RESUMO
Continued efforts are made on the development of earth-abundant metal catalysts for dehydrogenation/hydrolysis of amine boranes. In this study, complex [K-18-crown-6-ether][(NO)2Fe(µ-MePyr)(µ-CO)Fe(NO)2] (3-K-crown, MePyr = 3-methylpyrazolate) was explored as a pre-catalyst for the dehydrogenation of dimethylamine borane (DMAB). Upon evolution of H2(g) from DMAB triggered by 3-K-crown, parallel conversion of 3-K-crown into [(NO)2Fe(N,N'-MePyrBH2NMe2)]- (5) and an iron-hydride intermediate [(NO)2(CO)Fe(µ-H)Fe(CO)(NO)2]- (A) was evidenced by X-ray diffraction/nuclear magnetic resonance/infrared/nuclear resonance vibrational spectroscopy experiments and supported by density functional theory calculations. Subsequent transformation of A into complex [(NO)2Fe(µ-CO)2Fe(NO)2]- (6) is synchronized with the deactivated generation of H2(g). Through reaction of complex [Na-18-crown-6-ether][(NO)2Fe(η2-BH4)] (4-Na-crown) with CO(g) as an alternative synthetic route, isolated intermediate [Na-18-crown-6-ether][(NO)2(CO)Fe(µ-H)Fe(CO)(NO)2] (A-Na-crown) featuring catalytic reactivity toward dehydrogenation of DMAB supports a substrate-gated transformation of a pre-catalyst [(NO)2Fe(µ-MePyr)(µ-CO)Fe(NO)2]- (3) into the iron-hydride species A as an intermediate during the generation of H2(g).
RESUMO
Copper nitrite reductases (CuNiRs) catalyze the reduction of nitrite to form nitric oxide. In recent years, new classes of redox partner linked CuNiRs have been isolated and characterized by crystallographic techniques. Solution-state biophysical studies have shed light on the complex catalytic mechanisms of these enzymes and implied that protein dynamics may play a role in CuNiR catalysis. To investigate the structural, dynamical, and functional relationship of these CuNiRs, we have used protein reverse engineering and pulsed electron-electron double resonance (PELDOR) spectroscopy to determine their solution-state inter-copper distributions. Data show the multidimensional conformational landscape of this family of enzymes and the role of tethering in catalysis. The importance of combining high-resolution crystallographic techniques and low-resolution solution-state approaches in determining the structures and mechanisms of metalloenzymes is emphasized by our approach.
Assuntos
Cobre , Elétrons , Cobre/química , Nitrito Redutases/química , Nitrito Redutases/metabolismo , Oxirredução , Análise EspectralRESUMO
Diabetic neuropathy is associated with neuropathic pain in about 50% of diabetic subjects. Clinical management of neuropathic pain is complex and so far unsatisfactory. In this study, we analyzed the effects of the testosterone metabolites, dihydrotestosterone (DHT), and 3α-diol, on nociceptive and allodynia thresholds and on molecular and functional parameters related to pain modulation in the dorsal horns of the spinal cord and in the dorsal root ganglia of rats rendered diabetic by streptozotocin injection. Furthermore, the levels of DHT and 3α-diol were analyzed in the spinal cord. Diabetes resulted in a significant decrease in DHT levels in the spinal cord that was reverted by DHT or 3α-diol treatments. In addition, 3α-diol treatment resulted in a significant increase in 3α-diol in the spinal cord compared with control values. Both steroids showed analgesic properties on diabetic neuropathic pain, affecting different pain parameters and possibly by different mechanisms of action. Indeed, DHT counteracted the effect of diabetes on the mechanical nociceptive threshold, pre- and post-synaptic components, glutamate release, astrocyte immunoreactivity, and expression of interleukin-1ß (IL1ß), while 3α-diol was effective on tactile allodynia threshold, glutamate release, astrocyte immunoreactivity and the expression of substance P, toll-like receptor 4, tumor necrosis factor-α, transforming growth factor ß-1, IL1ß, and translocator protein. These results indicate that testosterone metabolites are potential agents for the treatment of diabetic neuropathic pain.
Assuntos
Neuropatias Diabéticas/metabolismo , Dor Nociceptiva/metabolismo , Testosterona/metabolismo , Animais , Neuropatias Diabéticas/genética , Di-Hidrotestosterona/metabolismo , Humanos , Interleucina-4/genética , Interleucina-4/metabolismo , Masculino , Dor Nociceptiva/genética , Ratos , Ratos Sprague-Dawley , Medula Espinal/metabolismoRESUMO
Progesterone is synthesized and actively metabolized in the central and peripheral nervous system, into neuroactive steroid metabolites, such as dihydroprogesterone, allopregnanolone and isopregnanolone. Progesterone and/or its metabolites exert a variety of effects acting as physiological regulators of neuronal and glial development and plasticity, controlling reproduction, neuroendocrine events, mood and affection. In addition, these neuroactive steroids maintain neural homeostasis and exert neuroprotective actions. In agreement, metabolic pathways of progesterone are affected by modifications in the level of gonadal hormones and by pathology or injury with a regional specificity and in a sex-dimorphic way. Therefore, observations here summarized may provide a background to design sex-specific therapies based on progesterone metabolites. On this point of view, considering that one of the major limits of a therapy based on neuroactive steroids could be modifications in their plasma levels and their consequent peripheral effects, pharmacological treatments aimed to increase their levels in the nervous system could provide an interesting therapeutic option.
Assuntos
Sistema Nervoso/metabolismo , Doenças Neurodegenerativas/metabolismo , Progesterona/metabolismo , Animais , Feminino , Humanos , Masculino , Caracteres SexuaisRESUMO
INTRODUCTION: Observations performed in a subset of subjects treated with finasteride (an inhibitor of the enzyme 5α-reductase) for male pattern hair loss seem to indicate that sexual dysfunction as well as anxious/depressive symptomatology may occur at the end of the treatment and continue after discontinuation. AIM: A possible hypothesis to explain depression symptoms after finasteride treatment might be impairment in the levels of neuroactive steroids. Therefore, neuroactive steroid levels were evaluated in paired plasma and cerebrospinal fluid samples obtained from male patients who received finasteride for the treatment of androgenic alopecia and who, after drug discontinuation, still show long-term sexual side effects as well as anxious/depressive symptomatology. METHODS: The levels of neuroactive steroids were evaluated by liquid chromatography-tandem mass spectrometry in three postfinasteride patients and compared to those of five healthy controls. MAIN OUTCOME MEASURES: Neuroactive steroid levels in plasma and cerebrospinal fluid of postfinasteride patients and healthy controls. RESULTS: At the examination, the three postfinasteride patients reported muscular stiffness, cramps, tremors, and chronic fatigue in the absence of clinical evidence of any muscular disorder or strength reduction. Severity and frequency of the anxious/depressive symptoms were quite variable; overall, all the subjects had a fairly complex and constant neuropsychiatric pattern. Assessment of neuroactive steroid levels in patients showed some interindividual differences. However, the most important finding was the comparison of their neuroactive steroid levels with those of healthy controls. Indeed, decreased levels of tetrahydroprogesterone, isopregnanolone and dihydrotestosterone and increased levels of testosterone and 17ß-estradiol were reported in cerebrospinal fluid of postfinasteride patients. Moreover, decreased levels of dihydroprogesterone and increased levels of 5α-androstane-3α,17ß-diol and 17ß-estradiol were observed in plasma. CONCLUSION: The present observations confirm that an impairment of neuroactive steroid levels, associated with depression symptoms, is still present in androgenic alopecia patients treated with finasteride despite the discontinuation of the treatment.
Assuntos
Inibidores de 5-alfa Redutase/efeitos adversos , Alopecia/tratamento farmacológico , Ansiedade/induzido quimicamente , Depressão/induzido quimicamente , Finasterida/efeitos adversos , Comportamento Sexual/efeitos dos fármacos , Esteroides/líquido cefalorraquidiano , Inibidores de 5-alfa Redutase/administração & dosagem , Inibidores de 5-alfa Redutase/líquido cefalorraquidiano , Adulto , Ansiedade/líquido cefalorraquidiano , Ansiedade/diagnóstico , Biomarcadores/líquido cefalorraquidiano , Estudos de Casos e Controles , Cromatografia Líquida , Depressão/líquido cefalorraquidiano , Depressão/diagnóstico , Esquema de Medicação , Feminino , Finasterida/administração & dosagem , Finasterida/líquido cefalorraquidiano , Humanos , Masculino , Esteroides/sangue , Espectrometria de Massas em Tandem , Fatores de Tempo , Resultado do TratamentoRESUMO
Physiological changes and pathological alterations in the nervous system of rodents are associated with modifications in the levels of neuroactive steroids in the brain, spinal cord and/or peripheral nerves. Measures of tissue levels of steroids in the nervous system present serious limitations for human studies and for longitudinal studies in animals. In this study we have explored whether levels of neuroactive steroids in plasma and the cerebrospinal fluid reflect their levels in neural tissues. To this aim, we have evaluated by liquid chromatography-tandem mass spectrometry the levels of several neuroactive steroids in plasma, cerebrospinal fluid, cerebral cortex, cerebellum, hippocampus, spinal cord and sciatic nerve of male and female rats. Data indicate that plasma and cerebrospinal fluid levels of steroids do not fully reflect their tissue levels. However, the interindividual variations in the levels of all the steroids assessed, with the exception of dehydroepiandrosterone, showed a positive correlation in plasma and cerebral cortex. Most steroids also showed a positive correlation in plasma and the cerebellum, the spinal cord and the sciatic nerve. In the hippocampus, the levels of tetrahydroprogesterone, testosterone and testosterone metabolites showed a significant positive correlation with their respective levels in plasma. The cerebrospinal fluid levels of some steroids, such as testosterone and dihydrotestosterone, showed a full correlation with tissue levels. In addition, cerebrospinal fluid levels of pregnenolone, progesterone, and 17ß-estradiol showed a positive correlation with their corresponding levels in the majority of the neural structures analyzed. These findings suggest that the levels of some neuroactive steroids in cerebrospinal fluid as well as in plasma may be valuable to predict their levels in the nervous system.
Assuntos
Química Encefálica , Neurotransmissores/sangue , Neurotransmissores/líquido cefalorraquidiano , Nervos Periféricos/química , Medula Espinal/química , Animais , Análise Química do Sangue/normas , Encéfalo/metabolismo , Cromatografia Líquida/normas , Feminino , Masculino , Nervos Periféricos/metabolismo , Ratos , Ratos Sprague-Dawley , Reprodutibilidade dos Testes , Medula Espinal/metabolismo , Esteroides/análise , Esteroides/metabolismo , Espectrometria de Massas em Tandem/normasRESUMO
Different experimental autoimmune encephalomyelitis models (EAE) have been developed. However, due to the different experimental conditions applied, observations simultaneously considering different pathological targets are still scarce. Using EAE induced in Dark Agouti rats with syngenic whole spinal cord homogenate suspended in incomplete Freund's adjuvant, we here analyze neurosteroidogenic machinery, cytokine levels, microglial cells, infiltration of inflammatory cells, myelin proteins and Na(+), K(+)-ATPase pump activity in the spinal cord. Data obtained in the acute phase of the disease confirmed that neurological signs were accompanied by the presence of perivascular infiltrating T cells (CD3(+) cells) and activated monocytic/microglial cells (ED1(+) and MHC-II(+)) in the spinal cord. In particular, the number of MHC-II(+) cells was significantly increased in association with increased expression of pro- (i.e., TNF-α, IL-1ß) and anti-inflammatory (i.e., TGF-ß) cytokines as well as with decreased expression of proteolipid protein and myelin basic protein. During the chronic phase of the disease, the number of MHC-II(+) cells was still increased, although less than in the acute phase. Changes in the number of MHC-II(+) cells were associated with decreased Na(+),K(+)-ATPase enzymatic activity. A general decrease in the levels of neuroactive steroids, with the exception of an increase in tetrahydroprogesterone and 17ß-estradiol, was detected in the acute phase. These changes were maintained or reverted in the chronic phase of EAE. In conclusion, we report that modifications in the neuroimmune response in the acute and chronic phases of EAE are associated with specific changes in myelin proteins, Na(+),K(+)-ATPase pump and in the levels of neuroactive steroids.
Assuntos
Encefalomielite Autoimune Experimental/patologia , Doença Aguda , Animais , Contagem de Células Sanguíneas , Doença Crônica , Citocinas/metabolismo , Progressão da Doença , Encefalomielite Autoimune Experimental/genética , Encefalomielite Autoimune Experimental/imunologia , Fluorometria , Genes MHC da Classe II/genética , Imuno-Histoquímica , Masculino , Espectrometria de Massas , Proteínas da Mielina/biossíntese , Proteínas da Mielina/genética , Neurônios/patologia , Infiltração de Neutrófilos/fisiologia , Ensaios de Proteção de Nucleases , Ratos , Reação em Cadeia da Polimerase em Tempo Real , Ribonucleases/metabolismo , Transdução de Sinais/fisiologia , ATPase Trocadora de Sódio-Potássio/metabolismo , Esteroides/farmacologia , Esteroides/uso terapêuticoRESUMO
Neuroactive steroid levels are decreased in the central nervous system (CNS) of streptozotocin (STZ) diabetic rats. In agreement, they exert protective effects in this experimental model, counteracting degenerative events occurring in the CNS. Therefore, an interesting therapeutic strategy could be to increase their levels directly in the CNS. In this study we have evaluated whether activation of translocator protein-18kDa (TSPO) or liver X receptors (LXRs) may affect the levels of neuroactive steroids present in the CNS of diabetic and non-diabetic animals. We observed that the treatment with either Ro5-4864 (i.e., a ligand of TSPO) or with GW3965 (i.e., a ligand of LXRs) induced an increase of neuroactive steroids in the spinal cord, the cerebellum and the cerebral cortex of STZ-rats, but not in the CNS of non-pathological animals. Interestingly, the pattern of induction was different among the three CNS areas analyzed and between the two pharmacological tools. In particular, the activation of LXRs might represent a promising neuroprotective strategy, because the treatment with GW3965, at variance to Ro5-4864 treatment, did not induce significant changes in the plasma levels of neuroactive steroids. This suggests that activation of LXRs may selectively increase the CNS levels of neuroactive steroids avoiding possible endocrine side effects exerted by the systemic treatment with these molecules. Interestingly GW3965 treatment induced an increase of dihydroprogesterone in the spinal cord of diabetic animals in association with an increase of myelin basic protein expression. Thus we demonstrated that LXR activation was able to rescue CNS symptoms of diabetes.
Assuntos
Proteínas de Transporte/metabolismo , Complicações do Diabetes/tratamento farmacológico , Sistemas de Liberação de Medicamentos , Degeneração Neural/tratamento farmacológico , Receptores Nucleares Órfãos/metabolismo , Receptores de GABA-A/metabolismo , Esteroides/sangue , Animais , Benzodiazepinonas/farmacologia , Complicações do Diabetes/metabolismo , Complicações do Diabetes/fisiopatologia , Modelos Animais de Doenças , Sistemas de Liberação de Medicamentos/métodos , Receptores X do Fígado , Masculino , Degeneração Neural/metabolismo , Degeneração Neural/fisiopatologia , Ratos , Ratos Sprague-Dawley , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/fisiologiaRESUMO
Clinical observations suggest a sex-dimorphism in the incidence and symptomatology of diabetic neuropathy, but this possible gender effect has never been investigated in detail in a well-characterized experimental model such as streptozotocin (STZ)-induced diabetes. Therefore, in this study we have compared with a multimodal set of tests the impact of diabetes on the sciatic nerve in male and female rats. To assess whether sex-dimorphism in peripheral diabetic neuropathy is dependent on gonadal hormones we have also analyzed the effect of ovariectomy and orchidectomy on the sciatic nerve of STZ-diabetic rats. Nerve conduction velocity (NCV), Na(+),K(+)-ATPase activity, expression of myelin proteins, thermal sensitivity and reactive oxygen species production were similarly affected in male and female animals by STZ. However, ovariectomy, but not orchidectomy, significantly counteracted STZ-induced alterations on NCV, Na(+),K(+)-ATPase activity, and expression of myelin proteins. This effect of ovariactomy was associated to an increase in the levels of neuroactive steroids, such as dehydroepiandrosterone, testosterone and dihydrotestosterone, in the sciatic nerve of diabetic rats. These neuroactive steroids have been demonstrated to be protective agents in this experimental model of diabetic neuropathy. However, their efficacy has been so far tested only in male animals. Therefore, the present data might represent an important background to evaluate their efficacy also in female diabetic animals.
Assuntos
Neuropatias Diabéticas/diagnóstico , Neuropatias Diabéticas/fisiopatologia , Fibras Nervosas Mielinizadas/metabolismo , Nervo Isquiático/fisiopatologia , Caracteres Sexuais , Esteroides/metabolismo , Animais , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/fisiopatologia , Neuropatias Diabéticas/metabolismo , Feminino , Masculino , Fibras Nervosas Mielinizadas/patologia , Orquiectomia , Ovariectomia , Ratos , Ratos Sprague-Dawley , Nervo Isquiático/metabolismo , Nervo Isquiático/patologiaRESUMO
Several reviews have so far pointed out on the relevant physiological and pharmacological role exerted by neuroactive steroids in the central nervous system. In the present review we summarize observations indicating that synthesis and metabolism of neuroactive steroids also occur in the peripheral nerves. Interestingly, peripheral nervous system is also a target of their action. Indeed, as here reported neuroactive steroids are physiological regulators of peripheral nerve functions and they may also represent interesting therapeutic tools for different types of peripheral neuropathy.
RESUMO
Our previous observations have shown that neuroactive steroid levels in the brain are affected by acute experimental autoimmune encephalomyelitis (EAE) with sex and regional specificity (Giatti et al. 2010). To better understand the effect of EAE on neuroactive steroids, we have here assessed the levels of pregnenolone, progesterone and its derivatives (i.e. dihydroprogesterone, tetrahydroprogesterone and isopregnanolone), testosterone and its derivatives (dihydrotestosterone and 5alpha-androstane-3alpha, 17beta-diol) in different CNS regions of male and female rats affected by chronic EAE. Data obtained by liquid chromatography tandem mass spectrometry revealed that chronic EAE results in sex and regional specific alterations in the levels of neuroactive steroids in the brain, which are in many cases different to those produced by acute EAE. The specific changes in neuroactive steroid levels after chronic EAE may be of relevance to design new possible therapeutic strategies for the disease.
Assuntos
Encefalomielite Autoimune Experimental/metabolismo , Encefalomielite Autoimune Experimental/fisiopatologia , Hormônios Esteroides Gonadais/metabolismo , Caracteres Sexuais , Animais , Doença Crônica , Modelos Animais de Doenças , Encefalomielite Autoimune Experimental/imunologia , Feminino , Hormônios Esteroides Gonadais/análise , Cobaias , Masculino , Esclerose Múltipla/imunologia , Esclerose Múltipla/metabolismo , Esclerose Múltipla/fisiopatologia , RatosRESUMO
Alterations in myelin membranes, as well as in the expression of myelin proteins have been reported in experimental models of diabetes. Data here reported show for the first time that the mRNA levels of two isoforms of myelin basic protein (MBP), 18.5 and 21.5 kDa, are decreased in the spinal cord of streptozotocin-treated rats and that treatment with a neuroactive steroid, such as progesterone (P), may counteract this effect. Interestingly, metabolism of progesterone into dihydroprogesterone (DHP) by the enzyme 5alpha-reductase seems to exert an important role in such an effect. As here demonstrated, 5alpha-reductase mRNA and DHP levels are reduced by diabetes in spinal cord, but treatment with P, is able to counteract these effects. Moreover, treatment with DHP is able to mimic the effect of P on MBP gene expression. Thus, the effects of P here observed are due to its enzymatic conversion into DHP. Because DHP, like P, interacts with P receptor (PR), the present results may suggest the importance to analyze the effects of PR modulators as tools of therapeutic strategies for diabetic complications occurring in nervous system.