Extracellular ATP effects on calcium signaling in cultured human non-pigmented ciliary body epithelium.

PURPOSE

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.