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Biomedical subjects

O Feron

Publications and source records attributed to O Feron.

At least 37 records · Page 2Linked 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

Thyroid status and postnatal changes in subsarcolemmal distribution and isoform expression of rat cardiac dihydropyridine receptors.

OBJECTIVE: The aim was to analyze the early postnatal changes in myocardial density, subsarcolemmal localization and isoform expression of dihydropyridine receptors in rat ventricle and the influence of thyroid status on these changes. METHODS: Newborn rats were treated from postnatal day 2 with L-triiodothyronine (T3) or 6-n-propyl-2-thiouracil )PTU) and ventricles were collected on day 1, 7 and 14. Radioligand binding and cell fractionation (density gradient centrifugation) techniques were used to determine the tissue density of various receptors and their subcellular localization. To analyze dihydropyridine receptor alpha 1 subunit isoform expression, cDNA fragments corresponding to a large portion of motif IV were amplified by reverse transcriptase-polymerase chain reaction and treated with appropriate restriction endonucleases to determine the frequency of splicing events at the level of motif IV. RESULTS: The myocardial density of dihydropyridine receptors increased 3-fold from day 1 to day 14 in control rats, and this increase occurred predominantly in membrane entities equilibrating at high densities in sucrose gradient, that is, presumably, in junctional structures (dyadic couplings). This maturation was delayed after PTU-treatment, and somewhat accelerated by excess T3. The proportion of mRNA variants typical of foetal heart (IVS3A variant and 'deleted' variant, showing a 33-nucleotide deletion at the level of the extracellular loop between IVS3 and IVS4) decreased with age in control rats. This reduction was delayed after treatment with PTU but was not influenced by excess T3. CONCLUSION: Hypothyroidism impaired the early postnatal maturation of dihydropyridine receptors as regards both their concentration into junctional structures and the decrease in the relative expression of alpha 1-subunit mRNA variants typical of foetal heart.

Analysis of Variance↗
PubMed1998

The action of calcium channel blockers on recombinant L-type calcium channel alpha1-subunits.

1. CHO cells expressing the alpha(1C-a) subunit (cardiac isoform) and the alpha(1C-b) subunit (vascular isoform) of the voltage-dependent L-type Ca2+ channel were used to investigate whether tissue selectivity of Ca2+ channel blockers could be related to different affinities for alpha1C isoforms. 2. Inward current evoked by the transfected alpha1 subunit was recorded by the patch-clamp technique in the whole-cell configuration. 3. Neutral dihydropyridines (nifedipine, nisoldipine, (+)-PN200-110) were more potent inhibitors of alpha(1C-)b-subunit than of alpha(1C-a)-subunit. This difference was more marked at a holding potential of -100 mV than at -50 mV. SDZ 207-180 (an ionized dihydropyridine) exhibited the same potency on the two isoforms. 4. Pinaverium (ionized non-dihydropyridine derivative) was 2 and 4 fold more potent on alpha(1C-a) than on alpha(1C-b) subunit at Vh of -100 mV and -50 mV, respectively. Effects of verapamil were identical on the two isoforms at both voltages. 5. [3H]-(+)-PN 200-110 binding experiments showed that neutral dihydropyridines had a higher affinity for the alpha(1C-b) than for the alpha(1C-a) subunit. SDZ 207-180 had the same affinity for the two isoforms and pinaverium had a higher affinity for the alpha(1C-a) subunit than for the alpha(1C-b) subunit. 6. These results indicate marked differences among Ca2+ channel blockers in their selectivity for the alpha(1C-a) and alpha(1C-b) subunits of the Ca2+ channel.

Animals↗
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↗
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

Action of the calcium channel blocker lacidipine on cardiac hypertrophy and endothelin-1 gene expression in stroke-prone hypertensive rats.

1. The tissue-protective effects of calcium channel blockers in hypertension are not well dissociated from their effect on systolic blood pressure (SBP). We have previously shown that lacidipine, a dihydropyridine-type calcium antagonist, reduced the cardiac hypertrophy and the cardiac endothelin-1 (ET-1) gene overexpression occurring in salt-loaded stroke-prone spontaneously hypertensive rats (SL-SHRSP), an effect occurring without systolic blood pressure (SBP) change. In the present study, we have examined whether this action was dose-related and if it could be associated with ET receptor changes. The action of lacidipine was also examined in control SHRSP and in Wistar Kyoto rats (WKY). 2. The daily dose of 0.3 mg kg-1 lacidipine which did not lower SBP but significantly prevented ventricle hypertrophy and cardiac preproET-1-mRNA expression in SL-SHRSP was inactive in control SHRSP. With the higher dose of lacidipine (1 mg kg-1 day-1), we observed a further reduction of cardiac hypertrophy and of ET-1 gene expression in SL-SHRSP and a significant effect on those parameters in control SHRSP but only a small reduction of SBP in both groups. 3. In WKY, salt loading did not induce change in SBP or increase of cardiac ET-1 gene expression and ventricle mass. In these normotensive rats, lacidipine (1 mg kg-1 day-1) did not modulate the basal preproET-1-mRNA expression and did not affect SBP or heart weight. 4. The maximum binding capacity (Bmax) and the dissociation constant (KD) of [125I]-ET-1 binding and the relative proportion of low- and high-affinity binding sites for ET-3 were not significantly affected by salt loading or lacidipine treatment in SHRSP. 5. These results show that lacidipine exerted a dose-related inhibition of ventricle hypertrophy and preproET-1-mRNA expression in SHRSP and indicate that this effect was unrelated to SBP changes. The dose-dependency of this inhibition suggests that salt-induced cardiac hypertrophy could be related to ET-1 gene overexpression. The results further show that ET receptor changes are not involved in the pathophysiological process studied here.

Animals↗
PubMed1995

Inhibition by lacidipine of salt-dependent cardiac hypertrophy and endothelin gene expression in stroke-prone spontaneously hypertensive rats.

Stroke-prone spontaneously hypertensive rats receiving a high salt diet were orally treated by the calcium antagonist lacidipine, at a dose which did not reduce systolic blood pressure. We observed that lacidipine inhibited the salt-induced cardiac hypertrophy and the concomitant increase of mRNA transcripts for preproendothelin-1 in ventricles. These data show that elevated blood pressure cannot necessarily account for cardiac hypertrophy and indicate that the therapeutic action of lacidipine is not only related to its haemodynamic properties, but also to the inhibition of the gene expression of growth factors such as endothelin.

Age Factors↗
PubMed1995

Influence of salt loading on the cardiac and renal preproendothelin-1 mRNA expression in stroke-prone spontaneously hypertensive rats.

Endothelin-1 is a potent vasoactive peptide which may play a role in the regulation of vascular resistance through its autocrine/paracrine effects. We have investigated the influence of salt loading on the renal and cardiac production of endothelin-1 in stroke prone spontaneously hypertensive rats, a classical model of hypertension. The results show that the dietary salt intake did not change systolic blood pressure nor the renal expression of the preproendothelin-1 mRNA but increased cardiac expression of the endothelin-1 gene transcript and a concomitant ventricular hypertrophy.

Animals↗
PubMed1995

Interaction of fluorescein derivatives with glibenclamide binding sites in rat brain.

In rat brain, [3H]glibenclamide binds with high affinity to sulfonylurea receptors associated with ATP-sensitive potassium (KATP) channels. KATP channels may play a modulatory role in neurotransmitter release and are involved in acute pathological events occurring in the brain. Fluorescein derivatives, which are suitable tools for the labelling of nucleotide binding sites, influence KATP channels and sulfonylurea receptors properties in insulinoma and cardiac cells. In this study, a negative allosteric action of fluorescein derivatives on glibenclamide binding sites has been shown in rat cortical neurons. This supports the hypothesis of interactions between nucleotide- and sulfonylurea-binding sites within the sulfonylurea receptor.

Animals↗
PubMed1995

Regulation of the L-type calcium channel alpha-1 subunit by chronic depolarization in the neuron-like PC12 and aortic smooth muscle A7r5 cell lines.

The regulation of L-type voltage-dependent Ca2+ channels by chronic depolarization was studied in the aortic smooth muscle A7r5 and neuron-type PC12 cell lines, by probing the expression and the functional state of their constitutive alpha-1 subunits. PC12 cells showed, after prolonged exposure to a high-K+ depolarizing solution, a 25% reduction of the functional Ca2+ channel density which was accompanied by a decrease of the alpha-1 subunit mRNA expression. In A7r5 cells submitted to a similar protocol of depolarization, 45Ca2+ uptake measurements revealed a fall in the functional activity of L-type Ca2+ channels which was not related to a modulation of their mRNA expression, but arose from a long-term voltage-dependent channel inactivation. Accordingly, the lag time and the mechanisms of recovery were different in the two cell types. In PC12 cells, when restoring physiological culture conditions, de novo synthesis of alpha-1 subunits allowed the recovery of the original density of L-type Ca2+ channels at the membrane surface. As for the A7r5 cells, we showed that after chronic depolarization, the complete restoration of the resting membrane potential and the related Ca2+ channel activity required a 2-day incubation in physiological medium and could probably be related to a normalization of the increased intracellular Ca2+ concentration. In contrast, it is noteworthy that, in PC12 cells, the only transient increase of intracellular Ca2+ content in the first hours of depolarization could account for the long-term down-regulation of L-type Ca2+ channels.

Adenosine Triphosphate↗
PubMed1995

Blood pressure-independent inhibition by lacidipine of endothelin-1-related cardiac hypertrophy in salt-loaded, stroke-prone spontaneously hypertensive rats.

We investigated the influence of salt loading on the renal and cardiac production of endothelin-1 (ET-1) in stroke-prone, spontaneously hypertensive rats (SHR-SP). The results show that the dietary salt intake did not change systolic blood pressure or the renal expression of the prepro-ET-1 mRNA but increased cardiac expression of the ET-1 gene transcript with concomitant ventricular hypertrophy. These changes were prevented by oral treatment with lacidipine, a long-lasting calcium antagonist, at a dose that did not reduce systolic blood pressure. This indicates that the cardioprotective properties of lacidipine may be dissociated from its blood pressure-lowering effect and could be related to inhibition of endothelin gene expression.

Animals↗
PubMed1994

Quantification of two splicing events in the L-type calcium channel alpha-1 subunit of intestinal smooth muscle and other tissues.

cDNA fragments encoding a representative region of the L-type calcium channel alpha-1 subunit of rabbit intestine smooth muscle were amplified by polymerase chain reaction (PCR). The nucleotide sequences of these intestine clones shared a high similarity with aorta, lung and heart calcium channels. However, in the extracellular loop between the third and fourth segments of domain IV and in the transmembrane IVS3 segment itself, we observed primary sequence variations corresponding to alternative splicing phenomenons. Since structural differences of L-type calcium channel alpha-1 subunits could result in functional variations, the respective expression frequency of these isoforms was determined in various tissues and species, and in the embryonic A7r5 cell line. The ontogeny of these splicing events was also examined from tissues of different ages. From this quantitative study, carried out by PCR of reverse-transcribed mRNA, it clearly appears that the observed splicing processes in the IVS3-IVS4 region are not only tissue-dependent but also regulated during development.

Amino Acid Sequence↗