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No description available.

To test whether araC interfered with NGF signalling, I examined the effect of araC treatment on signalling pathways both before and during apoptosis. Using a solid-phase kinase assay and antibodies to active forms of signalling proteins, I found that treatment of neurons with araC induced apoptosis without affecting NGF-maintained signalling. AraC caused no reduction in the activity of either mitogen activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) or protein kinase B/Akt, a kinase implicated in NGF-mediated survival. AraC also did not activate c-Jun N-terminal kinase (JNK) or cause c-Jun N-terminal phosphorylation, processes that are implicated in apoptosis induced by NGF withdrawal. In investigating a role for p53 in araC-induced apoptosis, I established that p53 protein levels were elevated before and during apoptosis by treatment with araC. There was, however, no increase in the levels of p53 protein after NGF withdrawal. Furthermore, neurons from p53 null mice were resistant to treatment with araC, although in response to NGF withdrawal, these neurons and neurons from wild type animals died at a similar rate. It was found previously that MAPK/ERK activity was not necessary for NGF-induced survival. I found, by contrast, that MAPK/ERK activity protected against araC-induced toxicity as inhibition of the MAPK pathway by PD98059 resulted in a significant increase in the rate of apoptosis induced by araC in the presence of NGF. Consistent with this finding, ciliary neurotrophic factor, which induces survival but not sustained activation of MAPK/ERK, failed to protect against araC toxicity. The data presented here suggests that araC does not inhibit NGF-mediated signalling, but instead causes apoptosis by a p53-dependent mechanism which can be inhibited by MAPK/ERK activity.

No description available.

No description available.

No description available.

No description available.

These studies have investigated the effects of fetal treatment with dexamethasone on the fetal cardiovascular, endocrine and metabolic responses to a 1h episode of acute hypoxaemia occurring both during, and 48 h following, the period of dexamethasone treatment in fetal sheep at 0.8 of gestation. Parallels have been drawn with the normal maturation of the cardiovascular, endocrine and metabolic responses to acute hypoxaemia by investigating developmental changes in the patterns of these responses close to term, and correlating them with the preparturient increase in fetal plasma glucocorticoid concentration. Mechanisms mediating the changes in the cardiovascular responses to acute hypoxaemia in the dexamethasone-treated and late gestation fetuses have been addressed by measuring the associated changes in fetal plasma concentrations of vasoconstrictor hormones (noradrenaline, adrenaline, arginine vasopressin, angiotensin II and neuropeptide Y), hormones associated with the hypothalamo-pituitary-adrenal axis (ACTH and cortisol), by assessing changes in the pressor and vasopressor responses to exogenous bolus doses of phenylephrine, arginine vasopressin and angiotensin II, and the chronotropic responses to exogenous isoprenaline, and by constructing chemoreflex and baroreflex function curves. Finally, to examine the possible role of changes in myocardial responsiveness to muscarinic cholinergic and β-adenergic stimulation in mediating alterations in the fetal heart rate response to acute hypoxaemia with glucocorticoid treatment or advancing gestational age, a novel Langendorff, biventricular, isolated, perfused fetal sheep heart preparation was developed.

Uncoupling proteins (UCPs) catalyse proton transport across the mitochondrial inner membrane, thereby uncoupling oxidative phosphorylation. UCP2 is expressed in several mammalian tissues including pancreatic β-cells. Since UCP2 activity uncouples the processes that link glucose metabolism (oxidation) to insulin secretion (phosphorylation), it is expected to attenuate glucose-stimulated insulin secretion (GSIS). Published studies using UCP2 over-expressing and UCP2 ablated β-cells support this hypothesis. Therefore, identifying regulators of β-cell UCP2 activity may help develop novel ways to treat type II diabetes. Furthermore, β-cells present a valuable system with which to study UCP2 activity <i>in situ</i>. Studies using isolated mitochondria have demonstrated that the superoxide radical and particular reactive alkenals activate UCP2, and that novel, mitochondrially-targeted antioxidants (mito-antioxidants) prevent activation of UCP2 by superoxide. Mito-antioxidants and reactive alkenals are thus useful tools to investigate pancreatic β-cell UCP2 activity <i>in situ. </i> I hypothesised that endogenously produced superoxide activates UCP2 in β-cells to negatively regulate GSIS, and tested this hypothesis by measuring UCP2 activity in isolated β-cell mitochondria and GSIS in intact β-cells. Mito-antioxidants prevented superoxide-activation of UCP2 in isolated β-cell mitochondria but, surprisingly, did not potentiate GSIS. These unexpected findings suggested that either UCP2 was endogenously inactive, or endogenous UCP2 activity is not inhibited by mito-antioxidants. I sought to distinguish between these possibilities using UCP2 overexpressing β-cells, and by investigating isolated mitochondria in which UCP2 appeared to be endogenously active. Certain reactive alkenals both activated UCP2 in β cell mitochondria and attenuated GSIS in intact cells, suggesting that may attenuate insulin secretion physiologically, or provide valuable tools to probe UCP2 activity in cells.

The vasorelaxant activities of cannabinoids were investigated in isolated segments of the left anterior descending coronary artery, and the third generation of superior mesenteric artery, of the rat. The well-known endocannabinoid anandamide caused endothelium-independent relaxation of precontracted coronary arteries by a novel mechanism that involved activation of large conductace Ca²⁺ -activated K⁺ channels (BK_ca) but was apparently independent of the cannabinoid CB₁ and CB₂ receptors or production of vasoactive metabolites. Interestingly, although anandamide is known to be an agonist of the vanilloid VR1 receptor, and activation of these receptors relaxed coronary artery, inhibitors of the VR1 receptor system did not affect anandamide-induced relaxation in this artery. The mechanisms by which anandamide increases BK_Ca activity remain unclear, however, it is unlikely that a direct interaction of anandamide with BK_Ca is involved. There is evidence that the synthetic cannabinoid R-(+)-WIN 55,212-2 also relaxes coronary arteries via mechanisms similar to those of anandamide. In the rat mesenteric artery, while anandamide relaxations were also independent of the endothelium, the major relaxant mechanism of anandamide is activation of VR1 receptors. However, additional mechanism including inhibition of Ca²⁺ influx to the vascular smooth muscle also play a role, indicating that the vasorelaxant actions of anandamide are complex. In fact, reduction of Ca²⁺ influx, perhaps via voltage-gated Ca²⁺ channels in the smooth muscle, formed the basis of the relaxation to the synthetic cannabinoids JWH 015 and R-(+)-WIN 55,212-2 in the mesenteric artery. However, as for rat coronary artery, there was no substantial evidence supporting the presence of CB₁ or CB₂ receptors, as classically defined, in the smooth muscle rat mesenteric arteries. Further studies using electrical field stimulation of mesenteric arteries, nevertheless, revealed that CB₁-like receptors might be present in the prejunctional sympathetic nerve terminals.