Carbachol-mediated pigment granule dispersion in retinal pigment epithelium requires Ca2+ and calcineurin.
BACKGROUND
Inside bluegill (Lepomis macrochirus) retinal pigment epithelial cells, pigment granules move in response to extracellular signals. During the process of aggregation, pigment motility is directed toward the cell nucleus; in dispersion, pigment is directed away from the nucleus and into long apical processes. A number of different chemicals have been found to initiate dispersion, and carbachol (an acetylcholine analog) is one example. Previous research indicates that the carbachol-receptor interaction activates a Gq-mediated pathway which is commonly linked to Ca2+ mobilization. The purpose of the present study was to test for involvement of calcium and to probe calcium-dependent mediators to reveal their role in carbachol-mediated dispersion.


RESULTS
Carbachol-induced pigment granule dispersion was blocked by the calcium chelator BAPTA. In contrast, the calcium channel antagonist verapamil, and incubation in Ca2+-free medium failed to block carbachol-induced dispersion. The calcineurin inhibitor cypermethrin blocked carbachol-induced dispersion; whereas, two protein kinase C inhibitors (staurosporine and bisindolylmaleimide II) failed to block carbachol-induced dispersion, and the protein kinase C activator phorbol 12-myristate 13-acetate failed to elicit dispersion.


CONCLUSIONS
A rise in intracellular calcium is necessary for carbachol-induced dispersion; however, the Ca2+ requirement is not dependent on extracellular sources, implying that intracellular stores are sufficient to enable pigment granule dispersion to occur. Calcineurin is a likely Ca2+-dependent mediator involved in the signal cascade. Although the pathway leads to the generation of diacylglycerol and calcium (both required for the activation of certain PKC isoforms), our evidence does not support a significant role for PKC.
Johnson
Adam S
AS
Biology Department, Texas State University-San Marcos, San Marcos, Texas 78666, USA. adammd00@yahoo.com
García
Dana M
DM
D000818:Animals; D019703:Calcineurin / Q000378:metabolism*; D065095:Calcineurin Inhibitors; D002118:Calcium / Q000378:metabolism*; D002217:Carbachol / Q000494:pharmacology; D018679:Cholinergic Agonists / Q000494:pharmacology; D066298:In Vitro Techniques; D017136:Ion Transport / Q000187:drug effects; D008858:Microscopy, Phase-Contrast; D010473:Perciformes / Q000378:metabolism; D010857:Pigment Epithelium of Eye / Q000502:physiology*; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism; D012160:Retina / Q000378:metabolism*; D015398:Signal Transduction
D065095:Calcineurin Inhibitors; D018679:Cholinergic Agonists; D002217:Carbachol; D011493:Protein Kinase C; D019703:Calcineurin; D002118:Calcium