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T Michel

Publications and source records attributed to T Michel.

At least 55 records · Page 3Linked to original sources
PubMed1998

The endothelial nitric-oxide synthase-caveolin regulatory cycle.

Nitric oxide production in the vascular endothelium is promoted by diverse agonists that transiently increase intracellular Ca2+ concentration and activate the endothelial nitric-oxide synthase (eNOS), a Ca2+/calmodulin-dependent enzyme. eNOS is acylated by the fatty acids myristate and palmitate and is targeted thereby to plasmalemmal signal-transducing domains termed caveolae. eNOS enzyme activity is markedly attenuated by its interactions with caveolin, the structural scaffolding protein of caveolae. We have discovered that in living cells, the eNOS-caveolin heteromeric complex undergoes cycles of dissociation and re-association modulated by Ca2+-mobilizing agonists. Calcium ionophore A23187 and the muscarinic cholinergic agonist carbachol both promote the dissociation of eNOS from caveolin in cultured cells, associated with translocation of eNOS from caveolae. As [Ca2+]i returns to basal levels, eNOS re-associates with caveolin, and the inhibited enzyme complex is then restored to caveolae, a process accelerated by palmitoylation of the enzyme. These data establish an eNOS-caveolin regulatory cycle, wherein enzyme activation is modulated by reversible protein-protein interactions controlled by Ca2+/calmodulin and by enzyme palmitoylation. Alterations in this cycle are likely to have an important influence on nitric oxide-dependent signaling in the vascular wall.

Animals↗
PubMed1998

Dynamic regulation of endothelial nitric oxide synthase: complementary roles of dual acylation and caveolin interactions.

N-Terminal myristoylation and thiopalmitoylation of the endothelial isoform of nitric oxide synthase (eNOS) are required for targeting the enzyme to specialized signal-transducing microdomains of plasma membrane termed caveolae. We have previously documented that the subcellular localization of eNOS is dynamically regulated by agonists such as bradykinin, which promotes enzyme depalmitoylation and translocation from caveolae. More recently, we have shown that association of eNOS with caveolin, the principal structural protein in caveolae, leads to enzyme inhibition, in a reversible process modulated by Ca2+-calmodulin (CaM). We now report studies of the respective roles of acylation and caveolin interaction for regulating eNOS activity. Using eNOS truncation and deletion mutants expressed in COS-7 cells, we have identified an obligatory role for the N-terminal half of eNOS in stabilizing its association with caveolin. By exploring the differential effects of detergents (CHAPS vs octyl glucoside), we have shown that this direct interaction between both proteins is facilitated by, but does not require, eNOS acylation, and, importantly, that treatment of intact aortic endothelial cells with the calcium ionophore A23187 leads to the rapid disruption of the eNOS-caveolin complexes. Finally, using transiently transfected COS-7 cells, we have observed that the myristoylation-deficient cytosol-restricted eNOS mutant (myr-) as well as the cytosolic fraction of the palmitoylation-deficient eNOS mutant (palm-) may both interact with caveolin; this association also leads to a marked inhibition of enzyme activity, which is completely reversed by addition of calmodulin. We conclude that the regulatory eNOS-caveolin association is independent of the state of eNOS acylation, indicating that agonist-evoked Ca2+/CaM-dependent disruption of the caveolin-eNOS complex, rather than agonist-promoted depalmitoylation of eNOS, relieves caveolin's tonic inhibition of enzyme activity. We therefore propose that caveolin may serve as an eNOS chaperone regulating NO production independently of the enzyme's residence within caveolae or its state of acylation.

Acylation↗
PubMed1998

Expression of endothelial nitric oxide synthase in human and rabbit gastrointestinal smooth muscle cells.

The aim of this study was to identify the nitric oxide synthase (NOS) isoform expressed in freshly dispersed rabbit gastric smooth muscle cells and in cultured rabbit gastric, human intestinal, and guinea pig taenia coli smooth muscle cells. RT-PCR products of the predicted size (354 bp) were obtained with endothelial NOS (eNOS)-specific primers, but not neuronal NOS (nNOS)- or inducible NOS (iNOS)-specific primers, in all smooth muscle preparations except guinea pig taenia coli. Control RT-PCR studies showed absence of the endothelial markers, platelet endothelial cell adhesion molecule-1 (PECAM-1) and vascular endothelial growth factor receptor (VEGFR), and the interstitial cell marker, c-kit, from cultures of smooth muscle cells. Cloning and sequence analysis showed that the predicted amino acid sequence (117 residues) in rabbit and human smooth muscle cells differed by only one residue from that of human eNOS. Northern blot analysis, using the PCR-generated and cloned eNOS cDNA from rabbits and humans as probes, demonstrated the expression of eNOS mRNA (4.4 kb) in both species. eNOS, but not nNOS or iNOS, transcripts were localized by in situ RT-PCR in single, freshly dispersed rabbit gastric smooth muscle cells; expression was evident in the majority of cells in each preparation. We conclude that eNOS is selectively expressed in rabbit gastric and human intestinal smooth muscle cells. The results confirm functional evidence for the existence of a constitutive NOS in smooth muscle cells of the gut in different species, except for guinea pig taenia coli.

Amino Acid Sequence↗
PubMed1997

Caveolin versus calmodulin. Counterbalancing allosteric modulators of endothelial nitric oxide synthase.

Nitric oxide is synthesized in diverse mammalian tissues by a family of calmodulin-dependent nitric oxide synthases. The endothelial isoform of nitric oxide synthase (eNOS) is targeted to the specialized signal-transducing membrane domains termed plasmalemmal caveolae. Caveolin, the principal structural protein in caveolae, interacts with eNOS and leads to enzyme inhibition in a reversible process modulated by Ca2+-calmodulin (Michel, J. B., Feron, O., Sacks, D., and Michel, T. (1997) J. Biol. Chem. 272, 15583-15586). Caveolin also interacts with other structurally distinct signaling proteins via a specific region identified within the caveolin sequence (amino acids 82-101) that appears to subserve the role of a "scaffolding domain." We now report that the co-immunoprecipitation of eNOS with caveolin is completely and specifically blocked by an oligopeptide corresponding to the caveolin scaffolding domain. Peptides corresponding to this domain markedly inhibit nitric oxide synthase activity in endothelial membranes and interact directly with the enzyme to inhibit activity of purified recombinant eNOS expressed in Escherichia coli. The inhibition of purified eNOS by the caveolin scaffolding domain peptide is competitive and completely reversed by Ca2+-calmodulin. These studies establish that caveolin, via its scaffolding domain, directly forms an inhibitory complex with eNOS and suggest that caveolin inhibits eNOS by abrogating the enzyme's activation by calmodulin.

Allosteric Regulation↗
PubMed1997

Dynamic targeting of the agonist-stimulated m2 muscarinic acetylcholine receptor to caveolae in cardiac myocytes.

In cardiac myocytes, as well as specialized conduction and pacemaker cells, agonist binding to muscarinic acetylcholine receptors (mAchRs) results in the activation of several signal transduction cascades including the endothelial isoform of nitric-oxide synthase (eNOS) expressed in these cells. Recent evidence indicates that, as in endothelial cells, eNOS in cardiac myocytes is localized to plasmalemma caveolae, specialized lipid microdomains that contain caveolin-3, a muscle-specific isoform of the scaffolding protein caveolin. In this report, using a detergent-free method for isolation of sarcolemmal caveolae from primary cultures of adult rat ventricular myocytes, we demonstrated that the muscarinic cholinergic agonist carbachol promotes the translocation of mAchR into low density gradient fractions containing most myocyte caveolin-3 and eNOS. Following isopycnic centrifugation, the different gradient fractions were exposed to the muscarinic radioligand [3H]quinuclidinyl benzilate (QNB), and binding was determined after membrane filtration or immunoprecipitation. In a direct radioligand binding assay, we found that [3H]QNB binding can be detected in caveolin-enriched fractions only when cardiac myocytes have been previously exposed to carbachol. Furthermore, most of this [3H]QNB binding can be specifically immunoprecipitated by an antibody to the m2 mAchR, indicating that the translocation of this receptor subtype is responsible for the [3H]QNB binding detected in the low density fractions. Moreover, the [3H]QNB binding could be quantitatively immunoprecipitated from the light membrane fractions with a caveolin-3 antibody (but not a control IgG1 antibody), confirming that the m2 mAchR is targeted to caveolae after carbachol treatment. Importantly, atropine, a muscarinic cholinergic antagonist, did not induce translocation of m2 mAchR to caveolae and prevented receptor translocation in response to the agonist carbachol. Thus, dynamic targeting of sarcolemmal m2 mAchR to caveolae following agonist binding may be essential to initiate specific downstream signaling cascades in these cells.

Animals↗
PubMed1997

Reciprocal regulation of endothelial nitric-oxide synthase by Ca2+-calmodulin and caveolin.

The endothelial nitric-oxide synthase (eNOS) is a key determinant of vascular homeostasis. Like all known nitric-oxide synthases, eNOS enzyme activity is dependent on Ca2+-calmodulin. eNOS is dynamically targeted to specialized cell surface signal-transducing domains termed plasmalemmal caveolae and interacts with caveolin, an integral membrane protein that comprises a key structural component of caveolae. We have previously reported that the association between eNOS and caveolin is quantitative and tissue-specific (Feron, O., Belhassen, L., Kobzick, L., Smith, T. W., Kelly, R. A., and Michel, T. (1996) J. Biol. Chem. 271, 22810-22814). We now report that in endothelial cells the interaction between eNOS and caveolin is importantly regulated by Ca2+-calmodulin. Addition of calmodulin disrupts the heteromeric complex formed between eNOS and caveolin in a Ca2+-dependent fashion. In addition, overexpression of caveolin markedly attenuates eNOS enzyme activity, but this inhibition is reversed by purified calmodulin. Caveolin overexpression does not affect the activity of the other NOS isoforms, suggesting eNOS-specific inhibition of NO synthase by caveolin. We propose a model of reciprocal regulation of eNOS in endothelial cells wherein the inhibitory eNOS-caveolin complex is disrupted by binding of Ca2+-calmodulin to eNOS, leading to enzyme activation. These findings may have broad implications for the regulation of Ca2+-dependent signal transduction in plasmalemmal caveolae.

Animals↗
PubMed1997

Regulation by cAMP of post-translational processing and subcellular targeting of endothelial nitric-oxide synthase (type 3) in cardiac myocytes.

Cardiac myocytes express the nitric-oxide synthase isoform originally identified in endothelial cells, termed eNOS or NOS3, where it plays a role in regulating myocyte responsiveness to both adrenergic and muscarinic cholinergic autonomic nervous system agonists. eNOS in endothelial cells has been shown to undergo extensive post-translational processing, and in cardiac myocytes as well as endothelial cells, eNOS has been shown to be targeted to plasmalemmal caveolae, a process that is dependent on myristoylation and palmitoylation. Other post-translational modifications essential for the correct subcellular targeting of eNOS have not been described previously. We demonstrate, using [35S]methionine pulse-chase experiments, that native eNOS in adult rat ventricular myocytes is initially translated as a nonpalmitoylated 150-kDa isoform, which is associated with cytosolic and intracellular membrane-enriched fractions. This is subsequently processed to a palmitoylated 135-kDa isoform, which is found only in a sarcolemma-enriched membrane fraction. Forskolin, an agent that elevates intracellular cAMP, rapidly inhibited processing of the 150-kDa isoform to the 135-kDa isoform and transport of eNOS to the sarcolemma, effects paralleled by protein kinase A-dependent phosphorylation of the larger eNOS isoform. Forskolin also decreased palmitoylation of the 135-kDa isoform, although it did not accelerate depalmitoylation of sarcolemmal eNOS, as determined by pulse-chase experiments with [3H]palmitate. Thus, post-translational processing of a 150-kDa isoform of myocyte eNOS appears to be necessary for intracellular trafficking of the enzyme to sarcolemmal caveolae. Both the post-translational processing and subcellular targeting of eNOS appear to be modified by changes in intracellular cAMP, an effect that may have important implications for cardiac myocyte responsiveness to autonomic agonists in vivo.

Acylation↗
PubMed1997

The role of palmitoyl-protein thioesterase in the palmitoylation of endothelial nitric oxide synthase.

Palmitoylation of eNOS is required for targeting to plasmalemmal caveolae and agonist-promoted depalmitoylation leads to eNOS translocation, modifying the agonist response. To date, one palmitoyl-protein thioesterase (PPT) has been purified and cloned. To explore the role of PPT in eNOS palmitoylation, we first established that PPT mRNA and protein are expressed in endothelial cells. Coexpression of PPT and eNOS in heterologous systems (COS and Sf-9 cells) resulted in a marked reduction in [3H]palmitate labeling of eNOS. We found, however, that co-expression did not alter subcellular targeting of eNOS, but that [3H]palmitate incorporation into cellular lipids, in particular palmitoyl-CoA, was significantly reduced. These results suggest that while PPT expression can significantly alter cellular lipid metabolism, it has no effect on eNOS palmitoylation.

Animals↗
PubMed1997

Ciliary neurotrophic factor (CNTF) genotypes: influence on choline acetyltransferase (ChAT) and acetylcholine esterase (AChE) activities and neurotrophin 3 (NT3) concentration in human post mortem brain tissue.

Cell culture and animal models suggest a significant influence of ciliary neurotrophic factor (CNTF) on cholinergic neurotransmitter systems. We therefore conducted an explorative pilot study to investigate the influence of a null mutation allele of the CNTF gene on ChAT (choline acetyltransferase) and AChE (acetylcholine esterase) activities in various regions of human post mortem brain tissue. Additionally, we determined NT3 (neurotrophin 3) levels, a factor which exhibits neurotrophic properties at cholinergic neurons, and the concentration of which in these brain regions varies with CNTF genotype. Homozygous carriers of the mutation lack CNTF completely, whereas heterozygotes have a CNTF level which is about half that of non-carriers. There was a trend toward lower ChAT and AChE activity levels in the cingulate cortex in individuals homozygous or heterozygous for the mutation when compared with non-mutant individuals. Additionally, higher NT3 concentrations were found in this region, as well as in the frontal cortex and caudate nucleus. ChAT and AChE activities in the frontal cortex and caudate nucleus were not significantly linked to CNTF genotype. These results are, however, preliminary and need to be further explored. The individuals investigated were heterogenous with respect to a range of parameters; nevertheless, the hypothesis that genetic variants for neurotrophic factors play a role in diseases of neural development and plasticity deserves further examination.

Acetylcholinesterase↗
PubMed1996

Endothelial nitric oxide synthase targeting to caveolae. Specific interactions with caveolin isoforms in cardiac myocytes and endothelial cells.

The endothelial isoform of nitric oxide synthase (eNOS) modulates cardiac myocyte function and is expressed in the particulate subcellular fraction. We have previously shown that eNOS is targeted to plasmalemmal caveolae in endothelial cells. Caveolae, specialized domains of the plasma membrane, may serve to sequester signaling proteins; a family of transmembrane proteins, the caveolins, form a key structural component of these microdomains. Caveolae in cardiac tissues contain the muscle-specific isoform caveolin-3, and caveolae in endothelial cells contain the widely expressed isoform caveolin-1, which shares limited sequence identity with caveolin-3. Our immunohistochemical analyses of rat cardiac muscle used isoform-specific caveolin antibodies to reveal prominent caveolin-3 staining in myocyte sarcolemmal membranes and at intercalated discs, whereas caveolin-1 staining was prominent in the vascular endothelium. Caveolin or eNOS antibodies were utilized to immunoprecipitate cardiac myocyte or cultured aortic endothelial cell lysates, which then were analyzed in immunoblots. In endothelial cells, we found that eNOS is quantitatively immunoprecipitated by antibodies to caveolin-1. In cardiac myocyte lysates, nearly all the eNOS is immunoprecipitated instead by antibodies to caveolin-3 and, conversely, eNOS antiserum immunoprecipitated primarily caveolin-3. These studies establish expression of eNOS in cardiac myocyte caveolae and document tissue-specific and quantitative associations of eNOS with caveolin. These findings may have important implications for the regulation of eNOS by caveolin isoforms and by other signaling proteins targeted to caveolae.

Animals↗
PubMed1996

Acylation targets emdothelial nitric-oxide synthase to plasmalemmal caveolae.

Endothelial nitric-oxide synthase (eNOS) generates the key signaling molecule nitric oxide in response to intralumenal hormonal and mechanical stimuli. We designed studies to determine whether eNOS is localized to plasmalemmal microdomains implicated in signal transduction called caveolae. Using immunoblot analysis, eNOS protein was detected in caveolar membrane fractions isolated from endothelial cell plasma membranes by a newly developed detergent-free method; eNOS protein was not found in noneaveolar plasma membrane. Similarly, NOS enzymatic activity was 9.4-fold enriched in caveolar membrane versus whole plasma membrane, whereas it was undetectable in non-caveolar plasma membrane. 51-86% of total NOS activity in postnuclear supernatant was recovered in plasma membrane, and 57-100% of activity in plasma membrane was recovered in caveolae. Immunoelectron microscopy showed that eNOS heavily decorated endothelial caveolae, whereas coated pits and smooth plasma membrane were devoid of gold particles. Furthermore, eNOS was targeted to caveolae in COS-7 cells transfected with wild-type eNOS cDNA. Studies with eNOS mutants revealed that both myristoylation and palmitoylation are required to target the enzyme to caveolae and that each acylation process enhances targeting by 10-fold. Thus, acylation targets eNOS to plasmalemmal caveolae. Localization to this microdomain is likely to optimize eNOS activation and the extracellular release of nitric oxide.

Acylation↗
PubMed1996

Explanted vein grafts with an intact endothelium demonstrate reduced focal expression of endothelial nitric oxide synthase specific to atherosclerotic sites.

The endothelial L-arginine:nitric oxide (NO) system is fundamental to vascular function. It is becoming evident that this system is compromised in aorto-coronary vein grafts, although it is not clear how it is affected. It was postulated that the development of intimal lesions in vein grafts may be associated with reduced expression or loss of endothelial NO synthase (eNOS). The immunocytochemical localization and quantitative expression of eNOS were therefore investigated in normal human saphenous veins (n = 6) and explanted vein grafts (n = 6). The vein grafts demonstrated marked morphological changes evident as fibro-intimal hyperplasia (FIH) and focal sites of atherosclerosis, often occurring along the same length of graft. Staining for eNOS was abundantly evident in the endothelium of normal veins but revealed a differential reduction in staining intensity in vein grafts. Staining intensity measurements revealed a significant reduction (P < 0.001) in the amount of eNOS present in areas of atherosclerosis as compared with normal veins and areas of vein graft with FIH changes alone. This reduction in the relative quantity of antigen was specific to eNOS, since the endothelial markers von Willebrand factor (vWf) and CD31 showed no such variations. These data support the view that vascular activity of NO is impaired in atherosclerosis and indicate that reduced expression of eNOS, and therefore by inference lower NO production, may make an important contribution to this phenomenon.

Arteriosclerosis↗
PubMed1996

Mapping protein-protein contact sites using cellulose-bound peptide scans.

We have characterized the interaction of two monoclonal antibodies with their respective antigens using cellulose-bound sets of overlapping peptides (peptide scans). Both antibodies CB/RS/5 and CB/MT/1 recognize discontinuous epitopes present in human interleukin-10 (IL-10) and tumor necrosis factor alpha (TNF-alpha). In addition, the interaction between TNF-alpha and its 55-kDa receptor (TNF-R) was investigated by the same approach. Both antibodies, as well as TNF-alpha, interacted with two or more regions of the peptide scans. Antibody-binding competition studies between the native antigens and peptides, covering single parts of the binding regions, enabled us to distinguish between binding to the paratope or other regions of the antibody. The combination of these experimental approaches allowed the identification of short antigen-derived sequences that are separated on the primary sequence but close in space on the surface of IL-10 and TNF-alpha, thus representing putative discontinuous epitopes. In the case of the TNF-R-derived peptide scans, two of the identified regions interact with the structurally similar TNF-beta in the TNF-beta-TNF-R complex. These data indicate that this approach should be generally applicable for mapping nonlinear protein-protein contact sites.

Amino Acid Sequence↗
PubMed1996

The role of the NO pathway in the control of cardiac function.

Nitric oxide (NO) acts as an autocrine- and paracrine-acting signaling autacoid that, among other functions, has been shown to regulate cardiac contractile responsiveness to beta-adrenergic and muscarinic cholinergic agonists. Nitric oxide (NO) is formed by the oxidation of one of two equivalent guanidino nitrogens in L-arginine by O2 to form NO and L-citrulline. This reaction is catalyzed by a family of enzymes termed NO synthases. Three distinct isoforms of NOS have been identified, each the product of a separate gene. Cellular constituents of cardiac muscle, including ventricular myocytes as well as microvascular endothelial cells, have been shown to express the "endothelial constitutive" isoform of NO synthase (ecNOS or NOS3) in vivo, and both cell types also express the NO synthase isoform induced by specific inflammatory cytokines (iNOS or NOS2) in vivo and in vitro. While NO-dependent intracellular signalling in cardiac myocytes clearly involves the activation of guanylate cyclase and downstream signalling by cGMP, there is accumulating evidence that non-cGMP-dependent regulatory signalling events are also initiated by NO. In addition, decreased contractile responsiveness of cardiac myocytes to beta-adrenergic agonists, following induction of NOS2 by inflammatory cytokines, requires the presence of insulin and the co-induction of enzymes responsible for production of tetrahydrobiopterin, a NOS co-factor. Inappropriate or excessive production of NO by cardiac myocytes and by microvascular endothelial cells likely contributes to the cardiac contractile dysfunction characteristic of the systemic inflammatory response syndrome and cardiac allograft rejection.

Animals↗
PubMed1995

Mutagenesis of palmitoylation sites in endothelial nitric oxide synthase identifies a novel motif for dual acylation and subcellular targeting.

The endothelial nitric oxide synthase (ec-NOS) plays a key role in the transduction of signals from the bloodstream to the underlying smooth muscle. ecNOS undergoes a complex series of covalent modifications, including myristoylation and palmitoylation, which appear to play a role in ecNOS membrane association. Mutagenesis of the myristoylation site, which prevents both myristoylation and palmitoylation, blocks ecNOS targeting to cell membranes. Further, as described for some G-protein alpha subunits, both membrane association and palmitoylation of ecNOS are dynamically regulated: in response to agonists, the enzyme undergoes partial redistribution to the cell cytosol concomitant with depalmitoylation. To clarify the role of palmitoylation in determining ecNOS subcellular localization, we have constructed palmitoylation-deficient mutants of ecNOS. Serine was substituted for cysteine at two potential palmitoylation sites (Cys-15 and Cys-26) by site-directed mutagenesis. Immunoprecipitation of ecNOS mutants following cDNA transfection and biosynthetic labeling with [3H]palmitate revealed that mutagenesis of either cysteine residue attenuated palmitoylation, whereas replacement of both residues completely eliminated palmitoylation. Analysis of N-terminal deletion mutations of ecNOS demonstrated that the region containing these two cysteine residues is both necessary and sufficient for enzyme palmitoylation. The cysteines thus identified as the palmitoylation sites for ecNOS are separated by an unusual (Gly-Leu)5 sequence and appear to define a sequence motif for dual acylation. We analyzed the subcellular distribution of ecNOS mutants by differential ultracentrifugation and found that mutagenesis of the ecNOS palmitoylation sites markedly reduced membrane association of the enzyme. These results document that ecNOS palmitoylation is an important determinant for the subcellular distribution of ecNOS and identify a new motif for the reversible palmitoylation of signaling proteins.

Acylation↗