Secretion From Individual Vesicles

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DESCRIPTION (provided by applicant): Chemical communication involves release of chemical messengers from biological cells, their diffusion to target cells, binding to specific receptors, and clearance from the extracellular space. Amperometry at a carbon-fiber microelectrode (with an applied potential sufficient to oxidize catecholamines) placed next to an isolated secretory cell can follow exocytosis as a series of current spikes composed of discrete packets of catecholamines detected by their electrooxidation. To evaluate events after exocytosis, carbon-fiber microelectrodes can be placed in tissue slices maintained in physiological buffer. The single cell measurements allow evaluation of individual exocytotic events while the tissue slice measurements provide key information on the diffusion and clearance of the released substances. In this proposal we set forth a plan to investigate some biochemical and physiochemical aspects that are central to chemical communication using the electrochemical approaches that we have developed. In these investigations we will probe the dynamics of release events both at single cells and in intact tissue slices. Thus, this proposal expands upon our prior work by providing a more complete view of the regulation of concentrations of chemical messengers from exocytosis to their removal from the extracellular fluid. Three specific proteins, each positioned at a central regulatory location, will be targets; for each protein, transgenically altered mice are available to us with deletions or additions of these proteins. The first is uncoupling protein 2 (UCP2), a mitochondrial protein found within neurons that is a key regulator of adenosine triphospate (ATP) production. The second is synapsin, a protein that is central to vesicular localization within nerve terminals. The third is the dopamine transporter (DAT), the protein responsible for reincorporation of dopamine back into its neurons. Thus, the specific aims of this proposal are: 1. Examine the role of UCP2, a mitochondrial protein that can diminish ATP production. We will examine its effects on neurotransmitter storage and release. 2. Examine the role of synapsin in determining the availability of vesicles for release. Synapsin, an abundant vesicular membrane protein, has been proposed to regulate the availability of vesicles for release. 3. Examine the extracellular lifetime of dopamine in the brains of mice that overexpress the DAT. We will examine the consequences of overexpression of this protein on dopaminergic neurotransmission. 4. Examine dopamine release in the brains of transgenic mice that express green-fluorescent protein (GFP) in neurons containing tyrosine hydroxylase (TH). 5. Evaluate control mechanisms of released catecholamines within the adrenal gland. Through precise placement of carbon-fiber microelectrodes within adrenal slices, we will monitor catecholamines from their exocytotic release to their transport to the blood vessels. The proposed research will provide an unprecedented view of chemical communication in the central nervous and neuroendocrine systems. PUBLIC HEALTH RELEVANCE: Chemical communication involves release of chemical messengers from biological cells, diffusion of secreted substances to target cells, binding to specific receptors, and clearance from the extracellular space. In the proposed research, these processes will be investigated with chemical sensing microelectrodes placed adjacent to isolated cells or implanted in slices of brain tissue.