Dr. James Porter, Department of Physiology & Developmental Biology
Specific Question 1. Can the down-regulation of epoxide hydrolase expression determined by microarray analysis be confirmed using RT-PCR?
We were able to use RT-PCR to confirm that mRNA expression for brain soluble epoxide hydrolase was significantly down-regulated. The magnitude of relative down-regulation was on the same order as that found using microarray analysis. This was an important finding since microarray data cannot be published unless corroborating evidence is found using a separate technology (these data were presented at the Experimental Biology 2010 meeting in April, 2010). We extended this question by asking if the soluble epoxide hydrolase protein, itself, was also down-regulated. This required extraction of total protein from brains of high salt (HS) and normal salt (NS) offspring, followed by Western Blot using an antibody specific for soluble epoxide hydrolase. We were surprised to find that protein expression was significantly increased. At this point, we are not sure why mRNA expression for soluble epoxide hydrolase is decreased in HS rats, but protein expression is increased. We hypothesize that the decrease in mRNA expression might be a negative feedback response to the increase in protein. If the protein is slower to be degraded than the mRNA, and such a negative feedback situation exits, we would expect to find the protein increased and the mRNA decreased. We were also interested in finding the localization of the soluble epoxide hydrolase protein within the brain. To do this, we employed immunohistochemistry. We found a strong signal for the protein in the arcuate nucleus (ARC) of the hypothalamus
(see figure). This is important because the
arcuate nucleus has been implicated in the
neural response to stress.
Dallin Dance, Tyson Finlinson, Stephen Adophson, Alan Brown, Brieann Emfield, Rachel Hill, Tim Jenkins, Jason Hunt, Christine Roach, Jeff Sorensen, Alan Sutak, Blaine White, Brandon Wilkinson, Robert Babbel, Bryce Coombs, Jennifer Heckmann, Elise Sienicki, Robby Wai, Clinton Johnson, Mike Petrini, Mike Rao, Kristy Rogers
Summer King, Clinton Johnson
Porter, J.P. and S. H. King*. Prenatal high salt programs enhanced sympathoadrenal activation of the cardiovascular response to restraint. Auton. Neurosci. Basic Clin. 150:140-143, 2009.
J.P. Porter, S. King*, M. Petrini†, and M. Rau†. The contribution of the sympathetic nervous system to the stress-induced pressor and tachycardic hyperresponsiveness programmed by prenatal high salt. Program 738.22, Experimental Biology, 2008, CD ROM.
J.P. Porter, C. Johnson†, K. Rogers†. Expression of soluble epoxide hydrolase (SEH) in brains of female offspring of rats fed high salt during pregnancy. Experimental Biology, 2010.
Johnson, C*. Mechanisms of prenatal high-salt “fetal programming” resulting in stress hyperresponsiveness in the adult female offspring in the Sprague Dawley rat. MS. Thesis, July, 2011, J.P. Porter Advisor.
* mentored graduate student
† mentored undergraduate student