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P2Y receptor-mediated stimulation of Müller glial DNA synthesis
Vanessa Moll1, Michael Weick, Ivan Milenkovic
1Department of Neurophysiology, Paul Flechsig Institute of Brain Research, University of Leipzig, Jahnallee 59, D-04109 Leipzig, Germany.
Purpose:
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.
Insights
Adenosine triphosphate (ATP) stimulates Müller glial cell proliferation by increasing DNA synthesis, a process dependent on intracellular calcium influx. This calcium influx is crucial for ATP-induced proliferation and is modulated by BK channel blockers.
Area of Science:
- Neuroscience
- Cell Biology
- Glial Cell Biology
Background:
- Müller glial cells play vital roles in retinal structure and function.
- Purinergic signaling, involving receptors like P2Y, is implicated in cellular processes.
- Understanding Müller cell proliferation is key to retinal repair and disease research.
Purpose of the Study:
- To investigate if P2Y receptor activation enhances Müller cell DNA synthesis.
- To determine if adenosine triphosphate (ATP)-induced Müller cell proliferation relies on intracellular calcium increase.
Main Methods:
- Primary guinea pig Müller cell cultures were treated with various substances.
- DNA synthesis rate was measured using a bromodeoxyuridine immunoassay.
- Intracellular calcium concentrations were monitored using fura-2 imaging.
Main Results:
- ATP and uridine triphosphate (UTP) significantly increased Müller cell DNA synthesis, mediated via P2Y receptors.
- ATP-induced proliferation required both intracellular calcium elevation and extracellular calcium influx.
- Blockers of calcium channels and BK channels inhibited ATP-stimulated DNA synthesis and shortened calcium transients.
Conclusions:
- ATP-induced Müller glial DNA synthesis is contingent upon extracellular calcium influx.
- BK channel blockers inhibit ATP-evoked DNA synthesis by impeding calcium influx.
- The magnitude of calcium influx correlates directly with the extent of ATP-induced proliferation.