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W Sadée

Publications and source records attributed to W Sadée.

At least 37 records · Page 2Linked to original sources
PubMed1999

Differential regulation of muscarinic M1 and M3 receptors by a putative phosphorylation domain.

A motif consisting of several serine residues flanked N-terminally by acidic residues occurs in the third intracellular loop of both muscarinic M1 and M3 receptors (287SerLeuThrSerSer291 and 349SerAlaSerSer352, respectively). We examined the role of these domains in modulating agonist-induced desensitization and receptor trafficking, and for the muscarinic M3 receptor, we assessed the contribution of phosphorylation to receptor regulation. Mutation of the above residues did not affect desensitization of phosphoinositide hydrolysis signaling for either the muscarinic M1 or M3 receptor and did not alter the agonist-induced phosphorylation state of the muscarinic M3 receptor. Mutation of this domain (349SerAlaSerSer352/349AlaAlaAlaAla352) in the muscarinic M3 receptor completely abrogated receptor internalization and subsequently, down-regulation. Mutation of the analogous domain (287SerLeuThrSerSer291/287AlaLeuAlaAlaAla291) in the muscarinic M1 receptor had no obvious effect on internalization, but led to a more rapid down-regulation. Thus, these serine-rich regions are not required for receptor desensitization, but are differentially involved in receptor trafficking for the muscarinic M1 and M3 receptors.

Animals↗
PubMed1999

3-Isobutyl-1-methylxanthine inhibits basal mu-opioid receptor phosphorylation and reverses acute morphine tolerance and dependence in mice.

Phosphorylation of the mu-opioid receptor may play a role in opioid tolerance and dependence. 3-Isobutyl-1-methylxanthine (IBMX) was found to inhibit basal mu-opioid receptor phosphorylation (IC50 < or = 10 microM) either upon acute treatment or after 8 h pre-treatment in HEK293 cells transfected with the mu-opioid receptor. In mice made acutely tolerant to and dependent on morphine, IBMX (30-100 nmol, i.c.v.) significantly attenuated the naloxone-induced withdrawal jumping and partially reversed morphine antinociceptive tolerance. IBMX also blocked changes to mu-opioid receptor signaling associated with chronic morphine treatment, specifically, the inverse agonist effect elicited by naloxone, in which naloxone paradoxically elevated the cAMP levels in cells previously exposed to morphine for > or = 12 h. These results suggest a new effect of IBMX in inhibiting basal mu-opioid receptor phosphorylation, and provide additional evidence for the involvement of receptor phosphorylation in the development of opioid tolerance and dependence.

1-Methyl-3-isobutylxanthine↗
PubMed1999

Sequestration of dopamine D2 receptors depends on coexpression of G-protein-coupled receptor kinases 2 or 5.

We examined the agonist-dependent sequestration/internalization of dopamine D2 receptor (the long form D2L and short form D2S), which were transiently expressed in COS-7 and HEK 293 cells with or without G-protein-coupled receptor kinases (GRK2 or GRK5). Sequestration was assessed quantitatively by loss of [3H] sulpiride-binding activity from the cell surface and by transfer of [3H] spiperone-binding activity from the membrane fraction to the light vesicle fraction in sucrose-density gradients. In COS-7 cells expressing D2 receptors alone, virtually no sequestration was observed with or without dopamine (< 4%). When GRK2 was coexpressed, 50% of D2S receptors and 36% of D2L receptors were sequestered by treatment with 10(-4) M dopamine for 2 h, whereas no sequestration was observed in cells expressing the dominant negative form of GRK2 (DN-GRK2). When GRK5 was coexpressed, 36% of D2S receptors were sequestered following the same treatment. The agonist-dependent and GRK2-dependent sequestration of D2S receptors was reduced markedly in the presence of hypertonic medium containing 0.45 M sucrose, suggesting that the sequestration follows the clathrin pathway. Internalization of D2S receptors was also assessed by immunofluorescence confocal microscopy. Translocation of D2 receptors from the cell membrane to intracellular vesicles was observed following the treatment with dopamine from HEK 293 cells only when GRK2 was coexpressed. D2S receptors expressed in HEK 293 cells were shown to be phosphorylated by GRK2 in an agonist-dependent manner. These results indicate that the sequestration of D2 receptors occurs only through a GRK-mediated pathway.

Animals↗
PubMed1999

Rapid measurements of intracellular calcium using a fluorescence plate reader.

Intracellular calcium is a universal second messenger that can serve as a broad-based measure of receptor activity. Recent developments in multi-well plate fluorescence readers facilitate measurement of intracellular free-calcium levels and reduce reliance on slower, more cumbersome or expensive data collection methods. In this report, we describe a rapid and sensitive method to assay intracellular calcium ions in human embryonic kidney (HEK293) and Chinese hamster ovary (CHO) cells from multi-well plates using a fluorometer equipped with on-line injectors. We examine the compatibility of visible-light excitable dyes Calcium Green-1 and Oregon Green 488 BAPTA-1. Using this assay, we were able to detect and quantify activity from muscarinic and beta-adrenergic receptors endogenous to HEK293 cells and detect calcium signals generated by activation of Gi-coupled recombinant mu-opioid and dopamine D2L receptors, and the Gs-coupled melanocortin subtype 4 (MC4) receptor. Fluorescence signals, stable in HEK293 cells, required the use of Oregon Green 488 BAPTA-1 and an inhibitor of organic anion transport in CHO cells. Under appropriate conditions, both cell types can be used to collect complete concentration-response data for a variety of receptors (including a recombinant muscarinic M1 receptor expressed in CHO cells) from a single plate of dye-loaded cells.

Animals↗
PubMed1999

Evaluation of individual and combined neurotoxicity of the immunosuppressants cyclosporine and sirolimus by in vitro multinuclear NMR spectroscopy.

Neurotoxicity, a crucial side effect of immunosuppressive therapy with cyclosporine, also has been demonstrated in vitro for sirolimus, a novel macrolide immunosuppressant, which is under clinical investigation in combination with cyclosporine. NMR spectroscopy was used to study the separate and combined effects of cyclosporine and sirolimus on cerebral metabolism, both in brain cells and in perfused rat brain slices. The high-energy phosphate metabolism was already affected significantly at cyclosporine concentrations as low as 100 micrograms/liter: phosphocreatine was reduced by 10 +/- 2% [half-maximal inhibition concentration (IC50) = 1850 +/- 600 micrograms/liter], and nucleoside triphosphate was reduced by 11 +/- 5% (IC50 = 1110 +/- 420 micrograms/liter; n = 4, P <.05). At 500 micrograms/liter cyclosporine, N-acetylaspartate and glutamate were decreased by 13 +/- 7% (IC50 = 1100 +/- 330 micrograms/liter) and 22 +/- 9% (IC50 = 360 +/- 220 micrograms/liter; n = 4, P <.05), respectively. As evaluated using an algorithm based on Loewe isobolograms, combination of cyclosporine and sirolimus resulted in a synergetic reduction of high-energy phosphate metabolites. Addition of sirolimus to the perfusion medium increased brain slice concentrations of cyclosporine. It is concluded that cyclosporine significantly reduced high-energy phosphate metabolism in brain tissue at in vivo relevant concentrations. Combination with sirolimus resulted in synergism, which, in part, is explained by a greater distribution of cyclosporine into the brain tissue in the presence of sirolimus.

Animals↗
PubMed1998

Translocation of human calcitonin in respiratory nasal epithelium is associated with self-assembly in lipid membrane.

We studied the mechanisms involved in the translocation of human calcitonin (hCT) through excised bovine nasal mucosa (net mucosal-to-serosal permeability approximately 10(-)5 cm s-1). To determine structural requirements for the suggested vesicular internalization two carboxyfluorescein-labeled (fl) hCT fragments, the C-terminal fragment [Nalpha-fl]hCT(9-32) and the N-terminal fragment [Lys(fl)18]hCT(1-24) were synthesized. In presence of the endocytosis inhibitor cytochalasin D mucosal-to-serosal and serosal-to-mucosal hCT permeabilities were equal. Pathway visualization by confocal laser scanning microscopy showed punctated fluorescence indicating vesicular internalization of both hCT and [Nalpha-fl]hCT(9-32). In contrast, the N-terminal fragment lacking the beta-sheet forming C-terminus (25-32) was not internalized. Circular dichroism showed that, when interacting with neutral and negatively charged liposomes, hCT adopts beta-sheet conformation. In a concentrated aqueous solution, beta-sheet formation induces hCT self-assembly and fibrillation. High partitioning of hCT into lipid bilayer membranes was reflected by an apparent partition coefficient log D(pH 7.4) = 2.5 (liposome-buffer equilibrium dialysis). We propose that the high lipid partitioning and beta-sheet formation result in C-terminus-restricted supramolecular self-assembly of hCT and [Nalpha-fl]hCT(9-32) in lipid membranes. Vesicular internalization is suggested to be associated with self-assembly induced perturbation of the lipid bilayer. Condensed hCT self-assemblies may explain the high capacity of net mucosal-to-serosal hCT permeation, which compares favorably with the low transport capacity of receptor-mediated endocytosis.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed1998

Membrane topology of the human dipeptide transporter, hPEPT1, determined by epitope insertions.

We have used epitope insertion to analyze the transmembrane topology of the human H+-dipeptide symporter hPEPT1. An epitope tag, EYMPME (EE), was inserted into different locations at amino acids 39, 78, 106, 412, and 708 of hPEPT1 by site-directed mutagenesis. The functional integrity of the tagged protein was tested by measuring its dipeptide transport activity in transfected Cos7 cells. Further, cells expressing hPEPT1 or EE-tagged hPEPT1 derivatives were labeled with an anti-EE-monoclonal antibody (anti-EE-mAb) or an antiserum raised against the carboxyl terminus of hPEPT1 (anti-hPEPT1) and examined by immunofluorescence confocal microscopy. EE106-, 412-, and 708-hPEPT1 transported the dipeptide tracer as well as wild-type hPEPT1. Tags at position 106 and 412 were shown to be extracellular because they were accessible to anti-epitope antibody in nonpermeabilized cells. In contrast, the carboxyl-terminal domain and EE708 were shown to be intracellular since they were only accessible to the antibodies in permeabilized cells. These results are consistent with a 12-transmembrane domain (TMD) topological model of PEPT1. Epitope insertions at regions linking the putative TMD1 and TMD2, and TMD2 and TMD3 (EE39- and EE78-hPEPT1), abolished the dipeptide transport into the cells. In transfected Cos7 cells, these tagged proteins remained largely intracellular rather than at the plasma membrane. These results suggest that the integrity of these regions is essential for transporter trafficking and/or function. Thus, the topology of the amino-terminal portion, including putative TMD1 and -2, remains to be clarified.

Amino Acid Sequence↗
PubMed1998

A synthetic human Agouti-related protein-(83-132)-NH2 fragment is a potent inhibitor of melanocortin receptor function.

Chemical synthesis of Agouti proteins - Agouti and Agouti-related proteins - is complicated by their large size and by multiple cysteine residues located in the carboxyl terminal regions. Three human Agouti-related protein (AGRP) fragments, two of which correspond to a proposed endoprotease cleavage site between amino acids 82 and 83, were synthesized and tested for anti-melanotropic activity using Xenopus laevis dermal melanophores. Amino-terminal fragments AGRP(25-51) and (54-82) were devoid of significant antagonist activity, whereas the amidated carboxyl-terminal AGRP fragment (83-132)-NH2 was potently active with an inhibitory equilibrium dissociation constant (Ki) of 0.7 nM. The ability to synthesize functionally active AGRP should help unravel its role in the central nervous system and its unusual properties with respect to interaction with the melanocortin family of G-protein coupled receptors.

Agouti Signaling Protein↗
PubMed1998

Internalization and down-regulation of human muscarinic acetylcholine receptor m2 subtypes. Role of third intracellular m2 loop and G protein-coupled receptor kinase 2.

Internalization and down-regulation of human muscarinic acetylcholine m2 receptors (hm2 receptors) and a hm2 receptor mutant lacking a central part of the third intracellular loop (I3-del m2 receptor) were examined in Chinese hamster ovary (CHO-K1) cells stably expressing these receptors and G protein-coupled receptor kinase 2 (GRK2). Agonist-induced internalization of up to 80-90% of hm2 receptors was demonstrated by measuring loss of [3H]N-methylscopolamine binding sites from the cell surface, and transfer of [3H]quinuclidinyl benzilate binding sites from the plasma membrane into the light-vesicle fractions separated by sucrose density gradient centrifugation. Additionally, translocation of hm2 receptors with endocytic vesicles were visualized by immunofluorescence confocal microscopy. Agonist-induced down-regulation of up to 60-70% of hm2 receptors was demonstrated by determining the loss of [3H]quinuclidinyl benzilate binding sites in the cells. The half-time (t1/2) of internalization and down-regulation in the presence of 10(-4) M carbamylcholine was estimated to be 9.5 min and 2.3 h, respectively. The rates of both internalization and down-regulation of hm2 receptors in the presence of 10(-6) M or lower concentrations of carbamylcholine were markedly increased by coexpression of GRK2. Agonist-induced internalization of I3-del m2 receptors was barely detectable upon incubation of cells for 1 h, but agonist-induced down-regulation of up to 40-50% of I3-del m2 receptors occurred upon incubation with 10(-4) M carbamylcholine for 16 h. However, the rate of down-regulation was lower compared with wild type receptors (t1/2 = 9.9 versus 2.3 h). These results indicate that rapid internalization of hm2 receptors is facilitated by their phosphorylation with GRK2 and does not occur in the absence of the third intracellular loop, but down-regulation of hm2 receptors may occur through both GRK2-facilitating pathway and third intracellular loop-independent pathways.

Animals↗
PubMed1998

An oligopeptide transporter is expressed at high levels in the pancreatic carcinoma cell lines AsPc-1 and Capan-2.

Carcinomas of the exocrine pancreas are poorly understood and have a poor prognosis because of their highly malignant nature. Using two human pancreatic cancer cell lines, AsPc-1 and Capan-2, we have investigated avenues that might be useful in targeting the delivery of antineoplastic agents to such cancers. Qualitative RNA PCRs established the presence of the oligopeptide transporter PEPT 1 in these pancreatic cell lines. Northern analysis confirmed the presence of a 3.3-kb transcript. The transporter is normally expressed primarily in small intestinal epithelial cells for nutrient absorption. It is also expressed in a human intestinal cell line, Caco-2. High levels of PEPT 1 protein expression in AsPc-1 and Capan-2, as multiple glycosylated forms (Mr approximately 90,000-120,000), were confirmed by Western immunoblotting, when compared with Caco-2 cell cultures. Absorption of the model dipeptide glycyl-L-sarcosine by AsPc-1 and Capan-2 cells was similar to glycyl-L-sarcosine absorption by Caco-2 cells and a Chinese hamster ovary cell line expressing human PEPT 1 (CHO-PEPT 1). Uptake was pH dependent and inhibited by several di/tripeptides and bestatin, but it remained unaffected by glycine and tetraglycine. Peptide solute transport by AsPc-1 and Capan-2 cells exhibited binding affinities (Kms) similar to those previously reported for PEPT 1, whereas the transport maximal velocity (Vmax) of the AsPc-1 cells was much greater than those of the Capan-2 and Caco-2 cells. Immunomicroscopy demonstrated PEPT 1 protein localized at the plasma membrane and in intracellular vesicular structures, similar to that observed for Caco-2 and CHO-PEPT 1 cells. These data suggest that the pancreatic cancer cells AsPc-1 and Capan-2 express surprisingly high levels of a solute transporter that was previously thought to be restricted in function to the absorption of nutrients from the small intestine.

Adenocarcinoma↗
PubMed1998

5'-Amino acid esters of antiviral nucleosides, acyclovir, and AZT are absorbed by the intestinal PEPT1 peptide transporter.

PURPOSE: General use of nucleoside analogues in the treatment of viral infections and cancer is often limited by poor oral absorption. Valacyclovir, a water soluble amino acid ester prodrug of acyclovir has been reported to increase the oral bioavailability of acyclovir but its absorption mechanism is unknown. This study characterized the intestinal absorption mechanism of 5' -amino acid ester prodrugs of the antiviral drugs and examined the potential of amino acid esters as an effective strategy for improving oral drug absorption. METHODS: Acyclovir (ACV) and Zidovudine (AZT) were selected as the different sugar-modified nucleoside antiviral agents and synthesized to L-valyl esters of ACV and AZT (L-Val-ACV and L-Val-AZT), D-valyl ester of ACV (D-Val-ACV) and glycly ester of ACV (Gly-ACV). The intestinal absorption mechanism of these 5' -amino acid ester prodrugs was characterized in three different experimental systems; in situ rat perfusion model, CHO/hPEPT1 cells and Caco-2 cells. RESULTS: Testing 5' -amino acid ester prodrugs of acyclovir and AZT, we found that the prodrugs increased the intestinal permeability of the parent nucleoside analogue 3- to 10-fold. The dose- dependent permeation enhancement was selective for L-amino acid esters. Competitive inhibition studies in rats and in CHO cells transfected with the human peptide transporter, hPEPT1, demonstrated that membrane transport of the prodrugs was mediated predominantly by the PEPT1 H+/dipeptide cotransporter even though these prodrugs did not possess a peptide bond. Finally, transport studies in Caco-2 cells confirmed that the 5' - amino acid ester prodrugs enhanced the transcellular transport of the parent drug. CONCLUSIONS: This study demonstrates that L-amino acid-nucleoside chimeras can serve as prodrugs to enhance intestinal absorption via the PEPT1 transporter, providing a novel strategy for improving oral therapy of nucleoside drugs.

Acyclovir↗
PubMed1997

Sequence alignments of the H(+)-dependent oligopeptide transporter family PTR: inferences on structure and function of the intestinal PET1 transporter.

PURPOSE: To study the structure and function of the intestinal H+/ peptide transporter PET1, we compared its amino acid sequence with those of related transporters belonging to the oligopeptide transporter family PTR, and with more distant transporter families. METHODS: We have developed a new approach to the sequence analysis of proteins with multiple transmembrane domains (TMDs) which takes into account the repeated TMD-loop topology. In addition to conventional analyses of the entire sequence, each TMD and its adjacent loop residues (= TMD segments) were analyzed separately as independent structural units. In combination with hydropathy analysis, this approach reveals any changes in the order of the TMD segments in the primary structure and permits TMD alignments among divergent structures even if rearrangements of the order of TMD segments have occurred in the course of evolution. RESULTS: Alignments of TMD segments indicate that the TMD order in PTR transporters may have changed in the process of evolution. Consideration of such changes permits the alignment of homologous TMD segments from PTR transporters belonging to distant akaryotic and eukaryotic phyla. Multiple alignments of TMDs reveal several highly conserved regions that may play a role in transporter function. In comparing the PTR transporters with other transporter gene families, alignment scores using the entire primary structure are too low to support a finding of probable homology. However, statistically significant alignments were observed among individual TMD segments if one disregards the order in which they occur in the primary structure. CONCLUSIONS: Our results support the hypothesis that the PTR transporters may have evolved by rearrangement, duplication, or insertions and deletions of TMD segments as independent modules. This modular structure suggests new alignment strategies for determining functional domains and testing relationship among distant transporter families.

Amino Acid Sequence↗
PubMed1997

Evolutionary relationships among proteins probed by an iterative neighborhood cluster analysis (INCA). Alignment of bacteriorhodopsins with the yeast sequence YRO2.

PURPOSE: Searching the existing databases for homologous sequences is essential to understanding a protein's structure and function. For a query sequence, its nearest neighbors can be identified by BLAST (basic local alignment search tool). However, a single query sequence is sufficient to define the entire neighborhood of related sequences, and multiple BLAST queries are needed. We describe here a program which permits automated and iterative BLAST analysis of an entire neighborhood of sequences and apply this to search for homologs of the bacteriorhodopsins outside the archaea phylum. METHODS: We have developed a Java program, 'Iterative Neighborhood Cluster Analysis' (INCA), which performs iterative BLAST searches, beginning with a single starter sequence, and proceeding with any other sequence achieving a predefined minimum alignment score. This results in a cluster of sequences where each sequence is related to at least one other sequence by the cutoff score, additional lists of more distantly related sequences for each member of cluster. RESULTS: Bacteriorhodopsins had not been previously aligned with any other protein family with scores indicative of probable homology. Using INCA, we identified a probable homolog in yeast, YRO2_YEAST, also containing seven putative transmembrane domains. A finding of probable homology was supported by additional alignment strategies. CONCLUSIONS: INCA is a useful tool to assess complete protein neighborhoods. With an increasing database, INCA can serve to detect the emergence of evolutionary links between even the most distantly related protein families. Identifying a homolog of the bacteriorhodopsins in yeast illustrates this approach but at the same time highlights the vast evolutionary distances between polytopic membrane proteins, such as the bacteriorhodopsins.

Amino Acid Sequence↗
PubMed1997

Dynorphin peptides: antagonists of melanocortin receptors.

PURPOSE: To identify possible targets that mediate the non-opioid effects of dynorphin-A (DynA), effects that include inflammation and aggravation of traumatic nerve injury. METHOD: We examined dynorphin peptides for functional interaction with the closely related melanocortin (MC) system. RESULTS: DynA-(1-13)NH2 and other related opioid dynorphin peptides antagonize the human MC1, MC3 and MC4 receptors, and an amphibian MC receptor, with dissociation constants (Kd's) of 40 to 150 nM. The affinity of dynorphin's interaction with MC receptors is therefore greater than with other previously proposed non-opioid targets of dynorphin, which require micromolar concentrations. Dynorphin also antagonizes the adrenocorticotropic hormone (ACTH; MC2) receptor and an MC-like receptor endogenous to COS-7 cells, but with lower efficacy. In contrast DynA had no effect on seven control receptors and was only weakly effective at two others. Metabolites of dynorphin derived from cleavage of the amino terminal Tyr residue, such as DynA(2-17), lack opioid activity yet still produce a number of well established non-opioid effects. These des-Tyr derivatives also antagonized each of the five MC receptors examined. CONCLUSIONS: DynA peptides were found to antagonize MC receptors in vitro with potencies that parallel those reported for pharmacological non-opioid effects of dynorphins in vivo. The combination of DynA and its active metabolites may reach levels sufficient to inhibit MC receptors physiologically. Dynorphin inhibition of MC receptors could prove to be an example of crosstalk between two distinct yet phylogenetically related neurotransmitter systems.

Adrenocorticotropic Hormone↗
PubMed1997

Residues specifically involved in down-regulation but not internalization of the m1 muscarinic acetylcholine receptor.

Human m1 muscarinic acetylcholine receptor mutants were screened to determine receptor domains and cellular pathways relevant to down-regulation. Mutations in the second intracellular loop and the junctions of the third intracellular loop of the receptor, where a role for receptor activation or internalization had been previously demonstrated in HEK293 cells, were selected for this study. To assess receptor down-regulation, the m1 receptor mutants were transfected into Chinese hamster ovary cells. Because receptor internalization is expected to precede down-regulation, mutants displaying intact internalization were selected to permit interpretation of mutational effects on down-regulation alone. Four mutations were identified that specifically impaired down-regulation without altering receptor internalization: V127A, I211A, E360A, and K362A. The results define new receptor domains in the second intracellular loop and the junctions of the third intracellular loop that are involved in down-regulation. These same four mutants were also defective in signaling via the phospholipase C and the adenylyl cyclase pathways and in G protein activation, as measured by [35S]GTP gamma S binding. However, the level of second messenger stimulation correlated poorly with the extent of down-regulation. In summary, several mutations of the m1 receptor selectively affect down-regulation, demonstrating that internalization and down-regulation represent distinct events driven by different cellular mechanisms.

Adenylyl Cyclases↗