Studies of [³H]-Noradrenaline and [¹4C]-Glutamate release from synaptosomes
McFadden, Susanne Christine
McFadden, Susanne Christine
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Date
2002
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Abstract
In the nerve ending, there are two types of synaptic vesicles, large dense-core vesicles (LDCV) and small synaptic vesicles (SSV). Glutamate (glu) and noradrenaline (NA) are both neurotransmitters found in the central nervous system that are released from SSV and LDCV respectively. We developed a novel protocol to load synaptosomes with both [ 3 H]-NA and [ 14 C]-glu and examine release of both neurotransmitters from the same sample. Recently, phosphoinositides have been identified as regulators of the vesicle cycle and the differences between release from LDCV and SSV. Inhibition of the synthesis of phosphatidylinositol 4-phosphate (PI4P) and phosphatidylinositol 4,5-bisphosphate (P14,5 P 2 ) by phenylarsine oxide, inhibits both [ 3 H]-NA and [ 14 C]-glu release from Streptolysin-O perforated synaptosomes. When PI 4 ,5P 2 is chelated by neomycin, only [ 3 H]-NA release is inhibited. When phosphatidic acid production is shunted into phosphatidylbutanol production by addition of 1-butanol or when phospholipase D is inhibited by C 2 -ceremide, [ 14 C]-glu release is unchanged while [ 3 H]-NA release is inhibited. Taken together, this evidence suggests that while PI4,5P 2 is not required for SSV fusion from the readily-releasable pool, it is necessary for LDCV exocytosis. Neuronal Calcium Sensor-1 (NCS-1) is part of a family of neuronal Ca 2+ binding proteins that have EF-hand Ca 2+ binding domains. Incubations with a peptide corresponding to the myristoylated N-terminal of NCS-1 caused a dose dependent decrease in release of both [ 3 H]-NA and [ 14 C]-glu. Antibodies against the N-terminal of NCS-1 caused significant increases in both basal and stimulated release while decreasing Ca 2+ -dependent release. Incubations with the antibody against the C-terminal of NCS-1 caused a decrease in Ca 2+ dependent release of both neurotransmitters. PI4K? is known to be upregulated by the C-terminal of NCS-1, so the antibody would inhibit this interaction and in so doing cause a decrease in release. These results show that NCS-1 is involved in regulating release from both LDCV and SSV.
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Subject
Subject(s)
Neurotransmitters
Neural transmission
Synaptosomes
Neural transmission
Synaptosomes
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Dissertation
Description
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ix, 121 leaves : illustrations
Department
Chemistry and Biochemistry