Mobilisation of intracellular calcium by P2Y2 receptors in cultured, non-transformed bovine ciliary epithelial cells

Abstract

PURPOSE. To examine extracellular ATP for its ability to mobilise intracellular calcium in bovine ciliary epithelial cells; to establish and characterise P2Y 2 receptor-mediated signal transduction in this tissue. METHODS. Bovine ciliary epithelial cells were isolated and cultured until confluence. The 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 calculated for intracellular calcium concentration. ATP, its analogues and other drugs were tested for their ability to mobilise intracellular calcium 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 Ca 2+, ATP, UTP or ADP induced a transient dose-dependent increase in intracellular calcium (maximum approx. 400%), which declined rapidly. The agonist potency order was UTP = ATP < ADP < AMP. Adenosine, a,ß-methylene-ATP and 2-methylthio-ATP were ineffective in mobilising intracellular calcium, as were adrenaline, noradrenaline, acetylcholine and carbachol. The response to ATP and UTP remained, in the absence of extracellular calcium or the presence of nickel. Desensitisation of the calcium response by repeated exposure to ATP was augmented by phorbol-myristate-acetate and abolished by staurosporine. The ATP response was abolished by preincubation with pertussis toxin. Microfluorimetric measurements on single cells established 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 protein. The results also suggest that this receptor activates phospholipase C, leading to mobilisation of calcium from intracellular stores.