Publication Date
2014
Document Type
Thesis
Committee Members
Adrian Corbett (Committee Member), Mark Rich (Committee Member), Christopher Wyatt (Advisor)
Degree Name
Master of Science (MS)
Abstract
It has previously been reported that AMP-activated protein kinase (AMPK) may be critical for hypoxic chemotransduction in carotid body type I cells (Wyatt et al, 2007). This study sought to determine the importance of the regulatory upstream kinase of AMPK, CamKKβ, in the acute response to hypoxia in isolated mouse type I cells.
Initial studies indicated several previously unreported artefacts associated with using the CamKKβ inhibitor STO-609 and calcium imaging techniques. Strong STO-609 autofluorescence meant that Fura-2 could not be used and X-Rhod-1 imaging revealed that STO-609 quenched fluorescence even in the absence of intracellular Ca2+. The whole-cell configuration of the perforated-patch clamp technique revealed for the first time that STO-609 (100μ) rapidly (seconds) inhibited outward macroscopic currents at +10 mV by 38.0% ± 7.4% (n = 5, P<0.03, mean ± SEM) and that this inhibition was abolished in the presence of the selective BKCa inhibitor paxilline (1 μM). Taken together these data suggest that STO-609 should be used with caution during Ca2+ imaging studies as it can directly interact with dyes. The rapid inhibitory effect of STO-609 on BKCa was unexpected as the majority of studies using this compound required an incubation of approximately 10 minutes to inhibit the kinase. Furthermore, as AMPK activation inhibits BKCa, inhibiting AMPK's upstream kinases would, if anything, be predicted to have the opposite effect on BKCa. Future work will determine if the inhibition of BKCa is via CamKKβ or via an off target action of STO-609 on the channel itself.
Page Count
80
Department or Program
Department of Neuroscience, Cell Biology & Physiology
Year Degree Awarded
2014
Copyright
Copyright 2014, all rights reserved. This open access ETD is published by Wright State University and OhioLINK.
