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M Bouvier

Publications and source records attributed to M Bouvier.

At least 37 records · Page 2Linked to original sources
PubMed2000

Newly synthesized human delta opioid receptors retained in the endoplasmic reticulum are retrotranslocated to the cytosol, deglycosylated, ubiquitinated, and degraded by the proteasome.

We have previously shown that only a fraction of the newly synthesized human delta opioid receptors is able to leave the endoplasmic reticulum (ER) and reach the cell surface (Petäjä-Repo, U. E, Hogue, M., Laperrière, A., Walker, P., and Bouvier, M. (2000) J. Biol. Chem. 275, 13727-13736). In the present study, we investigated the fate of those receptors that are retained intracellularly. Pulse-chase experiments revealed that the disappearance of the receptor precursor form (M(r) 45,000) and of two smaller species (M(r) 42,000 and 39,000) is inhibited by the proteasome blocker, lactacystin. The treatment also promoted accumulation of the mature receptor form (M(r) 55,000), indicating that the ER quality control actively routes a significant proportion of rescuable receptors for proteasome degradation. In addition, degradation intermediates that included full-length deglycosylated (M(r) 39,000) and ubiquitinated forms of the receptor were found to accumulate in the cytosol upon inhibition of proteasome function. Finally, coimmunoprecipitation experiments with the beta-subunit of the Sec61 translocon complex revealed that the receptor precursor and its deglycosylated degradation intermediates interact with the translocon. Taken together, these results support a model in which misfolded or incompletely folded receptors are transported to the cytoplasmic side of the ER membrane via the Sec61 translocon, deglycosylated and conjugated with ubiquitin prior to degradation by the cytoplasmic 26 S proteasomes.

Cell Line↗
PubMed2000

Export from the endoplasmic reticulum represents the limiting step in the maturation and cell surface expression of the human delta opioid receptor.

Synthesis and maturation of G protein-coupled receptors are complex events that require an intricate combination of processes that include protein folding, post-translational modifications, and transport through distinct cellular compartments. Relatively little is known about the nature and kinetics of specific steps involved in these processes. Here, the human delta opioid receptor expressed in human embryonic kidney 293S cells is used as a model to delineate these steps and to establish the kinetics of receptor synthesis, glycosylation, and transport. We found that the receptor is synthesized as a core-glycosylated M(r) 45,000 precursor that is converted to the fully mature M(r) 55,000 receptor with a half-time of about 120 min. In addition to trimming and processing of two N-linked oligosaccharides, maturation involves addition of O-glycans containing N-acetylgalactosamine, galactose, and sialic acid. In contrast to N-glycosylation, which is initiated co-translationally and is completed when the protein reaches the trans-Golgi network, O-glycosylation was found to occur only after the receptor exits from the endoplasmic reticulum (ER) and was terminated as early as the trans-Golgi cisternae. Once the carbohydrates are fully processed and the receptor reaches the trans-Golgi network, it is transported to the cell surface in about 10 min. The exit from the ER was found to be the limiting step in overall processing of the receptor. This indicates that early events in the folding of the receptor are probably rate-limiting and that receptor folding intermediates are retained in the ER until they can adopt the correct conformation. The overall low efficiency of receptor maturation, less than 50% of the precursor being processed to the fully glycosylated protein, further suggests that only a fraction of the synthesized receptors attain properly folded conformation that allows exit from the ER. This indicates that folding and ER export are key events in control of receptor cell surface expression. Whether or not the low efficiency of the ER export is a general feature among G protein-coupled receptors remains to be investigated.

Biological Transport↗
PubMed2000

Detection of beta 2-adrenergic receptor dimerization in living cells using bioluminescence resonance energy transfer (BRET).

Heptahelical receptors that interact with heterotrimeric G proteins represent the largest family of proteins involved in signal transduction across biological membranes. Although these receptors generally were believed to be monomeric entities, a growing body of evidence suggests that they may form functionally relevant dimers. However, a definitive demonstration of the existence of G protein-coupled receptor (GPCR) dimers at the surface of living cells is still lacking. Here, using bioluminescence resonance energy transfer (BRET), as a protein-protein interaction assay in whole cells, we unambiguously demonstrate that the human beta(2)-adrenergic receptor (beta(2)AR) forms constitutive homodimers when expressed in HEK-293 cells. Receptor stimulation with the hydrophilic agonist isoproterenol led to an increase in the transfer of energy between beta(2)AR molecules genetically fused to the BRET donor (Renilla luciferase) and acceptor (green fluorescent protein), respectively, indicating that the agonist interacts with receptor dimers at the cell surface. Inhibition of receptor internalization did not prevent agonist-promoted BRET, demonstrating that it did not result from clustering of receptors within endosomes. The notion that receptor dimers exist at the cell surface was confirmed further by the observation that BS3, a cell-impermeable cross-linking agent, increased BRET between beta(2)AR molecules. The selectivity of the constitutive interaction was documented by demonstrating that no BRET occurred between the beta(2)AR and two other unrelated GPCR. In contrast, the well characterized agonist-dependent interaction between the beta(2)AR and the regulatory protein beta-arrestin could be monitored by BRET. Taken together, the data demonstrate that GPCR exist as functional dimers in vivo and that BRET-based assays can be used to study both constitutive and hormone-promoted selective protein-protein interactions.

Animals↗
PubMed2000

Characterization of lipopolysaccharide-induced emesis in conscious piglets: effects of cervical vagotomy, cyclooxygenase inhibitors and a 5-HT(3) receptor antagonist.

The emetic response to intraperitoneal (i.p., 0.5, 2, 8 mg kg(-1)) and intravenous (i.v., 200 microg kg(-1)) administration of bacterial lipopolysaccharides (LPS) was characterized in conscious piglets observed for 4 h. The latencies and the incidence of the emetic response to LPS (i.p.) decreased and increased, respectively, in a dose-dependent manner. In 14 additional piglets, a bilateral vagotomy performed 4 h prior to LPS administration abolished the vomiting induced by i.p. LPS (2 mg kg(-1)), and decreased its incidence by 77% in the i.v. injected animals. Sham-operated animals (n=6) exhibited a similar emetic pattern to the controls injected intraperitoneally with LPS (2 mg kg(-1)). In 7 piglets, the administration of granisetron, a 5-HT(3) receptor antagonist (i.v., 2 mg kg(-1)), 30 min prior to the i.p. LPS injection (2 mg kg(-1)) failed to reduce significantly the emetic activity; whereas, in 6 animals, a combination of meloxicam (0.3 mg kg(-1)) and indomethacin (5 mg kg(-1)), two cyclooxygenase (COX) inhibitors, administered per os 1.5 h prior to the i.p. LPS (2 mg kg(-1)) abolished the emetic response to endotoxins. The present results show that the activation of the medullary "vomiting centre" in response to i.p. administration of LPS is mediated via vagal afferents and is likely to involve prostaglandins.

Animals↗
PubMed2000

Pharmacological chaperones: a new twist on receptor folding.

Protein misfolding is at the root of several genetic human diseases. These diseases do not stem from mutations within the active domain of the proteins, but from mutations that disrupt their three-dimensional conformation, which leads to their intracellular retention by the quality control apparatus of the cell. Facilitating the escape of the mutant proteins from the quality control system by lowering the temperature of the cells or by adding chemicals that assist folding (chemical chaperones) can result in proteins that are fully functional despite their mutation. The discovery that ligands with pharmacological selectivity (pharmacological chaperones) can rescue the proper targeting and function of misfolded proteins, including receptors, might help to develop new treatments for 'conformational diseases'.

Animals↗
PubMed2000

Functional significance of oligomerization of G-protein-coupled receptors.

In contrast to other families of cell surface receptors for which dimerization is an integral part of the activation process, G-protein-coupled receptors (GPCRs) were thought, until recently, to function as monomeric units. However, a growing body of evidence indicates that GPCRs could exist and be active as oligomeric complexes. Because they are major pharmacological targets, their existence as homo- or heterodimers could have important implications for the development and screening of new drugs.

Animals↗
PubMed2000

Dynamic anal endosonography may challenge defecography for assessing dynamic anorectal disorders: results of a prospective pilot study.

BACKGROUND AND STUDY AIMS: The diagnosis of anorectal dynamic disorders and perineal insufficiency has been mainly based up to now on the use of defecography. Here, we performed a prospective blinded study to evaluate the accuracy of a new procedure, dynamic anorectal endosonography (DAE), by comparing it with defecography. PATIENTS AND METHODS: A total of 43 women (mean age 53), presenting with outlet obstruction, were prospectively enrolled in our study. The DAE was performed with a 7.5-Mhz linear probe (Toshiba, Tokyo, Japan) with basal and straining recordings. None of the DAE or defecography operators was informed about the results of the respective other investigation. RESULTS: Defecography showed a descending perineum in 29 patients (68%), a rectocele in 25 patients (58%), and a rectal intussusception in eight patients (18%). The rate of concordance between the diagnosis of descending urinary bladder at DAE and descending perineum at defecography was 35/43 (80%), and that between the descending puborectal muscle at DAE and descending perineum at defecography was 40/43 (93 %). The rates of concordance between DAE and defecography for the diagnosis of rectocele and rectal intussusception were 27/43 patients (57%) and 34/43 patients (80%), respectively. In the last 15 patients, the DAE technique was adapted in order to improve the accuracy for diagnosing rectocele by filling the rectum with water. This improved sensitivity from 36 to 86% and accuracy from 57 to 87%. CONCLUSIONS: DAE is a new imaging approach to anorectal dynamic disorders, providing a highly reliable means of diagnosing perineal insufficiency as well as rectocele. DAE should be substituted for previous methods since it makes it possible at the same time to assess the anal sphincters and to avoid pelvic irradiation.

Adult↗
PubMed2000

Pharmacological chaperones rescue cell-surface expression and function of misfolded V2 vasopressin receptor mutants.

Over 150 mutations within the coding sequence of the V2 vasopressin receptor (V2R) gene are known to cause nephrogenic diabetes insipidus (NDI). A large number of these mutant receptors fail to fold properly and therefore are not routed to the cell surface. Here we show that selective, nonpeptidic V2R antagonists dramatically increase cell-surface expression and rescue the function of 8 mutant NDI-V2Rs by promoting their proper folding and maturation. A cell-impermeant V2R antagonist could not mimic these effects and was unable to block the rescue mediated by a permeant agent, indicating that the nonpeptidic antagonists act intracellularly, presumably by binding to and stabilizing partially folded mutants. In addition to opening new therapeutic avenues for NDI patients, these data demonstrate that by binding to newly synthesized mutant receptors, small ligands can act as pharmacological chaperones, promoting the proper folding and maturation of receptors and their targeting to the cell surface.

Animals↗
PubMed1999

Activation of the beta(2)-adrenergic receptor-Galpha(s) complex leads to rapid depalmitoylation and inhibition of repalmitoylation of both the receptor and Galpha(s).

Palmitoylation is unique among lipid modifications in that it is reversible. In recent years, dynamic palmitoylation of G protein alpha subunits and of their cognate receptors has attracted considerable attention. However, very little is known concerning the acylation/deacylation cycle of the proteins in relation to their activity status. In particular, the relative contribution of the activation and desensitization of the signaling unit to the regulation of the receptors and G proteins palmitoylation state is unknown. To address this issue, we took advantage of the fact that a fusion protein composed of the stimulatory alpha subunit of trimeric G protein (Galpha(s)) covalently attached to the beta(2)-adrenergic receptor (beta(2)AR) as a carboxyl-terminal extension (beta(2)AR-Galpha(s)) can be stimulated by agonists but does not undergo rapid inactivation, desensitization, or internalization. When expressed in Sf9 cells, both the receptor and the Galpha(s) moieties of the fusion protein were found to be palmitoylated via thioester linkage. Stimulation with the beta-adrenergic agonist isoproterenol led to a rapid depalmitoylation of both the beta(2)AR and Galpha(s) and inhibited repalmitoylation. The extent of depalmitoylation induced by a series of agonists was correlated (0.99) with their intrinsic efficacy to stimulate the adenylyl cyclase activity. However, forskolin-stimulated cAMP production did not affect the palmitoylation state of beta(2)AR-Galpha(s), indicating that the agonist-promoted depalmitoylation is linked to conformational changes and not to second messenger generation. Given that, upon activation, the fusion protein mimics the activated receptor-G protein complex but cannot undergo desensitization, the data demonstrate that early steps in the activation process lead to the depalmitoylation of both receptor and G protein and that repalmitoylation requires later events that cannot be accommodated by the activated fusion protein.

Animals↗
PubMed1999

Beta(2)-adrenergic receptor down-regulation. Evidence for a pathway that does not require endocytosis.

Sustained activation of most G protein-coupled receptors causes a time-dependent reduction of receptor density in intact cells. This phenomenon, known as down-regulation, is believed to depend on a ligand-promoted change of receptor sorting from the default endosome-plasma membrane recycling pathway to the endosome-lysosome degradation pathway. This model is based on previous studies of epidermal growth factor (EGF) receptor degradation and implies that receptors need to be endocytosed to be down-regulated. In stable clones of L cells expressing beta(2)-adrenergic receptors (beta(2)ARs), sustained agonist treatment caused a time-dependant decrease in both beta(2)AR binding sites and immuno-detectable receptor. Blocking beta(2)AR endocytosis with chemical treatments or by expressing a dominant negative mutant of dynamin could not prevent this phenomenon. Specific blockers of the two main intracellular degradation pathways, lysosomal and proteasome-associated, were ineffective in preventing beta(2)AR down-regulation. Further evidence for an endocytosis-independent pathway of beta(2)AR down-regulation was provided by studies in A431 cells, a cell line expressing both endogenous beta(2)AR and EGF receptors. In these cells, inhibition of endocytosis and inactivation of the lysosomal degradation pathway did not block beta(2)AR down-regulation, whereas EGF degradation was inhibited. These data indicate that, contrary to what is currently postulated, receptor endocytosis is not a necessary prerequisite for beta(2)AR down-regulation and that the inactivation of beta(2)ARs, leading to a reduction in binding sites, may occur at the plasma membrane.

Adrenergic beta-2 Receptor Agonists↗
PubMed1999

Nitric oxide modulates beta(2)-adrenergic receptor palmitoylation and signaling.

To determine whether nitric oxide (NO) modulates the beta-adrenergic signaling pathway, we treated cells expressing beta(2)-adrenergic receptors (beta(2)AR) with the NO donors, 3-morpholinosydnonimine (SIN-1) and 1,2,3,4-oxatriazolium, 5-amino-3-(3-chloro-2-methylphenyl)chloride and determined the intracellular production of cAMP after exposure to beta-adrenergic receptor agonists, cholera toxin and forskolin. NO significantly decreased the potency of the beta-adrenergic agonist, isoproterenol, to stimulate cAMP production without affecting the stimulatory action of forskolin and cholera toxin, which directly activate adenylyl cyclase and G(s), respectively. Treatment with the NO donor increased the guanyl nucleotide-sensitive high affinity constant for the agonist, isoproterenol, thus suggesting that it reduced functional coupling between the receptor and G(s). Stimulation of endogenous NO production by lipopolysaccharide in RAW 264.7 macrophages also caused a significant increase in the EC(50) for isoproterenol-stimulated cAMP production. SIN-1 treatment also led to a reduction in both basal and isoproterenol-stimulated incorporation of [(3)H]palmitate into the beta(2)AR. Signaling through the nonpalmitoylated, Gly(341)beta(2)AR mutant was unchanged by SIN-1 treatment. Given the link between beta(2)AR palmitoylation and its responsiveness to agonist, these results suggest that the primary action of NO was depalmitoylation of the beta(2)AR resulting in decreased signaling through the beta(2)AR.

Adenylyl Cyclases↗
PubMed1999

Sleep and wakefulness modulation of the neuronal firing in the auditory cortex of the guinea pig.

Sleep-related changes-including modification in sensory processing-that influence brain and body functions, occur during both slow wave and paradoxical sleep. Our aim was to investigate how cortical auditory neurons behave during the sleep/waking cycle, and to study cell firing patterns in relation to the processing of auditory information without the interference of anesthetic drugs. We recorded single cells in the A region of the auditory cortex in restrained, chronically-implanted guinea pigs, and compared their evoked and spontaneous activity during sleep stages and quiet wakefulness. A new classification of the unit's responses to simple sound during wakefulness is presented. Moreover, a number of the neurons in the primary auditory cortex exhibited significant quantitative changes in their evoked or spontaneous firing rates. These changes could be correlated to sleep stages or wakefulness in 42.2% to 58.3% of the sampled neurons. A similar population did not show behavioral related changes in firing rates. Our results indicate that the responsiveness of the auditory system during sleep may be considered partially preserved. An important result was that spontaneous and evoked activity may vary in opposite directions, i.e. , the evoked activity could increase while the spontaneous activity decrease or vice versa. Then, a general question was proposed: is the increased spontaneous activity in the auditory cortex, particularly during PS, related to auditory hypnic 'images'? The studied cortical auditory neurons exhibit changes in their firing rates in correlation to stages of sleep and wakefulness. This is consistent with the hypothesis that a general shift in the neuronal networks involved in sensory processing occurs during sleep.

Acoustic Stimulation↗
PubMed1999

Comparative binding study of rat natriuretic peptide receptor-A.

The natriuretic peptide receptor-A (NPR-A) is involved in blood pressure and body fluid regulation in order to help maintain cardiovascular homeostasis. It has been shown that these biological effects are mediated through the natriuretic peptide family of hormones, which bind NPR-A according to the rank order ANP>BNP>>CNP. Previous studies performed with rat kidney papillary tissue suggested the existence of an heterologous NPR-A population since two binding components were obtained for pBNP32, one of high affinity (pK 9.4 +/- 0.1) and the other of lower affinity (pK 7.5 +/- 0.1), while in the same preparation rANP28 binding displayed the expected affinity (pK 10.22 +/- 0.01) and was best fitted with a model involving a single class of binding sites. This apparent heterogeneity of NPR-A in rat kidney papillae could be explained by the presence of two receptor isoforms or of monomeric and oligomeric forms of the same receptor. To investigate the NPR-A binding heterogeneity, we have cloned the rat NPR-A from PC12 cells and compared its pharmacological profile with that of the papillae. Our results with rat NPR-A transfected Cos-P cells show an equivalent pharmacological profile as with the rat tissue, i.e. a high affinity for rANP28 (pK 10.4 +/- 0.1) and two distinctive affinities for pBNP32 (pK 9.74 +/- 0.05 and 7.8 +/- 0.1). Although multiple receptor glycoforms were sometimes detectable by western blotting, only one molecular form was obtained by cross-linking with 125I-rANP28. It thus appears that NPR-A alone can account for the two binding components found in the rat papillae and that a single molecular form of the protein is implicated. We therefore propose that the oligomerization state of the receptors could be responsible for the apparent binding heterogeneity of rat NPR-A.

Amino Acid Sequence↗
PubMed1999

Effects of alverine citrate on cat intestinal mechanoreceptor responses to chemical and mechanical stimuli.

BACKGROUND: Alverine citrate is commonly used in the treatment of painful affections of the colon. AIM: To determine whether alverine citrate acts on the vagal sensory endings. METHODS: Unitary recordings were performed at the level of the vagal fibres in the nodose ganglion of anaesthetized cats using extracellular glass microelectrodes, and the patterns of response to chemical and mechanical stimuli applied to identified vagal intestinal mechanoreceptors were studied. RESULTS: The intestinal mechanoreceptors located at the endings of type C vagal fibres responded mainly to mechanical stimuli (distension and contraction), but also responded to chemical substances (cholecystokinin and substance P). The most conspicuous effect of alverine (2 mg/kg) was that it significantly inhibited the pattern of vagal activity produced in response to either cholecystokinin (5-10 microg/kg), substance P (5-10 microg/kg) or phenylbiguanide (5-10 microg/kg), a 5-HT3 receptor agonist. On the other hand, the unitary vagal response to the mechanical distension was slightly enhanced by alverine, as was any spontaneous activity present. CONCLUSIONS: Based on the present data, alverine citrate can be said to decrease the sensitivity of the intestinal mechanoreceptors, which is consistent with its previously established anti-spasmodic effects.

Animals↗
PubMed1999

Evolution of the hepatitis C virus second envelope protein hypervariable region in chronically infected patients receiving alpha interferon therapy.

Sustained hepatitis C virus (HCV) RNA clearance is achieved in 8 to 12% of patients with chronic HCV infection treated with alpha interferon (IFN-alpha) at the approved dose of 3 MU three times a week for 6 months and in about 25% of those receiving this treatment for 12 months. We used single-strand conformation polymorphism analysis combined with cloning and sequencing strategies to characterize the genetic evolution of HCV second envelope gene hypervariable region 1 (HVR1) quasispecies during and after IFN therapy in patients who failed to clear HCV RNA. Sustained HCV RNA clearance was achieved in 6% of patients. Profound changes in HVR1 quasispecies major variants were estimated to occur in 70% of the patients during and after therapy. These changes were evolutionary and were characterized by shifts in the virus population, related to selection and subsequent diversification of minor pretreatment variants. The quasispecies changes appeared to be induced by changes in the host environment likely resulting from the IFN-induced enhancement and post-IFN attenuation of neutralizing and possibly cytotoxic responses against HVR1. The remaining patients had no apparent changes in HVR1 quasispecies major variants, suggesting selection of major pretreatment variants, but some changes were observed in other genomic regions. We conclude that IFN-alpha administration and withdrawal profoundly alters the nature of circulating HCV quasispecies, owing to profound changes in virus-host interactions, in patients in whom sustained HCV RNA clearance fails to occur. These changes are associated with profound alterations of the natural outcome of HCV-related liver disease, raising the hypothesis of a causal relationship.

Adolescent↗
PubMed1998

Crystal structures of HLA-A*0201 complexed with antigenic peptides with either the amino- or carboxyl-terminal group substituted by a methyl group.

The crystal structures of class I major histocompatibility complex (MHC) molecules complexed with antigenic peptides revealed a network of hydrogen bonds between the charged amino- and carboxyl-termini of the peptides and conserved MHC residues at both ends of the peptide binding site. These interactions were shown to contribute substantially to the stability of class I MHC/peptide complexes by thermal denaturation studies using synthetic peptides in which either the amino- or carboxyl-terminal group is substituted by a methyl group. Here we report crystal structures of HLA-A*0201 complexed with these terminally modified synthetic peptides showing that they adopt the same bound conformation as antigenic peptides. A number of variations in peptide conformation were observed for the terminally modified peptides, including in one case, a large conformational difference in four central peptide residues that is apparently caused by the lattice contact. This is reminiscent of the way binding a T-cell receptor changed the conformation of central residues of an MHC-bound peptide. The structures determined identify which conserved hydrogen bonds are eliminated in terminally substituted peptides and suggest an increased energetic importance of the interactions at the peptide termini for MHC-peptide stability.

Crystallography, X-Ray↗
PubMed1998

Evidence for supraspinal nervous control of external anal sphincter motility in the cat.

The aim of this study was to investigate the role of noradrenergic descending nervous pathways in external anal sphincter motility. For this purpose, the effects of intravenously injected adrenoceptor antagonist and agonist on the tonic electrical activity of this sphincter were studied in anesthetized cats. The effects of stimulating the region of the locus coeruleus and the effects of intravenous, intracerebroventricular and intrathecal injection of the above drugs on the electromyographic responses of this muscle to pudendal nerve stimulation were also investigated. The tonic sphincteric activity and the reflex response triggered by electrically stimulating pudendal afferent nerve fibers were inhibited by alpha1-adrenoceptor antagonist nicergoline and enhanced by alpha1-adrenoceptor agonist phenylephrine. Stimulation of the locus coeruleus area either inhibited or enhanced the reflex responses. Intracerebroventricular and intrathecal injection of the alpha2-adrenoceptor agonists, morphine and leu-enkephalin decreased the amplitude of these reflex responses. All the effects of opioids were blocked by naloxone and by spinalization performed at the cervical and lumbar levels. The direct response elicited by stimulating the sphincteric motor axons was not affected either by these drugs or by the brainstem stimulation. These results suggests the existence of a pontine neuronal network controlling the motility of the external anal sphincter via noradrenergic and opioid neurons.

Adrenergic alpha-2 Receptor Agonists↗