Extracellular ATP effects on calcium signaling in cultured human non-pigmented ciliary body epithelium.
OBJECTIVE
To determine the effects of extracellular ATP on calcium signaling in cultured human non-pigmented ciliary body epithelium (HNPE).


METHODS
Intracellular calcium (Ca(2+)(i)) was measured using spectrofluorescence video microscopy in isolated HNPE cells loaded with the fluorescent dye Fura-2.


RESULTS
Nucleotides caused a transient oscillatory increase in Ca(2+)(i) with a potency order of ATP = UTP > ADP > AMP> alpha,beta-methylene-ATP. Treatment with thapsigargin (100 nM), an inhibitor of endoplasmic Ca(2+)-ATPase pumps, produced a sustained increase in Ca(2+)(i). Subsequent exposure to ATP caused a rapid reduction in Ca(2+)(i) and this effect was reduced by pre-exposure to vanadate and to a lesser extent in sodium free solution. Prolonged exposure to ATP in the presence of thapsigargin caused a transient spike increase in Ca(2+)(i) which was prevented by exposure to low extracellular Ca(2+) (1 nmol/l), verapamil, nifedipine or the microfilament disrupting agent, cytochalasin B.


CONCLUSIONS
These results provide evidence for ATP mobilisation of Ca(2+) from intracellular stores via P2Y2 receptor activation in HNPE cells. ATP also primarily activates a vanadate-sensitive Ca(2+ )-ATPase pump, in addition to having a smaller effect on the Na( +)/ Ca(2+) exchanger in terminating the calcium signal. Capacitative calcium entry, possibly via an L-type Ca(2+) channel, is implicated in generating a calcium signal following emptying of intracellular stores and is sensitive to cytoskeleton disruption. ATP can thus regulate a potent intracellular signal for secretion, suggest-ing that purinergic receptors may provide a therapeutic target in glaucoma.
Cullinane
A B
AB
Wellcome Trust Cellular Physiology Research Unit, Department of Physiology, National University of Ireland, Cork, Ireland. abcullinane@hotmail.com
Coca-Prados
M
M
Harvey
B J
BJ
D000255:Adenosine Triphosphate / Q000494:pharmacology*; D002118:Calcium / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D002478:Cells, Cultured; D002924:Ciliary Body / Q000166:cytology*; D005456:Fluorescent Dyes / Q000378:metabolism; D016257:Fura-2 / Q000378:metabolism; D006801:Humans; D008856:Microscopy, Fluorescence; D010857:Pigment Epithelium of Eye / Q000187:drug effects* / Q000378:metabolism; D018048:Receptors, Purinergic P2 / Q000378:metabolism; D058473:Receptors, Purinergic P2Y2; D015398:Signal Transduction; D019831:Sodium-Calcium Exchanger / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D019284:Thapsigargin / Q000494:pharmacology
D005456:Fluorescent Dyes; C546385:P2RY2 protein, human; D018048:Receptors, Purinergic P2; D058473:Receptors, Purinergic P2Y2; D019831:Sodium-Calcium Exchanger; D019284:Thapsigargin; D000255:Adenosine Triphosphate; D000252:Calcium-Transporting ATPases; D002118:Calcium; D016257:Fura-2