Publication Date

2012-05-04

Availability

Open access

Embargo Period

2012-05-04

Degree Name

Master of Science (MS)

Department

Marine Biology and Fisheries (Marine)

Date of Defense

2012-04-03

First Committee Member

Danielle McDonald

Second Committee Member

Douglas Crawford

Third Committee Member

Diego Lirman

Fourth Committee Member

Chris Wood

Fifth Committee Member

Lynne Fieber

Abstract

The Gulf toadfish (Opsanus beta) is a facultative ureotele in which urea production via the ornithine-urea cycle (O-UC) and pulsatile urea excretion are initiated by high cortisol levels, but a permissive drop in cortisol is required for the activation of the toadfish urea transporter (tUT) to occur. The goal of this study is to determine the exact relationship between cortisol and regulation of the serotonin 2A (5-HT2A) receptor, which is believed to be responsible for the activation of tUT. A better understanding of the interactions between cortisol and the 5-HT2 receptor family could provide some answers or new treatment methods for humans who suffer from hypercortisol and hypocortisol conditions like Cushing’s Syndrome and Addison’s Disease, respectively. The hypothesis of the study was that chronically elevated cortisol levels will result in an increase in the gill basolateral membrane population of 5-HT2A receptors through membrane insertion with no significant corresponding change in gill 5-HT2A receptor mRNA expression. Cortisol levels were elevated after one week of crowding compared to uncrowded controls and were reduced by treatment with the cortisol production blocker, metyrapone, compared to saline-treated controls. Crowded fish showed no difference in plasma 5-HT yet had elevated plasma urea concentrations compared to uncrowded fish; in contrast, plasma urea levels were not different between control and metyrapone -injected fish. Furthermore, no significant difference was measured in gill or brain 5-HT2A mRNA expression levels among uncrowded and crowded, control- or metyrapone-treated fish. A change in [3H] serotonin binding kinetics or in the competition of the 5-HT2 agonist, α-methyl 5-HT, against [3H] serotonin was also not seen in isolated gill basolateral membranes of uncrowded or crowded toadfish. However, binding kinetics of the 5-HT2A receptor agonist, [3H] ketanserin, was significantly different between the four groups of fish. Specifically, the binding affinity (KD) values for uncrowded, saline-injected, and metyrapone injected fish were similar to mammalian values for the 5-HT2A receptor, while crowded fish had a KD that was two-fold higher, suggesting a slight upregulation in a lower affinity receptor in this group of fish. Furthermore, we found the binding maximum (Bmax) of metyrapone-injected fish to be the greatest followed in descending order by control, crowded, and uncrowded fish. Our results suggest that chronic cortisol elevations may increase 5-HT2A receptor populations on the basolateral membrane. Interestingly, metyrapone-treated fish excreted approximately 2-fold more urea compared to controls when injected with α-methyl 5-HT suggesting that lowering endogenous levels of cortisol on a relatively acute time frame may remove a subsequent inhibition of cortisol on the 5-HT2A receptor or stimulate 5-HT2A recruitment to the basolateral membrane.

Keywords

Cortisol; tUT; serotonin; urea transport; nitrogen excretion; Gulf toadfish

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