Colocalization of eNOS and the Catalytic Subunit of PKA in Endothelial Cell Junctions : A Clue for Regulated NO Production
Thrombosis and Haemostasis Laboratory (HFGH,SW,J-WNA), and Department of Cell Biology, (HFGH,SW,JWS), University Medical Center Utrecht, Utrecht, The Netherlands; Institute for Biomembranes (HFGH,SW,J-WNA,JWS), Utrecht, The Netherlands; and National Jewish Medical Center (JDC,RPB), Denver, Colorado
Correspondence to: Dr. Harry F.G. Heijnen, Thrombosis and Haemostasis Laboratory, University Medical Center Utrecht, Heidelberglaan 100, 3584 CX Utrecht, The Netherlands. E-mail: h.f.g.heijnen{at}azu.nl
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Summary |
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Key Words: eNOS PKA caveolin-1 NO production
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Introduction |
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We here present data on the subcellular steady-state distribution of eNOS in relation to two potential regulatory proteins, caveolin-1 and the catalytic subunit of PKA (PKA-c), in rat aorta endothelium. We demonstrate that an important pool of cell surface eNOS is outside caveolae and principally located in endothelial cell junctions, where it is closely associated with PKA-c, and not with caveolin-1. Distinct pools of PKA-c were also associated with cell surface caveolae and with the trans site of the Golgi. These restricted sites of colocalization may provide mechanisms for PKA-dependent eNOS regulation in different endothelial subcellular compartments.
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Materials and Methods |
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Cell Culture and Tissue Preparation
The procedures involving the use of animals followed the Institute of Laboratory Animal Resources (1996) Guide for the Care and Use of Laboratory Animals. Rats were anesthetized and perfusion-fixed through the left ventricle of the heart with a mixture of 2% paraformaldehyde (PFA) and 0.2% glutaraldehyde (GA) in 0.1 M sodium phosphate buffer (pH 7.4). After perfusion, small aorta rings were excised, postfixed for 2 hr at room temperature, and stored in 1% PFA at 4C. After washing, the samples were embedded in 10% gelatin, cooled in ice, and cut into 1-mm3 blocks in the cold room. The blocks were infused with 2.3 M sucrose at 4C for 24 hr, frozen in liquid nitrogen, and stored until cryoultramicrotomy. Primary BAECs were grown in EGM BulletKit medium (Clonetics) according to the manufacturer's instructions, fixed in 2% PFA and 0.2% GA in 0.1 M sodium phosphate buffer (pH 7.4), infused with 2.3 M sucrose at 4C, and frozen in liquid nitrogen.
Immunofluorescence
Semithin cryosections were prepared from the luminal area of the aorta. The sections were transferred to microscope slides and incubated with primary antibodies for 30 min, followed by incubation with Cy3-conjugated secondary antibodies. In a similar fashion, BAECs were permeabilized, and immunostained for 60 min with primary antibodies, washed three times with PBS, followed by a 60-min incubation with Cy3-conjugated secondary antibodies. Samples were analyzed by a Leica TCS 4D confocal laser-scanning microscope (Leica; Lasertechnik, Heidelberg, Germany).
Immunoelectron Microscopy
Cryosections 50 nm thick were cut at 120C using an Ultracut S ultramicrotome (Leica). The sections were collected on formvar-coated grids using a mixture of 1.8% methylcellulose and 2.3 M sucrose (Liou et al. 1996) and incubated with primary antibodies and protein Agold (Slot et al. 1991
). For monoclonal antibodies (MAbs), rabbit anti-mouse was used as bridging antibody. Immunogold double labeling was performed using 10-nm (eNOS) and 15-nm (Cav-1 and PKA-c) gold particles (Slot and Geuze 1985
). After labeling, the sections were fixed with 1% glutaraldehyde, counterstained with uranyl acetate, and embedded in methyl celluloseuranyl acetate. The sections were viewed in a JEOL 1200CX electron microscope.
Quantitative Analysis
For quantitative evaluation, cryosections were prepared from three different areas of the aorta lumen and 25 electron micrographs were taken randomly from the endothelium. The endothelial cell surface was subdivided into luminal, basal, and lateral membranes and the peripheral junctional membranes (lamellipodia). Gold particles over these categories were attributed to caveolar and non-caveolar membranes. The relative membrane length was determined using a transparent overlay with lines on the electron micrographs and counting the number of membrane intersections over the categories. The linear labeling density, defined as the ratio of gold particles and membrane intersections, was expressed per µm membrane. The intracellular gold label distribution of eNOS was determined over endoplasmic reticulum, Golgi complex, non-identified membranes, Weibel-Palade bodies, and cytosol. eNOS surface density was defined as the ratio of gold particles and random points over the compartments, and expressed per µm2. Random points were counted using a transparent overlay with points. The relative distribution of PKA-c was determined over caveolar and non-caveolar plasma membrane, peripheral cytoskeletal network, and intracellular membranes. Gold particles within 20 nm of a membrane were considered membrane-associated. Statistical ANOVA of linear label density was performed using the Student's t-test.
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Results |
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Discussion |
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There is increasing evidence that eNOS activity is regulated by PKA-mediated phosphorylation (Michell et al. 2001,2002
; Boo et al. 2002a
,b
). Several PKA target sites have been identified in bovine eNOS (Ser 1179, Ser 635, and Ser 617). Selectivity of PKA signaling is mainly provided by its subcellular location. Targeting of PKA catalytic activity to restricted intracellular locations depends on the binding of the PKA-RII subunit to the so-called A-kinase anchoring proteins (Edwards and Scott 2000
; Skalhegg and Tasken 2000
). The diverse locations of PKA-c that we found in our present study are consistent with the concept that PKA-c action is compartmentalized in restricted areas in the aortic endothelial cell, suggesting that different PKA pools may be operational in the regulation of eNOS. eNOS and PKA-c are in close proximity in the peripheral lamellipodia, at endothelial cellcell contacts, and in the Golgi area. The 2.5-fold enrichment of eNOS in the lamellipodial membranes and the absence of caveolin-1 therein indicate that the local eNOS activity in these peripheral contact areas is not regulated by caveolin-1 interaction. The topological proximity of PKA-c and eNOS instead highlights a possible PKA-dependent regulation of eNOS activity in these locations. Our finding that a proportion of PKA-c is associated with caveolae is not quite unexpected. In vitro, caveolin-1 can interact with the catalytic subunit of PKA and negatively regulate PKA-c action (Razani et al. 1999
; Razani and Lisanti 2001
). This may have implications for the shear-dependent eNOS activation in these domains. Luminal caveolae are believed to be the mechanical sensors of blood flow variations (Rizzo et al, 1998
). Shear-induced alterations in the interaction of caveolin-1 with PKA-c may affect the phosphorylation state of eNOS in caveolae and have downstream effects on the eNOS activity in these domains. Support for such a mechanism is obtained from recent studies showing that shear-induced phosphorylation of eNOS at Ser 1179 and Ser 635 is fully PKA-dependent (Boo et al. 2002a
,b
). However, the peripheral lamellipodia are also luminally oriented, and changes in pulsatile blood flow may therefore be sensed in these membrane domains as well. Because eNOS activity and PKA catalytic activity are probably not regulated by caveolin-1 interaction in these locations, the shear-induced regulation of eNOS may be directly under the control of PKA. Our data further suggest that at steady state, a portion of eNOS is phosphorylated in the Golgi area. A site-specific role of PKA in the Golgi may particularly be connected to the regulation of functional eNOS at the cell surface. cAMP-raising agents such as forskolin prevent protein export from the trans-Golgi network (Muniz et al. 1996
), and more specifically prevent the trafficking of mature eNOS to plasma membrane caveolae (Belhassen et al. 1997
).
In conclusion, we have demonstrated that, in rat aorta endothelium, a large portion of cell surface eNOS is located outside caveolae, in distinct endothelial cell junctions. Our findings are in agreement with those reported in rat endocardiac endothelium, where eNOS was predominantly found on peripheral cell borders (Andries et al. 1998). The absence of the inhibitor caveolin-1 and the close proximity of the catalytic form of PKA in these locations suggest a site-specific regulation of the enzyme independent of caveolin-1. In addition to luminal caveolae, changes in vascular tone and blood flow may also be sensed in these specialized endothelial cell junctions and hence regulate the flow-responsive NO production in a PKA-dependent manner. Such a mechanism is consistent with recent findings in cultured endothelial cells where eNOS localization and NO production have been reported to dramatically depend on the existence of cellcell contacts (Sowa et al. 1999
; Govers et al. 2002a
). Further studies are required to determine which pool of PKA is responsible for the shear-related NO production.
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Acknowledgments |
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Footnotes |
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