Cardiovascular toxicity is defined as a severe and potentially fatal adverse reaction to certain drugs. It is one of the leading causes of attrition in drug development. Cardiovascular toxicity research has primarily focused on the role of cardiomyocytes in functional and structural cardiovascular toxicity. However, there is a growing awareness that non-myocyte cells may contribute to cardiovascular toxicity. The heart is a highly vascularised organ. Endothelial cells from the heart were compared between rat and human, as well as between different vascular beds with the use of human cardiac microvascular endothelial cells (HCMECs) and human dermal microvascular endothelial cells (HDMECs). This data demonstrated ligand induced activation of EGFR-1 in HCMEC indicating the presence of functional EGFR1. Analysis of mRNA expression of EGFR1-4 revealed higher expression of both EGFR1 and EGFR2 in HCMEC compared with HDMEC. The role of EGFR-2 (Her2) in drug-induced cardiovascular toxicity was analysed with use of Herceptin® and doxorubicin treatment. Herceptin® and doxorubicin are known to induce cardiovascular toxicity in the clinic. The effect of these drugs on the endothelial tight junction barrier was tested, revealing that Herceptin® and doxorubicin are able to induce barrier perturbment and decreased barrier function in HCMEC. Herceptin® treatment had no effect on the tight junction barrier function in HDMECs. Previous work in the group has identified a role for extracellular-signal-regulated kinase 5 (ERK5) in regulation of endothelial cell survival. The role of ERK5 in endothelial tight junction regulation was investigated using small molecule inhibitors, siRNA transient gene silencing and adenoviral–mediated overexpression of ERK5 to reveal that ERK5 plays an important role in tight junction regulation and endothelial barrier function. Statins are clinically used to lower plasma LDL-cholesterol levels in patients via inhibition of 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMG-CoA reductase). Simvastatin activated ERK5 in endothelial cells via a pathway requiring MEKK3 and MEK5 leading to increased tight junction formation and increased barrier function, providing a possible mechanism for the pleiotropic effects of statins on endothelial cells. Analysis of the effects of a range of anti-cancer drugs with known cardiovascular toxicity liability revealed these drugs could disrupt tight junctions and decrease barrier function. Pre-incubation with simvastatin protected the endothelial cells from drug induced perturbment of endothelial tight junction formation and the associated decrease in barrier function. The data shows the importance of drug-induced endothelial injury in cardiovascular toxicity and highlights potential for therapeutically targeting vasculature to protect against drug-induced vascular injury.

The long-term administration or abuse of analgesics and therapeutic doses of non-steroidal anti-inflammatory drugs have been implicated in renal papillary necrosis (RPN). Several drugs implicated in RPN undergo metabolic activation, via prostaglandin synthetase (PGS) and / or lipoxygenase (LO) systems in papilla, forming potentially damaging free radicals, providing an attractive theory to account for the necrosis. This thesis demonstrated interspecies and intrarenal variation in terms of renal PGS and LO activities. PGS was primarily responsible for arachidonic acid (AA)-dependent metabolism of xenobiotics in rabbit, while in a combination of PGS and LO catalysed drug cooxidation in rat kidney. In contrast in man, AA-dependent cooxidation of drugs in renal tissue occurred via the 5-lipoxygenase, which was predominately located in the papilla. These results indicated that the commonly used laboratory animals, rabbit and rat, are not suitable models for studying RPN in man. These studies also offer a potential explanation for a long-standing anomaly. PGS was thought to be central to the development of RPN but it has been difficult to equate the involvement of PGS with the fact that many papillotoxic compounds are also PGS inhibitors (eg, indomethacin, mefenamic acid). However, this study suggests that 5-LO may be responsible for RPN, either by metabolic activation of drugs or production of pro-inflammatory 5-LO products, altering the renal eicosanoid balance and subsequently renal haemodynamics, thereby precipitating necrosis. This hypothesis was further strengthened by the increased activity of renal 5-LO noted in animal models of both diabetic- and chemically-induced papillotoxicity. Unfortunately, administration of 5-LO inhibitors did not protect against this chemically-induced papillotoxicity. It is possible that inhibitors of 5-LO may provide protection against, or reverse, drug- or disease-induced nephropathy.

In this thesis, evidence has been presented that the two penta-peptides Met- and Leu-enkephalin are synthesized locally in the myenteric plexus longitudinal muscle preparation of the guinea-pig ileum. The local synthesis of Met- and Leu-enkephalin probably occurs through a mechanism involving ribosomal assembly of the peptides at the 80 S ribosomes of the rough endoplasmic reticulum in the neurone perikarion. The ribosomal assembly did not involve 70 S ribosomes present in mitochondria. That the incorporation of {3H}-tyrosine into enkephalins was inhibited when the antibiotic puromycin was present during the labelling and chase period, that such an effect was absent when the antibiotic was present only during the chase period and the presence of a lag period of 1-2 h followed by a linear increase in the appearance of radiolabelled Met- and Leu-enkephalin during the time course of incorporation of the labelled amino acid into the peptides, all suggest that the enkephalins are synthesized at the ribosomes in an inactive form or precursor of higher molecular weight. This is later processed to the final product, enkephalin, through a maturation mechanism that seems to require 1-2 h. The nature of this pro-enkephalin polypeptide and the processing or post-translational modification of it still remain unknown. Levorphanol but not dextrorphan at relatively low concentration (27 nM) decreased the in vitro incorporation of {3H}-tyrosine into Met- and Leu-enkephalin in the myenteric plexus preparation. This effect was not due to an inhibition of protein synthesis at the ribosomal level. However, a higher concentration of levorphanol (108 nM) did not produce the same effect. Naloxone, 100 nM; D-Ala2 - Met5-enkephalin, 80 nM; tetrodotoxin, 1.5 M and hexamethonium, 70 M did not affect the incorporation of labelled tyrosine into enkephalin or proteins of the myenteric plexus preparation. An increase in potassium concentration (50 muM) produced release of "enkephalin-like material" from the superfused crude synaptosomal fraction of rat brain; the amount of enkephalin released represented 2.3% of the total tissue content measured at the end of the super-fusion. The release of "enkephalin-like material" was calcium dependent, since the release of enkephalin was abolished when calcium was omitted and EDTA was added to the superfusing fluid. Release of enkephalin could not be detected by direct measurement after electrical stimulation of the guinea-pig small intestine, possibly due to the rapid inactivation of the enkephalins. When an indirect method to measure the release of enkephalin from the guinea-pig myenteric plexus preparation was developed, it was shown that electrical stimulation does release enkephalin from this tissue. The release of enkephalins from the myenteric plexus preparation depends on the total number of pulses rather than the frequency used. The amount released was 4-5 fmol g-1 per pulse and the fractional release per pulse was 1.1 x 10-5 and 1.3 x 10-5 for frequencies of 1 and 10 Hz, respectively. The release evoked by electrical stimulation was blocked by tetrodotoxin. The amount of enkephalin present in the myenteric plexus preparation after electrical field stimulation, carried out in the presence and absence of an inhibitor of the synthesis of enkephalins was used to calculate the enkephalin turnover. The myenteric plexus preparation showed a turnover of about 310 pmol g-1 Met-enkephalin during electrical stimulation for 2 h at 1 Hz and 190 pmol g-1 during stimulation for 1 h at 10 Hz. The acute in vivo administration of naloxone or morphine or the chronic exposure to morphine did not change the total enkephalin content of the mouse brain. Thus the present results provide additional direct evidence that Met- and Leu-enkephalin may act as neurotransmitters in the central and peripheral nervous system.

The agonist and antagonist properties of narcotic analgesic drugs were examined in the mouse isolated vas deferens. The receptor characteristics of this preparation were then compared with those of other morphine-sensitive tissues. In general, the receptors appeared to resemble those of the guinea-pig isolated ileum, although minor differences were noted. There was a good correlation between the relative agonist potencies of both morphine-like and dual-action compounds in the two peripheral tissues. Dual-action compounds did, however, exhibit flat dose-response curves in the moxise vas deferens, and it was necessary to take special precautions in order to determine their true agonist activities in this preparation. The Ke values of naloxone, and other pure antagonists, to antagonise the actions of normorphine, agreed with those obtained in the ileum within a factor of 2. On the other hand, the antagonist potencies of dual-action compounds were significantly lower in 'the mouse vas deferens. An assessment was made of a new group of benzomorphan agonists which are unusual in that they do not produce a morphine-like dependence syndrome in monkeys. The relative agonist potencies of these compounds were considerably lower in the mouse vas deferens than in the guinea-pig ileum. Furthermore, they required 3 to 5 times more naloxone to antagonise their agonist actions than did morphine-like compounds. The mouse vas deferens was found to be a good predictive model of the clinical analgesic and antagonist activities of opiate drugs. In addition, there was a close relationship between the relative receptor affinities of 'non-dependence producing' benzo-morphans, as measured by inhibition of 3H-dihydromorphine binding to membrane fragments of rat brain, and their relative agonist activities in this isolated preparation. These observations suggest that there is a certain similarity between the receptor populations of brain and vas deferens. The rates of onset and decline of the actions of morphine-like agonists were inversely related to their lipid solubility; this effect may result from secondary binding at lipid-rich sites. Ho relationship was found between drug potency and lipid solubility. These findings are in agreement with those obtained in guinea-pig ileum, but contrast with those obtained in vivo. As an assay preparation, the mouse vas deferens is less robust and consistent in its responses than the guinea-pig ileum. This tissue does, however, exhibit certain features, in particular a high sensitivity to the antagonist properties of dual-action compounds, which make it an invaluable supplementary test system for determining opiate activity. Competitive affinity studies were undertaken in order to compare the binding characteristics of morphine-like and 'non-dependence producing' agonists in guinea-pig brain, ileum and mouse vas deferens homogenates. Comparative analysis of the "binding properties of brain and ileum homogenatss indicated that the receptors being monitored in these preparations were virtually identical in nature. The receptor affinity of each test compound, as measured by 3 inhibition of 3H-naloxone binding, was markedly reduced in the presence of sodium, 'Non-dependence producing' benzomorphans appeared to have lower sensitivities to this inhibitory effect of sodium than did morphine-like agonists. However, a wide variation was found in the sodium shifts of individual morphine-like compounds. Detailed analysis of this sodium effect revealed the existence of a complex relationship between ionic concentration and opiate receptor binding, the sodium' shift of each compound being directly correlated to the slope of the regression line: log IC50 vs log [NaC1]. The Hill coefficients of many of the morphine-like agonists were considerably lower than the theoretical value of unity. This finding suggests that these compounds interact with more than one receptor in order to inhibit the binding of 3H-naloxone. It seems unlikely that these different binding sites represent "sodium" and "no-sodium" conformations of the same receptor. There was a close relationship between the relative receptor affinities of the morphine-like agonists in the absence of sodium, and their relative pharmacological activities in "both the mouse vas deferens and the guinea-pig ileum. In the absence of sodium, the relative activities of the 'non-dependence producing' benzomorphans were similar to their relative agonist potencies in the mouse vas deferens, rather than the guinea-pig ileum. There was a better correlation between the receptor affinities of these compounds and their agonist activities in -!he latter preparation, when binding studies were performed in the presence of 12 mM sodium. Two compounds emerged whose relative potencies to inhibit 3H-naloxone binding, both in the presence and absence of sodium, were considerably lower than their relative agonist potencies in either of the isolated preparations. Of these anomalous compounds, one belonged to the group of morphine-lilce agonists, while the other was a 1non-dependence producing' benzomorphan. The full significance of these findings is discussed, as is the predictive value of competitive affinity studies. Proposals fox future research are outlined.

No description available.

Garlic (Allium sativum) contains a wide range of organosulfur compounds which show a variety of biological effects including broad spectrum antibacterial, antifungal and antiviral activity, as well as selective anticancer activity. One highly bioactive class of compounds from garlic are diallyl polysulfides (DAS), containing one to six sulfur atoms in a linear chain. The bioactivity of DAS has been shown to increase with increasing sulfur chain length up to DAS4 and in this study the even higher bioactivity of DAS5 and DAS6 was demonstrated. The bioactivity of DAS is believed to be initiated following initial reaction with intracellular low molecular weight (LMW) and protein thiols. In this study the interaction between DAS and LMW thiols was investigated and for the first time the reduced DAS metabolites allyl hydropolysulfides have been detected in vitro and in vivo in the Gram positive bacterium Bacillus subtilis. Additionally, formation of mixed polysulfides between DAS and LMW thiols with up to five sulfur atoms was observed in vitro. Proteomic studies revealed a large number of proteins in B. subtilis that formed mixed di- and trisulfides with DAS. Therefore multiple points of DAS attack have been proven and the disturbance of the cellular redox status through lowering the pool of reduced LMW thiols was established in two different organisms (B. subtilis and the nematode Steinernema feltiae). To exploit the polysulfide chemistry for the development of a “green” nematicide, the nematicidal activity of DAS was investigated in bioassays as well as the efficacy of DAS formulations towards plant pathogenic nematodes (Meloidogyne spp. and Globodera spp.) in potato and carrot field trials. It was demonstrated that the DAS derived nematicides form an equally effective alternative compared to synthetic nematicides at a much lower environmental and health risk.

The aim of the thesis was to examine how Ca2+ activates the ERK pathway and the contribution of Ca2+ released from intracellular stores in physiological and pathophysiological conditions using isolated nerve terminals (synaptosomes) in a presynaptic model. The Ca2+-dependent phosphorylation/activation of ERK1 and ERK2 stimulated by depolarisation of the plasma membrane or by Ca2+ influx mediated by the ionophore ionomycin was significantly reduced by the removal of external Ca2+. Intrasynaptosomal Ca2+ contribution to the Ca2+-dependent component of ERK1 and ERK2 phosphorylation/activation was indicated by the depletion of intrasynaptosomal Ca2+ or inhibition of the smooth endoplasmic reticulum Ca2+-ATPase pump. Two main pathways were found to lead to the release of Ca2+ from intrasynaptosomal stores. Firstly, external Ca2+ influx directly activated ryanodine receptors (RyRs) to mediate Ca2+-induced Ca2+ release (CICR). Secondly, Ca2+ influx or activation of GPCRs coupled to Gq/11 activated phospholipase C (PLC) to effect PIP2 metabolism and IP3 production, with consequent activation of IP3-induced Ca2+ release (IPCR). The activation of group I metabotropic glutamate receptor (mGluR1/5) stimulation supported IPCR. Intriguingly, inhibition of Ca2+ influx through voltage-dependent calcium channels (VDCCs) by stimulating GABAB, group III mGluRs, 5-HT1A and A1 receptors was suppressed by prior depletion of the smooth endoplasmic reticulum. Mitochondria and acidic compartments also appear to store Ca2+ intrasynaptosomally, with mitochondrial depolarisation resulting in a transient increase in ERK1 and ERK2 phosphorylation/activation. Finally, a pathophysiological model of nerve terminal ischemia showed that intrasynaptosomal Ca2+ release contributes to the Ca2+-dependent component of phosphorylation/activation of ERK1 and ERK2 occurring when Na+/K+- ATPase is inhibited. In conclusion, extracellular Ca2+ influx and intracellular Ca2+ store release together support Ca2+ mediated stimulation of the ERK pathway in synaptosomes. This has important implications in the cross-talk of signalling pathways to ERK1 and ERK2 phosphorylation/activation and neurotransmitter release from nerve terminals in physiological and pathophysiological conditions.

No description available.

No description available.

No description available.