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. 2016 Jul 12;111(1):208-21.
doi: 10.1016/j.bpj.2016.05.034.

Shear-Induced Nitric Oxide Production by Endothelial Cells

Krishna Sriram  1 , Justin G Laughlin  1 , Padmini Rangamani  2 , Daniel M Tartakovsky  3
Affiliations

Shear-Induced Nitric Oxide Production by Endothelial Cells

Krishna Sriram et al. Biophys J. .

Abstract

We present a biochemical model of the wall shear stress-induced activation of endothelial nitric oxide synthase (eNOS) in an endothelial cell. The model includes three key mechanotransducers: mechanosensing ion channels, integrins, and G protein-coupled receptors. The reaction cascade consists of two interconnected parts. The first is rapid activation of calcium, which results in formation of calcium-calmodulin complexes, followed by recruitment of eNOS from caveolae. The second is phosphorylation of eNOS by protein kinases PKC and AKT. The model also includes a negative feedback loop due to inhibition of calcium influx into the cell by cyclic guanosine monophosphate (cGMP). In this feedback, increased nitric oxide (NO) levels cause an increase in cGMP levels, so that cGMP inhibition of calcium influx can limit NO production. The model was used to predict the dynamics of NO production by an endothelial cell subjected to a step increase of wall shear stress from zero to a finite physiologically relevant value. Among several experimentally observed features, the model predicts a highly nonlinear, biphasic transient behavior of eNOS activation and NO production: a rapid initial activation due to the very rapid influx of calcium into the cytosol (occurring within 1-5 min) is followed by a sustained period of activation due to protein kinases.

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Figures

Figure 1
Figure 1
Reaction network for shear-induced NO production.
Figure 2
Figure 2
Temporal variability of the concentrations of (A) cytosolic calcium, [Ca2+]c and (B) stored calcium, [Ca2+]s, and the complexes (C) Ca4CaM, [Ca4CaM] and (D) eNOS-CaM, [eNOS−CaM] for WSS τ = 8, 16, and 24 dynes/cm2.
Figure 3
Figure 3
Temporal variability of (A) [NO], (B) AKT-phosphorylated eNOS concentration, [eNOS∗], (C) caveolin-bound eNOS concentration, [eNOScav], and (D) [cGMP] for three levels of WSS, τ = 8, 16, and 24 dynes/cm2.
Figure 4
Figure 4
(A) Predicted (line) and observed (symbols) dependence of NO production rate on WSS. Predicted NO production rates are given by the QNO term in Eq. 22, which at steady state is equal to the rate of release of NO by ECs and formation of NO metabolites in the surrounding blood/media (because NO consumption by the ECs themselves was found to be negligible). Experimental data are from the following sources: squares are from column C of Table 1 in Kuchan and Frangos (26), where NO production rates were estimated using measurements of NOx accumulation rates; circles are from Fig. 4C in Kaur et al. (64), where NO production rates were estimated from nitrite accumulation rates; and triangles are from Fig. 8 in Kanai et al. (76), where NO production rates were estimated from direct measurements of moles of NO released per unit time. Each experimental data set was normalized to the rate at τ = 0, except for Kanai et al. (76), where the values were normalized to the lowest nonzero measurement, at τ = 0.2 dynes/cm2; the simulation results were normalized with the predicted rate at τ = 0. (B) The predicted (lines) and observed (symbols) cumulative release of NO to the media/bloodstream as a function of time. The experimental data are from the top panel of Fig. 1 in Tsao et al. (54), showing normalized increase (above baseline measurement) of NOx accumulation in conditioned media. Both experimental and model data are normalized against cumulative NO/NOx release at 12 dynes/cm2 after 24 h.
Figure 5
Figure 5
(A) The predicted and observed NO concentration at three levels of WSS τ (in dynes/cm2). The experimental data are from Mashour and Boock (55). (B) The predicted and observed changes in NO concentration from its basal levels for three values of WSS (in dynes/cm2). The experimental data are from Andrews et al. (57).
Figure 6
Figure 6
Impact of modulation of protein kinase activity on NO production. (A) The predicted and observed eNOS phosphorylation by AKT, [eNOS∗], at normal and completely inhibited kinase activity (PI3K and PI3K−). Also shown is the corresponding effect on cGMP, with and without PI3K inhibition after 1 and 2 h. The experimental data are from Dimmeler et al. (25). (B) The predicted changes in steady state [NO], at three values of WSS, in response to elimination of PI3K activation (PI3K−) and elimination of phosphorylation of eNOS by either AKT (AKT−) or PKC (PKC−). Also shown is the impact on steady-state [NO] of the simultaneous elimination of eNOS phosphorylation by both AKT and PKC, as well as of the increase in AKT activity with (AKT+) or without (AKT+/PKC−) PKC.
Figure 7
Figure 7
(A) Effect of external calcium concentration on NO production. At high external calcium concentration, we observe a biphasic dynamics of NO. When external calcium is depleted, the first phase of NO is unchanged (as this is largely driven by rapid release of calcium from internal stores) but the second phase of NO production is lost. (B) Effect of calmodulin concentration on NO production. When sufficient CaM is present, NO production displays biphasic kinetics. When CaM is depleted, a smaller first phase of NO is observed, but the second phase is abolished.
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