In HT29 human colonic epithelial carcinoma cells, the activation of the M3 muscarinic receptor by carbachol (CCh) leads to a Ca2+ response with a characteristic prolonged plateau phase due to Ca2+ influx following Ca2+ store activation. The activation of the P2Y2 purinergic receptor by UTP, however, does not show this plateau phase. The lack of a plateau phase during P2Y2 stimulation may result from the inhibition of Ca2+ influx. Hence, the aim of this study was to investigate whether UTP inhibited store-operated Ca2+ influx.
Fura-2 imaging techniques were used to monitor changes in intracellular Ca2+ concentration in HT29 cells.
We first used the rate of quenching of intracellular fura-2 by exogenous Mn2+ to estimate the activity of the store-operated Ca2+ channels. We found that the rate of Mn2+ influx during prolonged UTP stimulation was 34% lower than during prolonged CCh stimulation, consistent with UTP inhibiting the activity of the Ca2+ influx channels. We then estimated the activity of these channels by using the rate of increase of intracellular Ca2+ concentration during re-admission of extracellular Ca2+ to cells in which the intracellular Ca2+ stores had been depleted by exposure to thapsigargin in Ca2+-free medium. We found that UTP reduced the rate of Ca2+ influx under these conditions by 45% compared to the rate of Ca2+ influx during re-admission of Ca2+ alone, and by 34% compared to the rate observed during the re-admission of Ca2+ in the presence of CCh. This also suggested that UTP inhibits store-operated Ca2+ influx.
We conclude that UTP inhibits store-activated Ca2+ influx channels.