Kinetics of intracellular calcium release by inositol 1, 4, 5-trisphosphate and extracellular ATP in porcine cultured aortic endothelial cells

Abstract

Quantitative, time-resolved measurements have been made of intracellular Ca ion release by inositol 1,4,5-trisphosphate (InsP$_{3}$) and extracellular ATP in porcine aortic endothelial cells in tissue culture. Intracellular free [Ca] was detected with the calcium dye fluo-3 and InsP$_{3}$ released intracellularly by photolysis of `caged' InsP$_{3}$ in whole-cell voltage-clamped aortic endothelial cells. A rise of [Ca] was recorded at InsP$_{3}$ concentrations greater than 0.2 $\mu $M. The timecourse at low InsP$_{3}$ concentrations comprised a delay of mean 300 ms (range 266-330 ms), a peak in 2-3 s before declining with a half-time of 5-10 s. The delay and time-to-peak decreased with increasing concentrations of InsP$_{3}$ over the range 0.2-5 $\mu $M. At very high concentrations of InsP$_{3}$ (> 5 $\mu $M), the delay in the Ca response was short, always less than 20 ms. The results are consistent with a direct binding and gating action of InsP$_{3}$ on the Ca channel of the cellular store. Following InsP$_{3}$ action there is a refractoriness of the InsP$_{3}$ Ca release process which recovers with a timecourse of half-time about 30 s. A comparison can be made between the timecourse of InsP$_{3}$ and extracellular ATP actions. High concentrations of ATP (500 $\mu $M) acted with a delay of mean 1.8 s (range 1.2-2.5 s), whereas even moderate concentrations of InsP$_{3}$ acted much more quickly, suggesting that there are slow coupling steps before or during the production of InsP$_{3}$ in response to extracellular ATP. Both ATP and InsP$_{3}$ evoked an increase in membrane conductance to K$^{+}$, probably via Ca.