Somatostatin (SRIF) exerts its antiproliferative actions through membrane bound receptors, for which there are currently five distinct receptor genes cloned (sst<SUB>1.5</SUB>). The main objectives of this study were to (a) characterise the effects of activation of the SIRF<SUB>1</SUB> group of receptors (namely the sst<SUB>2</SUB>, sst<SUB>3</SUB> and sst<SUB>5</SUB> receptors) on cell proliferation on CHO-K1 cells, and (b) investigate the possible involvement of particular intracellular signalling molecules in the antiproliferative effect of somatostatin through human somatostatin receptors. The pharmacology of the clinically used somatostatin analogue, angiopeptin (BIM-23014; sst<SUB>2</SUB>/sst<SUB>3</SUB> selective), was examined throughout the course of this study. Somatostatin did not modulate basal proliferation of CHO-K1 cells selectively expressing human somatostatin SRIF<SUB>1</SUB> receptors. Further experiments using rat recombinant somatostatin receptors illustrated species differences with angiopeptin comparatively more efficacious at rat receptors. The major conclusions from this study are (a) species differences may explain the poor clinical benefit reported for angiopeptin in the treatment of restenosis in view of initial encouraging animal data, (b) the intrinsic activity and potency estimates for angiopeptin at an individual receptor type changed dramatically depending on the functional response measured, and (c) the antiproliferative effect of somatostatin through the human sst<SUB>2</SUB> and sst<SUB>5</SUB> receptors is mediated <I>via</I> a sustained stimulation of MAP kinase resulting in increased expression of p21. Both the duration and the strength of MAP kinase activation appears to be crucial to the resultant effects on cell growth. These findings have important implications for understanding agonist activity and the use of animal models for predicting clinical utility.

This thesis shows that secondary activation of the P2X₁ receptor represents a significant pathway by which a number of major platelet can elicit a rapid [Ca²⁺]_I increase, particularly at weak agonist levels. P2X₁ receptor activation was able to amplify the early [Ca²⁺]_I increase evoked by collagen, thrombin, thromboxane A₂ and ADP, without requiring synergy wit co-activated P2Y receptors. Moreover, contrasting the prostacyclin-sensitivity of the GPCR-coupled Ca²⁺-mobilisation pathways that are activated by thrombin, thromboxane A₂ and ADP, the P2X₁ receptor was completely insensitive to prostacyclin – a major natural platelet inhibitor that is released from the endothelium and acts to increase interplatelet cyclic AMP (cAMP). Furthermore, the early collagen-evoked [Ca²⁺]_I response consisted of both cAMP-sensitive and –insensitive Ca²⁺- mobilisation components, with a Ca²⁺ influx through the activated P2X₁ receptor representing the major contributor to the cAMP-insensitive component. Evidence suggested that early collagen-evoked ATP secretion was responsible for activating the P2X₁ receptor, particularly since this nucleotide is known to be stored at high concentrations in secretory granules and activates this ionotropic receptor with high efficacy (EC₅₀ <100nM). Whilst the P2X₁ receptor was unable to amplify the early collagen-evoked ATP secretion, it augmented the reversible collagen-stimulated shape change response monitored by standard turbidimetric measurements. A study of platelets stored under Blood Bank conditions found that P2X₁ receptor signalling was still functional up to 14 days <i>ex vivo</i>, indicating that the P2X₁ concentrates into a patient and that stored platelets could be a useful source of tissue to study this Ca²⁺-elevating receptor.

Noxious stimulation increases the levels of AP-1 and other immediate early gene (IEG) proteins in the spinal cord. These transcription enhancers regulate the expression of a second set of genes which have the potential to cause long-term changes in neuronal phenotype and function. In the spinal cord, such changes are manifested as hypersensitivity to stimulation leading to chronic pain states. One putative target of IEG control is the gene encoding the opioid peptide dynorphin. Robust increases in this gene are seen in the spinal cord in animal models of pain and its presence has been correlated with alteration of neuronal function. This study has used the rat formalin test as a model for gene activation in the spinal cord. The stimulus-specificity of changes in gene expression caused by hind-paw injection of dilute formalin was compared with similar injection of the neurotoxin capsaicin. The contribution of different neurotransmitters to the IEG response was assessed by pharmacological means in formalin-injected animals. Finally, the presence of functional IEG proteins in the spinal cord of formalin-injected animals was assessed by gel shift assay. Formalin and capsaicin produced different physiological and behavioural responses; formalin provoked long-lasting inflammation and prolonged biphasic nociceptive behaviour, whilst capsaicin was non-inflammatory and without a profound effect on behaviour. However, both treatments caused similar short-term upregulation in IEG expression in the spinal cord followed by an increase in preprodynorphin mRNA. Thus even brief noxious stimulation, as caused by capsaicin, was capable of significantly altering neuronal gene expression. Pharmacological investigation of the formalin test showed that both phases of the behavioural response could be selectively inhibited by non-NMDA and NMDA glutamate antagonists (NBQX and HA966).

Human platelet cytosolic phospholipase A<SUB>2</SUB> (cPLA<SUB>2</SUB>) mediates the release of arachidonic acid (AA) from membrane phospholipids. AA is the precursor for eicosanoids, such as thromboxane A<SUB>2</SUB> (TxA<SUB>2</SUB>), which can contribute to platelet positive feedback. Collagen and ionomycin (a Ca<SUP>2+</SUP>ionophore) each provoked a release of AA from human platelets which was independent of TxA<SUB>2</SUB> production and ADP secretion, but required at least cytosolic Ca<SUP>2+</SUP> levels. Collagen-stimulated AA release and TxB<SUB>2</SUB> production correlated with the rise in intracellular-free Ca<SUP>2+</SUP> concentration ([Ca<SUP>2+</SUP>]<SUB>i</SUB>), and tyrosine phosphorylation. Ionomycin-stimulated AA release and TxB<SUB>2</SUB> production correlated with PKC activation and tyrosine phosphorylation, but not with the rise in [Ca<SUP>2+</SUP>]<SUB>i</SUB>. Ro31-8220 (a PKC inhibitor) and genistein (a protein tyrosine kinase (PTK) inhibitor) were found to potentiate and suppress collagen-stimulated AA release respectively. Ro31-8220 and genistein each suppressed by 50%, and in combination almost abolished, ionomycin-stimulated AA release. Therefore, PKC is necessary for full AA release from ionomycin-stimulated platelets, but acts to suppress AA release in response to collagen. Furthermore, PTKs appear to mediate both collagen- and ionomycin-stimulated AA release. When platelets were activated by collagen, cPLA<SUB>2</SUB> became phosphorylated upon tyrosine, and associated with several other phosphotyrosine-containing proteins. Moreover, cPLA<SUB>2</SUB> was a good <I>in vitro</I> substrate for the PTK pp60<SUP>c-src</SUP>, which is abundant in platelets. To conclude, cPLA<SUB>2</SUB> is likely regulated by both direct tyrosine phosphorylation, possibly mediated by pp60<SUP>c-src</SUP>, and indirect tyrosine phosphorylation of closely associated proteins.

The aims of my project were (i) to elucidate the molecular mechanism of Na<SUP>+</SUP>-dependent activation of GIRK channel, and (ii) to investigate the effect of intracellular acidification on GIRK channel activity. Intracellular Na<SUP>+</SUP> dose-dependently activated the wild type GIRK2 homomeric and GIRK1/GIRK2 heteromeric channels. The proximal C-terminal region of the GIRK2 subunit was shown to mediate the Na<SUP>+</SUP>-dependent activation of both homomeric and heteromeric channels. Within this region, GIRK2 has an aspartate at position 226, whereas GIRK1 has an asparagine at the equivalent position (217). A single point mutation, D226N, in GIRK2, abolished the Na<SUP>+</SUP>-dependent activation of both the homomeric and heteromeric channels without affecting their ability to be activated via the m<SUB>2</SUB>-muscarinic receptors. Neutralizing a nearby charge, E234S had no effect. A reverse mutation in the GIRK1 subunit, N217D, was sufficient to restore the Na<SUP>+</SUP>-dependent activation of the GIRK1N217D/GIRK2D226N heteromeric channels. Thus, the aspartate 226 in GIRK2 plays a crucial role in Na<SUP>+</SUP>-dependent activation of both the GIRK2 homomeric and GIRK1/GIRK2 heteromeric channels. Interestingly, intracellular Na<SUP>+</SUP> slowed the time-course of inhibition by PIP<SUB>2</SUB>-specific antibody to a similar extent as the neutralization of the aspartate 226 in GIRK2 did. This suggests that intracellular Na<SUP>2+ </SUP>binds to the GIRK2 subunit to neutralize the aspartate 226 and to promote the binding of PIP<SUB>2</SUB> to a nearby region of the C-terminus. This activates the GIRK2 homomeric and GIRK1/GIRK2 heteromeric channels.

In Chapter 1 a review of the 5-HT_1B receptor is conducted, describing physiological and pathological roles as well as known ligands. The structural biology of Family A GPCRs is reviewed to provide insights into the behaviour of 5-HT_1B itself. In Chapter 2, a multiple sequence alignment is used to study residue conservation in human monoamine GPCRs and to elucidate areas of functional and structural significance. Patterns are identified that are consistent with current knowledge of GPCR function while also producing novel insights into 5-HT_1B receptor structure. In Chapter 3, homology modelling based upon both bovine rhodopsin and the human β2-adrenergic receptor is employed to construct models of 5-HT_1B. In Chapter 4, molecular dynamics is then used to refine these structures, verify the intramolecular interactions identified in Chapter 2 and investigate conformation changes during receptor activation. In Chapter 5, existing ligands are studied and novel pharmacophores for the receptor are derived. Higher ligand recall with tighter constraints is achieved by introducing virtual sits into the pharmacophores. These pharmacophores are then integrated into the receptor models to compare them, to identify binding interactions and to investigate the dynamic behaviour of pharmacophores. From the conjunction of ligand and receptor-derived information a model for ligand efficacy is derived. In Chapter 6, the design of a novel series of 5-HT_1b antagonists using and verifying the previously-derived models is described.

No description available.

No description available.

Five different plant polyphenols (resveratrol, hesperetin, quercetin, daidzein and silymarin) were shown to interact with BCRP expressed in MCF7/MR and K562/BCRP cells, significantly increasing the accumulation of mitoxantrone and bodipy prazosin above control levels. They also stimulated the BCRP-associated ATPase activity in vesicles derived from <i>Lactococcus lactis </i>bacteria transformed with BCRP cDNA. None of these polyphenols had any effects on the long-term expression of BCRP in MCF7/MR cells. Inhaled corticosteroids have revolutionized the treatment of chronic obstructive pulmonary disease but cause long term suppression of the hypothalamic-pituitary-adrenal (HPA) axis, leading to low cortisol levels. It has been suggested that P-gp at the BBB may influence suppression of the HPA by oral corticosteroids. Five inhaled corticosteroids were tested for their ability to modulate P-gp and BCRP function. Beclomethasone dipropionate, mometasone furoate and the newer ciclesonide used at 5 and 10 <i>μ</i>M increased the accumulation of mitoxantrone in BCRP-expressing MCF7/MR cells, and the accumulation of calcein in P-gp expressing SW620/R cells, though triamcinolone acetonide and budesonide had no inhibitory effects. Beclomethasone, mometasone and ciclesonide at 5 <i>μ</i>M enhanced the cytotoxicity of doxorubicin in SW620/R cells. These three compounds as well as budesonide stimulated the BCRP-associated ATPase activity in <i>L. lactis </i>vesicles expressing BCRP. Both the plant polyphenols and the three corticosteroids examined in this study could either be ‘fast diffusing’ or competitive substrates of BCRP, inhibiting the efflux of another substrate while stimulating the BCRP-associated ATPase activity. These results establish that BCRP is present at the BBB and may modulate the access of both dietary and therapeutic steroidal compounds into the brain.

The two glial transporters, GLAST (<SUB>L</SUB>-glutamate/L-aspartate transporter) and GLT-1 (<SUB>L</SUB>-glutamate transporter), are critical in regulating extracellular glutamate levels, being responsible for the majority of glutamate reuptake. The present study investigated GLAST and GLT-1 mRNA and protein levels in two animal models of inherited epilepsy: the EL (epileptic) mouse, a convulsive seizure model, and the GAERS (genetic absence epilepsy rat from Strasbourg) rat, a model of absence epilepsy. Additionally, tissue glutamate concentrations were determined in these animals by high pressure liquid chromatography (HPLC). Initially, polyclonal antibodies specific to mouse and rat GLT-1 and GLAST were generated and characterised. These antibodies revealed distribution patterns for the two transporters confirming those previously reported. <I>In situ</I> hybridisation and Western blot analysis revealed widespread reductions in GLT-1 mRNA in the brains of EL mice compared with control animals, accompanied by a decrease in GLT-1 protein in the parietal cortex, a region crucial to seizure initiation in this model. An increase in tissue glutamate concentration in the parietal cortex of EL mice was additionally observed. GLAST mRNA was also reduced in various brain regions of EL mice, with a reduction in protein observed in the hippocampus, a region essential for seizure generalisation. Glial glutamate transporter downregulation in these two regions may therefore play a role in seizure initiation and generalisation in the EL mouse. Similar analyses in GAERS rats revealed upregulation of GLT-1 and GLAST mRNA in thalamic and cortical regions, respectively, when compared with controls.