Effect of dopamine on synchronous neuronal oscillations in the globus pallidus-subthalamic nucleus network

Changes in the pattern of activity of neurones within the basal ganglia are relevant in the pathophysiology and symptoms of Parkinson’s disease. The globus pallidus (GP) – subthalamic nucleus (STN) network has been proposed to form a pacemaker driving regenerative synchronous bursting activity. In order to test whether this activity can be sustained in vitro a 20o parasagittal slice of mouse midbrain was developed which preserved functional connectivity between the STN and GP. Mouse STN and GP cells were characterised electrophysiologically by the presence or absence of a voltage sag in response to hyperpolarising current steps indicative of Ih and the presence of rebound depolarisations. The presence of evoked and spontaneous post-synaptic GABA and glutamatergic currents indicated functional connectivity between the STN and GP. In control slices, STN cells fired action potentials at a regular rate, activity which was unaffected by bath application of the GABAA receptor antagonist picrotoxin (50 μM) or the glutamate receptor antagonist CNQX (10 μM). Paired extracellular recordings of STN cells showed uncorrelated firing. Oscillatory burst activity was induced pharmacologically using the glutamate receptor agonist, NMDA (20 μM), in combination with the potassium channel blocker apamin (50 -100 nM). The burst activity was unaffected by bath application of picrotoxin or CNQX while paired STN recordings showed uncorrelated activity indicating that the activity is not produced by the neuronal network. Thus, no regenerative activity is evident in this mouse brain preparation, either in control slices or when bursting is pharmacologically induced, suggesting the requirement of other afferent inputs that are not present in the slice. Using single-unit extracellular recording, dopamine (30 μM) produced an excitation of STN cells. This excitation was independent of synaptic transmission and was mimicked by both the Dl-like receptor agonist SKF38393 (10 μM) and the D2-like receptor agonist quinpirole (10 μM). However, the excitation was partially reduced by the D1-like antagonist SCH23390 (2 μM) but not by the D2-like antagonists sulpiride (10 μM) and eticlopride (10 μM). Using whole-recordings, dopamine was shown to induce membrane depolarisation. This depolarisation was caused either by a D1-like receptor mediated increase in a conductance which reversed at -34 mV, consistent with a non-specific cation conductance, or a D2-like receptor mediated decrease in conductance which reversed around -100 mV, consistent with a potassium conductance. Bath application of dopamine altered the pattern of the burst-firing produced by NMDA an apamin towards a more regular pattern. This effect was associated with a decrease in amplitude and ll1crease in frequency of TTX-resistant plateau potentials which underlie the burst activity.

Mediators of gram-positive septic shock

Septic shock can occur as a result of Gram-negative or Gram-positive infection and involves a complex interaction between bacterial factors and the host immune system producing a systemic inflammatory state that may progress to multiple organ failure and death. Gram-positive bacteria are increasingly becoming more prevalent especially Staphylococcus epidermidis in association with indwelling devices. Lipopolysaccaride (LPS) is the key Gram-negative component involved in this process, but it is not clear which components of Gram-positive bacteria are responsible for progression of this often fatal disease. The aim of this thesis was to investigate the effect of bacterial components on the immune systems. Lipid S, a short chain form of lipoteichoic acid (LTA) found to be excreted from bacteria during growth in culture medium was examined along with other Gram-positive cell wall components: LTA, peptidoglycan (PG) and wall teichoic acids (WTA) and LPS from Gram-negative bacteria. Lipid S, LTA, PG and LPS but not WTA all stimulated murine macrophages and cell lines to produce significant amounts of NO, TNF-a, IL-6 and IL-1 and would induce fever and tissue damage seen in inflammatory diseases. Lipid S proved to be the most potent out of the Gram-positive samples tested. IgG antibodies in patients serum were found to bind to and cross react with lipid S and LTA. Anti-inflammatory antibiotics, platelet activating factor (PAF), PAF receptor antagonists and monoclonal antibodies (mAbs) directed to LTA, CD14 and toll-like receptors were utilised to modulate cytokine and NO production. In cell culture the anti-LTA and the anti-CD14 mAbs failed to markedly attenuate the production of NO, TNF-a, IL-6 or IL-1, the anti-TLR4 antibody did greatly inhibit the ability of LPS to stimulate cytokine production but not lipid S. The tetracyclines proved to be the most effective compounds, many were active at low concentrations and showed efficacy to inhibit both lipid S and LPS stimulated macrophages to produce NO.

An insight into the activation mechanism of the calcatonin-gene-related peptide receptor

The calcitonin-gene- related peptide (CGRP) receptor is unique among G-protein coupled receptors (GPCRs) as it consists of at least three proteins: calcitonin receptor like receptor (CLR), receptor activity modifying protein (RAMP)1 and receptor component protein (RCP). An endogenous agonist for this curious receptor is aCGRP, which is a sensory nerve-derived peptide made up of 37 amino acids. aCGRP acts as a potent vasodilator having pronounced effects on arterioles and capillaries. Understanding the pharmacodynamics of the CGRP receptor may have pharmaceutical benefit as the receptor has been associated with the onset of migraines and implicated in Raynauds syndrome. The primary aim of this thesis was to identify functionally important residues in the extracellular face of the CGRP receptor. Three areas of interest were selected including the extreme N-terminus of the CLR, extracellular loop 1 (ECL1) of the CLR and its associated transmembrane (TM) regions, and finally extracellular loop 3 (ECL3) of the CLR and its juxtamembrane regions. A site-directed mutagenesis (SDM) strategy was used to investigate these regions, primarily substituting the innate residues of CLR with alanine and assessing the mutation on multiple criteria including a functional cAMP assay, cell-surface expression, total expression, agonist-mediated internalisation and aCGRP binding. The results are interpreted and discussed taking into consideration contemporary concepts surrounding Secretin-like GPCRs. Moreover, the thesis also contains details of RAMP purification. Overall the thesis provides novel data that furthers insight into the complex phenomenon of CGRP receptor activation. Site-directed mutants have been identified that affect aCGRP binding, receptor signal transduction, the CLR/RAMP1 interface and the integrity of the protein complex structure.

Physiological effects of oxidized phospholipids and their cellular signaling mechanisms in inflammation

Oxidized phospholipids, such as the products of the oxidation of 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine by nonenzymatic radical attack, are known to be formed in a number of inflammatory diseases. Interest in the bioactivity and signaling functions of these compounds has increased enormously, with many studies using cultured immortalized and primary cells, tissues, and animals to understand their roles in disease pathology. Initially, oxidized phospholipids were viewed largely as culprits, in line with observations that they have proinflammatory effects, enhancing inflammatory cytokine production, cell adhesion and migration, proliferation, apoptosis, and necrosis, especially in vascular endothelial cells, macrophages, and smooth muscle cells. However, evidence has emerged that these compounds also have protective effects in some situations and cell types; a notable example is their ability to interfere with signaling by certain Toll-like receptors (TLRs) induced by microbial products that normally leads to inflammation. They also have protective effects via the stimulation of small GTPases and induce up-regulation of antioxidant enzymes and cytoskeletal rearrangements that improve endothelial barrier function. Oxidized phospholipids interact with several cellular receptors, including scavenger receptors, platelet-activating factor receptors, peroxisome proliferator-activated receptors, and TLRs. The various and sometimes contradictory effects that have been observed for oxidized phospholipids depend on their concentration, their specific structure, and the cell type investigated. Nevertheless, the underlying molecular mechanisms by which oxidized phospholipids exert their effects in various pathologies are similar. Although our understanding of the actions and mechanisms of these mediators has advanced substantially, many questions do remain about their precise interactions with components of cell signaling pathways.

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.

Adrenomedullin and calcitonin gene-related peptide receptors in endocrine-related cancers:opportunities and challenges

Adrenomedullin (AM), adrenomedullin 2 (AM2/intermedin) and calcitonin gene-related peptide (CGRP) are members of the calcitonin family of peptides. They can act as growth or survival factors for a number of tumours, including those that are endocrine-related. One mechanism through which this occurs is stimulating angiogenesis and lymphangiogenesis. AM is expressed by numerous tumour types and for some cancers, plasma AM levels can be correlated with the severity of the disease. In cancer models, lowering AM content or blocking AM receptors can reduce tumour mass. AM receptors are complexes formed between a seven transmembrane protein, calcitonin receptor-like receptor and one of the two accessory proteins, receptor activity-modifying proteins (RAMPs) 2 or 3 to give the AM1 and AM2 receptors respectively. AM also has affinity at the CGRP receptor, which uses RAMP1. Unfortunately, due to a lack of selective pharmacological tools or antibodies to distinguish AM and CGRP receptors, the precise receptors and signal transduction pathways used by the peptides are often uncertain. Two other membrane proteins, RDC1 and L1/G10D (the 'ADMR'), are not currently considered to be genuine CGRP or AM receptors. In order to properly evaluate whether AM or CGRP receptor inhibition has a role in cancer therapy, it is important to identify which receptors mediate the effects of these peptides. To effectively distinguish AM1 and AM2 receptors, selective receptor antagonists need to be developed. The development of specific CGRP receptor antagonists suggests that this is now feasible.

The activation of the CGRP receptor

The CGRP (calcitonin gene-related peptide) receptor is a family B GPCR (G-protein-coupled receptor). It consists of a GPCR, CLR (calcitonin receptor-like receptor) and an accessory protein, RAMP1 (receptor activity modifying protein 1). RAMP1 is needed for CGRP binding and also cell-surface expression of CLR. CLR is an example of a family B GPCR. Unlike family A GPCRs, little is known about how these receptors are activated by their endogenous ligands. This review considers what is known about the activation of family B GPCRs and then considers how this might be applied to CLR, particularly in light of new knowledge of the crystal structures of family A GPCRs.

The role of ECL2 in CGRP receptor activation:a combined modelling and experimental approach

The calcitonin gene-related peptide (CGRP) receptor is a complex of a cal-citonin receptor-like receptor (CLR), which is a family B G-protein-coupled receptor (GPCR) and receptor activity modifying protein 1. The role of the second extracellular loop (ECL2) of CLR in binding CGRP and coupling to Gs was investigated using a combination of mutagenesis and modelling. An alanine scan of residues 271-294 of CLR showed that the ability of CGRP to produce cAMP was impaired by point mutations at 13 residues; most of these also impaired the response to adrenomedullin (AM). These data were used to select probable ECL2-modelled conformations that are involved in agonist binding, allowing the identification of the likely contacts between the peptide and receptor. The implications of the most likely structures for receptor activation are discussed. © 2013 The Authors.

Direct evidence for endothelial vascular endothelial growth factor receptor-1 function in nitric oxide-mediated angiogenesis

Vascular endothelial growth factor-A (VEGF) is critical for angiogenesis but fails to induce neovascularization in ischemic tissue lesions in mice lacking endothelial nitric oxide synthase (eNOS). VEGF receptor-2 (VEGFR-2) is critical for angiogenesis, although little is known about the precise role of endothelial VEGFR-1 and its downstream effectors in this process. Here we have used a chimeric receptor approach in which the extracellular domain of the epidermal growth factor receptor was substituted for that of VEGFR-1 (EGLT) or VEGFR-2 (EGDR) and transduced into primary cultures of human umbilical vein endothelial cells (HUVECs) using a retroviral system. Activation of HUVECs expressing EGLT or EGDR induced rapid phosphorylation of eNOS at Ser1177, release of NO, and formation of capillary networks, similar to VEGF. Activation of eNOS by VEGFR-1 was dependent on Tyr794 and was mediated via phosphatidylinositol 3-kinase, whereas VEGFR-2 Tyr951 was involved in eNOS activation via phospholipase Cgamma1. Consistent with these findings, the VEGFR-1-specific ligand placenta growth factor-1 activated phosphatidylinositol 3-kinase and VEGF-E, which is selective for VEGFR-2-activated phospholipase Cgamma1. Both VEGFR-1 and VEGFR-2 signal pathways converged on Akt, as dominant-negative Akt inhibited the NO release and in vitro tube formation induced following activation of EGLT and EGDR. The identification Tyr794 of VEGFR-1 as a key residue in this process provides direct evidence of endothelial VEGFR-1 in NO-driven in vitro angiogenesis. These studies provide new sites of modulation in VEGF-mediated vascular morphogenesis and highlight new therapeutic targets for management of vascular diseases.

Vascular endothelial growth factor-induced endothelial cell proliferation is regulated by interaction between VEGFR-2, SH-PTP1 and eNOS

VEGF receptor-2 plays a critical role in endothelial cell proliferation during angiogenesis. However, regulation of receptor activity remains incompletely explained. Here, we demonstrate that VEGF stimulates microvascular endothelial cell proliferation in a dose-dependent manner with VEGF-induced proliferation being greatest at 5 and 100 ng/ml and significantly reduced at intermediate concentrations (>50% at 20 ng/ml). Neutralization studies confirmed that signaling occurs via VEGFR-2. In a similar fashion, ERK/MAPK is strongly activated in response to VEGF stimulation as demonstrated by its phosphorylation, but with a decrease in phosphoryation at 20 ng/ml VEGF. Immunoblotting analysis revealed that VEGF did not cause a dose-dependent change in expression of VEGFR-2 but instead resulted in reduced phosphorylation of VEGFR-2 when cells were exposed to 10 and 20 ng/ml of VEGF. VEGFR-2 dephosphorylation was associated with an increase in the protein tyrosine phosphatase, SH-PTP1, and endothelial nitric oxide synthase (eNOS). Immunoprecipitation and selective immunoblotting confirmed the association between VEGFR-2 dephosphorylation and the upregulation of SH-PTP1 and eNOS. Transfection of endothelial cells with antisense oligonucleotide against VEGFR-2 completely abolished VEGF-induced proliferation, whereas anti SH-PTP1 dramatically increased VEGF-induced proliferation by 1 and 5-fold at 10 and 200 ng/ml VEGF, respectively. Suppression of eNOS expression only abolished endothelial cell proliferation at VEGF concentrations above 20 ng/ml. Taken together, these results indicate that activation of VEGFR-2 by VEGF enhances SH-PTP1 activity and eNOS expression, which in turn lead to two diverse events: one is that SH-PTP1 dephosphorylates VEGFR-2 and ERK/MAPK, which weaken VEGF mitogenic activity, and the other is that eNOS increases nitric oxide production which in turn lowers SH-PTP1 activity via S-nitrosylation.

Heterodimers and family-B GPCRs:RAMPs, CGRP and adrenomedullin

RAMPs (receptor activity-modifying proteins) are single-pass transmembrane proteins that associate with certain family-B GPCRs (G-protein-coupled receptors). Specifically for the CT (calcitonin) receptor-like receptor and the CT receptor, this results in profound changes in ligand binding and receptor pharmacology, allowing the generation of six distinct receptors with preferences for CGRP (CT gene-related peptide) adrenomedullin, amylin and CT. There are three RAMPs: RAMP1-RAMP3. The N-terminus appears to be the main determinant of receptor pharmacology whereas the transmembrane domain contributes to association of the RAMP with the GPCR. The N-terminus of all members of the RAMP family probably contains two disulphide bonds; a potential third disulphide is found in RAMP1 and RAMP3. The N-terminus appears to be in close proximity to the ligand and plays a key role in its binding, either directly or indirectly. BIBN4096BS, a CGRP antagonist, targets RAMP1 and this gives the compound very high selectivity for the human CGRP(1) receptor.

Interaction of calcitonin-gene-related peptide with its receptors

The receptor for calcitonin-gene-related peptide (CGRP) is a heterodimer formed by calcitonin-receptor-like receptor (CRLR), a type II (family B) G-protein-coupled receptor, and receptor-activity-modifying protein 1 (RAMP1), a single-membrane-pass protein. It is likely that the first seven or so amino acids of CGRP (which form a disulphide-bonded loop) interact with the transmembrane domain of CRLR to cause receptor activation. The rest of the CGRP molecule falls into three domains. Residues 28-37 and 8-18 are normally required for high-affinity binding, while residues 19-27 form a hinge region. The 28-37 region is almost certainly in direct contact with the receptor; 8-18 may make additional receptor contacts or may stabilize an appropriate conformation of 28-37. It is likely that these regions of CGRP interact both with CRLR and with the extracellular domain of RAMP1.

Orexin-stimulated MAP kinase cascades are activated through multiple G-protein signalling pathways in human H295R adrenocortical cells: diverse roles for orexins A and B

Orexins A and B (ORA and ORB) are neuropeptide hormones found throughout the central nervous system and periphery. They are required for a host of physiological processes including mitogen-activated protein kinase (MAPK) regulation, steroidogenesis, appetite control and energy regulation. While some signalling mechanisms have been proposed for individual recombinant orexin receptors in generic mammalian cell types, it is clear that the peripheral effects of orexin are spatially and temporally complex. This study dissects the different G-protein signalling and MAPK pathways activated in a pluripotent human adrenal H295R cell line capable of all the physiological steps involved in steroidogenesis. Both extracellular receptor kinase 1/2 (ERK1/2) and p38 were phosphorylated rapidly with a subsequent decline, in a time- and dose-dependent manner, in response to both ORA and ORB. Conversely, there was little or no direct activation of the ERK5 or JNK pathway. Analysis using signalling and MAPK inhibitors as well as receptor-specific antagonists determined the precise mediators of the orexin response in these cells. Both ERK1/2 and p38 activation were predominantly Gq- and to a lesser extent Gs-mediated; p38 activation even had a small Gi-component. Effects were broadly comparable for both orexin sub-types ORA and ORB and although most of the effects were transmitted through the orexin receptor-1 subtype, we did observe a role for orexin receptor-2-mediated activation of both ERK1/2 and p38. Cortisol secretion also differed in response to ORA and ORB. These data suggest multiple roles for orexin-mediated MAPK activation in an adrenal cell-line, this complexity may help to explain the diverse biological actions of orexins with wide-ranging consequences for our understanding of the mechanisms initiated by these steroidogenic molecules.

A novel extracellular role for tissue transglutaminase in matrix-bound VEGF-mediated angiogenesis

The importance of tissue transglutaminase (TG2) in angiogenesis is unclear and contradictory. Here we show that inhibition of extracellular TG2 protein crosslinking or downregulation of TG2 expression leads to inhibition of angiogenesis in cell culture, the aorta ring assay and in vivo models. In a human umbilical vein endothelial cell (HUVEC) co-culture model, inhibition of extracellular TG2 activity can halt the progression of angiogenesis, even when introduced after tubule formation has commenced and after addition of excess vascular endothelial growth factor (VEGF). In both cases, this leads to a significant reduction in tubule branching. Knockdown of TG2 by short hairpin (shRNA) results in inhibition of HUVEC migration and tubule formation, which can be restored by add back of wt TG2, but not by the transamidation-defective but GTP-binding mutant W241A. TG2 inhibition results in inhibition of fibronectin deposition in HUVEC monocultures with a parallel reduction in matrix-bound VEGFA, leading to a reduction in phosphorylated VEGF receptor 2 (VEGFR2) at Tyr1214 and its downstream effectors Akt and ERK1/2, and importantly its association with b1 integrin. We propose a mechanism for the involvement of matrix-bound VEGFA in angiogenesis that is dependent on extracellular TG2-related activity. © 2013 Macmillan Publishers Limited. All rights reserved.

RAMPs and CGRP receptors

Receptor activity modifying protein 1 (RAMP1) forms a complex with calcitonin receptor-like receptor (CLR) to produce the receptor for calcitonin gene-related peptide (CGRP). RAMP1 has two main roles. It facilitates the cell-surface expression of CLR. It is also essential for the binding of CGRP to the receptor. It seems likely that Y66, F93, H97 and F101, amongst other residues, form a binding site for CLR. These cluster together on the same face of the extracellular portion of RAMP1, probably close to where it enters the plasma membrane. Residues at the other end of RAMP1 are most likely to be involved in CGRP recognition, although it is currently unclear how they do this. Within this area, W74 is important for the binding of the nonpeptide antagonist, BIBN4096BS, although it does not seem to be involved in the binding of CGRP itself. It has been shown that there is an epitope within residues 23-60 of CLR that are essential for RAMP recognition. Under some circumstances, changes in the expression of RAMP1 can alter the sensitivity of cells to CGRP, demonstrating that regulation of its levels may be of physiological or pathophysiological importance.