How do G-protein-coupled receptors work? The case of metabotropic glutamate and GABA receptors

G-protein coupled receptors (GPCRs) represent the largest membrane proteins family in animal genomes. Being the receptors for most hormones and neurotransmitters, these proteins play a central role in intercellular communication. GPCRs can be classified into several groups based on the sequence similarity of their common structural feature: the heptahelical domain. The metabotropic receptors for the main neurotransmitters glutamate and gamma-aminobutyric acid (GABA) belong to the class III of GPCRs, together with others receptors for Ca2+, for sweet and amino acid taste compounds and for some pheromones, as well as for odorants in fish. Besides their transmembrane heptahelical domain responsible for G-protein activation, most of class III receptors possess a large extracellular domain responsible for ligand recognition. The recent resolution of the structure of this binding domain of one of these receptors, the mGlu1 receptor, together with the recent demonstration that these receptors are dimers, revealed an original mechanism of activation for these GPCRs. Such data open new possibilities to develop drugs aimed at modulating these receptors, and raised a number of interesting questions on the activation mechanism of other GPCRs.     

Probiotic-mediated biotransformation of monosodium glutamate to γ-aminobutyric acid: differential production in complex and minimal media and kinetic modelling

Lactic cultures were screened for their ability to accumulate γ-aminobutyric acid (GABA) in the culture medium. Lactobacillus bulgaricus CFR 2028 produced the highest yields of GABA (22.7 mM) from the substrate monosodium glutamate (MSG). Instrumental characterization by high-performance liquid chromatography and structural characterization by mass spectrometry confirmed GABA production by L. bulgaricus CFR 2028. The differential production of GABA in complex and minimal media indicated that minimal medium (modified tryptone, yeast extract and glucose; TYG) supported the highest production of GABA (37 mM), thereby signifying that the acidic pH alone could have a significant bearing on the yield of GABA. This observation opens up avenues for the optimization of the medium components for yield maximization. The biotransformation kinetics of MSG was examined in batch experiments by varying initial MSG concentration (1–5 %). The Monod model was fitted to determine the kinetic parameters under the MSG uninhibited domain, and the MSG inhibited domain was represented well by Briggs–Haldane model.     

Control of βAR- and N-methyl-D-aspartate (NMDA) Receptor-Dependent cAMP Dynamics in Hippocampal Neurons

Norepinephrine, a neuromodulator that activates β-adrenergic receptors (βARs), facilitates learning and memory as well as the induction of synaptic plasticity in the hippocampus. Several forms of long-term potentiation (LTP) at the Schaffer collateral CA1 synapse require stimulation of both βARs and N-methyl-D-aspartate receptors (NMDARs). To understand the mechanisms mediating the interactions between βAR and NMDAR signaling pathways, we combined FRET imaging of cAMP in hippocampal neuron cultures with spatial mechanistic modeling of signaling pathways in the CA1 pyramidal neuron. Previous work implied that cAMP is synergistically produced in the presence of the βAR agonist isoproterenol and intracellular calcium. In contrast, we show that when application of isoproterenol precedes application of NMDA by several minutes, as is typical of βAR-facilitated LTP experiments, the average amplitude of the cAMP response to NMDA is attenuated compared with the response to NMDA alone. Models simulations suggest that, although the negative feedback loop formed by cAMP, cAMP-dependent protein kinase (PKA), and type 4 phosphodiesterase may be involved in attenuating the cAMP response to NMDA, it is insufficient to explain the range of experimental observations. Instead, attenuation of the cAMP response requires mechanisms upstream of adenylyl cyclase. Our model demonstrates that Gs-to-Gi switching due to PKA phosphorylation of βARs as well as Gi inhibition of type 1 adenylyl cyclase may underlie the experimental observations. This suggests that signaling by β-adrenergic receptors depends on temporal pattern of stimulation, and that switching may represent a novel mechanism for recruiting kinases involved in synaptic plasticity and memory.     

Role of prions in neuroprotection and neurodegeneration: A mechanism involving glutamate receptors?

There is increasing evidence that cellular prion protein plays important roles in neurodegeneration and neuroprotection. One of the possible mechanism by which this may occur is a functional inhibition of ionotropic glutamate receptors, including N-Methyl-D-Aspartate (NMDA) receptors. Here we review recent evidence implicating a possible interplay between NMDA receptors and prions in the context of neurodegenerative disorders. Such is a functional link between NMDA receptors and normal prion protein, and therefore possibly between these receptors and pathological prion isoforms, raises interesting therapeutic possibilities for prion diseases.Key words: NMDA, NR2D, glutamate, neuroprotection, calciumPrions are most often discussed in the context of transmissible spongiform encephalopathies (TSEs) which encompass a range of neurological disorders that include human Creutzfeldt-Jakob disease (among others), sheep scrapie and bovine spongiform encephalopathy.^1,2 It is well established that these disorders arise from a progressive conversion of the normal, mainly helical form of cellular prion protein (PrP^C) into a different PrP^Sc protein conformation with a high beta sheet content.³ In their PrP^Sc form, prions act as templates that catalyze misfolding of PrP^C to produce increasing levels of PrP^Sc, which likely represents several or even many different conformational states of the same source protein, resulting in diverse clinical phenotypes. This in turn leads to accumulation of PrP^Sc deposits in the brain that can appear as aggregates and amyloid-like plaques⁴ and which disrupt normal neurophysiology.⁵ While the neuropathology of TSE''s has been explored in great detail dating back to the 1920s,⁶ less effort has perhaps been expended on understanding the cellular and physiological function of PrP^C which is ubiquitously expressed, and found even in simple organisms.^5,7,8 A number of mouse lines either lacking PrP^C or overexpressing PrP^C have been created, including the widely used Zurich I PrP^C knockout strain.^9,10 Despite the wide distribution of PrP^C in the mammalian CNS, it perhaps surprisingly has only a relatively mild behavioral phenotype that appears to include some deficits in spatial learning at the behavioral level^11,12 as well as alterations in long term potentiation at the cellular level.^13–17 In addition, it has been shown that these mice show an increased excitability of hippocampal neurons.^13,18–20 In contrast, deletion of certain parts of the PrP^C protein in vivo can have serious physiological consequences. For example, deletion of a stretch of amino acids between just upstream of the octarepeat copper binding motifs produces a lethal phenotype, that can be rescued by overexpression of increasing levels of normal PrP^C.^21,22 Of particular note, these deletion mutants show degeneration of axons and myelin, both in the CNS and in peripheral nerves; indeed some mutants show a predilection for axomyelinic degeneration with little neuronal pathology,²¹ suggesting that certain mutated forms of PrP have a direct toxic effect on oligodendrocytes and/or myelin.²³ Moreover, activation of the Dpl1 gene in mice lacking PrP^C leads to an ataxic phenotype, that is not observed in the presence of PrP^C.²⁴ Collectively, this indicates that PrP^C may act in a protective capacity and in contrast, certain abnormal forms of PrP are “toxic”, promoting much more injury to various elements of the CNS and PNS than outright absence of wild-type PrP^C.This notion is further corroborated by a number of studies in PrP^C knockout mice, both in vivo and in cell culture models. Cultured hippocampal neurons from PrP^C null mice display greater apoptosis during oxidative stress.²⁵ Moreover, overexpression of PrP^C in rats protects them from neuronal damage during ischemic stroke, whereas PrP^C null mice show greater damage.^27–29 When PrP^C null mice are subjected to different types of seizure paradigms, they showed increased mortality and increased numbers of seizures.³⁰ This increased neuronal damage can be diminished by the NMDA receptor blocker MK-801,³¹ potentially implicating glutamate receptors in this process. Finally, it was recently shown that the absence of PrP^C protein protects neurons from the deleterious effects of beta amyloid, a protein involved in Alzheimer disease.³² It is important to note that NMDA receptors have been implicated in seizure disorders and in cell death during ischemic stroke.^33–35 Indeed, our own work has shown that NMDA receptors expressed endogenously in myelin contribute to myelin damage and may be one of the first steps leading to demyelination.³⁶ Furthermore, the NMDA receptor blocker memantine is used to treat Alzheimer disease, implicating NMDA receptors. The observations above suggest that there may be an interplay between NMDA receptor activity and the physiological function of PrP^C. In support of this hypothesis, our recent work has directly identified a common functional and molecular link between NMDA receptors and PrP^C.³⁷ Brain slices obtained from Zurich I PrP^C null mice showed an increased excitability of hippocampal slices, which could be ablated by blocking NMDA receptor activity with amino-5-phosphonovaleric acid. Removal of extracellular magnesium ions to enhance NMDA receptor activity resulted in stronger pro-excitatory effects in slices and cultured neurons from PrP^C null mice compared with those from normal animals. Synaptic recordings indicate that the amplitude and duration of NMDA mediated miniature synaptic currents is increased in PrP^C null mouse neurons, and evoked NMDA receptor currents show a dramatic slowing of deactivation kinetics in PrP^C null mouse neurons. The NMDA current kinetics observed in these neurons were qualitatively consistent with NMDA receptors containing the NR2D subunit.³⁸ Consistent with a possible involvement of NR2D containing receptors, siRNA knockdown of NR2D normalized current kinetics in PrP-null mouse neurons. Furthermore, a selective co-immunoprecipitation between PrP^C and the NR2D, but not NR2B subunits, was observed. This then may suggest the possibility that under normal circumstances, PrP^C serves to suppress NR2D function, but when PrP^C is absent, NR2D containing receptors become active, and because of their slow kinetics, may contribute to calcium overload under circumstances where excessive (or even normal) levels of glutamate are present. This would include conditions such as epileptic seizures, ischemia and Alzheimer disease, thus providing a possible molecular explanation for the link between PrP^C and neuroprotection under pathophysiological conditions. Indeed, NMDA promoted greater toxicity in PrP^C null mouse neurons, and upon injection into brains of PrP^C null mice. It is interesting to note that one of the major NMDA receptor subtypes expressed in myelin is NR2D, thus bridging the observations of Micu et al.³⁶ of NMDA receptor mediated cell death in ischemic white matter, and those of Baumann and colleagues²¹ showing that PrP^C deletion mutants can cause damage to myelin.How might PrP^C deletion mutants affect neuronal survival? One possibility may be that these deletion mutants compete with normal PrP^C for NMDA receptors, but are unable to functionally inhibit them. Alternatively, it is possible that the PrP^C deletion mutants, by virtue of binding to the receptors, may in fact increase receptor activity, thus causing increased cell death. In both cases, increasing the expression of normal PrP^C would be expected to outcompete the deletion variants, thus reestablishing the protective function. A similar mechanism could perhaps apply to TSEs. It is possible that the PrP^Sc form, perhaps in a manner reminiscent of the PrP^C deletion mutants, may be unable to inhibit NMDAR function, or perhaps would even enhance it. Any excess glutamate that may be released as a result of cell damage due to PrP^Sc aggregates, or even normally released amounts glutamate during the course of physiological neuronal signaling, could be sufficient to cause NMDAR mediated cell death and neuronal degeneration. In this context, it is interesting to note that chronic administration of the weakly NR2D selective inhibitor memantine delays death as a consequence of scrapie infection in mice.³⁹ In the context of Alzheimer disease, binding of PrP^C to beta amyloid may prevent the inhibitory action of PrP^C on NMDA receptor function, thus increasing NMDA receptor activity and promoting cell death. This then may perhaps explain the beneficial effects of memantine in the treatment of Alzheimer disease.In summary, despite the fact that PrP^C is one of the most abundantly expressed proteins in the mammalian CNS, its physiological role is uncertain. Recent observations from our labs have established an unequivocal functional link between normal prion protein and the ubiquitous excitatory NMDA receptor. Thus, one of the key physiological roles of PrP^C may be regulation of NMDA receptor activity. The presence of abnormal species of prion protein, whether acquired via “infection”, spontaneous conformational conversion or genetically inherited, may in turn alter normal function and regulation of NMDA receptors, leading to chronic “cytodegeneration” of elements in both gray and white matter regions of the CNS. This key functional link between PrP and glutamate receptors may provide our first opportunity for rational therapeutic design against the devastating spongiform encephalopathies and potentially other neurodegenerative disorders not traditionally considered as TSE''s.     

Speciation of binary complexes of Co(II), Ni(II) and Cu(II) with L-aspartic acid in propylene glycol–water mixtures

Abstract

Speciation of binary complexes of Co(II), Ni(II) and Cu(II) with L-aspartic acid in (0-60% v/v) propylene glycol-water mixtures was studied pH metrically at 303.0±0.1 K and at an ionic strength of 0.16 mol L^-1. The binary species refined were ML, ML₂, ML₂H₂, ML₂H₃ and ML₂H₄. The stabilities of the complexes followed the Irving-Williams order i.e.Co(II) <Ni(II) < Cu(II). The linear variation of stability constants as a function of dielectric constant of the medium indicated the dominance of electrostatic forces over non-electrostatic forces. Some species were stabilised due to electrostatic interactions and some were destabilised due to the decreased dielectric constant. The order of ingredients influencing the magnitudes of stability constants due to incorporation of errors in their concentrations was alkali > acid > ligand > metal. Equilibria for the formation of binary complexes were proposed based on the forms of the ligand and their existence at different pH values.     

Surface receptors: Are they involved in transformation of spermatozoa of Ascaris?

Exposure of the spheroidal spermatozoa of Ascaris suum to an extract of the male accessory gland causes their transformation into ameboid cells. We have investigated the mechanism of this transformation, also termed activation, by labeling the proteins of accessory gland extracts with fluorescein isothiocyanate (FITC) or [125I], followed by qualitative localization of the sperm activating substances (SAS) and quantitative measurements of [125I]-SAS binding. Fluorescent patches of FITC-conjugated SAS were localized at the spermatozoan surface and were concentrated primarily at the posterior region. Few fluorescent patches were detectable in the region of the newly formed pseudopodia following transformation. Although spermatozoan transformation occurs within 2-5 min after exposure to SAS, the fluorescent patches became more distinct after a minimum of 8 min and reached maximum density at 15-30 min. Spermatozoa activated with [125I]-SAS became radioactively labeled in direct proportion to the amount of available [125I]-SAS until a saturation level was reached. SDS-polyacrylamide gel electrophoresis combined with autoradiography indicated that the cells bind two SAS components, of small (9,000 MW) and large (56,000 MW) sizes. These same two components were also detectable in a membrane fraction, obtained by differential centrifugation, of the spermatozoa after incubation with [125I]-SAS. binding of the two SAS components was not inhibited by preincubation of the spermatozoa with trypsin or Concanavalin A; however, the 56,000 MW component of SAS was not detectable in autoradiograms of spermatozoa incubated with periodic acid (1.6-10 mM) treated SAS. Such cells also failed to transform into ameboid spermatozoa. These results indicate that the two components of SAS that bind to the spermatozoan surface are possibly responsible for inducing the cell transformations associated with activation.     

Estrogen receptor α and β are involved in the activation of lordosis behavior in estradiol-primed rats

The present study was designed to assess the participation of estrogen receptors alpha (ERα) and beta (ERβ) in the short-term facilitation of lordosis behavior in ovariectomized (ovx), estradiol (E₂) primed rats. In experiment 1, dose response curves for PPT and DPN (ERα and ERβ agonists, respectively) facilitation of lordosis behavior (lordosis quotient and lordosis score) were established by infusing these agonists into the right lateral ventricle (icv) in female rats injected 40 h previously with 5 μg of E₂ benzoate. PPT doses of 0.08 and 0.4 ng produced high lordosis quotients starting at 30 min and continuing at 120 and 240 min post-injection. DPN induced high levels of lordosis behavior at all times tested. However, the intensity of lordosis induced by both agonists was weak. In experiment 2, we tested the involvement of each ER in facilitation of lordosis by icv infusion of MPP (ERα-selective antagonist) or PHTPP (ERβ-selective antagonist) prior to infusion of 2 ng of free E₂. Icv infusion of either MPP or PHTPP 30 min before free E2 significantly depressed E₂ facilitation of lordosis. The results suggest that both forms of ER are involved in the short-latency facilitation of lordosis behavior in E₂-primed rats.     

Are isocitrate dehydrogenases and 2-oxoglutarate involved in the regulation of glutamate synthesis?

In plants, nitrogen assimilation into amino acids relies on the availability of the reduced form of nitrogen, ammonium. The glutamine synthetase–glutamate synthase pathway, which requires carbon skeletons in the form of 2-oxoglutarate, achieves this. To date, the exact enzymatic origin of 2-oxoglutarate for plant ammonium assimilation is unknown. Isocitrate dehydrogenases synthesize 2-oxoglutarate. Recent efforts have concentrated on evaluating the involvement of different isocitrate dehydrogenases, distinguished by co-factor specificity and sub-cellular localization. Furthermore, several observations indicate that 2-oxoglutarate is likely to be a metabolic signal that regulates the coordination of carbon:nitrogen metabolism. This is discussed in the context of recent advances in bacterial signalling processes.     

Hippocampal synaptic plasticity and NMDA receptors: a role in information storage?

There has recently been renewed interest in the idea that alterations in synaptic efficacy may be the neural basis of information storage. Particular attention has been focused upon long-term potentiation (LTP), a long-lasting, but experimentally induced synaptic change whose physiological properties point to it being a candidate memory mechanism. However, considerations of storage capacity and the possibility of concomitant activity-dependent synaptic depression make it unlikely that individual learning experiences will give rise to gross changes in field potentials similar to those that occur in LTP, even if learning and LTP utilize common neural mechanisms. One way of investigating the functional significance of LTP is to use selective antagonists of those excitatory amino acid receptors whose activation is essential for its induction. This paper discusses various design requirements for such experiments and reviews work indicating that the N-methyl-D-aspartate receptor antagonist AP5 causes a behaviourally selective learning impairment having certain common features to the behavioural profile seen after hippocampal lesions. Two new studies are described whose results show that AP5 has no effect upon the retrieval of previously established memories, and that the dose-response profile of the impairment of spatial learning occurs across a range of extracellular concentrations in hippocampus for which receptor selectivity exists. These experiments show that activation of NMDA receptors is essential for certain kinds of learning.     

Are G-protein-coupled receptors involved in mediating larval settlement and metamorphosis of coral planulae?

Larvae of the scleractinian coral Pocillopora damicornis are induced to settle and metamorphose by the presence of marine bacterial biofilms, and the larvae of Montipora capitata respond to a combination of filamentous and crustose coralline algae. The primary goal of this study was to better understand metamorphosis of cnidarian larvae by determining what types of receptors and signal-transduction pathways are involved during stimulation of metamorphosis of P. damicornis and M. capitata. Evidence from studies on larvae of hydrozoans suggests that G-protein-coupled receptors (GPCRs) are good candidates. Settlement experiments were conducted in which competent larvae were exposed to neuropharmacological agents that affect GPCRs and their associated signal-transduction pathways, AC/cAMP and PI/DAG/PKC. On the basis of the results of these experiments, we conclude that GPCRs and these pathways do not mediate settlement and metamorphosis in either coral species. Two compounds that had an effect on both species, forskolin and phorbol-12-myristate-13-acetate (TPA), may be acting on other cellular processes not related to GPCRs. This study strengthens our understanding of the underlying physiological mechanisms that regulate metamorphosis in coral larvae.     

Experimental evidence that maleic acid markedly compromises glutamate oxidation through inhibition of glutamate dehydrogenase and α-ketoglutarate dehydrogenase activities in kidney of developing rats

Molecular and Cellular Biochemistry - This study was aimed to explore the role of C1q/TNF-related protein 9 (CTRP9) on atherosclerotic lesion formation. A recombinant lentiviral vector carrying...     

The insulin sensitizing effects of PPAR-γ agonist are associated to changes in adiponectin index and adiponectin receptors in Zucker fatty rats

The adiponectin high molecular weight isoform (HMW-adp) and its relation with the other adiponectin isoforms (adiponectin index, S(A)), have been identified as essential for the adiponectin insulin sensitizing effects. The objective of this study is to gain further insight on the effect of the insulin sensitizing agents, PPAR-γ agonists, on the distribution of the adiponectin isoforms and the adiponectin receptors, adipoR1 and adipoR2 in an animal model of obesity and insulin resistance. To achieve the objective, Zucker fatty rats were treated with pioglitazone, rosiglitazone or placebo for six weeks. At the end of the treatment, total adiponectin, adiponectin isoforms and adiponectin receptors expression were measured. In order to see the possible relation with insulin sensitivity parameters, HOMA-IR, muscle insulin-stimulated glucose transport, muscle GLUT4 and plasma free fatty acids were also measured. The two glitazones improved insulin sensitivity and both muscle insulin-stimulated glucose transport and GLUT4 total content. Total plasma adiponectin and visceral fat HMW-adp were increased only by pioglitazone. On the other hand, both glitazones changed the distribution of adiponectin isoforms in plasma, leading to an increase in the S(A) of 21% by pioglitazone and 31% by rosiglitazone. Muscle adipoR1 expression was increased by both glitazones whereas liver adipoR2 expression was increased by rosiglitazone and tended to increase in the pioglitazone group. The insulin sensitizing action of glitazones is mediated, at least in part, by their effect on muscle insulin-stimulated glucose transport and by their direct influence on the adiponectin index and the adiponectin receptors expression.     

The role of γ-aminobutyric acid and its receptors in the nucleus of basal optic root in pigeons

The effects of γ-aminobutyric acid (GABA) and its antagonists bicuculline and 2-hydroxysaclofen on neuronal firings in the nucleus of basal optic root (nBOR) in pigeons were studied by using extracellular recording and microiontophoretic techniques. The results suggest that GABA may be an inhibitory neurotransmitter or modulator within nBOR, functioning by means of main mediation of GABAA receptors and of minor mediation of GABAB receptors. Furthermore, GABA and its GABAA receptors are involved in the modulation of directional selectivity in part of nBOR neurons.     

Are polyamines involved in the induction and regulation of the Crassulacean acid metabolism?

Leaves of plants with Crassulacean acid metabolism (CAM) were analyzed for variation in the content of polyamines in connection with the metabolism of malic acid in the dark and in the light, and with the induction of full-CAM activity. Under conditions (long days) resulting in extremely low CAM activity, young leaves of K. blossfeldiana have very low content in the polyamine-precursor arginine and in putrescine. The content in these two substances was increased dramatically by full-CAM induction with short days. During the course of the night/day cycle two peaks of putrescine content were observed in leaves of Kalanchoe blossfeldiana Poelln. Tom Thumb performing full-CAM operation: a large increase occurs toward the end of the day and the first half of the night, and its kinetics corresponds to the increase in the rate of malic acid synthesis; another peak, very sharp, appears during the first hours of the day, concomitant with the time of release of malic acid from the vacuole into the cytoplasm. In the case of Bryophyllum daigremontianum Berger similar variations were observed for the content in spermidine. These results support the hypothesis that polyamines could be involved in countering the tendency toward acidification of the cytoplasm at those moments of CAM operation at which the local concentration of malic acid is increased (i.e., during active synthesis in the dark and during the efflux from the vacuole in the light).Abbreviation CAM Crassulacean acid metabolism     

The role of thrombin and thrombin receptors in ischemic,hemorrhagic and traumatic brain injury: deleterious or protective?

In the last two decades it has become apparent that thrombin has many extravascular effects that are mediated by a family of protease-activated receptors (PARs). PAR-1, -3 and -4 are activated via cleavage by thrombin. The importance of extravascular thrombin in modulating ischemic, hemorrhagic and traumatic injury in brain has recently become clear. Thus, in vitro, thrombin at low concentration protects neurons and astrocytes from cell death caused by a number of different insults. In vivo, pretreating the brain with a low dose of thrombin (thrombin preconditioning), attenuates the brain injury induced by a large dose of thrombin, an intracerebral hemorrhage or by focal cerebral ischemia. Thrombin may also be an important mediator of ischemic preconditioning. In contrast, high doses of thrombin kill neurons and astrocytes in vitro and cause disruption of the blood-brain barrier, brain edema and seizures in vivo. This review examines the role of thrombin in brain injury and the molecular mechanisms and signaling cascades involved.