Semester

Spring

Date of Graduation

2012

Document Type

Dissertation

Degree Type

PhD

College

School of Medicine

Department

Physiology, Pharmacology & Neuroscience

Committee Chair

Gregory M. Dick.

Abstract

Bisphenol A (BPA) exposure has recently become a public health concern, and the safety of the utilization of BPA has attracted the attention of the scientific community, politicians, and the general population. Although BPA is banned from baby products in a few countries because infants and young children are particularly susceptible to endocrine disruption, recent studies have consistently indicated the association between urinary BPA concentration and cardiovascular diseases in adults. Therefore, adult exposure to this xenoestrogen is becoming a healthcare issue that cannot be ignored. We selected large conductance Ca2+/voltage sensitive potassium (BK) channels as one of the target proteins for BPA effects because the function and expression of BK channels are regulated by estrogen. Additionally, BK channels play an important role in vascular function and changes in BK expression are associated with disease. We hypothesized that BPA would differently affect BK channel function and expression via separate non-genomic and genomic mechanisms. The goals of our studies were 1) to characterize a novel, membrane-permeable, and economical antagonist called penitrem A to facilitate the study of BK channels, 2) to determine the mechanisms by which BPA increases BK channel activity and, 3) to determine the mechanisms by which BPA reduces vascular BK channel expression.;Current from patches of HEK 293 cells transfected with hSlo alpha or alpha + beta1 were blocked >95% by penitrem A (IC50 6.4 vs. 64.4 nM; p<0.05). Further, penitrem A inhibited BK channels in inside-out and cell-attached patches, whereas iberiotoxin could not. Inhibitory effects of penitrem A on whole-cell K+ currents were equivalent to iberiotoxin in canine coronary smooth muscle cells. Penitrem A enhanced sensitivity to K+-induced contraction in canine coronary arteries by 23 +/- 5% (p<0.05) and increased the blood pressure response to phenylephrine in anesthetized mice by 36 +/- 11% (p<0.05).;In cultured canine coronary artery smooth muscle cells (CASMC), acute BPA (10 microM) exposure increased BK channel currents 179 +/- 8% ( p<0.05). Inside out recordings from native canine CASMCs showed that acute BPA increased BK channel open probability (Po) from 0.11 +/- 0.03 to 0.47 +/- 0.09 at +40 mV (p<0.05). HEK cells transfected with BK alpha subunits alone were insensitive to acute BPA (currents at +100 mV were 101 +/- 13% of control) while acute BPA increased BK channel currents by 53 +/- 12% (p<0.05) when cells were transfected with alpha and beta1 subunits. Similarly, single channel recordings indicated NPo was 123 +/- 20% of control in the BK alpha subunits alone with BPA, however, acute BPA increased NPo 201 +/- 41% in patches from cells expressing BK alpha and beta1 subunits. Thus, BK beta1 subunits facilitated BPA-induced BK channel activation and the effects of BPA were non-genomic, as these effects were observed in cell-free patches.;In contrast to the acute effects of BPA on BK channel activity, 48-72 hrs of BPA (10 microM) exposure decreased BK channel expression in A7r5 cells and cultured rat aorta. Western blot data indicated that BK alpha subunit protein content was 33 +/- 8% of control in BPA-treated aorta (p<0.05). Patch clamp recording showed that whole cell current densities at +100 mV were 57 +/- 7% lower in aortic SMC treated with 10 microM BPA (p<0.05) while whole cell current densities from aortic SMC treated with BPA + ICI 182780 (100 nM; an estrogen receptor blocker) were not different (101 +/- 14% of control). Penitrem A-sensitive current densities at 100 mV were 53.9 +/- 14.8, 12.0 +/- 3.0, and 48.4 +/- 10.1 pA/pF in DMSO, BPA and BPA + ICI 182780, respectively (p<0.05). Thus, chronic BPA exposure decreased BK channel expression by estrogen receptor mediated signaling.;Our data indicate: 1) penitrem A is an economical alternative to iberiotoxin for BK channel analysis; 2) acute BPA exposure activates BK channel activity and BK beta1 subunits play a role in BPA induced BK channel activation; 3) chronic BPA exposure decreases vascular BK channel expression via estrogen receptor-dependent mechanisms. Although acute BPA exposure activates the BK channel through a non-genomic mechanism that could be beneficial for heart due to coronary dilation, longer BPA exposure decreases the number of functional BK channels through a genomic mechanism that could be detrimental to the heart. Thus, the overall effect of BPA exposure on vascular function may be harmful due to disruption of BK channel-mediated vascular regulation. Taken together, these results indicated BPA induced altered BK channel function and expression may be one of the mechanisms for the association between BPA exposure levels and cardiovascular diseases seen in epidemiological studies.

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