Determination of time-dependent inositol-1,4,5-trisphosphate concentrations during calcium release in a smooth muscle cell.
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.
Fink
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CC
Department of Physiology and Center for Biomedical Imaging Technology, University of Connecticut Health Center, Farmington, Connecticut 06030, USA.
Slepchenko
B
B
Loew
L M
LM
D000251:Adenosine Triphosphatases / Q000378:metabolism; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002460:Cell Line; D004721:Endoplasmic Reticulum / Q000378:metabolism; D005456:Fluorescent Dyes; D005470:Fluorometry; D015544:Inositol 1,4,5-Trisphosphate / Q000031:analogs & derivatives* / Q000378:metabolism; D007295:Inositol Phosphates / Q000378:metabolism; D007700:Kinetics; D008845:Microinjections; D018613:Microscopy, Confocal; D008962:Models, Theoretical; D009131:Muscle, Smooth, Vascular / Q000378:metabolism*; D009930:Organic Chemicals; D051381:Rats
C087840:1-(glycerophosphoryl)-myo-inositol 4,5-diphosphate P(4(5))-1-(2-nitrophenyl)ethyl ester; D005456:Fluorescent Dyes; D007295:Inositol Phosphates; D009930:Organic Chemicals; C061868:inositol 1,4,5-trisphosphate 1-(2-nitrophenyl)ethyl ester; C071336:calcium green; D015544:Inositol 1,4,5-Trisphosphate; D000251:Adenosine Triphosphatases; D002118:Calcium