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  • Acetylcholine induces Ca2+ signaling in chicken retinal pigmented epithelial cells during dedifferentiation.

Acetylcholine induces Ca2+ signaling in chicken retinal pigmented epithelial cells during dedifferentiation.

American journal of physiology. Cell physiology (2009-02-27)
Mariko Sekiguchi-Tonosaki, Masakatsu Obata, Akira Haruki, Toshiyuki Himi, Jun Kosaka
ABSTRACT

Retinal pigmented epithelial cells exchange their cellular phenotypes into lens cells and neurons, via depigmented and non-epithelial-shaped dedifferentiated intermediates. Because these dedifferentiated cells can either revert to pigmented epithelial cells or transdifferentiate into lens cells and/or neurons, they are recognized as candidates for lens and retinal cell regeneration. The purpose of the present study was to elucidate the signal transduction pathways between chicken retinal pigmented epithelial cells and their dedifferentiated intermediates. We monitored intracellular Ca(2+) concentrations using Fluo-4-based Ca(2+) optical imaging and focused on cellular responses to the neurotransmitter acetylcholine. Muscarinic Ca(2+) mobilization was observed both in retinal pigmented epithelial cells and in dedifferentiated cells, and was inhibited by atropine. The muscarine-dependent acetylcholine response depended on Ca(2+) release from intracellular Ca(2+) stores, which was completely blocked by thapsigargin. In contrast, the nicotine-dependent acetylcholine response that led to Ca(2+) influx through L-type Ca(2+) channels was inhibited by alpha-bungarotoxin and attenuated by nifedipine, and it was detected only in the dedifferentiated intermediates. Application of (S)-(-)-BayK8644 elevated intracellular Ca(2+) both in retinal pigmented epithelial cells and in dedifferentiated intermediates; however, the nicotinic response was not observed in pigmented epithelial cells. Another L-type Ca(2+) channel blocker, diltiazem, also blocked the nicotine-dependent acetylcholine response in dedifferentiated cells and maintained the epithelial-like morphology of retinal pigmented epithelial cells. Our results indicate that an alternative acetylcholine signaling pathway is used during the dedifferentiation process of retinal pigmented epithelial cells.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
(+)-cis-Diltiazem hydrochloride, ≥99% (HPLC)
Sigma-Aldrich
Thapsigargin, ≥98% (HPLC), solid film
Sigma-Aldrich
Atropine sulfate salt monohydrate, ≥97% (TLC), crystalline
Sigma-Aldrich
(S)-(−)-Bay K8644, ≥98% (HPLC), solid
Sigma-Aldrich
Acetylcholine chloride, ≥99% (TLC)
Sigma-Aldrich
(+)-Muscarine chloride, ~95% (TLC), powder
Sigma-Aldrich
Acetylcholine chloride, ≥99% (TLC), free-flowing, Redi-Dri
Sigma-Aldrich
Nifedipine, ≥98% (HPLC), powder
Sigma-Aldrich
Tubocurarine hydrochloride pentahydrate, ≥97%