RESUMEN
Amyloid-ß (Aß) and hyperphosphorylated tau (P-tau) are Alzheimer's disease (AD) biomarkers that interact in a complex manner to induce most of the cognitive and brain alterations observed in this disease. Since the neuronal cytoskeleton is a common downstream pathological target of tau and Aß, which mostly lead to augmented microtubule instability, the administration of microtubule stabilizing agents (MSAs) can protect against their pathological actions. However, the effectiveness of MSAs is still uncertain due to their state-dependent negative effects; thus, evaluating their specific actions in different pathological or physiological conditions is required. We evaluated whether epothilone-D (Epo-D), a clinically used MSA, rescues from the functional and behavioral alterations produced by intracerebroventricular injection of Aß, the presence of P-tau, or their combination in rTg4510 mice. We also explored the side effects of Epo-D. To do so, we evaluated hippocampal-dependent spatial memory with the Hebb-Williams maze, hippocampal CA1 integrity and the intrinsic and synaptic properties of CA1 pyramidal neurons with the patch-clamp technique. Aß and P-tau mildly impaired memory retrieval, but produced contrasting effects on intrinsic excitability. When Aß and P-tau were combined, the alterations in excitability and spatial reversal learning (i.e., cognitive flexibility) were exacerbated. Interestingly, Epo-D prevented most of the impairments induced Aß and P-tau alone and combined. However, Epo-D also exhibited some side effects depending on the prevailing pathological or physiological condition, which should be considered in future preclinical and translational studies. Although we did not perform extensive histopathological evaluations or measured microtubule stability, our findings show that MSAs can rescue the consequences of AD-like conditions but otherwise be harmful if administered at a prodromal stage of the disease.
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Neuron-microglia communication through the Cx3cr1-Cx3cl1 axis is essential for the development and refinement of neural circuits, which determine their function into adulthood. In the present work we set out to extend the behavioral characterization of Cx3cr1-/- mice evaluating innate behaviors and spatial navigation, both dependent on hippocampal function. Our results show that Cx3cr1-deficient mice, which show some changes in microglial and synaptic terminals morphology and density, exhibit alterations in activities of daily living and in the rapid encoding of novel spatial information that, nonetheless, improves with training. A neural substrate for these cognitive deficiencies was found in the form of synaptic dysfunction in the CA3 region of the hippocampus, with a marked impact on the mossy fiber (MF) pathway. A network analysis of the CA3 microcircuit reveals the effect of these synaptic alterations on the functional connectivity among CA3 neurons with diminished strength and topological reorganization in Cx3cr1-deficient mice. Neonatal population activity of the CA3 region in Cx3cr1-deficient mice shows a marked reorganization around the giant depolarizing potentials, the first form of network-driven activity of the hippocampus, suggesting that alterations found in adult subjects arise early on in postnatal development, a critical period of microglia-dependent neural circuit refinement. Our results show that interruption of the Cx3cr1-Cx3cl1/neuron-microglia axis leads to changes in CA3 configuration that affect innate and learned behaviors.
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Conducta Animal , Receptor 1 de Quimiocinas CX3C , Comunicación Celular , Quimiocina CX3CL1 , Microglía , Neuronas , Actividades Cotidianas , Animales , Conducta Animal/fisiología , Receptor 1 de Quimiocinas CX3C/genética , Receptor 1 de Quimiocinas CX3C/metabolismo , Comunicación Celular/genética , Comunicación Celular/fisiología , Quimiocina CX3CL1/genética , Quimiocina CX3CL1/metabolismo , Modelos Animales de Enfermedad , Hipocampo/metabolismo , Humanos , Ratones , Ratones Noqueados , Microglía/metabolismo , Neuronas/metabolismoRESUMEN
Chronic kidney disease (CKD) is a multifactorial pathology that progressively leads to the deterioration of metabolic functions and results from deficient glomerular filtration and electrolyte imbalance. Its economic impact on public health is challenging. Mexico has a high prevalence of CKD that is strongly associated with some of the most common metabolic disorders like diabetes and hypertension. The gradual loss of kidney functions provokes an inflammatory state and endocrine alterations affecting several systems. High serum levels of prolactin have been associated with CKD progression, inflammation, and olfactory function. Also, the nutritional status is altered due to impaired renal function. The decrease in calorie and protein intake is often accompanied by malnutrition, which can be severe at advanced stages of the disease. Nutrition and olfactory functioning are closely interconnected, and CKD patients often complain of olfactory deficits, which ultimately can lead to deficient food intake. CKD patients present a wide range of deficits in olfaction like odor discrimination, identification, and detection threshold. The chronic inflammatory status in CKD damages the olfactory epithelium leading to deficiencies in the chemical detection of odor molecules. Additionally, the decline in cognitive functioning impairs the capacity of odor differentiation. It is not clear whether peritoneal dialysis and hemodialysis improve the olfactory deficits, but renal transplants have a strong positive effect. In the present review, we discuss whether the olfactory deficiencies caused by CKD are the result of the induced inflammatory state, the hyperprolactinemia, or a combination of both.
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BACKGROUND: Deficits in odor detection and discrimination are premature symptoms of Alzheimer's disease (AD) that correlate with pathological signs in the olfactory bulb (OB) and piriform cortex (PCx). Similar olfactory dysfunction has been characterized in AD transgenic mice that overproduce amyloid-ß peptide (Aß), which can be prevented by reducing Aß levels by immunological and pharmacological means, suggesting that olfactory dysfunction depends on Aß accumulation and Aß-driven alterations in the OB and/or PCx, as well as on their activation. However, this possibility needs further exploration. OBJECTIVE: To characterize the effects of Aß on OB and PCx excitability/coupling and on olfaction. METHODS: Aß oligomerized solution (containing oligomers, monomers, and protofibrils) or its vehicle were intracerebroventricularlly injected two weeks before OB and PCx excitability and synchrony were evaluated through field recordings in vivo and in brain slices. Synaptic transmission from the OB to the PCx was also evaluated in slices. Olfaction was assessed through the habituation/dishabituation test. RESULTS: Aß did not affect lateral olfactory tract transmission into the PCx but reduced odor habituation and cross-habituation. This olfactory dysfunction was related to a reduction of PCx and OB network activity power in vivo. Moreover, the coherence between PCx-OB activities was also reduced by Aß. Finally, Aß treatment exacerbated the 4-aminopyridine-induced excitation in the PCx in slices. CONCLUSION: Our results show that Aß-induced olfactory dysfunction involves a complex set of pathological changes at different levels of the olfactory pathway including alterations in PCx excitability and its coupling with the OB. These pathological changes might contribute to hyposmia in AD.
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Péptidos beta-Amiloides/toxicidad , Trastornos del Olfato/inducido químicamente , Trastornos del Olfato/fisiopatología , Bulbo Olfatorio/fisiopatología , Vías Olfatorias/fisiopatología , Fragmentos de Péptidos/toxicidad , Corteza Piriforme/fisiopatología , Péptidos beta-Amiloides/administración & dosificación , Animales , Ratones , Microinyecciones/métodos , Bulbo Olfatorio/efectos de los fármacos , Vías Olfatorias/efectos de los fármacos , Técnicas de Cultivo de Órganos , Fragmentos de Péptidos/administración & dosificación , Corteza Piriforme/efectos de los fármacosRESUMEN
Chronic intermittent hypoxia (CIH) is the most distinct feature of obstructive sleep apnea (OSA), a common breathing and sleep disorder that leads to several neuropathological consequences, including alterations in the hippocampal network and in seizure susceptibility. However, it is currently unknown whether these alterations are permanent or remit upon normal oxygenation. Here, we investigated the effects of CIH on hippocampal spontaneous network activity and hyperexcitability in vitro and explored whether these alterations endure or fade after normal oxygenation. Results showed that applying CIH for 21 days to adult rats increases gamma-band hippocampal network activity and aggravates 4-Aminopyridine-induced epileptiform activity in vitro. Interestingly, these CIH-induced alterations remit after 30 days of normal oxygenation. Our findings indicate that hippocampal network alterations and increased seizure susceptibility induced by CIH are not permanent and can be spontaneously reverted, suggesting that therapeutic interventions against OSA in patients with epilepsy, such as surgery or continuous positive airway pressure (CPAP), could be favorable for seizure control.
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4-Aminopiridina/toxicidad , Ritmo Gamma/fisiología , Hipocampo/fisiopatología , Hipoxia Encefálica/fisiopatología , Red Nerviosa/fisiopatología , Animales , Enfermedad Crónica , Ritmo Gamma/efectos de los fármacos , Hipocampo/efectos de los fármacos , Hipoxia Encefálica/complicaciones , Masculino , Red Nerviosa/efectos de los fármacos , Técnicas de Cultivo de Órganos , Bloqueadores de los Canales de Potasio/toxicidad , Ratas , Ratas Wistar , Convulsiones/inducido químicamente , Convulsiones/etiología , Convulsiones/fisiopatologíaRESUMEN
Transgenic mice have been used to make valuable contributions to the field of neuroscience and model neurological diseases. The simultaneous functional analysis of hippocampal cell activity combined with hippocampal dependent innate task evaluations provides a reliable experimental approach to detect fine changes during early phases of neurodegeneration. To this aim, we used a merge of patch-clamp with two hippocampal innate behavior tasks. With this experimental approach, whole-cell recordings of CA1 pyramidal cells, combined with hippocampal-dependent innate behaviors, have been crucial for evaluating the early mechanism of neurodegeneration and its consequences. Here, we present our protocol for ex vivo whole-cell recordings of CA1 pyramidal cells and hippocampal dependent innate behaviors in an adolescent (p30) mice.
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Hydrogen peroxide (H2O2) is a messenger involved in both damaging neuroinflammatory responses and physiological cell communication. The ventrolateral medulla, which regulates several vital functions including breathing and blood pressure, is highly influenced by hydrogen peroxide, whose extracellular levels could be determined by hypoxia and microglial activity, both of which modulate ventrolateral medulla function. Therefore, in this study we aimed to test whether different patterns of hypoxia and/or putative microglial modulators change extracellular hydrogen peroxide in the ventrolateral medulla by using an enzymatic reactor online sensing procedure specifically designed for this purpose. With this new technique, we detected extracellular levels of hydrogen peroxide in the ventrolateral medulla in vitro, which spontaneously fluctuated. These fluctuations are reduced by minocycline, a putative microglial inhibitor, and by the microglial toxin liposomal clodronate. Suitably, lipopolysaccharide increases extracellular hydrogen peroxide, while minocycline and liposomal clodronate reduce this increase. Application of blue light to slices with microglia expressing channelrhodopsin-2 also increases extracellular hydrogen peroxide. Moreover, long-lasting and intermittent hypoxia (as well as subsequent reoxygenation) increase extracellular hydrogen peroxide to similar levels, which is partially prevented by minocycline. The effect of long-lasting hypoxia was reproduced in vivo. Overall, our data show that changes in oxygen concentration, and possibly microglial function, modulate extracellular H2O2 levels in the ventrolateral medulla, which could influence the function of this neural circuit under normal and pathological conditions related to inflammation and/or hypoxia.
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Líquido Extracelular/metabolismo , Peróxido de Hidrógeno/metabolismo , Hipoxia/fisiopatología , Bulbo Raquídeo/citología , Potenciales de Acción/efectos de los fármacos , Potenciales de Acción/genética , Animales , Receptor 1 de Quimiocinas CX3C/genética , Receptor 1 de Quimiocinas CX3C/metabolismo , Channelrhodopsins/genética , Channelrhodopsins/metabolismo , Líquido Extracelular/efectos de los fármacos , Hipoxia/tratamiento farmacológico , Lidocaína/farmacología , Lipopolisacáridos/farmacología , Masculino , Ratones , Ratones Transgénicos , Microglía/efectos de los fármacos , Microglía/fisiología , Minociclina/uso terapéutico , Oxígeno/farmacología , Técnicas de Placa-Clamp , ARN no Traducido/genética , ARN no Traducido/metabolismo , Ratas Wistar , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Tau hyperphosphorylation at several sites, including those close to the microtubule domain region (MDr), is considered a key pathological event in the development of Alzheimer's disease (AD). Recent studies indicate that at the very early stage of this disease, increased phosphorylation in Tau's MDr domain correlates with reduced levels of neuronal excitability. Mechanistically, we show that pyramidal neurons and some parvalbumin-positive interneurons in 1-month-old triple-transgenic AD mice accumulate hyperphosphorylated Tau protein and that this accumulation correlates with changes in theta oscillations in hippocampal neurons. Pyramidal neurons from young triple-transgenic AD mice exhibited less spike accommodation and power increase in subthreshold membrane oscillations. Furthermore, triple-transgenic AD mice challenged with the potassium channel blocker 4-aminopyridine had reduced theta amplitude compared with 4-aminopyridine-treated control mice and, unlike these controls, displayed no seizure-like activity after this challenge. Collectively, our results provide new insights into AD pathogenesis and suggest that increases in Tau phosphorylation at the initial stages of the disease represent neuronal responses that compensate for brain circuit overexcitation.
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Potenciales de Acción , Enfermedad de Alzheimer/patología , Modelos Animales de Enfermedad , Hipocampo/patología , Células Piramidales/patología , Ritmo Teta/fisiología , Proteínas tau/fisiología , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/metabolismo , Animales , Células Cultivadas , Hipocampo/metabolismo , Masculino , Ratones , Ratones Transgénicos , Fosforilación , Células Piramidales/metabolismoRESUMEN
The identification of pathways necessary for retinal pigment epithelium (RPE) function is fundamental to uncover therapies for blindness. Prolactin (PRL) receptors are expressed in the retina, but nothing is known about the role of PRL in RPE. Using the adult RPE 19 (ARPE-19) human cell line and mouse RPE, we identified the presence of PRL receptors and demonstrated that PRL is necessary for RPE cell survival via anti-apoptotic and antioxidant actions. PRL promotes the antioxidant capacity of ARPE-19 cells by reducing glutathione. It also blocks the hydrogen peroxide-induced increase in deacetylase sirtuin 2 (SIRT2) expression, which inhibits the TRPM2-mediated intracellular Ca(2+) rise associated with reduced survival under oxidant conditions. RPE from PRL receptor-null (prlr(-/-)) mice showed increased levels of oxidative stress, Sirt2 expression and apoptosis, effects that were exacerbated in animals with advancing age. These observations identify PRL as a regulator of RPE homeostasis.
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Envejecimiento/fisiología , Prolactina/metabolismo , Epitelio Pigmentado de la Retina/citología , Sirtuina 2/metabolismo , Canales Catiónicos TRPM/metabolismo , Animales , Apoptosis/efectos de los fármacos , Femenino , Glutatión/metabolismo , Humanos , Masculino , Ratones , Prolactina/genética , Receptores de Prolactina/genética , Receptores de Prolactina/metabolismo , Epitelio Pigmentado de la Retina/metabolismo , Sirtuina 2/genética , Canales Catiónicos TRPM/genéticaRESUMEN
Soluble amyloid beta peptide (A ß ) is responsible for the early cognitive dysfunction observed in Alzheimer's disease. Both cholinergically and glutamatergically induced hippocampal theta rhythms are related to learning and memory, spatial navigation, and spatial memory. However, these two types of theta rhythms are not identical; they are associated with different behaviors and can be differentially modulated by diverse experimental conditions. Therefore, in this study, we aimed to investigate whether or not application of soluble A ß alters the two types of theta frequency oscillatory network activity generated in rat hippocampal slices by application of the cholinergic and glutamatergic agonists carbachol or DHPG, respectively. Due to previous evidence that oscillatory activity can be differentially affected by different A ß peptides, we also compared Aß 25-35 and Aß 1-42 for their effects on theta rhythms in vitro at similar concentrations (0.5 to 1.0 µ M). We found that Aß 25-35 reduces, with less potency than Aß 1-42, carbachol-induced population theta oscillatory activity. In contrast, DHPG-induced oscillatory activity was not affected by a high concentration of Aß 25-35 but was reduced by Aß 1-42. Our results support the idea that different amyloid peptides might alter specific cellular mechanisms related to the generation of specific neuronal network activities, instead of exerting a generalized inhibitory effect on neuronal network function.
RESUMEN
Oscillatory activity in the entorhinal cortex has been associated with several cognitive functions. Accordingly, Alzheimer Disease-associated cognitive decline has been related to amyloid beta-induced disturbances in several of these oscillatory patterns. We have previously shown that acute application of amyloid beta inhibits the generation of slow frequency oscillations (7-20 Hz). In contrast, alterations in faster oscillations recorded in Alzheimer Disease-transgenic mice that over-express amyloid beta have been controversial. Since transgenic mice may produce complex responses due to compensatory mechanisms, we tested the effect of acute application of amyloid beta on fast oscillations (beta-gamma bursts) generated by entorhinal cortex slices in vitro in a Mg2+ -ree solution. We also explored the participation of the enzyme glycogen synthase kinase 3 (GSK-3) in this effect. Our results show that bath application of a clinically relevant concentration of amyloid beta (10 nM) activates GSK-3 and reduces the power of beta-gamma bursts in the entorhinal cortex. The reduction of beta-gamma bursts by amyloid beta is blocked by inhibiting GSK-3 either with lithium or with SB 216763. Our results suggest that amyloid beta-induced inhibition of entorhinal cortex beta-gamma activity involves GSK-3 activation, which may provide a molecular mechanism for amyloid beta-induced neural network disruption and support the use of GSK-3 inhibitors to treat Alzheimer Disease.
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Péptidos beta-Amiloides/farmacología , Corteza Entorrinal/efectos de los fármacos , Glucógeno Sintasa Quinasa 3/metabolismo , Neuronas/efectos de los fármacos , Fragmentos de Péptidos/farmacología , Potenciales Sinápticos/efectos de los fármacos , Animales , Corteza Entorrinal/fisiología , Neuronas/fisiología , Fosforilación/efectos de los fármacos , Ratas , Ratas Wistar , Potenciales Sinápticos/fisiologíaRESUMEN
Breathing and the activity of its generator (the pre-Bötzinger complex; pre-BötC) are highly regulated functions. Among neuromodulators of breathing, somatostatin (SST) is unique: it is synthesized by a subset of glutamatergic pre-BötC neurons, but acts as an inhibitory neuromodulator. Moreover, SST regulates breathing both in normoxic and in hypoxic conditions. Although it has been implicated in the neuromodulation of breathing, neither the locus of SST modulation, nor the receptor subtypes involved have been identified. In this study, we aimed to fill in these blanks by characterizing the SST-induced regulation of inspiratory rhythm generation in vitro and in vivo. We found that both endogenous and exogenous SST depress all preBötC-generated rhythms. While SST abolishes sighs, it also decreases the frequency and increases the regularity of eupnea and gasping. Pharmacological experiments showed that SST modulates inspiratory rhythm generation by activating SST receptor type-2, whose mRNA is abundantly expressed in the pre-Bötzinger complex. In vivo, blockade of SST receptor type-2 reduces gasping amplitude and consequently, it precludes auto-resuscitation after asphyxia. Based on our findings, we suggest that SST functions as an inhibitory neuromodulator released by excitatory respiratory neurons when they become overactivated in order to stabilize breathing rhythmicity in normoxic and hypoxic conditions.
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Asfixia/fisiopatología , Inhalación/efectos de los fármacos , Neurotransmisores/farmacología , Centro Respiratorio/efectos de los fármacos , Somatostatina/farmacología , Animales , Asfixia/metabolismo , Asfixia/mortalidad , Fenómenos Electrofisiológicos , Hipoxia/metabolismo , Hipoxia/mortalidad , Hipoxia/fisiopatología , Inhalación/fisiología , Ratones , Neuronas/efectos de los fármacos , Neuronas/patología , Neuronas/fisiología , Neurotransmisores/fisiología , Periodicidad , Pletismografía , Receptores de Somatostatina/agonistas , Receptores de Somatostatina/genética , Centro Respiratorio/fisiología , Somatostatina/fisiología , Tasa de Supervivencia , Regulación hacia ArribaRESUMEN
Alzheimer disease (AD) patients show alterations in both neuronal network oscillations and the cognitive processes associated to them. Related to this clinical observation, it has been found that amyloid beta protein (Abeta) differentially affects some hippocampal network activities, reducing theta and gamma oscillations, without affecting sharp waves and ripples. Beta-like oscillations is another cognitive-related network activity that can be evoked in hippocampal slices by several experimental manipulations, including bath application of kainate and increasing extracellular potassium. Here, we tested whether or not different Abeta peptides differentially affect beta-like oscillatory patterns. We specifically tested the effects of fresh dissolved Abeta(25-35) and oligomerized Abeta(1-42) and found that kainate-induced oscillatory network activity was affected, in a slightly concentration dependent-manner, by both fresh dissolved (mostly monomeric) Abeta(25-35) and oligomeric Abeta(1-42). In contrast, potassium-induced oscillatory activity, which is reduced by oligomeric Abeta(1-42), is not affected by monomeric Abeta(25-35) at any of the concentrations tested. Our results support the idea that different amyloid peptides might alter specific cellular mechanisms related to the generation of specific neuronal network activities, instead of a generalized inhibitory effect of Abeta peptides on neuronal network function.
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Péptidos beta-Amiloides/metabolismo , Hipocampo/fisiopatología , Red Nerviosa/fisiopatología , Transmisión Sináptica/efectos de los fármacos , Enfermedad de Alzheimer/fisiopatología , Animales , Técnicas Electroquímicas , Agonistas de Aminoácidos Excitadores/farmacología , Hipocampo/efectos de los fármacos , Humanos , Técnicas In Vitro , Ácido Kaínico/farmacología , Red Nerviosa/efectos de los fármacos , Concentración Osmolar , Fragmentos de Péptidos/metabolismo , Perfusión , Potasio/farmacología , Ratas , Ratas WistarRESUMEN
Early cognitive deficit characteristic of early Alzheimer's disease seems to be produced by the soluble forms of beta-amyloid protein. Such cognitive deficit correlates with neuronal network dysfunction that is reflected as alterations in the electroencephalogram of both Alzheimer patients and transgenic murine models of such disease. Correspondingly, recent studies have demonstrated that chronic exposure to betaAP affects hippocampal oscillatory properties. However, it is still unclear if such neuronal network dysfunction results from a direct action of betaAP on the hippocampal circuit or it is secondary to the chronic presence of the protein in the brain. Therefore, we aimed to explore the effect of acute exposure to betaAP(25-35) on hippocampal network activity both in vitro and in vivo, as well as on intrinsic and synaptic properties of hippocampal neurons. We found that betaAP(25-35), reversibly, affects spontaneous hippocampal population activity in vitro. Such effect is not produced by the inverse sequence betaAP(35-25) and is reproduced by the full-length peptide betaAP(1-42). Correspondingly betaAP(25-35), but not the inverse sequence betaAP(35-25), reduces theta-like activity recorded from the hippocampus in vivo. The betaAP(25-35)-induced disruption in hippocampal network activity correlates with a reduction in spontaneous neuronal activity and synaptic transmission, as well as with an inhibition in the subthreshold oscillations produced by pyramidal neurons in vitro. Finally, we studied the involvement of Fyn-kinase on the betaAP(25-35)-induced disruption in hippocampal network activity in vitro. Interestingly, we found that such phenomenon is not observed in slices obtained from Fyn-knockout mice. In conclusion, our data suggest that betaAP acutely affects proper hippocampal function through a Fyn-dependent mechanism. We propose that such alteration might be related to the cognitive impairment observed, at least, during the early phases of Alzheimer's disease.
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Péptidos beta-Amiloides/metabolismo , Hipocampo/fisiología , Fragmentos de Péptidos/metabolismo , Proteínas Proto-Oncogénicas c-fyn/metabolismo , Potenciales de Acción/fisiología , Animales , Región CA1 Hipocampal/enzimología , Región CA1 Hipocampal/fisiología , Potenciales Evocados/fisiología , Hipocampo/enzimología , Técnicas In Vitro , Ratones , Ratones Noqueados , Vías Nerviosas/enzimología , Vías Nerviosas/fisiología , Neuroglía/enzimología , Neuroglía/fisiología , Neuronas/enzimología , Neuronas/fisiología , Periodicidad , Proteínas Proto-Oncogénicas c-fyn/genética , Células Piramidales/enzimología , Células Piramidales/fisiología , Ratas , Ratas Wistar , Transmisión Sináptica/fisiologíaRESUMEN
Non-selective cationic channels (NSCC) are a heterogeneous family of channels, widely expressed in non-excitable and excitable cells, that share several functional characteristics but have diverse molecular origin. NSCC can be formed by transient receptor potential (TRP) channels, calcium activated non-selective channels, hyperpolarization activated cation currents, acid-sensitive cationic channels (ASIC), etc. As a result of its wide expression, as well as to the fact that the activation of such currents produce a persistent membrane depolarization, NSCC have been involved in a variety of neuronal processes such as signal transduction, firing pattern (including plateau potentials and bursting mechanisms) as well as synaptic transmission. Due to the relevance of such channels, alterations in their normal function have been associated with the pathophysiology of several nervous system diseases. Over the last years several blockers of such channels have been discovered. Here we review the pharmacology of NSCC blockers including trivalent cations, verapamil derivates, flufenamic acid, the "typical" TRP blockers 2-APB, ACA and SKF 96365 as well as ASIC blockers. This review focuses on the pharmacological properties of such drugs and their potential use for the understanding of the nervous system as well as for the treatment of neurological diseases.
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Canales Iónicos/antagonistas & inhibidores , Sistema Nervioso/efectos de los fármacos , Ácidos/metabolismo , Animales , Cationes , Canales Iónicos/metabolismo , Sistema Nervioso/metabolismo , Sensibilidad y EspecificidadRESUMEN
TRPC5 forms Ca2+-permeable nonselective cation channels important for neurite outgrowth and growth cone morphology of hippocampal neurons. Here we studied the activation of mouse TRPC5 expressed in Chinese hamster ovary and human embryonic kidney 293 cells by agonist stimulation of several receptors that couple to the phosphoinositide signaling cascade and the role of calmodulin (CaM) on the activation. We showed that exogenous application of 10 microM CaM through patch pipette accelerated the agonist-induced channel activation by 2.8-fold, with the time constant for half-activation reduced from 4.25 +/- 0.4 to 1.56 +/- 0.85 min. We identified a novel CaM-binding site located at the C terminus of TRPC5, 95 amino acids downstream from the previously determined common CaM/IP3R-binding (CIRB) domain for all TRPC proteins. Deletion of the novel CaM-binding site attenuated the acceleration in channel activation induced by CaM. However, disruption of the CIRB domain from TRPC5 rendered the channel irresponsive to agonist stimulation without affecting the cell surface expression of the channel protein. Furthermore, we showed that high (>5 microM) intracellular free Ca2+ inhibited the current density without affecting the time course of TRPC5 activation by receptor agonists. These results demonstrated that intracellular Ca2+ has dual and opposite effects on the activation of TRPC5. The novel CaM-binding site is important for the Ca2+/CaM-mediated facilitation, whereas the CIRB domain is critical for the overall response of receptor-induced TRPC5 channel activation.
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Canales de Calcio/metabolismo , Calcio/metabolismo , Calmodulina/metabolismo , Proteínas de Transporte de Catión/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión , Células CHO , Canales de Calcio/genética , Proteínas de Transporte de Catión/agonistas , Proteínas de Transporte de Catión/genética , Membrana Celular/metabolismo , Cricetinae , Humanos , Ratones , Datos de Secuencia Molecular , Unión Proteica , Estructura Terciaria de Proteína , Sistemas de Mensajero Secundario/fisiología , Canales Catiónicos TRPCRESUMEN
Exposure of cultured cerebellar granule neurons (24 h serum-starved) during 3 min to 30% hyposmotic medium activated the tyrosine kinase receptor ErbB4 in the absence of its ligand. Hyposmolarity also activated the non-receptor tyrosine kinases, Src, focal adhesion kinase (FAK), extracellular signal-regulated protein kinase (ERK)1/2, and the tyrosine kinase target phosphatidyl-inositol-3-kinase (PI3K). The hyposmotic-induced activation of these kinases required the prior phosphorylation of ErbB4 as shown by the effect of ErbB4 blockade with AG213 reducing by 85-95% the phosphorylation of FAK and ERK1/2, by 74% and 36% that of PI3K and Src, respectively. These results suggest a key role of ErbB4 as a signal integrator of events associated with hyposmolarity. PI3K seems to be an important connecting element in the signaling network evoked by the hyposmolarity/ErbB4 activation as: (i) the p85 regulatory subunit of PI3K co-immunoprecipitates with ErbB4 and with FAK; (ii) PI3K blockade with wortmannin reduced the hyposmotic activation of FAK (90%) and ERK1/2 (84-91%). Inhibition of Src with PP2 reduced ErbB4 phosphorylation and inhibited the subsequent cytosolic kinase activation with the same potency as ErbB4 blockade. These results point to Src and ErbB4 and as early targets of the hyposmotic stimulus and osmosignaling. The functional significance for cell volume regulation of the ErbB4-Src-PI3K signaling cascade is indicated by the 48-66% decrease of the hyposmotic taurine efflux observed by inhibition of these kinases.
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Cerebelo/metabolismo , Receptores ErbB/metabolismo , Neuronas/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Animales , Células Cultivadas , Cerebelo/citología , Cerebelo/enzimología , Medios de Cultivo/farmacología , Activación Enzimática , Quinasa 1 de Adhesión Focal , Proteína-Tirosina Quinasas de Adhesión Focal , Adhesiones Focales/fisiología , Concentración Osmolar , Fosfatidilinositol 3-Quinasas/metabolismo , Fosforilación/efectos de los fármacos , Ratas , Receptor ErbB-4 , Taurina/metabolismo , Factores de Tiempo , Familia-src Quinasas/metabolismoRESUMEN
A decrease in external osmolarity results in cell swelling and the immediate activation of a mechanism to restore cell volume, known as regulatory volume decrease (RVD). When exposed to a gradual osmolarity decrease (GODE), some cells do not swell. This reflects the operation of an active regulatory process known as isovolumetric regulation (IVR). The mechanisms underlying IVR appear similar to those activated during RVD, namely the extrusion of K+, Cl-, amino acids, and other organic molecules. A previous study has documented IVR in cerebellar granule neurons, parallel to an early efflux of taurine and Cl-, whereas K+ efflux is delayed. In this work we briefly review the importance of amino acids in the mechanisms of cell volume control in the brain, with emphasis on IVR. We also present experiments showing the response to GODE in cerebellar astrocytes. The currents activated during GODE, recorded in the whole-cell configuration of the patch clamp technique, indicate the early activation of an anion current, followed by a more delayed cation current. A correlation between the time course of amino acid efflux during GODE and the occurrence or not of IVR in various cell types, suggest the importance of these osmolytes in the volume regulatory process in this model.
Asunto(s)
Tamaño de la Célula , Concentración Osmolar , Transducción de SeñalRESUMEN
Exposure of cultured Swiss 3T3 fibroblasts to 35% hyposmotic solution activated epidermal growth factor receptor (EGFR) phosphorylation to a greater extent than the ligand, EGF. Concanavalin A (Con A) and wheat-germ agglutinin (WGA) had the same effect. EGFR phosphorylation seems to be involved in the transduction signalling for hyposmotically induced taurine release, as suggested by the latter's reduction when EGFR phosphorylation was blocked by 50 microM AG213 or AG112 and, conversely, its potentiation by EGF (200 ng/ml). The relationship between hyposmotically induced taurine efflux and reduced osmolarity showed saturable kinetics, following a sigmoidal function. EGF shifted the relationship to the left, implying an increase in sensitivity to hyposmolarity. EGF increased taurine efflux only marginally under isosmotic conditions. EGF and agglutinins also potentiated the hyposmotically induced release of 86Rb but, in contrast to taurine, the efflux was unaffected by EGFR inhibition. EGF and agglutinins markedly increased 86Rb release under isosmotic conditions. The EGF-evoked isosmotic 86Rb release, together with the hyposmotic efflux, accounted fully for the observed potentiation by EGF, raising the possibility of an overlapping of these two effects, rather than a true potentiation. A link between EGFR, phosphatidylinositide-3-kinase (PI3K) and hyposmotically induced taurine (but not 86Rb) release is suggested by the increase in PI3K activity elicited by hyposmolarity, which was fully prevented by EGFR inhibition, and by a marked reduction of hyposmotically induced taurine (but not 86Rb) release, by wortmannin. The present findings, together with results showing EGF activation of osmosensitive Cl- fluxes implicate EGFR as an important modulator of osmolyte efflux pathways.
Asunto(s)
Receptores ErbB/metabolismo , Fibroblastos/metabolismo , Transducción de Señal/fisiología , Animales , Relación Dosis-Respuesta a Droga , Factor de Crecimiento Epidérmico/metabolismo , Factor de Crecimiento Epidérmico/farmacología , Receptores ErbB/fisiología , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Ratones , Concentración Osmolar , Fosforilación/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Células 3T3 SwissRESUMEN
G protein-coupled inwardly rectifying K(+) channels (GIRK or Kir3) form functional heterotetramers gated by Gbetagamma subunits. GIRK channels are critical for functions as diverse as heart rate modulation and neuronal post-synaptic inhibition. GIRK5 (Kir3.5) is the oocyte homologue of the mammalian GIRK subunits that conform the K(ACh) channel. It has been claimed that even when the oocytes express GIRK5 proteins they do not form functional channels. However, the GIRK5 gene shows three initiation sites that suggest the existence of three isoforms. In a previous work we demonstrated the functionality of homomultimers of the shortest isoform overexpressed in the own oocytes. Remarkably, the basal GIRK5-Delta25 inward currents were not coupled to the activation of a G-protein receptor in the oocytes. These results encouraged us to study this channel in another expression system. In this work we show that Sf21 insect cells can be successfully transfected with this channel. GIRK5-Delta25 homomultimers produce time-dependent inward currents only with GTPgammaS in the recording pipette. Therefore, alternative modes of stimulus input to heterotrimeric G-proteins should be present in the oocytes to account for these results.