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R.C. Splittgerber, M.E. Andison, C.E. Strang, K.T. Keyser; Expression and Function of nAChRs in Human Vascular Endothelial Cells . Invest. Ophthalmol. Vis. Sci. 2006;47(13):5338.
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© ARVO (1962-2015); The Authors (2016-present)
Neuronal nicotinic acetylcholine receptors (nAChRs) are widely expressed in neural and non–neural tissues of the eye and have been reported to play a role in neovascularization. Increased choroidal neovascularization has been reported in response to chronic exposure to nicotine (Suner et al, 2004). Although nAChR subunits have been identified in non–neuronal ocular tissues, the subunits that combine to form functional nAChRs, their mechanisms of action, and the downstream signaling pathways have yet to be determined. Since some nAChR subtypes have high Ca2+ permeability, we are investigating the possible role of Ca2+ in activating signaling cascades that underlie microvascular outgrowth.
RT–PCR was performed using cultured human endothelial cell (EC) RNA and primers for human neuronal nAChR subunits. For calcium imaging, cells were loaded with Fluo–3 AM calcium indicator dye, examined, and quantified with confocal microscopy and imaging software. Ca2+ levels at different time points were tested in response to nicotinic agonist and antagonist applications by ANOVA followed by Tukey–Kramer’s multiple comparisons test.
Human EC RNA contained message for all nAChR subunits except, α2, α6 and ß3. Sequence homology ranged from 90–100%. Nicotine treatment significantly increased Ca2+ as compared to controls. Pretreatment with MLA, an nAChR antagonist that is non–specific at micromolar concentrations, followed by nicotine, blocked the increase in calcium dye fluorescence. Treatment with α–bungarotoxin, an α7 specific antagonist, significantly decreased, but did not abolish, Fluo–3 reactivity compared to nicotine treatment alone. The residual signal was significantly higher than that in controls. Inhibition of nicotine–induced Ca2+ release from intracellular stores using antagonists for IP3 and ryanodine receptors in addition to nicotine resulted in a significantly lower amount of fluorescence.
Nicotine induced a rapid and sustained increase in intracellular Ca2+. This raises the possibility that the nicotine–induced increase in intracellular Ca2+ concentration may potentially trigger signaling cascades, in part using intracellular Ca2+ stores, and that nAChRs are involved in vascular outgrowth.
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