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Kinetic characterisation of the plasma membrane transport of excitatory sulphur-containing amino acids in cultured brain cells and isolated nerve endings

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AngusGrieveMScthesis1993_original_C.pdf (14.81Mb)
Date
1993
Author
Grieve, Angus
Supervisor
Griffiths, Roger
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Abstract
The endogenous neuroexcitatory sulphur-containing amino acids (SAAs) are generally regarded as putative transmitter substances. Of many criteria that need to be satisfied before acceptance as a transmitter, one is that a mechanism exists for termination of post-synaptic receptor activation. For amino acids such inactivation normally requires participation of a plasma membrane transport system for rapid and efficient removal of transmitter from the extracellular environment. This thesis reports on the kinetic characterisation of the plasma membrane transport of the SAAs, namely L-cysteine sulphinate (CSA), L-cysteate (CA), L-homocysteine sulphinate (HCSA) and L-homocysteate (HCA) in (i) synaptosome fractions from rat cerebral cortex and (ii) primary cultures of neurons from distinct brain regions and astrocytes from prefrontal cortex. It was shown that each SAA acted as a substrate for the plasma membrane transporter in the different brain preparations studied with CSA and CA exhibiting a high-affinity for uptake (Kₘ200< 100μM) HCSA and HCA exhibiting a low-affinity for uptake (Kₘ200 > 100μM). The plasma membrane carrier specificity of the SAAs which was studied using cerebrocortical synaptosome fractions was established following comparison with other high-affinity neurotransmitter systems. The results obtained strongly suggest that the SAAs share a common synaptosomal transport system with L-glutamate and L- and D-aspartate. A detailed kinetic analysis of the inhibition by the proposed selective inhibitor of HCA uptake, β-p-chlorophenylglutamate (chlorpheg), on HCA and D-aspartate uptake using (i) cerebrocortical synaptosomes and (ii) primary cultures of cerebellar granule cells and cortical astrocytes has been undertaken. The results from this kinetic inhibition study show clearly a non-selective competitive inhibition of D-aspartate and L-HCA uptake by chlorpheg in all the brain preparations studied. In a tangential HPLC study, the cellular localisation of HCA and HCSA was investigated using ethanolic amino acid extracts prepared from primary cultures of cerebellar neurons and astrocytes. The results suggest a predominant astrocytic, rather than neuronal, localisation of HCA and HCSA.
Type
Thesis, MSc Master of Science
Collections
  • Biology Theses
URI
http://hdl.handle.net/10023/21881

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