RESUMEN
Cyclic guanosine monophosphate (cGMP) is a second messenger produced by the NO-sensitive guanylyl cyclase (NO-GC). The NO-GC/cGMP pathway in platelets has been extensively studied. However, its role in regulating the biomechanical properties of platelets has not yet been addressed and remains unknown. We therefore investigated the stiffness of living platelets after treatment with the NO-GC stimulator riociguat or the NO-GC activator cinaciguat using scanning ion conductance microscopy (SICM). Stimulation of human and murine platelets with cGMP-modulating drugs decreased cellular stiffness and downregulated P-selectin, a marker for platelet activation. We also quantified changes in platelet shape using deep learning-based platelet morphometry, finding that platelets become more circular upon treatment with cGMP-modulating drugs. To test for clinical applicability of NO-GC stimulators in the context of increased thrombogenicity risk, we investigated the effect of riociguat on platelets from human immunodeficiency virus (HIV)-positive patients taking abacavir sulfate (ABC)-containing regimens. Our results corroborate a functional role of the NO-GC/cGMP pathway in platelet biomechanics, indicating that biomechanical properties such as stiffness or shape could be used as novel biomarkers in clinical research.
Increased platelet activation and development of thrombosis has been linked to a dysfunctional NO-GC/cGMP signaling pathway. How this pathway affects platelet stiffness, however, has not been studied yet. For the first time, we used novel microscopy techniques to investigate stiffness and shape of platelets in human and murine blood samples treated with cGMP modifying drugs. Stiffness contains information about biomechanical properties of the cytoskeleton, and shape quantifies the spreading behavior of platelets. We showed that the NO-GC/cGMP signaling pathway affects platelet stiffness, shape, and activation in human and murine blood. HIV-positive patients are often treated with medication that may disrupt the NO-GC/cGMP signaling pathway, leading to increased cardiovascular risk. We showed that treatment with cGMP-modifying drugs altered platelet shape and aggregation in blood from HIV-negative volunteers but not from HIV-positive patients treated with medication. Our study suggests that platelet stiffness and shape can be biomarkers for estimating cardiovascular risk.
Asunto(s)
Plaquetas , Transducción de Señal , Humanos , Ratones , Animales , Fenómenos Biomecánicos , Plaquetas/metabolismo , Guanilato Ciclasa/metabolismo , Guanilato Ciclasa/farmacología , Activación Plaquetaria , GMP Cíclico/metabolismo , GMP Cíclico/farmacología , Óxido Nítrico/metabolismo , Agregación PlaquetariaRESUMEN
This toxicology study was conducted to assess the impact of formaldehyde, a common air pollutant found in Chinese gymnasiums, on the brain function of athletes. In this research, a total of 24 Balb/c male mice of SPF-grade were divided into four groups, each consisting of six mice. The mice were exposed to formaldehyde at different concentrations, including 0 mg/m3, 0.5 mg/m3, 3.0 mg/m3, and 3.0 mg/m3 in combination with an injection of L-NMMA (NG-monomethyl-L-arginine), which is a nitric oxide synthase antagonist. Following a one-week test period (8 h per day, over 7 days), measurements of biomarkers related to the nitric oxide (NO)/cGMP-cAMP signaling pathway were carried out on the experimental animals post-treatment. The study found that: (1) Exposure to formaldehyde can lead to brain cell apoptosis and neurotoxicity; (2) Additionally, formaldehyde exposure was found to alter the biomarkers of the NO/cGMP-cAMP signaling pathway, with some changes being statistically significant (p < 0.05 or p < 0.01); (3) The use of L-NMMA, an antagonist of the NO/cGMP-cAMP signaling pathway, was found to prevent these biomarker changes and had a protective effect on brain cells. The study suggests that the negative impact of formaldehyde on the brain function of mice is linked to the regulation of the NO/cGMP-cAMP signaling pathway.
Asunto(s)
GMP Cíclico , Óxido Nítrico , Hipersensibilidad Respiratoria , Humanos , Masculino , Ratones , Animales , omega-N-Metilarginina/farmacología , Óxido Nítrico/metabolismo , Ratones Endogámicos BALB C , GMP Cíclico/farmacología , Formaldehído/toxicidad , Transducción de Señal , Encéfalo/metabolismo , BiomarcadoresRESUMEN
This study aimed to examine the endothelial dependence of vasodilation induced by the phosphodiesterase inhibitor theophylline in isolated rat thoracic aortas and elucidate the underlying mechanism, with emphasis on endothelial nitric oxide (NO). The effects of various inhibitors and endothelial denudation on theophylline-induced vasodilation, and the effect of theophylline on vasodilation induced by NO donor sodium nitroprusside, cyclic guanosine monophosphate (cGMP) analog bromo-cGMP, and ß-agonist isoproterenol in endothelium-denuded aorta were examined. The effects of theophylline and sodium nitroprusside on cGMP formation were also examined. We examined the effect of theophylline on endothelial nitric oxide synthase (eNOS) phosphorylation and intracellular calcium levels. Theophylline-induced vasodilation was greater in endothelium-intact aortas than that in endothelium-denuded aortas. The NOS inhibitor, NW-nitro-L-arginine methyl ester; non-specific guanylate cyclase (GC) inhibitor, methylene blue; and NO-sensitive GC inhibitor, 1H-[1,2,4]oxadiazolo[4,3-a] quinoxalin-1-one inhibited theophylline-induced vasodilation in endothelium-intact aortas. Theophylline increased the vasodilation induced by sodium nitroprusside, bromo-cGMP, and isoproterenol. Theophylline increased cGMP formation in endothelium-intact aortas, and sodium nitroprusside-induced cGMP formation in endothelium-denuded aortas. Moreover, theophylline increased stimulatory eNOS (Ser1177) phosphorylation and endothelial calcium levels, but decreased the phosphorylation of inhibitory eNOS (Thr495). These results suggested that theophylline-induced endothelium-dependent vasodilation was mediated by increased endothelial NO release and phosphodiesterase inhibition.
Asunto(s)
Óxido Nítrico , Vasodilatación , Ratas , Animales , Teofilina/farmacología , Isoproterenol/farmacología , Nitroprusiato/farmacología , Hidrolasas Diéster Fosfóricas/farmacología , Calcio , Aorta Torácica , Aorta , Óxido Nítrico Sintasa de Tipo III , GMP Cíclico/farmacología , GMP Cíclico/fisiología , Endotelio VascularRESUMEN
Volatile anesthetics alter presynaptic function through effects on Ca2+ influx and neurotransmitter release. These actions are proposed to play important roles in their pleiotropic neurophysiological effects including immobility, unconsciousness and amnesia. Nitric oxide and cyclic guanosine monophosphate (NO/cGMP) signaling has been implicated in presynaptic mechanisms, and disruption of NO/cGMP signaling has been shown to alter sensitivity to volatile anesthetics in vivo. We investigated volatile anesthetic actions NO/cGMP signaling in relation to presynaptic function in cultured rat hippocampal neurons using pharmacological tools and genetically encoded biosensors and sequestering probes of cGMP levels. Using the fluorescent cGMP biosensor cGull, we found that electrical stimulation-evoked NMDA-type glutamate receptor-independent presynaptic cGMP transients were inhibited 33.2% by isoflurane (0.51 mM) and 26.4% by sevoflurane (0.57 mM) (p < 0.0001) compared to control stimulation without anesthetic. Stimulation-evoked cGMP transients were blocked by the nonselective inhibitor of nitric oxide synthase N-ω-nitro-l-arginine, but not by the selective neuronal nitric oxide synthase inhibitor N5-(1-imino-3-butenyl)-l-ornithine. Isoflurane and sevoflurane inhibition of stimulation-evoked increases in presynaptic Ca2+ concentration, measured with synaptophysin-GCaMP6f, and of synaptic vesicle exocytosis, measured with synaptophysin-pHlourin, was attenuated in neurons expressing the cGMP scavenger protein sponge (inhibition of exocytosis reduced by 54% for isoflurane and by 53% for sevoflurane). The anesthetic-induced reduction in presynaptic excitability was partially occluded by inhibition of HCN channels, a cGMP-modulated excitatory ion channel that can facilitate glutamate release. We propose that volatile anesthetics depress presynaptic cGMP signaling and downstream effectors like HCN channels that are essential to presynaptic function and excitability. These findings identify novel mechanisms by which volatile anesthetics depress synaptic transmission via second messenger signaling involving the NO/cGMP pathway in hippocampal neurons.
Asunto(s)
Anestésicos por Inhalación , Isoflurano , Ratas , Animales , Isoflurano/farmacología , Sinaptofisina/metabolismo , Sevoflurano/farmacología , Ratas Sprague-Dawley , Neuronas , Anestésicos por Inhalación/farmacología , Ácido Glutámico/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Hipocampo , GMP Cíclico/farmacologíaRESUMEN
Aim: This study investigated the effect of allografting umbilical cord blood mononuclear cells (UCBMCs) into the scrotum on sexual function in male elderly mice. Methods: UCBMCs were injected once into the scrotal sheath cavity of elderly mice. Results: The transplanted UCBMCs survived in the scrotal sheath cavity for 1 month. The mice had significantly increased blood testosterone concentrations, cyclic guanosine monophosphate (cGMP) levels and total nitric oxide synthase (T-NOS) activity in the corpus cavernosum and an increase in the number of mouse matings within 30 min (all p = 0.000). Conclusion: Scrotum-implanted UCBMCs improve the sexual function of male elderly mice through testosterone production and the NOS/cGMP pathway, which may provide an innovative transplantation approach for the treatment of erectile dysfunction.
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Disfunción Eréctil , Sangre Fetal , Humanos , Ratones , Masculino , Animales , Anciano , Sangre Fetal/metabolismo , Escroto/metabolismo , Disfunción Eréctil/metabolismo , Pene/metabolismo , GMP Cíclico/metabolismo , GMP Cíclico/farmacología , Testosterona/metabolismo , Testosterona/farmacologíaRESUMEN
Staphylococcus epidermidis are common bacteria associated with biofilm related infections on implanted medical devices. Antibiotics are often used in combating such infections, but they may lose their efficacy in the presence of biofilms. Bacterial intracellular nucleotide second messenger signaling plays an important role in biofilm formation, and interference with the nucleotide signaling pathways provides a possible way to control biofilm formation and to increase biofilm susceptibility to antibiotic therapy. This study synthesized small molecule derivates of 4-arylazo-3,5-diamino-1 H-pyrazole (named as SP02 and SP03) and found these molecules inhibited S. epidermidis biofilm formation and induced biofilm dispersal. Analysis of bacterial nucleotide signaling molecules showed that both SP02 and SP03 significantly reduced cyclic dimeric adenosine monophosphate (c-di-AMP) levels in S. epidermidis at doses as low as 25 µM while having significant effects on multiple nucleotides signaling including cyclic dimeric guanosine monophosphate (c-di-GMP), c-di-AMP, and cyclic adenosine monophosphate (cAMP) at high doses (100 µM or greater). We then tethered these small molecules to polyurethane (PU) biomaterial surfaces and investigated biofilm formation on the modified surfaces. Results showed that the modified surfaces significantly inhibited biofilm formation during 24 h and 7-day incubations. The antibiotic ciprofloxacin was used to treat these biofilms and the efficacy of the antibiotic (2 µg/mL) was found to increase from 94.8% on unmodified PU surfaces to > 99.9% on both SP02 and SP03 modified surfaces (>3 log units). Results demonstrated the feasibility of tethering small molecules that interfere with nucleotide signaling onto polymeric biomaterial surfaces and in a way that interrupts biofilm formation and increases antibiotic efficacy for S. epidermidis infections.
Asunto(s)
Ciprofloxacina , Staphylococcus epidermidis , Ciprofloxacina/farmacología , Nucleótidos , Biopelículas , Antibacterianos/farmacología , GMP Cíclico/metabolismo , GMP Cíclico/farmacología , Materiales Biocompatibles/farmacología , Adenosina MonofosfatoRESUMEN
The inherited eye disease retinitis pigmentosa (RP) causes the loss of photoreceptors by a still unknown cell death mechanism. During this degeneration, cyclic guanosine-3',5'-monophosphate (cGMP) levels become elevated, leading to over-activation of the cGMP-binding protein cGMP-dependent protein kinase (PKG). cGMP analogs selectively modified to have inhibitory actions on PKG have aided in impeding photoreceptor death, and one such cGMP analog is Rp-8-Br-PET-cGMPS. However, cGMP analogs have previously been shown to interact with numerous targets, so to better understand the therapeutic action of Rp-8-Br-PET-cGMPS, it is necessary to elucidate its target-selectivity and hence what potential cellular mechanism(s) it may affect within the photoreceptors. Here, we, therefore, applied affinity chromatography together with mass spectrometry to isolate and identify Rp-8-Br-PET-cGMPS interactors from retinas derived from three different murine RP models (i.e., rd1, rd2, and rd10 mice). Our findings revealed that Rp-8-Br-PET-cGMPS bound seven known cGMP-binding proteins, including PKG1ß, PDE1ß, PDE1c, PDE6α, and PKA1α. Furthermore, an additional 28 proteins were found to be associated with Rp-8-Br-PET-cGMPS. This latter group included MAPK1/3, which is known to connect with cGMP/PKG in other systems. However, in organotypic retinal cultures, Rp-8-Br-PET-cGMPS had no effect on photoreceptor MAPK1/3 expression or activity. To summarize, Rp-8-Br-PET-cGMPS is more target specific compared to regular cGMP.
Asunto(s)
GMP Cíclico , Retina , Ratones , Animales , GMP Cíclico/metabolismo , GMP Cíclico/farmacología , Retina/metabolismo , Proteínas Quinasas Dependientes de GMP Cíclico/metabolismoRESUMEN
Cyclic guanosine 3',5'-monophosphate (cGMP) is a ubiquitous important second messenger involved in various physiological functions. Here, intracellular cGMP (cGMPi) was visualized in chemotactic Dictyostelium cells using the fluorescent probe, D-Green cGull. When wild-type cells were stimulated with a chemoattractant, fluorescence transiently increased, but guanylate cyclase-null cells did not show a change in fluorescence, suggesting that D-Green cGull is a reliable indicator of cGMPi. In the aggregation stage, the responses of cGMPi propagated in a wave-like fashion from the aggregation center. The oscillation of the cGMPi wave was synchronized almost in phase with those of other second messengers, such as the intracellular cAMP and Ca2+. The phases of these waves preceded those of the oscillations of actomyosin and cell velocity, suggesting that these second messengers are upstream of the actomyosin and chemotactic migration. An acute increase in cGMPi concentration released from membrane-permeable caged cGMP induced a transient shuttle of myosin II between the cytosol and cell cortex, suggesting a direct link between cGMP signaling and myosin II dynamics.
Asunto(s)
Dictyostelium , Dictyostelium/fisiología , Quimiotaxis/fisiología , Actomiosina , GMP Cíclico/farmacología , GMP Cíclico/fisiología , Miosina Tipo IIRESUMEN
Rewarming from accidental hypothermia could be complicated by acute cardiac dysfunction but providing supportive pharmacotherapy at low core temperatures is challenging. Several pharmacological strategies aim to improve cardiovascular function by increasing cAMP in cardiomyocytes as well as cAMP and cGMP levels in vascular smooth muscle, but it is not clear what effects temperature has on cellular elimination of cAMP and cGMP. We therefore studied the effects of differential temperatures from normothermia to deep hypothermia (37 °C-20 °C) on cAMP levels in embryonic H9c2 cardiac cells and elimination of cAMP and cGMP by PDE-enzymes and ABC-transporter proteins. Our experiments showed significant elevation of intracellular cAMP in H9c2-cells at 30 °C but not 20 °C. Elimination of both cAMP and cGMP through ABC transport-proteins and PDE-enzymes showed a temperature dependent reduction. Accordingly, the increased cardiomyocyte cAMP-levels during moderate hypothermia appears an effect of preserved production and reduced elimination at 30 °C. This correlates with earlier in vivo findings of a positive inotropic effect of moderate hypothermia.
Asunto(s)
Hipotermia , Humanos , AMP Cíclico/metabolismo , Criopreservación/métodos , Recalentamiento , Miocitos Cardíacos/metabolismo , GMP Cíclico/metabolismo , GMP Cíclico/farmacologíaRESUMEN
Beside its mechanical roles in controlling posture and locomotion, skeletal muscle system, the largest insulin and steroid hormones target tissue, plays a key role in influencing thermoregulation, secondary sexual characteristics, hormones metabolism, and glucose uptake and storage, as well as energetic metabolism. Indeed, in addition to insulin, several hormones influence the skeletal muscle metabolism/function and/or are influenced by skeletal muscles activity (i.e., physical exercise). Particularly, steroid hormones play a key role in modulating many biological processes in muscles, essential for overall muscle's function and homeostasis, both at rest and during all physical activities (i.e., physical exercise, muscular work). Phosphodiesterase type 5 (PDE5) is the enzyme engaged to hydrolyze cyclic guanosine monophosphate (cGMP) in inactive 5'-GMP form. Therefore, through the inhibition of this enzyme, the intracellular level of cGMP increases, and the cGMP-related cellular responses are prolonged. Different drugs inhibiting PDE5 (PDE5i) exist, and the commercially available PDE5i are sildenafil, vardenafil, tadalafil, and avanafil. The PDE5i tadalafil may influence cellular physiology and endocrine-metabolic pathways in skeletal muscles and exerts its functions both by activating the cell signaling linked to the insulin-related metabolic pathways and modulating the endocrine responses, protein catabolism and hormone-related anabolism/catabolism during and after physical exercise-related stress. Based on recent in-vivo and in-vitro findings, in this narrative review the aim was to summarize the available evidence describing the interactions between the PDE5i tadalafil and steroid hormones in skeletal muscle tissue and physical exercise adaptation, focusing our interest on their possible synergistic or competitive action(s) on muscle metabolism and function.
Asunto(s)
Insulinas , Inhibidores de Fosfodiesterasa 5 , Tadalafilo/farmacología , Tadalafilo/metabolismo , Inhibidores de Fosfodiesterasa 5/farmacología , Inhibidores de Fosfodiesterasa 5/metabolismo , Carbolinas/metabolismo , Carbolinas/farmacología , Músculo Esquelético/metabolismo , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 5/metabolismo , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 5/farmacología , GMP Cíclico/metabolismo , GMP Cíclico/farmacología , Hormonas/metabolismo , Hormonas/farmacología , Insulinas/metabolismo , Insulinas/farmacologíaRESUMEN
Cilostamide, a phosphodiesterase 3A (Pde3A) inhibitor, is known to increase intraoocyte cyclic adenosine monophosphate (cAMP) level which is involved in sustaining meiotic arrest of the oocytes. To explore the mechanisms involved in the cilostamide-mediated meiotic arrest of the oocytes, the present study describes the effects of cilostamide on cAMP level and related factors involved in maturation of the oocytes at its different meiotic stages; diplotene, metaphase I (MI) and metaphase II (MII). The oocytes from these three stages were collected from rat ovary and incubated with 10 µM cilostamide for 3 h in CO2 incubator. The levels of cAMP, cyclic guanosine monophosphate (cGMP) and the key players of maintaining meiotic arrest during oocyte maturation; Emi2, Apc, Cyclin B1, and Cdk1, were analyzed in diplotene, MI and MII stages. Pde3A was found to be expressed at all three stages but with the lowest level in MI oocyte. As compared to the control sets, the cAMP concentration was found to be highest in MII whereas cGMP was highest in the diplotene stage of cilostamide-treated group. The treated group showed declined reactive oxygen species level as compared with the control counterparts. Relatively increased levels of the Emi2, Cyclin B1, and phosphorylated thr161 of Cdk1 versus declined levels of phosphorylated thr14/tyr15 of Cdk1 in diplotene and MII stage oocytes are known to be involved in maintaining meiotic arrest and all these factors were found to undergo similar pattern of change due to the treatment with cilostamide. The findings thus suggest that cilostamide treatment promotes meiotic arrest by Pde3A inhibition led increase of both cAMP and cGMP level vis-a-vis modulation of the related regulatory factors such as Emi2, CyclinB1, and phosphorylated status of Cdk1 in diplotene and MII stage oocytes. Such a mechanism of meiotic arrest could allow the oocyte to prepare itself for meiotic maturation and thereby to improve oocyte quality.
Asunto(s)
Factor Promotor de Maduración , Inhibidores de Fosfodiesterasa , Femenino , Ratas , Animales , Ciclina B1 , Inhibidores de Fosfodiesterasa/farmacología , Meiosis , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 3 , Oocitos , AMP Cíclico/farmacología , GMP Cíclico/farmacología , Adenosina Monofosfato/farmacologíaRESUMEN
BACKGROUND: Staphylococcus aureus is a leading cause for morbidity and mortality associated with skin and burn wound infections. Therapeutic options for methicillin-resistant S. aureus (MRSA) have dwindled and therefore alternative treatments are urgently needed. In this study, the immuno-stimulating and anti-MRSA effects of cyclic di-guanosine monophosphate (c-di-GMP), a uniquely bacterial second messenger and immuno-modulator, were investigated in HaCaT human epidermal keratinocytes and a murine skin wound infection model. RESULTS: Stimulation of HaCaT cells with 125 µM c-di-GMP for 12 h prior to MRSA challenge resulted in a 20-fold reduction in bacterial colonization compared with untreated control cells, which was not the result of a direct c-di-GMP toxic effect, since bacterial viability was not affected by this dose in the absence of HaCaT cells. C-di-GMP-stimulated or MRSA-challenged HaCaT cells displayed enhanced secretion of the antimicrobial peptides human ß-defensin 1 (hBD-1), hBD-2, hBD-3 and LL-37, but for hBD1 and LL-37 the responses were additive in a c-di-GMP-dose-dependent manner. Secretion of the chemokines CXCL1 and CXCL8 was also elevated after stimulation of HaCaT cells with lower c-di-GMP doses and peaked at a dose of 5 µM. Finally, pre-treatment of mice with a 200 nmol dose of c-di-GMP 24 h before a challenge with MRSA in skin wound infection model resulted in a major reduction (up to 1,100-fold by day 2) in bacterial CFU counts recovered from challenged skin tissue sections compared PBS-treated control animals. Tissue sections displayed inflammatory cell infiltration and enhanced neutrophil influx in the c-di-GMP pre-treated animals, which might account for the reduced ability of MRSA to colonize c-di-GMP pre-treated mice. CONCLUSIONS: These results demonstrate that c-di-GMP is a potent immuno-modulator that can stimulate anti-MRSA immune responses in vivo and might therefore be a suitable alternative prophylactic or therapeutic agent for MRSA skin or burn wound infections.
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Adyuvantes Inmunológicos , GMP Cíclico/análogos & derivados , Inmunidad Innata , Staphylococcus aureus Resistente a Meticilina , Infecciones Cutáneas Estafilocócicas , Adyuvantes Inmunológicos/farmacología , Adyuvantes Inmunológicos/uso terapéutico , Animales , Quemaduras/complicaciones , GMP Cíclico/farmacología , GMP Cíclico/uso terapéutico , Modelos Animales de Enfermedad , Humanos , Inmunidad Innata/efectos de los fármacos , Queratinocitos/efectos de los fármacos , Staphylococcus aureus Resistente a Meticilina/efectos de los fármacos , Ratones , Infecciones Cutáneas Estafilocócicas/tratamiento farmacológicoRESUMEN
AIM: This study aimed to investigate the effects of Rosa damascena Mill. essential oil on the vascular activity of rat thoracic aorta and its underlying mechanisms. METHODS: Experiments were performed using the isolated tissue bath model and Wistar rats. 0.1, 1, 10, and 100 µg/mL concentrations of rose oil were administered in all groups. To determine the vasoactive effects of rose oil, submaximal contractions were conducted by applying 10-5 M PE and 45 mM KCl separately in both endothelium-intact and -denuded segments. Time-matched distilled water groups were formed for control. To evaluate the role of endothelium-derived vasodilative factors, endothelium-intact segments were incubated with nitric oxide synthase inhibitor L-NAME, soluble guanylate cyclase inhibitor ODQ, and a non-selective cyclooxygenase inhibitor INDO. The statistical significance level was considered as p < 0.05. RESULTS: 1, 10, and 100 µg/mL rose oil doses led to vasorelaxation in thoracic aortas precontracted with 10-5 M PE (p: 0.029, p: 0.000, p: 0.000, respectively). In precontracted thoracic aortas with 45 mM KCl, the significant effect of rose oil persisted, albeit slightly diminished. When the endothelium was removed, the relaxant effect of rose oil was partially reduced, but still significant (p: 0.035, p: 0.028, p: 0.000, respectively). Preincubations with L-NAME and ODQ significantly attenuated rose oil-induced relaxation of endothelium-intact aortas precontracted with 10-5 M PE. In contrast, preincubation INDO did not modulate rose oil-induced relaxation. CONCLUSION: In conclusion, it was shown for the first time that rose oil can significantly mediate vasorelaxation in both PE and KCl precontracted rat thoracic aortas. Rose oil induced vasodilation with or without endothelium in a concentration-dependent manner. It was also shown that rose oil-induced vasorelaxant effects were reduced by L-NAME or ODQ pretreatment, but not modulated by INDO. These results demonstrated that rose oil-induced endothelium-dependent vasodilation is mediated by the NO-cGMP-dependent pathway.
Asunto(s)
Aceites Volátiles , Rosa , Animales , Aorta Torácica/metabolismo , GMP Cíclico/metabolismo , GMP Cíclico/farmacología , Inhibidores de la Ciclooxigenasa/farmacología , Endotelio Vascular , NG-Nitroarginina Metil Éster/farmacología , Óxido Nítrico/metabolismo , Óxido Nítrico Sintasa/metabolismo , Aceites Volátiles/metabolismo , Aceites Volátiles/farmacología , Ratas , Ratas Wistar , Rosa/metabolismo , Guanilil Ciclasa Soluble/metabolismo , Guanilil Ciclasa Soluble/farmacología , Vasodilatación , Vasodilatadores/farmacologíaRESUMEN
Our previous study suggested that inhibition of Phosphodiesterase 2 ameliorates memory loss upon exposure to oxidative stress. While whether memory enhancing effects of PDE2 inhibition on Alzheimer's disease mouse model are involved in antioxidant defense and neuronal remodeling, are largely unexplored. The present study addressed whether and how PDE2 inhibitor Bay 60-7550 rescued Aß oligomers (Aßo)-induced neuronal damage and memory impairment. The results suggested that exposure of primary cortical neurons to Aßo induced neuronal cells damage and increased PDE2 expression, which were paralleled to an increase in the oxidative parameter malondialdehyde (MDA) level and cellular apoptosis. However, this Aßo-induced oxidative damage was blocked by pre-treatment with protein kinase A or G (PKA or PKG) inhibitor, suggesting the involvement of cAMP/cGMP signaling. Moreover, microinjection of Aßo into the prefrontal cortex of mice increased the MDA level; while Bay 60-7550 reversed this effect and increased antioxidant and anti-apoptotic factors, i.e. increased trolox-equivalent-antioxidant capacity and Bcl-2/Bax ratio. Bay 60-7550 also rescued Aßo-induced synaptic atrophy and memory deficits, as evidenced by the increased synaptic proteins' levels and spine density in the prefrontal cortex, and improved cognitive behaviors by decreased working memory errors in the eight-arm maze and increased discrimination index in the novel object recognition test. These findings suggest that inhibition of PDE2 contributes to antioxidant defense and neuronal remodeling by regulation of cAMP/cGMP signaling, which provide a theoretical basis for the future use of PDE2 inhibitors as the anti-AD drugs.
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Enfermedad de Alzheimer , Inhibidores de Fosfodiesterasa , Enfermedad de Alzheimer/tratamiento farmacológico , Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/metabolismo , Animales , Antioxidantes/farmacología , GMP Cíclico/farmacología , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 2 , Hipocampo , Trastornos de la Memoria/tratamiento farmacológico , Ratones , Ratones Endogámicos ICR , Neuronas , Fragmentos de Péptidos , Inhibidores de Fosfodiesterasa/farmacología , Inhibidores de Fosfodiesterasa/uso terapéuticoRESUMEN
Although the association of elevated homocysteine level with cardiac hypertrophy has been reported, the molecular mechanisms by which homocysteine induces cardiac hypertrophy remain inadequately understood. In this study we aim to uncover the roles of cyclic nucleotide phosphodiesterase 1 (PDE1) and endoplasmic reticulum (ER) stress and their relationship to advance the mechanistic understanding of homocysteine-induced cardiac cell hypertrophy. H9c2 cells and primary neonatal rat cardiomyocytes are exposed to homocysteine with or without ER stress inhibitor TUDCA or PDE1-specific inhibitor Lu AF58027, or transfected with siRNAs targeting PDE1 isoforms prior to homocysteine-exposure. Cell surface area is measured and ultrastructure is examined by transmission electron microscopy. Hypertrophic markers, PDE1 isoforms, and ER stress molecules are detected by q-PCR and western blot analysis. Intracellular cGMP and cAMP are measured by ELISA. The results show that homocysteine causes the enlargement of H9c2 cells, increases the expressions of hypertrophic markers ß-MHC and ANP, upregulates PDE1A and PDE1C, promotes the expressions of ER stress molecules, and causes ER dilatation and degranulation. TUDCA and Lu AF58027 downregulate ß-MHC and ANP, and alleviate cell enlargement. TUDCA decreases PDE1A and PDE1C levels. Silencing of PDE1C inhibits homocysteine-induced hypertrophy, whereas PDE1A knockdown has minor effect. Both cAMP and cGMP are decreased after homocysteine-exposure, while only cAMP is restored by Lu AF58027 and TUDCA. TUDCA and Lu AF58027 also inhibit cell enlargement, downregulate ANP, ß-MHC and PDE1C, and enhance cAMP level in homocysteine-exposed primary cardiomyocytes. ER stress mediates homocysteine-induced hypertrophy of cardiac cells via upregulating PDE1C expression Cyclic nucleotide, especially cAMP, is the downstream mediator of the ER stress-PDE1C signaling axis in homocysteine-induced cell hypertrophy.
Asunto(s)
Fosfodiesterasas de Nucleótidos Cíclicos Tipo 1 , Estrés del Retículo Endoplásmico , Homocisteína , Animales , Factor Natriurético Atrial/genética , Factor Natriurético Atrial/metabolismo , Cardiomegalia/metabolismo , GMP Cíclico/metabolismo , GMP Cíclico/farmacología , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 1/metabolismo , Estrés del Retículo Endoplásmico/efectos de los fármacos , Activación Enzimática/efectos de los fármacos , Homocisteína/farmacología , Miocitos Cardíacos/metabolismo , Hidrolasas Diéster Fosfóricas/genética , Hidrolasas Diéster Fosfóricas/metabolismo , Ratas , Ácido Tauroquenodesoxicólico/farmacologíaRESUMEN
Tetrabromobisphenol A (TBBPA) is a flame retardant that can contaminate the environment and human being, acting as an endocrine disruptor. Several studies propose a correlation between TBBPA exposure and adverse health outcomes, however, at vascular level TBBPA effects are still poorly understood. Thus, considering that the vascular tonus is regulated by vasoactive substances (serotonin and histamine) which are involved in some pathological processes, this work aimed to analyse the direct effects and the 24 h exposure of TBBPA on the human umbilical artery (HUA) and to investigate its signalling pathway. Using organ bath technique, endothelium-denuded HUA rings were contracted with serotonin (5-HT, 1 µM), histamine (His, 10 µM) and potassium chloride (KCl, 60 mM), and the exposure (0-24 h) of different concentrations of TBBPA (1, 10 and 50 µM) were evaluated. Besides, the vascular mode of action of TBBPA was studied through the analysis of cyclic guanosine monophosphate and calcium channels activity, pathways involved in relaxation and contraction of HUA, respectively. Our results demonstrated that the direct effects of TBBPA induce a vasorelaxation of HUA. The maximum relaxant effect was observed at 100 µM of TBBPA with 63.74%, 64.24% and 30.05%, for 5-HT-, His- or KCl-contracted arteries respectively. The 24 h TBBPA exposure altered the vasorelaxant response pattern of sodium nitroprusside and nifedipine. This effect is due to the involvement of TBBPA with the NO/sGC/cGMP/PKG pathway and the interference in calcium influx. Furthermore, using the real-time quantitative polymerase chain reaction, TBBPA clearly modulates L-type calcium and large-conductance Ca2+ 1.1 α- and ß1 -subunit channels, and soluble guanylyl cyclase and protein Kinase G. So, at vascular level TBBPA induces changes in HUA after TBBPA exposure.
Asunto(s)
Calcio , Donantes de Óxido Nítrico , Calcio/metabolismo , GMP Cíclico/metabolismo , GMP Cíclico/farmacología , Histamina/farmacología , Humanos , Óxido Nítrico/metabolismo , Donantes de Óxido Nítrico/farmacología , Bifenilos Polibrominados , Canales de Potasio/farmacología , Serotonina , Vasodilatación/fisiologíaRESUMEN
Alzheimer's disease (AD), one of the greatest threats to human health, is characterized by declined cognition and changed behavior. Cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) that play an important role in learning and memory are hydrolyzed by phosphodiesterases (PDEs). Most PDE isoforms are highly expressed in the brain, and the inhibition of PDEs is beneficial to counteract AD. Thus, targeting PDEs represents a therapeutic potential for this disease. So far, a variety of PDE inhibitors have been discovered with significant cognitive enhancement effects in animal models and more than ten agents have entered into clinical trials. In this review, we summarize PDE mediated cyclic nucleotide signaling pathways, PDE family members involved in AD and recent advance of PDE inhibitors in preclinical and clinical studies, trying to provide an outlook of PDE inhibitors for the treatment of AD in future.
Asunto(s)
Enfermedad de Alzheimer , Inhibidores de Fosfodiesterasa , 3',5'-AMP Cíclico Fosfodiesterasas/metabolismo , Enfermedad de Alzheimer/tratamiento farmacológico , Animales , Cognición , GMP Cíclico/farmacología , Inhibidores de Fosfodiesterasa/farmacología , Inhibidores de Fosfodiesterasa/uso terapéutico , Hidrolasas Diéster Fosfóricas/metabolismoRESUMEN
Cyclic Guanosine-Monophosphate (cGMP) is implicated as second messenger in a plethora of pathways and its effects are executed mainly by cGMP-dependent protein kinases (PKG). It is involved in both peripheral (cardiovascular regulation, intestinal secretion, phototransduction, etc.) and brain (hippocampal synaptic plasticity, neuroinflammation, cognitive function, etc.) processes. Stimulation of hippocampal cGMP signaling have been proved to be beneficial in animal models of aging, Alzheimer's disease or hepatic encephalopathy, restoring different cognitive functions such as passive avoidance, object recognition or spatial memory. However, even when some inhibitors of cGMP-degrading enzymes (PDEs) are already used against peripheral pathologies, their utility as neurological treatments is still under clinical investigation. Additionally, it has been demonstrated a list of cGMP roles as not second but first messenger. The role of extracellular cGMP has been specially studied in hippocampal function and cognitive impairment in animal models and it has emerged as an important modulator of neuroinflammation-mediated cognitive alterations and hippocampal synaptic plasticity malfunction. Specifically, it has been demonstrated that extracellular cGMP decreases hippocampal IL-1ß levels restoring membrane expression of glutamate receptors in the hippocampus and cognitive function in hyperammonemic rats. The mechanisms implicated are still unclear and might involve complex interactions between hippocampal neurons, astrocytes and microglia. Membrane targets for extracellular cGMP are still poorly understood and must be addressed in future studies.
Asunto(s)
GMP Cíclico , Hiperamonemia , Animales , GMP Cíclico/metabolismo , GMP Cíclico/farmacología , Hipocampo/metabolismo , Hiperamonemia/metabolismo , Microglía/metabolismo , Ratas , Transducción de Señal/fisiologíaRESUMEN
The identification of agonists of the stimulator of interferon genes (STING) pathway has been an area of intense research due to their potential to enhance innate immune response and tumor immunogenicity in the context of immuno-oncology therapy. Initial efforts to identify STING agonists focused on the modification of 2',3'-cGAMP (1) (an endogenous STING activator ligand) and other closely related cyclic dinucleotides (CDNs). While these efforts have successfully identified novel CDNs that have progressed into the clinic, their utility is currently limited to patients with solid tumors that STING agonists can be delivered to intratumorally. Herein, we report the discovery of a unique class of non-nucleotide small-molecule STING agonists that demonstrate antitumor activity when dosed intratumorally in a syngeneic mouse model.
Asunto(s)
Proteínas de la Membrana/agonistas , Animales , Cristalografía por Rayos X , AMP Cíclico/química , AMP Cíclico/farmacología , GMP Cíclico/química , GMP Cíclico/farmacología , Femenino , Humanos , Inmunidad Innata/efectos de los fármacos , Inmunoterapia/métodos , Proteínas de la Membrana/química , Ratones , Ratones Endogámicos BALB C , Modelos Moleculares , Neoplasias/inmunología , Transducción de Señal/efectos de los fármacos , Bibliotecas de Moléculas PequeñasRESUMEN
Cyclic nucleotides (cAMP and cGMP) and corresponding protein kinases, protein kinase A (PKA) and protein kinase G (PKG), are the main intracellular mediators of endothelium-derived platelet inhibitors. Pharmacological PKA/PKG inhibitors are often used to discriminate between these two kinase activities and to analyze their underlying mechanisms. Previously we showed that all widely used PKG inhibitors (KT5823, DT3, RP isomers) either did not inhibit PKG or inhibited and even activated platelets independently from PKG. In this study, we examined several PKA inhibitors as well as inhibitors of adenylate and guanylate cyclases to reveal their effects on platelets and establish whether they are mediated by PKA/PKG. The commonly used PKA inhibitor H89 inhibited both PKA and PKG but PKA-independently inhibited thrombin-induced platelet activation. In our experiments, KT5720 did not inhibit PKA and had no effect on platelet activation. PKI inhibited PKA activity in platelets but also strongly PKA-independently activated platelets. Inhibition of adenylate and guanylate cyclases may be an alternative approach to analyze PKA/PKG function. Based on our previous and presented data, we conclude that all results where the mentioned PKA inhibitors were used for the analysis of PKA activity in intact platelets should be considered with caution.