Title

The Effects of Growth Hormone-Releasing Hormone on Nitric Oxide Production in Human Coronary Arterial Endothelial Cells

Date of Award

5-2020

Degree Type

Thesis

Degree Name

Master of Science in Biomedical Sciences

First Advisor

Richard E. White, PhD

Second Advisor

Shu Zhu, MD, PhD

Third Advisor

Shafik Habal, MD

Fourth Advisor

Lori Redmond, PhD

Abstract

Objective: Cardiovascular disease (CVD) and type 2 diabetes mellitus (T2DM) are the leading causes of morbidity and mortality worldwide. An early hallmark sign of diabetes-induced CVD is vascular endothelium dysfunction, indicated by decreased nitric oxide (NO) bioavailability and decreased phosphorylation of eNOS at the serine1177 (ser1177) residue. eNOS ser1177 can be phosphorylated in a cyclic adenosine monophosphate (cAMP) manner, primarily through the activity of protein kinase A (PKA). The hypothalamic growth hormone-releasing hormone (GHRH) regulates the release of growth hormone (GH) by pituitary somatotrophs but also exerts separate cardioprotective actions on endothelial cells through GHRH receptors (GHRH-R) in a cAMP-dependent manner. However, its role in NO production by p-eNOS ser1177 activity in non-diabetic and diabetic human coronary arterial endothelial cells (HCAECs) remains elusive.

Methods and Results: By Western blot analysis, we demonstrated for the first time the presence of GHRH receptors and its splice variant, SV1, in HCAECs. We also provided evidence to support the downregulation of p-eNOS ser1177 seen in diabetes, and treatment with GHRH agonist MR-409 (5μM) for 30 minutes, can elevate p-eNOS ser1177 levels. To support this evidence, we also treated the HCAECs in a similar fashion with an adenylyl cyclase agonist forskolin (10μM), which elevates cAMP, and saw similar results. To measure NO production upon a 30-minute treatment of MR-409, fluorescence microscopy and 4-amino-5-methylamino-2′,7′-difluorofluorescein diacetate (DAF-FM DA; 2μM) were used. MR-409 significantly elevated DAF fluorescence in T2DM HCAECs and non-diabetic HCAECs (p < 0.01, p < 0.0001, respectively). Forskolin also significantly elevated DAF fluorescence in T2DM and non-diabetic HCAECs (p

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