Determination of Time-Dependent inositol-1,4,5-trisphosphate Concentrations During Calcium Release in a Smooth Muscle Cell

Biophys J. 1999 Jul;77(1):617-28. doi: 10.1016/S0006-3495(99)76918-3.

Abstract

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.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Adenosine Triphosphatases / metabolism
  • Animals
  • Calcium / metabolism*
  • Cell Line
  • Endoplasmic Reticulum / metabolism
  • Fluorescent Dyes
  • Fluorometry
  • Inositol 1,4,5-Trisphosphate / analogs & derivatives*
  • Inositol 1,4,5-Trisphosphate / metabolism
  • Inositol Phosphates / metabolism
  • Kinetics
  • Microinjections
  • Microscopy, Confocal
  • Models, Theoretical
  • Muscle, Smooth, Vascular / metabolism*
  • Organic Chemicals
  • Rats

Substances

  • 1-(glycerophosphoryl)-myo-inositol 4,5-diphosphate P(4(5))-1-(2-nitrophenyl)ethyl ester
  • Fluorescent Dyes
  • Inositol Phosphates
  • Organic Chemicals
  • inositol 1,4,5-trisphosphate 1-(2-nitrophenyl)ethyl ester
  • calcium green
  • Inositol 1,4,5-Trisphosphate
  • Adenosine Triphosphatases
  • Calcium