M. Shahidullah et Ws. Wilson, MOBILIZATION OF INTRACELLULAR CALCIUM BY P2Y(2) RECEPTORS IN CULTURED, NONTRANSFORMED BOVINE CILIARY EPITHELIAL-CELLS, Current eye research, 16(10), 1997, pp. 1006-1016
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.