases Proteína-Quinases Ativadas por AMP , Tecido Adiposo Branco/efeitos dos fármacos , Animais , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/química , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/metabolismo , Dieta , Intolerância à Glucose/prevenção & controle , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Camundongos , Modelos Moleculares , Músculo Esquelético/efeitos dos fármacos , NAD/metabolismo , Obesidade/prevenção & controle , Proteínas Quinases/metabolismo , Resveratrol , Rolipram/administração & dosagem , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Sirtuína 1/metabolismoRESUMO
Sleep deprivation (SD) is widely acknowledged as a significant risk factor for cognitive impairment. In this study, intraperitoneal caffeine administration significantly ameliorated the learning and memory (L/M) deficits induced by SD and reduced aggressive behaviors in adult zebrafish. SD led to a reduction in protein kinase A (PKA) phosphorylation, phosphorylated-cAMP response element-binding protein (p-CREB), and c-Fos expression in zebrafish brain. Notably, these alterations were effectively reversed by caffeine. In addition, caffeine mitigated neuroinflammation induced by SD, as evident from suppression of the SD-mediated increase in glial fibrillary acidic protein (GFAP) and nuclear factor-κB (NF-κB) activation. Caffeine restored normal O-GlcNAcylation and O-GlcNAc transferase (OGT) levels while reversing the increased expression of O-GlcNAcase (OGA) in zebrafish brain after SD. Intriguingly, rolipram, a selective phosphodiesterase 4 (PDE4) inhibitor, effectively mitigated cognitive deficits, restored p-CREB and c-Fos levels, and attenuated the increase in GFAP in brain induced by SD. In addition, rolipram reversed the decrease in O-GlcNAcylation and OGT expression as well as elevation of OGA expression following SD. Treatment with H89, a PKA inhibitor, significantly impaired the L/M functions of zebrafish compared with the control group, inducing a decrease in O-GlcNAcylation and OGT expression and, conversely, an increase in OGA expression. The H89-induced changes in O-GlcNAc cycling and L/M dysfunction were effectively reversed by glucosamine treatment. H89 suppressed, whereas caffeine and rolipram promoted O-GlcNAc cycling in Neuro2a cells. Our collective findings underscore the interplay between PKA signaling and O-GlcNAc cycling in the regulation of cognitive function in the brain, offering potential therapeutic targets for cognitive deficits associated with SD.NEW & NOTEWORTHY Our observation highlights the intricate interplay between cAMP/PKA signaling and O-GlcNAc cycling, unveiling a novel mechanism that potentially governs the regulation of learning and memory functions. The dynamic interplay between these two pathways provides a novel and nuanced perspective on the molecular foundation of learning and memory regulation. These insights open avenues for the development of targeted interventions to treat conditions that impact cognitive function, including SD.
Assuntos
Disfunção Cognitiva , Isoquinolinas , Privação do Sono , Sulfonamidas , Animais , Privação do Sono/tratamento farmacológico , Peixe-Zebra/metabolismo , Cafeína/farmacologia , Rolipram , Acetilglucosamina/metabolismo , Processamento de Proteína Pós-Traducional , Cognição , Disfunção Cognitiva/tratamento farmacológico , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , N-Acetilglucosaminiltransferases/genética , N-Acetilglucosaminiltransferases/metabolismoRESUMO
Autosomal Dominant Osteopetrosis type II (ADO2) is a rare bone disease of impaired osteoclastic bone resorption caused by heterozygous missense mutations in the chloride channel 7 (CLCN7). Adenylate cyclase, which catalyzes the formation of cAMP, is critical for lysosomal acidification in osteoclasts. We found reduced cAMP levels in ADO2 osteoclasts compared to wild-type (WT) osteoclasts, leading us to examine whether regulating cAMP would improve ADO2 osteoclast activity. Although forskolin, a known activator of adenylate cyclase and cAMP levels, negatively affected osteoclast number, it led to an overall increase in ADO2 and WT osteoclast resorption activity in vitro. Next, we examined cAMP hydrolysis by the phosphodiesterase 4 (PDE4) proteins in ADO2 versus WT osteoclasts. QPCR analysis revealed higher expression of the three major PDE4 subtypes (4a, 4b, 4d) in ADO2 osteoclasts compared in WT, consistent with reduced cAMP levels in ADO2 osteoclasts. In addition, we found that the PDE4 antagonists, rolipram and roflumilast, stimulated ADO2 and WT osteoclast formation in a dose-dependent manner. Importantly, roflumilast and rolipram displayed a concentration-dependent increase in osteoclast resorption activity which was greater in ADO2 than WT osteoclasts. Moreover, treatment with roflumilast rescued cAMP levels in ADO2 OCLs. The key findings from our studies demonstrate that osteoclasts from ADO2 mice exhibit reduced cAMP levels and PDE4 inhibition rescues cAMP levels and ADO2 osteoclast activity dysfunction in vitro. The mechanism of action of PDE4 inhibitors and their ability to reduce the high bone mass of ADO2 mice in vivo are currently under investigation. Importantly, these studies advance the understanding of the mechanisms underlying the ADO2 osteoclast dysfunction which is critical for the development of therapeutic approaches to treat clinically affected ADO2 patients.
Assuntos
Aminopiridinas , Benzamidas , Reabsorção Óssea , Inibidores da Fosfodiesterase 4 , Humanos , Camundongos , Animais , Rolipram/farmacologia , Rolipram/metabolismo , Inibidores da Fosfodiesterase 4/farmacologia , Inibidores da Fosfodiesterase 4/metabolismo , Osteoclastos/metabolismo , Adenilil Ciclases/metabolismo , Reabsorção Óssea/tratamento farmacológico , Reabsorção Óssea/metabolismo , Canais de Cloreto/genética , CiclopropanosRESUMO
Activation and transdifferentiation of hepatic stellate cells (HSC) into migratory myofibroblasts is a key process in liver fibrogenesis. Cell migration requires an active remodeling of the cytoskeleton, which is a tightly regulated process coordinated by Rho-specific guanine nucleotide exchange factors (GEFs) and the Rho family of small GTPases. Rho-associated kinase (ROCK) promotes assembly of focal adhesions and actin stress fibers by regulating cytoskeleton organization. GEF exchange protein directly activated by cAMP 1 (EPAC1) has been implicated in modulating TGFß1 and Rho signaling; however, its role in HSC migration has never been examined. The aim of this study was to evaluate the role of cAMP-degrading phosphodiesterase 4 (PDE4) enzymes in regulating EPAC1 signaling, HSC migration, and fibrogenesis. We show that PDE4 protein expression is increased in activated HSCs expressing alpha smooth muscle actin and active myosin light chain (MLC) in fibrotic tissues of human nonalcoholic steatohepatitis cirrhosis livers and mouse livers exposed to carbon tetrachloride. In human livers, TGFß1 levels were highly correlated with PDE4 expression. TGFß1 treatment of LX2 HSCs decreased levels of cAMP and EPAC1 and increased PDE4D expression. PDE4 specific inhibitor, rolipram, and an EPAC-specific agonist decreased TGFß1-mediated cell migration in vitro. In vivo, targeted delivery of rolipram to the liver prevented fibrogenesis and collagen deposition and decreased the expression of several fibrosis-related genes, and HSC activation. Proteomic analysis of mouse liver tissues identified the regulation of actin cytoskeleton by the kinase effectors of Rho GTPases as a major pathway impacted by rolipram. Western blot analyses confirmed that PDE4 inhibition decreased active MLC and endothelin 1 levels, key proteins involved in cytoskeleton remodeling and contractility. The current study, for the first time, demonstrates that PDE4 enzymes are expressed in hepatic myofibroblasts and promote cytoskeleton remodeling and HSC migration. © 2023 The Pathological Society of Great Britain and Ireland.
Assuntos
Actinas , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4 , Animais , Humanos , Camundongos , Actinas/metabolismo , Movimento Celular , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/genética , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/metabolismo , Citoesqueleto/metabolismo , Citoesqueleto/patologia , Fibrose , Células Estreladas do Fígado/metabolismo , Cirrose Hepática/patologia , Proteômica , Rolipram/metabolismoRESUMO
AIM: Circulated histones play a crucial role in the pathogenesis of infectious diseases and severe trauma, and it is one of the potential molecular targets for therapeutics. Recently, we reported that histone is one of the causative agents for urinary L-FABP increase. However, the mechanism is still unclear, especially in severe cases. We further investigated the mechanism of urinary L-FABP increase using a more severe mouse model with histone-induced kidney injury. This study also aims to evaluate the therapeutic responsiveness of urinary L-FABP as a preliminary study. METHODS: Human L-FABP chromosomal transgenic mice were administrated 30 mg/kg histone from a tail vein with a single dose. We also performed a comparative study in LPS administration model. For the evaluation of the therapeutic responsiveness of urinary L-FABP, we used heparin and rolipram. RESULTS: The histological change with cast formation as a characteristic of the models was observed in proximal tubules. Urinary L-FABP levels were significantly elevated and these levels tended to be higher in those with more cast formation. Heparin and rolipram had the ameliorative effect of the cast formation induced by histone and urinary L-FABP levels significantly decreased. CONCLUSION: Histone is one of the causative agents for the increase of urinary L-FABP at an early stage of AKI. In addition, it suggested that urinary L-FABP may be useful as a subclinical AKI marker reflecting kidney damage induced by histone. Furthermore, urinary L-FABP reflected the degree of the damage after the administration of therapeutic agents such as heparin and PDE4 inhibitor.
Assuntos
Injúria Renal Aguda , Histonas , Camundongos , Animais , Humanos , Preparações Farmacêuticas , Rolipram , Rim/patologia , Injúria Renal Aguda/induzido quimicamente , Injúria Renal Aguda/diagnóstico , Camundongos Transgênicos , Proteínas de Ligação a Ácido Graxo/genética , Proteínas de Ligação a Ácido Graxo/urina , Biomarcadores/urina , Heparina , FígadoRESUMO
BACKGROUND: Alzheimer disease (AD) and depression often cooccur, and inhibition of phosphodiesterase-4 (PDE4) has been shown to ameliorate neurodegenerative illness. Therefore, we explored whether PDE4 inhibitor rolipram might also improve the symptoms of comorbid AD and depression. METHODS: APP/PS1/tau mice (10 months old) were treated with or without daily i.p. injections of rolipram for 10 days. The animal groups were compared in behavioral tests related to learning, memory, anxiety, and depression. Neurochemical measures were conducted to explore the underlying mechanism of rolipram. RESULTS: Rolipram attenuated cognitive decline as well as anxiety- and depression-like behaviors. These benefits were attributed at least partly to the downregulation of amyloid-ß, Amyloid precursor protein (APP), and Presenilin 1 (PS1); lower tau phosphorylation; greater neuronal survival; and normalized glial cell function following rolipram treatment. In addition, rolipram upregulated B-cell lymphoma-2 (Bcl-2) and downregulated Bcl-2-associated X protein (Bax) to reduce apoptosis; it also downregulated interleukin-1ß, interleukin-6, and tumor necrosis factor-α to restrain neuroinflammation. Furthermore, rolipram increased cAMP, PKA, 26S proteasome, EPAC2, and phosphorylation of ERK1/2 while decreasing EPAC1. CONCLUSIONS: Rolipram may mitigate cognitive deficits and depression-like behavior by reducing amyloid-ß pathology, tau phosphorylation, neuroinflammation, and apoptosis. These effects may be mediated by stimulating cAMP/PKA/26S and cAMP/exchange protein directly activated by cAMP (EPAC)/ERK signaling pathways. This study suggests that PDE4 inhibitor rolipram can be an effective target for treatment of comorbid AD and depression.
Assuntos
Doença de Alzheimer , Inibidores da Fosfodiesterase 4 , Camundongos , Animais , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Precursor de Proteína beta-Amiloide/farmacologia , Rolipram/farmacologia , Camundongos Transgênicos , Inibidores da Fosfodiesterase 4/farmacologia , Doenças Neuroinflamatórias , Presenilina-1/metabolismo , Presenilina-1/farmacologia , Depressão/tratamento farmacológico , Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Transtornos da Memória/tratamento farmacológico , Apoptose , Modelos Animais de DoençasRESUMO
Rolipram is a selective phosphodiesterase-4 (PDE4) inhibitor. The effect of rolipram on the metastasis of choriocarcinoma is barely known. Here, we evaluated the role of rolipram in the migration and invasion of human choriocarcinoma cells in vitro. Human choriocarcinoma cells lines JEG3 and JAR were used in this study. The expression profile of PDE4 subfamily members in choriocarcinoma cells was evaluated using real-time PCR. The migration and invasion properties of choriocarcinoma cells before and after inhibition of PDE4 by rolipram or RNAi-directed knockdown were evaluated in vitro. Expression levels of MMP9, TIMP1, E-cadherin, vimentin, TGFß1, SMAD1, and SMAD4 of choriocarcinoma cells were compared before and after rolipram treatment, RNAi-directed knockdown of PDE4D, and overexpression of PDE4D. We found PDE4D was the most commonly expressed isoform of PDE4 both in JEG3 and JAR cells. Rolipram and knockdown of PDE4D were efficient to inhibit the migration and invasion of choriocarcinoma cells in vitro, accompanied by decreased expression of MMP9 and TIMP1. Furthermore, rolipram and knockdown of PDE4D promoted the expression of E-cadherin but reduced the expression of vimentin in choriocarcinoma cells, and overexpression of PDE4D decreased the expression of E-cadherin but promoted the expression of vimentin. Rolipram suppressed migration and invasion of human choriocarcinoma cells in vitro, possibly by inhibiting epithelial-mesenchymal transition through PDE4 inhibition.
Assuntos
Coriocarcinoma , Inibidores da Fosfodiesterase 4 , Gravidez , Feminino , Humanos , Rolipram/farmacologia , Rolipram/metabolismo , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/genética , Metaloproteinase 9 da Matriz/genética , Vimentina , Linhagem Celular Tumoral , Transição Epitelial-Mesenquimal , Inibidores da Fosfodiesterase 4/farmacologiaRESUMO
Diabetic neuropathy (DN) is the most prevalent complication of diabetes. Pharmacological treatments for DN are often limited in efficacy, so the development of new agents to alleviate DN is essential. The aim of this study was to evaluate the effects of rolipram, a selective phosphodiesterase-4 inhibitor (PDE-4I), and pentoxifylline, a general PDE inhibitor, using a rat model of DN. In this study, a diabetic rat model was established by i.p. injection of STZ (55 mg/kg). Rats were treated with rolipram (1 mg/kg), pentoxifylline (100 mg/kg), and combination of rolipram (0.5 mg/kg) and pentoxifylline (50 mg/kg), orally for 5 weeks. After treatments, sensory function was assessed by hot plate test. Then rats were anesthetized and dorsal root ganglion (DRG) neurons isolated. Cyclic adenosine monophosphate (cAMP), adenosine triphosphate (ATP, adenosine diphosphate and mitochondrial membrane potential (MMP) levels, Cytochrome c release, Bax, Bcl-2, caspase-3 proteins expression in DRG neurons were assessed by biochemical and ELISA methods, and western blot analysis. DRG neurons were histologically examined using hematoxylin and eosin (H&E) staining method. Rolipram and/or pentoxifylline significantly attenuated sensory dysfunction by modulating nociceptive threshold. Rolipram and/or pentoxifylline treatment dramatically increased the cAMP level, prevented mitochondrial dysfunction, apoptosis and degeneration of DRG neurons, which appears to be mediated by inducing ATP and MMP, improving cytochrome c release, as well as regulating the expression of Bax, Bcl-2, and caspase-3 proteins, and improving morphological abnormalities of DRG neurons. We found maximum effectiveness with rolipram and pentoxifylline combination on mentioned factors. These findings encourage the use of rolipram and pentoxifylline combination as a novel experimental evidence for further clinical investigations in the treatment of DN.
Assuntos
Diabetes Mellitus , Neuropatias Diabéticas , Pentoxifilina , Ratos , Animais , Pentoxifilina/farmacologia , Pentoxifilina/uso terapêutico , Rolipram/farmacologia , Rolipram/metabolismo , Rolipram/uso terapêutico , Neuropatias Diabéticas/metabolismo , Caspase 3/metabolismo , Citocromos c/metabolismo , Gânglios Espinais/metabolismo , Proteína X Associada a bcl-2/metabolismo , Inibidores de Fosfodiesterase/farmacologia , Inibidores de Fosfodiesterase/metabolismo , Inibidores de Fosfodiesterase/uso terapêutico , Apoptose , Neurônios/metabolismo , Trifosfato de Adenosina/metabolismo , Mitocôndrias , Diabetes Mellitus/metabolismoRESUMO
Spinal cord injury (SCI) results in immediate axonal damage and cell death, as well as a prolonged secondary injury consist of a cascade of pathophysiological processes. One important aspect of secondary injury is activation of phosphodiesterase 4 (PDE4) that leads to reduce cAMP levels in the injured spinal cord. We have developed an amphiphilic copolymer, poly (lactide-co-glycolide)-graft-polyethylenimine (PgP) that can deliver Rolipram, the PDE4 inhibitor. The objective of this work was to investigate the effect of rolipram loaded PgP (Rm-PgP) on secondary injury and motor functional recovery in a rat moderate contusion SCI model. We observed that Rm-PgP can increase cAMP level at the lesion site, and reduce secondary injury such as the inflammatory response by macrophages/microglia, astrogliosis by activated astrocytes and apoptosis as well as improve neuronal survival at 4 weeks post-injury (WPI). We also observed that Rm-PgP can improve motor functional recovery after SCI over 4 WPI.
Assuntos
Contusões , Nanopartículas , Traumatismos da Medula Espinal , Ratos , Animais , Rolipram/farmacologia , Rolipram/uso terapêutico , Ratos Sprague-Dawley , Recuperação de Função Fisiológica , Traumatismos da Medula Espinal/tratamento farmacológico , Contusões/tratamento farmacológicoRESUMO
Ineffective cancer treatment is implicated in metastasis, recurrence, resistance to chemotherapy and radiotherapy, and evasion of immune surveillance. All these failures occur due to the persistence of cancer stem cells (CSCs) even after rigorous therapy, thereby rendering them as essential targets for cancer management. Contrary to the quiescent nature of CSCs, a gene profiler array disclosed that phosphatidylinositol-3-kinase (PI3K), which is known to be crucial for cell proliferation, differentiation, and survival, was significantly upregulated in CSCs. Since PI3K is modulated by cyclic adenosine 3',5' monophosphate (cAMP), analyses of cAMP regulation revealed that breast CSCs expressed increased levels of phosphodiesterase 4 (PDE4) in contrast to normal stem cells. In accordance, the effects of rolipram, a PDE4 inhibitor, were evaluated on PI3K regulators and signaling. The efficacy of rolipram was compared with paclitaxel, an anticancer drug that is ineffective in obliterating breast CSCs. Analyses of downstream signaling components revealed a switch between cell survival and death, in response to rolipram, specifically of the CSCs. Rolipram-mediated downregulation of PDE4A levels in breast CSCs led to an increase in cAMP levels and protein kinase A (PKA) expression. Subsequently, PKA-mediated upregulation of phosphatase and tensin homolog antagonized the PI3K/AKT/mTOR pathway and led to cell cycle arrest. Interestingly, direct yet noncanonical activation of mTOR by PKA, circumventing the influence of PI3K and AKT, temporally shifted the fate of CSCs toward apoptosis. Rolipram in combination with paclitaxel indicated synergistic consequences, which effectively obliterated CSCs within a tumor, thereby suggesting combinatorial therapy as a sustainable and effective strategy to abrogate breast CSCs for better patient prognosis.
Assuntos
Neoplasias da Mama , Inibidores da Fosfodiesterase 4 , Humanos , Feminino , Inibidores da Fosfodiesterase 4/farmacologia , Inibidores da Fosfodiesterase 4/metabolismo , Rolipram/farmacologia , Rolipram/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Paclitaxel/farmacologia , Células-Tronco Neoplásicas/metabolismoRESUMO
5- HT2A receptor is a member of the family A G-protein-coupled receptor. It is involved in many psychiatric disorders, such as depression, addiction and Parkinson's disease. 5-HT2AR targeted drugs play an important role in regulating cognition, memory, emotion and other physiological function by coupling G proteins, and their most notable function is stimulating the serotonergic hallucination. However, not all 5-HT2AR agonists exhibit hallucinogenic activity, such as lisuride. Molecular mechanisms of these different effects are not well illustrated. This study suggested that 5-HT2AR coupled both Gs and Gq protein under hallucinogenic agonists DOM and 25CN-NBOH stimulation, but nonhallucinogenic agonist lisuride and TBG only activates Gq signaling. Moreover, in head twitch response (HTR) model, we found that cAMP analogs 8-Bromo-cAMP and PDE4 inhibitor Rolipram could increase HTR, while Gs protein inhibitor Melittin could reduce HTR. Collectively, these results revealed that Gs signaling is a key signaling pathway that may distinguish hallucinogenic agonists and nonhallucinogenic agonists.
Assuntos
Subunidades alfa Gs de Proteínas de Ligação ao GTP/metabolismo , Alucinógenos/farmacologia , Movimentos da Cabeça/efeitos dos fármacos , Agonistas do Receptor 5-HT2 de Serotonina/farmacologia , Animais , AMP Cíclico/metabolismo , Células HEK293 , Movimentos da Cabeça/fisiologia , Humanos , Lisurida/farmacologia , Masculino , Meliteno/farmacologia , Camundongos Endogâmicos C57BL , Rolipram/farmacologia , Transdução de Sinais/efeitos dos fármacosRESUMO
RATIONALE: The stress response of heart rate, which is determined by the plasticity of the sinoatrial node (SAN), is essential for cardiac function and survival in mammals. As an RNA-binding protein, CIRP (cold-inducible RNA-binding protein) can act as a stress regulator. Previously, we have documented that CIRP regulates cardiac electrophysiology at posttranscriptional level, suggesting its role in SAN plasticity, especially upon stress conditions. OBJECTIVE: Our aim was to clarify the role of CIRP in SAN plasticity and heart rate regulation under stress conditions. METHODS AND RESULTS: Telemetric ECG monitoring demonstrated an excessive acceleration of heart rate under isoprenaline stimulation in conscious CIRP-KO (knockout) rats. Patch-clamp analysis and confocal microscopic Ca2+ imaging of isolated SAN cells demonstrated that isoprenaline stimulation induced a faster spontaneous firing rate in CIRP-KO SAN cells than that in WT (wild type) SAN cells. A higher concentration of cAMP-the key mediator of pacemaker activity-was detected in CIRP-KO SAN tissues than in WT SAN tissues. RNA sequencing and quantitative real-time polymerase chain reaction analyses of single cells revealed that the 4B and 4D subtypes of PDE (phosphodiesterase), which controls cAMP degradation, were significantly decreased in CIRP-KO SAN cells. A PDE4 inhibitor (rolipram) abolished the difference in beating rate resulting from CIRP deficiency. The mechanistic study showed that CIRP stabilized the mRNA of Pde4b and Pde4d by direct mRNA binding, thereby regulating the protein expression of PDE4B and PDE4D at posttranscriptional level. CONCLUSIONS: CIRP acts as an mRNA stabilizer of specific PDEs to control the cAMP concentration in SAN, maintaining the appropriate heart rate stress response.
Assuntos
Proteínas e Peptídeos de Choque Frio/metabolismo , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/metabolismo , Frequência Cardíaca , Miócitos Cardíacos/metabolismo , Proteínas de Ligação a RNA/metabolismo , Agonistas Adrenérgicos beta/farmacologia , Animais , Células Cultivadas , Proteínas e Peptídeos de Choque Frio/genética , AMP Cíclico/metabolismo , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/genética , Isoproterenol/farmacologia , Masculino , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/fisiologia , Inibidores de Fosfodiesterase/farmacologia , Estabilidade de RNA , Proteínas de Ligação a RNA/genética , Ratos , Ratos Sprague-Dawley , Rolipram/farmacologia , Nó Sinoatrial/citologia , Nó Sinoatrial/metabolismo , Nó Sinoatrial/fisiologia , Estresse FisiológicoRESUMO
ABSTRACT: In human hearts, muscarinic receptors (M-R) are expressed in ventricular and atrial tissue, but the acetylcholine-activated potassium current (IK,ACh) is expressed mainly in the atrium. M-R activation decreases force and increases electrical stability in human atrium, but the impact of IK,ACh to both effects remains unclear. We used a new selective blocker of IK,ACh to elaborate the contribution of IK,ACh to M-R activation-mediated effects in human atrium. Force and action potentials were measured in rat atria and in human right atrial trabeculae. Cumulative concentration-effect curves for norepinephrine-induced force and arrhythmias were measured in the presence of carbachol (CCh; 1 µM) or CCh together with the IK,ACh -blocker XAF-1407 (1 µM) or in time-matched controls. To investigate the vulnerability to arrhythmias, we performed some experiments also in the presence of cilostamide (0.3 µM) and rolipram (1 µM), inhibiting PDE3 and PDE4. In rat atria and human right atrial trabeculae, CCh shortened the action potential duration persistently. However, the direct negative inotropy of CCh was only transient in human, but stable in rat atria. In rat and human atria, the negative inotropic effect was insensitive to blockage of IK,ACh by XAF-1407. In the presence of cilostamide and rolipram about 40% of trabeculae developed arrhythmias when exposed to norepinephrine. CCh prevented these concentration-dependent norepinephrine-induced arrhythmias, again insensitive to XAF-1407. Maximum catecholamine-induced force was not depressed by CCh. In human atrium, the direct and the indirect negative inotropic effect of CCh are independent of IK,ACh. The same applies to the CCh-mediated suppression of norepinephrine/PDE-inhibition-induced arrhythmias.
Assuntos
Acetilcolina , Átrios do Coração , Acetilcolina/farmacologia , Animais , Arritmias Cardíacas/induzido quimicamente , Humanos , Norepinefrina/farmacologia , Ratos , Receptores Muscarínicos/fisiologia , Rolipram/farmacologiaRESUMO
Diabetic neuropathy (DN) is the most challenging microvascular complication of diabetes and there is no suitable treatment for it, so the development of new agents to relieve DN is urgently needed. Since oxidative stress and inflammation play an essential role in the development of DN, clearance of these factors are good strategies for the treatment of this disease. According to key role of cyclic adenosine monophosphate (cAMP) in the regulation of oxidative stress and inflammatory pathways, it seems that phosphodiesterase inhibitors (PDEIs) can be as novel drug targets for improving DN through enhancement of cAMP level. The aim of this study was to evaluate the effects of rolipram, a selective PDE4 inhibitor, and pentoxifylline, a general PDE inhibitor on experimental model of DN and also to determine the possible mechanisms involved in the effectiveness of these agents. We investigated the effects of rolipram (1 mg/kg) and pentoxifylline (100 mg/kg) and also combination of rolipram (0.5 mg/kg) and pentoxifylline (50 mg/kg), orally for five weeks in rats that became diabetic by STZ (55 mg/kg, i.p.). After treatments, motor function was evaluated by open-field test, then rats were anesthetized and dorsal root ganglion (DRG) neurons isolated. Next, oxidative stress biomarkers and inflammatory factors were assessed by biochemical and ELISA methods, and RT-PCR analysis in DRG neurons. Rolipram and/or pentoxifylline treatment significantly attenuated DN - induced motor function deficiency by modulating distance moved and velocity. Rolipram and/or pentoxifylline treatment dramatically increased the cAMP level, as well as suppressed DN - induced oxidative stress which was associated with decrease in LPO and ROS and increase in TAC, total thiol, CAT and SOD in DRG neurons. On the other hand, the level of inflammatory factors (TNF-α, NF-kB and COX2) significantly decreased following rolipram and/or pentoxifylline administration. The maximum effectiveness was with rolipram and/or pentoxifylline combination on mentioned factors. These findings provide novel experimental evidence for further clinical investigations on rolipram and pentoxifylline combination for the treatment of DN.
Assuntos
Neuropatias Diabéticas , Pentoxifilina , Inibidores da Fosfodiesterase 4 , Animais , Ratos , Rolipram/farmacologia , Rolipram/uso terapêutico , Pentoxifilina/farmacologia , Pentoxifilina/uso terapêutico , Neuropatias Diabéticas/tratamento farmacológico , Gânglios Espinais/metabolismo , Inibidores da Fosfodiesterase 4/farmacologia , Fator de Necrose Tumoral alfa/metabolismo , Espécies Reativas de Oxigênio , NF-kappa B/metabolismo , Ciclo-Oxigenase 2/metabolismo , Estresse Oxidativo , Neurônios/metabolismo , Biomarcadores/metabolismo , Compostos de Sulfidrila , Monofosfato de Adenosina/metabolismo , Superóxido Dismutase/metabolismoRESUMO
Extracts from Drosera rotundifolia are traditionally used to treat cough symptoms during a common cold. The present study aimed to investigate the impact of extracts from D. rotundifolia and active compounds on the respiratory tract. Tracheal slices of C57BL/6N mice were used ex vivo to examine effects on airway smooth muscle (ASM) and ciliary beat frequency (CBF). Phosphodiesterase (PDE) inhibition assays were carried out to test whether PDE1 or PDE4 are targeted by the active compounds. An ethanol-water extract, as well as an aqueous fraction of this extract, exerted antispasmodic properties against acetylcholine-induced contractions. In addition, contractions induced by 60 mM K+ were abrogated by the aqueous fraction. Effects on ASM could be attributed to the flavonoids quercetin, 2â³-O-galloylhyperoside and hyperoside. Moreover, the Drosera extract and the aqueous fraction increased the CBF of murine tracheal slices. Quercetin and 2â³-O-galloylhyperoside were identified as active compounds involved in the elevation of CBF. Both compounds inhibited PDE1A and PDE4D. The elevation of CBF was mimicked by the subtype-selective PDE inhibitor rolipram (PDE4) and by 8-methoxymethyl-IBMX. In summary, our study shows, for the first time, that a Drosera extract and its flavonoid compounds increase the CBF of murine airways while antispasmodic effects were transferred to ASM.
Assuntos
Drosera , 1-Metil-3-Isobutilxantina/farmacologia , Acetilcolina/farmacologia , Animais , Etanol/farmacologia , Flavonoides/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Músculo Liso , Parassimpatolíticos/farmacologia , Diester Fosfórico Hidrolases/farmacologia , Quercetina/farmacologia , Rolipram/farmacologia , Traqueia , Água/farmacologiaRESUMO
Increased lung vascular permeability and neutrophilic inflammation are hallmarks of acute lung injury. Alveolar macrophages (AMÏ), the predominant sentinel cell type in the airspace, die in massive numbers while fending off pathogens. Recent studies indicate that the AMÏ pool is replenished by airspace-recruited monocytes, but the mechanisms instructing the conversion of recruited monocytes into reparative AMÏ remain elusive. Cyclic AMP (cAMP) is a vascular barrier protective and immunosuppressive second messenger in the lung. Here, we subjected mice expressing GFP under the control of the Lysozyme-M promoter (LysM-GFP mice) to the LPS model of rapidly resolving lung injury to address the impact of mechanisms determining cAMP levels in AMÏ and regulation of mobilization of the reparative AMÏ-pool. RNA-seq analysis of flow-sorted MÏ identified phosphodiesterase 4b (PDE4b) as the top LPS-responsive cAMP-regulating gene. We observed that PDE4b expression markedly increased at the time of peak injury (4 h) and then decreased to below the basal level during the resolution phase (24 h). Activation of transcription factor NFATc2 was required for the transcription of PDE4b in MÏ. Inhibition of PDE4 activity at the time of peak injury, using intratracheal rolipram, increased cAMP levels, augmented the reparative AMÏ pool, and resolved lung injury. This response was not seen following conditional depletion of monocytes, thus establishing airspace-recruited PDE4b-sensitive monocytes as the source of reparative AMÏ. Interestingly, adoptive transfer of rolipram-educated AMÏ into injured mice resolved lung edema. We propose suppression of PDE4b as an effective approach to promote reparative AMÏ generation from monocytes for lung repair.
Assuntos
Lesão Pulmonar Aguda/patologia , AMP Cíclico/metabolismo , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/metabolismo , Macrófagos Alveolares/citologia , Monócitos/citologia , Fatores de Transcrição NFATC/metabolismo , Transferência Adotiva/métodos , Animais , Permeabilidade Capilar/fisiologia , Diferenciação Celular/fisiologia , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/genética , Feminino , Inflamação , Lipopolissacarídeos/farmacologia , Macrófagos Alveolares/transplante , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neutrófilos/imunologia , Inibidores da Fosfodiesterase 4/farmacologia , Rolipram/farmacologia , Ativação Transcricional/genéticaRESUMO
The expansion of primordial germ cells (PGCs), the precursors for the oocytes and spermatozoa, is a key challenge in reproductive biology/medicine. Using a chemical screening exploiting PGC-like cells (PGCLCs) induced from mouse embryonic stem cells (ESCs), we here identify key signaling pathways critical for PGCLC proliferation. We show that the combinatorial application of Forskolin and Rolipram, which stimulate cAMP signaling via different mechanisms, expands PGCLCs up to ~50-fold in culture. The expanded PGCLCs maintain robust capacity for spermatogenesis, rescuing the fertility of infertile mice. Strikingly, during expansion, PGCLCs comprehensively erase their DNA methylome, including parental imprints, in a manner that precisely recapitulates genome-wide DNA demethylation in gonadal germ cells, while essentially maintaining their identity as sexually uncommitted PGCs, apparently through appropriate histone modifications. By establishing a paradigm for PGCLC expansion, our system reconstitutes the epigenetic "blank slate" of the germ line, an immediate precursory state for sexually dimorphic differentiation.
Assuntos
Diferenciação Celular , Proliferação de Células , Células-Tronco Embrionárias/fisiologia , Epigênese Genética , Células Germinativas/crescimento & desenvolvimento , Animais , Colforsina/metabolismo , Células Germinativas/efeitos dos fármacos , Camundongos , Rolipram/metabolismo , Transdução de SinaisRESUMO
INTRODUCTION: Over 300 million people in the world live with asthma, resulting in 500,000 annual global deaths with future increases expected. It is estimated that around 50-80% of asthma exacerbations are due to viral infections. Currently, a combination of long-acting beta agonists (LABA) for bronchodilation and glucocorticoids (GCS) to control lung inflammation represent the dominant strategy for the management of asthma, however, it is still sub-optimal in 35-50% of moderate-severe asthmatics resulting in persistent lung inflammation, impairment of lung function, and risk of mortality. Mechanistically, LABA/GCS combination therapy results in synergistic efficacy mediated by intracellular cyclic adenosine monophosphate (cAMP). HYPOTHESIS: Increasing intracellular cAMP during LABA/GCS combination therapy via inhibiting phosphodiesterase 4 (PDE4) and/or blocking the export of cAMP by ATP Binding Cassette Transporter C4 (ABCC4), will potentiate anti-inflammatory responses of mainstay LABA/GCS therapy. METHODS: Expression and localization experiments were performed using in situ hybridization and immunohistochemistry in human lung tissue from healthy subjects, while confirmatory transcript and protein expression analyses were performed in primary human airway epithelial cells and cell lines. Intervention experiments were performed on the human airway epithelial cell line, HBEC-6KT, by pre-treatment with combinations of LABA/GCS with PDE4 and/or ABCC4 inhibitors followed by Poly I:C or imiquimod challenge as a model for viral stimuli. Cytokine readouts for IL-6, IL-8, CXCL10/IP-10, and CCL5/RANTES were quantified by ELISA. RESULTS: Using archived human lung and human airway epithelial cells, ABCC4 gene and protein expression were confirmed in vitro and in situ. LABA/GCS attenuation of Poly I:C or imiquimod-induced IL-6 and IL-8 were potentiated with ABCC4 and PDE4 inhibition, which was greater when ABCC4 and PDE4 inhibition was combined. Modulation of cAMP levels had no impact on LABA/GCS modulation of Poly I:C-induced CXCL10/IP-10 or CCL5/RANTES. CONCLUSION: Modulation of intracellular cAMP levels by PDE4 or ABCC4 inhibition potentiates LABA/GCS efficacy in human airway epithelial cells challenged with viral stimuli. The data suggest further exploration of the value of adding cAMP modulators to mainstay LABA/GCS therapy in asthma for potentiated anti-inflammatory efficacy.
Assuntos
Agonistas de Receptores Adrenérgicos beta 2/farmacologia , Budesonida/farmacologia , AMP Cíclico/metabolismo , Células Epiteliais/efeitos dos fármacos , Fumarato de Formoterol/farmacologia , Glucocorticoides/farmacologia , Pulmão/efeitos dos fármacos , Aminopiridinas/farmacologia , Benzamidas/farmacologia , Benzotiazóis/farmacologia , Linhagem Celular , Quimiocinas/metabolismo , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/metabolismo , Ácidos Cicloexanocarboxílicos/farmacologia , Ciclopropanos/farmacologia , Sinergismo Farmacológico , Quimioterapia Combinada , Células Epiteliais/metabolismo , Humanos , Pulmão/metabolismo , Proteínas Associadas à Resistência a Múltiplos Medicamentos/antagonistas & inibidores , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Nitrilas/farmacologia , Inibidores da Fosfodiesterase 4/farmacologia , Rolipram/farmacologia , Sistemas do Segundo Mensageiro , Triazóis/farmacologiaRESUMO
Inhibitors of cAMP-phosphodiesterase 4 (PDE4) exert a number of promising therapeutic benefits, but adverse effects, in particular emesis and nausea, have curbed their clinical utility. Here, we show that PAN-selective inhibition of PDE4, but not inhibition of PDE3, causes a time- and dose-dependent accumulation of chow in the stomachs of mice fed ad libitum without changing the animals' food intake or the weight of their intestines, suggesting that PDE4 inhibition impairs gastric emptying. Indeed, PDE4 inhibition induced gastric retention in an acute model of gastric motility that traces the passage of a food bolus through the stomach over a 30 minutes time period. In humans, abnormal gastric retention of food is known as gastroparesis, a syndrome predominated by nausea (>90% of cases) and vomiting (>80% of cases). We thus explored the abnormal gastric retention induced by PDE4 inhibition in mice under the premise that it may represent a useful correlate of emesis and nausea. Delayed gastric emptying was produced by structurally distinct PAN-PDE4 inhibitors including Rolipram, Piclamilast, Roflumilast, and RS25344, suggesting that it is a class effect. PDE4 inhibitors induced gastric retention at similar or below doses commonly used to induce therapeutic benefits (e.g., 0.04 mg/kg Rolipram), thus mirroring the narrow therapeutic window of PDE4 inhibitors in humans. YM976, a PAN-PDE4 inhibitor that does not efficiently cross the blood-brain barrier, induced gastroparesis only at significantly higher doses (≥1 mg/kg). This suggests that PDE4 inhibition may act in part through effects on the autonomic nervous system regulation of gastric emptying and that PDE4 inhibitors that are not brain-penetrant may have an improved safety profile. The PDE4 family comprises four subtypes, PDE4A, B, C, and D. Selective ablation of any of these subtypes in mice did not induce gastroparesis per se, nor did it protect from PAN-PDE4 inhibitor-induced gastroparesis, indicating that gastric retention may result from the concurrent inhibition of multiple PDE4s. Thus, potentially, any of the four PDE4 subtypes may be targeted individually for therapeutic benefits without inducing nausea or emesis. Acute gastric retention induced by PDE4 inhibition is alleviated by treatment with the widely used prokinetic Metoclopramide, suggesting a potential of this drug to alleviate the side effects of PDE4 inhibitors. Finally, given that the cause of gastroparesis remains largely idiopathic, our findings open the possibility that a physiologic or pathophysiologic downregulation of PDE4 activity/expression may be causative in a subset of patients.
Assuntos
Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/metabolismo , Gastroparesia/induzido quimicamente , Inibidores da Fosfodiesterase 4/efeitos adversos , Aminopiridinas/efeitos adversos , Animais , Benzamidas/efeitos adversos , Ciclopropanos/efeitos adversos , Modelos Animais de Doenças , Feminino , Camundongos , Camundongos Nus , Piridinas/efeitos adversos , Pirimidinonas/efeitos adversos , Rolipram/efeitos adversosRESUMO
Chemical manufacturing is conducted using either batch systems or continuous-flow systems. Flow systems have several advantages over batch systems, particularly in terms of productivity, heat and mixing efficiency, safety, and reproducibility. However, for over half a century, pharmaceutical manufacturing has used batch systems because the synthesis of complex molecules such as drugs has been difficult to achieve with continuous-flow systems. Here we describe the continuous-flow synthesis of drugs using only columns packed with heterogeneous catalysts. Commercially available starting materials were successively passed through four columns containing achiral and chiral heterogeneous catalysts to produce (R)-rolipram, an anti-inflammatory drug and one of the family of γ-aminobutyric acid (GABA) derivatives. In addition, simply by replacing a column packed with a chiral heterogeneous catalyst with another column packed with the opposing enantiomer, we obtained antipole (S)-rolipram. Similarly, we also synthesized (R)-phenibut, another drug belonging to the GABA family. These flow systems are simple and stable with no leaching of metal catalysts. Our results demonstrate that multistep (eight steps in this case) chemical transformations for drug synthesis can proceed smoothly under flow conditions using only heterogeneous catalysts, without the isolation of any intermediates and without the separation of any catalysts, co-products, by-products, and excess reagents. We anticipate that such syntheses will be useful in pharmaceutical manufacturing.