Specific binding of triatiated neurotensin to rat brain membranes: Characterization and regional distribution

Goedert, Michel; Pittaway, K.; Williams, Betty J.; Emson, Piers C.

doi:10.1016/0006-8993(84)90862-x

Cited by 97 publications

(9 citation statements)

References 40 publications

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“…The equilibrium binding studies suggest the presence of a single population of sites with an apparent K D of 3.3 nM and a B,,, of 350 fmol/mg protein. This K D value is in agreement with that obtained in similar studies in human cortical membranes (Kanba et al, 1986) or rat brain membranes (Kitabgi et al, 1977;Uhl et al, 1977;Goedert et al, 1984).…”

Section: Binding Characteristics Of [3h]neurotensin In Intact Membranessupporting

confidence: 91%

“…Radiolabelled ligand binding studies have established the presence of specific neurotensin-binding sites in brain homogenates of a number of mammalian species, including rat (Kitabgi et al, 1977;Laza-rus et al, 1977;Uhl et al, 1977;Goedert et al, 1984), guinea pig (Sadoul et al, 1984a), mouse (Nakagawa et al, 1984), and human (Sadoul et al, 19846;Kanba et al, 1986), as well as in the membranes of neuronal (Nakagawa et al, 1984: Poustis et al, 1984 and certain non-neuronal cell lines (Kitabgi et al, 1980). It is tempting to think that these sites mark specific neurotensin receptor proteins of the membrane.…”

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confidence: 99%

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Characterization of Neurotensin Binding Sites in Intact and Solubilized Bovine Brain Membranes

Mills

Demoliou-Mason

Barnard

1988

Journal of Neurochemistry

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Analysis of the equilibrium binding of [3H]-neurotensin(1-13) at 25 degrees C to its receptor sites in bovine cortex membranes indicated a single population of sites with an apparent equilibrium dissociation constant (KD) of 3.3 nM and a density (Bmax) of 350 fmol/mg protein (Hill coefficient nH = 0.97). Kinetic dissociation studies revealed the presence of a second class of sites comprising less than 10% of the total. KD values of 0.3 and 2.0 nM were obtained for the higher and lower affinity classes of sites, respectively, from association-dissociation kinetic studies. The binding of [3H]neurotensin was decreased by cations (monovalent and divalent) and by a nonhydrolysable guanine nucleotide analogue. Competition studies gave a potency ranking of [Gln4]neurotensin greater than neurotensin(8-13) greater than neurotensin(1-13). Smaller neurotensin analogues and neurotensin-like peptides were unable to compete with [3H]neurotensin. Stable binding activity for [3H]neurotensin in detergent solution (Kd = 5.5 nM, Bmax = 250 fmol/mg protein, nH = 1.0) was obtained in 2% digitonin/1 mM Mg2+ extracts of membranes which had been preincubated (25 degrees C, 1 h) with 1 mM Mg2+ prior to solubilization. Association-dissociation kinetic studies then revealed the presence of two classes of sites (KD1 = 0.5 nM, KD2 = 3.6 nM) in a similar proportion to that found in the membranes. The solubilized [3H]-neurotensin activity retained its sensitivity to cations and guanine nucleotide.

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Section: Binding Characteristics Of [3h]neurotensin In Intact Membranessupporting

confidence: 91%

mentioning

confidence: 99%

Characterization of Neurotensin Binding Sites in Intact and Solubilized Bovine Brain Membranes

Mills

Demoliou-Mason

Barnard

1988

Journal of Neurochemistry

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“…Reactions were carried out in 50 mM Tris-HCl (pH 7.4) containing 0.04% bacitracin, 0.1% bovine serum albumin, and 1 mM Na 2 EDTA at 25°C for 60 min. Neurotensin (1.0 M) was used to define nonspecific binding (36,37).…”

Section: Receptor Binding Assaysmentioning

confidence: 99%

Isolation and Characterization of a Novel ConusPeptide with Apparent Antinociceptive Activity

McIntosh

Corpuz

Layer

et al. 2000

Journal of Biological Chemistry

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Cone snails are tropical marine mollusks that envenomate prey with a complex mixture of neuropharmacologically active compounds. We report the discovery and biochemical characterization of a structurally unique peptide isolated from the venom of Conus marmoreus. The new peptide, mr10a, potently increased withdrawal latency in a hot plate assay (a test of analgesia) at intrathecal doses that do not produce motor impairment as measured by rotarod test. The sequence of mr10a is NGVCCGYKLCHOC, where O is 4-trans-hydroxyproline. This sequence is highly divergent from all other known conotoxins. Analysis of a cDNA clone encoding the toxin, however, indicates that it is a member of the recently described T-superfamily. Total chemical synthesis of the three possible disulfide arrangements of mr10a was achieved, and elution studies indicate that the native form has a disulfide connectivity of Cys1-Cys4 and Cys2-Cys3. This disulfide linkage is unprecedented among conotoxins and defines a new family of Conus peptides.Conus is a genus of predatory marine gastropods that envenomate their prey. Prey capture is accomplished through a sophisticated arsenal of peptides that target specific ion channel and receptor subtypes. Each Conus venom appears to contain a unique set of 50 -200 peptides. The structure and function of only a small minority of these peptides have been determined to date. For peptides where function has been determined, three classes of targets have been elucidated: voltage-gated ion channels, ligand-gated ion channels, and G-protein-linked receptors.Conus peptides that target voltage-gated ion channels include those that delay the inactivation of sodium channels as well as blockers specific for sodium channels, calcium channels, and potassium channels. Peptides that target ligand-gated ion channels include antagonists of N-methyl-D-aspartate and serotonin receptors as well as competitive and non-competitive nicotinic receptor antagonists. Peptides that act on G protein receptors include neurotensin and vasopressin receptor agonists. The unprecedented targeting selectivity of conotoxins derives from specific disulfide bond frameworks combined with hypervariable amino acids within disulfide loops (see Ref. 1 for review). Due to the high potency and exquisite selectivity of the conopeptides, several are in various stages of clinical development for treatment of human disorders (2).In this report we describe the isolation of a new peptide from the venom of the marble cone, Conus marmoreus. C. marmoreus is found in the Indo-Pacific, from India to the Marshall Islands and Fiji. It preys upon various gastropods including other cone snails (3). We previously reported the isolation and characterization of a peptide from this venom that potently inhibits voltage-gated sodium channels (4). In this report, we describe the isolation of a novel peptide that appears antinociceptive and likely represents a defining member of a new family of Conus peptides. EXPERIMENTAL PROCEDURES Materials and HPLC 1 ConditionsThe venom of C...

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“…Nevertheless, the consistency of our estimates with those of others for a high-affinity binding site for NT is noteworthy, since our experiments were conducted in a physiological medium. Several laboratories concur that cations, especially sodium ions at physiological concentrations, significantly decrease the affinity of NT for its specific binding site (Ahmad et al, 1987;Goedert et al, 1984b;Mills et al, 1988). Therefore, other ions present in the medium must counteract the inhibitory effect of sodium.…”

Section: Discussionmentioning

confidence: 98%

Neurotensin binding sites in porcine jejunum: Biochemical Characterization and intramural Localization

Seybold¹,

Treder²,

Aaonsen

et al. 1990

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Neurotensin is present in high concentrations in the mammalian gut, especially in enteroendocrine cells of the mucosa. Exogenous neurotensin has been shown to alter ion transport by the mucosa and contractile activity of intestinal smooth muscle. The purpose of this study was to determine the distribution of neurotensin binding sites within the intestinal wall. Initially, biochemical characteristics of [125I]neurotensin binding sites were determined within two preparations of the distal porcine jejunum: (1) the mucosa and submucosa, and (2) the circular and longitudinal muscle with their intramural plexuses. Ligand binding data for the preparation including the mucosa and submucosa indicated that [125I]neurotensin bound specifically to two sites having apparent equilibrium dissociation constants of approximately 0.046 and 0.37 nM. A binding site with a dissociation constant of approximately 0.38 nM was confirmed for the preparation of muscle and associated intramural plexuses. Xenopsin and neurotensin were equipotent to neurotensin in competing for these binding sites; neuromedin N was approximately 40 times less potent in the preparation of mucosa and submucosa. Receptor autoradiography was used to determine the distribution of [125I]neurotensin binding sites within the wall of the jejunum. Autoradiograms of [125I]neurotensin bound to cross sections of the proximal and distal jejunum showed that the highest densities of silver grains were associated with the internal submucosal ganglia, external submucosal plexus and myenteric ganglia. A moderate density of silver grains was associated with the circular muscle. The localization of neurotensin binding sites to submucosal ganglia is consistent with observations that neurotensin effects on active anion secretion by the mucosa are blocked by tetrodotoxin. Immunohistochemical localization of neurotensin in the porcine jejunum demonstrated a limited population of neurotensin immunoreactive cells within the mucosal epithelium. It is possible that neurotensin released from these cells in the mucosa as well as neurotensin-related peptides released from enteric neurons may be the endogenous ligands for the binding sites visualized in this study.

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Specific binding of triatiated neurotensin to rat brain membranes: Characterization and regional distribution

Cited by 97 publications

References 40 publications

Characterization of Neurotensin Binding Sites in Intact and Solubilized Bovine Brain Membranes

Characterization of Neurotensin Binding Sites in Intact and Solubilized Bovine Brain Membranes

Isolation and Characterization of a Novel ConusPeptide with Apparent Antinociceptive Activity

Neurotensin binding sites in porcine jejunum: Biochemical Characterization and intramural Localization

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