Wilson, Rachael-Anh
(2019)
In vivo Measurement of Ectopeptidase Activity Using Electroosmotic Perfusion- Microdialysis.
Doctoral Dissertation, University of Pittsburgh.
(Unpublished)
This is the latest version of this item.
Abstract
Insulin regulated aminopeptidase (IRAP, EC 3.4.11.3) is a promising drug target for treatment of cognitive impairment. This membrane-bound ectopeptidase is densely concentrated in areas of the brain associated with cognition and is known to hydrolyze neuropeptides involved in memory and learning in vitro. A hypothesis is that inhibition of IRAP may extend the lifetime of beneficial neuropeptides in the extracellular space, mitigating the effects of diseases such as Alzheimer’s. A challenge in testing this hypothesis is the lack of available methods for quantitatively measuring ectopeptidase activity in vivo.
This work describes the development of analytical techniques to aid in the understanding of ectopeptidases and the fate of neuropeptides in the extracellular space. We have done this by enhancing the sensitivity and quantitative capabilities of detection methods for online collection of neuropeptides and by the fabrication and development of a novel sampling technique called electroosmotic perfusion-microdialysis (EOP-MD). With EOP-MD, substrate peptides are perfused using electroosmotic flow and hydrolysis products along with unhydrolyzed substrate are collected at the microdialysis probe. The advantage of using this approach compared to conventional microdialysis is that the volume of substrate and the residence time in the tissue can be controlled using current. Additionally, spatial resolution is dependent on the distance between the EOP and MD probes, which is approximately 100 micron. We have demonstrated this approach by measuring leucine enkephalin hydrolysis in the anesthetized rat brain and observed a dose-dependent reduction in hydrolysis with the IRAP inhibitor HFI-419. As far as we are aware, this is the first sampling method capable of quantifying ectopeptidase activity in vivo, particularly at a spatial resolution of 100 micron.
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Details
| Item Type: |
University of Pittsburgh ETD
|
| Status: |
Unpublished |
| Creators/Authors: |
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| ETD Committee: |
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| Date: |
25 September 2019 |
| Date Type: |
Publication |
| Defense Date: |
29 April 2019 |
| Approval Date: |
25 September 2019 |
| Submission Date: |
8 July 2019 |
| Access Restriction: |
No restriction; Release the ETD for access worldwide immediately. |
| Number of Pages: |
159 |
| Institution: |
University of Pittsburgh |
| Schools and Programs: |
Dietrich School of Arts and Sciences > Chemistry |
| Degree: |
PhD - Doctor of Philosophy |
| Thesis Type: |
Doctoral Dissertation |
| Refereed: |
Yes |
| Uncontrolled Keywords: |
peptide, brain, liquid chromatography, mass spectrometry |
| Date Deposited: |
25 Sep 2019 19:23 |
| Last Modified: |
25 Sep 2019 19:23 |
| URI: |
http://d-scholarship.pitt.edu/id/eprint/37435 |
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In vivo Measurement of Ectopeptidase Activity Using Electroosmotic Perfusion- Microdialysis. (deposited 25 Sep 2019 19:23)
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