长链非编码RNA NR2F1-AS1靶向微小RNA-145-5p调控结肠癌细胞增殖、迁移和侵袭的分子机制

目的 探讨长链非编码RNA(lnc RNA)核受体亚族2F组成员1反义RNA(NR2F1-AS1)对结肠癌细胞增殖、迁移和侵袭的影响及作用机制.方法 收集2015年3月至2017年5月青海省第五人民医院外科手术切除并经病理证实为原发性结肠腺癌组织25例,另留取距肿瘤边缘3 cm的癌旁组织(正常结肠组织).实时荧光定量P...     

Mutations at F637 in the NMDA receptor NR2A subunit M3 domain influence agonist potency, ion channel gating and alcohol action

BACKGROUND AND PURPOSE: NMDA receptors are important molecular targets of ethanol action in the CNS. Previous studies have identified a site in membrane-associated domain 3 (M3) of the NR1 subunit and two sites in M4 of the NR2A subunit that influence alcohol action; the sites in NR2A M4 also regulate ion channel gating. The purpose of this study was to determine whether mutations at the site in the NR2A subunit corresponding to the NR1 M3 site influence alcohol action and ion channel gating. EXPERIMENTAL APPROACH: We investigated the effects of mutations at phenylalanine (F) 637 of the NR2A subunit using whole-cell and single-channel patch-clamp electrophysiological recording in transiently-transfected HEK 293 cells. KEY RESULTS: Mutations at F637 in the NR2A subunit altered peak and steady-state glutamate EC(50) values, maximal steady-state to peak current ratios (I(ss):I(p)), mean open time, and ethanol IC(50) values. Differences in glutamate potency among the mutants were not due to changes in desensitization. Ethanol IC(50) values were significantly correlated with glutamate EC(50) values, but not with maximal I(ss):I(p) or mean open time. Ethanol IC(50) values were linearly and inversely related to molecular volume of the substituent. CONCLUSIONS AND IMPLICATIONS: These results demonstrate that NR2A(F637) influences NMDA receptor affinity, ion channel gating, and ethanol sensitivity. The changes in NMDA receptor affinity are likely to be the result of altered ion channel gating. In contrast to the cognate site in the NR1 subunit, the action of ethanol does not appear to involve occupation of a critical volume at NR2A(F637).     

Functional crosstalk of CAR-LXR and ROR-LXR in drug metabolism and lipid metabolism

Nuclear receptor crosstalk represents an important mechanism to expand the functions of individual receptors. The liver X receptors (LXR, NR1H2/3), both the α and β isoforms, are nuclear receptors that can be activated by the endogenous oxysterols and other synthetic agonists. LXRs function as cholesterol sensors, which protect mammals from cholesterol overload. LXRs have been shown to regulate the expression of a battery of metabolic genes, especially those involved in lipid metabolism. LXRs have recently been suggested to play a novel role in the regulation of drug metabolism. The constitutive androstane receptor (CAR, NR1I3) is a xenobiotic receptor that regulates the expression of drug-metabolizing enzymes and transporters. Disruption of CAR alters sensitivity to toxins, increasing or decreasing it depending on the compounds. More recently, additional roles for CAR have been discovered. These include the involvement of CAR in lipid metabolism. Mechanistically, CAR forms an intricate regulatory network with other members of the nuclear receptor superfamily, foremost the LXRs, in exerting its effect on lipid metabolism. Retinoid-related orphan receptors (RORs, NR1F1/2/3) have three isoforms, α, β and γ. Recent reports have shown that loss of RORα and/or RORγ can positively or negatively influence the expression of multiple drug-metabolizing enzymes and transporters in the liver. The effects of RORs on expression of drug-metabolizing enzymes were reasoned to be, at least in part, due to the crosstalk with LXR. This review focuses on the CAR-LXR and ROR-LXR crosstalk, and the implications of this crosstalk in drug metabolism and lipid metabolism.     

Subtype-selective antagonism of N-methyl-D-aspartate receptor ion channels by synthetic conantokin peptides

Conantokin-G (con-G), conantokin-T (con-T), a truncated conantokin-R (con-R[1-17]), that functions the same as wild-type con-R, and variant sequences of con-T, were chemically synthesized and employed to investigate their selectivities as antagonists of glutamate/glycine-evoked ion currents in human embryonic kidney-293 cells expressing various combinations of NMDA receptor (NMDAR) subunits (NR), viz., NR1a/2A, NR1a/2B, NR1b/2A and NR1b/2B. Con-G did not substantially affect ion flow into NR1a,b/NR2A-transfected cells, but potently inhibited cells expressing NR1a,b/NR2B, showing high NR2B selectivity. Con-T and con-R served as non-selective antagonists of all of four NMDAR subunit combinations. C-terminal truncation variants of the 21-residue con-T were synthesized and examined in this regard. While NMDAR ion channel antagonist activity, and the ability to adopt the Ca(2+)-induced alpha-helical conformation, diminished as a function of shortening the COOH-terminus of con-T, NMDAR subtype selectivity was enhanced in the con-T[1-11], con-T[1-9], and con-T[1-8] variants toward NR2A, NR1b, and NR1b/2A, respectively. Receptor subtype selectivity was also obtained with Met-8 sequence variants of con-T. Con-T[M8A] and con-T[M8Q] displayed selectivity with NR2B-containing subunits, while con-T[M8E] showed enhanced activity toward NR1b-containing NMDAR subtypes. Of those studied, the most highly selective variant was con-T[M8I], which showed maximal NMDAR ion channel antagonism activity toward the NR1a/2A subtype. These studies demonstrate that it is possible to engineer NMDAR subtype antagonist specificity into con-T. Since the subunit composition of the NMDAR varies temporally and spatially in developing brain and in various disease states, conantokins with high subtype selectivities are potentially valuable drugs that may be used at specific stages of disease and in selected regions of the brain.     

Regulation of NMDA receptor subunit expression and its implications for LTD, LTP, and metaplasticity

NMDA-type glutamate receptors (NMDARs) mediate many forms of synaptic plasticity. These tetrameric receptors consist of two obligatory NR1 subunits and two regulatory subunits, usually a combination of NR2A and NR2B. In the neonatal neocortex NR2B-containing NMDARs predominate, and sensory experience facilitates a developmental switch in which NR2A levels increase relative to NR2B. In this review, we clarify the roles of NR2 subunits in synaptic plasticity, and argue that a primary role of this shift is to control the threshold, rather than determining the direction, for modifying synaptic strength. We also discuss recent studies that illuminate the mechanisms regulating NR2 subunits, and suggest that the NR2A/NR2B ratio is regulated by multiple means, which may control the ratio both locally at individual synapses and globally in a cell-wide manner. Finally, we use the visual cortex as a model system to illustrate how activity-dependent modifications in the NR2A/NR2B ratio may contribute to the development of cortical functions.     

GABAergic disinhibition induced pain hypersensitivity by upregulating NMDA receptor functions in spinal dorsal horn

Intense noxious stimuli impair GABAergic inhibition in spinal dorsal horn, which has been proposed as a critical contributor to pathological pain. However, how the reduced inhibition exacerbates the transfer of nociceptive information at excitatory glutamatergic synapses is still poorly understood. The present study demonstrated that one of the striking consequences of GABAergic disinhibition was to enhance the function of N-methyl-D-aspartate subtype glutamate receptors (NMDARs), a well-characterized player in central sensitization. We found that intrathecal application of bicuculline, a GABA_A receptor antagonist, to remove the inhibition readily elicited mechanical allodynia in naive mice, which could be dose-dependently attenuated by NMDARs antagonist D-APV. Biochemical analysis demonstrated that bicuculline did not affect the total expression levels of the obligatory NMDARs subunit NR1 and the regulatory subunit NR2A and NR2B. However, bicuculline promoted NR1 phosphorylation at Serine 897 (NR1-S897) by cAMP-dependent protein kinase (PKA). This PKA-mediated phosphorylation incorporated NR1 along with NR2B into synapses. When PKA inhibitor H-89 was intrathecally applied, it totally eliminated bicuculline-induced NMDARs phosphorylation, synaptic redistribution as well as pain sensitization. Importantly, the reduced inhibition also operated to enhance NMDARs functions after peripheral inflammation, because spinal injection of diazepam to rescue the inhibition in inflamed mice greatly depressed PKA phosphorylation of NR1-S897, reduced the synaptic concentration of NR1/NR2B and meanwhile, alleviated the inflammatory pain. These data suggested that removal of GABAergic inhibition allowed for PKA-mediated NMDARs phosphorylation and synaptic accumulation, thus exaggerating NMDARs-dependent nociceptive transmission and behavioral sensitization.     

Evolution and function of the NR1I nuclear hormone receptor subfamily (VDR, PXR, and CAR) with respect to metabolism of xenobiotics and endogenous compounds

The NR1I subfamily of nuclear hormone receptors includes the 1,25-(OH)(2)-vitamin D(3) receptor (VDR; NR1I1), pregnane X receptor (PXR; NR1I2), and constitutive androstane receptor (CAR; NR1I3). PXR and VDR are found in diverse vertebrates from fish to mammals while CAR is restricted to mammals. Current evidence suggests that the CAR gene arose from a duplication of an ancestral PXR gene, and that PXR and VDR arose from duplication of an ancestral gene, represented now by a single gene in the invertebrate Ciona intestinalis. Aside from the high-affinity effects of 1,25-(OH)(2)-vitamin D(3) on VDRs, the NR1I subfamily members are functionally united by the ability to bind potentially toxic endogenous compounds with low affinity and initiate changes in gene expression that lead to enhanced metabolism and elimination (e.g., induction of cytochrome P450 3A4 expression in humans). The detoxification role of VDR seems limited to sensing high concentrations of certain toxic bile salts, such as lithocholic acid, whereas PXR and CAR have the ability to recognize structurally diverse compounds. PXR and CAR show the highest degree of cross-species variation in the ligand-binding domain of the entire vertebrate nuclear hormone receptor superfamily, suggesting adaptation to species-specific ligands. This review examines the insights that phylogenetic and experimental studies provide into the function of VDR, PXR, and CAR, and how the functions of these receptors have expanded to evolutionary advantage in humans and other animals.     

Synthesis and pharmacology of N1-substituted piperazine-2,3-dicarboxylic acid derivatives acting as NMDA receptor antagonists

The binding site for competitive NMDA receptor antagonists is on the NR2 subunit, of which there are four types (NR2A-D). Typical antagonists such as (R)-AP5 have a subunit selectivity of NR2A > NR2B > NR2C > NR2D. The competitive NMDA receptor antagonist (2R,3S)-(1-biphenylyl-4-carbonyl)piperazine-2,3-dicarboxylic acid (PBPD, 16b) displays an unusual selectivity with improved relative affinity for NR2C and NR2D vs NR2A and NR2B. Analogues of 16b bearing aroyl or aryl substituents attached to the N(1) position of piperazine-2,3-dicarboxylic acid have been synthesized to probe the structural requirements for NR2C/NR2D selectivity. A phenanthrenyl-2-carbonyl analogue, 16e, had >60-fold higher affinity for NR2C and NR2D and showed 3-5-fold selectivity for NR2C/NR2D vs NR2A/NR2B. The phenanthrenyl-3-carbonyl analogue (16f) was less potent but more selective, having 5- and 7-fold selectivity for NR2D vs NR2A and NR2B, respectively. Thus, antagonists bearing bulky hydrophobic residues have a different NR2 subunit selectivity than that of typical antagonists.     

Synthesis and evaluation of an 18F‐labeled derivative of F3 for targeting surface‐expressed nucleolin in cancer and tumor endothelial cells

The surface overexpression of nucleolin provides an anchor for the specific attachment of biomolecules to cancer and angiogenic endothelial cells. The peptide F3 is a high‐affinity ligand of the nucleolin receptor (NR) that has been investigated as a carrier to deliver biologically active molecules to tumors for both therapeutic and imaging applications. A site‐specific PEGylated F3 derivative was radiolabeled with [¹⁸F]Al‐F. The binding affinity and cellular distribution of the compound was assessed in tumor (H2N) and tumor endothelial (2H‐11) cells. Specific uptake via the NR was demonstrated by the siRNA knockdown of nucleolin in both cell lines. The partition and the plasma stability of the compound were assessed at 37°C. The enzyme‐mediated site‐specific modification of F3 to give NODA‐PEG‐F3 (NP‐F3) was achieved. Radiolabeling with [¹⁸F]Al‐F gave ¹⁸F‐NP‐F3. ¹⁸F‐NP‐F3 demonstrated high affinity for cancer and tumor endothelial cells. The siRNA knockdown of nucleolin resulted in a binding affinity reduction of 50% to 60%, confirming cell surface binding via the NR. NP‐F3 was stable in serum for 2 h. ¹⁸F‐NP‐F3 is reported as the first ¹⁸F‐labeled F3 derivative. It was obtained in a site‐specific, high‐yield, and efficient manner and binds to surface NR in the low nanomolar range, suggesting it has potential as a tumor and angiogenesis tracer.     

Subunit-specific agonist activity at NR2A-, NR2B-, NR2C-, and NR2D-containing N-methyl-D-aspartate glutamate receptors

The four N-methyl-d-aspartate (NMDA) receptor NR2 subunits (NR2A-D) have different developmental, anatomical, and functional profiles that allow them to serve different roles in normal and neuropathological situations. Identification of subunit-selective NMDA receptor agonists, antagonists, or modulators could prove to be both valuable pharmacological tools as well as potential new therapeutic agents. We evaluated the potency and efficacy of a wide range of glutamate-like compounds at NR1/NR2A, NR1/NR2B, NR1/NR2C, and NR1/NR2D receptors. Twenty-five of 53 compounds examined exhibited agonist activity at the glutamate binding site of NMDA receptors. Concentration-response relationships were determined for these agonists at each NR2 subunit. We find consistently higher potency at the NR2D subunit for a wide range of dissimilar structures, with (2S,4R)-4-methylglutamate (SYM2081) showing the greatest differential potency between NR2A- and NR2D-containing receptors (46-fold). Analysis of chimeric NR2A/D receptors suggests that enhanced agonist potency for NR2D is controlled by residues in both of the domains (Domain1 and Domain2) that compose the bilobed agonist binding domain. Molecular dynamics (MD) simulations comparing a crystallography-based hydrated NR1/NR2A model with a homology-based NR1/NR2D hydrated model of the agonist binding domains suggest that glutamate exhibits a different binding mode in NR2D compared with NR2A that accommodates a 4-methyl substitution in SYM2081. Mutagenesis of functionally divergent residues supports the conclusions drawn based on the modeling studies. Despite high homology and conserved atomic contact residues within the agonist binding pocket of NR2A and NR2D, glutamate adopts a different binding orientation that could be exploited for the development of subunit selective agonists and competitive antagonists.     

Role of NMDA receptor functional domains in excitatory cell death

The mechanisms by which the NMDA receptor (NMDAR) induces excitotoxicity were investigated using a novel assay. We quantitated the capacity of wild type and mutant receptors for cell killing in CHO cells and cultured cortical neurons by measuring the activity of a co-transfected firefly luciferase expression plasmid. NR1 subunit pore mutations that block Ca(2+) influx, and deletion of the NR1 cytoplasmic C-terminal domain, which functions in Ca(2+) regulation of receptor currents, decreased NMDAR mediated cell killing. We also transfected the NR1 pore mutants and C-terminal truncations in the presence of co-expressed exogenous wild type subunits. The pore and C-terminal truncation mutants acted in a dominant negative fashion and increased the survival of NMDAR-expressing CHO cells. Although physiological studies of similar NMDA receptor mutants have been carried out in heterologous cell lines, their functions in neurons remain relatively unknown. We show that expression of pore mutants and specific C terminal truncation mutants in cultured cortical neurons also exerts dominant negative function and protects these primary cells from endogenous receptor induced excitotoxic death. These results implicate positive actions of the selectivity filter and of the NR1 C-terminal domain in a Ca(2+)-dependent mechanism for NMDAR excitotoxicity. They also indicate that the mutant receptors which show diminished excitotoxicity and dominant negative action in heterologous cells can co-assemble with endogenous subunits in primary neurons and block NMDAR-dependent excitotoxic death.     

Role of altered structure and function of NMDA receptors in development of alcohol dependence

Long-term alcohol exposure gives rise to development of physical dependence on alcohol in consequence of changes in certain neurotransmitter functions. Accumulating evidence suggests that the glutamatergic neurotransmitter system, especially the N-methyl-D-aspartate (NMDA) type of glutamate receptors is a particularly important site of ethanol's action, since ethanol is a potent inhibitor of the NMDA receptors (NMDARs) and prolonged ethanol exposition leads to a compensatory "upregulation" of NMDAR mediated functions supposedly contributing to the occurrence of ethanol tolerance, dependence as well as the acute and delayed signs of ethanol withdrawal.Recently, expression of different types of NMDAR subunits was found altered after long-term ethanol exposure. Especially, the expression of the NR2B and certain splice variant forms of the NR1 subunits were increased in primary neuronal cultures treated intermittently with ethanol. Since NMDA ion channels with such an altered subunit composition have increased permeability for calcium ions, increased agonist sensitivity, and relatively slow closing kinetics, the abovementioned alterations may underlie the enhanced NMDAR activation observed after long-term ethanol exposure. In accordance with these changes, the inhibitory potential of NR2B subunit-selective NMDAR antagonists is also increased, demonstrating excellent potency against alcohol withdrawal-induced in vitro cytotoxicity. Although in vivo data are few with these compounds, according to the effectiveness of the classic NMDAR antagonists in attenuation, not only the physical symptoms, but also some affective and motivational components of alcohol withdrawal, novel NR2B subunit selective NMDAR antagonists may offer a preferable alternative in the pharmacotherapy of alcohol dependence.     

Subunit specificity and mechanism of action of NMDA partial agonist D-cycloserine

Recently, we have shown that 1-aminocyclopropanecarboxylic acid (ACPC) acts simultaneously as a high affinity full glycine agonist and a low affinity glutamate site competitive antagonist for NMDA receptor channels. In this paper, we have attempted to determine the subunit specificity and mechanism of action of a different putative cyclic partial agonist, D-cycloserine (DCS). NMDA receptor currents were measured utilizing the two-electrode voltage clamp technique on Xenopus oocytes injected with NR1-1a cRNA and either NR2A, NR2B or NR2C cRNA. Efficacies of DCS were 35-68% of glycine controls for channels containing NR1-1a and NR2A or NR2B subunits, but channels containing NR2C subunits had efficacies greater than glycine controls (192%). Unlike ACPC, DCS efficacy does not increase with increasing NMDA concentration; however, the lowered efficacy elicited by DCS results solely through its interaction with the glycine binding site. The efficacy of DCS was pH sensitive for NR2A or NR2B-containing channels, but not for channels containing NR2C. From this, we suggest that the protonated and deprotonated forms of DCS when bound, probably open NMDA channels with different efficiency. Two models compatible with these results are presented.     

Metabolism and functional roles of endogenous D-serine in mammalian brains

It has now been well established that D-serine, a coagonist for the N-methyl-D-aspartate (NMDA) glutamate receptors (NR1/NR2 type), is maintained at a high concentration in mammalian brains for life and shows a brain-selective and NMDA receptor R2B subunit-related distribution, overturning the hitherto generally accepted theory that D-amino acid is not always present in mammalian tissues. D-Serine in the brain has been shown to be contained in both the glia and neurons and to have specific processes of biosynthesis, extracellular release, uptake, and degradation. Moreover, the selective elimination of D-serine reduces the NMDA receptor-mediated intracellular signaling and long-term potentiation of synaptic connections. Together with the anti-psychotic and anti-ataxic property of D-serine and the pivotal roles of the NMDA receptor in divergent higher brain functions, these observations support the view that the D-amino acid may be involved as an endogenous modulator for the NMDA receptor in various neuropsychiatric functions and their pathological conditions.