Molecular pharmacology of human NMDA receptors

N-methyl-d-aspartate (NMDA) receptors are ionotropic glutamate receptors that mediate excitatory neurotransmission. NMDA receptors are also important drug targets that are implicated in a number of pathophysiological conditions. To facilitate the transition from lead compounds in pre-clinical animal models to drug candidates for human use, it is important to establish whether NMDA receptor ligands have similar properties at rodent and human NMDA receptors. Here, we compare amino acid sequences for human and rat NMDA receptor subunits and discuss inter-species variation in the context of our current knowledge of the relationship between NMDA receptor structure and function. We summarize studies on the biophysical properties of human NMDA receptors and compare these properties to those of rat orthologs. Finally, we provide a comprehensive pharmacological characterization that allows side-by-side comparison of agonists, un-competitive antagonists, GluN2B-selective non-competitive antagonists, and GluN2C/D-selective modulators at recombinant human and rat NMDA receptors. The evaluation of biophysical properties and pharmacological probes acting at different sites on the receptor suggest that the binding sites and conformational changes leading to channel gating in response to agonist binding are highly conserved between human and rat NMDA receptors. In summary, the results of this study suggest that no major detectable differences exist in the pharmacological and functional properties of human and rat NMDA receptors.     

Characterisation of kynurenine pathway metabolism in human astrocytes and implications in neuropathogenesis

Abstract

The role of astrocytes in the production of the neurotoxin quinolinic acid (QUIN) and other products of the kynurenine pathway (KP) is controversial. Using cytokine-stimulated human astrocytes, we assayed key enzymes and products of the KP. We found that astrocytes lack kynurenine-hydroxylase so that large amounts of kynurenine (KYN) and kynurenic acid (KYNA) were produced, while minor amounts of QUIN were synthesised that were completely degraded. We then showed that kynurenine added to macrophages led to significant production of QUIN. These results suggest that astrocytes alone are neuroprotective by minimising QUIN production and maximising synthesis of KYNA. However, it is likely that, in the presence of macrophages and/or microglia, astrocytes are neurotoxic by producing large concentrations of KYN that can be metabolised by neighbouring monocytic cells to QUIN.     

Aging and surface expression of hippocampal NMDA receptors

Aging is known to alter many physiological processes within the brain including synaptic responses, long-term potentiation, learning, and memory. Aging has also been shown to alter the expression and distribution of N-methyl-d-aspartate (NMDA) receptors in many different brain regions, including the hippocampus. Additionally, we have recently reported that young adult rats show an activity-dependent increase in the surface expression of NMDA receptors. We have extended these observations in the present study in aged animals and have found that aged Fischer 344 rats fail to show activity-dependent changes in the surface distribution of NMDA receptors. In conjunction with this observation we have also noted that aged rats show an expression deficit in the C2 splice variant of the NR1 subunit. This subunit is preferentially shifted to the surface following stimulation in young adult animals. As the NMDA receptor is thought to play an important role in neuronal signaling, these observations suggest possible new areas of dysfunction in this receptor that might underlie age-related deficits in neuronal physiology.     

Characterisation of kynurenine pathway metabolism in human astrocytes and implications in neuropathogenesis

The role of astrocytes in the production of the neurotoxin quinolinic acid (QUIN) and other products of the kynurenine pathway (KP) is controversial. Using cytokine-stimulated human astrocytes, we assayed key enzymes and products of the KP. We found that astrocytes lack kynurenine-hydroxylase so that large amounts of kynurenine (KYN) and kynurenic acid (KYNA) were produced, while minor amounts of QUIN were synthesised that were completely degraded. We then showed that kynurenine added to macrophages led to significant production of QUIN. These results suggest that astrocytes alone are neuroprotective by minimising QUIN production and maximising synthesis of KYNA. However, it is likely that, in the presence of macrophages and/or microglia, astrocytes are neurotoxic by producing large concentrations of KYN that can be metabolised by neighbouring monocytic cells to QUIN.     

Characterisation of CGRP receptors in the human isolated middle meningeal artery

Although the understanding of migraine pathophysiology is still incomplete, there seems to be little doubt that dilatation of cranial blood vessels, including meningeal arteries, is involved in the headache phase of migraine. Since calcitonin gene-related peptide (CGRP) has been implicated in this vasodilatation, the present study set out to compare the relaxant effects of the endogenous ligand h-alphaCGRP, and [ethylamide-Cys(2,7)]h-alphaCGRP ([Cys(Et)(2,7)]h-alphaCGRP), a CGRP(2) receptor agonist, on human isolated middle meningeal artery segments, precontracted with KCl. Classical Schild plot analysis was used to characterise the receptor population in this artery using BIBN4096BS and h-alphaCGRP(8-37) as antagonists. h-alphaCGRP relaxed arterial segments more potently than [Cys(Et)(2,7)]h-alphaCGRP (pEC(50): 8.51+/-0.16 and 7.48+/-0.24, respectively), while the maximal responses to these agonists were not significantly different. BIBN4096BS equipotently blocked the relaxations induced by both agonists with a pA(2) of approximately 10 and with a Schild plot slope not significantly different from unity. h-alphaCGRP(8-37) also antagonised the response to h-alphaCGRP with a pA(2) of 6.46+/-0.16 and a Schild plot slope not different from unity. Furthermore, the results obtained from RT-PCR studies confirmed the presence of all the essential components required for a functional CGRP(1) receptor in these arteries. Considering the high antagonist potency of BIBN4096BS, coupled to the lower agonist potency of [Cys (Et)(2,7)]h-alphaCGRP, it is reasonable to suggest a predominant role of CGRP(1) receptors in the human middle meningeal artery. This view is reinforced by Schild plot analysis, which revealed a slope of unity in all experiments, giving further evidence for a homogeneous CGRP receptor population in this vascular preparation.     

Characterisation of receptors for 1,25-dihydroxyvitamin D3 in the human testis

Specific high affinity receptors for 1,25-dihydroxyvitamin D3 have been demonstrated in the human testes. The mean binding affinity (Kd +/- SD) of the receptor for 1,25-dihydroxyvitamin D3 was 1.75 +/- 0.32 x 10(-10) M but the binding capacity was low (mean Nmax +/- SD = 0.53 +/- 0.18 fmol/mg protein). Binding was time- and temperature-dependent, with a maximum binding achieved after 1 h at 25 degrees C. Although binding also took place at 4 and 37 degrees C, higher and more rapid binding was found at 25 degrees C. Furthermore, the binding between the ligand and the receptor was specific since only unlabelled 1,25-dihydroxyvitamin D3 competed with the labelled ligand. Binding of 1,25-dihydroxyvitamin D3 was abolished by trypsin and heat. Sucrose density gradient centrifugation revealed a sedimentation coefficient of 3.6S.     

Store Ca2+ depletion enhances NMDA responses in cultured human astrocytes.

NMDA produced whole-cell membrane currents in cultured human astrocytes. The currents were not inhibited by the selective NMDA receptor antagonist, APV, while they were partially inhibited by the broad G-protein inhibitor, GDPbetaS. NMDA-induced currents were enhanced by either the microsomal Ca2+/ATPase inhibitors, thapsigargin and cyclopiazonic acid, or the ATP-uncoupler, dinitrophenol (DNP). In the Ca2+ assay, NMDA increased intracellular calcium concentration. The increase was inhibited by 26% in Ca2+-free extracellular solution, and it was not inhibited by APV. The results of the present study suggest that NMDA responses in human astrocytes are regulated by store Ca2+ depletion-associated signal.     

Dopamine receptors regulate NMDA receptor surface expression in prefrontal cortex neurons

Interactions between dopamine (DA) and glutamate systems in the prefrontal cortex (PFC) are important in addiction and other psychiatric disorders. Here, we examined DA receptor regulation of NMDA receptor surface expression in postnatal rat PFC neuronal cultures. Immunocytochemical analysis demonstrated that surface expression (synaptic and non-synaptic) of NR1 and NR2B on PFC pyramidal neurons was increased by the D1 receptor agonist SKF 81297 (1 microM, 5 min). Activation of protein kinase A (PKA) did not alter NR1 distribution, indicating that PKA does not mediate the effect of D1 receptor stimulation. However, the tyrosine kinase inhibitor genistein (50 microM, 30 min) completely blocked the effect of SKF 81297 on NR1 and NR2B surface expression. Protein cross-linking studies confirmed that SKF 81297 (1 microM, 5 min) increased NR1 and NR2B surface expression, and further showed that NR2A surface expression was not affected. Genistein blocked the effect of SKF 81297 on NR1 and NR2B. Surface-expressed immunoreactivity detected with a phospho-specific antibody to tyrosine 1472 of NR2B also increased after D1 agonist treatment. Our results show that tyrosine phosphorylation plays an important role in the trafficking of NR2B-containing NMDA receptors in PFC neurons and the regulation of their trafficking by DA receptors.     

Apolipoprotein D expression in human brain reactive astrocytes.

Astrocytosis is a hallmark of damage that frequently occurs during aging in human brain. Astrocytes proliferate in elderly subjects, becoming hypertrophic and highly immunoreactive for glial fibrillary acidic protein (GFAP). These cells are one type that actively responds in the repair and reorganization of damage to the neural parenchyma and are a source of several peptides and growth factors. One of these biomolecules is apolipoprotein D (apo D), a member of the lipocalin family implicated in the transport of small hydrophobic molecules. Although the role of apo D is unknown, increments in brain apo D expression have been observed in association with aging and with some types of neuropathology. We have found an overexpression of apo D mRNA in reactive astrocytes by in situ hybridization in combination with immunohistochemistry for apo D in normal aged human brains. The number of double-labeled cells varied according to the cerebral area and the gliosis grade. The possible significance of this increased synthesis of apo D in reactive astrocytes is discussed in relation to the role of apo D in aging and in glial function.     

NMDA receptors are movin' in

Dynamic modulation of the number of postsynaptic glutamate receptors is considered one of the main mechanisms for altering the strength of excitatory synapses in the central nervous system (CNS). However, until recently N-methyl-d-aspartate (NMDA) receptors were considered relatively stable once in the plasma membrane, especially in comparison with alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors that are internalized at a high rate. A series of recent studies has changed this viewpoint by revealing that NMDA receptors are subject to constitutive as well as agonist-induced internalization through clathrin-mediated endocytosis. Surprisingly, agonist-induced internalization is not dependent on current flow through the NMDA channel, and the receptors are primed for this type of internalization by selective stimulation of the glycine site but not of the glutamate site. Endocytosis of NMDA receptors provides a fundamental mechanism for dynamic regulation of the number of NMDA receptors at synapses, which might be important for physiological and pathological functioning of the CNS.     

NMDA receptors expressed in oligodendrocytes

Oligodendrocytes are known to express (Ca2+)-permeable glutamate receptors and to have low resistance to oxidative stress, two factors that make them potentially susceptible to injury. Oligodendrocyte injury is intrinsic to the loss of function experienced in conditions ranging from cerebral palsy to spinal cord injury, focal ischaemia and multiple sclerosis. NMDA receptors, a subtype of glutamate receptors, are vital to the remodeling of synaptic connections during postnatal development and associative learning abilities in adults and possibly in improvements in oligodendrocyte function. Previous studies had failed to detect NMDA receptor mRNA or current in oligodendrocytes but three new papers demonstrate NMDA receptor expression in oligodendrocytes and discuss its implications for ischaemia therapy.     

Ethanol sensitivity of NMDA receptors

NMDA receptors are ionotropic glutamate receptors assembled of subunits of the NR1 and of the NR2 family (NR2A–NR2D). The subunit diversity largely affects the pharmacological properties of NMDA receptors and, hence, gives rise to receptor heterogeneity. As an overall result of studies on recombinant and native NMDA receptors, ethanol inhibits the function of receptors containing the subunits NR2A and/or NR2B to a greater extent than those containing NR2C or NR2D. For example, in rat cultured mesencephalic neurons, NR2C expression was developmentally increased, whereas expression of NR2A and NR2B was decreased. These changes coincided with a developmental loss of sensitivity of NMDA responses to ethanol and ifenprodil, a non-competitive NMDA receptor antagonist that shows selectivity for NR2B-containing receptors. Also in rat locus coeruleus neurons, the low ethanol sensitivity of somatic NMDA receptors could be explained by a prominent expression of NR2C. The inhibitory site of action for ethanol on the NMDA receptor is not yet known. Patch–clamp studies suggest a target site exposed to or only accessible from the extracellular environment. Apparently, amino acid residue Phe⁶³⁹, located in the TM3 domain of NR1, plays a crucial role in the inhibition of NMDA receptor function by ethanol. Since this phenylalanine site is common to all NMDA and non-NMDA receptor (AMPA/kainate receptor) subunits, this observation is consistent with accumulating evidence for a similar ethanol sensitivity of a variety of NMDA and non-NMDA receptors, but it cannot explain the differences in ethanol sensitivity observed with different NR2 subunits.     

Induction of human beta-defensin-2 expression in human astrocytes by lipopolysaccharide and cytokines

Defensins are cationic peptides with broad-spectrum antimicrobial activity. They are members of a supergene family consisting of alpha and beta subtypes and each subtype is comprised of a number of different isoforms. For example, human alpha-defensin (HAD) has six isoforms, which are expressed by polymorphonuclear leukocytes and Paneth cells. In contrast, human beta-defensin (HBD) has two isoforms that are expressed by epithelial cells of the skin, gut, respiratory and urogenital tracts. Recently, HBD-1 was detected in human brain biopsy tissue. However, little is known about the expression of HBD-1 or HBD-2 in the CNS and whether neural cells can secrete these peptides. For the present study, human astrocyte, microglial, meningeal fibroblast and neuronal cultures were probed for the expression of HBD-1 and HBD-2 mRNA and protein. Each cell type was either maintained in tissue culture medium alone or in medium containing lipopolysaccharide (LPS) at concentrations ranging from 0.1 to 1 microgram/mL, interleukin-1 beta (IL-1beta) at 1-50 ng/mL, or tumor necrosis factor alpha (TNF-alpha) at the same concentrations. The expression of HBD-1 and HBD-2 mRNAs was monitored by RT-PCR. The cDNA products were sequenced to characterize the gene product. HBD-2 protein was detected by immunoblot, immunoprecipitation and immunocytochemistry. Results of these studies showed that HBD-1 mRNA was detected in all cell cultures except in those enriched for neurons. In contrast, HBD-2 mRNA was detected only in astrocyte cultures that were treated with LPS, IL-1beta or TNF-alpha. The detection of the respective proteins correlated positively with the mRNA results. As such, these data represent the first demonstration of HBD-2 expression by astrocytes and suggest that this peptide may play a role in host defense against bacterial CNS pathogenesis.     

NMDA receptor expression and roles in human articular chondrocyte mechanotransduction

Mechanical forces influence articular cartilage structure by regulating chondrocyte activity. Mechanical stimulation results in activation of an alpha5beta1 integrin dependent intracellular signal cascade involving focal adhesion kinase and protein kinase C, triggering the release of interleukin-4 from the cell. In normal HAC the response to physiological mechanical stimulation is characterised by increased levels of aggrecan mRNA and a decrease in levels of mRNA for matrix metalloproteinase 3 (MMP-3), the net result of which would be to maintain and optimise cartilage structure and function. This protective/anabolic response is not seen when chondrocytes from osteoarthritic cartilage are subjected to an identical mechanical stimulation regime. Following the observation that the neurotransmitter substance P is involved in chondrocyte mechanotransduction the present study was undertaken to establish potential roles for glutamate receptors in the control of chondrocyte mechanical responses. Using immunohistochemistry and RTPCR normal and OA chondrocytes are shown to express NR1 and NR2a subunits of the NMDA receptor. Addition of NMDA receptor agonists to chondrocytes in primary culture resulted in changes in membrane potential consistent with expression of functional receptors. NMDA receptor antagonists inhibited the hyperpolarisation response of normal chondrocytes to mechanical stimulation but had no effect on the depolarisation response of osteoarthritic chondrocytes to mechanical stimulation. These studies indicate that at least one subset of the NMDA receptor family of molecules is expressed in cartilage and may have important modulatory effects on mechanotransduction and cellular responses following mechanical stimulation. Indeed the results suggest that there is an alteration of NMDA receptor signalling in OA chondrocytes, which may be critical in the abnormal response of OA chondrocytes to mechanical stimulation. Thus NMDA receptors appear to be involved in the regulation of human articular chondrocyte responses to mechanical stimulation, and in OA, mechanotransduction pathways may be modified as a result of altered activation and function of these receptors.     

Extracellular mild acidosis decreases the Ca2+ permeability of the human NMDA receptors

NMDA receptors (NMDARs) are glutamate-gated ion channels involved in excitatory synaptic transmission and in others physiological processes such as synaptic plasticity and development. The overload of Ca²⁺ ions through NMDARs, caused by an excessive activation of receptors, leads to excitotoxic neuronal cell death. For this reason, the reduction of Ca²⁺ flux through NMDARs has been a central focus in finding therapeutic strategies to prevent neuronal cell damage.Extracellular H⁺ are allosteric modulators of NMDARs. Starting from previous studies showing that extracellular mild acidosis reduces NMDA-evoked whole cell currents, we analyzed the effects of this condition on the NMDARs Ca²⁺ permeability, measured as “fractional calcium current” (P_f, i.e. the percentage of the total current carried by Ca²⁺ ions), of human NMDARs NR1/NR2A and NR1/NR2B transiently transfected in HeLa cells. Extracellular mild acidosis significantly reduces P_f of both human NR1/NR2A and NR1/NR2B NMDARs, also decreasing single channel conductance in outside out patches for NR1/NR2A receptor. Reduction of Ca²⁺ flux through NMDARs was also confirmed in cortical neurons in culture. A comparative analysis of both NMDA evoked Ca²⁺ transients and whole cell currents showed that extracellular H⁺ differentially modulate the permeation of Na⁺ and Ca²⁺ through NMDARs.Our data highlight the synergy of two distinct neuroprotective mechanisms during acidosis: Ca²⁺ entry through NMDARs is lowered due to the modulation of both open probability and Ca²⁺ permeability. Furthermore, this study provides the proof of concept that it is possible to reduce Ca²⁺ overload in neurons modulating the NMDAR Ca²⁺ permeability.     

NMDA receptors in immune competent cells

This review presents analysis of literature data indicating the presence of NMDA-type glutamate receptors in several types of immune competent cells such as thymocytes, lymphocytes, and neutrophils. The possible role of these receptors in the function of these cells is discussed. The interaction of the receptors with certain ligands circulating in the blood-stream and their role in modulation of immune function is described. It is suggested that homocysteine acts as modulator of these receptors, and its toxicity is largely explained by hyperactivation of the NMDA-type glutamate receptors.     

Mechanism of human immunodeficiency virus-induced complement expression in astrocytes and neurons

The cerebral complement system is hypothesized to contribute to neurodegeneration in the pathogenesis of AIDS-associated neurological disorders. Our former results have shown that the human immunodeficiency virus (HIV) strongly induces the synthesis of complement factor C3 in astrocytes. This upregulation explains in vivo data showing elevated complement levels in the cerebrospinal fluid of patients with AIDS-associated neurological symptoms. Since inhibition of complement synthesis and activation in the brain may represent a putative therapeutic goal to prevent virus-induced damage, we analyzed in detail the mechanisms of HIV-induced modulation of C3 expression. HIV-1 increased the C3 levels in astrocyte culture supernatants from 30 to up to 400 ng/ml; signal transduction studies revealed that adenylate cyclase activation with upregulation of cyclic AMP is the central signaling pathway to mediate that increase. Furthermore, activity of protein kinase C is necessary for HIV induction of C3, since inhibition of protein kinase C by prolonged exposure to the phorbol ester tetradecanoyl phorbol acetate partly abolished the HIV effect. The cytokines tumor necrosis factor alpha and gamma interferon were not involved in mediating the HIV-induced C3 upregulation, since neutralizing antibodies had no effect. Besides whole HIV virions, the purified viral proteins Nef and gp41 are biologically active in upregulating C3, whereas Tat, gp120, and gp160 were not able to modulate C3 synthesis. Further experiments revealed that neurons were also able to respond on incubation with HIV with increased C3 synthesis, although the precise pattern was slightly different from that in astrocytes. This strengthens the hypothesis that HIV-induced complement synthesis represents an important mechanism for the pathogenesis of AIDS in the brain.