RESUMEN
Hydrogen sulfide (H2S) has been extensively studied as a signal molecule in the body for the past 30 years. Researchers have conducted studies using both natural and synthetic sources of H2S, known as H2S donors, which have different characteristics in terms of how they release H2S. These donors can be inorganic salts or have various organic structures. In recent years, certain types of sulfur compounds found naturally in foods have been characterized as H2S donors and explored for their potential health benefits. These compounds are referred to as "sulfanutraceuticals," a term that combines "nutrition" and "pharmaceutical". It is used to describe products derived from food sources that offer additional health advantages. By introducing the terms "sulfaceuticals" and "sulfanutraceuticals," we categorize sulfur-containing substances based on their origin and their use in both preclinical and clinical research, as well as in dietary supplements.
Asunto(s)
Sulfuro de Hidrógeno , Compuestos de Azufre/uso terapéutico , AzufreRESUMEN
OBJECTIVES: A prolonged vasoconstriction/hypoperfusion/hypoxic event follows self-terminating focal seizures. The ketogenic diet (KD) has demonstrated efficacy as a metabolic treatment for intractable epilepsy and other disorders but its effect on local brain oxygen levels is completely unknown. This study investigated the effects of the KD on tissue oxygenation in the hippocampus before and after electrically elicited (kindled) seizures and whether it could protect against a seizure-induced learning impairment. We also examined the effects of the ketone ß-hydroxybutyrate (BHB) as a potential underlying mechanism. METHODS: Male and female rats were given access to one of three diet protocols 2 weeks prior to the initiation of seizures: KD, caloric restricted standard chow, and ad libitum standard chow. Dorsal hippocampal oxygen levels were measured prior to initiation of diets as well as before and after a 10-day kindling paradigm. Male rats were then tested on a novel object recognition task to assess postictal learning impairments. In a separate cohort, BHB was administered 30 min prior to seizure elicitation to determine whether it influenced oxygen dynamics. RESULTS: The KD increased dorsal hippocampal oxygen levels, ameliorated postictal hypoxia, and prevented postictal learning impairments. Acute BHB administration did not alter oxygen levels before or after seizures. INTERPRETATION: The ketogenic diet raised brain oxygen levels and attenuated severe postictal hypoxia likely through a mechanism independent of ketosis and shows promise as a non-pharmacological treatment to prevent the postictal state.
Asunto(s)
Encéfalo/metabolismo , Dieta Cetogénica/métodos , Hipoxia/metabolismo , Discapacidades para el Aprendizaje/metabolismo , Oxígeno/metabolismo , Convulsiones/metabolismo , Animales , Femenino , Hipoxia/dietoterapia , Cetosis/inducido químicamente , Cetosis/metabolismo , Discapacidades para el Aprendizaje/dietoterapia , Discapacidades para el Aprendizaje/prevención & control , Masculino , Neuroprotección/fisiología , Ratas , Ratas Long-Evans , Convulsiones/dietoterapiaRESUMEN
Epilepsy therapy is based on antiseizure drugs that treat the symptom, seizures, rather than the disease and are ineffective in up to 30% of patients. There are no treatments for modifying the disease-preventing seizure onset, reducing severity or improving prognosis. Among the potential molecular targets for attaining these unmet therapeutic needs, we focused on oxidative stress since it is a pathophysiological process commonly occurring in experimental epileptogenesis and observed in human epilepsy. Using a rat model of acquired epilepsy induced by electrical status epilepticus, we show that oxidative stress occurs in both neurons and astrocytes during epileptogenesis, as assessed by measuring biochemical and histological markers. This evidence was validated in the hippocampus of humans who died following status epilepticus. Oxidative stress was reduced in animals undergoing epileptogenesis by a transient treatment with N-acetylcysteine and sulforaphane, which act to increase glutathione levels through complementary mechanisms. These antioxidant drugs are already used in humans for other therapeutic indications. This drug combination transiently administered for 2 weeks during epileptogenesis inhibited oxidative stress more efficiently than either drug alone. The drug combination significantly delayed the onset of epilepsy, blocked disease progression between 2 and 5 months post-status epilepticus and drastically reduced the frequency of spontaneous seizures measured at 5 months without modifying the average seizure duration or the incidence of epilepsy in animals. Treatment also decreased hippocampal neuron loss and rescued cognitive deficits. Oxidative stress during epileptogenesis was associated with de novo brain and blood generation of high mobility group box 1 (HMGB1), a neuroinflammatory molecule implicated in seizure mechanisms. Drug-induced reduction of oxidative stress prevented HMGB1 generation, thus highlighting a potential novel mechanism contributing to therapeutic effects. Our data show that targeting oxidative stress with clinically used drugs for a limited time window starting early after injury significantly improves long-term disease outcomes. This intervention may be considered for patients exposed to potential epileptogenic insults.
Asunto(s)
Acetilcisteína/farmacología , Epilepsia/prevención & control , Glutatión/metabolismo , Isotiocianatos/farmacología , Estrés Oxidativo/efectos de los fármacos , Animales , Astrocitos/metabolismo , Biomarcadores/metabolismo , Estudios de Casos y Controles , Recuento de Células , Disfunción Cognitiva/complicaciones , Disfunción Cognitiva/prevención & control , Modelos Animales de Enfermedad , Estimulación Eléctrica , Epilepsia/complicaciones , Proteína HMGB1/sangre , Hipocampo/metabolismo , Humanos , Masculino , Neuronas/metabolismo , Neuronas/patología , Ratas , Estado Epiléptico/complicaciones , Estado Epiléptico/metabolismo , Estado Epiléptico/prevención & control , SulfóxidosRESUMEN
Autism spectrum disorder (ASD) is a highly prevalent neurodevelopmental disorder that exhibits a common set of behavioral and cognitive impairments. Although the etiology of ASD remains unclear, mitochondrial dysfunction has recently emerged as a possible causative factor underlying ASD. The ketogenic diet (KD) is a high-fat, low-carbohydrate diet that augments mitochondrial function, and has been shown to reduce autistic behaviors in both humans and in rodent models of ASD. The aim of the current study was to examine mitochondrial bioenergetics in the BTBR mouse model of ASD and to determine whether the KD improves mitochondrial function. We also investigated changes in mitochondrial morphology, which can directly influence mitochondrial function. We found that BTBR mice had altered mitochondrial function and exhibited smaller more fragmented mitochondria compared to C57BL/6J controls, and that supplementation with the KD improved both mitochondrial function and morphology. We also identified activating phosphorylation of two fission proteins, pDRP1S616 and pMFFS146, in BTBR mice, consistent with the increased mitochondrial fragmentation that we observed. Intriguingly, we found that the KD decreased pDRP1S616 levels in BTBR mice, likely contributing to the restoration of mitochondrial morphology. Overall, these data suggest that impaired mitochondrial bioenergetics and mitochondrial fragmentation may contribute to the etiology of ASD and that these alterations can be reversed with KD treatment.
Asunto(s)
Trastorno del Espectro Autista/etiología , Trastorno del Espectro Autista/metabolismo , Dieta Cetogénica , Susceptibilidad a Enfermedades , Mitocondrias/genética , Mitocondrias/metabolismo , Animales , Trastorno del Espectro Autista/diagnóstico , Trastorno del Espectro Autista/dietoterapia , Biomarcadores , Manejo de la Enfermedad , Modelos Animales de Enfermedad , Ratones , Mitocondrias/ultraestructura , Dinámicas Mitocondriales , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , Neuronas/metabolismo , Neuronas/patologíaRESUMEN
OBJECTIVE: Status epilepticus (SE) is a life-threatening and commonly drug-refractory condition. Novel therapies are needed to rapidly terminate seizures to prevent mortality and morbidity. Monoacylglycerol lipase (MAGL) is the key enzyme responsible for the hydrolysis of the endocannabinoid 2-arachidonoylglycerol (2-AG) and a major contributor to the brain pool of arachidonic acid (AA). Inhibiting of monoacylglycerol lipase modulates synaptic activity and neuroinflammation, 2 mediators of excessive neuronal activation underlying seizures. We studied the effect of a potent and selective irreversible MAGL inhibitor, CPD-4645, on SE that was refractory to diazepam, its neuropathologic sequelae, and the mechanism underlying the drug's effects. METHODS: Diazepam-resistant SE was induced in adult mice fed with standard or ketogenic diet or in cannabinoid receptor type 1 (CB1) receptor knock-out mice. CPD-4645 (10 mg/kg, subcutaneously) or vehicle was dosed 1 and 7 h after status epilepticus onset in video-electroencephalography (EEG) recorded mice. At the end of SE, mice were examined in the novel object recognition test followed by neuronal cellloss analysis. RESULTS: CPD-4645 maximal plasma and brain concentrations were attained 0.5 h postinjection (half-life = 3.7 h) and elevated brain 2-AG levels by approximately 4-fold. CPD-4645 administered to standard diet-fed mice progressively reduced spike frequency during 3 h postinjection, thereby shortening SE duration by 47%. The drug immediately abrogated SE in ketogenic diet-fed mice. CPD-4645 rescued neuronal cell loss and cognitive deficit and reduced interleukin (IL)-1ß and cyclooxygenase 2 (COX-2) brain expression resulting from SE. The CPD-4645 effect on SE was similar in mice lacking CB1 receptors. SIGNIFICANCE: MAGL represents a novel therapeutic target for treating status epilepticus and improving its sequelae. CPD-4645 therapeutic effects appear to be predominantly mediated by modulation of neuroinflammation.
Asunto(s)
Carbamatos/uso terapéutico , Monoacilglicerol Lipasas/antagonistas & inhibidores , Piperidinas/uso terapéutico , Estado Epiléptico , Sulfonamidas/uso terapéutico , Animales , Encéfalo/metabolismo , Encéfalo/patología , Ondas Encefálicas/efectos de los fármacos , Ondas Encefálicas/fisiología , Carbamatos/química , Carbamatos/farmacocinética , Trastornos del Conocimiento/tratamiento farmacológico , Trastornos del Conocimiento/etiología , Diazepam/efectos adversos , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Epilepsia Refractaria/inducido químicamente , Epilepsia Refractaria/enzimología , Epilepsia Refractaria/terapia , Electroencefalografía , Agonistas de Aminoácidos Excitadores/toxicidad , Fluoresceínas/metabolismo , Ácido Kaínico/toxicidad , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Monoacilglicerol Lipasas/metabolismo , Neuronas/efectos de los fármacos , Neuronas/patología , Piperidinas/química , Piperidinas/farmacología , Distribución Aleatoria , Receptor Cannabinoide CB1/deficiencia , Receptor Cannabinoide CB1/genética , Reconocimiento en Psicología/efectos de los fármacos , Estado Epiléptico/inducido químicamente , Estado Epiléptico/complicaciones , Estado Epiléptico/enzimología , Estado Epiléptico/terapia , Sulfonamidas/química , Sulfonamidas/farmacocinética , Factores de TiempoRESUMEN
Epilepsy therapy is based on antiseizure drugs that treat the symptom, seizures, rather than the disease and are ineffective in up to 30% of patients. There are no treatments for modifying the disease-preventing seizure onset, reducing severity or improving prognosis. Among the potential molecular targets for attaining these unmet therapeutic needs, we focused on oxidative stress since it is a pathophysiological process commonly occurring in experimental epileptogenesis and observed in human epilepsy. Using a rat model of acquired epilepsy induced by electrical status epilepticus, we show that oxidative stress occurs in both neurons and astrocytes during epileptogenesis, as assessed by measuring biochemical and histological markers. This evidence was validated in the hippocampus of humans who died following status epilepticus. Oxidative stress was reduced in animals undergoing epileptogenesis by a transient treatment with N-acetylcysteine and sulforaphane, which act to increase glutathione levels through complementary mechanisms. These antioxidant drugs are already used in humans for other therapeutic indications. This drug combination transiently administered for 2 weeks during epileptogenesis inhibited oxidative stress more efficiently than either drug alone. The drug combination significantly delayed the onset of epilepsy, blocked disease progression between 2 and 5 months post-status epilepticus and drastically reduced the frequency of spontaneous seizures measured at 5 months without modifying the average seizure duration or the incidence of epilepsy in animals. Treatment also decreased hippocampal neuron loss and rescued cognitive deficits. Oxidative stress during epileptogenesis was associated with de novo brain and blood generation of disulfide high mobility group box 1 (HMGB1), a neuroinflammatory molecule implicated in seizure mechanisms. Drug-induced reduction of oxidative stress prevented disulfide HMGB1 generation, thus highlighting a potential novel mechanism contributing to therapeutic effects. Our data show that targeting oxidative stress with clinically used drugs for a limited time window starting early after injury significantly improves long-term disease outcomes. This intervention may be considered for patients exposed to potential epileptogenic insults.
Asunto(s)
Acetilcisteína/farmacología , Acetilcisteína/uso terapéutico , Epilepsia/tratamiento farmacológico , Dominios HMG-Box/efectos de los fármacos , Proteína HMGB1/sangre , Proteína HMGB1/metabolismo , Isotiocianatos/uso terapéutico , Estrés Oxidativo/efectos de los fármacos , Animales , Astrocitos/metabolismo , Biomarcadores/sangre , Biomarcadores/metabolismo , Disfunción Cognitiva/complicaciones , Disfunción Cognitiva/tratamiento farmacológico , Modelos Animales de Enfermedad , Quimioterapia Combinada , Epilepsia/metabolismo , Proteína HMGB1/biosíntesis , Hipocampo/metabolismo , Isotiocianatos/farmacología , Masculino , Degeneración Nerviosa/dietoterapia , Neuronas/metabolismo , Ratas , SulfóxidosRESUMEN
Here, by using in vitro and ex vivo approaches, we elucidate the impairment of the hydrogen sulfide (H2S) pathway in vascular complications associated with metabolic syndrome (MetS). In the in vitro model simulating hyperlipidemic/hyperglycemic conditions, we observe significant hallmarks of endothelial dysfunction, including eNOS/NO signaling impairment, ROS overproduction, and a reduction in CSE-derived H2S. Transitioning to an ex vivo model using db/db mice, a genetic MetS model, we identify a downregulation of CBS and CSE expression in aorta, coupled with a diminished L-cysteine-induced vasorelaxation. Molecular mechanisms of eNOS/NO signaling impairment, dissected using pharmacological and molecular approaches, indicate an altered eNOS/Cav-1 ratio, along with reduced Ach- and Iso-induced vasorelaxation and increased L-NIO-induced contraction. In vivo treatment with the H2S donor Erucin ameliorates vascular dysfunction observed in db/db mice without impacting eNOS, further highlighting a specific action on smooth muscle component rather than the endothelium. Analyzing the NO signaling pathway in db/db mice aortas, reduced cGMP levels were detected, implicating a defective sGC/cGMP signaling. In vivo Erucin administration restores cGMP content. This beneficial effect involves an increased sGC activity, due to enzyme persulfidation observed in sGC overexpressed cells, coupled with PDE5 inhibition. In conclusion, our study demonstrates a pivotal role of reduced cGMP levels in impaired vasorelaxation in a murine model of MetS involving an impairment of both H2S and NO signaling. Exogenous H2S supplementation through Erucin represents a promising alternative in MetS therapy, targeting smooth muscle cells and supporting the importance of lifestyle and nutrition in managing MetS.
Asunto(s)
GMP Cíclico , Sulfuro de Hidrógeno , Síndrome Metabólico , Ratones Endogámicos C57BL , Guanilil Ciclasa Soluble , Animales , Sulfuro de Hidrógeno/metabolismo , Sulfuro de Hidrógeno/farmacología , GMP Cíclico/metabolismo , Síndrome Metabólico/metabolismo , Ratones , Masculino , Guanilil Ciclasa Soluble/metabolismo , Vasodilatación/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Óxido Nítrico Sintasa de Tipo III/metabolismo , Humanos , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/metabolismo , Óxido Nítrico/metabolismo , Aorta/efectos de los fármacos , Aorta/metabolismo , Enfermedades Vasculares/metabolismo , Modelos Animales de EnfermedadRESUMEN
Comorbidities during the period between seizures present a significant challenge for individuals with epilepsy. Despite their clinical relevance, the pathophysiology of the interictal symptomatology is largely unknown. Postictal severe hypoxia (PIH) in those brain regions participating in the seizure has been indicated as a mechanism underlying several negative postictal manifestations. It is unknown how repeated episodes of PIH affect interictal symptoms in epilepsy. Using a rat model, we observed that repeated seizures consistently induced episodes of PIH that become increasingly severe with each seizure occurrence. Additionally, recurrent seizure activity led to decreased levels of oxygen in the hippocampus during the interictal period. However, these reductions were prevented when we repeatedly blocked PIH using either the COX-inhibitor acetaminophen or the L-type calcium channel antagonist nifedipine. Moreover, we found that interictal cognitive deficits caused by seizures were completely alleviated by repeated attenuation of PIH events. Lastly, mitochondrial dysfunction may contribute to the observed pathological outcomes during the interictal period. These findings provide evidence that seizure-induced hypoxia may play a crucial role in several aspects of epilepsy. Consequently, developing and implementing treatments that specifically target and prevent PIH could potentially offer significant benefits for individuals with refractory epilepsy.
Asunto(s)
Trastornos del Conocimiento , Disfunción Cognitiva , Animales , Ratas , Disfunción Cognitiva/etiología , Hipoxia/complicaciones , Convulsiones , OxígenoRESUMEN
Prolonged severe hypoxia follows brief seizures and represents a mechanism underlying several negative postictal manifestations without interventions. Approximately 50% of the postictal hypoxia phenomenon can be accounted for by arteriole vasoconstriction. What accounts for the rest of the drop in unbound oxygen is unclear. Here, we determined the effect of pharmacological modulation of mitochondrial function on tissue oxygenation in the hippocampus of rats after repeatedly evoked seizures. Rats were treated with mitochondrial uncoupler 2,4 dinitrophenol (DNP) or antioxidants. Oxygen profiles were recorded using a chronically implanted oxygen-sensing probe, before, during, and after seizure induction. Mitochondrial function and redox tone were measured using in vitro mitochondrial assays and immunohistochemistry. Postictal cognitive impairment was assessed using the novel object recognition task. Mild mitochondrial uncoupling by DNP raised hippocampal oxygen tension and ameliorated postictal hypoxia. Chronic DNP also lowered mitochondrial oxygen-derived reactive species and oxidative stress in the hippocampus during postictal hypoxia. Uncoupling the mitochondria exerts therapeutic benefits on postictal cognitive dysfunction. Finally, antioxidants do not affect postictal hypoxia, but protect the brain from associated cognitive deficits. We provided evidence for a metabolic component of the prolonged oxygen deprivation that follow seizures and its pathological sequelae. Furthermore, we identified a molecular underpinning of this metabolic component, which involves excessive oxygen conversion into reactive species. Mild mitochondrial uncoupling may be a potential therapeutic strategy to treat the postictal state where seizure control is absent or poor.
Asunto(s)
Antioxidantes , Hipoxia , Ratas , Animales , Especies Reactivas de Oxígeno/metabolismo , Antioxidantes/farmacología , Hipoxia/metabolismo , Oxígeno/metabolismo , Mitocondrias , Convulsiones/metabolismo , Desacopladores/metabolismo , Desacopladores/farmacologíaRESUMEN
Epilepsy is at times a fatal disease. Sudden unexpected death in epilepsy (SUDEP) is the leading cause of epilepsy-related mortality in people with intractable epilepsy and is defined by exclusion; non-accidental, non-toxicologic, and non-anatomic causes of death. While SUDEP often follows a bilateral tonic-clonic seizure, the mechanisms that ultimately lead to terminal apnea and then asystole remain elusive and there is a lack of preventative treatments. Based on the observation that discrete seizures lead to local and postictal vasoconstriction, resulting in hypoperfusion, hypoxia and behavioural disturbances in the forebrain we reasoned those similar mechanisms may play a role in SUDEP when seizures invade the brainstem. Here we tested this neurovascular-based hypothesis of SUDEP in awake non-anesthetized mice by pharmacologically preventing seizure-induced vasoconstriction, with cyclooxygenase-2 or L-type calcium channel antagonists. In both acute and chronic mouse models of seizure-induced premature mortality, ibuprofen and nicardipine extended life while systemic drug levels remained high enough to be effective. We also examined the potential role of spreading depolarization in the acute model of seizure-induced premature mortality. These data provide a proof-of-principle for the neurovascular hypothesis of SUDEP rather than spreading depolarization and the use of currently available drugs to prevent it.
Asunto(s)
Epilepsia , Muerte Súbita e Inesperada en la Epilepsia , Ratones , Animales , Muerte Súbita e Inesperada en la Epilepsia/prevención & control , Epilepsia/tratamiento farmacológico , Epilepsia/complicaciones , Convulsiones/prevención & control , Convulsiones/complicaciones , Hipoxia/complicaciones , Muerte Súbita/etiología , Muerte Súbita/prevención & controlRESUMEN
Flucytosine (5-fluorocytosine, 5-FC) is a fluorinated analogue of cytosine currently approved for the systemic treatment of fungal infections, which has recently demonstrated a very promising antivirulence activity against the bacterial pathogen Pseudomonas aeruginosa. In this work, we propose novel inhalable hyaluronic acid (HA)/mannitol composite dry powders for repositioning 5-FC in the local treatment of lung infections, including those affecting cystic fibrosis (CF) patients. Different dry powders were produced in one-step by spray-drying. Powder composition and process conditions were selected after in depth formulation studies aimed at selecting the 5-FC/HA/mannitol formulation with convenient aerosolization properties and drug release profile in simulated lung fluids. The optimized 5-FC/HA/mannitol powder for inhalation (HyaMan_FC#3) was effectively delivered from different breath-activated dry powder inhalers (DPI) already available to CF patients. Nevertheless, the aerodynamic assessment of fine particles suggested that the developed formulation well fit with a low-resistance DPI. HyaMan_FC#3 inhibited the growth of the fungus Candida albicans and the production of the virulence factor pyoverdine by P. aeruginosa at 5-FC concentrations that did not affect the viability of both wild type (16HBE14o-) and CF (CFBE41o-) human bronchial epithelial cells. Finally, pharmacokinetics of HyaMan_FC#3 inhalation powder and 5-FC solution after intratracheal administration in rats were compared. In vivo results clearly demonstrated that, when formulated as dry powder, 5-FC levels in both bronchoalveolar lavage fluid and lung tissue were significantly higher and sustained over time as compared to those obtained with the 5-FC solution. Of note, when the same 5-FC amount was administered intravenously, no significant drug amount was found in the lung at each time point from the injection. To realize a 5-FC lung concentration similar to that obtained by using HyaMan_FC#3, a 6-fold higher dose of 5-FC should be administered intravenously. Taken together, our data demonstrate the feasibility to deliver 5-FC by the pulmonary route likely avoiding/reducing the well-known side effects associated to the high systemic 5-FC doses currently used in humans. Furthermore, our results highlight that an appropriate formulation design can improve the persistence of the drug at lungs, where microorganisms causing severe infections are located.
Asunto(s)
Antibacterianos/administración & dosificación , Antifúngicos/administración & dosificación , Reposicionamiento de Medicamentos , Inhaladores de Polvo Seco , Flucitosina/administración & dosificación , Ácido Hialurónico/química , Manitol/química , Administración por Inhalación , Aerosoles/química , Animales , Antibacterianos/farmacocinética , Antibacterianos/farmacología , Antifúngicos/farmacocinética , Antifúngicos/farmacología , Candida albicans/efectos de los fármacos , Candidiasis/tratamiento farmacológico , Flucitosina/farmacocinética , Flucitosina/farmacología , Humanos , Pulmón/microbiología , Enfermedades Pulmonares Fúngicas/tratamiento farmacológico , Masculino , Tamaño de la Partícula , Polvos , Infecciones por Pseudomonas/tratamiento farmacológico , Pseudomonas aeruginosa/efectos de los fármacos , Ratas WistarRESUMEN
Vascular smooth muscle cells (VSMCs) that accumulate in neointima after angioplastic injury show different phenotypic characteristics from those of medial layer and an impaired reactivity to contracting agents. The aim of the study was to correlate the vascular hyporesponsiveness to the changes in intracellular calcium concentration [Ca(2+)](i) and the expression of proteins necessary for its utilization in mechanically injured rat carotid arteries (IC) at 14 and 28 days after angioplastic balloon. IC showed a significant reduction (P<0.01) to PE- or KCl-induced contraction as compared to uninjured carotid (UC). Fura-2AM-loaded VSMCs isolated from IC revealed that this hyporeactivity to PE or KCl was accompanied by the impairment of the increase in [Ca(2+)](i) induced by contracting agents in both Ca(2+)-free or -containing medium. Similar results were observed following the ryanodine challenge in VSMC. Western blot analysis showed a significant (P<0.05) reduction in myosin heavy chain (MHC) and IP(3)-type III receptor expression in IC isolated at 14 days from injury compared to UC, while an improvement of these proteins expression was observed at 28 days after damage. On the other hand, in IC tissue, SERCA2 and alpha-actin expression, compared to UC was significantly higher at 14 days than at 28 days. These data indicate that vascular hyporeactivity induced by mechanical injury may be due to alterations of either [Ca(2+)](i) or contractile proteins. These modifications could be related to the changes of VSMC phenotypic characteristics, as supported by the observed modifications in MHC, SERCA2 and alpha-actin expression, proteins considered as biological markers of cellular differentiation.
Asunto(s)
Traumatismos de las Arterias Carótidas/fisiopatología , Arteria Carótida Externa , Músculo Liso Vascular/fisiopatología , Vasoconstricción/fisiología , Vasoconstrictores/farmacología , Actinas/metabolismo , Angioplastia de Balón/efectos adversos , Animales , Western Blotting , Calcio/metabolismo , Canales de Calcio/metabolismo , ATPasas Transportadoras de Calcio/metabolismo , Traumatismos de las Arterias Carótidas/metabolismo , Traumatismos de las Arterias Carótidas/patología , Arteria Carótida Externa/efectos de los fármacos , Arteria Carótida Externa/patología , Arteria Carótida Externa/fisiopatología , Células Cultivadas , Modelos Animales de Enfermedad , Técnicas In Vitro , Inositol 1,4,5-Trifosfato/metabolismo , Receptores de Inositol 1,4,5-Trifosfato , Masculino , Músculo Liso Vascular/efectos de los fármacos , Músculo Liso Vascular/metabolismo , Cadenas Pesadas de Miosina/metabolismo , Fenotipo , Fenilefrina/farmacología , Cloruro de Potasio/farmacología , Ratas , Ratas Wistar , Receptores Citoplasmáticos y Nucleares/metabolismo , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico , Espectrometría de Fluorescencia , Vasoconstricción/efectos de los fármacosRESUMEN
Apomorphine is used in the erectile dysfunction therapy and its action has been ascribed to the stimulation of central dopamine receptor. At the present stage, very little is known about the peripheral action of apomorphine on human corpus cavernosum (HCC). We have investigated the peripheral action of apomorphine and the role of dopamine receptors in HCC. We here demonstrate that both D1 and D2 receptors were expressed in the HCC, D1 receptors were two-fold more abundant than D2 and that both receptors were mainly localized on the smooth muscle cell component. Apomorphine in vitro exerted an anti-alpha1 adrenergic activity in human cavernosal strips since it prevented contraction induced by phenylephrine (PE), but not by U46619 or endothelin. Apomorphine elicited endothelium-independent and concentration-dependent relaxation of the strips contracted by PE, U46619 or endothelin. The EC50 values (microM) for apomorphine, in the presence and absence of endothelium, were 51.0+/-16 and 16.0+/-14, 120+/-19 and 150+/-18, 59.0+/-15 and 140+/-50 on PE-, U46619- or endothelin-induced contraction, respectively. Selective dopamine receptor agonist A-68930 (D1-like), but not quinpirole (D2-like), caused concentration-dependent relaxation of the cavernosal strips, which was partially prevented by endothelium removal or by treatment with an inhibitor of nitric oxide (NO) synthase. In conclusion, we show that (1) apomorphine has a peripheral relaxant direct effect as well as an antiadrenergic activity, (2) HCC possesses more D1-like (D1 and D5) than D2-like (D2, D3 and D4) receptors, (3) both D1- and D2-like receptors are mainly localized on smooth muscle cells and (4) the relaxant activity is most probably mediated by D1-like receptor partially through NO release from endothelium.
Asunto(s)
Apomorfina/farmacología , Cromanos/farmacología , Agonistas de Dopamina/farmacología , Relajación Muscular/efectos de los fármacos , Pene/efectos de los fármacos , Ácido 15-Hidroxi-11 alfa,9 alfa-(epoximetano)prosta-5,13-dienoico/farmacología , Relación Dosis-Respuesta a Droga , Endotelinas/farmacología , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/metabolismo , Humanos , Técnicas In Vitro , Masculino , Contracción Muscular/efectos de los fármacos , Músculo Liso/efectos de los fármacos , Músculo Liso/metabolismo , Fenilefrina/farmacología , Quinpirol/farmacología , Receptores de Dopamina D1/agonistas , Receptores de Dopamina D1/metabolismo , Receptores de Dopamina D2/agonistas , Receptores de Dopamina D2/metabolismoRESUMEN
BACKGROUND AND PURPOSE: Hydrogen sulphide (H2S) is a gaseous mediator strongly involved in cardiovascular homeostasis, where it provokes vasodilatation. Having previously shown that H2 S contributes to testosterone-induced vasorelaxation, here we aim to uncover the mechanisms underlying this effect. EXPERIMENTAL APPROACH: H2 S biosynthesis was evaluated in rat isolated aortic rings following androgen receptor (NR3C4) stimulation. Co-immunoprecipitation and surface plasmon resonance analysis were performed to investigate mechanisms involved in NR3C4 activation. KEY RESULTS: Pretreatment with NR3C4 antagonist nilutamide prevented testosterone-induced increase in H2S and reduced its vasodilator effect. Androgen agonist mesterolone also increased H2S and induced vasodilatation; effects attenuated by the selective cystathionine-γ lyase (CSE) inhibitor propargylglycine. The NR3C4-multicomplex-derived heat shock protein 90 (hsp90) was also involved in this effect; its specific inhibitor geldanamycin strongly reduced testosterone-induced H2S production. Neither progesterone nor 17-ß-oestradiol induced H2S release. Furthermore, we demonstrated that CSE, the main vascular H2S-synthesizing enzyme, is physically associated with the NR3C4/hsp90 complex and the generation of such a ternary system represents a key event leading to CSE activation. Finally, H2S levels in human blood collected from male healthy volunteers were higher than those in female samples. CONCLUSIONS AND IMPLICATIONS: We demonstrated that selective activation of the NR3C4 is essential for H2S biosynthesis within vascular tissue, and this event is based on the formation of a ternary complex between cystathionine-γ lyase, NR3C4and hsp90. This novel molecular mechanism operating in the vasculature, corroborated by higher H2S levels in males, suggests that the L-cysteine/CSE/H2S pathway may be preferentially activated in males leading to gender-specific H2S biosynthesis.
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Sulfuro de Hidrógeno/metabolismo , Receptores Androgénicos/metabolismo , Testosterona/farmacología , Vasodilatación/efectos de los fármacos , Adulto , Alquinos/farmacología , Antagonistas de Andrógenos/farmacología , Animales , Aorta Torácica/efectos de los fármacos , Aorta Torácica/metabolismo , Femenino , Glicina/análogos & derivados , Glicina/farmacología , Humanos , Imidazolidinas/farmacología , Masculino , Persona de Mediana Edad , Ratas , Ratas Wistar , Receptores Androgénicos/efectos de los fármacos , Factores Sexuales , Vasodilatadores/farmacologíaRESUMEN
We have investigated the role of ATP-sensitive potassium (K(ATP)) channels in an experimental model of a delayed phase of vascular hyporeactivity induced by lipopolysaccharide (LPS) in rats. After 24 h, from LPS treatment, in anaesthetized rats the bolus injection of phenylephrine (PE) produced an increase in mean arterial pressure (MAP) significantly (P<0.05) reduced in LPS-treated rats compared to the vehicle-treated rats. This reduction was prevented by pre-treatment of rats with glibenclamide (GLB), a selective inhibitor of K(ATP) channels. GLB administration did not affect the MAP in vehicle-treated rats but produced an increase of MAP in rats treated with LPS. Cromakalim (CRK), a selective K(ATP) channel opener, produced a reduction of MAP that was significantly (P<0.05) higher in LPS- than in vehicle-treated rats. In contrast, the hypotension induced by glyceryl trinitrate (GTN) in LPS-treated rats was not distinguishable from that produced in vehicle-treated rats. Experiments in vitro were conducted on aorta rings collected from rats treated with vehicle or LPS 24 h before sacrifice. The concentration-dependent curve to PE was statistically (P<0.005) reduced in aorta rings collected from LPS- compared to vehicle-treated rats. This difference was totally abolished by tetraethylammonium (TEA), a non-selective inhibitor of K+ channels. CRK produced a relaxation of PE precontracted aorta rings higher in rings from LPS- than in vehicle-treated rats. GLB inhibited CRK-induced relaxation in both tissues, abolishing the observed differences. In conclusion, our results indicate an involvement of K(ATP) channels to the hyporesponsiveness of vascular tissue after 24 h from a single injection of LPS in rats. We can presume an increase in the activity of K(ATP) channels on vascular smooth muscle cells but we cannot exclude an increase of K(ATP) channel number probably due to the gene expression activation.
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Adenosina Trifosfato/fisiología , Lipopolisacáridos/farmacología , Músculo Liso Vascular/efectos de los fármacos , Canales de Potasio/fisiología , Animales , Aorta Torácica/efectos de los fármacos , Aorta Torácica/fisiología , Presión Sanguínea/efectos de los fármacos , Cromakalim/farmacología , Inhibidores Enzimáticos/farmacología , Gliburida/farmacología , Frecuencia Cardíaca/efectos de los fármacos , Técnicas In Vitro , Masculino , Contracción Muscular/efectos de los fármacos , Músculo Liso Vascular/fisiología , NG-Nitroarginina Metil Éster/farmacología , Fenilefrina/farmacología , Ratas , Ratas Wistar , Tetraetilamonio/farmacología , Vasodilatadores/farmacologíaRESUMEN
(1) Septic shock represents an important risk factor for patients critically ill. This pathology has been largely demonstrated to be a result of a myriad of events. Glucocorticoids represent the main pharmacological therapy used in this pathology. (2) Previously we showed that ATP-sensitive potassium (KATP) channels are involved in delayed vascular hyporeactivity in rats (24 h after Escherichia coli lipopolysaccharide (LPS) injection). In LPS-treated rats, we observed a significant hyporeactivity to phenylephrine (PE) that was reverted by glybenclamide (GLB), and a significant increase in cromakalim (CRK)-induced hypotension. (3) We evaluated the effect of dexamethasone (DEX 8 mg kg-1 i.p.) whether on hyporeactivity to PE or on hyperreactivity to CRK administration, in vivo, in a model of LPS (8 x 106 U kg-1 i.p.)-induced endotoxemia in urethane-anaesthetised rats. (4) DEX treatment significantly reduced, in a time-dependent manner, the increased hypotensive effect induced by CRK in LPS-treated rats. This effect was significantly (P<0.05) reverted by the glucocorticoid receptor antagonist RU38486 (6.6 mg kg-1 i.p.). (5) GLB-induced hypertension (40 mg kg-1 i.p.), in LPS-treated rats, was significantly inhibited by DEX if administered at the same time of LPS. (6) Simultaneous administration of DEX and LPS to rats completely abolished the hyporeactivity to PE observed after 24 h from LPS injection. (7) In conclusion, our results suggest that the beneficial effect of DEX in endotoxemia could be ascribed, at least in part, to its ability to interfere with KATP channel activation induced by LPS. This interaction may explain the improvement of vascular reactivity to PE, mediated by DEX, in LPS-treated rats, highlighting a new pharmacological activity to the well-known anti-inflammatory properties of glucocorticoids.
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Adenosina Trifosfato/metabolismo , Dexametasona/uso terapéutico , Hipotensión/tratamiento farmacológico , Lipopolisacáridos/toxicidad , Canales de Potasio/metabolismo , Animales , Dexametasona/farmacología , Hipotensión/inducido químicamente , Hipotensión/metabolismo , Masculino , Ratas , Ratas Wistar , Choque Séptico/inducido químicamente , Choque Séptico/tratamiento farmacológico , Choque Séptico/metabolismo , Vasoconstrictores/farmacologíaRESUMEN
The effect of dexamethasone on hypotension induced by mu-, kappa- and delta-opioid receptor agonists was investigated in pentobarbital-anaesthetised rats. Morphine (nonselective opioid receptor agonist), DAGO (D-Ala2-N-methyl-[Phe4-Gly5-ol]enkephalin; mu-opioid receptor-selective agonist), U50-488H (trans(+/-)-3,4-dichloro-N-methyl-N-(2[1pyrrolidynyl]cyclohexyl)-benzeneacetamide; kappa-opioid receptor-selective agonist) and deltorphin II (delta-opioid receptor-selective agonist), given intravenously, 5 micromol/kg, induced hypotension in rats. This hypotension was characterised by a fall in mean arterial blood pressure in 1-2 min that recovered in 30 min for morphine and U50-488H and in 5 or 20 min for DAGO and deltorphin II, respectively. Dexamethasone per se at a dose of 7.5 micromol/kg, i.v. did not significantly modify the mean arterial blood pressure of animals. Dexamethasone administration 90 min, but not 30 or 60 min, before the opioid agonists injection, prevented the hypotension induced by morphine or U50-488H, but not that induced by DAGO or deltorphin II. Pretreatment with RU-38486 (mifepristone; 7.5 micromol/kg, i.v.), a glucocorticoid receptor antagonist, 15 min before the steroid, prevented dexamethasone inhibition of hypotension induced by morphine and U50-488H. Furthermore, pretreatment with cycloheximide, a protein synthesis inhibitor (3.5 micromol/kg, i.v.), was also able to abolish the effects of dexamethasone on morphine- and U50-488H-induced hypotension. Results of the present study indicate that dexamethasone inhibited kappa-opioid receptor-mediated hypotension in rats, indicating a further important functional interaction between corticosteroids and the opioid system at kappa receptors. The ability of cycloheximide and RU-38486 to block dexamethasone effects indicates that steroid interference with kappa-opioid receptor-mediated hypotension involves a protein synthesis-dependent mechanism via glucocorticoid receptors.
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Antiinflamatorios/farmacología , Dexametasona/farmacología , Hipotensión/prevención & control , Narcóticos , Animales , Antiinflamatorios/uso terapéutico , Presión Sanguínea/efectos de los fármacos , Cicloheximida/farmacología , Dexametasona/antagonistas & inhibidores , Dexametasona/uso terapéutico , Encefalina Ala(2)-MeFe(4)-Gli(5) , Frecuencia Cardíaca/efectos de los fármacos , Hipotensión/inducido químicamente , Masculino , Mifepristona/farmacología , Morfina , Oligopéptidos , Ratas , Ratas Wistar , Receptores de Glucocorticoides/antagonistas & inhibidores , Receptores Opioides kappa/antagonistas & inhibidoresRESUMEN
In this study we investigated, for the first time in vivo, the effect of cloricromene, a cumarine derivative, on NF-kappaB activation in endotoxin-treated rats. Endotoxemia was induced in male rats by the intravenous injection of Salmonella typhosa lipopolysaccharide (LPS; 2 mg/kg/i.v.). In vivo treatment with cloricromene (2 mg/kg/i.v.) 30 min before lipopolysaccharide administration reversed the LPS-induced loss in tone of the aortic rings, improved their reactivity to phenylephrine, decreased both nitric oxide (NO) and TNF-alpha serum levels by inhibiting LPS-induced inducible NO synthase and TNF-alpha mRNA expression, and interestingly inhibited LPS-induced NF-kappaB activation. Our data suggest that cloricromene protects rats from LPS by blocking LPS-induced NF-kappaB activation, leading to inhibition of NO and TNF-alpha overproduction and thereby reversing the LPS-induced vascular hyporeactivity.
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Cromonar/análogos & derivados , Cromonar/uso terapéutico , Endotoxemia/tratamiento farmacológico , FN-kappa B/antagonistas & inhibidores , Salmonella typhi , Animales , Aorta Torácica/efectos de los fármacos , Aorta Torácica/metabolismo , Aorta Torácica/fisiología , Ensayo de Cambio de Movilidad Electroforética , Endotoxemia/metabolismo , Endotoxemia/fisiopatología , Técnicas In Vitro , Masculino , Contracción Muscular/efectos de los fármacos , Músculo Liso Vascular/efectos de los fármacos , Músculo Liso Vascular/fisiología , FN-kappa B/genética , FN-kappa B/metabolismo , Nitratos/sangre , Óxido Nítrico Sintasa/antagonistas & inhibidores , Óxido Nítrico Sintasa/genética , Óxido Nítrico Sintasa de Tipo II , Nitritos/sangre , Fenilefrina , ARN Mensajero/antagonistas & inhibidores , Ratas , Ratas Wistar , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Factor de Necrosis Tumoral alfa/genéticaRESUMEN
The main problem of using human corpus cavernosum (HCC) tissue to perform bioassay is linked to its limited availability further complicated by the heterogeneous source of the tissues used. Here, we show that gender reassignment is a reliable source of human tissue without major ethical problems. Indeed, the entire corpus cavernosum is obtained from the surgery procedure, which allows creating a standardized procedure to prepare HCC strip. In addition, human tissue, if kept in the fridge in the condition described, does not loose its ability to contract to phenylephrine (PE; alpha agonist), angiotensin II (AG II) and KCl up to 4 days. Furthermore, once contracted with PE, HCC relaxes to acetylcholine (endothelium-dependent mechanism); sodium nitroprusside (endothelium-independent mechanism); cromakalim (CRK), a K(ATP) channel opener; or alprostadil, a synthetic PGE2 (ALPR). In conclusion, we have standardized a procedure that allows the use of HCC strips to evaluate drug activity and/or to study pathophysiological mechanisms with an intact functional human tissue up to 4 days from the surgery procedure.
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Bioensayo/métodos , Contracción Muscular/fisiología , Relajación Muscular/fisiología , Músculo Liso/fisiología , Pene/fisiología , Acetilcolina/farmacología , Alprostadil/farmacología , Angiotensina II/farmacología , Bioensayo/normas , Cromakalim/farmacología , Humanos , Masculino , Contracción Muscular/efectos de los fármacos , Relajación Muscular/efectos de los fármacos , Músculo Liso/efectos de los fármacos , Músculo Liso/cirugía , Nitroprusiato/farmacología , Pene/efectos de los fármacos , Pene/cirugía , Fenilefrina/farmacología , Cloruro de Potasio/farmacología , Transexualidad/cirugíaRESUMEN
This study was carried out with the aim to optimize the pharmacological profile of gliquidone (GLI)--a poorly bioavailable hypoglycaemic agent sparingly soluble in water--through complexation with cyclodextrins. In order to increase the apparent solubility of GLI, two cyclodextrins, namely beta-cyclodextrin (betaCD) and hydroxypropyl-beta-cyclodextrin (HPbetaCD), were tested. The effect of cyclodextrin addition on the aqueous solubility of GLI was evaluated by the phase solubility method at different pH values. The amount of GLI in solution increased upon CD addition according to A type plots. The aqueous solubility of GLI was enhanced more at higher pH values and using HPbetaCD. On the basis of its performance, HPbetaCD was selected as host to prepare GLI oral formulations. GLI/HPbetaCD solid systems were prepared at 1:2 molar ratio by co-grinding, spray-drying and freeze-drying and characterized by DSC, FTIR and X-ray powder diffractometry. Powders were amorphous and showed an improved dissolution rate in comparison with GLI. GLI/HPbetaCD co-ground and freeze-dried products were the most interesting systems, since they dissolved 62 and 94% of total drug after 15 min, respectively. The hypoglycaemic effect of the most rapidly dissolving binary systems was evaluated after oral administration in fasted rats by measuring plasma glucose level in the time interval 0.5-36 h and compared to free GLI. Our findings indicate that cyclodextrin-containing formulations not only provide an onset of hypoglycaemic effect faster than GLI, but also enhance significantly the pharmacological effect due to improved biopharmaceutics. The association GLI/HPbetaCD allows a reduction of the oral dose and is expected to provide a better control over drug side effects, contributing to improve safety and efficacy of GLI.