Evidence of alpha 7 nicotinic acetylcholine receptor expression in retinal pigment epithelial cells

Abstract

Some evidence suggests that retinal pigment epithelium (RPE) can express nicotinic acetylcholine receptors (nAChRs) as described for other epithelial cells, where nAChRs have been involved in processes such as cell development, cell death, cell migration, and angiogenesis. This study is designed to determine the expression and activity of a7 nAChRs in RPE cells. Reverse transcriptase (RT)-PCR was performed to test the expression of nicotinic a7 subunit in bovine RPE cells. Protein expression was determined by Western blot and by immunocytochemistry. Expression of nicotinic a7 subunits was also analyzed in cryostat sections of albino rat retina. Changes in protein expression were tested under hypoxic conditions. Functional nAChRs were studied by examining the Ca 2+ transients elicited by nicotine and acetylcholine stimulation in fura-2-loaded cells. Expression of endogenous modulators of nAChRs was analyzed by RT-PCR and Western blot in retina and RPE. Cultured bovine RPE cells expressed nicotinic receptors containing a7 subunit. RT-PCR amplified the expected specific a7 fragment. Western blotting showed expression at the protein level, with a specific band being found at 57 kDa in both cultured and freshly isolated RPE cells. Expression of nAChRs was confirmed for cultured cells by immunofluorescence. Immunohistochemistry confirmed a7 receptor expression in rat RPE retina. a7 receptor expression was down-regulated by long-term hypoxia. A small subpopulation of RPE cultured cells showed functional nAChRs, as evidenced by the selective response elicited by nicotine and acetylcholine stimulation. Expression of the endogenous nicotinic receptors' modulator lynx1 was confirmed in bovine retina and RPE, and expression of lynx1 and other endogenous nicotinic receptor modulators (SLURP1 and RGD1308195) were also confirmed in rat retina. These results suggest that nAChRs could have a significant role in RPE, which may not be related to the traditional role in nerve transmission but could more likely be related to the nonneuronal cholinergic system in the eye.

Localization of Acetylcholine-Related Molecules in the Retina: Implication of the Communication from Photoreceptor to Retinal Pigment Epithelium

“…Several reports have confirmed that RPE expresses α7 nAChR [10], [24]. Thus, we speculated that acetylcholine might also be enriched in photoreceptor OS.…”

“…Although nAChRs act as signal transducers in neurons [6], they have different roles in nonexcitable cells where they contribute to homeostasis [7]–[9]. Maneu et al [10] investigated the expression of α7 nAChR in retinal pigment epithelium (RPE) cells, and reported that immunoreactivity of α7 nAChR was observed in RPE cells, especially in the apical surface of RPE cells adjacent to photoreceptor outer segments (OS). In contrast, photoreceptor OS showed no α7 nAChR immunoreactivity.…”

Nicotine reduces VEGF-secretion and phagocytotic activity in porcine RPE

“…We were able to show that the resistance of the RPE against nicotine is not limited to cell lines but holds true for primary RPE cells, and is valid after even after prolonged exhibition (up to 1 week). The authors suggested the resistance of ARPE-19 cells to be explained by an absence of nicotine receptors on these; however, RPE cells do express nicotinic acetylcholine receptors, and as such are able to specifically respond to nicotine [23,24].…”

Evidence of BrdU-positive retinal neurons after application of an Alpha7 nicotinic acetylcholine receptor agonist

“…This result would dispute the possibility that BrdU positive cells are initiated from RGCs that contain α7 nAChRs (Thompson et al, 2006). However, it does not rule out the possibility that the α7 nAChR agonist binds to receptors in the retinal pigment epithelium (Maneu et al, 2010; Matsumoto et al, 2012; Fuhrmann et al, 2014), which then secrete unknown signaling factors that initiates the division of Müller glia. Further experiments are required to examine this possibility.…”