P2Y receptor-mediated stimulation of Müller glial DNA synthesis.
OBJECTIVE
To determine whether activation of P2Y receptors may increase the DNA synthesis rate of cultured Müller cells and to investigate whether adenosine 5'-triphosphate (ATP)-induced Müller cell proliferation is mediated by an intracellular calcium increase.


METHODS
Primary cultures of Müller cells of the guinea pig were treated with test substances for 16 hours. The DNA synthesis rate was assessed by a bromodeoxyuridine immunoassay, and ATP-induced elevations of the intracellular calcium concentration were recorded by fura-2 imaging.


RESULTS
ATP or uridine triphosphate (UTP) increased the DNA synthesis rate whereas alpha,beta-methylene-ATP, 2-methyl-thio-ATP, and adenosine were ineffective, indicating that the action of ATP was through P2Y receptors. The effect of ATP was dose dependent, with an EC(50) of 5.9 microM. The mitogenic effect of ATP required an elevation of the intracellular calcium and a calcium influx into Müller cells. Blockers of calcium-permeable channels (nickel ions) or of calcium-dependent potassium (BK) channels (iberiotoxin, charybdotoxin) inhibited the ATP-stimulated DNA synthesis. In calcium-imaging experiments, ATP-evoked intracellular calcium transients were significantly shortened in the presence of extracellular nickel ions or of iberiotoxin. A correlation was found between the duration of the ATP-evoked calcium transients and the basal proliferation rate of the cultures.


CONCLUSIONS
The results indicate that the ATP-induced elevation of Müller glial DNA synthesis is dependent on an influx of calcium ions from the extracellular space and that the inhibiting effect of BK channel blockers on ATP-evoked DNA synthesis is caused by an inhibition of this influx. The amount of the calcium influx seems to be directly correlated to the strength of the ATP-evoked proliferation.
Moll
Vanessa
V
Department of Neurophysiology, Paul Flechsig Institute of Brain Research, University of Leipzig, Jahnallee 59, D-04109 Leipzig, Germany.
Weick
Michael
M
Milenkovic
Ivan
I
Kodal
Hannes
H
Reichenbach
Andreas
A
Bringmann
Andreas
A
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D002118:Calcium / Q000378:metabolism; D002121:Calcium Channel Blockers / Q000494:pharmacology; D015220:Calcium Channels / Q000378:metabolism; D002455:Cell Division / Q000187:drug effects; D002478:Cells, Cultured; D004247:DNA / Q000096:biosynthesis*; D004305:Dose-Response Relationship, Drug; D016257:Fura-2; D006168:Guinea Pigs; D009457:Neuroglia / Q000187:drug effects / Q000378:metabolism*; D018048:Receptors, Purinergic P2 / Q000378:metabolism*; D014544:Uridine Triphosphate / Q000494:pharmacology
D002121:Calcium Channel Blockers; D015220:Calcium Channels; D018048:Receptors, Purinergic P2; D000255:Adenosine Triphosphate; D004247:DNA; D002118:Calcium; D016257:Fura-2; D014544:Uridine Triphosphate