Endothelial [Ca2+]i and caveolin-1 antagonistically regulate eNOS activity and microvessel permeability in rat venules - PubMed (original) (raw)

Endothelial [Ca2+]i and caveolin-1 antagonistically regulate eNOS activity and microvessel permeability in rat venules

Xueping Zhou et al. Cardiovasc Res. 2010.

Abstract

Aims: In this study, we investigated the mechanisms by which caveolin-1 (CAV) inhibits increases in permeability induced by platelet activating factor (PAF) and elucidated the relationship between the endothelial intracellular Ca(2+) concentration ([Ca(2+)](i)) and CAV in regulating endothelial nitric oxide synthase (eNOS) activity and microvessel permeability in intact microvessels.

Methods and results: Experiments were conducted in individually perfused mesenteric venules in Sprague-Dawley rats. Permeability was determined by measuring hydraulic conductivity (Lp). Endothelial [Ca(2+)](i) and nitric oxide (NO) production were measured in fura-2- and DAF-2-loaded microvessels. Perfusion of the CAV scaffolding domain, AP-CAV, at 1 microM for 30 min did not affect PAF-induced increases in endothelial [Ca(2+)](i) but significantly attenuated PAF-induced NO production from 143 +/- 2 to 110 +/- 3% of control fluorescence intensity (FI). The PAF-induced Lp increase was correlatively reduced from a mean peak value of 7.5 +/- 0.9 to 1.9 +/- 0.5 times that of the control. Increasing extracellular [Ca(2+)] that potentiated PAF-induced peak [Ca(2+)](i) from 500 to 1225 nM augmented NO production to 193 +/- 13% and further increased Lp to 17.3 +/- 1.6 times the control value. More importantly, enhanced Ca(2+) influx restored the reduced NO production and Lp by AP-CAV with NO FI at 149% and Lp at 7.7 +/- 1.1 times the control value.

Conclusion: Our results indicate that eNOS inhibition and reduced NO production contribute to the inhibitory action of AP-CAV on PAF-induced increases in permeability. CAV and endothelial [Ca(2+)](i) antagonistically regulate eNOS activity in intact microvessels, and the level of produced NO is the key determinant of the degree of permeability increases during inflammation.

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Figures

Figure 1

AP-CAV attenuated PAF-induced Lp increases. (A) PAF induced a transient increase in Lp in the absence of AP-CAV. (B) PAF-induced Lp changes after AP-CAV (open symbols) or AP-CAV-X (filled symbols) perfusion from two individual experiments. Perfusion of 1 µM AP-CAV for 30 min abolished PAF-induced Lp increase and AP-CAP-X showed no effect.

Figure 2

AP-CAV attenuated PAF-induced NO production without affecting endothelial [Ca2+]i. (A) PAF-induced transient increases in endothelial [Ca2+]i in the absence of AP-CAV. (B) PAF induced a similar magnitude increase in endothelial [Ca2+]i in a AP-CAV pre-perfused vessel, indicating that AP-CAV has no effect on PAF-induced Ca2+ influx. (C) PAF-induced NO production in the presence or absence of AP-CAV in two of the experiments. PAF-induced cumulative DAF-2 FI, an indication of increased NO production, in the presence (open circle) or absence (filled circle) of AP-CAV is shown as a function of time (left y_-axis). The differential conversion of the cumulative FI (d_f/d_t_, right _y_-axis) represents the NO production rate (solid line: PAF alone; dotted line: with AP-CAV perfusion).

Figure 3

Increasing extracellular Ca2+, [Ca2+]o, potentiated PAF-induced increases in endothelial [Ca2+]i, NO production, and microvessel permeability. (A) Endothelial [Ca2+]i measurements from two of the experiments showing that increasing [Ca2+]o potentiates PAF-induced increases in endothelial [Ca2+]i. (B) High [Ca2+]o further increased PAF-induced NO production, as indicated by DAF-2 cumulative FI curves (open triangle vs. filled circle) and NO production rate (dashed line vs. solid line). (D) Lp measurements from two of the experiments showing a potentiated Lp increase relative to that measured with normal [Ca2+]o, which correlated with high magnitude increases in endothelial [Ca2+]i and NO production.

Figure 4

AP-CAV and endothelial [Ca2+]i antagonistically regulate PAF-induced NO production and Lp increases. (A) Fluorescence images from two of the experiments showing the difference in PAF-induced NO production (DAF-2 FI changes relative to control) in the absence (left panel) or presence of applied AP-CAV (right panel). (B) Images from two of the experiments demonstrating the magnitude of the differences in NO production under normal and high [Ca2+]o. The left panel indicates that high [Ca2+]o potentiated PAF-induced NO production and the right panel shows that high [Ca2+]o partially restored AP-CAV-mediated NO reduction to a level comparable to that shown in the left panel of (A). The third image in the left panel shows further increases in FI after superfusion with NO donor sodium nitroprusside (SNP), which is the routine at end of each NO measurement to check the dye loading status. (C) The NO quantification from the images shown in (B) demonstrates that PAF stimulated cumulative DAF-2 FI and the calculated NO production rates as a function of time under high [Ca2+]o, with or without AP-CAV application. (D) Lp measurements showing that AP-CAV perfusion resulted in a moderate Lp increase under high [Ca2+]o, indicating that attenuated NO production by AP-CAV can override the high Ca2+ signal serving as a key determinant of Lp increases.

Figure 5

A summary of results demonstrating PAF-induced changes among endothelial [Ca2+]i, NO production, and microvessel Lp when the relative levels of endothelial CAV and [Ca2+]o varies. (A) PAF-induced changes in endothelial [Ca2+]i under either normal (2 mM, n = 4 for each group) or high (10 mM) [Ca2+]o, without (n = 6) or with (n = 7) AP-CAV (1 µM) application. (B) PAF-induced DAF-2 FI changes under either normal or high [Ca2+]o, in the absence or presence of AP-CAV (n = 5 for each group). (C) PAF-induced increases in Lp under normal [Ca2+]o (n = 6 for PAF alone; n = 2 for AP-CAV at 0.1 µM; n = 5 for AP-CAV at 1 µM; n = 4 for AP-CAV-X) or high [Ca2+]o (n = 5 for each group), in the absence or presence of AP-CAV. (D) The magnitude of PAF-induced increases in Lp, showing a linear correlation with the level of stimulated NO production under different experimental conditions.

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Endothelial [Ca2+]i and caveolin-1 antagonistically regulate eNOS activity and microvessel permeability in rat venules - PubMed (original) (raw)