Determination of time-dependent inositol-1,4,5-trisphosphate concentrations during calcium release in a smooth muscle...

The level of [InsP3]cyt required for calcium release in A7r5 cells, a smooth muscle cell line, was determined by a new set of procedures using quantitative confocal microscopy to measure release of InsP3 from cells microinjected with caged InsP3. From these experiments, the [InsP3]cyt required to evoke a half-maximal calcium response is 100 nM. Experiments with caged glycerophosphoryl-myo-inositol 4, 5-bisphosphate (GPIP2), a slowly metabolized analogue of InsP3, gave a much slower recovery and a half-maximal response of an order of magnitude greater than InsP3. Experimental data and highly constrained variables were used to construct a mathematical model of the InsP3-dependent [Ca2+]cyt changes; the resulting simulations show high fidelity to experiment. Among the elements considered in constructing this model were the mechanism of the InsP3-receptor, InsP3 degradation, calcium buffering in the cytosol, and refilling of the ER stores via sarcoplasmic endoplasmic reticulum ATPase (SERCA) pumps. The model predicts a time constant of 0.8 s for InsP3 degradation and 13 s for GPIP2. InsP3 degradation was found to be a prerequisite for [Ca2+]cyt recovery to baseline levels and is therefore critical to the pattern of the overall [Ca2+]cyt signal. Analysis of the features of this model provides insights into the individual factors controlling the amplitude and shape of the InsP3-mediated calcium signal.