All ETDs from UAB

Advisory Committee Chair

Kent T Keyser

Advisory Committee Members

Craig Elmets

Paul Gamlin

Alecia Gross

Lori McMahon-Wakefield

Document Type


Date of Award


Degree Name by School

Doctor of Philosophy (PhD) School of Optometry


Neuronal nicotinic acetylcholine receptors (nAChRs) are widely expressed in neural and non-neural tissues and have been reported to play a role in neovascularization and vascular diseases of the eye, specifically age-related macular degeneration (ARMD). However, the subunit composition of functional nAChRs, their mechanisms of action, and the downstream signaling pathways in non-neuronal tissues are not well understood. Since some nAChR subtypes have high Ca2+ permeability, we investigated the possible role of Ca2+ in activating signaling cascades that underlie the affects of nAChR activation in Human Dermal Microvascular Endothelial Cells (HDMECs). RT-PCR experiments revealed message for nearly all of the known nicotinic receptor subunits and muscarinic receptors. The effects of nAChR activation were examined using fluorescent Ca2+ imaging. The Ca2+ indicator dye, Fluo-3, revealed a significant increase in intracellular Ca2+ influx after exposure to nicotine. This increase was significantly reduced or eliminated by nAChR antagonists. The effect of nAChR activation on intracellular signaling cascades was assessed by measuring the phosphorylation of signaling proteins. Phosphorylation of ERK1/2 was increased by exposure to nicotine for five minutes but decreased after 60 minutes of exposure. ERK1/2 phosphorylation after nicotine exposure was significantly reduced by both specific and non-specific nAChR antagonists and binding of Ca2+ by the Ca2+ chelator BAPTA-AM, demonstrating that the phosphorylation was a result of calcium dependent nAChR activation. As a member of iv the mitogen-activating protein kinase pathway, it is hypothesized that ERK1/2 activation leads to activation of transcription factors targeting angiogenesis-related genes. Real-time RT-PCR was used to analyze gene expression changes due to acute or chronic exposure to nicotine. At short durations nicotine exhibits anti-angiogenic effects and chronic exposure promotes a pro-angiogenic vascular environment via transcription of inflammatory molecules, adhesion molecules, and growth factors. These results were significantly decreased using nAChR antagonists. Our results suggest nicotinic activation of receptors in non-neuronal cells results in intracellular Ca2+ influx and phosphorylation of signaling kinases, leading to modified transcription of angiogenesis-related genes which we hypothesize affects cell proliferation or angiogenesis.

Included in

Optometry Commons



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