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M Bouvier

Publications and source records attributed to M Bouvier.

At least 109 records · Page 6Linked to original sources
PubMed1994

Desensitization, phosphorylation and palmitoylation of the human dopamine D1 receptor.

The regulation and post-translational modifications of the human dopamine D1 receptor were studied in the baculovirus-eukaryotic cell expression system. Baculovirus constructs containing either the DNA encoding the dopamine D1 receptor or a DNA encoding a c-myc epitope tagged dopamine D1 receptor (c-myc-dopamine D1 receptor) were used to infect Spodoptera frugiperda (Sf9) insect cells. Expressed dopamine D1 and c-myc-dopamine D1 receptors bound agonists and antagonists with affinities and a rank order of potency characteristic of a classical dopamine D1 receptor pharmacological profile. In membrane preparations from cells expressing c-myc-dopamine D1 receptor, the photoaffinity label [125I](3-methyl-2-[4'-azidophenyl]-2,3,5-tetrahydro-2H-3-benzazepine) ([125I]MAB) bound specifically upon photolysis. A major broad band of approximately 48 kDa was detected. This species was identified in immunoblots by the monoclonal antibody raised against the c-myc epitope of c-myc-dopamine D1 receptor was isolated by immunoprecipitation from whole cells and was shown to be post-translationally modified by phosphorylation and palmitoylation. Exposure of cells expressing c-myc-dopamine D1 receptor to dopamine for 15 min resulted in a reduction in the maximal dopamine stimulated adenylyl cyclase activity, which was accompanied by an increased phosphorylation of the receptor and a rapid redistribution of surface c-myc-dopamine D1 receptor as detected by in situ immunofluorescence. Dopamine exposure also resulted in an increased level of incorporation of [3H]palmitic acid into the receptor. Thus, we provide the first evidence that the human dopamine D1 receptor undergoes agonist-dependent desensitization, phosphorylation and palmitoylation.

2,3,4,5-Tetrahydro-7,8-dihydroxy-1-phenyl-1H-3-ben↗
PubMed1994

Ethical issues involved in establishing a registry for familial Alzheimer's disease.

In January 1992, the IMAGE Project extended the establishment of its registry of familial Alzheimer cases to all areas of the province of Quebec, for collection of epidemiological and clinical data, as well as biological samples. The aim is to study genetic transmission patterns of Alzheimer's disease (AD) and to provide a sampling framework for further etiologic and risk factor studies. The IMAGE registry already includes data of a population-based study in the Saguenay-Lac-St-Jean area; the project to collect data on familial AD (FAD) cases across the province of Quebec is known as the ALGENE Initiative. The registry is thus a collection of "AD families" for both familial and sporadic cases. The establishment of the registry involves several steps in the field work: recruitment and selection of families; collection of information on family medical history; selection of informative families and genetic testing for AD/FAD by linkage analysis. As AD is not homogeneous in its etiology and since we do not know if, in the event that genetics is involved in AD whether or not penetrance of the gene(s) is high, we must be aware of the "genetic horizons" of AD in collecting and conserving data on families of cases, and in the genetic testing for AD/FAD by linkage analysis. Families who choose genetic testing must be aware of the implications of our undertaking, assured of the confidentiality of the test and, at the same time, they must understand its limitations. The experimental nature of our research project raises ethical dilemmas. This article examines these initial considerations of the field work involved in developing a registry pertaining to genetic testing for AD/FAD by linkage analysis and offers some preliminary observations on the experience of the first year of this project.

Aged↗
PubMed1994

Beta-adrenergic receptor desensitization in the early stage of hereditary cardiomyopathy in hamsters.

The beta-adrenergic receptor (beta AR)/adenylyl cyclase signalling pathway was examined in cardiac membranes from cardiomyopathic Syrian hamsters. Three stages were examined during the progression of this hereditary cardiomyopathy (30 days old, prenecrotic phase; 60 days old, necrotic phase; and 120 days old, compensatory phase). Isoproterenol-stimulated adenylyl cyclase activity was decreased by 32 +/- 16% in 30-day-old cardiomyopathic hamsters, compared with age-matched controls. This was not accompanied by any change in the fluoride- or forskolin-stimulated activities, suggesting that the decrease reflects a perturbation of the receptor-mediated stimulation. Neither the density nor the subcellular distribution of the beta AR, as assessed by [125I]iodocyanopindolol binding assays, was affected in these animals. However, the agonist binding properties of the beta AR were significantly affected. Indeed, the effect of guanyl nucleotides on isoproterenol binding was decreased in 30-day-old cardiomyopathic hamsters. Given that guanyl nucleotide sensitivity is correlated with the ability of the beta AR to productively interact with Gs protein, these results suggest that the decreased beta-adrenergic-stimulated adenylyl cyclase activity results from a functional uncoupling of the beta AR with no change in receptor density. The desensitization of the beta-adrenergic-stimulated adenylyl cyclase was transient, since no change in isoproterenol-stimulated adenylyl cyclase was detected in 60- and 120-day-old hamsters, compared with age-matched controls. Similarly, the receptor number and distribution were not affected at those ages.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenylyl Cyclases↗
PubMed1994

Characterization, expression, and hormonal control of a thymic beta 2-adrenergic receptor.

In the present study, we have characterized the beta 2-adrenergic receptor (beta 2-AR)-adenosine 3',5'-cyclic monophosphate (cAMP) system of the rat thymus gland and examined the hormonal regulation of the thymic beta 2-AR gene expression under physiological or pharmacological conditions accompanied by marked alterations of the sex steroid hormone milieu. We report here that membrane preparations of female rat thymic tissue contain iodocyanopindolol binding sites that exhibit pharmacological properties typical of a beta-AR. Detailed analysis by computer modeling of the binding potencies of a large series of beta 1- and beta 2-adrenergic agonists and antagonists revealed predominantly the beta 2-AR subtype (78%) in rat thymus. This inference from radioligand binding studies was corroborated functionally by the rank order of potencies of a series of adrenergic agonists to stimulate the production of cAMP. Northern blot analysis, using a human beta 2-AR cDNA as a probe, revealed the presence of a mRNA of 2.3 kb, which is consistent with the size of the beta 2-AR mRNA found in other rat tissues. Physiological regulation of specific beta 2-AR in the rat thymus was indicated by significant increases in both receptor concentration and steady-state levels of beta 2-AR mRNA during the diestrous 2 and proestrous phases of the rat estrous cycle and pregnancy, whereas castration sharply reduced beta 2-AR numbers and transcript levels within the thymus. The modulation of the thymic beta 2-AR-cAMP signaling system by the preexisting sex steroid milieu, coupled with the sex-dependent adrenergic modulation of thymic cell-mediated immune response, may contribute to the various sex-related alterations in immune responsiveness and could play a role in sexually related immune disorders.

Adenylyl Cyclases↗
PubMed1994

Inverse agonist activity of beta-adrenergic antagonists.

Agonist-independent properties of the human beta 2-adrenergic receptor (beta 2AR) were studied using the baculovirus expression system in Sf9 cells. In the absence of agonist but in the presence of GTP, membranes from cells expressing the beta 2AR exhibited higher levels of cAMP production than did membranes from uninfected cells or from cells infected with wild-type baculovirus. The increase in cAMP production was proportional to the number of beta 2AR expressed, up to 40 pmol/mg of membrane protein, and it could be inhibited in a dose-dependent manner by beta AR antagonists. The increase and its reversal both were independent of the possible presence of contaminating catecholamines in the culture medium and thus appear to reflect spontaneous beta 2AR activity and direct antagonist-receptor interactions, respectively. The maximal level of inhibition varied among the beta AR ligands tested, to yield the following rank order of "inverse efficacy"; timolol > or = propranolol > alprenolol > or = pindolol > labetalol > dichloroisoproterenol. The same rank order was observed using membranes prepared from Chinese hamster fibroblasts expressing beta 2AR. The effect of timolol was partly blocked by labetalol and dichloroisoproterenol, in an apparently competitive manner. The intracellular cAMP content of Sf9 cells cultured in serum-free medium was also increased by the expression of beta 2AR, and that increase was reversed by timolol and propranolol, consistent with observations in membrane preparations. The properties revealed by the expression of the beta 2AR in Sf9 cells suggest two agonist-independent traits of G protein-linked receptors, i.e., 1) that unliganded receptors are able to activate G proteins both in membrane preparations and in whole cells and 2) that antagonists may mediate their effects not only by preventing the binding of agonists but also by decreasing the propensity of the receptor to assume an active state.

Adrenergic beta-Agonists↗
PubMed1993

Human serotonin1B receptor expression in Sf9 cells: phosphorylation, palmitoylation, and adenylyl cyclase inhibition.

Analysis of the primary protein structure of the human serotonin1B (5-HT1B) receptor reveals consensus sites for phosphorylation and a putative site for palmitoylation. To investigate these posttranslational modifications, we have expressed a c-myc epitope-tagged 5-HT1B (m5-HT1B) receptor in Sf9 cells. This strategy enabled receptors to be detected by immunoblot analysis and purified by immunoprecipitation using a monoclonal antibody, 9E10, specific for the c-myc epitope. Agonist radioligand [3H]5-HT binding studies showed that the expressed 5-HT1B and m5-HT1B receptors displayed the characteristic pharmacological profile of the neuronal 5-HT1B receptor. The expressed receptors displayed both high- and low-affinity states for [3H]5-HT, suggesting that the receptors were coupled to endogenous G-proteins. Indeed, agonist binding to the high-affinity receptor state was regulated in the presence of GTP gamma S, Gpp(NH)p, and pertussis toxin. [32P]ADP-ribosylation experiments identified a major approximately 41-kDa ADP-ribosylated protein present in Sf9 membranes that comigrated with partially purified bovine brain Gi alpha/G(o) alpha subunits. Measurements of adenylyl cyclase activity in membranes from cells expressing m5-HT1B receptors showed that serotonergic agonists mediated the inhibition of adenylyl cyclase activity with a rank order of potency comparable to their affinity constants. Immunoblot analysis of membranes prepared from cells expressing m5-HT1B receptors and photoaffinity labeling of the immunoprecipitated material revealed photolabeled species at approximately 95 and at approximately 42 kDa.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Diphosphate Ribose↗
PubMed1993

Mutation of tyrosine-350 impairs the coupling of the beta 2-adrenergic receptor to the stimulatory guanine nucleotide binding protein without interfering with receptor down-regulation.

Long-term stimulation of the beta 2-adrenergic receptor (beta 2AR) leads to an internalization and degradation of the receptor. This down-regulation of the beta 2AR number contributes to the desensitization of the adenylyl cyclase activity induced by chronic exposure to agonists. It was proposed that two tyrosine residues (Tyr-350 and Tyr-354) located in the cytoplasmic tail of the beta 2AR play a crucial role in agonist-induced down-regulation. In addition to perturbation of the down-regulation, the substitution of these tyrosines for alanines also led to a functional uncoupling of the receptor from Gs [Valiquette et al. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 5089-5093]. To further characterize the relative contribution of Tyr-350 and Tyr-354 to the receptor interaction with Gs and agonist-promoted down-regulation, both tyrosines were individually replaced by alanines and mutant receptors expressed in CHW cells. We show here that mutation of Tyr-350 but not that of Tyr-354 significantly decreased the ability of the beta 2AR to be functionally coupled to Gs and thereby to stimulate the adenylyl cyclase. Moreover, in contrast to the double tyrosine mutation, neither of the single-point mutations affected the agonist-induced down-regulation pattern. These data suggest that the presence of either Tyr-350 or Tyr-354 is sufficient to maintain normal agonist-induced down-regulation whereas the integrity of Tyr-350 is required for an appropriate coupling to Gs.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenylyl Cyclases↗
PubMed1993

Altered phosphorylation and desensitization patterns of a human beta 2-adrenergic receptor lacking the palmitoylated Cys341.

Exposure of beta 2-adrenergic receptors to agonists causes a rapid desensitization of the receptor-stimulated adenylyl cyclase, associated with an increased phosphorylation of the receptor. Agonist-promoted phosphorylation of the beta 2-adrenergic receptor (beta 2AR) by protein kinase A and the beta-adrenergic receptor kinase (beta ARK) is believed to promote a functional uncoupling of the receptor from the guanyl nucleotide regulatory protein Gs. More recently, palmitoylation of Cys341 of the receptor has also been proposed to play an important role in the coupling of the beta 2-adrenergic receptor to Gs. Here we report that substitution of the palmitoylated cysteine by a glycine (Gly341 beta 2 AR) using site directed mutagenesis leads to a receptor being highly phosphorylated and largely uncoupled from Gs. In Chinese hamster fibroblasts (CHW), stably transfected with the human receptor cDNAs, the basal phosphorylation level of Gly341 beta 2AR was found to be approximately 4 times that of the wild type receptor. This elevated phosphorylation level was accompanied by a depressed ability of the receptor to stimulate the adenylyl cyclase and to form a guanyl nucleotide-sensitive high affinity state for agonists. Moreover, exposure of this unpalmitoylated receptor to an agonist did not promote any further phosphorylation or uncoupling. A modest desensitization of the receptor-stimulated adenylyl cyclase response was observed but resulted from the agonist-induced sequestration of the unpalmitoylated receptor and could be blocked by concanavalin A. This contrasts with the agonist-promoted phosphorylation and uncoupling of the wild type receptor.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenylyl Cyclases↗
PubMed1993

Cellular characterization of the pharmacological selectivity and tachyphylactic properties of denopamine for the human beta adrenergic receptors.

The pharmacological properties of the cardiotonic agent denopamine toward human beta-1 and beta-2 adrenergic receptors (AR) were assessed in a heterologous expression system. In whole cells radiolabeled with the nonselective beta antagonist [125I]iodopindolol, denopamine displayed a 7-fold lower inhibition constant for the beta-1 than for the beta-2 AR. Similarly, denopamine exhibited a 7-fold greater potency to stimulate the adenylyl cyclase activity in cells expressing the beta-1 AR than in cells expressing the beta-2 subtype, which confirmed the subtype selectivity of this drug. The maximal stimulation of adenylyl cyclase activity by denopamine was less than 10% of that produced by isoproterenol at both receptor subtypes, which indicated that denopamine is a weak partial agonist. The extent of beta-1 AR desensitization induced by denopamine was then compared with that induced by the full agonist isoproterenol. By contrast with isoproterenol, preincubation with denopamine for 20 min did not induce any decrease in the responsiveness of the receptor to subsequent stimulation nor did it promote any sequestration of the receptor. A 24-hr pretreatment with denopamine produced a time-dependent reduction in the number of beta-1 AR with a rate of down-regulation slower than that produced by isoproterenol. These data therefore indicate that denopamine is a beta-1 adrenergic selective agonist with low intrinsic activity that is less prone than full agonists to cause desensitization. This may explain the long-lasting cardiotonic effects of denopamine compared with those of full agonists.

Adenylyl Cyclases↗
PubMed1993

[Aseptic osteomyelitis].

Based on a series of eight personal cases and a review of the literature, this study tries to define the diagnostic elements of aseptic osteomyelitis. Although sternocostoclavicular sites are immediately suggestive of the diagnosis, lesions of the pelvis and spine and long bones of the limbs are particularly difficult to diagnose. Inflammatory type pain occurred in episodesover a number of years and responded to NSAIs and possibly diphosphonates. Laboratory abnormalities were usually confined to a raised ESR, but alterations of the IgA levels, similar to those observed in ankylosing spondylitis were observed in four cases. Hyperostosis occurred late in the course, preceded by signs of osteomyelitis, periosteitis or enthesitis. Histology does not provide any formal conclusions. The most suggestive lesions are those of nonspecific aseptic osteomyelitis, followed by a Paget-like appearance. The interpretation of these findings may be complicated by the presence of eosinophils, giant cells or mast cells. Two elements are very valuable for establishing the diagnosis: the presence of infraradiological anterior thoracic increased uptake on bone scan and the presence of a skin disorder, either severe acne or, more especially, palmoplantar pustulosis.

Adolescent↗
PubMed1993

The human beta 3-adrenergic receptor is resistant to short term agonist-promoted desensitization.

The human beta 3-adrenergic receptor (beta 3AR) lacks most of the structural determinants that, in the beta 2AR, contribute to agonist-induced receptor desensitization. To evaluate the effect of these structural differences on the beta 3AR desensitization profile, the human beta 2- and beta 3AR were stably expressed in Chinese hamster fibroblasts (CHW) and murine Ltk- cells (L cells). Incubation of CHW-beta 2 or L-beta 2 cells with 10 microM isoproterenol for 30 min induced a decrease in the maximal agonist-stimulated adenylyl cyclase activity and a cAMP-dependent reduction in the potency of isoproterenol to stimulate the receptor. In addition, this pretreatment impaired the formation of the high affinity heterotrimeric agonist-receptor-guanine nucleotide-binding protein complex and induced the sequestration of approximately 30% of the beta 2AR away from the cell surface. In contrast, similar treatment of CHW-beta 3 and L-beta 3 cells did not affect the maximal receptor-stimulated adenylyl cyclase activity, nor did it induce any significant sequestration of the beta 3AR. In fact, only a modest cAMP-independent decrease in the potency of isoproterenol to stimulate the receptor could be observed after isoproterenol treatment. The rapid desensitization pattern of a chimeric beta 3AR, in which the third cytoplasmic loop and the carboxyl-terminal tail were exchanged with those of the beta 2AR (which include potential phosphorylation sites and other possible molecular determinants of desensitization), was found to be intermediate between those of the two original receptor subtypes. These results demonstrate that (i) the beta 3AR is less prone than the beta 2AR to undergo rapid agonist-promoted desensitization and, (ii) in addition to the phosphorylation sites located in the third cytoplasmic loop and the carboxyl-terminal tail of the beta 2AR, other molecular determinants contribute to short term desensitization.

Adenylyl Cyclases↗
PubMed1992

The glial cell glutamate uptake carrier countertransports pH-changing anions.

Uptake into glial cells helps to terminate glutamate's neurotransmitter action and to keep its extracellular concentration, [Glu]o, below neurotoxic levels. The accumulative power of the uptake carrier stems from its transport of inorganic ions such as sodium (into the cell) and potassium (out of the cell). There is controversy over whether the carrier also transports a proton (or pH-changing anion). Here we show that the carrier generates an alkalinization outside and an acidification inside glial cells, and transports anions out of the cells, suggesting that there is a carrier cycle in which two Na+ accompany each glutamate anion into the cell, while one K+ and one OH- (or HCO3-) are transported out. This stoichiometry predicts a minimum [Glu]o of 0.6 microM normally (tonically activating presynaptic autoreceptors and post-synaptic NMDA receptors), and 370 microM during brain anoxia (high enough to kill neurons). Transport of OH-/HCO3- on the uptake carrier generates significant pH changes, and may provide a mechanism for neuron-glial interaction.

Acetazolamide↗