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-i(2+)) 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-i(2+) with a potency order of ATP = UTP > ADP > AMP > alpha,beta-methylene-ATP. Treatment with thapsigargin (100 nM), an inhibitor of endoplasmic Ca2+-ATPase pumps, produced a sustained increase in Ca2+. Subsequent exposure to ATP caused a rapid reduction in Ca-i(2+) 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-i(2+) which was prevented by exposure to low extracellular Ca2+ (1 nmol/l), verapamil, nifedipine or the microfilament disrupting agent, cytochalasin B. Conclusions. These results provide evidence for ATP mobilisation of Ca2+ from intracellular stores via P2Y2 receptor activation in HNPE cells. ATP also primarily activates a vanadate-sensitive Ca2+-ATPase pump, in addition to having a smaller effect on the Na+/Ca2+ exchanger in terminating the calcium signal. Capacitative calcium entry, possibly via an L-type Ca2+ 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, suggesting that purinergic receptors may provide a therapeutic target in glaucoma.