BKCa Channel Expression and Functional Regulation In Type 1 Diabetic Human Pulmonary Artery Smooth Muscle Cells

Date of Award

2016

Degree Type

Thesis

Degree Name

Master of Science (MS)

First Advisor

Shu Zhu, MD, PhD

Second Advisor

Abigail Hielscher, PhD

Third Advisor

Richard E White, PhD, FAHA

Fourth Advisor

Brian Matayoshi, PhD

Abstract

Pulmonary arterial hypertension (PAH) is a progressive vascular disease that results in right-side heart failure and death and is characterized by increased pulmonary blood pressure and vascular resistance. Large-conductance, calcium- and voltage-activated potassium (BKCa) channels play a crucial role in the regulation of membrane potential and contractility of pulmonary arterial smooth muscle cells (PASMCs). Under normal conditions, cAMP-elevating agents activate BKCa channels in PASMCs and cause vasodilatation, while pulmonary vasoconstriction caused by inhibition (closing) of BKCa channels has been considered as one of the key factors that contribute to the development of pulmonary hypertension. Clinical evidence suggests that there is a higher chance of developing pulmonary hypertension in patients with diabetes. However, little information is known about the expression and potential signaling mechanisms of BKCa channels in the pulmonary vasculature in the diabetic state. In our preliminary studies, western blots showed that both normal and diabetic porcine PASMCs express both BKCa channel α-subunits and β1-subunits with a greater expression of α-subunits in diabetic PASMC. Electrophysiological studies using inside-out cell patches from normal PASMC also demonstrated that BKCa channels were activated by 100 μM Ca2+, which was blocked by 1 mM tetraethylammonium (TEA, a selective BKCa channel inhibitor at this concentration). In cell-attached patches, forskolin (10 μM), an activator of adenylyl cyclase which increases cAMP concentration, increased BKCa channel activity significantly in control PASMC. Basal cAMP levels in diabetic porcine PASMC were lower than in control PASMC, and foskolin increased cAMP generation to a lesser extent in diabetic PASMC compared to control PASMC. Therefore, we assessed BKCa channel expression and signaling regulation in human pulmonary artery smooth muscle cells (HPASMC) from normal and type 1 diabetic human cell lines. Western blots showed that both normal and type 1 diabetic HPASMC (D1HPASMC) express both BKCa channel α-subunits and β1-subunits with a decreased expression of both α- and β1-subunits in D1HPASMC. Subsequently, cAMP and cGMP generation was measured in both control and D1PASMCs. Basal cAMP levels in D1HPASMC were drastically lower than in normal HPASMCs, and foskolin increased cAMP to a lesser extent in D1HPASMCs compared to normal HPASMCs. Collectively, these results indicate that signaling pathways involving BKCa channels and cyclic nucleotides are altered in D1HPASMCs, which suggests potential differences in pulmonary vasoreactivity between the normal and diabetic states.

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