Disruption Of Caveolae Research Articles

Caveolae Differentially Control Phosphorylation of Sarcoplasmic Reticular Proteins Following β2 Adrenoceptor Stimulation in the Adult Cardiac Myocyte

Caveolae, small flask-like lipid rafts, play a key role in shaping the spatial characteristics of the β2-adrenoceptor cAMP signal and confining this to the sarcolemmal compartment in the adult cardiac myocyte. Here we determine the consequences of disrupting caveolae for the ability of β2 signalling to target sarcoplasmic reticular proteins phospholamban (PLB) and the ryanodine receptor (RyR). Experiments were performed with dissociated adult rat ventricular myocytes. Selective β2 adrenoceptor stimulation was achieved with 10 μM zinterol in the presence of 300 nM CGP20712A (CGP). Disruption of caveolae (using the cholesterol depleting agent methyl-β-cyclodextrin, MBCD) resulted in inotropic and lusitropic responses to β2 stimulation (70.2 ± 9.7% increase in shortening; 13.3 ± 1.3 % decrease in time to half relaxation) which were absent in control cells (n=12-20 myocytes, P<0.001). PLB contributes to inotropic and lusitropic responses via protein kinase A (PKA)-dependent phosphorylation at Ser16. In agreement with functional data, MBCD-treated myocytes showed a marked 561 ± 144% increase in Ser16-phosphorylated PLB in response to β2 stimulation (relative to that in cells exposed to CGP alone) which was absent in control cells (93 ± 31% of that with CGP alone) (n=4, P<0.05). By contrast, we saw no significant increase (P>0.05) in phosphorylation of one of the PKA-targeted sites of RyR, Ser2809, in either control (112 ± 11%) or MBCD-treated (116 ± 18%) myocytes in response to β2 stimulation (n=5). These preliminary data suggest that caveolae selectively control cAMP signals even within the same broad (sarcoplasmic reticular) compartment of the adult cardiac myocyte. Disruption of caveolae allows β2 cAMP-dependent signalling to access a sub-compartment of the sarcoplasmic reticulum which contains PLB, but not one which contains RyR.

Little caves ameliorate hepatic insulin signaling. Focus on “Caveolin gene transfer improves glucose metabolism in diabetic mice”

CAVEOLAE, DISCOVERED BY ELECTRON microscopy in 1950s, are membrane invaginations that accommodate various molecules involved in cellular signaling. Caveolin, a major component of caveolae, has been shown to inhibit the function of multiple caveolar proteins, including kinases, involved in cell differentiation and proliferation. Interestingly, insulin signal seems exempted from this inhibition and would rather require caveolin for proper signaling. In this issue of American Journal of Physiology-Cell Physiology, Otsu and coworkers (16) show that the insulin resistance state of obese and diabetic mice can be reversed upon caveolin-3 overexpression in liver. These interesting findings point to a novel role of caveolin in controlling whole insulin sensitivity and glucose homeostasis in mice. Caveolae, or “little caves,” first identified approximately 50 years ago when electron microscopy started, were described as morphologically distinct flasks, suggested to function as endocytic vesicles (17). In 1992, a major finding was the discovery of caveolin (now called caveolin-1) as a protein marker of caveolae. Caveolin is a small protein of 22 kDa with a hydrophobic region near the COOH terminus which anchors the protein to the membrane (10, 22). When caveolin is overexpressed in cells devoid of caveolae, the typical caveolar pattern is formed, implying that caveolin is the principal structural component of caveolae. In subsequent years, two other caveolin isoforms were identified, caveolin-2 and caveolin-3, which are the products of different genes. While the amino acid sequence homology is high between caveolin-1 and -3, their tissue distribution is quite different. Caveolin-1 is widely expressed in adipocytes, endothelial cells, and epithelial cells, whereas caveolin-3 is expressed in cardiac and in skeletal myocytes. Caveolin-2 is mostly coexpressed with caveolin-1, and the two subtypes may form functional hetero-oligomeric complexes. Because caveolin expression was thought, until recently, to be necessary and sufficient to form caveolae, the discovery of caveolins paved the way for functional studies that sought to define the role of caveolae. Indirect approaches to manipulate caveolin function first involved the introduction into cells of a small stretch of a peptide derived from the caveolin scaffolding domain. This revealed a role for caveolins in a number of signaling pathways and led to the conclusion that caveolae orchestrate signaling by organizing protein interactions at the plasma membrane (for review see Ref. 14). Further confirmation came by other experiments, such as by ablation of caveolae by altering the composition of the plasma membrane. Indeed, caveolar membranes closely resemble lipid rafts, enriched with particular lipid species such as sphingolipids and cholesterol. Thus, agents that destroy lipid rafts cause the disappearance of caveolae. From the use of such approaches, it also appeared that signaling was not the only membraneassociated event modified by alterations in caveolae. The endocytic delivery of a number of molecules was also altered by disruption of caveolae, thus emphasizing their function as endocytic portals (see Ref. 18 for review). Considering caveolins from a “trafficking” point of view also suggested that the function of caveolins and caveolae might be linked to cellular lipid transport and homeostasis. Caveolin can directly bind lipids, such as cholesterol or fatty acids, and can dynamically associate with cytoplasmic lipid droplets that provide intracellular lipid stores (11).

Effect of β-cyclodextrin and its derivatives on caveolae disruption, relationships with their cholesterol extraction capacities

Endothelial cells (HUVEC) were treated with β-cyclodextrin (β-CD) and hydroxypropylated or methylated derivatives solutions to confirm their lack of affinity with phospholipids and their specificity towards cholesterol. Further studies were performed on bovine aortic endothelial cells to assess the effect of β-CDs (mainly methylated derivatives) on membrane microdomains (lipid rafts or caveolae), by detecting the caveolae marker caveolin-1 in fractions of sucrose gradients. A displacement from the lighter to the heavier fractions, characteristic of caveolae disruption, was observed using CDs. The strongest effect was obtained with dimethyl-β-CD, for which an accumulation of caveolin-1 was observed in the bottom of the gradient. Crysmeb® and trimethyl-β-CD seemed to have the weaker effects as a significative amount of caveolin-1 was still detected in the light fraction corresponding to caveolae. β-CD and CDs having a degree of methylation a bit lower than 2 showed intermediate effects. The results of the present study on microdomains seem in good correlation with the cell cholesterol extraction capacities of CDs previously determined.

Muscarinic receptor-mediated bronchoconstriction is coupled to caveolae in murine airways

Cholinergic bronchoconstriction is mediated by M(2) and M(3) muscarinic receptors (MR). In heart and urinary bladder, MR are linked to caveolin-1 or -3, the structural proteins of caveolae. Caveolae are cholesterol-rich, omega-shaped invaginations of the plasma membrane. They provide a scaffold for multiple G protein receptors and membrane-bound enzymes, thereby orchestrating signaling into the cell interior. Hence, we hypothesized that airway MR signaling pathways are coupled to caveolae as well. To address this issue, we determined the distribution of caveolin isoforms and MR subtype M2R in murine and human airways and investigated protein-protein associations by fluorescence resonance energy transfer (FRET)-confocal laser scanning microscopy (CLSM) analysis in immunolabeled murine tissue sections. Bronchoconstrictor responses of murine bronchi were recorded in lung-slice preparations before and after caveolae disruption by methyl-β-cyclodextrin, with efficiency of this treatment being validated by electron microscopy. KCl-induced bronchoconstriction was unaffected after treatment, demonstrating functional integrity of the smooth muscle. Caveolae disruption decreased muscarine-induced bronchoconstriction in wild-type and abolished it in M2R(-/-) and M3R(-/-) mice. Thus M2R and M3R signaling pathways require intact caveolae. Furthermore, we identified a presumed skeletal and cardiac myocyte-specific caveolin isoform, caveolin-3, in human and murine bronchial smooth muscle and found it to be associated with M2R in situ. In contrast, M2R was not associated with caveolin-1, despite an in situ association of caveolin-1 and caveolin-3 that was detected. Here, we demonstrated that M2R- and M3R-mediated bronchoconstriction is caveolae-dependent. Since caveolin-3 is directly associated with M2R, we suggest caveolin-3 as novel regulator of M2R-mediated signaling.

Caveolin-1 mediates endotoxin inhibition of endothelin-1-induced endothelial nitric oxide synthase activity in liver sinusoidal endothelial cells

Endothelin-1 (ET-1) plays a key role in the regulation of endothelial nitric oxide synthase (eNOS) activation in liver sinusoidal endothelial cells (LSECs). In the presence of endotoxin, an increase in caveolin-1 (Cav-1) expression impairs ET-1/eNOS signaling; however, the molecular mechanism is unknown. The objective of this study was to investigate the molecular mechanism of Cav-1 in the regulation of LPS suppression of ET-1-mediated eNOS activation in LSECs by examining the effect of caveolae disruption using methyl-beta-cyclodextrin (CD) and filipin. Treatment with 5 mM CD for 30 min increased eNOS activity (+255%, P < 0.05). A dose (0.25 microg/ml) of filipin for 30 min produced a similar effect (+111%, P < 0.05). CD induced the perinuclear localization of Cav-1 and eNOS and stimulated NO production in the same region. Readdition of 0.5 mM cholesterol to saturate CD reversed these effects. Both the combined treatment with CD and ET-1 (CD + ET-1) and with filipin and ET-1 stimulated eNOS activity; however, pretreatment with endotoxin (LPS) abrogated these effects. Following LPS pretreatment, CD + ET-1 failed to stimulate eNOS activity (+51%, P > 0.05), which contributed to the reduced levels of eNOS-Ser1177 phosphorylation and eNOS-Thr495 dephosphorylation, the LPS/CD-induced overexpression and translocation of Cav-1 in the perinuclear region, and the increased perinuclear colocalization of eNOS with Cav-1. These results supported the hypothesis that Cav-1 mediates the action of endotoxin in suppressing ET-1-mediated eNOS activation and demonstrated that the manipulation of caveolae produces significant effects on ET-1-mediated eNOS activity in LSECs.

Compartmentalizing VEGF-Induced ERK2/1 Signaling in Placental Artery Endothelial Cell Caveolae: A Paradoxical Role of Caveolin-1 in Placental Angiogenesis in Vitro

On vascular endothelial growth factor (VEGF) stimulation, both VEGF R1 and R2 receptors were phosphorylated in ovine fetoplacental artery endothelial (oFPAE) cells. Treatment with VEGF stimulated both time- and dose-dependent activation of ERK2/1 in oFPAE cells. VEGF-induced ERK2/1 activation was mediated by VEGFR2, but not VEGFR1, and was linked to intracellular calcium, protein kinase C, and Raf-1. VEGF stimulated oFPAE cell proliferation, migration, and tube formation in vitro. Blockade of ERK2/1 pathway attenuated VEGF-induced cell proliferation and tube formation but failed to inhibit migration in oFPAE cells. Disruption of caveolae by cholesterol depletion with methyl-beta-cyclodextrin or by down-regulation of its structural protein caveolin-1 blunted VEGF-induced ERK2/1 activation, proliferation, and tube formation in oFPAE cells, indicating an essential role of integral caveolae in these VEGF-induced responses. Adenoviral overexpression of caveolin-1 and addition of a caveolin scaffolding domain peptide also inhibited VEGF-stimulated ERK2/1 activation, cell proliferation, and tube formation in oFPAE cells. Furthermore, molecules comprising the ERK2/1 signaling module, including VEGFR2, protein kinase Calpha, Raf-1, MAPK kinase 1/2, and ERK2/1, resided with caveolin-1 in caveolae. VEGF transiently stimulated ERK2/1 activation in the caveolae similarly as in intact cells. Caveolae disruption greatly diminished ERK2/1 activation by VEGF in oFPAE cell caveolae. We conclude that caveolae function as a platform for compartmentalizing the VEGF-induced ERK2/1 signaling module. Caveolin-1 and caveolae play a paradoxical role in regulating VEGF-induced ERK2/1 activation and in vitro angiogenesis as evidenced by the similar inhibitory effects of down-regulation and overexpression of caveolin-1 and disruption of caveolae in oFPAE cells.

Cardiac myocyte‐specific caveolin‐3 overexpression modulates ANP production and attenuates cardiac hypertrophy in vivo

IntroductionThe organization of signaling molecules in cardiac hypertrophy are incompletely understood. Caveolae, invaginations of the sarcolemmal membrane localize signaling molecules involved in cardiac hypertrophy. Atrial naturetic peptide (ANP) regulates endothelin‐1 release and cardiac hypertrophy. Furthermore, ANP is secreted via and stored within caveolae. We hypothesized that cardiac myocyte‐specific overexpression of Cav‐3 (Cav‐3 OE) modulates ANP production attenuating cardiac hypertrophy in vivo.MethodsCav‐3 OE and littermate controls (C) were subjected to transverse aortic constriction (TAC) for 4 weeks. Echocardiography, histology and molecular analysis were performed.ResultsCav‐3OE mice had reduced heart weights post TAC compared to C. Furthermore, Cav‐3 OE and C mice had similar fractional shortening (FS) pre TAC but had greater FS compared to C post TAC. mRNA levels of ANP were 7‐fold greater in Cav‐3 OE at baseline compared to C, resulting in increased protein expression of ANP in the cardiac myocyte and blood. Disruption of caveolae with colchicine in adult cardiac myocytes isolated from Cav‐3 OE resulted in a decrease in ANP.ConclusionWe show that Cav‐3 OE alters ANP production in cardiac myocytes blunting cardiac hypertrophy while preserving cardiac function post TAC. This suggests a therapeutic role for overexpression of Cav‐3 in pathological cardiac hypertrophy.

CANNABINOR, A NOVEL PERIPHERALLY ACTING CANNABINOID-2-RECEPTOR AGONIST REDUCES NONVOIDING-CONTRACTIONS IN RATS WITH PARTIAL URETHRAL OBSTRUCTION

potentially contribute to bladder dysfunction. This study investigates if changes in caveolae or caveolar elements occur after bladder outlet obstruction (BOO), and whether these changes affect the responsiveness to agonists. METHODS: BOO was created for 1, 2 and 4 weeks by placing a ligature around the proximal urethra of male rats. Differences in bsm caveolae after BOO were visualized by electron microscopy. Changes in caveolin (Cav) expression and distribution after BOO were determined by real time rt-PCR and immunofluorescence microscopy. Longitudinal tissue from obstructed bladder was stretched in organ bath at 37oC. Contractile responses to physiologic agonists were repeated before and after disruption of caveolae (by methyl-s-cyclodextrin, mscd), and compared with non obstructed controls. RESULTS: After 2 weeks of BOO, bladder weight markedly increased compared to controls. The number of caveolae significantly decreased after 1 wk of BOO compared with non-obstructed bladders. Cav-1 immunoreactivity diminished after 1 wk of BOO and further decreased after 2 wks of BOO. Cav-2 immunoreactivity decreased after 1 wk of BOO but was less attenuated after 2-wks. Cav-2 gene expression was significantly up-regulated after 2wks of BOO. Following 2 wks of BOO, the contractile response to bradykinin (BK) did not change after caveolar depletion compared with non-obstructed animals, in which m cd significantly increased the BK-induced contraction; however in BOO animals the amplitude of BK contraction was greater at baseline compared with non obstructed controls. In non obstructed bladders, the contractile response to 5HT was diminished by caveolar depletion. In BOO, the 5HT induced response was slightly diminished by m cd after 2 weeks, but after 4 weeks the contractile response was not affected by m cd. CONCLUSIONS: This study linked the loss of caveolar elements in BOO with functional alteration of specific agonist-induced responses. These data are consistent with reduced negative and positive modulation of BK and 5HT induced contractions respectively, which normally is imparted by caveolae, but is progressively lost during BOO as caveolae gradually disappear from the bsm membrane. Thus, alterations in caveolae may play a role in bladder dysfunction induced by BOO.

Cholesterol depletion induces anoikis‐like apoptosis via FAK down‐regulation and caveolae internalization

Caveolae (lipid rafts), microdomains of the plasma membrane, are known to contain various signalling molecules and consequently are involved in the regulation of many biological functions. To investigate the role of the caveolae in cell survival and adhesion, we disrupted the caveolae by depletion of cholesterol, a major lipid component of the caveolae, with methyl-beta cyclodextrin (MbetaCD) treatment of A431 cells. We found that cholesterol depletion induced an anoikis-like cell death involving actin reorganization, resulting in a decrease in cell spreading and an increase in cell detachment, which was reversed by cholesterol addition. Disruption of caveolae led to the down-regulation of FAK, Src activation, tyrosine phosphorylation of caveolin-1 and mobilization of caveolae markers, GM1 and caveolin-1, from the cell surface to the cytoplasm, which were also recovered by cholesterol addition. The expression of dominant-active FAK was able to delay caveolae internalization and apoptosis and attenuated Akt inactivation by MbetaCD, whereas dominant-negative FAK expression resulted in enhanced apoptosis. Moreover, FAK down-regulation by si-RNA resulted in Akt inactivation and thus increased cell death by MbetaCD treatment. Our results suggest that the cholesterol content and/or surface levels of the caveolae affect the activity of FAK, which in turn regulates caveolae internalization and cell survival.

Extraction of membrane cholesterol disrupts caveolae and impairs serotonergic (5-HT2A) and histaminergic (H1) responses in bovine airway smooth muscle: role of Rho-kinase

Some receptors and signaling molecules, such as Rho-kinase (ROCK), localize in caveolae. We asked whether the function of histamine receptors (H(1)) and 5-hydroxytryptamine (serotonin) receptors (5-HT(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-beta-cyclodextrin (MbetaCD), used to deplete membrane cholesterol, was shown to disrupt caveolae and diminish sustained contractions to histamine (approximately 80%), 5-HT (100%), alpha-methyl-5-HT (100%), and KCl (approximately 30%). Cholesterol-loaded MbetaCD (CL-MbetaCD) restored the responses to KCl and partially restored the responses to agonists. ROCK inhibition by Y-27632 diminished contractions to histamine (approximately 85%) and 5-HT (approximately 59%). 5-HT or histamine stimulation augmented ROCK activity. These increases were reduced by MbetaCD and partially reestablished by CL-MbetaCD. The increase in intracellular Ca(2+) that was induced by both agonists was reduced by MbetaCD. The presence of caveolin-1 (Cav-1), H1, 5-HT(2A), and ROCK1 was corroborated by immunoblotting of membrane fractions from sucrose gradients and by confocal microscopy. H(1) receptors coimmunoprecipitated with Cav-1 in caveolar and noncaveolar membrane fractions, whereas 5-HT(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(1) and 5-HT(2A) receptors through their Rho/ROCK signaling.

The rapid activation of N-Ras by α-thrombin in fibroblasts is mediated by the specific G-protein Gα i2–Gβ 1–G γ5 and occurs in lipid rafts

α-thrombin is a potent mitogen for fibroblasts and initiates a rapid signal transduction pathway leading to the activation of Ras and the stimulation of cell cycle progression. While the signaling events downstream of Ras have been studied in significant detail and appear well conserved across many species and cell types, the precise molecular events beginning with thrombin receptor activation and leading to the activation of Ras are not as well understood. In this study, we examined the immediate events in the rapid response to α-thrombin, in a single cell type, and found that an unexpected degree of specificity exists in the pathway linking α-thrombin to Ras activation. Specifically, although IIC9 cells express all three Ras isoforms, only N-Ras is rapidly activated by α-thrombin. Further, although several Gα subunits associate with PAR1 and are released following stimulation, only Gα i2 couples to the rapid activation of Ras. Similarly, although IIC9 cells express many Gβ and Gγ subunits, only a subset associates with Gα i2, and of those, only a single Gβγ dimer, Gβ 1γ 5, participates in the rapid activation of N-Ras. We then hypothesized that co-localization into membrane microdomains called lipid rafts, or caveolae, is at least partially responsible for this degree of specificity. Accordingly, we found that all components localize to lipid rafts and that disruption of caveolae abolishes the rapid activation of N-Ras by α-thrombin. We thus report the molecular elucidation of an extremely specific and rapid signal transduction pathway linking α-thrombin stimulation to the activation of Ras.

Enhanced myogenic constriction of mesenteric artery in heart failure relates to decreased smooth muscle cell caveolae numbers and altered AT1‐ and epidermal growth factor‐receptor function

We previously showed that enhanced myogenic constriction (MC) of peripheral resistance arteries involves active AT(1) receptors in chronic heart failure (CHF). Recent data suggest both transactivation of EGF receptors and caveolae-like microdomains to be implicated in the activity of AT(1) receptors. Thus, we assessed their roles in increased MC in mesenteric arteries of CHF rats. Male Wistar rats underwent myocardial infarction to induce CHF and were sacrificed after 12 weeks. The number of caveolae in smooth muscle cells (SMC) of mesenteric arteries of CHF rats was decreased by 43.6 +/- 4.0%, this was accompanied by increased MC, which was fully normalized to the level of sham by antagonists of the AT(1)-receptor (losartan) or EGF-receptor (AG1478). Acute disruption of caveolae in sham rats affected caveolae numbers and MC to a similar extent as CHF, however MC was only reversed by the antagonist of the EGF-receptor, but not by the AT(1)-receptor antagonist. Further, in sham rats, MC was increased by a sub-threshold concentration of angiotensin II and reversed by both AT(1)- as well as EGF-receptor inhibition. In contrast, increased MC by a sub-threshold concentration of EGF was only reversed by EGF receptor inhibition. These findings provide the first evidence that decreased SMC caveolae numbers are involved in enhanced MC in small mesenteric arteries, by affecting AT(1)- and EGF-receptor function. This suggests a novel mechanism involved in increased peripheral resistance in CHF.

Caveolae Act As Membrane Reserves Which May Limit ICl,swell Activation During Cardiac Myocyte Swelling

The channel responsible for swelling-activated chloride current (ICl,swell) is a mechanosensor which responds to changes in membrane tension during cell swelling, and regulates cell volume. It has been proposed that the ICl,swell channel (or elements that regulate this) are dependent on caveolae, and we have previously shown that disrupting caveolae increases the rate of hyposmotic cardiac myocyte swelling. Here we test the hypothesis that the role of caveolae as a membrane reserve limits activation of ICl,swell. Rat ventricular myocytes were treated with methyl- β-cyclodextrin (MBCD) to disrupt caveolae and exposed to 0.02T solution (until cell lysis) or 0.64T solution for 10-15 min (swelling). Maximum cell volume achieved prior to lysis was calculated from a video image. Swollen cells (0.64T) were fixed for electron microscopy, and the negative inotropic response to swelling used as an index of ICl,swell activation. Following disruption of caveolae, the time to lysis in 0.02T solution was significantly reduced compared with control cells. In cells fixed for EM, caveolae were defined as invaginations or closed subsarcolemmal vesicles with a diameter of ≈50-100 nm. MBCD and 0.064T hyposmotic swelling significantly reduced the total number of caveolae by 75 and 50% respectively. Both ‘open’ and ‘closed’ caveolae were reduced by MBCD, but swelling only affected the ‘closed’ population. The negative inotropic response observed 6 and 10 min after exposure to 0.064T solution was blocked by the ICl,swell inhibitor DIDS but enhanced by disruption of caveolae. Our data suggest that swelling causes flattening of ‘open’ caveolae, in tandem with sarcolemmal incorporation of ‘closed’ caveolae. We propose that disrupting caveolae removes essential membrane reserves, thereby speeding cell swelling in hyposmotic conditions and promoting activation of mechanosensitive ICl,swell channels. Supported by CRISTAL and the British Heart Foundation.

L-arginine and Tetrahydrobiopterin Inhibit Mitochondrial Permeability Transition Pore by Preventing ROS Formation by Mitochondrial Nitric Oxide Synthase

Background: The functional role of mitochondrial nitric oxide synthase (mtNOS) in heart has remained a matter of debate. Methods: We used laser scanning confocal microscopy in combination with fluorescent dyes to characterize mitochondrial NO and ROS production and the permeability transition pore (PTP) activity in permeabilized cat ventricular myocytes. Results: Stimulation of mitochondrial calcium uptake resulted in a dose-dependent increase in mitochondrial NO production when L-arginine, a substrate for mtNOS, was present. The potential contribution of the caveolae-located eNOS and SR-targeted nNOS was ruled out based on the fact that disruption of caveolae with methyl-βeτα-cyclodextrin or prevention of SR uptake with thapsigargin did not affect calcium-induced NO production. Collapsing the mitochondrial membrane potential, blocking the mitochondrial calcium uniporter and respiratory chain abolished mitochondrial NO production. In the absence of L-arginine, calcium-induced NO production was significantly decreased; however an increased ROS production was observed. Inhibition of mitochondrial arginase (which limits L-arginine availiability) resulted in 50% inhibition of calcium-induced ROS production. Both mitochondrial NO and ROS production were blocked by the nNOS inhibitor (4S)-N-(4-amino-5[aminoethyl]aminopentyl]-N′-nitroguanidine and the calmodulin antagonist W-7, while the eNOS inhibitor L-NIO or the iNOS inhibitor 1400W had no effect. The superoxide dismutase mimetic MnTBAP abolished calcium-induced ROS generation and increased NO production threefold. In the absence of L-arginine, mitochondrial calcium uptake induced opening of the mitochondrial PTP, which was blocked by cyclosporin A, MnTBAP and reversed by L-arginine. The essential mtNOS co-factor tetrahydrobiopterin also inhibited mitochondrial ROS generation and PTP opening at a concentration of 100 μM, while 10 μM tetrahydrobiopterin had no effect. Conclusion: Our data demonstrate the importance of L-arginine and tetrahydrobiopterin for the regulation of mitochondrial oxidative stress and modulation of PTP opening by mtNOS.

Phosphorylation of caveolin-1 is anti-apoptotic and promotes cell attachment during oxidative stress of kidney cells

Caveolin-1 (cav1) is reported to have both cell survival and pro-apoptotic characteristics. This may be explained by its localisation or phosphorylation in injured cells. This study investigated the role of cav1 in kidney cells of different nephron origin and developmental state after oxidative stress. Renal MCDK distal tubular, HK2 proximal tubular epithelial cells and HEK293T renal embryonic cells were treated with 1 mM hydrogen peroxide. Apoptosis, loss of cell adhesion, and cell survival were compared with expression of cav1 in its non-phosphorylated and phosphorylated (p-cav1) forms. Cav1 was transfected into the HEK293T cells, or caveolae were disrupted with filipin or nystatin in HK2 cells, to investigate functions of cav1 and p-cav1. Oxidative stress induced more apoptosis in HK2s than MDCKs (p < 0.05). HK2s had lower endogenous cav1 and p-cav1 than MDCKs (p < 0.05). Both cell lines had increased p-cav1, but not cav1, with oxidative stress. This increase was greatest in MDCKs (p < 0.01). Cav1 was located mainly in the plasma membrane of untreated cells and translocated to the cytoplasm with oxidative stress in both cell lines, more so in MDCKs. Disruption of caveolae caused cytoplasmic translocation of cav1 in HK2s, but did not alter high levels of oxidative stress-induced apoptosis. When HEK293Ts lacking endogenous cav1 were transfected with cav1, oxidant-induced apoptosis and loss of cell adhesion was decreased (p < 0.01), and p-cav1 was induced by treatment. Cav1 expression and localisation in kidney cells is not anti-apoptotic, but increased expression of p-cav1 may promote cell survival after oxidative stress.

Endothelial Cell Protein C Receptor Cellular Localization and Trafficking

Endothelial cell protein C receptor (EPCR) is the cellular receptor for protein C (PC) and activated protein C (APC). Recent studies from others and us showed that EPCR also acts as a cellular binding site for factor VII (FVII) and activated factor VII (FVIIa). Although much is know about the biochemistry and pathophysiological importance of EPCR, little is know how EPCR interaction with its ligands on cell surfaces affects its expression and facilitate internalization of the ligands. The present study was undertaken to characterize cellular localization and trafficking of EPCR and investigate how FVIIa or APC binding to EPCR influences these processes. The studies employed two cell model systems - primary cultured human umbilical endothelial cells (HUVEC) and CHO cells stably transfected with EPCR. Cellular localization of EPCR and its trafficking was analyzed by immunofluorescence confocal microscopy or monitoring the expression of EPCR tagged with green fluorescence protein (GFP). EPCR endocytosis was evaluated by biotinylation of cell surface proteins with NHS-SS- biotin, followed by monitoring the protection of biotinylated EPCR from a membrane-impermeable reducing agent. FVIIa and APC internalization and recycling was evaluated by monitoring the uptake/release of 125I-labeled ligands or following the intracellular routing of fluorescent dye (AF488)-conjugated ligands added to EPCR expressing cells. Data from these studies showed that a majority of EPCR is localized on the cell surface and distributed in a patchy and punctuate manner. Immunostaining of HUVEC and CHO-EPCR cells with EPCR mAb and caveolin-1 antibodies showed a high degree of co-localization of EPCR and caveolin-1. Depletion of cholesterol from the plasma membrane, which disrupts caveolae, by ß methyl cyclodextrin reduced the extent EPCR and caveloin-1 colocalization. These data indicate EPCR on the cell surface predominantly localizes in caveolae. Inside the cell, EPCR is mainly localized in a small perinuclear structure, which is the site of centrosome. Colocalization of EPCR with tubulin (a marker for centrosome) and rab 11 (a marker of recycling compartment, REC) revealed that EPCR is localized actually in the REC and not in the centrosome. A small fraction of EPCR is also localized in endosomes as evident from colocalization of EPCR with EEA1 and Rab5, early endosome markers. Chasing the cell surface biotinylated proteins showed no significant increase in the biotinylated EPCR in the intracellular pool of proteins, which indicate that EPCR is not actively endocytosed constitutively or that the internalized EPCR is immediately recycled back to the cell surface. FVIIa or APC binding to EPCR promoted the EPCR endocytosis. The endocytosed receptors were first observed in proximity of the plasma membrane after 10 min and by 30 to 60 min most of the endocytosed EPCR was accumulated in the REC. The internalized FVIIa or APC appeared to follow the same route of the endocytosed EPCR. Proteolytically inactive FVIIa or APC behaved same as FVIIa or APC in promoting EPCR endocytosis and its trafficking. EPCR-dependent FVIIa or APC internalization is a dynamin-dependent process as the inhibition of the GTPase activity of dynamin by a specific inhibitor (Dynasore) completely abrogated their internalization and accumulation in the REC. Additional studies revealed that disruption of coated-pit pathway by potassium depletion blocked the endocytosis of transferrin, a classic marker for endocytosis via clathrin-dependent coated-pit pathway, but had no effect on EPCR-dependent FVIIa or APC internalization. In contrast, disruption of caveolae by cholesterol depletion blocked the internalization of FVIIa but not transferrin. Rab11 dominant negative mutant form (S25N) prevented the passage of the endocytosed EPCR or the internalized FVIIa or APC into the REC. After peaking at 30 min, the amount of both the ligands and the receptor in the REC gradually decreased, and some of the internalized ligand re-appeared at the cell surface. Overall the data provided herein suggest that FVIIa or APC binding to EPCR, independent of their protease activity, promotes EPCR endocytosis via the dynamin-dependent caveolar pathway and the activation of rab11 by GTP is required for exit of the endocytosed receptor or the ligands from sorting endosomes to the recycling compartment.

Caveolin-3 Associates with and Affects the Function of Hyperpolarization-Activated Cyclic Nucleotide-Gated Channel 4

Targeting of ion channels to caveolae, a subset of lipid rafts, allow cells to respond efficiently to extracellular signals. Hyperpolarization-activated cyclic nucleotide-gated channel (HCN) 4 is a major subunit for the cardiac pacemaker. Caveolin-3 (Cav3), abundantly expressed in muscle cells, is responsible for forming caveolae. P104L, a Cav3 mutant, has a dominant negative effect on wild type (WT) Cav3 and associates with limb-girdle muscular dystrophy and cardiomyopathy. HCN4 was previously shown to localize to lipid rafts, but how caveolae regulate the function of HCN4 is unknown. We hypothesize that Cav3 associates with HCN4 and regulates the function of HCN4 channel. In this study, we applied whole-cell patch clamp analysis, immunostaining, biotinylation, and immunoprecipitation methods to investigate this hypothesis. The immunoprecipitation results indicated an association of HCN4 and Cav3 in the heart and in HEK293 cells. Our immunostaining results showed that HCN4 colocalized with Cav3 but only partially colocalized with P104L in HEK293 cells. Transient expression of Cav3, but not P104L, in HEK 293 cells stably expressing HCN4 caused a 45% increase in HCN4 current (IHCN4) density. Transient expression of P104L caused a two-fold increase in the activation time constant for IHCN4 and shifted the voltage of the steady-state inactivation to a more negative potential. We conclude that HCN4 associates with Cav3 to form a HCN4 macromolecular complex. Our results indicated that disruption of caveolae using P104L alters HCN4 function and could cause a reduction of cardiac pacemaker activity.

Smooth muscle NOS, colocalized with caveolin‐1, modulates contraction in mouse small intestine

Neuronal nitric oxide synthase (nNOS) in myenteric neurons is activated during peristalsis to produce nitric oxide which relaxes intestinal smooth muscle. A putative nNOS is also found in the membrane of intestinal smooth muscle cells in mouse and dog. In this study we studied the possible functions of this nNOS expressed in mouse small intestinal smooth muscle colocalized with caveolin-1(Cav-1). Cav-1 knockout mice lacked nNOS in smooth muscle and provided control tissues. 60 mM KCl was used to increase intracellular [Ca2+] through L-type Ca2+ channel opening and stimulate smooth muscle NOS activity in intestinal tissue segments. An additional contractile response to LNNA (100 μM, NOS inhibitor) was observed in KCl-contracted tissues from control mice and was almost absent in tissues from Cav-1 knockout mice. Disruption of caveolae with 40 mM methyl-β cyclodextrin in tissues from control mice led to the loss of Cav-1 and nNOS immunoreactivity from smooth muscle as shown by immunohistochemistry and a reduction in the response of these tissues to N-ω-nitro-L-arginine (LNNA). Reconstitution of membrane cholesterol using water soluble cholesterol in the depleted segments restored the immunoreactivity and the response to LNNA added after KCl. Nicardipine (1 μM) blocked the responses to KCl and LNNA confirming the role of L-type Ca2+ channels. ODQ (1 μM, soluble guanylate cyclase inhibitor) had the same effect as inhibition of NOS following KCl. We conclude that the activation of nNOS, localized in smooth muscle caveolae, by calcium entering through L-type calcium channels triggers nitric oxide production which modulates muscle contraction by a cGMP-dependent mechanism.

Vanishing Act

Potassium channels play an important role in the regulation of vascular smooth muscle tone and, thus, contribute to the regulation of blood pressure, blood flow, and microvascular exchange.1 These channels importantly participate in the determination of vascular smooth muscle cell (VSMC) membrane potential,1,2 which, in turn, controls Ca2+ influx through voltage-gated Ca2+ channels1,2 and has been implicated in the control of Ca2+ release and Ca2+ sensitivity of VSMCs.1 VSMCs express a diverse array of K+ channels that contribute to the regulation of VSMC function,1 including ≥1 type of vascular ATP-sensitive K+ (KATP) channels.2,3 KATP channels consist of a tetramer of α-pore–forming subunits from the KIR6.X family of inwardly rectifying K+ channels, along with complimentary sulfonylurea receptor (SUR) subunits that are members of the ATP-binding cassette family of proteins.2 The SUR subunits are essential for normal trafficking of KATP channels, modulate channel function, and are the binding sites for sulfonylurea antagonists of these channels, such as glibenclamide.2 Vascular smooth muscle KATP channels appear to be composed of KIR6.1 and SUR2B subunits,2 although some VSMCs may also express KATP channels composed of KIR6.2/SUR2B.3 As originally described, KATP channels open during hypoxia or ischemic conditions when cellular ATP levels fall, decreasing cell excitability and protecting energy-limited cells.2 However, both in vitro and in vivo studies suggest that VSMC KATP channels may be open under resting conditions and contribute to the regulation of VSMC membrane potential and vascular tone.1,2 Importantly, the activity of KATP channels can be modulated by a number of physiologically relevant vasoactive substances and conditions. As their name implies, KATP channels may be activated by decreases in intracellular …

Calcium extrusion by plasma membrane calcium pump is impaired in caveolin-1 knockout mouse small intestine

Plasma membrane calcium ATPase (PMCA) is an important calcium extrusion mechanism in smooth muscle cells. PMCA4 is the predominant isoform operating in conditions of high intracellular calcium during contraction. PMCA appears to be localized in lipid rafts and caveolae. In this study we examined the effects of the PMCA4-selective inhibitor caloxin 1c2 (5 µM) in intestine of caveolin-1 knockout mice and in bovine tracheal smooth muscle after caveolae disruption on PMCA4 function. Small intestinal tissues from control mice treated with caloxin 1c2 showed a higher contractile response of the longitudinal smooth muscle to Carbachol (10 µM) when compared to control tissues treated with a similar concentration of a control peptide. This effect of caloxin 1c2 was not found in tissues from caveolin-1 knockout mice. Immunohistochemistry and Western blotting of membrane fractions showed that PMCA was co-localized with caveolin-1 in smooth muscle plasma membrane in control tissues. One of the PMCA4 splice variant bands was missing in the lipid raft-enriched fraction prepared from caveolin-1 knockout tissue. In bovine tracheal smooth muscle tissue, caveolae disruption by cholesterol depletion led to the diminution of caveolin-1 and PMCA4b immunoreactivities, previously co-localized in the smooth muscle plasma membrane, and to the loss of the increase in Carbachol-induced contraction by caloxin 1c2. Our results suggest that the calcium removal function of PMCA4 in smooth muscle cells is dependent on its presence in intact caveolae. We suggest that this is due to the close spatial arrangement that allows calcium extrusion from a privileged cytosolic space between caveolae and sarcoplasmic reticulum.