A New Paradigm
Calcium Independent and Caveolae Internalization Dependent Release of Nitric Oxide by the Endothelial Nitric Oxide Synthase
See related article, pages 870–877
Caveolae, cholesterol rich microdomain platforms localized mostly at the cytoplasmic membrane of endothelial cells as well as other cell types, are importantly involved in cell signaling and enzymatic activity.1–6 Caveolae also contain cationic amino acid transporter (CAT) which provide L-Arginine to the eNOS-Ca2+-Calmodulin complex toward the production of nitric oxide (NO).1–6
Caveolin-1, a major caveolae-localized protein, represses markedly eNOS-dependent NO production by interfering with NADPH-dependent electron flux.3 Alteration in caveolin abundance or its subcellular location leads to the control of NO production.5,6 Thus, it is clear, that the more the caveolin the less the NO, and inversely.3
The conventional paradigm supports the concept that eNOS–caveolin-1 interaction promotes inhibition of NO production, whereas increased intracellular Ca2+ activates Ca2+-Calmodulin which in turn disrupts the eNOS–caveolin-1 interaction and thus liberates the enzyme. This in turn promotes NO production.1–6
A new paradigm is now suggested by Miniatis and colleagues7, who elegantly demonstrated that NO production in pulmonary endothelial cells is significantly mediated by caveolae internalization and is independent of the increase of intracellular Ca2+.7 Furthermore, these authors clearly show that the albumin-binding protein gp60 is importantly involved in both caveolae internalization and consequently in the increase of the endothelial NOS dependent NO release.
In their study, Miniatis et al, adopted a strategy demonstrating, under specific experimental conditions, that the NO release following activation of eNOS activity by gp60 was Ca2+-independent.7 The fact that the intracellular loaded Ca2+ chelator BAPTA had no effect on NO production when compared with control supports quite well the concept of a Ca2+-independent eNOS activation of pulmonary endothelial cells by gp60.
Miniatis and colleagues also made a very good case in support of their mechanistic hypothesis by demonstrating gp60-induced dissociation of eNOS from caveolin-1 and the involvement of Src kinase and Akt in albumin-induced NO production. Importantly, the same authors also argued in their manuscript that albumin binding proteins such as gp60 interact with several intracellular pathways besides activating Src dependent phosphorylation of dynamin-2 and caveolin-1.
In support of this latter concept, Spisni et al8 previously demonstrated that prostacyclin synthase and caveolin are colocalized in endothelial cells. It is therefore possible, that infusing albumin (to activate gp60) in intact vascular circuits untreated with NSAIDS (nonsteroid antiinflammatory drugs), may alter the production of vasoactive eicosanoids triggered by peptides such as endothelin-1.9 This confounding factor should be taken into account in future studies using albumin binding protein activation in intact vessels. Indeed mediators other than NO will modulate vascular tone of pulmonary vasculature and of high resistance vessels. Maniatis and colleagues also put forward several arguments to explain the difference between their results, where gp60-induced caveolae endocytosis was reduced in caveolin-1 null lungs, compared with those reported by Drab et al,10 who showed, in the same transgenic mice model, the vasodilator response to acetylcholine to be enhanced. Among these arguments, vascular heterogeneity (pulmonary microvasculature versus aortic rings) and distinct agents used to induce eNOS activation (gp60 versus acetylcholine) are contemplated by the authors.7
The results and conclusion reported in the Maniatis et al, article will definitely provoke a discussion between proponents of the conventional paradigm1–6 and those who are likely to also adopt the new paradigm. Certainly, some believers of both the accepted paradigm and the new paradigm will find an explanation for the exaggerated high production of NO in some pathological and experimental settings. This new paradigm may simply complement the more conventional concept of eNOS regulation and NO production.
Meanwhile, nonbelievers and skeptics will await answers to some important questions prompted by the observations made by Miniatis et al. Some of the more important could be: 1) Because the density of caveolae is remarkably lower in cultured cells when compared with native endothelial cells,3 is it possible to correlate results obtained on in vitro cell culture experiments with those obtained using intact vascular beds; 2) Is 1 μmol/L of Ca2+ chelator BAPTA sufficient to buffer all free and bound Ca2+ in the cytosol, endoplasmic reticulum, mitochondria and nucleus when bound calcium can reach near the Molar range; 3) Is it possible that the new paradigm only applies to pulmonary microvascular endothelial cells or it extends to other cell types such as cardiomyocytes11? Are there also interspecies variations; 4) Can the new paradigm be modulated by cholesterol-disrupting drugs; and 5) If in accordance to the new paradigm NO production is Ca2+ independent, is it also calmodulin independent and insensitive to calmodulin inhibitors?
Importantly, Figure 3 of Maniatis et al, may also inform the reader with regard to the fact that eNOS is also present at the nuclear level (although this concept is not raised by the authors). eNOS12 as well as receptors known to modulate its activity and NO production, such as endothelin-1 ETB receptors13,14 and phosphatiolic acid (LPA) LPA1 receptor12 are present in the nucleus. In this case, is it possible that a part of the NO production, reported by Maniatis et al, is because of nuclear NO production?
Future research on the conventional and the new paradigms is likely to be very fruitful to the field of caveolae-eNOS regulation of NO production.
Sources of Funding
This work is supported by Canadian Institutes of Health research (to G.B., P.D.J. and D.J.). D.J. is a recipient of a scholarship from Heart and Stroke Foundation of Canada. The authors apologize for citing reviews and only key references, because of space limitations.
The opinions expressed in this editorial are not necessarily those of the editors or of the American Heart Association.
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