MOBILIZATION OF INTRACELLULAR CALCIUM BY P2Y(2) RECEPTORS IN CULTURED, NONTRANSFORMED BOVINE CILIARY EPITHELIAL-CELLS

Purpose. To examine extracellular ATP for its ability to mobilise intr acellular calcium in bovine ciliary epithelial cells to establish and characterise P2Y(2) receptor-mediated signal transduction in this tiss ue. Methods. Bovine ciliary epithelial cells were isolated and culture d until confluence. Thee cells were reseeded on sterile coverslips and grown to obtain monolayers, then loaded with fura 2. Fluorescence was measured by a computer-controlled spectrofluorimeter and values calcu lated for intracellular calcium concentration. ATP, its analogues and other drugs were tested for their ability to mobilise intracellular ca lcium by adding them to the bathing solution. Results. Basal cytosolic calcium in bovine ciliary epithelium was 138.4 +/- 0.8 nM (n = 274). In the presence of extracellular Ca2+, ATP, UTP or ADP induced a trans ient dose-dependent increase in intracellular calcium (maximum approx. 400%), which declined rapidly The agonist potency order was UTP = ATP > ADP > AMP. Adenosine, alpha,beta-methylene-ATP and 2-methylthio-ATP were ineffective in mobilising intracellular calcium, as were adrenal ine, noradrenaline, acetylcholine and carbachol. The response to ATP a nd UTP remained, in the absence of extracellular calcium or the presen ce of nickel. Desensitisation of the calcium response by repeated expo sure to ATP was augmented by phorbol-myristate-acetate and abolished b y staurosporine. The ATP response was abolished by preincubation with pertussis toxin. Microfluorimetric measurements on single cells establ ished that both pigmented and non-pigmented epithelia responded to ATP or UTP similarly. Conclusions. In the bovine ciliary epithelium, ATP stimulates P2Y(2) receptors coupled to a pertussis toxin-sensitive G p rotein. The results also suggest that this receptor activates phosphol ipase C, leading to mobilisation of calcium from intracellular stores.