Ca2+ dynamics affect many critical cellular processes. In the bag cell neurons of Aplysia californica, intracellular Ca2+ is elevated during a prolonged period of firing known as the afterdischarge. This consists of a fast and slow phase of firing, which triggers peptide secretion and culminates in egg-laying. The present study examines how Ca2+ influx and release shape neurosecretion and membrane activity. Using capacitance tracking as an index of secretion, a 5 Hz, 1 min train, to mimic the fast phase, induced a clear elevation in the membrane surface area of cultured bag cell neurons. The capacitance change was abolished by replacing external Ca2+ with Ba2+ or addition of the Ca2+ channel blocker, Ni2+. Additionally, the response was reduced by either strong buffering of intracellular Ca2+ or pretreatment with N-ethylmaleimide, an alkylating agent that disrupts vesicular transport. Depleting mitochondrial Ca2+ with the protonophore, carbonyl cyanide-p-trifluoromethoxyphenyl-hydrazone (FCCP), also elevated capacitance, while depleting endoplasmic reticulum Ca2+ with the Ca2+-ATPase inhibitor, cyclopiazonic acid, did not. Similarly, FCCP alone depolarized bag cell neurons. In a concentration-dependent manner, FCCP elicited an inward current that was insensitive to Ni2+, associated with an increase in conductance, and a linear current/voltage relationship that reversed around -40 mV. Removal of extracellular Ca2+ reduced the current and left-shifted the reversal, consistent with opening a Ca2+-permeable, voltage-independent, non-selective cation channel. The current was decreased when intracellular Ca2+ was strongly buffered, while fura-imaging demonstrated that FCCP elevated intracellular Ca2+ with a similar time course, suggesting a dependence on intracellular Ca2+. Although both oligomycin A and bafilomycin A, inhibitors of mitochondrial ATP sythetase and V-type H+-ATPase, respectively, gradually increased Ca2+, neither produced a current. The FCCP-induced Ca2+ elevation and the current were also diminished by disabling the mitochondrial permeability transition pore with N-ethylmaleimide. The data suggests that a cation current is preferentially gated by Ca2+ released from the mitochondria, rather than disruption of ATP production. This current could provide depolarizing drive for the afterdischarge. While Ca2+ entry appears to be responsible for initiating neurosecretion, mitochondrial Ca2+ may support prolonged peptide release during and subsequent to the afterdischarge. / Thesis (Master, Physiology) -- Queen's University, 2009-09-18 19:30:32.957
| Identifer | oai:union.ndltd.org:LACETR/oai:collectionscanada.gc.ca:OKQ.1974/5166 |
| Date | 19 September 2009 |
| Creators | Hickey, Charlene |
| Contributors | Queen's University (Kingston, Ont.). Theses (Queen's University (Kingston, Ont.)) |
| Source Sets | Library and Archives Canada ETDs Repository / Centre d'archives des thèses électroniques de Bibliothèque et Archives Canada |
| Language | English, English |
| Detected Language | English |
| Type | Thesis |
| Format | 1204427 bytes, application/pdf |
| Rights | This publication is made available by the authority of the copyright owner solely for the purpose of private study and research and may not be copied or reproduced except as permitted by the copyright laws without written authority from the copyright owner. |
| Relation | Canadian theses |
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