Confocal imaging of Ca2+ signaling in cultured rat retinal pigment epithelial cells during mechanical and pharmacologic stimulation.
OBJECTIVE
The purpose of this study was to investigate the mechanism of Ca2+ signaling in rat retinal pigment epithelial (RPE) cells during mechanical and pharmacologic stimulation and to analyze the pathway of intercellular communication during mechanical stimulation.


METHODS
Subconfluent monolayers of RPE cells cultured for 3 to 7 days after isolation from 5- to 8-day-old Long Evans rats were analyzed using the fluorescent Ca2+ dye fluo-3 and confocal laser microscopy. Mechanical stimulation of a single cell was applied by a slight, brief touch with a glass capillary.


RESULTS
Pharmacologic agents [Arg8]-vasopressin, carbachol, adenosine triphosphate, substance P, fetal calf serum, and histamine all elicited, to some extent, a biphasic [Ca2+]i transient by releasing Ca2+ from InsP3-sensitive Ca2+ stores. Stretching cellular membranes by touching them with the glass capillary increased free intracellular calcium concentration ([Ca2+]i) in the mechanically stimulated cell. After a time delay of up to 1.5 seconds, it increased free intracellular calcium concentration as well in the two to three adjacent layers of neighboring cells, resulting in the centrifugal propagation of an intercellular Ca2+ wave. In the absence of external Ca2+, neither a [Ca2+]i rise in the mechanically stimulated cell nor a propagating Ca2+ wave in the neighboring cells was found. The Ca2+ influx was not inhibited by verapamil or by K+ depolarization, but the addition of Ni2+ reduced the influx, suggesting the involvement of cation channels sensitive to Ni2+. Ca2+ entry during mechanical stimulation was followed by Ca2+ release from intracellular Ca2+ stores. This release did not occur after pretreatment of the cells with thapsigargin and was reduced by exposure to ryanodine. The propagation of the Ca2+ wave could be blocked by the gap-junction blocker halothane and partially by K(+)-rich solution. It can be assumed, therefore, that cultured RPE cells express functional gap junctions.


CONCLUSIONS
Mechanical stimulation triggers a Ca2+ influx that is followed by intracellular Ca2+ release. Intercellular communication in RPE cells depends on the presence of functional gap junctions. Pharmacologic stimulation relies primarily on the release of Ca2+ from intracellular Ca2+ stores.
Stalmans
P
P
Laboratory of Physiology, O&N Gasthuisberg, Katholieke Universiteit Leuven, Belgium.
Himpens
B
B
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000814:Aniline Compounds / Q000378:metabolism; D000818:Animals; D000831:Animals, Newborn; D001127:Arginine Vasopressin / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D002217:Carbachol / Q000494:pharmacology; D002450:Cell Communication / Q000502:physiology; D002469:Cell Separation; D002478:Cells, Cultured; D005456:Fluorescent Dyes / Q000378:metabolism; D017629:Gap Junctions / Q000502:physiology; D006632:Histamine / Q000494:pharmacology; D018613:Microscopy, Confocal / Q000379:methods; D004364:Pharmaceutical Preparations; D010812:Physical Stimulation; D010857:Pigment Epithelium of Eye / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D051381:Rats; D015398:Signal Transduction; D013373:Substance P / Q000494:pharmacology; D014966:Xanthenes / Q000378:metabolism
D000814:Aniline Compounds; D005456:Fluorescent Dyes; D004364:Pharmaceutical Preparations; D014966:Xanthenes; D001127:Arginine Vasopressin; C059715:Fluo-3; D013373:Substance P; D006632:Histamine; D000255:Adenosine Triphosphate; D002217:Carbachol; D002118:Calcium