Functional significance of differential eNOS translocation

Abstract

Nitric oxide (NO) regulates flow and permeability. ACh and platelet-activating factor (PAF) lead to endothelial NO synthase (eNOS) phosphorylation and NO release. While ACh causes only vasodilation, PAF induces vasoconstriction and hyperpermeability. The key differential signaling mechanisms for discriminating between vasodilation and hyperpermeability are unknown. We tested the hypothesis that differential translocation may serve as a regulatory mechanism of eNOS to determine specific vascular responses. We used ECV-304 cells permanently transfected with eNOS-green fluorescent protein (ECVeNOS-GFP) and demonstrated that the agonists activate eNOS and reproduce their characteristic endothelial permeability effects in these cells. We evaluated eNOS localization by lipid raft analysis and immunofluorescence microscopy. After PAF and ACh, eNOS moves away from caveolae. eNOS distributes both in the plasma membrane and Golgi in control cells. ACh (10(-5) M, 10(-4) M) translocated eNOS preferentially to the trans-Golgi network (TGN) and PAF (10(-7) M) preferentially to the cytosol. We suggest that PAF-induced eNOS translocation preferentially to cytosol reflects a differential signaling mechanism related to changes in permeability, whereas ACh-induced eNOS translocation to the TGN is related to vasodilation.

Fig. 1

Expression of endothelial nitric oxide synthase (eNOS) in ECV-304 cells permanently transfected with eNOS-green fluorescent protein (ECVeNOS-GFP) and in dermal microvascular endothelial cells (DMVEC). A: Western blot of protein extracted from ECVeNOS-GFP cells. The eNOS-GFP protein is expressed in the transfected cells but not in the control. B shows high expression of eNOS-GFP protein in cell membrane and Golgi in ECVeNOS-GFP. C shows similar eNOS distribution in DMVEC.

Fig. 2

ECVeNOS-GFP cells express functional receptor-mediated eNOS activation. Cell lysates from control, ACh-, and platelet-activating factor (PAF)-treated cells were immunoblotted against phospho-eNOS (p-eNOS) and β-actin. A: ACh and PAF activate eNOS by Ser1177 phosphorylation and Thr495 dephosphorylation. B: inhibition of muscarinic ACh and PAF receptors (PAF-r) blocks Ser1177 phosphorylation. C: ECVeNOS-GFP cells express muscarinic M3 ACh-receptors (M3-r) and PAF-r. Atr, atropine; ABT, PAF-receptor inhibitor.

Fig. 3

PAF increases permeability in ECVeNOS-GFP cells. Monolayers of ECVeNOS-GFP cells were treated with PAF (A) or ACh (B). Permeability to FITC-Dextran-70 was measured. Data are expressed as permeability coefficients (means ± SE) for control, PAF-, and ACh-stimulated monolayers. *P < 0.05; PAF, n = 6; ACh, n = 5.

Fig. 4

ACh and PAF move eNOS away from the caveolae. Isolation of lipid raft domains was done in ACh- or PAF-treated cells. Fractions were probed against caveolin-1 and eNOS. A: ACh treatment. B: PAF treatment. Blots represent 3 independent experiments.

Fig. 5

A: influence of ACh and PAF on the distribution of eNOS-GFP was assessed by fluorescence of GFP. B: colocalization between eNOS and zonula occludens-1 (ZO-1). Confocal images were obtained in ECVeNOS-GFP cells treated with ACh and PAF. Arrows point to disappearance of eNOS from plasma membrane. Images are representative of 3 independent experiments.

Fig. 6

A: colocalization of eNOS and TGN46 (a marker for the trans-Golgi network) in ECVeNOS-GFP cells treated with ACh and PAF. B: immunofluorescence microscopy in DMVEC treated with ACh and PAF, respectively. Red, eNOS; green, caveolin. Images are representative of 3 independent experiments.

Fig. 7

PAF stimulates eNOS translocation in vivo. PAF (10⁻⁷ M) was applied to the hamster cheek pouch and subsequently processed for Western blot. eNOS band intensities were evaluated by integrated optical density (IOD). *P < 0.05, n = 5.