Studies of neurotransmitter release from permeabilized synaptosomesShow full item record
Title | Studies of neurotransmitter release from permeabilized synaptosomes |
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Author | Zheng, Xu |
Date | 1997 |
Genre | Dissertation |
Degree | Doctor of Philosophy |
Abstract | We examined $\lbrack\sp3$H) -noradrenaline $(\lbrack\sp3$H) -NA) release from streptolysin-O (SLO) permeated rat brain cortical synaptosomes under different conditions. Lowering temperature to 20$\sp\circ$C decreased basal release, but Ca$\sp{2+}$-induced release increased slightly. The maximal ratio of Ca$\sp{2+}$-induced release to basal release occured at longer times with decreasing temperature. Permeation at 20$\sp\circ$C allowed $\lbrack\gamma$-$\sp{32}$P) ATP and cAMP-dependent protein kinase (PKA) catalytic subunit to rapidly enter the synaptosomes to phosphorylate synapsins. Lactate dehydrogenase (LDH) efflux was time- and SLO-concentration dependent. The fact that 0.1mM Cd$\sp{2+}$ did not inhibit release from permeabilized synaptosomes indicated that permeabilization was complete. Synaptosomes were successfully perforated with high concentrations ($\ge$400U/ml) of Staphylococcus aureus $\alpha$-toxin. The free Ca$\sp{2+}$-concentration dependence of ($\sp3$H) -NA release was similar to that observed for PC 12 and chromaffin cells. Release from the $\alpha$-toxin perforated synaptosomes was not significantly inhibited by $\omega$-conotoxin GVIA. LDH efflux from $\alpha$-toxin-perforated synaptosomes was not different than efflux from control synaptosomes, and an antibody to N-ethylmaleimide-sensitive fusion protein (NSF) did not enter the synaptosomes. In SLO perforated synaptosomes, Ca$\sp{2+}$-induced release was resolved into at least two sequential steps: a MgATP-dependent priming step and a MgATP-independent, Ca$\sp{2+}$-triggered step. Vesicle depriming was detected when the synaptosomes were perforated and incubated for 2 min in the absence of MgATP before increasing Ca$\sp{2+}$ to promote release. Polyclonal antibodies against NSF and Hrs-2 blocked Ca$\sp{2+}$-induced release at both early and late phases, indicating involvement of both NSF and Hrs-2 in ATP-independent and ATP-dependent stages of neurosecretion. One mM N-ethylmaleimide (NEM) inhibited both MgATP-dependent and MgATP-independent release; this suggests a role for NSF after ATP hydrolysis and vesicle priming, possibly in the membrane fusion step. We also examined the subcellular distribution of syntaxin1 in rat brain cortical synaptosomes and its role in neuronal exocytosis. Different synaptosomal subfractions were isolated by sucrose-gradient centrifugation and the distribution of syntaxin1 was determined by immunoassay. Our results showed that syntaxin1 is associated with presynaptic membranes and synaptic vesicles. Several SDS-resistant, syntaxinl containing complexes also were detected in the presynaptic membrane fraction, but not in the synaptic vesicle fraction, suggesting that syntaxinl exists in distinct forms in different pools. Monoclonal anti-syntaxin Fab fragments inhibited both Ca$\sp{2+}$-dependent and Ca$\sp{2+}$-independent $\sp3$H-NA release when introduced into permeabilized synaptosomes, suggesting an active role of syntaxin1 in neurotransmitter release. |
Link | https://repository.tcu.edu/handle/116099117/31823 |
Department | Chemistry and Biochemistry |
Advisor | Bobich, Joseph A. |
This item appears in the following Collection(s)
- Doctoral Dissertations [1526]
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