Glycogen synthase kinases 3α and 3β in cardiac myocytes: regulation and consequences of their inhibition

Inhibition of glycogen synthase kinase 3β (GSK3β) as a consequence of its phosphorylation by protein kinase B/Akt (PKB/Akt) has been implicated in cardiac myocyte hypertrophy in response to endothelin-1 or phenylephrine. We examined the regulation of GSK3α (which we show to constitute a significant proportion of the myocyte GSK3 pool) and GSK3β in cardiac myocytes. Although endothelin increases phosphorylation of GSK3 and decreases its activity, the response is less than that induced by insulin (which does not promote cardiac myocyte hypertrophy). GSK3 phosphorylation induced by endothelin requires signalling through the extracellular signal-regulated kinase 1/2 (ERK1/2) cascade and not the PKB/Akt pathway, whereas the reverse is true for insulin. Cardiac myocyte hypertrophy involves changes in morphology, and in gene and protein expression. The potent GSK3 inhibitor 1-azakenpaullone increases myocyte area as a consequence of increased cell length whereas phenylephrine increases both length and width. Azakenpaullone or insulin promotes AP1 transcription factor binding to an AP1 consensus oligonucleotide, but this was significantly less than that induced by endothelin and derived principally from increased binding of JunB protein, the expression of which was increased. Azakenpaullone promotes significant changes in gene expression (assessed by Affymetrix microarrays), but the overall response is less than with endothelin and there is little overlap between the genes identified. Thus, although GSK3 may contribute to cardiac myocyte hypertrophy in some respects (and presumably plays an important role in myocyte metabolism), it does not appear to contribute as significantly to the response induced by endothelin as has been maintained.

Insulin, glucagon-like peptide 1, glucose-dependent insulinotropic polypeptide and insulin-like growth factor I as putative mediators of the hypolipidemic effect of oligofructose in rats

The addition of oligofructose as a dietary fiber decreases the serum concentration and the hepatic release of VLDL-triglycerides in rats. Because glucose, insulin, insulin-like growth factor I (IGF-I) and gut peptides [i.e., glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1)]) are factors involved in the metabolic response to nutrients, this paper analyzes their putative role in the hypolipidemic effect of oligofructose. Male Wistar rats were fed a nonpurified diet with or without 10% oligofructose for 30 d. Glucose, insulin, IGF-I and GIP concentrations were measured in the serum of rats after eating. GIP and GLP-1 contents were also assayed in small intestine and cecal extracts, respectively. A glucose tolerance test was performed in food-deprived rats. Serum insulin level was significantly lower in oligofructose-fed rats both after eating and in the glucose tolerance test, whereas glycemia was lower only in the postprandial state. IGF-I serum level did not differ between groups. GIP concentration was significantly higher in the serum of oligofructose-fed rats. The GLP-1 cecal pool was also significantly higher. In this study, we have shown that cecal proliferation induced by oligofructose leads to an increase in GLP-1 concentration. This latter incretin could be involved in the maintenance of glycemia despite a lower insulinemia in the glucose tolerance test in oligofructose-fed rats. We discuss also the role of hormonal changes in the antilipogenic effect of oligofructose.

Effects of the Allosteric Antagonist 1-(4-Chlorophenyl)-3-[3-(6-pyrrolidin-1-ylpyridin-2-yl)phenyl]urea (PSNCBAM-1) on CB1 Receptor Modulation in the Cerebellum

PSNCBAM-1 has recently been described as a cannabinoid CB1 receptor allosteric antagonist associated with hypophagic effects in vivo; however, PSNCBAM-1 effects on CB1 ligand-mediated modulation of neuronal excitability remain unknown. Here, we investigate PSNCBAM-1 actions on CB1 receptor-stimulated [35S]GTPγS binding in cerebellar membranes and on CB1 ligand modulation of presynaptic CB1 receptors at inhibitory interneurone-Purkinje cell (IN-PC) synapses in the cerebellum using whole-cell electrophysiology. PSNCBAM-1 caused non-competitive antagonism in [35S]GTPγS binding studies, with higher potency against the CB receptor agonist CP55940 than for WIN55,212-2 (WIN55). In electrophysiological studies, WIN55 and CP55940 reduced miniature inhibitory postsynaptic currents (mIPSCs) frequency, but not amplitude. PSNCBAM-1 application alone had no effect on mIPSCs; however, PSNCBAM-1 pre-treatment revealed agonist-dependent functional antagonism, abolishing CP55940-induced reductions in mIPSC frequency, but having no clear effect on WIN55 actions. The CB1 antagonist/inverse agonist AM251 increased mIPSC frequency beyond control, this effect was reversed by PSNCBAM-1. PSNCBAM-1 pre-treatment also attenuated AM251 effects. Thus, PSNCBAM-1 reduced CB1 receptor ligand functional efficacy in the cerebellum. The differential effect of PSNCBAM-1 on CP55940 versus WIN55 actions in [35S]GTPγS binding and electrophysiological studies and the attenuation of AM251 effects are consistent with the ligand-dependency associated with allosteric modulation. These data provide the first description of functional PSNCBAM-1 allosteric antagonist effects on neuronal excitability in the mammalian CNS. PSNCBAM-1 allosteric antagonism may provide viable therapeutic alternatives to orthosteric CB1 antagonists/inverse agonists in the treatment of CNS disease.

Preparation of transition metal complexes of bicyclo[2.2.1]hept-5-ene-2-carboxylic acid (C7H9CO2H) and X-ray crystal structure of [Cu2?(±)-endo-μ-O2CC7H9?4 (CH3OH)2]·2CH3OH

Metathesis reactions were used to prepare a range of dicopper(II), monocopper(I), diruthenium(II, III), dimolybdenum(II,II) and dirhodium(II,II) complexes of either racemic or resolved forms of endo- and exo-bicyclo[2.2.1]hept-5-ene-2-carboxylic acid (C7H9CO2H). The X-ray crystal structure of [Cu2{(±)-endo-μ-O2CC7H9}4(CH3OH)2]·2CH3OH shows the two copper(II) ions bridged by two (+)-endo-bicyclo[2.2.1]hept-5-ene-2-carboxylate anions and two (−)-endo-bicyclo[2.2.1]hept-5-ene-2-carboxylate anions. Methanol molecules occupy the two trans axial sites, and there are also two methanol molecules hydrogen bonded to opposite carboxyl oxygens.

Synthesis and X-ray crystal structure of the copper(I) dicarboxylic acid complex [Cu(η1-bdoaH)(PPh3)3] (bdoaH2 = benzene-1,2- dioxyacetic acid)

The copper(II) complex [Cu(bdoa)(H2O)2] (bdoaH2 = benzene-1,2-dioxyacetic acid) reacts with triphenylphosphine (1:4 mol ratio) to give the colourless copper(I) complex [Cu(η1-bdoaH)(PPh3)3] (1) in good yield. The X-ray crystal structure of the complex shows the copper atom at the centre of a distorted tetrahedron, and is ligated by the phosphorus atoms of the three triphenylphosphines and one carboxylate oxygen atom of the bdoaH− ligand. Significant intermolecular hydrogen-bonding exists between the pendant carboxylate OH function of one molecule and the uncoordinated “ketonic” oxygen of a neighbouring molecule. Complex 1 is non-conducting in chloroform but ionizes readily in acetonitrile. The cyclic voltammogram of an acetonitrile solution of 1 shows a single irreversible anodic peak for the oxidation of the PPh3 ligands and the copper(I) centre, and a single irreversible cathodic peak for the reduction of the bdoaH− ion. IR and mass spectral data for 1 are given.

Copper(II) complexes of benzene-1,2-dioxyacetic acid (bdoaH2): X-ray crystal structure of [Cu2(bdoa)(phen)4]bdoa·13H2O (phen = 1,10-phenanthroline)

Copper(II) acetate reacts with benzene-1,2-dioxyacetic acid (bdoaH2) in aqueous media to give [Cu(bdoa)(H2O)2] (1). Complex 1 reacts with the N-donor ligands pyridine (py), ammonia and 1,10-phenanthroline (phen) to give [Cu(bdoa)(NH3)2]·H2O (2), [Cu(bdoa)(py)2]·H2O (3) and [Cu2(bdoa)(phen)4]bdoa·13H2O (4), respectively. The X-ray crystal structure of the dicopper(II,II) complex 4 shows each copper atom at the centre of a distorted trigonal bipyramid comprising four nitrogen atoms from two chelating phen ligands and a single oxygen atom from one of the carboxylate moieties of the bridging bdoa2− ligand. The cyclic voltammogram of 4 shows a single reversible wave for the Cu2+/Cu+ couple at E = + 115 mV (vs Ag/AgCl). Spectroscopic and magnetic data for the complexes are given.

Combined transcriptomic-(1)H NMR metabonomic study reveals yhat monoethylhexyl phthalate stimulates adipogenesis and glyceroneogenesis in human adipocytes

Adipose tissue is a major storage site for lipophilic environmental contaminants. The environmental metabolic disruptor hypothesis postulates that some pollutants can promote obesity or metabolic disorders by activating nuclear receptors involved in the control of energetic homeostasis. In this context, monoethylhexyl phthalate (MEHP) is of particular concern since it was shown to activate the peroxisome proliferator-activated receptor γ (PPARγ) in 3T3-L1 murine preadipocytes. In the present work, we used an untargeted, combined transcriptomic-(1)H NMR-based metabonomic approach to describe the overall effect of MEHP on primary cultures of human subcutaneous adipocytes differentiated in vitro. MEHP stimulated rapidly and selectively the expression of genes involved in glyceroneogenesis, enhanced the expression of the cytosolic phosphoenolpyruvate carboxykinase, and reduced fatty acid release. These results demonstrate that MEHP increased glyceroneogenesis and fatty acid reesterification in human adipocytes. A longer treatment with MEHP induced the expression of genes involved in triglycerides uptake, synthesis, and storage; decreased intracellular lactate, glutamine, and other amino acids; increased aspartate and NAD, and resulted in a global increase in triglycerides. Altogether, these results indicate that MEHP promoted the differentiation of human preadipocytes to adipocytes. These mechanisms might contribute to the suspected obesogenic effect of MEHP.

Expression-system-dependent modulation of HIV-1 envelope glycoprotein antigenicity and immunogenicity.

Recombinant expression systems differ in the type of glycosylation they impart on expressed antigens such as the human immunodeficiency virus type 1 (HIV-1) envelope glycoproteins, potentially affecting their biological properties. We performed head-to-head antigenic, immunogenic and molecular profiling of two distantly related Env surface (gp120) antigens produced in different systems: (a) mammalian (293 FreeStyle cells; 293F) cells in the presence of kifunensine, which impart only high-mannose glycans; (b) insect cells (Spodoptera frugiperda, Sf9), which confer mainly paucimannosidic glycans; (c) Sf9 cells recombinant for mammalian glycosylation enzymes (Sf9 Mimic), which impart high-mannose, hybrid and complex glycans without sialic acid; and (d) 293F cells, which impart high-mannose, hybrid and complex glycans with sialic acid. Molecular models revealed a significant difference in gp120 glycan coverage between the Sf9-derived and wild-type mammalian-cell-derived material that is predicted to affect ligand binding sites proximal to glycans. Modeling of solvent-exposed surface electrostatic potentials showed that sialic acid imparts a significant negative surface charge that may influence gp120 antigenicity and immunogenicity. Gp120 expressed in systems that do not incorporate sialic acid displayed increased ligand binding to the CD4 binding and CD4-induced sites compared to those expressed in the system that do, and imparted other more subtle differences in antigenicity in a gp120 subtype-specific manner. Non-sialic-acid-containing gp120 was significantly more immunogenic than the sialylated version when administered in two different adjuvants, and induced higher titers of antibodies competing for CD4 binding site ligand-gp120 interaction. These findings suggest that non-sialic-acid-imparting systems yield gp120 immunogens with modified antigenic and immunogenic properties, considerations that should be considered when selecting expression systems for glycosylated antigens to be used for structure-function studies and for vaccine use.

Riluzole neuroprotection in a parkinson’s disease model involves suppression of reactive astrocytosis but not GLT-1 regulation

Background Riluzole is a neuroprotective drug used in the treatment of motor neurone disease. Recent evidence suggests that riluzole can up-regulate the expression and activity of the astrocyte glutamate transporter, GLT-1. Given that regulation of glutamate transport is predicted to be neuroprotective in Parkinson's disease, we tested the effect of riluzole in parkinsonian rats which had received a unilateral 6-hydroxydopamine injection into the median forebrain bundle. Results Rats were treated with intraperitoneal riluzole (4 mg/kg or 8 mg/kg), 1 hour before the lesion then once daily for seven days. Riluzole produced a modest but significant attenuation of dopamine neurone degeneration, assessed by suppression of amphetamine-induced rotations, preservation of tyrosine hydroxylase positive neuronal cell bodies in the substantia nigra pars compacta and attenuation of striatal tyrosine hydroxylase protein loss. Seven days after 6-hydroxydopamine lesion, reactive astrocytosis was observed in the striatum, as determined by increases in expression of glial fibrillary acidic protein, however the glutamate transporter, GLT-1, which is also expressed in astrocytes was not regulated by the lesion. Conclusions The results confirm that riluzole is a neuroprotective agent in a rodent model of parkinson’s disease. Riluzole administration did not regulate GLT-1 levels but significantly reduced GFAP levels, in the lesioned striatum. Riluzole suppression of reactive astrocytosis is an intriguing finding which might contribute to the neuroprotective effects of this drug.

Mozzarella-type curd made from buffalo, cows’ and ultrafiltered cows’ milk. 1. Rheology and microstructure

Rennet-induced curd was made from both natural buffalo and cows’ milk, and ultrafiltered cows’ milk (cows’ milk was concentrated such that it had a chemical composition approximately equivalent to that of the buffalo milk). These milk samples were compared on the basis of their rheology, physicochemical characteristics and curd microstructure. The ionic and soluble calcium contents were found to be similar in all milk samples studied. The total and casein bound calcium were higher in concentrated cows’ milk than in standard cows’ milk. Both cows’ milk types were found to have lower total and casein bound calcium than the buffalo milk. This is probably due to concentration of the colloidal part of milk (casein), during the ultrafiltration (UF) process. The rennet coagulation time was similar in UF cows’ and buffalo milk while both were shorter when compared with that of the cows’ milk. The dynamic moduli (G′, G″) values were higher in both the buffalo and UF cows’ milk than in the cows’ milk after 90 min coagulation. The loss tangent, however, was found to be similar in both the UF cows’ and buffalo milk curds and was lower than that observed for the cows’ milk (0.42, 0.42 and 0.48, respectively). The frequency profile of each type of curd was recorded 90 min after the enzyme addition (0.1–10 Hz); all samples were found to be “weak” viscoelastic, frequency dependent gels. The yield stress was also measured 95 min after the enzyme addition, and a higher value was observed in buffalo milk curd when compared with other curd samples made from both the natural cows’ milk and the UF cows’ milk. The cryo-scanning electron and confocal laser scanning micrographs showed that curd structure appeared to be more “dense” and less porous in buffalo milk than cows’ milk even after concentration to equivalent levels of protein/total solids to those found in the buffalo milk.

Endothelin-converting enzyme-1 regulates trafficking and signalling of the neurokinin 1 receptor in endosomes of myenteric neurones

Neuropeptide signalling at the plasma membrane is terminated by neuropeptide degradation by cell-surface peptidases, and by beta-arrestin-dependent receptor desensitization and endocytosis. However, receptors continue to signal from endosomes by beta-arrestin-dependent processes, and endosomal sorting mediates recycling and resensitization of plasma membrane signalling. The mechanisms that control signalling and trafficking of receptors in endosomes are poorly defined. We report a major role for endothelin-converting enzyme-1 (ECE-1) in controlling substance P (SP) and the neurokinin 1 receptor (NK(1)R) in endosomes of myenteric neurones. ECE-1 mRNA and protein were expressed by myenteric neurones of rat and mouse intestine. SP (10 nM, 10 min) induced interaction of NK(1)R and beta-arrestin at the plasma membrane, and the SP-NK(1)R-beta-arrestin signalosome complex trafficked by a dynamin-mediated mechanism to ECE-1-containing early endosomes, where ECE-1 can degrade SP. After 120 min, NK(1)R recycled from endosomes to the plasma membrane. ECE-1 inhibitors (SM-19712, PD-069185) and the vacuolar H(+)ATPase inhibitor bafilomycin A(1), which prevent endosomal SP degradation, suppressed NK(1)R recycling by >50%. Preincubation of neurones with SP (10 nM, 5 min) desensitized Ca(2+) transients to a second SP challenge after 10 min, and SP signals resensitized after 60 min. SM-19712 inhibited NK(1)R resensitization by >90%. ECE-1 inhibitors also caused sustained SP-induced activation of extracellular signal-regulated kinases, consistent with stabilization of the SP-NK(1)R-beta-arrestin signalosome. By degrading SP and destabilizing endosomal signalosomes, ECE-1 has a dual role in controlling endocytic signalling and trafficking of the NK(1)R: promoting resensitization of G protein-mediated plasma membrane signalling, and terminating beta-arrestin-mediated endosomal signalling.

Protein phosphatase 2A mediates resensitization of the neurokinin 1 receptor

Activated G protein-coupled receptors (GPCRs) are phosphorylated and interact with beta-arrestins, which mediate desensitization and endocytosis. Endothelin-converting enzyme-1 (ECE-1) degrades neuropeptides in endosomes and can promote recycling. Although endocytosis, dephosphorylation, and recycling are accepted mechanisms of receptor resensitization, a large proportion of desensitized receptors can remain at the cell surface. We investigated whether reactivation of noninternalized, desensitized (phosphorylated) receptors mediates resensitization of the substance P (SP) neurokinin 1 receptor (NK(1)R). Herein, we report a novel mechanism of resensitization by which protein phosphatase 2A (PP2A) is recruited to dephosphorylate noninternalized NK(1)R. A desensitizing concentration of SP reduced cell-surface SP binding sites by only 25%, and SP-induced Ca(2+) signals were fully resensitized before cell-surface binding sites started to recover, suggesting resensitization of cell-surface-retained NK(1)R. SP induced association of beta-arrestin1 and PP2A with noninternalized NK(1)R. beta-Arrestin1 small interfering RNA knockdown prevented SP-induced association of cell-surface NK(1)R with PP2A, indicating that beta-arrestin1 mediates this interaction. ECE-1 inhibition, by trapping beta-arrestin1 in endosomes, also impeded SP-induced association of cell-surface NK(1)R with PP2A. Resensitization of NK(1)R signaling required both PP2A and ECE-1 activity. Thus, after stimulation with SP, PP2A interacts with noninternalized NK(1)R and mediates resensitization. PP2A interaction with NK(1)R requires beta-arrestin1. ECE-1 promotes this process by releasing beta-arrestin1 from NK(1)R in endosomes. These findings represent a novel mechanism of PP2A- and ECE-1-dependent resensitization of GPCRs.

Endosomal endothelin-converting enzyme-1: a regulator of beta-arrestin-dependent ERK signaling

Neuropeptide signaling at the cell surface is regulated by metalloendopeptidases, which degrade peptides in the extracellular fluid, and beta-arrestins, which interact with G protein-coupled receptors (GPCRs) to mediate desensitization. beta-Arrestins also recruit GPCRs and mitogen-activated protein kinases to endosomes to allow internalized receptors to continue signaling, but the mechanisms regulating endosomal signaling are unknown. We report that endothelin-converting enzyme-1 (ECE-1) degrades substance P (SP) in early endosomes of epithelial cells and neurons to destabilize the endosomal mitogen-activated protein kinase signalosome and terminate signaling. ECE-1 inhibition caused endosomal retention of the SP neurokinin 1 receptor, beta-arrestins, and Src, resulting in markedly sustained ERK2 activation in the cytosol and nucleus, whereas ECE-1 overexpression attenuated ERK2 activation. ECE-1 inhibition also enhanced SP-induced expression and phosphorylation of the nuclear death receptor Nur77, resulting in cell death. Thus, endosomal ECE-1 attenuates ERK2-mediated SP signaling in the nucleus to prevent cell death. We propose that agonist availability in endosomes, here regulated by ECE-1, controls beta-arrestin-dependent signaling of endocytosed GPCRs.

Endothelin-converting enzyme 1 promotes re-sensitization of neurokinin 1 receptor-dependent neurogenic inflammation

BACKGROUND AND PURPOSE: The metalloendopeptidase endothelin-converting enzyme 1 (ECE-1) is prominently expressed in the endothelium where it converts big endothelin to endothelin-1, a vasoconstrictor peptide. Although ECE-1 is found in endosomes in endothelial cells, the role of endosomal ECE-1 is unclear. ECE-1 degrades the pro-inflammatory neuropeptide substance P (SP) in endosomes to promote recycling and re-sensitization of its neurokinin 1 (NK(1)) receptor. We investigated whether ECE-1 regulates NK(1) receptor re-sensitization and the pro-inflammatory effects of SP in the endothelium. EXPERIMENTAL APPROACH: We examined ECE-1 expression, SP trafficking and NK(1) receptor re-sensitization in human microvascular endothelial cells (HMEC-1), and investigated re-sensitization of SP-induced plasma extravasation in rats. KEY RESULTS: HMEC-1 expressed all four ECE-1 isoforms (a-d), and fluorescent SP trafficked to early endosomes containing ECE-1b/d. The ECE-1 inhibitor SM-19712 prevented re-sensitization of SP-induced Ca2+ signals in HMEC-1 cells. Immunoreactive ECE-1 and NK(1) receptors co-localized in microvascular endothelial cells in the rat. SP-induced extravasation of Evans blue in the urinary bladder, skin and ears of the rat desensitized when the interval between two SP injections was 10 min, and re-sensitized after 480 min. SM-19712 inhibited this re-sensitization. CONCLUSIONS AND IMPLICATIONS: By degrading endocytosed SP, ECE-1 promotes the recycling and re-sensitization of NK(1) receptors in endothelial cells, and thereby induces re-sensitization of the pro-inflammatory effects of SP. Thus, ECE-1 inhibitors may ameliorate the pro-inflammatory actions of SP.

Endothelin-converting enzyme-1 degrades internalized somatostatin-14

Agonist-induced internalization of somatostatin receptors (ssts) determines subsequent cellular responsiveness to peptide agonists and influences sst receptor scintigraphy. To investigate sst2A trafficking, rat sst2A tagged with epitope was expressed in human embryonic kidney cells and tracked by antibody labeling. Confocal microscopical analysis revealed that stimulation with sst and octreotide induced internalization of sst2A. Internalized sst2A remained sequestrated within early endosomes, and 60 min after stimulation, internalized sst2A still colocalized with beta-arrestin1-enhanced green fluorescence protein (EGFP), endothelin-converting enzyme-1 (ECE-1), and rab5a. Internalized (125)I-Tyr(11)-SST-14 was rapidly hydrolyzed by endosomal endopeptidases, with radioactive metabolites being released from the cell. Internalized (125)I-Tyr(1)-octreotide accumulated as an intact peptide and was released from the cell as an intact peptide ligand. We have identified ECE-1 as one of the endopeptidases responsible for inactivation of internalized SST-14. ECE-1-mediated cleavage of SST-14 was inhibited by the specific ECE-1 inhibitor, SM-19712, and by preventing acidification of endosomes using bafilomycin A(1). ECE-1 cleaved SST-14 but not octreotide in an acidic environment. The metallopeptidases angiotensin-1 converting enzyme and ECE-2 did not hydrolyze SST-14 or octreotide. Our results show for the first time that stimulation with SST-14 and octreotide induced sequestration of sst2A into early endosomes and that endocytosed SST-14 is degraded by endopeptidases located in early endosomes. Furthermore, octreotide was not degraded by endosomal peptidases and was released as an intact peptide. This mechanism may explain functional differences between octreotide and SST-14 after sst2A stimulation. Moreover, further investigation of endopeptidase-regulated trafficking of neuropeptides may result in novel concepts of neuropeptide receptor inactivation in cancer diagnosis.