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Aberrant regulation of eNOS and associated NO release are directly linked with various vascular diseases. Caveolin-1 (Cav-1), the main coat protein of caveolae, is highly expressed in endothelial cells. Its scaffolding domain serves as an endogenous negative regulator of eNOS function. Structure-function analysis of Cav-1 has shown that phenylalanine 92 (F92) is critical for the inhibitory actions of Cav-1 toward eNOS. Herein, we show that F92A-Cav-1 and a mutant cell-permeable scaffolding domain peptide called Cavnoxin can increase basal NO release in eNOS-expressing cells. Cavnoxin reduced vascular tone ex vivo and lowered blood pressure in normal mice. In contrast, similar experiments performed with eNOS- or Cav-1-deficient mice showed that the vasodilatory effect of Cavnoxin is abolished in the absence of these gene products, which indicates a high level of eNOS/Cav-1 specificity. Mechanistically, biochemical assays indicated that noninhibitory F92A-Cav-1 and Cavnoxin specifically disrupted the inhibitory actions of endogenous Cav-1 toward eNOS and thereby enhanced basal NO release. Collectively, these data raise the possibility of studying the inhibitory influence of Cav-1 on eNOS without interfering with the other actions of endogenous Cav-1. They also suggest a therapeutic application for regulating the eNOS/Cav-1 interaction in diseases characterized by decreased NO release., Introduction NO is well known to actively regulate vascular tone and blood pressure (BP). eNOS is the main source of vascular NO, and aberrant regulation of eNOS activity is linked [...]

Some receptors and signaling molecules, such as Rho-kinase (ROCK), localize in caveolae. We asked whether the function of histamine receptors ([H.sub.1]) and 5-hydroxytryptamine (serotonin) receptors ([5-HT.sub.2A]) in bovine tracheal smooth muscle are modified after caveolae disruption and if so, whether the altered ROCK activity plays a role in this modification. Methyl-β-cyclodextrin (MβCD), used to deplete membrane cholesterol, was shown to disrupt caveolae and diminish sustained contractions to histamine (~80%), 5-HT (100%), a-methyl-5-HT (100%), and KCl (~30%). Cholesterol-loaded MβCD (CL-MβCD) restored the responses to KCl and partially restored the responses to agonists. ROCK inhibition by Y-27632 diminished contractions to histamine (~85%) and 5-HT (~59%). 5-HT or histamine stimulation augmented ROCK activity. These increases were reduced by MβCD and partially reestablished by CL-MβCD. The increase in intracellular [Ca.sup.2+] that was induced by both agonists was reduced by MβCD. The presence of caveolin-1 (Cav-1), [H.sub.1], [5-HT.sub.2A], and ROCK' was corroborated by immunoblotting of membrane fractions from sucrose gradients and by confocal microscopy. [H.sub.1] receptors coimmunoprecipitated with Cav-1 in caveolar and noncaveolar membrane fractions, whereas [5- HT.sub.2A] receptors appeared to be restricted to noncaveolar membrane fractions. We conclude that caveolar and cholesterol integrity are indispensable for the proper functionality of the [H.sub.1] and [5-HT.sub.2A] receptors through their Rho/ROCK signaling. Key words: airway smooth muscle, caveolae, lipid rafts, histamine [H.sub.1] receptor, [5-HT.sub.2A] receptor, Rho- kinase activity. On trouve certains recepteurs et certaines molecules de signalisation, telle la rho-kinase (ROCK), dans les caveoles. Nous avons examine si les fonctions des recepteurs [H.sub.1] et [5-HT.sub.2A] dans le muscle lisse tracheal bovin changent aprs la destruction des caveoles et si l'activite modifiee de la ROCK participe a ces changements. L'emploi de la methyl-β-cyclodextrine (MβCD), pour epuiser le cholesterol membranaire, a detruit les caveoles et diminue les contractions soutenues induites par l'histamine (80 %), la 5-HT (100 %), l'a-methyl 5-HT (100 %) et le KCl (30 %). La MβCD chargee en cholesterol (MβCD-CC) a retabli partiellement les reponses au KCl et aux agonistes. L'inhibition de la ROCK par Y-27672 a diminue les contractions induites par l'histamine (85 %) et la 5-HT (59 %). La stimulation par la 5-HT ou l'histamine a augmente l'activite de la ROCK. Ces augmentations ont ete diminuees par la MβCD et partiellement retablies par la MβCD-CC. L'augmentation de la concentration de [C.sup.2+] intracellulaire induite par les deux agonistes a ete reduite par la MβCD-CC. La presence de caveoline-1 (Cav-1), de [H.sub.1], de [5-HT.sub.2A] et de ROCK' a ete attestee par immunobuvardage de fractions de membranes sur gradients de sucrose et par microscopie confocale. Les recepteurs [H.sub.1] et la Cav-1 coimmunoprecipitent dans les fractions caveolaires et noncaveolaires, alors que les recepteurs [5-HT.sub.2A] ont ete observes dans les fractions noncaveolaires. Nous concluons que l'integrite des caveoles et du cholesterol est indispensable au bon fonctionnement des recepteurs [H.sub.1] et [5-HT.sub.2A] dans la signalisation liee a Rho/ROCK. Mots-cles : caveoles des muscles lisses des voies aeriennes, radeaux lipidiques, recepteur [H.sub.1] de l'histamine, recepteur [5-HT.sub.2A], activite Rho-kinase. [Traduit par la Redaction], Introduction Most agonist-induced contractions in airway smooth muscle are initiated by the activation of specific G protein-coupled receptors (GPCR). Histamine and 5-hydroxytryptamine (serotonin) (5-HT) activate [H.sub.1] and [5-HT.sub.2A] receptors that [...]

Endothelial nitric oxide (NO) synthase (eNOS) is thought to regulate microvascular permeability via NO production. We tested the hypotheses that the expression of eNOS and eNOS endocytosis by caveolae are fundamental for appropriate signaling mechanisms in inflammatory endothelial permeability to macromolecules. We used bovine coronary postcapillary venular endothelial cells (CVECs) because these cells are derived from the microvascular segment responsible for the transport of macromolecules in inflammation. We stimulated CVECs with platelet-activating factor (PAF) at 100 nM and measured eNOS phosphorylation, NO production, and CVEC monolayer permeability to FITC-dextran 70 KDa (Dx-70). PAF translocated eNOS from plasma membrane to cytosol, induced changes in the phosphorylation state of the enzyme, and increased NO production from 4.3 [+ or -] 3.8 to 467 [+ or -] 22.6 nM. PAF elevated CVEC monolayer permeability to FITC-Dx-70 from 3.4 [+ or -] 0.3 x [10.sup.-6] to 8.5 [+ or -] 0.4 x [10.sup.-6] cm/s. The depletion of endogenous eNOS with small interfering RNA abolished PAF-induced hyperpermeability, demonstrating that the expression of eNOS is required for inflammatory hyperpermeability responses. The inhibition of the caveolar internalization by blocking caveolar scission using transfection of dynamin dominant-negative mutant, dyn2K44A, inhibited PAF-induced hyperpermeability to FITC-Dx-70. We interpret these data as evidence that 1) eNOS is required for hyperpermeability to macromolecules and 2) the internalization of eNOS via caveolae is an important mechanism in the regulation of endothelial permeability. We advance the novel concept that eNOS internalization to cytosol is a signaling mechanism for the onset of microvascular hyperpermeability in inflammation. endothelial nitric oxide; endothelial cells; microvascular permeability; endothelial nitric oxide synthase translocation; acetylcholine; platelet-activating factor

Caveolin-3 (Cav3), the primary protein component of caveolae in muscle cells, regulates numerous signaling pathways including insulin receptor signaling and facilitates free fatty acid (FA) uptake by interacting with several FA transport proteins. We previously reported that Cav3 knockout mice (Cav3KO) develop cardiac hypertrophy with diminished contractile function; however, the effects of Cav3 gene ablation on cardiac substrate utilization are unknown. The present study revealed that the uptake and oxidation of FAs and glucose were normal in hypertrophic Cav3KO hearts. Real-time PCR analysis revealed normal expression of lipid metabolism genes including FA translocase (CD36) and carnitine palmitoyl transferase-1 in Cav3KO hearts. Interestingly, myocardial cAMP content was significantly increased by 42%; however, this had no effect on PKA activity in Cav3KO hearts. Microarray expression analysis revealed a marked increase in the expression of genes involved in receptor trafficking to the plasma membrane, including Rab4a and the expression of WD repeat/FYVE domain containing proteins. We observed a fourfold increase in the expression of cellular retinol binding protein-III and a 3.5-fold increase in 17[beta]-hydroxysteroid dehydrogenase type 11, a member of the short-chain dehydrogenase/ reductase family involved in the biosynthesis and inactivation of steroid hormones. In summary, a loss of Cav3 in the heart leads to cardiac hypertrophy with normal substrate utilization. Moreover, a loss of Cav3 mRNA altered the expression of several genes not previously linked to cardiac growth and function. Thus we have identified a number of new target genes associated with the pathogenesis of cardiac hypertrophy. cardiomyopathy; caveolae; fatty acids; isolated perfused hearts; adenosine 3',5'-cyclic monophosphate

Transient receptor potential vanilloid 5 (TRPV5) constitutes the apical entry pathway for transepithelial [Ca.sup.2+] reabsorption in kidney. Many hormones alter renal [Ca.sup.2+] reabsorption at least partly by regulating TRPV5. The mechanism for acute regulation of TRPV5 by phospholipase C-coupled hormones is largely unknown. Here, we found that protein kinase C (PKC) activator 1-oleoyl-acetyl-sn-glycerol (OAG) increased TRPV5 current density and surface abundance in cultured cells. The OAG-mediated increase of TRPV5 was prevented by preincubation with specific PKC inhibitors. Coexpression with a dominant-negative dynamin increased the basal TRPV5 current density and prevented the increase by OAG. Knockdown of caveolin-1 by small interference RNA (siRNA) prevented the increase of TRPV5 by OAG. In contrast, knockdown of clathrin heavy chain had no effects. OAG had no effect on TRPV5 expressed in caveolin-1 null cells derived from caveolin-1 knockout mice. Forced expression of recombinant caveolin-1 restored the regulation of TRPV5 by OAG in caveolin-1 knockout cells. Mutations of serine-299 and/or serine-654 of TRPV5 (consensus residues for phosphorylation by PKC) abolished the regulation by OAG. Parathyroid hormone (PTH) increased TRPV5 current density in cells coexpressing TRPV5 and type 1 PTH receptor. The increase caused by PTH was prevented by PKC inhibitor, mutation of serine-299/serine-654, or by knockdown of caveolin-1. Thus, TRPV5 undergoes constitutive caveolae-mediated endocytosis. Activation of PKC increases cell surface abundance of TRPV5 by inhibiting the endocytosis. This mechanism of regulation by PKC may contribute to the acute stimulation of TRPV5 and renal [Ca.sup.2+] reabsorption by PTH. dynamin; caveolin; parathyroid hormone; phospholipase C; Madin-Darby canine kidney cells

We developed a two-dimensional model of transport-dependent intracellular calcium signaling in endothelial cells (ECs). Our purpose was to evaluate the effects of spatial colocalization of endothelial nitric oxide synthase (eNOS) and capacitative calcium entry (CCE) channels in caveolae on eNOS activation in response to ATP. Caveolae are specialized microdomains of the plasma membrane that contain a variety of signaling molecules to optimize their interactions and regulate their activity. In ECs, these molecules include CCE channels and eNOS. To achieve a quantitative understanding of the mechanisms of microdomain calcium signaling and the preferential sensitivity of eNOS to calcium entering the cell through CCE channels, we constructed a mathematical model incorporating the cell morphology and cellular physiological processes. The model predicts that the spatial segregation of calcium channels in ECs can create transport-dependent sharp gradients in calcium concentration within the cell. The calcium concentration gradient is affected by channel density and cell geometry. This transport-dependent calcium signaling specificity effect is enhanced in ECs by increasing the spatial segregation of the caveolar signaling domains. Our simulation significantly advances the understanding of how [Ca.sup.2+], despite its many potential actions, can mediate selective activation of signaling pathways. We show that diffusion-limited calcium transport allows functional compartmentalization of signaling pathways based on the spatial arrangements of [Ca.sup.2+] sources and targets. capacitative calcium entry; caveolae; endothelial cells: endothelial nitric oxide synthase; mathematical model