Participación de rafts en enfermedades neurológicas / Role of rafts in neurological disorders

Introducción: Los rafts constituyen nano-dominios estructurales de naturaleza lipoproteica que propician la eficiente transducción de señales y la modulación de procesos fisiológicos asociados a la membrana plasmática. En el sistema nervioso, la alteración de estos dominios se ha asociado con el desarrollo de diversos padecimientos. Desarrollo: En el presente artículo se revisa el concepto de rafts, los procesos del sistema nervioso en los cuales están involucrados y su papel en distintas afectaciones, entre las que se destacan las enfermedades de Parkinson, Alzheimer y Huntington. Conclusiones: Dadas las evidencias de su participación en diversas neuropatologías, la preservación y/o reconstitución de los rafts se vislumbran como una atractiva estrategia terapéutica.(AU)

Introduction: Rafts are function-structural cell membrane nano-domains. They contribute to explain the efficiency of signal transduction at the low physiological membrane concentrations of the signaling partners by their clustering inside specialized signaling domains. Development: In this article, we review the current model of the membrane rafts and their physio-pathological relevance in the nervous system, including their role in Parkinson, Alzheimer, and Huntington diseases. Conclusions: Rafts disruption/dysfunction has been shown to relate diverse neurological diseases. Therefore, it has been suggested that preservation of membrane rafts may represent a strategy to prevent or delay neuronal dysfunctions in several diseases.(AU)

Role of rafts in neurological disorders.

INTRODUCTION: Rafts are protein-lipid structural nanodomains involved in efficient signal transduction and the modulation of physiological processes of the cell plasma membrane. Raft disruption in the nervous system has been associated with a wide range of disorders. DEVELOPMENT: We review the concept of rafts, the nervous system processes in which they are involved, and their role in diseases such as Parkinson's disease, Alzheimer disease, and Huntington disease. CONCLUSIONS: Based on the available evidence, preservation and/or reconstitution of rafts is a promising treatment strategy for a wide range of neurological disorders.

Role of rafts in neurological disorders. / Participación de rafts en enfermedades neurológicas.

INTRODUCTION: Rafts are function-structural cell membrane nano-domains. They contribute to explain the efficiency of signal transduction at the low physiological membrane concentrations of the signaling partners by their clustering inside specialized signaling domains. DEVELOPMENT: In this article, we review the current model of the membrane rafts and their physio-pathological relevance in the nervous system, including their role in Parkinson, Alzheimer, and Huntington diseases. CONCLUSIONS: Rafts disruption/dysfunction has been shown to relate diverse neurological diseases. Therefore, it has been suggested that preservation of membrane rafts may represent a strategy to prevent or delay neuronal dysfunctions in several diseases.

Molecular mechanisms and physiological relevance of RGK proteins in the heart.

The primary route of Ca2+ entry into cardiac myocytes is via 1,4-dihydropyridine-sensitive, voltage-gated L-type Ca2+ channels. Ca2+ influx through these channels influences duration of action potential and engages excitation-contraction (EC) coupling in both the atria and the myocardium. Members of the RGK (Rad, Rem, Rem2 and Gem/Kir) family of small GTP-binding proteins are potent, endogenously expressed inhibitors of cardiac L-type channels. Although much work has focused on the molecular mechanisms by which RGK proteins inhibit the CaV 1.2 and CaV 1.3 L-type channel isoforms that expressed in the heart, their impact on greater cardiac function is only beginning to come into focus. In this review, we summarize recent findings regarding the influence of RGK proteins on normal cardiac physiology and the pathological consequences of aberrant RGK activity.

Significados construidos y parálisis cerebral: una mirada cualitativa a la comprensión de la condición de salud de un grupo de adolescentes / Constructed meaning and cerebral palsy: a qualitative look at how they understand their health condition, with a group of adolescents

Introduction: Cerebral Palsy (CP) is the most prevalent diagnosis in Teleton Institutes in Chile. The syndrome is characterized by a non progressive brain lesion that causes an impact in different areas of development. It is our interest to study how adolescents with CP symbolize this experience, given that identity formation and self representation are key aspects during this developmental stage. Objective: To describe meanings build in mild hemiplegic CP, from 12 to 16 years of age, attending Teleton Institute Temuco. Subjects and Method: Qualitative study, exploratory descriptive type, phenomenological. Data were gathered through semi-structured interviews applied to 10 adolescents, and a focus group with 4 of these, both experiences included patients from 12 to 16 years with mild hemiplegic CP and intellectual capacity to answer medium complexity questions. Results: Adolescents describe hemiplegia as a condition characterized by physical differences, as a disability and a challenge, they recognize its origins and the beliefs they perceive in others. It is not considered an illness and as implications they count experiences, needs and problems that are part of this condition, however, they are able to reformulate this experience, holding a positive and resilient view towards their condition. Conclusion: Adolescents express similitudes between the concept of hemiplegia and disability; they point it as a complex experience that brings negative consequences as well as learning experiences. They are finally able to reformulate it and experience it as a challenge.

Introducción: La parálisis cerebral (PC) es el diagnóstico con mayor incidencia en los Institutos Teletón (IT) del país. Este síndrome está determinado por una lesión cerebral de carácter no progresivo, que puede tener implicancias en los distintos ámbitos del desarrollo. El cómo simbolizan esta experiencia adolescentes con PC tipo hemiparesia, es de nuestro interés, considerando que la formación de la identidad y la representación de sí mismos en esta etapa son claves. Objetivo: Describir los significados construidos en torno a la PC tipo hemiparesia leve, de adolescentes de 12 a 16 años de ITTemuco. Usuarios y Método: Investigación cualitativa de tipo exploratorio descriptivo, fenomenológica. Recolección de datos a través de entrevista semi estructurada aplicada a 10 usuarios y un grupo focal realizado con 4 participantes, en ambas instancias con la participación de jóvenes de 12 a 16 años con PC de tipo hemiparesia leve y capacidad cognitiva para responder a preguntas de mediana complejidad. Resultados: Los jóvenes definen la hemiparesia como una condición caracterizada por diferencias físicas, como una discapacidad y un desafío, reconocen sus orígenes y las creencias que perciben en otros. No es considerada como enfermedad y como implicancias indican vivencias, experiencias, necesidades y problemas que conlleva esta condición, no obstante, logran re-significar esta experiencia manteniendo una mirada positiva y resiliente en relación a su condición. Conclusión: Los adolescentes expresan similitudes entre el concepto de hemiparesia y discapacidad, las significan como una experiencia compleja que trae consecuencias negativas así como aprendizajes, logrando re-significarla y vivenciarla finalmente como un desafío.

Differential effects of RGK proteins on L-type channel function in adult mouse skeletal muscle.

Work in heterologous systems has revealed that members of the Rad, Rem, Rem2, Gem/Kir (RGK) family of small GTP-binding proteins profoundly inhibit L-type Ca(2+) channels via three mechanisms: 1), reduction of membrane expression; 2), immobilization of the voltage-sensors; and 3), reduction of Po without impaired voltage-sensor movement. However, the question of which mode is the critical one for inhibition of L-type channels in their native environments persists. To address this conundrum in skeletal muscle, we overexpressed Rad and Rem in flexor digitorum brevis (FDB) fibers via in vivo electroporation and examined the abilities of these two RGK isoforms to modulate the L-type Ca(2+) channel (CaV1.1). We found that Rad and Rem both potently inhibit L-type current in FDB fibers. However, intramembrane charge movement was only reduced in fibers transfected with Rad; charge movement for Rem-expressing fibers was virtually identical to charge movement observed in naïve fibers. This result indicated that Rem supports inhibition solely through a mechanism that allows for translocation of CaV1.1's voltage-sensors, whereas Rad utilizes at least one mode that limits voltage-sensor movement. Because Rad and Rem differ significantly only in their amino-termini, we constructed Rad-Rem chimeras to probe the structural basis for the distinct specificities of Rad- and Rem-mediated inhibition. Using this approach, a chimera composed of the amino-terminus of Rem and the core/carboxyl-terminus of Rad inhibited L-type current without reducing charge movement. Conversely, a chimera having the amino-terminus of Rad fused to the core/carboxyl-terminus of Rem inhibited L-type current with a concurrent reduction in charge movement. Thus, we have identified the amino-termini of Rad and Rem as the structural elements dictating the specific modes of inhibition of CaV1.1.

Potent inhibition of L-type Ca2+ currents by a Rad variant associated with congestive heart failure.

Ca(2+) influx via L-type voltage-gated Ca(2+) channels supports the plateau phase of ventricular action potentials and is the trigger for excitation-contraction (EC) coupling in the myocardium. Rad, a member of the RGK (Rem, Rem2, Rad, Gem/Kir) family of monomeric G proteins, regulates ventricular action potential duration and EC coupling gain through its ability to inhibit cardiac L-type channel activity. In this study, we have investigated the potential dysfunction of a naturally occurring Rad variant (Q66P) that has been associated with congestive heart failure in humans. Specifically, we have tested whether Rad Q66P limits, or even eliminates, the inhibitory actions of Rad on CaV1.2 and CaV1.3, the two L-type channel isoforms known to be expressed in the heart. We have found that mouse Rad Q65P (the murine equivalent of human Rad Q66P) inhibits L-type currents conducted by CaV1.2 or CaV1.3 channels as potently as wild-type Rad (>95% inhibition of both channels). In addition, Rad Q65P attenuates the gating movement of both channels as effectively as wild-type Rad, indicating that the Q65P substitution does not differentially impair any of the three described modes of L-type channel inhibition by RGK proteins. Thus, we conclude that if Rad Q66P contributes to cardiomyopathy, it does so via a mechanism that is not related to its ability to inhibit L-type channel-dependent processes per se. However, our results do not rule out the possibility that decreased expression, mistargeting or altered regulation of Rad Q66P may reduce the RGK protein's efficacy in vivo.

RGS2 blocks slow muscarinic inhibition of N-type Ca(2+) channels reconstituted in a human cell line.

1. Native N-type Ca(2+) channels undergo sustained inhibition through a slowly activating pathway linked to M1 muscarinic acetylcholine receptors and Galphaq/11 proteins. Little is known concerning the regulation of this slow inhibitory pathway. We have reconstituted slow muscarinic inhibition of N-type channels in HEK293 cells (a human embryonic kidney cell line) by coexpressing cloned alpha1B (Ca(V)2.2) Ca(2+) channel subunits and M1 receptors. Expressed Ca(2+) currents were recorded using standard whole-cell, ruptured-patch techniques. 2. Rapid application of carbachol produced two kinetically distinct components of Ca(2+) channel inhibition. The fast component of inhibition had a time constant of < 1 s, whereas the slow component had a time constant of 5-40 s. Neither component of inhibition was reduced by pertussis toxin (PTX) or staurosporine. 3. The fast component of inhibition was selectively blocked by the Gbetagamma-binding region of beta-adrenergic receptor kinase 1, suggesting that fast inhibition is mediated by Gbetagamma released from Galphaq/11. 4. The slow component of inhibition was selectively blocked by regulator of G protein signalling 2 (RGS2), which preferentially interacts with Galphaq/11 proteins. RGS2 also attenuated channel inhibition produced by intracellular dialysis with non-hydrolysable GTPgammaS. Together these results suggest that RGS2 selectively blocked slow inhibition by functioning as an effector antagonist, rather than as a GTPase-accelerating protein (GAP). 5. These experiments demonstrate that slow muscarinic inhibition of N-type Ca(2+) channels can be reconstituted in non-neuronal cells, and that RGS2 can selectively block slow muscarinic inhibition while leaving fast muscarinic inhibition intact. These results identify RGS2 as a potential physiological regulator of the slow muscarinic pathway.

Muscarinic stimulation of alpha1E Ca channels is selectively blocked by the effector antagonist function of RGS2 and phospholipase C-beta1.

Neuronal alpha1E Ca channel subunits are widely expressed in mammalian brain, where they are thought to form R-type Ca channels. Recent studies have demonstrated that R-type channels contribute to neurosecretion and dendritic Ca influx, but little is known concerning their modulation. Here we show that alpha1E channels are strongly stimulated, and only weakly inhibited, through M1 muscarinic acetylcholine receptors. Both forms of channel modulation are mediated by pertussis toxin-insensitive G-proteins. Channel stimulation is blocked by regulator of G-protein signaling 2 (RGS2) or the C-terminal region of phospholipase C-beta1 (PLCbeta1ct), which have been previously shown to function as GTPase-activating proteins for Galphaq. In contrast, RGS2 and PLCbeta1ct do not block inhibition of alpha1E through M1 receptors. Inhibition is prevented, however, by the C-terminal region of beta-adrenergic receptor kinase 1, which sequesters Gbetagamma dimers. Thus, stimulation of alpha1E is mediated by a pertussis toxin-insensitive Galpha subunit (e.g., Galphaq), whereas inhibition is mediated by Gbetagamma. The ability of RGS2 and PLCbeta1ct to selectively block stimulation indicates these proteins functioned primarily as effector antagonists. In support of this interpretation, RGS2 prevented stimulation of alpha1E with non-hydrolyzable guanosine 5'-0-(3-thiotriphosphate). We also report strong muscarinic stimulation of rbE-II, a variant alpha1E Ca channel that is insensitive to voltage-dependent inhibition. Our results predict that Galphaq-coupled receptors predominantly stimulate native R-type Ca channels. Receptor-mediated enhancement of R-type Ca currents may have important consequences for neurosecretion, dendritic excitability, gene expression, or other neuronal functions.

Biphasic, opposing modulation of cloned neuronal alpha1E Ca channels by distinct signaling pathways coupled to M2 muscarinic acetylcholine receptors.

Neuronal alpha1E subunits are thought to form R-type Ca channels. When expressed in human embryonic kidney cells with M2 muscarinic acetylcholine receptors, Ca channels encoded by rabbit alpha1E exhibit striking biphasic modulation. Receptor activation first produces rapid inhibition of current amplitude and activation rate. However, in the continued presence of agonist, alpha1E currents subsequently increase. Kinetic slowing persists during this secondary stimulation phase. After receptor deactivation, kinetic slowing is quickly relieved, and current amplitude over-recovers before returning toward control levels. These features indicate that inhibition and stimulation of alpha1E are separate processes, with stimulation superimposed on inhibition. Pertussis toxin eliminates inhibition without affecting stimulation, demonstrating that inhibition and stimulation involve distinct signaling pathways. Neither inhibition nor stimulation is altered by coexpression of Ca channel beta2a or beta3 subunits. Stimulation is abolished by staurosporine and reduced by intracellular 5'-adenylylimidodiphosphate, suggesting that phosphorylation is required. However, stimulation does not seem to involve cAMP-dependent protein kinase, protein kinase C, cGMP-dependent protein kinase, tyrosine kinases, or phosphoinositide 3-kinases. Stimulation does not require a Ca signal, because it is not specifically altered by varying intracellular Ca buffering or by substituting Ba as the charge carrier. In contrast to those formed by alpha1E, Ca channels formed by alpha1A or alpha1B display only inhibition and no stimulation during prolonged activation of M2 receptors. The dual modulation of alpha1E may confer unique physiological properties on native R-type Ca channels. As one possibility, R-type channels may continue to mediate Ca influx during steady inhibition of N-type and P/Q-type channels by muscarinic or other receptors.

Regulators of G protein signaling attenuate the G protein-mediated inhibition of N-type Ca channels.

Regulators of G protein signaling (RGS) proteins bind to the alpha subunits of certain heterotrimeric G proteins and greatly enhance their rate of GTP hydrolysis, thereby determining the time course of interactions among Galpha, Gbetagamma, and their effectors. Voltage-gated N-type Ca channels mediate neurosecretion, and these Ca channels are powerfully inhibited by G proteins. To determine whether RGS proteins could influence Ca channel function, we recorded the activity of N-type Ca channels coexpressed in human embryonic kidney (HEK293) cells with G protein-coupled muscarinic (m2) receptors and various RGS proteins. Coexpression of full-length RGS3T, RGS3, or RGS8 significantly attenuated the magnitude of receptor-mediated Ca channel inhibition. In control cells expressing alpha1B, alpha2, and beta3 Ca channel subunits and m2 receptors, carbachol (1 microM) inhibited whole-cell currents by approximately 80% compared with only approximately 55% inhibition in cells also expressing exogenous RGS protein. A similar effect was produced by expression of the conserved core domain of RGS8. The attenuation of Ca current inhibition resulted primarily from a shift in the steady state dose-response relationship to higher agonist concentrations, with the EC50 for carbachol inhibition being approximately 18 nM in control cells vs. approximately 150 nM in RGS-expressing cells. The kinetics of Ca channel inhibition were also modified by RGS. Thus, in cells expressing RGS3T, the decay of prepulse facilitation was slower, and recovery of Ca channels from inhibition after agonist removal was faster than in control cells. The effects of RGS proteins on Ca channel modulation can be explained by their ability to act as GTPase-accelerating proteins for some Galpha subunits. These results suggest that RGS proteins may play important roles in shaping the magnitude and kinetics of physiological events, such as neurosecretion, that involve G protein-modulated Ca channels.

G-Protein-dependent facilitation of neuronal alpha1A, alpha1B, and alpha1E Ca channels.

Modulation of neuronal voltage-gated Ca channels has important implications for synaptic function. To investigate the mechanisms of Ca channel modulation, we compared the G-protein-dependent facilitation of three neuronal Ca channels. alpha1A, alpha1B, or alpha1E subunits were transiently coexpressed with alpha2-deltab and beta3 subunits in HEK293 cells, and whole-cell currents were recorded. After intracellular dialysis with GTPgammaS, strongly depolarized conditioning pulses facilitated currents mediated by each Ca channel type. The magnitude of facilitation depended on current density, with low-density currents being most strongly facilitated and high-density currents often lacking facilitation. Facilitating depolarizations speeded channel activation approximately 1.7-fold for alpha1A and alpha1B and increased current amplitudes by the same proportion, demonstrating equivalent facilitation of G-protein-inhibited alpha1A and alpha1B channels. Inactivation typically obscured facilitation of alpha1E current amplitudes, but the activation kinetics of alpha1E currents showed consistent and pronounced G-protein-dependent facilitation. The onset and decay of facilitation had the same kinetics for alpha1A, alpha1B, and alpha1E, suggesting that Gbeta gamma dimers dissociate from and reassociate with these Ca channels at very similar rates. To investigate the structural basis for N-type Ca channel modulation, we expressed a mutant of alpha1B missing large segments of the II-III loop and C terminus. This deletion mutant exhibited undiminished G-protein-dependent facilitation, demonstrating that a Gbeta gamma interaction site recently identified within the C terminus of alpha1E is not required for modulation of alpha1B.

Induction of classical lactotropes by epidermal growth factor in rat pituitary cell cultures.

Long term incubation of pituitary tumor GH3 cultures with epidermal growth factor (EGF) induces reciprocal changes in PRL and GH production. However, it is not known whether EGF alters the cellular composition of these cultures. Another unanswered question is whether chronic treatment with EGF stimulates PRL secretion from nonneoplastic pituitary cells. In this study, GH3 cells and pituitary cells from neonatal (10-day-old) rats were cultured for 6 and 2 days, respectively, in the absence or presence of 5 nM EGF. Cells containing PRL and/or GH were then enumerated using light microscopic immunocytochemistry. In addition, neonatal pituitary cells were subjected to reverse hemolytic plaque assays for PRL. EGF treatment drastically increased the proportion of classical lactotropes (cells that secrete only PRL) in the GH3 cultures, from about 0.5% to 8% of all cells, without modifying the percentage of GH-positive cells. A similar action of EGF was observed in the primary cultures. Moreover, EGF enhanced by 240% the amount of PRL secreted from the neonatal lactotrope population during 1-h incubations under basal conditions. This effect was mediated by a selective increase in the relative number of PRL secretors forming large plaques. The results suggest that EGF promotes the differentiation of classical lactotropes in both GH3 cultures and pituitary cultures from neonatal rats, and that these cells are characterized by a high basal rate of PRL secretion.

Long-term regulation of calcium channels in clonal pituitary cells by epidermal growth factor, insulin, and glucocorticoids.

In rat pituitary GH3 cells, epidermal growth factor (EGF) and insulin stimulate prolactin production, whereas glucocorticoids exert the opposite effect. In the present study, GH3 cells were subjected to whole-cell patch clamp to assess the chronic actions of such regulatory factors on voltage-dependent calcium currents. Before the electrical recording, cells were grown 5-6 d either under standard conditions or in the presence of 5 nM EGF, 100 nM insulin, 1 microM dexamethasone or 5 microM cortisol. EGF induced a twofold selective increase in high-threshold calcium current density. Insulin and glucocorticoids, on the other hand, specifically regulated low-threshold Ca channels. Current density through these channels increased by 70% in insulin-treated cells, and decreased by 50% in cells exposed to dexamethasone or cortisol. Other Ca channel properties investigated (conductance-voltage curves, deactivation rates, time course and voltage dependence of low-threshold current inactivation) were unaffected by the chemical messengers. The alterations in current density persisted for many hours after removing the regulatory factors from the culture medium. In fact, the stimulatory action of EGF on high-threshold current lasted > 3 d. The results suggest that the control of prolactin production by the factors tested involves regulation of the surface density of functional Ca channels in the plasma membrane.

Effects of the acute and chronic administration of indapamide on systemic and renal haemodynamics in essential hypertension.

The effects of acute and chronic administration of indapamide on systemic and renal haemodynamics were studied in 10 patients with arterial essential hypertension. No important changes were observed on mean arterial blood pressure, cardiac index and total peripheral resistance during acute administration of the drug. Chronic administration produced a significant reduction in both mean arterial blood pressure and total peripheral resistance whereas the cardiac index did not change. Glomerular filtration rate diminished significantly during the acute administration of the drug, while renal plasma flow remained unchanged. Urinary volume did not change whereas osmotic clearance increased and free water clearance decreased evidently. Both urinary sodium and potassium increased significantly during acute administration of indapamide. Long-term administration of this substance had no effect on renal haemodynamics.