Date of Award

2017

Degree Type

Thesis

Degree Name

Master of Science in Pharmaceutical Sciences

Specialization

Pharmacology and Toxicology

Department

Biomedical and Pharmaceutical Sciences

First Advisor

Robert L. Rodgers

Abstract

The actions of glucagon have been extensively studied for many years. In heart, it is well established that glucagon is capable of causing inotropy. This action is mediated by glucagon binding to a G-protein coupled receptor (GPCR) which activates adenylate cyclase, increasing the intracellular concentrations of cAMP. The studies that were able to show this effect used concentrations of glucagon that were considered to be much higher than what is normally found in blood. A recent report, by Harney and Rodgers (2008), described the actions and signaling mechanisms of physiological concentrations of glucagon (<10-10 M) on perfused rat hearts. These investigators were also able to show that a peak physiological concentration of glucagon (10-10 M) increased glucose uptake in the same preparation. It was also found that this action was mediated by the activation of the PI3K/AKT signaling pathway. The missing piece of this mechanism to fully show glucagon’s action is the receptor responsible for glucagon binding and initiation of the reported downstream signaling effects. We hypothesize that one of two situations would occur: 1) the glucagon receptor that mediates glucose uptake is a GPCR or 2) there is a novel glucagon receptor responsible for the effects observed at low concentrations. Hearts were removed from male Sprague Dawley rats (n=1-6/grp) and isolated heart perfusion methods, either Langendorff or working mode, were used to deliver a physiological solution to the heart. Specific concentrations of hormone, one which represents peak physiological glucagon levels and another that represents a concentration of glucagon too high to be normally found in blood, were added to the physiological solution. An anti-glucagon receptor antibody was obtained which was used to block the known glucagon receptor. Hearts were treated with no hormone or with the following treatments: 1) 10-10 M glucagon; 2) 4x10-10 M insulin; 3) 10-8 M glucagon; 4) 10-8 M glucagon plus antibody and 5) 10-10 M glucagon plus antibody. Immediately after perfusion, hearts were processed and probed for AKT activation. It was found that glucagon concentrations at 10-10 M did not induce an inotropic response during Langendorff perfusion, while glucagon concentrations at 10-8 M was able to. For working mode, an inotropic response was not observed when hearts were perfused with 10-10 M glucagon and 10-8 M glucagon. There was no observable activation in AKT after physiological glucagon treated hearts in either Langendorff or working mode perfusion methods. In conclusion, the receptor that mediates glucagon’s actions at physiological concentrations was not able to be characterized from these experiments. Experimental strategy should be revised to optimize ex vivo heart perfusion method and include additional in vitro experiments to conclude the identity of the glucagon receptor.

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