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

The level of [InsP(3)](oyt) 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 InsP(3) from cells microinjected with caged InsP(3). From these experiments, the [InsP(3)](cyt) required to evoke a half-maximal calcium response is 100 nM. Experiments with caged glycerophosphoryl-myo-inositol 4,5-bisphosphate (GPIP(2)), a slowly metabolized analogue of InsP(3), gave a much slower recovery and a half-maximal response of an order of magnitude greater than InsP(3). Experimental data and highly constrained variables were used to construct a mathematical model of the InsP(3)-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 InsP(3)-receptor, InsP(3) 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 InsP(3) degradation and 13 s for GPIP(2). InsP(3) 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 InsP(3)-mediated calcium signal.