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Cycloleucine (CL) is a very potent inhibitor of S-adenosyl methionine transferase causing a rapid decline in CNS S-adenosyl methionine levels and increase in methionine in the nervous system. CL interrupts the transmethylation reaction in the CNS similar to that caused by deficiency of vitamin B12 or folic acid. CL was administered as a single dose intraperitoneally (2mg/g body weight) to mice of varying ages (21 days - 5 months). The 21 day old mice given CL 2mg/g body weight showed evidence of toxicity within 24 hours and thereafter developed progressive muscle weakness and ataxia. Animals did not survive longer than 1 week. Light and electron microscopic examination o f the central and peripheral nervous systems, from 12 hours to 7 days post-injection showed that intramyelinic vacuolation developed in the white matter of brain and cord within 12 hours but no myelin vacuolation occurred in peripheral nerves. Axonal lesions in the distal parts of motor nerves occurred within 1 2 - 2 4 hours resulting in degeneration of intramuscular nerve fibres and terminals. Motor end-plates became denervated. There was no degeneration of motor or sensory nerves in the muscle spindles. Later there was evidence of axonal degeneration in tibial and sciatic nerves. Many dorsal root ganglion cells became vacuolated or necrotic three days after administration of CL and numerous degenerated fibres were noted in the white matter of the spinal cord, especially in funiculus gracilis. The intramyelinic vacuolation in the white matter of brain and cord persisted and became more severe during the course of the experiment. In adult mice (6 weeks - 5 months) the pathology consisted mainly of distal motor axonal degeneration which developed at 1 - 2 days. Only slight intramyelinic vacuolation in white matter was noted. At 3 days later, numerous degenerated fibres were seen in the posterior columns of cervical cord, especially in the funiculus gracilis. In a second series of experiments valine (3 mg/g body weight) was given every 12 hours for 6 doses beginning either 24 or 48 hours after CL. Valine (VL) is a very effective antidote against cycloleucine and reversed its toxicity. The intramyelinic vacuolation disappeared from the white matter in the CNS leaving little residual pathology. Regenerating axons and remyelinated fibres were found in previously degenerated distal peripheral nerves and denervated motor end-plates. The reinnervation took place more rapidly in animals given VL from 24 hours after CL than 48 hours after CL. In conclusion it can be stated that CL causes a distal motor axonopathy and sensory ganglion cell necrosis in addition to the previously reported myelinic vacuolation in the CNS. The mechanism of toxicity of CL is likely to be related to the failure of transmethylation processes affecting particularly myelin basic protein and perhaps membrane turnover at active sites of transmitter release such as the neuromuscular junction.

Cycloleucine (CL) is a synthetic annino acid which inhibits the vitamin B12/folate dependent methyl-transfer reaction. A single dose of CL (2mg/g body weight) was administered intraperitoneally to 21-day-old and adult mice. Animals were allowed to survive for between 12 hours and 7 days. The 21-day-old mice showed paralysis of the hindlimbs within 24 hours, whereas this symptom became apparent in the adult mice at 48 hours. In the adult mice the twitch and tetanic responses of extensor digitorum longus (Edl) and soleus evoked by neural stimulation fell dramatically within 24 hours. In both the 21-day-old and the adult mice intracellular recordings made at 24 hours in soleus and Edl revealed that a significant number of end-plates were denervated, while other end-plates demonstrated intermittent failures in transmission and end- plate potentials (epps) with prolonged latencies. End-plates with abnormally high frequencies of miniature end-plate potentials (mepps) were commonly encountered in soleus and Edl, of 21-day-old mice at 12 hours, and in the adult mice at 24 hours. Morphological abnormalities in both the intramuscular nerves and the neuromuscular junctions of soleus and Edl were seen in both the young and adult mice at 24 hours. These abnormalities included areas of electron lucent axoplasm and swollen degenerative mitochondria and nerve terminals lacking synaptic vesicles. The innervation of proximal muscles was unaffected at this time. Over the next 2-3 days further reductions in the number of soleus and Edl fibres demonstrating mepps or epps occurred in both young and adult mice. The muscle spindles in soleus were found to be both functionally and structurally intact. At 7 days a limited recovery of function occurred in both soleus and Edl of young and adult mice. In biceps brachii of young mice, however, denervated end-plates and abnormally high mepp frequencies were found at 7 days. It is suggested that the distal motor axonopathy induced by CL is caused by the failure of the methyl-transfer pathway which leads to abnormalities in the phospholipid composition of the axolemma at the neuromuscular junction. These changes are believed to cause an increase in microviscosity of the axolemma and hence a decrease in efficiency of ion channels/pumps which are responsible for maintaining electrochemical gradients essential for the structural and functional integrity of the neuromuscular junction.

The homeostatic regulation of amino acid concentrations is crucial for optimal brain function and development. Different amino acid transporters at cell membranes work together to facilitate the movement of amino acids into and out of the brain. Despite countless in vitro and in vivo research on these amino acids' activities, many fundamental concerns about their metabolic function in different brain areas and pathophysiological conditions remain unanswered. In the framework of this thesis, the effects of exogenous administration of several non-essential amino acids and the participation of their specific transporters in brain metabolism were investigated in Guinea pig cortical brain slices and mouse brain tissues using a targeted neuropharmacological and metabolomic strategy. Alterations in brain metabolism were analyzed using 1H and 13C nuclear magnetic resonance spectroscopy to evaluate changes in metabolite pools and 13C-enriched substrates. All the amino acid transporters mentioned in this study were addressed by the existing solute carrier (SLC) gene nomenclature system for amino acid transporters. The effect of exogenous L-aspartate, L-ornithine, and their salt, L-aspartate-L-ornithine (LOLA), on brain metabolism was investigated with or without an intact blood-brain barrier (BBB). The results indicated that neither L-aspartate, L-ornithine, nor LOLA, affected brain metabolism with an intact BBB. In cortical tissue slices L-aspartate increased brain metabolism concentration-dependently, L-ornithine significantly slowed it at higher concentrations (100 μmol/L), and the effects of LOLA was largely dependent on the balance of its two constituent amino acids. D-aspartate, another isoform of aspartate, produced a range of metabolic impacts, particularly on glutamatergic and GABAergic systems, with varying concentrations. In principal component analysis, the effects of D-aspartate were clearly distinguished from those of L-aspartate, indicating a metabolic pattern distinct from that of excitatory mechanisms. L-Proline administration significantly inhibited brain metabolism in Guinea pig cortical tissue slices, indicating a GABA-like effect; however, it was not a significant metabolic substrate. While it was actively taken up by cells in a concentration-dependent manner but was not completely metabolized. The metabolic pattern revealed that L-proline's effects clustered with 3-aminopropyl(methyl)phosphinic acid (SKF 97541), GABA, 1,5,6-tetrahydropyridin-4-yl)methylphosphinic acid (TPMPA) and (5,6-dihydro-5-methyl-6-oxo-4H-imidazo[1,5-a]thieno[2,3-f][1,4]diazepine-3-carboxylic acid) 1,1-dimethylethyl ester (RO194603) at lower concentrations (10 μmol/L) and with vigabatrin and RO194603 at higher concentrations (100 μmol/L); indicating that proline may act as a GABAB receptor agonist or GABAArho antagonist. Deletion of SLC6A17/NTT4 (neurotransmitter transporter 4) gene significantly impaired glutamate-glutamine cycle, reduced incorporation of 13C into Krebs cycle intermediates, and increased incorporation into lactate in the brain of mice lacking the gene. NTT4 knockout also altered several important metabolites in glutamatergic neurones, implying that it is a crucial transporter for maintaining brain amino acid homeostasis. Investigation of glutamine transport in cerebellum demonstrated that system A dominates glutamine transport in the cerebellum, with contributions from system N, which is inhibited by histidine and 2-(Methylamino)-2-methylpropionic acid (MeAIB) exerting the most metabolic influence. Inhibition of systems A and L by L-γ-Glutamyl-p-nitroanilide (GPNA) and 2-amino-4-bis(aryloxybenzyl)aminobutanoic acid (AABA) did not influence glutamine transport due to their low affinity for the transporters. Inhibition of systems L and B0 by 2-Aminobicyclo[2.2.1]heptane-2-carboxylic acid (BCH) showed little effect on fluxes from [1-13C]D-glucose but increased the flux of [1,2-13C]acetate into Glu C4,5 and Gln C4,5. Effects of cycloleucine were comparable to BCH but less powerful. This study provided new insight into the role of several non-essential amino acids in brain metabolism and also showed how brain metabolism is regulated in different brain regions.

The antitumour bifunctional alkylating agent nitrogen mustard (HN2) inhibited the unidirectional influx of the potassium congener, 86 rubidium, into murine PC6A plasmacytoma cells and L1210 leukaemia cells. The proliferation of L1210 cells in vitro was characterised and shown to be sentitive to HN2. 86Rubidium influx into cells from rapidly-dividing cultures was more sensitive to inhibition by HN2 than that of cells from stationary cultures. Three components of unidirectional 86Rb+ & K+ influx into proliferating L1210 cells were identified pharmacologically: approximately 40% was active to the Na+ K+ ATPase inhibitor ouabain (10-3M), 40% was sensitive to the `loop' diuretics bumetanide (10-4M) and furosemide (10-3M) and the remainder was insensitive to both ouabain and furosemide. HN2 (10-5M) selectively inhibited the diuretic-sensitive component, which was entirely dependent upon extracellular Na+ and Cl- ions, and was presumed to represent Na+ K+ Cl- cotransport activity. The system did not mediate K+ /K+ exchange or unidirectional 86Rb+ efflux; accordingly, 86Rb+ efflux was insensitive to HN2. Inhibition of 86Rb & K+ influx by 10-5M HN2 was accompanied by approximately 35% of cell volume under isosmotic conditions; thus intracellular Na+ and K+ concentrations remained unchanged. These effects followed lethal damage to the cells but preceded actual cell death; other cellular functions were maintained including accumulation of cycloleucine, transmembrane potential, permeability to trypan blue, intracellular pH, total intracellular glutathione and calcium concentrations. No evidence was found that elevated cAMP levels or reduced ATP levels were involved in modulation of 86Rb+ & K+ influx. However, the Na+ - depedent transport of an amino acid was inhibited in a manner which appeared to be independent of 86Rb & K+ influx. An HN2-resistant L1210R cell line was also resistant to furosemide, and lacked a component of 86Rb+ & K+ influx which was sensitive to furosemide (10-3M). The results strongly suggest that the Na+ K+ Cl- costransporter of L1210 cells is a cellular target for HN2. This lesion is discussed with reference to the cytotoxic effects of the agent.

This investigation involved the synthesis of 1-amino-2-hydroxycyclopentanecarboxylic acid, a potential structural analog of the natural amino acids, serine and threonine. The title compound also includes the structural features present in an established antitumor agent, cycloleucine.