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Kinetic analysis of receptor-activated phosphoinositide turnover.

Xu C, Watras J, Loew LM - J. Cell Biol. (2003)

Bottom Line: Phosphatidylinositol-4,5-bisphosphate (PIP2) decreased over the first 30 s, and then recovered over the following 2-3 min.This was subsequently confirmed experimentally.Furthermore, this analysis could help to resolve a controversy over whether the translocation of PH-GFP from membrane to cytosol is due to a decrease in PIP2 on the membrane or an increase in InsP3 in cytosol; by computationally clamping the concentrations of each of these compounds, the model shows how both contribute to the dynamics of probe translocation.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, University of Connecticut Health Center, Farmington, CT 06030, USA.

ABSTRACT
We studied the bradykinin-induced changes in phosphoinositide composition of N1E-115 neuroblastoma cells using a combination of biochemistry, microscope imaging, and mathematical modeling. Phosphatidylinositol-4,5-bisphosphate (PIP2) decreased over the first 30 s, and then recovered over the following 2-3 min. However, the rate and amount of inositol-1,4,5-trisphosphate (InsP3) production were much greater than the rate or amount of PIP2 decline. A mathematical model of phosphoinositide turnover based on this data predicted that PIP2 synthesis is also stimulated by bradykinin, causing an early transient increase in its concentration. This was subsequently confirmed experimentally. Then, we used single-cell microscopy to further examine phosphoinositide turnover by following the translocation of the pleckstrin homology domain of PLCdelta1 fused to green fluorescent protein (PH-GFP). The observed time course could be simulated by incorporating binding of PIP2 and InsP3 to PH-GFP into the model that had been used to analyze the biochemistry. Furthermore, this analysis could help to resolve a controversy over whether the translocation of PH-GFP from membrane to cytosol is due to a decrease in PIP2 on the membrane or an increase in InsP3 in cytosol; by computationally clamping the concentrations of each of these compounds, the model shows how both contribute to the dynamics of probe translocation.

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Experimental and simulated time courses of bradykinin-induced changes of PIP2 and PIP in N1E-115 cells. [3H]inositol-prelabeled N1E-115 cells were incubated in the presence of 1 μM bradykinin for the indicated times. Membrane lipids were extracted and analyzed as described in Materials and methods. Each data point is the mean ± SEM of three to five experiments. All experiments were performed at RT. Black diamonds represent our initial determination of PIP2 changes; purple triangles are the PIP data. These data, along with our prior study of InsP3 dynamics in this cell, were used to constrain a model that produced the respective black and purple solid curves. The pathways that were modeled are shown in Fig. 2, and the model equations and parameters are described in the . The prediction of the model that there was an initial increase in [PIP2] led us to determine the change in PIP2 at 5 s, shown as a red diamond. The model calculation of the change in [InsP3] is shown in the inset.
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fig1: Experimental and simulated time courses of bradykinin-induced changes of PIP2 and PIP in N1E-115 cells. [3H]inositol-prelabeled N1E-115 cells were incubated in the presence of 1 μM bradykinin for the indicated times. Membrane lipids were extracted and analyzed as described in Materials and methods. Each data point is the mean ± SEM of three to five experiments. All experiments were performed at RT. Black diamonds represent our initial determination of PIP2 changes; purple triangles are the PIP data. These data, along with our prior study of InsP3 dynamics in this cell, were used to constrain a model that produced the respective black and purple solid curves. The pathways that were modeled are shown in Fig. 2, and the model equations and parameters are described in the . The prediction of the model that there was an initial increase in [PIP2] led us to determine the change in PIP2 at 5 s, shown as a red diamond. The model calculation of the change in [InsP3] is shown in the inset.

Mentions: On addition of a 1-μM concentration of bradykinin that is maximal for the bradykinin receptor-mediated InsP3 generation (Fink et al., 1999a, 2000), there was a relative decrease of PIP2 (Fig. 1, black diamonds), which was detectable by 10 s, and reached a minimum (−36.0 ± 6.0%, n = 5) by 20 s. Levels of [3H]PIP2 recovered to basal levels in ∼150 s in the continued presence of bradykinin. The rate of recovery of PIP2 was much slower than that of InsP3, which reaches its maximum before 10 s and returns to basal levels by 30 s at 37°C (Fink et al., 1999a, 2000). Also shown in Fig. 1 are the experimental data for PIP, which show relatively little change after bradykinin stimulation.


Kinetic analysis of receptor-activated phosphoinositide turnover.

Xu C, Watras J, Loew LM - J. Cell Biol. (2003)

Experimental and simulated time courses of bradykinin-induced changes of PIP2 and PIP in N1E-115 cells. [3H]inositol-prelabeled N1E-115 cells were incubated in the presence of 1 μM bradykinin for the indicated times. Membrane lipids were extracted and analyzed as described in Materials and methods. Each data point is the mean ± SEM of three to five experiments. All experiments were performed at RT. Black diamonds represent our initial determination of PIP2 changes; purple triangles are the PIP data. These data, along with our prior study of InsP3 dynamics in this cell, were used to constrain a model that produced the respective black and purple solid curves. The pathways that were modeled are shown in Fig. 2, and the model equations and parameters are described in the . The prediction of the model that there was an initial increase in [PIP2] led us to determine the change in PIP2 at 5 s, shown as a red diamond. The model calculation of the change in [InsP3] is shown in the inset.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2199375&req=5

fig1: Experimental and simulated time courses of bradykinin-induced changes of PIP2 and PIP in N1E-115 cells. [3H]inositol-prelabeled N1E-115 cells were incubated in the presence of 1 μM bradykinin for the indicated times. Membrane lipids were extracted and analyzed as described in Materials and methods. Each data point is the mean ± SEM of three to five experiments. All experiments were performed at RT. Black diamonds represent our initial determination of PIP2 changes; purple triangles are the PIP data. These data, along with our prior study of InsP3 dynamics in this cell, were used to constrain a model that produced the respective black and purple solid curves. The pathways that were modeled are shown in Fig. 2, and the model equations and parameters are described in the . The prediction of the model that there was an initial increase in [PIP2] led us to determine the change in PIP2 at 5 s, shown as a red diamond. The model calculation of the change in [InsP3] is shown in the inset.
Mentions: On addition of a 1-μM concentration of bradykinin that is maximal for the bradykinin receptor-mediated InsP3 generation (Fink et al., 1999a, 2000), there was a relative decrease of PIP2 (Fig. 1, black diamonds), which was detectable by 10 s, and reached a minimum (−36.0 ± 6.0%, n = 5) by 20 s. Levels of [3H]PIP2 recovered to basal levels in ∼150 s in the continued presence of bradykinin. The rate of recovery of PIP2 was much slower than that of InsP3, which reaches its maximum before 10 s and returns to basal levels by 30 s at 37°C (Fink et al., 1999a, 2000). Also shown in Fig. 1 are the experimental data for PIP, which show relatively little change after bradykinin stimulation.

Bottom Line: Phosphatidylinositol-4,5-bisphosphate (PIP2) decreased over the first 30 s, and then recovered over the following 2-3 min.This was subsequently confirmed experimentally.Furthermore, this analysis could help to resolve a controversy over whether the translocation of PH-GFP from membrane to cytosol is due to a decrease in PIP2 on the membrane or an increase in InsP3 in cytosol; by computationally clamping the concentrations of each of these compounds, the model shows how both contribute to the dynamics of probe translocation.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, University of Connecticut Health Center, Farmington, CT 06030, USA.

ABSTRACT
We studied the bradykinin-induced changes in phosphoinositide composition of N1E-115 neuroblastoma cells using a combination of biochemistry, microscope imaging, and mathematical modeling. Phosphatidylinositol-4,5-bisphosphate (PIP2) decreased over the first 30 s, and then recovered over the following 2-3 min. However, the rate and amount of inositol-1,4,5-trisphosphate (InsP3) production were much greater than the rate or amount of PIP2 decline. A mathematical model of phosphoinositide turnover based on this data predicted that PIP2 synthesis is also stimulated by bradykinin, causing an early transient increase in its concentration. This was subsequently confirmed experimentally. Then, we used single-cell microscopy to further examine phosphoinositide turnover by following the translocation of the pleckstrin homology domain of PLCdelta1 fused to green fluorescent protein (PH-GFP). The observed time course could be simulated by incorporating binding of PIP2 and InsP3 to PH-GFP into the model that had been used to analyze the biochemistry. Furthermore, this analysis could help to resolve a controversy over whether the translocation of PH-GFP from membrane to cytosol is due to a decrease in PIP2 on the membrane or an increase in InsP3 in cytosol; by computationally clamping the concentrations of each of these compounds, the model shows how both contribute to the dynamics of probe translocation.

Show MeSH
Related in: MedlinePlus