Caveolin-1, the primary coat protein of caveolae, has been implicated as a regulator of signal transduction through binding of its "scaffolding domain" to key signaling molecules. However, the physiological importance of caveolin-1 in regulating signaling has been difficult to distinguish from its traditional functions in caveolae assembly, transcytosis, and cholesterol transport. To directly address the importance of the caveolin scaffolding domain in vivo, we generated a chimeric peptide with a cellular internalization sequence fused to the caveolin-1 scaffolding domain (amino acids 82-101). The chimeric peptide was efficiently taken up into blood vessels and endothelial cells, resulting in selective inhibition of acetylcholine (Ach)-induced vasodilation and nitric oxide (NO) production, respectively. More importantly, systemic administration of the peptide to mice suppressed acute inflammation and vascular leak to the same extent as a glucocorticoid or an endothelial nitric oxide synthase (eNOS) inhibitor. These data imply that the caveolin-1 scaffolding domain can selectively regulate signal transduction to eNOS in endothelial cells and that small-molecule mimicry of this domain may provide a new therapeutic approach.

Injection of carrageenan 1% (50 μl) in the mouse paw causes a biphasic response: an early inflammatory response that lasts 6 h and a second late response that peaks at 72 h, declining at 96 h. Only mice 7‐ or 8‐week old, weighing 32–34 g, displayed a consistent response in both phases. In 8‐week‐old mice, myeloperoxidase (MPO) levels are significantly elevated in the early phase at 6 h and reach their maximum at 24 h to decline to basal value at 48 h. Nitrate+nitrite (NOx) levels in the paw are maximal after 2 h and slowly decline thereafter in contrast to prostaglandin E2 levels that peak in the second phase at the 72 h point. Western blot analysis showed that inducible nitric oxide synthase (iNOS) is detectable at 6 h and cyclooxygenase 2 (COX‐2) at 24 h point, respectively. Analysis of endothelial nitric oxide synthase (eNOS), iNOS and COX‐2 expression at 6 and 24 h in 3–8‐week‐old mice demonstrated that both eNOS and iNOS expressions are dependent upon the age–weight of mice, as opposite to COX‐2 that is present only in the second phase of the oedema and is not linked to mouse age–weight. Subplantar injection of carrageenan to C57BL/6J causes a biphasic oedema that is significantly reduced by about 20% when compared to CD1 mice. Interestingly, in these mice, iNOS expression is absent up to 6 h, as opposite to CD1, and becomes detectable at the 24 h point. Cyclooxygenase (COX‐1) expression is upregulated between 4 and 24 h after carrageenan injection, whereas in CD1 mice COX‐1 remains unchanged after irritant agent injection. MPO levels are maximal at the 24 h point and they are significantly lower, at 6 h point, than MPO levels detected in CD1 mice. In conclusion, mouse paw oedema is biphasic and age‐weight dependent. The present results are the first report on the differential expressions of eNOS, iNOS, COX‐1 and COX‐2 in response to carrageenan injection in the two phases of the mouse paw oedema. British Journal of Pharmacology (2004) 142, 331–338. doi:

Objective-Recent studies have demonstrated that hydrogen sulfide (H 2 S) is produced within the vessel wall from L-cysteine regulating several aspects of vascular homeostasis. H 2 S generated from cystathione ␥-lyase (CSE) contributes to vascular tone; however, the molecular mechanisms underlying the vasorelaxing effects of H 2 S are still under investigation. Methods and Results-Using isolated aortic rings, we observed that addition of L-cysteine led to a concentrationdependent relaxation that was prevented by the CSE inhibitors DL-propargylglyicine (PAG) and ␤-cyano-L-alanine (BCA). Moreover, incubation with PAG or BCA resulted in a rightward shift in sodium nitroprusside-and isoproterenol-induced relaxation. Aortic tissues exposed to PAG or BCA contained lower levels of cGMP, exposure of cells to exogenous H 2 S or overexpression of CSE raised cGMP concentration. RNA silencing of CSE expression reduced intracellular cGMP levels confirming a positive role for endogenous H 2 S on cGMP accumulation. The ability of H 2 S to enhance cGMP levels was greatly reduced by the nonselective phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine. Finally, addition of H 2 S to a cell-free system inhibited both cGMP and cAMP breakdown. Conclusion-These findings provide direct evidence that H 2 S acts as an endogenous inhibitor of phosphodiesterase activity and reinforce the notion that this gasotransmitter could be therapeutically exploited. Key Words: endothelium Ⅲ hypertension Ⅲ signal transduction Ⅲ vascular muscle Ⅲ vasodilation Ⅲ cystathione ␥-lyase Ⅲ hydrogen sulfide Ⅲ cAMP Ⅲ cGMP Ⅲ phosphodiesterase N itric oxide (NO) is believed to account for most of the endothelium-derived relaxing factor activity released within the vessel wall, at least in some vessels. 1 On muscarinic stimulation, NO is produced following the conversion of L-arginine to NO by endothelial nitric oxide synthase (eNOS). 2 NO diffuses from the endothelium to the underlying smooth muscle cell layer, where it stimulates soluble guanylate cyclase to produce cGMP. cGMP in turn activates protein kinase G (PKG), which initiates a cascade of events leading to relaxation. 2,3 Hydrogen sulfide (H 2 S) is emerging as a new gaseous signaling molecule in the cardiovascular system. 4,5 Vascular endothelial cells express cystathionine ␥-lyase (CSE) and produce measurable amounts of this gasotransmitter. 6 Recent evidence suggests that H 2 S exhibits endothelium-derived relaxing factor activity. 6 In addition, it has been shown that muscarinic stimulation leads to CSE activation in the endothelium, triggering the conversion of L-cysteine to H 2 S and that CSE, like eNOS, is a calcium/calmodulindependent enzyme. Therefore, within the vascular wall, these 2 pathways coexist and serve a similar function. The relative amounts of NO versus H 2 S likely depend on the vascular bed studied 7 or on the state of the tissue, eg, healthy versus diseased. 5,8 Mice with targeted disruption of the CSE locus (CSE null mice) exhibit hypertension, similarly to what is observe...