The development and growth of tissues derived from cranial neural crest and primitive mesoderm is dependent on the ligation status of retinoic acid receptor γ:evidence that retinoic acid receptor γ functions to maintain stem/progenitor cells in the absence of retinoic acid

Retinoic acid (RA) signaling is important to normal development. However, the function of the different RA receptors (RARs)-RARα, RARβ, and RARγ-is as yet unclear. We have used wild-type and transgenic zebrafish to examine the role of RARγ. Treatment of zebrafish embryos with an RARγ-specific agonist reduced somite formation and axial length, which was associated with a loss of hoxb13a expression and less-clear alterations in hoxc11a or myoD expression. Treatment with the RARγ agonist also disrupted formation of tissues arising from cranial neural crest, including cranial bones and anterior neural ganglia. There was a loss of Sox 9-immunopositive neural crest stem/progenitor cells in the same anterior regions. Pectoral fin outgrowth was blocked by RARγ agonist treatment. However, there was no loss of Tbx-5-immunopositive lateral plate mesodermal stem/progenitor cells and the block was reversed by agonist washout or by cotreatment with an RARγ antagonist. Regeneration of the caudal fin was also blocked by RARγ agonist treatment, which was associated with a loss of canonical Wnt signaling. This regenerative response was restored by agonist washout or cotreatment with the RARγ antagonist. These findings suggest that RARγ plays an essential role in maintaining stem/progenitor cells during embryonic development and tissue regeneration when the receptor is in its nonligated state.

A study of the regulatory role of retinoic acid receptor gamma in Zebrafish development

Retinoic acid (RA) is thought to signal through retinoic acid receptors (RARs), i.e. RARα, β, and γ to play important roles in embryonic development and tissue regeneration. In this thesis, the zebrafish (Danio rario) was used as a vertebrate model organism to examine the role of RARγ. Treatment of zebrafish embryos with a RARγ specific agonist reduced the axial length of developing embryos, associated with reduced somite number and loss of hoxb13a expression. There were no clear alterations in hoxc11a or myoD expression. Treatment with the RARγ agonist disrupted the formation of anterior structures of the head, the cranial bones and the anterior lateral line ganglia, associated with a loss of sox9 immunopositive cells in the same regions. Pectoral fin outgrowth was blocked by treatment with the RARγ agonist; however, this was not associated with loss of tbx5a immunopositive lateral plate cells and was reversed by wash out of the RARγ agonist or co-treatment with a RARγ antagonist. Regeneration of the transected caudal fin was also blocked by RARγ agonist treatment and restored by agonist washout or antagonist co-treatment; this phenotype was associated with a localised reduction in canonical Wnt signalling. Conversely, elevated canonical Wnt signalling after RARγ treatment was seen in other tissues, including ectopically in the notochord. Furthermore, some phenotypes seen in the RARγ treated embryos were present in mutant zebrafish embryos in which canonical Wnt signalling was constitutively increased. These data suggest that RARγ plays an essential role in maintaining neural crest and mesodermal stem/progenitor cells during normal embryonic development and tissue regeneration when the receptor is in its non-ligated state. In addition, this work has provided evidence that the activation status of RARγ may regulate hoxb13a gene expression and canonical Wnt signalling. Further research is required to confirm such novel regulatory roles.

Investigation of aspects of reactive gliosis in human astrocytoma cell lines:application for toxicity assessment

The process of astrogliosis, or reactive gliosis, is a typical response of astrocytes to a wide range of physical and chemical injuries. The up-regulation of the astrocyte specific glial fibrillary acidic protein (GFAP) is a hallmark of reactive gliosis and is widely used as a marker to identify the response. In order to develop a reliable, sensitive and high throughput astrocyte toxicity assay that is more relevant to the human response than existing animal cell based models, the U251-MG, U373-MG and CCF-STTG 1 human astrocytoma cell lines were investigated for their ability to exhibit reactive-like changes following exposure to ethanol, chloroquine diphosphate, trimethyltin chloride and acrylamide. Cytotoxicity analysis showed that the astrocytic cells were generally more resistant to the cytotoxic effects of the agents than the SH-SY5Y neuroblastoma cells. Retinoic acid induced differentiation of the SH-SY5Y line was also seen to confer some degree of resistance to toxicant exposure, particularly in the case of ethanol. Using a cell based ELISA for GFAP together with concurrent assays for metabolic activity and cell number, each of the three cell lines responded to toxicant exposure by an increase in GFAP immunoreactivity (GFAP-IR), or by increased metabolic activity. Ethanol, chloroquine diphosphate, trimethyltin chloride and bacterial lipopolysaccharide all induced either GFAP or MTT increases depending upon the cell line, dose and exposure time. Preliminary investigations of additional aspects of astrocytic injury indicated that IL-6, but not TNF-α. or nitric oxide, is released following exposure to each of the compounds, with the exception of acrylamide. It is clear that these human astrocytoma cell lines are capable of responding to toxicant exposure in a manner typical of reactive gliosis and are therefore a valuable cellular model in the assessment of in vitro neurotoxicity.

Transglutaminase 2 interaction with small heat shock proteins mediate cell survival upon excitotoxic stress

Transglutaminase 2 has been postulated to be involved in the pathogenesis of central nervous system neurodegenerative disorders. However, its role in neuronal cell death remains to be elucidated. Excitotoxicity is a common event underlying neurodegeneration. We aimed to evaluate the protein targets for transglutaminase 2 in cell response to NMDA-induced excitotoxic stress, using SH-SY5Y neuroblastoma cells which express high tranglutaminase 2 levels upon retinoic acid-driven differentiation toward neurons. NMDA-evoked calcium increase led to transglutaminase 2 activation that mediated cell survival, as at first suggested by the exacerbation of NMDA toxicity in the presence of R283, a synthetic competitive inhibitor of transglutaminase active site. Assays of R283-mediated transglutaminase inhibition showed the involvement of enzyme activity in NMDA-induced reduction in protein basal levels of pro-apoptotic caspase-3 and the stress protein Hsp20. However, this occurred in a way different from protein cross-linking, given that macromolecular assemblies were not observed in our experimental conditions for both proteins. Co-immunoprecipitation experiments provided evidence for the interaction, in basal conditions, between transglutaminase 2 and Hsp20, as well as between Hsp20 and Hsp27, a major anti-apoptotic protein promoting caspase-3 inactivation and degradation. NMDA treatment disrupted both these interactions that were restored upon transglutaminase 2 inhibition with R283. These results suggest that transglutaminase 2 might be protective against NMDA-evoked excitotoxic insult in neuronal-like SH-SY5Y cells in a way, independent from transamidation that likely involves its interaction with the complex Hsp20/Hsp27 playing a pro-survival role. © 2011 Springer-Verlag.

Structure-function analysis of amino acid 74 of human RAMP1 and RAMP3 and its role in peptide interactions with adrenomedullin and calcitonin gene-related peptide receptors

The receptors for calcitonin gene-related peptide (CGRP) and adrenomedullin (AM) are complexes of the calcitonin receptor-like receptor (CLR) and receptor activity-modifying proteins (RAMP). The CGRP receptor is a CLR/RAMP1 pairing whereas CLR/RAMP2 and CLR/RAMP3 constitute two subtypes of AM receptor: AM(1) and AM(2), respectively. Previous studies identified Glu74 in RAMP3 to be important for AM binding and potency. To further understand the importance of this residue and its equivalent in RAMP1 (Trp74) we substituted the native amino acids with several others. In RAMP3, these were Trp, Phe, Tyr, Ala, Ser, Thr, Arg and Asn; in RAMP1, Glu, Phe, Tyr, Ala and Asn substitutions were made. The mutant RAMPs were co-expressed with CLR in Cos7 cells; receptor function in response to AM, AM(2)/intermedin and CGRP was measured in a cAMP assay and cell surface expression was determined by ELISA. Phe reduced AM potency in RAMP3 but had no effect in RAMP1. In contrast, Tyr had no effect in RAMP3 but enhanced AM potency in RAMP1. Most other substitutions had a small effect on AM potency in both receptors whereas there was little impact on CGRP or AM(2) potency. Overall, these data suggest that the geometry and charge of the residue at position 74 contribute to how AM interacts with the AM(2) and CGRP receptors and confirms the role of this position in dictating differential AM pharmacology at the AM(2) and CGRP receptors.

The role of the 8-18 helix of CGRP 8-37 in mediating high affinity binding to CGRP receptors; coulombic and steric interactions

1. The role of individual residues in the 8-18 helix of CGRP 8-37 in promoting high-affinity binding to CGRP 1 receptors expressed on rat L6 and human SK-N-MC cells has been examined. The relative potencies of various derivatives were estimated from their ability to inhibit the human αCGRP-mediated increase in cyclic AMP production and the binding of [ 125I]-human αCGRP. 3. Arg 11 and Arg 18 were replaced by serines to give [Ser 11.18]CGRP 8-37. These bound with pKi values <6 to SK-N-MC cells and had apparent pA 2 values of 5.81 ± 0.04 and 5.31 ± 0.11 on SK-N-MC and L6 cells. CGRP 8-37 had a pKi of 8.22 on SK-N-MC cells and pK b values on the above cell lines of 8.95±0.04 and 8.76±0.04. 3. The arginines were replaced with glutamic acid residues. [Glu 11]CGRP 8-37 had a pK b of 7.14±0.14 on SK-N-MC cells (pKi=7.05±0.05) and 6.99±0.08 on L6 cells. [Glu 18]CGRP 8-37 had a pK b of 7.10±0.0.08 on SK-N-MC cells (pKi=6.91±0.23) and 7.12±0.09 on L6 cells. 4. Leu 12, Leu 15 and Leu 16 were replaced by benzoyl-phenylalanine (bpa) residues. On SK-N-MC cells, the apparent pA 2 values of [bpa 12]-, [bpa 15]- and [bpa 16]CGRP 8-37 were respectively 7.43±0.23, 8.34±0.11 and 5.66±0.16 (pKi values of 7.14±0.17, 7.66±0.21 and <6): on L6 cells they were 7.96±0.36, 8.28±0.21 and 6.09±0.04 (all n=3). 5. It is concluded that the Arg 11 and Arg 18 are involved in specific electrostatic interactions with other residues, either on the CGRP 1 receptors or elsewhere on CGRP 8-37. Leu 16 is in a conformationally restricted site when CGRP 8-37 binds to CGRP 1 receptors, unlike Leu 12 and Leu 15.

The role of cannabinoid receptors in modulation of GABAergic neurotransmission in the rat medial entorhinal cortex in vitro

Type 1 cannabinoid receptors (CB1R) have a well established role in modulating GABAergic signalling with the central nervous system, and are thought to be the only type present at GABAergic presynaptic terminals. In the medial entorhinal cortex (mEC), some cortical layers show high levels of ongoing GABAergic signalling (namely layer II) while others show relatively low levels (layer V). Using whole-cell patch clamp techniques, I have, for the first time, demonstrated the presence of functional CB1R in both deep and superficial layers of the mEC. Furthermore, using a range of highly specific ligands for both CB1R and CB2R, I present strong pharmacological evidence for CB2Rs being present in both deep and superficial layers of the mEC in the adult rat brain. In brain slices taken at earlier points in CNS development (P8-12), I have shown that while both CB1R and CB2R specific ligands do modulate GABAergic signalling at early developmental stages, antagonists/ inverse agonists and full agonists have similar effects, and serve only to reduce GABAergic signalling. These data suggest that the full cannabinoid signalling mechanisms at this early stage in synaptogenesis are not yet in place. During these whole-cell studies, I have developed and refined a novel recording technique, using an amantidine derivative (IEM1460) which allows inhibitory postsynaptic currents to be recorded under conditions in which glutamate receptors are not blocked and network activity remains high. Finally I have shown that bath applied CB1 and CB2 receptor antagonists/ inverse agonists are capable of modulating kainic acid induced persistent oscillatory activity in mEC. Inverse agonists suppressed oscillatory activity in the superficial layers of the mEC while it was enhanced in the deeper layers. It seems likely that cannabinoid receptors modulate the inhibitory neuronal activity that underlies network oscillations.

Non-neuronal, slow GABA signalling in the ventrobasal thalamus targets δ-subunit-containing GABA(A) receptors

The rodent ventrobasal (VB) thalamus contains a relatively uniform population of thalamocortical (TC) neurons that receive glutamatergic input from the vibrissae and the somatosensory cortex, and inhibitory input from the nucleus reticularis thalami (nRT). In this study we describe ?-aminobutyric acid (GABA)(A) receptor-dependent slow outward currents (SOCs) in TC neurons that are distinct from fast inhibitory postsynaptic currents (IPSCs) and tonic currents. SOCs occurred spontaneously or could be evoked by hypo-osmotic stimulus, and were not blocked by tetrodotoxin, removal of extracellular Ca(2+) or bafilomycin A1, indicating a non-synaptic, non-vesicular GABA origin. SOCs were more common in TC neurons of the VB compared with the dorsal lateral geniculate nucleus, and were rarely observed in nRT neurons, whilst SOC frequency in the VB increased with age. Application of THIP, a selective agonist at d-subunit-containing GABA(A) receptors, occluded SOCs, whereas the benzodiazepine site inverse agonist ß-CCB had no effect, but did inhibit spontaneous and evoked IPSCs. In addition, the occurrence of SOCs was reduced in mice lacking the d-subunit, and their kinetics were also altered. The anti-epileptic drug vigabatrin increased SOC frequency in a time-dependent manner, but this effect was not due to reversal of GABA transporters. Together, these data indicate that SOCs in TC neurons arise from astrocytic GABA release, and are mediated by d-subunit-containing GABA(A) receptors. Furthermore, these findings suggest that the therapeutic action of vigabatrin may occur through the augmentation of this astrocyte-neuron interaction, and highlight the importance of glial cells in CNS (patho) physiology.

Novel peptide antagonists of adrenomedullin and calcitonin gene-related peptide receptors:identification, pharmacological characterization, and interactions with position 74 in receptor activity-modifying protein 1/3

Human adrenomedullin (AM) is a 52-amino acid peptide belonging to the calcitonin peptide family, which also includes calcitonin gene-related peptide (CGRP) and AM2. The two AM receptors, AM(1) and AM(2), are calcitonin receptor-like receptor (CL)/receptor activity-modifying protein (RAMP) (RAMP2 and RAMP3, respectively) heterodimers. CGRP receptors comprise CL/RAMP1. The only human AM receptor antagonist (AM(22-52)) is a truncated form of AM; it has low affinity and is only weakly selective for AM(1) over AM(2) receptors. To develop novel AM receptor antagonists, we explored the importance of different regions of AM in interactions with AM(1), AM(2), and CGRP receptors. AM(22-52) was the framework for generating further AM fragments (AM(26-52) and AM(30-52)), novel AM/alphaCGRP chimeras (C1-C5 and C9), and AM/AM(2) chimeras (C6-C8). cAMP assays were used to screen the antagonists at all receptors to determine their affinity and selectivity. Circular dichroism spectroscopy was used to investigate the secondary structures of AM and its related peptides. The data indicate that the structures of AM, AM2, and alphaCGRP differ from one another. Our chimeric approach enabled the identification of two nonselective high-affinity antagonists of AM(1), AM(2), and CGRP receptors (C2 and C6), one high-affinity antagonist of AM(2) receptors (C7), and a weak antagonist selective for the CGRP receptor (C5). By use of receptor mutagenesis, we also determined that the C-terminal nine amino acids of AM seem to be responsible for its interaction with Glu74 of RAMP3. We provide new information on the structure-activity relationship of AM, alphaCGRP, and AM2 and how AM interacts with CGRP and AM(2) receptors.

Modulation of interleukin-6 and matrix metalloproteinase 2 expression in human fibroblast-like synoviocytes by functional ionotropic glutamate receptors

Objective. Patients with rheumatoid arthritis (RA) have increased concentrations of the amino acid glutamate in synovial fluid. This study was undertaken to determine whether glutamate receptors are expressed in the synovial joint, and to determine whether activation of glutamate receptors on human synoviocytes contributes to RA disease pathology. Methods. Glutamate receptor expression was examined in tissue samples from rat knee joints and in human fibroblast-like synoviocytes (FLS). FLS from 5 RA patients and 1 normal control were used to determine whether a range of glutamate receptor antagonists influenced expression of the proinflammatory cytokine interleukin-6 (IL-6), enzymes involved in matrix degradation and cytokine processing (matrix metalloproteinase 2 [MMP-2] and MMP-9), and the inhibitors of these enzymes (tissue inhibitor of metalloproteinases 1 [TIMP-1] and TIMP-2). IL-6 concentrations were determined by enzyme-linked immunosorbent assay, MMP activity was measured by gelatin zymography, and TIMP activity was determined by reverse zymography. Fluorescence imaging of intracellular calcium concentrations in live RA FLS stimulated with specific antagonists was used to reveal functional activation of glutamate receptors that modulated IL-6 or MMP-2. Results. Ionotropic and metabotropic glutamate receptor subunit mRNA were expressed in the patella, fat pad, and meniscus of the rat knee and in human articular cartilage. Inhibition of N-methyl-D-aspartate (NMDA) receptors in RA FLS increased proMMP-2 release, whereas non-NMDA ionotropic glutamate receptor antagonists reduced IL-6 production by these cells. Stimulation with glutamate, NMDA, or kainate (KA) increased intracellular calcium concentrations in RA FLS, demonstrating functional activation of specific ionotropic glutamate receptors. Conclusion. Our findings indicate that activation of NMDA and KA glutamate receptors on human synoviocytes may contribute to joint destruction by increasing IL-6 expression. © 2007, American College of Rheumatology.

GABAB receptor-mediated activation of astrocytes by gamma-hydroxybutyric acid

The gamma-aminobutyric acid (GABA) metabolite gamma-hydroxybutyric acid (GHB) shows a variety of behavioural effects when administered to animals and humans, including reward/addiction properties and absence seizures. At the cellular level, these actions of GHB are mediated by activation of neuronal GABAB receptors (GABABRs) where it acts as a weak agonist. Because astrocytes respond to endogenous and exogenously applied GABA by activation of both GABAA and GABABRs, here we investigated the action of GHB on astrocytes on the ventral tegmental area (VTA) and the ventrobasal (VB) thalamic nucleus, two brain areas involved in the reward and proepileptic action of GHB, respectively, and compared it with that of the potent GABABR agonist baclofen. We found that GHB and baclofen elicited dose-dependent (ED50: 1.6 mM and 1.3 µM, respectively) transient increases in intracellular Ca2+ in VTA and VB astrocytes of young mice and rats, which were accounted for by activation of their GABABRs and mediated by Ca2+ release from intracellular store release. In contrast, prolonged GHB and baclofen exposure caused a reduction in spontaneous astrocyte activity and glutamate release from VTA astrocytes. These findings have key (patho)physiological implications for our understanding of the addictive and proepileptic actions of GHB.

Differential impact of amino acid substitutions on critical residues of the human glucagon-like peptide-1 receptor involved in peptide activity and small-molecule allosterys

The glucagon-like peptide-1 receptor (GLP-1R) is a class B G protein-coupled receptor that has a critical role in the regulation of glucose homeostasis, principally through the regulation of insulin secretion. The receptor systemis highly complex, able to be activated by both endogenous [GLP-1(1-36)NH2, GLP-1(1-37), GLP-1(7-36)NH2, GLP-1(7-37), oxyntomodulin], and exogenous (exendin-4) peptides in addition to small-molecule allosteric agonists (compound 2 [6,7-dichloro-2-methylsulfonyl-3-tertbutylaminoquinoxaline], BETP [4-(3-benzyloxy)phenyl)-2-ethylsulfinyl-6-(trifluoromethyl)pyrimidine]). Furthermore, the GLP-1R is subject to single-nucleotide polymorphic variance, resulting in amino acid changes in the receptor protein. In this study, we investigated two polymorphic variants previously reported to impact peptidemediated receptor activity (M149) and small-molecule allostery (C333). These residues were mutated to a series of alternate amino acids, and their functionality was monitored across physiologically significant signaling pathways, including cAMP, extracellular signal-regulated kinase 1 and 2 phosphorylation, and intracellular Ca2+ mobilization, in addition to peptide binding and cell-surface expression. We observed that residue 149 is highly sensitive to mutation, with almost all peptide responses significantly attenuated at mutated receptors. However, most reductions in activity were able to be restored by the small-molecule allosteric agonist compound 2. Conversely, mutation of residue 333 had little impact on peptide-mediated receptor activation, but this activity could not be modulated by compound 2 to the same extent as that observed at the wild-type receptor. These results provide insight into the importance of residues 149 and 333 in peptide function and highlight the complexities of allosteric modulation within this receptor system.

Pyroligneous acid:a farmer’s flexible friend?

This article is a review of the findings of key studies into the potential benefits of pyroligneous acid, arising from charcoal production, to the agricultural industry. Through a review of bioassay studies conducted on known plant and human pathogens (e.g., Agrobacterium tumefacien and Xanthomonas campestris) and arthropods, and germination studies on selected crops, the article highlights a number of potential benefits of smoke recovery in the production of charcoal. In addition, the article calls for further research into the impact, if any, of its long-term use on environmental receptors/humans and for the development of a methodology to guarantee consistency in product composition, quality, and efficacy. In doing so, it is hoped that its widespread use as part of sustainable management practices adopted in the agricultural industry will be secured.

GPCR-styrene maleic acid lipid particles (GPCR-SMALPs):their nature and potential

G-protein-coupled receptors (GPCRs) form the largest class of membrane proteins and are an important target for therapeutic drugs. These receptors are highly dynamic proteins sampling a range of conformational states in order to fulfil their complex signalling roles. In order to fully understand GPCR signalling mechanisms it is necessary to extract the receptor protein out of the plasma membrane. Historically this has universally required detergents which inadvertently strip away the annulus of lipid in close association with the receptor and disrupt lateral pressure exerted by the bilayer. Detergent-solubilized GPCRs are very unstable which presents a serious hurdle to characterization by biophysical methods. A range of strategies have been developed to ameliorate the detrimental effect of removing the receptor from the membrane including amphipols and reconstitution into nanodics stabilized by membrane scaffolding proteins (MSPs) but they all require exposure to detergent. Poly(styrene-co-maleic acid) (SMA) incorporates into membranes and spontaneously forms nanoscale poly(styrene-co-maleic acid) lipid particles (SMALPs), effectively acting like a 'molecular pastry cutter' to 'solubilize' GPCRs in the complete absence of detergent at any stage and with preservation of the native annular lipid throughout the process. GPCR-SMALPs have similar pharmacological properties to membrane-bound receptor, exhibit enhanced stability compared with detergent-solubilized receptors and being non-proteinaceous in nature, are fully compatible with downstream biophysical analysis of the encapsulated GPCR.