© 2004 by European Society of Cardiology
Copyright © 2004, European Society of Cardiology
Caveolae and EDHF production
Cardiovascular Division, King's College London, Guy's Campus, London SE1 1UL, UK
* Tel.: +44 20 7848 6210; fax: +44 20 7848 6220. Email address: jeremy.pearson{at}kcl.ac.uk
Received 10 August 2004; accepted 18 August 2004
See article by Graziani et al. [8] (pages 234–242) in this issue.
It has been recognised for just over 50 years that endothelial cells possess abundant invaginated plasma membrane structures with a limited size range (50–100 nm diameter), first characterised by electron microscopy and suspected to be vesicles involved in transcytosis, delivering macromolecules from plasma to the subendothelium. With the recognition of the specific architecture of the vesicles they were named caveolae ("little caves") and the major protein responsible for maintaining the basket or flask shape of the vesicles was identified in the early 1990s and named caveolin-1 [1]. Caves are well known in fairy tales to contain treasure, and the unfolding story of caveolae has already yielded many treasures.
The plasma membrane lipid composition in caveolae is compartmentalised and distinct from that of the general plasma membrane. Caveolae are thus a particularly visible example of lipid rafts–cholesterol- and sphingolipid-enriched areas of the membrane–and this in turn means that they are preferentially associated with certain transmembrane proteins and glycosylphosphatidylinositol (GPI)-linked external proteins, while on the cytoplasmic face of the membrane they preferentially localise cytosolic proteins with fatty acid side chain modifications (e.g., palmitoylation or myristoylation) [2].
The number of proteins that has been claimed to be preferentially associated with endothelial caveolae and/or caveolin is steadily increasing, and caveolae are now regarded by many authors as sites where molecular complexes can be reversibly assembled in a compartmentalised fashion, and therefore serve as important structures for transducing external signals into cellular responses [2]. There is little doubt that in endothelial cells caveolae are indeed sites for macromolecular uptake and trafficking, since they are associated with the necessary molecular components for endocytosis, and caveolin-1 knockout mice (which lack caveolar structures) have impaired uptake of molecules such as albumin [3]. There is also good evidence that in both endothelial and smooth muscle cells caveolae are preferential sites for calcium entry in response to store depletion [4]. Components of signal transduction pathways, notably those triggered by G protein coupled receptors, including phospholipase C
and calmodulin, together with downstream target enzymes including adenylyl cyclase, prostacyclin synthase and NO synthase are concentrated in the caveolar fraction [1,2]. Most recently, overexpression or knockout of caveolin-1 has been shown to modulate angiogenic properties of endothelium in vitro, perhaps as a consequence of effects on NO synthase activity [5].
The role of endothelial caveolae as integrating centres for endothelial NO production has been studied extensively. Direct interaction of caveolin-1 with NO synthase inhibits enzyme activity, and this interaction is modulated by calmodulin and other proteins, for example in response to Ca2+ entry via ion channels also co-localised in caveolae. The caveolin-1 knockout mouse, missing this tonic control of NO synthase, has substantially increased basal NO production [3]. Furthermore, the arginine transporter CAT-1 and salvage pathway enzymes recycling citrulline to arginine are also localised in caveolae [6]. This compartmentation provides an explanation for the "arginine paradox" whereby NO synthesis is sensitive to altered external arginine concentrations despite the high level of cytosolic arginine, since the enzyme preferentially uses substrate that is channeled through the nearby caveolar membrane or locally recycled. Recently, oxidised low density lipoproteins have been shown to lead to depletion of cholesterol from caveolae and disruption of their functions, including impairment of NO synthase activity [7].
Caveolae have thus been shown to be critically involved in the control of synthesis of 2 important endothelium-derived vasodilators, NO and prostacyclin. In this issue of Cardiovascular Research, Graziani et al. [8] provide the first evidence that caveolae are also important for the synthesis of a third endothelium-derived vasodilator, endothelium-derived hyperpolarising factor (EDHF). As readers of this journal are undoubtedly aware, EDHF is unlikely to be a single chemical entity, and in the last few years several candidates have been proposed including K+, H2O2, and cytochrome P450 epoxygenase products derived from arachidonic acid (epoxyeicosatrienoic acids, EETs) [9–11], together with arguments unifying the mode of action of EDHF in terms of the flux of ions or small signalling molecules such as cyclic AMP through gap junctions linking endothelial and smooth muscle cells [12].
In the current paper [8] experiments were carried out on isolated porcine coronary arteries, where Fleming [11] has previously implicated EETs as EDHF, and cultured porcine aortic endothelial cells. Membrane cholesterol depletion was carried out by the standard technique of incubation in the presence of methyl-β-cyclodextrin (MCD). This led to inhibition of EDHF-mediated relaxation of the arterial rings induced by bradykinin or by the calcium ionophore ionomycin, without affecting endothelium-independent relaxation, suggesting that EDHF production is impaired when cholesterol-enriched membrane domains including caveolae are disrupted. EDHF activity was reduced, as expected, by phospholipase (PL) A2 inhibition, consistent with the view that EDHF in this tissue is a product of arachidonic acid derived from hydrolysis of phospholipids, and the effects of MCD were partially reversed by supplementation with arachidonic acid.
In the cultured cells, MCD treatment did not reduce bradykinin-induced Ca2+ signalling (surprisingly in the light of the known association of bradykinin receptors and Ca2+ entry with caveolae [2]). MCD treatment did reduce arachidonate release in response to ionomycin, though the authors did not test bradykinin-induced arachidonate release. The authors fractionated cell membranes by density gradient centrifugation: caveolin-1 was restricted mainly to a low density fraction in control cells; PLA2 was found in several fractions including the caveolin-containing fraction. After MCD treatment, both caveolin-1 and PLA2 were redistributed into higher density fractions, but while some caveolin-1 remained in the low density fraction no PLA2 was detectable. An association between caveolin-1 and PLA2 was confirmed by immunoprecipitation, and this association was reduced after bradykinin stimulation, though not altered after MCD treatment.
On the basis of these results the authors propose that control of PLA2 activity in caveolae is pivotal for regulation of EET (i.e., EDHF) synthesis, just as control of NO synthesis in caveolae is pivotal for regulation of vascular tone by NO. This would provide a novel additional example of the role of caveolae as important integrated signal transduction sites in endothelium, adding a new treasure to those already mined from these little caves.
Further studies are therefore now needed to strengthen the authors' conclusions. They have provided evidence in favour of association of at least a fraction of PLA2 with caveolae, confirming a single previous recent report of an inhibitory association of PLA2 with caveolin-1 in neurones [13]. However, the class IV Ca2+-dependent PLA2 responsible for arachidonic acid mobilisation in response to external stimuli is mainly located in the cytosol and translocates to intracellular membrane compartments on activation by Ca2+ and serine phosphorylation (e.g., by p42/p44 ERK) [14], so it is not yet clear how association of a fraction of PLA2 with caveolae is critical for EET synthesis (which occurs in the endoplasmic reticulum). In addition, the authors did not measure EET production in their experiments. Corroborative evidence, e.g., by immunolocalisation, for a caveolar location of PLA2 in intact cells and for its translocation away from caveolae on stimulation or after treatment with MCD (which disrupts all cholesterol-enriched membrane domains) needs to be obtained, and experiments to elucidate the biochemical control of PLA2 activity by caveolin or other caveolar-associated proteins are required.
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