Pregnenolone sulfate (PS), an endogenously occurring neurosteroid, has been shown to modulate the activity of several neurotransmitter-gated channels, including the NMDA receptor (NMDAR). NMDARs are glutamate-gated ion channels involved in excitatory synaptic transmission, synaptic plasticity, and excitotoxicity. In this study, we analyzed the effects of PS on calcium signaling in cultured hippocampal neurons and HEK293 cells expressing NMDAR. The cells were loaded with the Ca(2+) sensor Fura-2. In agreement with previous electrophysiological experiments, PS potentiated the increases in intracellular Ca(2+) induced by an exogenous application of glutamate; however, PS also increased intracellular Ca(2+) in the absence of exogenous NMDA agonist. The agonist-independent effect of PS was induced in all neurons studied and in HEK293 cells expressing GluN1/GluN2A-B receptors in a neurosteroid-specific manner. We conclude that PS is an endogenous NMDA agonist that activates the GluN1/GluN2A-B receptors.
- MeSH
- gating iontového kanálu fyziologie MeSH
- HEK293 buňky MeSH
- lidé MeSH
- pregnenolon metabolismus MeSH
- receptory N-methyl-D-aspartátu metabolismus MeSH
- vápník metabolismus MeSH
- vápníková signalizace fyziologie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
N-methyl-d-aspartate (NMDA) receptors (NMDARs) are highly expressed in the CNS and mediate the slow component of excitatory transmission. The present study was aimed at characterizing the temperature dependence of the kinetic properties of native NMDARs, with special emphasis on the deactivation of synaptic NMDARs. We used patch-clamp recordings to study synaptic NMDARs at layer II/III pyramidal neurons of the rat cortex, recombinant GluN1/GluN2B receptors expressed in human embryonic kidney (HEK293) cells, and NMDARs in cultured hippocampal neurons. We found that time constants characterizing the deactivation of NMDAR-mediated excitatory postsynaptic currents (EPSCs) were similar to those of the deactivation of responses to a brief application of glutamate recorded under conditions of low NMDAR desensitization (whole-cell recording from cultured hippocampal neurons). In contrast, the deactivation of NMDAR-mediated responses exhibiting a high degree of desensitization (outside-out recording) was substantially faster than that of synaptic NMDA receptors. The time constants characterizing the deactivation of synaptic NMDARs and native NMDARs activated by exogenous glutamate application were only weakly temperature sensitive (Q(10)=1.7-2.2), in contrast to those of recombinant GluN1/GluN2B receptors, which are highly temperature sensitive (Q(10)=2.7-3.7). Ifenprodil reduced the amplitude of NMDAR-mediated EPSCs by approximately 50% but had no effect on the time course of deactivation. Analysis of GluN1/GluN2B responses indicated that the double exponential time course of deactivation reflects mainly agonist dissociation and receptor desensitization. We conclude that the temperature dependences of native and recombinant NMDAR are different; in addition, we contribute to a better understanding of the molecular mechanism that controls the time course of NMDAR-mediated EPSCs. Copyright 2010 IBRO. Published by Elsevier Ltd. All rights reserved.
- MeSH
- antagonisté excitačních aminokyselin farmakologie MeSH
- buněčné linie MeSH
- excitační postsynaptické potenciály fyziologie účinky léků MeSH
- hipokampus fyziologie účinky léků MeSH
- kinetika MeSH
- krysa rodu rattus MeSH
- kultivované buňky MeSH
- kyselina glutamová metabolismus MeSH
- lidé MeSH
- mozková kůra fyziologie účinky léků MeSH
- nervový přenos fyziologie účinky léků MeSH
- piperidiny farmakologie MeSH
- potkani Wistar MeSH
- pyramidové buňky fyziologie účinky léků MeSH
- receptory N-methyl-D-aspartátu metabolismus MeSH
- synapse fyziologie účinky léků MeSH
- techniky in vitro MeSH
- teplota MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- lidé MeSH
- zvířata MeSH
- Publikační typ
- práce podpořená grantem MeSH
Pregnenolone sulfate (PS), an endogenously occurring neurosteroid, has been shown to modulate the activity of several neurotransmitter-gated channels, including the N-methyl-D-aspartate receptor (NMDAR). NMDARs are glutamate-gated ion channels involved in excitatory synaptic transmission, synaptic plasticity, and excitotoxicity. To determine the mechanism that controls PS sensitivity of NMDARs, we measured NMDAR responses induced by exogenous agonist application in voltage-clamped HEK293 cells expressing NR1/NR2B NMDARs and cultured rat hippocampal neurons. We report that PS potentiates the amplitude of whole-cell recorded NMDAR responses in cultured hippocampal neurons and HEK293 cells; however, the potentiating effect of PS on NMDAR in outside-out patches isolated from cultured hippocampal neurons and HEK293 cells was lost within 2 min after patch isolation in a neurosteroid-specific manner. The rate of diminution of the PS potentiating effect was slowed by protein phosphatase inhibitors. Treatment of cultured hippocampal neurons with a nonspecific protein kinase inhibitor and a specific protein kinase A (PKA) inhibitor diminished PS-induced potentiation, which was recovered by adding a PKA, but not a protein kinase C (PKC), activator. These results suggest that the effect of PS on NMDARs is controlled by cellular mechanisms that are mediated by dephosphorylation/phosphorylation pathways.
- MeSH
- buněčné linie MeSH
- fosforylace MeSH
- hipokampus fyziologie MeSH
- krysa rodu rattus MeSH
- kultivované buňky MeSH
- lidé MeSH
- membránové potenciály MeSH
- metoda terčíkového zámku MeSH
- neurony fyziologie MeSH
- pregnenolon metabolismus MeSH
- proteinfosfatasy antagonisté a inhibitory MeSH
- proteinkinasa C metabolismus MeSH
- proteinkinasy závislé na cyklickém AMP metabolismus MeSH
- receptory N-methyl-D-aspartátu agonisté metabolismus MeSH
- vápník metabolismus MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- lidé MeSH
- zvířata MeSH
- Publikační typ
- práce podpořená grantem MeSH
The synthesis of several novel 5alpha- and 5beta-20-oxo-pregnane derivatives substituted in the position 3 and 7 of the steroid skeleton is described. Activity of synthesized compounds was studied in voltage-clamped cultured rat hippocampal neurons. Substituted derivatives inhibited NMDA-elicited neuronal activity. The relationship between biological activity and structure is discussed.
- MeSH
- elektrická vodivost MeSH
- financování organizované MeSH
- hipokampus cytologie MeSH
- inhibiční koncentrace 50 MeSH
- krysa rodu rattus MeSH
- kultivované buňky MeSH
- metoda terčíkového zámku MeSH
- neurony metabolismus účinky léků MeSH
- pregnany farmakologie chemická syntéza chemie MeSH
- receptory N-methyl-D-aspartátu antagonisté a inhibitory MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- zvířata MeSH
Většina excitačního synaptického přenosu v savčí centrální nervové soustavě je zprostředkována ionotropními glutamátovými receptory. NMDA receptory, tvořící jednu ze tří tříd této skupiny chemicky aktivovaných iontových kanálů, se podílejí na tvorbě paměti a procesu učení a hrají roli i v etiologii nejrůznějších neurodegenerativních onemocnění. Činnost NMDA receptoru je ovlivňována mnoha faktory, jako jsou teplota, pH nebo různé modulační látky. Patří mezi ně i neurosteroidy, deriváty progesteronu syntetizované přímo v nervové tkáni z cholesterolu nebo ze steroidních prekurzorů pocházejících z periferních zdrojů. Cílem tohoto článku je shrnout výsledky pokusů zabývajících se modulací funkce NMDA receptoru steroidními látkami. Tyto studie byly prováděny v uplynulých letech na oddělení buněčné neurofyziologie Fyziologického ústavu AV ČR, v.v.i. (http://www2.biomed.cas.cz/d331/index.html), které je součástí Centra neuropsychiatrických studií (http://www.pcp.lf3.cuni.cz/cns/).
Excitatory synaptic transmission in mammalian CNS is mostly mediated by ionotropic glutamate receptors. NMDA receptors, one of three subclasses of this ligand-activated ion channels family, are involved in memory formation and learning and also play a role in the pathogenesis of various neurodegenerative diseases. NMDA receptors function is influenced by several factors, including temperature, pH or various chemical modulators. Among others, this group includes neurosteroids; progesterone derivatives, synthesised in neural tissue from cholesterol or different steroid precursors from peripheral sources. The aim of this article is to sum up the results of experiments designed to study the modulation of NMDA receptors function by steroid compounds. This work has been done at the Department of Cellular Neurophysiology of the Institute of Physiology AS CR, v.v.i. (http://www2.biomed.cas.cz/d331/eng/index_eng. html), a member of the Centre of Neuropsychiatric Studies (http://www.pcp.lf3.cuni.cz/cns/).
Ionotropic glutamate receptors function can be affected by neurosteroids, both positively and negatively. N-methyl-D-aspartate (NMDA) receptor responses to exogenously applied glutamate are potentiated or inhibited (depending on the receptor subunit composition) by pregnenolone sulphate (PS) and inhibited by pregnanolone sulphate (3alpha5betaS). While PS effect is most pronounced when its application precedes that of glutamate, 3alpha5betaS only binds to receptors already activated. Synaptically activated NMDA receptors are inhibited by 3alpha5betaS, though to a lesser extent than those tonically activated by exogenous glutamate. PS, on the other hand, shows virtually no effect on any of the models of synaptically activated NMDA receptors. The site of neurosteroid action at the receptor molecule has not yet been identified, however, the experiments indicate that there are at least two distinct extracellularly located binding sites for PS mediating its potentiating and inhibitory effects respectively. Experiments with chimeric receptors revealed the importance of the extracellular loop connecting the third and the fourth transmembrane domain of the receptor NR2 subunit for the neurosteroid action. alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA)/kainate receptors are inhibited by both PS and 3alpha5betaS. These neurosteroids also affect AMPA receptors-mediated synaptic transmission, however, in a rather indirect way, through presynaptically located targets of action.
- MeSH
- AMPA receptory metabolismus MeSH
- excitační postsynaptické potenciály účinky léků MeSH
- glutamátové receptory metabolismus MeSH
- lidé MeSH
- mozek metabolismus MeSH
- nervový přenos účinky léků MeSH
- pregnenolon chemie farmakologie MeSH
- receptory N-methyl-D-aspartátu chemie metabolismus MeSH
- vazba proteinů MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- přehledy MeSH
N-methyl-D-aspartate (NMDA) receptors are highly expressed in the CNS, mediate the slow component of excitatory transmission and play key roles in synaptic plasticity and excitotoxicity. These ligand-gated ion channels are heteromultimers composed of NR1 and NR2 subunits activated by glycine and glutamate. In this study, patch-clamp recordings were used to study the temperature sensitivity of recombinant NR1/NR2B receptors expressed in human embryonic kidney (HEK) 293 cells. Rate constants were assessed by fitting a six-state kinetic scheme to time courses of transient macroscopic currents induced by glutamate at 21.9-46.5 degrees C. Arrhenius transformation of the rate constants characterizing NMDA receptor channel activity indicates that the most sensitive were the rate constants of desensitization (temperature coefficient Q(10)=10.3), resensitization (Q(10)=4.6) and unbinding (Q(10)=3.6). Other rate constants and the amplitude of single-channel currents were less temperature sensitive. Deactivation of responses mediated by NR1/NR2B receptors after a brief application of glutamate was best fit by a double exponential function (tau(fast): Q(10)=3.7; tau(slow): Q(10)=2.7). From these data, we conclude that desensitization/resensitization of the NMDA receptor and glutamate unbinding are especially temperature sensitive and imply that at physiological temperatures the channel kinetics play an important role in determining amplitude and time course of NMDA receptor-mediated postsynaptic currents and these receptors mediated synaptic plasticity.
- MeSH
- algoritmy MeSH
- buněčné linie MeSH
- elektrofyziologie MeSH
- financování organizované MeSH
- gating iontového kanálu fyziologie MeSH
- kinetika MeSH
- kyselina glutamová metabolismus MeSH
- ledviny cytologie metabolismus MeSH
- lidé MeSH
- metoda terčíkového zámku MeSH
- receptory N-methyl-D-aspartátu fyziologie genetika účinky léků MeSH
- rekombinantní proteiny MeSH
- statistické modely MeSH
- techniky in vitro MeSH
- teplota MeSH
- transfekce MeSH
- Check Tag
- lidé MeSH
- MeSH
- finanční podpora výzkumu jako téma MeSH
- Publikační typ
- abstrakty MeSH
