Domains involved in the specificity of G protein activation in phospholipase C-coupled metabotropic glutamate receptors.

G protein-coupled glutamate receptors (mGluR) have recently been characterized. These receptors have seven putative transmembrane domains, but display no sequence homology with the large family of G protein-coupled receptors. They constitute therefore a new family of receptors.

Activity of oxyntomodulin on gastric acid secretion induced by histamine or a meal in the rat.

Conscious rats with chronic gastric fistula were trained for drinking a 14-ml milk meal. The activity of an intestinal hormone, oxyntomodulin (OXM), was studied in this model and compared to that observed when histamine was the stimulus. Under histamine (0.

Localization and characterization of vasopressin binding sites in the rat brain using an iodinated linear AVP antagonist.

The binding characteristics and central distribution of 125I-Linear AVP antagonist, a new ligand for vasopressin binding sites, are described in the following studies. Saturation studies performed on rat brain septal membranes demonstrated that 125I-Linear AVP antagonist binds to a single class of sites with high affinity (55 pM) and limited capacity (88 fmol/mg protein). In autoradiographic studies, 125I-Linear AVP antagonist labeled brain areas known to contain vasopressin receptors without binding to neurophysins.

NMDA-dependent superoxide production and neurotoxicity.

Neuronal injury resulting from acute brain insults and some neurodegenerative diseases implicates N-methyl-D-aspartate (NMDA) glutamate receptors. The fact that antioxidants reduce some types of brain damage suggests that oxygen radicals may have a role. It has been shown that mutations in Cu/Zn-superoxide dismutase (SOD), an enzyme catalysing superoxide (O2.

Glucagon-like peptide-1-(7-36) amide, oxyntomodulin, and glucagon interact with a common receptor in a somatostatin-secreting cell line.

Glucagon-like peptide-1(7-36)amide (tGLP-1), oxyntomodulin (OXM), and glucagon are posttranslational end products of the glucagon gene expressed in intestinal L-cells. In vivo, these peptides are potent inhibitors of gastric acid secretion via several pathways, including stimulation of somatostatin release. We have examined the receptors through which these peptides stimulate somatostatin secretion using the somatostatin-secreting cell line RIN T3.

Glutamate stimulates glucagon secretion via an excitatory amino acid receptor of the AMPA subtype in rat pancreas.

The effect of L-glutamate was studied on glucagon secretion from rat isolated pancreas perfused with 2.8 mM glucose. L-Glutamate (3.

GEP31, a new gastric epithelial protein, early expressed during ontogenesis.

A set of monoclonal antibodies directed against various gastric markers was produced in order to study the developmental expression of the gastric mucosa. A previously described monoclonal antibody, mab 146.14, labeled the proton pump (H+, K+) ATPase specifically located in gastric parietal cells.

Stimulation by glutamate receptors of arachidonic acid release depends on the Na+/Ca2+ exchanger in neuronal cells.

In primary cultures of striatal neurons, stimulation of N-methyl-D-aspartic acid (NMDA) receptors or associative activation (but not separate activation) of (RS)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors and metabotropic glutamate receptors (mGluR) strongly increased arachidonic acid (AA) release via activation of phospholipase A2 (PLA2). Depolarizing agents, such as veratridine, were as potent as NMDA in stimulating AA release. However, increasing the intracellular Ca2+ concentration via voltage-sensitive Ca2+ channels did not result in a significant stimulation of PLA2.

Cholecystokinin increases intracellular Ca2+ concentration in the Human JURKAT T Lymphocyte cell line.

We have recently demonstrated the presence of specific high-affinity cholecystokinin binding sites of the central type on the Human JURKAT T Lymphocyte Cell line. In this paper, changes in intracellular Ca2+ concentration ([Ca2+]i) in response to CCK (cholecystokinin) peptides were measured in the Human JURKAT T Lymphocyte Cell line by fura-2 fluorometry. CCK-8 (the C-terminal octapeptide of CCK), the potent CCK analog Boc-[Nle28,31]CCK-7, stimulated ([Ca2+]i) mobilization in a dose-dependent manner in cells preloaded with fura-2 AM with an EC50 of 2.

Molecular modeling of coiled-coil alpha-tropomyosin: analysis of staggered and in register helix-helix interactions.

In register and staggered models of tropomyosin coiled-coil were built from X-ray C alpha coordinates and refined via molecular dynamics. The two models show similar structural features with the X-ray structure of GCN4 leucine zipper. Empirical energetic methods used to compare the in register and staggered models indicate that both are equally probable.

Deglycosylation and fragmentation of purified rat liver angiotensin II receptor: application to the mapping of hormone-binding domains.

We report new structural data about the rat liver angiotensin II receptor, which belongs to the AT1 subclass. This receptor has been purified at analytical or semi-preparative levels by a previously described strategy involving its photolabelling with a biotinylated azido probe and selective adsorption of the covalent probe-receptor complexes to immobilized streptavidin [Marie, Seyer, Lombard, Desarnaud, Aumelas, Jard and Bonnafous (1990) Biochemistry 29, 8943-8950]. Chemical or enzymic deglycosylation of the purified receptor has shown a shift in its molecular mass from 65 kDa to 40 kDa.

Non-classical glutamate receptors, blocked by both NMDA and non-NMDA antagonists, stimulate nitric oxide production in neurons.

In striatal neurons in primary culture, kainate and domoate stimulated cGMP production, whereas two other analogs of glutamate which act at non-NMDA receptors, alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionate (AMPA) and quisqualate were ineffective. However, both agonists stimulated cGMP accumulation on neurons pretreated with concanavalin A, a lectin which is known to prevent desensitization of AMPA receptors. We show here that such a treatment also potentiated the kainate-stimulated cGMP production.

Characterization of homologous 5-hydroxytryptamine4 receptor desensitization in colliculi neurons.

Exposure of mouse colliculi neurons to selective 5-hydroxytryptamine (5-HT)4 agonists was accompanied by a rapid desensitization of the receptor-stimulated adenylyl cyclase response. Half-maximal desensitization occurred after 2 min. Only exposure of neurons to selective 5-HT4 agonists led to a potent desensitization of the 5-HT4-mediated response.

Measurement of gamma-enolase release, a new method for selective quantification of neurotoxicity independently from glial lysis.

We have developed a sensitive enzymatic-immunoassay to quantify the level of gamma-enolase (a specific neuronal enzyme) which is released from cultured cells after exposure to various toxins. We show that this method can estimate selectively neuronal cell death without significantly interfering with glial cell death. Indeed, no gamma-enolase is released when glial cells are killed with free-radical producing agents.

Differential coupling of 5-HT1A receptors occupied by 5-HT or 8-OH-DPAT to adenylyl cyclase.

Human serotonin (5-hydroxytryptamine, 5-HT)-1A receptors have been transfected in NIH-3T3 cells, and their coupling to adenylyl cyclase was analysed depending on 1) the number of receptor expressed, 2) the experimental conditions used, 3) the nature of the agonists. Two monoclonal cell lines were used, expressing low (45 fmol/mg) and high (500 fmol/mg) levels of 5-HT1A receptor. Two methods were tested to study the negative coupling of the transfected 5-HT1A receptors to adenylyl cyclase: 1) measurement of cAMP production in intact cells, 2) measurement of adenylyl cyclase activity in vitro on membrane preparations.

Kinetics of amide proton exchange in parvalbumin studied by 1H 2-D NMR. A comparison of the calcium and magnesium loaded forms.

The amide proton exchange rates have been measured for the pike parvalbumin loaded either with calcium (PaCa2) or with magnesium (PaMg2) by using 2-D total correlation spectroscopy experiments. The differences in the exchange rates observed between these two species were unexpected when compared with the small conformational changes induced in parvalbumin by the Ca/Mg exchange. With the calcium-loaded protein (PaCa2), a significant difference was observed for the amide proton exchange rates of residues located in the N-terminal domain AB in contrast to the slower exchange rates that were observed in the CD and EF domains.

Activation of 5-HT1A receptors expressed in NIH-3T3 cells induces focus formation and potentiates EGF effect on DNA synthesis.

NIH-3T3 fibroblasts have been transfected with human serotonin 5-HT1A receptors. Clonal cell lines expressed between 40 and 500 fmol receptor/mg. 5-HT1A agonists strongly inhibited nonstimulated- as well as forskolin- or isoproterenol-stimulated adenylyl cyclase.

Both enantiomers of 1-aminocyclopentyl-1,3-dicarboxylate are full agonists of metabotropic glutamate receptors coupled to phospholipase C.

We tested the effects of two enantiomers of a glutamate analogue, (trans)-1-aminocyclopentyl-1,3-dicarboxylate (t-ACPD), in striatal and cerebellar neurons in primary culture, as well as in Xenopus oocytes injected with cerebellar rat RNA. In the presence of MK-801, to avoid N-methyl-D-aspartate receptor activation, and 3 microM tetrodotoxin, both enantiomers [(1R,3S)- and (1S,3R)-t-ACPD] stimulated inositol phosphate (InsP) formation both in striatal neurons after 9-11 days in vitro [EC50, 3.7 +/- 1.

Protein purification using combined streptavidin (or avidin)-Sepharose and thiopropyl-Sepharose affinity chromatography.

The major problem usually encountered in the application of the (strept)avidin-biotin system to the purification of proteins (or other biological molecules) lies in the difficult reversion of the interaction between immobilized (strept)avidin and the adsorbed biotinylated protein. Among the proposed solutions is the selective biotinylation of the entity to be purified by a disulphide-containing biotinylated reagent which allows its recovery from (strept)avidin gels by dithiothreitol (DTT) treatment. As emphasized by the example of angiotensin II receptor purification, achieved using this strategy, optimum reduction of this disulphide bridge may require improvement of its accessibility using denaturating agents such as sodium dodecyl sulphate or urea.

Transfection of human 5-hydroxytryptamine1A receptors in NIH-3T3 fibroblasts: effects of increasing receptor density on the coupling of 5-hydroxytryptamine1A receptors to adenylyl cyclase.

Human serotonin [5-hydroxytryptamine (5-HT)1A] receptors have been transfected in NIH-3T3 cells, and their pharmacology and coupling to adenylyl cyclase have been analyzed. Three cellular preparations were used, 1) monoclonal cell lines (clones 6, 2B, and 4B), expressing 45, 280, and 500 fmol of 5-HT1A receptors/mg of protein, respectively; 2) clones 6, 2B, and 4B in which the concentration of 5-HT1A receptors was increased after stimulation of the glucocorticoid-inducible promoter with dexamethasone; and 3) polyclonal cell lines that expressed an increasing amount of 5-HT1A receptor as a function of cell passage. The transfected 5-HT1A receptors inhibited basal, forskolin-stimulated, and isoproterenol-stimulated adenylyl cyclase.

Oxyntomodulin-like immunoreactivity: diurnal profile of a new potential enterogastrone.

The biological specificity of oxyntomodulin toward the gastric mucosa results from its C-terminal octapeptide. A RIA using a specific antibody raised against this region permitted quantification of the whole set of proglucagon-derived peptides that interact with the oxyntomodulin recognition systems, corresponding to the new concept of oxyntomodulin-like-immunoreactivity (OLI). The present report describes the physiological 24-h OLI profile in human plasma (eight men and eight women; mean age, 45 yr; range, 20-77 yr).

Evidence for a glutamate receptor of the AMPA subtype which mediates insulin release from rat perfused pancreas.

1. The effect of L-glutamate has been studied on insulin secretion by the isolated perfused pancreas of the rat. The glutamate receptor subtype involved has been characterized.

Pharmacological studies on CCKB receptors in guinea pig synaptoneurosomes.

Preliminary studies on CCK receptors in the central nervous system were carried out on guinea pig cerebral cortical synaptoneurosome preparations. In binding assays, the range of affinity of CCK-8, Boc-[Nle28,Nle31]CCK-7, a potent CCK analog, Boc-[Leu31]CCK-4 and of the two benzodiazepine CCK receptor antagonists L-365,260 and MK-329, is in agreement with the presence of CCKB receptors on this model. The effects of Boc-[Nle28,Nle31]CCK-7 on inositol phosphates, cAMP accumulation and 45Ca2+ efflux were investigated.