Distribution of opioid receptors in canine small intestine: implications for function

1989 ◽  
Vol 256 (6) ◽  
pp. G966-G974 ◽  
Author(s):  
H. D. Allescher ◽  
S. Ahmad ◽  
P. Kostka ◽  
C. Y. Kwan ◽  
E. E. Daniel
Keyword(s):  
Small Intestine ◽  
Binding Sites ◽  
Myenteric Plexus ◽  
Plasma Membranes ◽  
Binding Capacity ◽  
Opiate Receptors ◽  
Circular Muscle ◽  
Maximum Binding Capacity ◽  
Opiate Binding ◽  
Subtype Selective

Distribution of the binding sites for [3H]diprenorphine, a non-selective opiate ligand, was studied in membrane fractions from longitudinal muscle/myenteric plexus and circular muscle containing deep muscular plexus. [3H]saxitoxin was used as a marker for neuronal plasma membranes and 5'-nucleotidase as a marker for smooth muscle plasma membranes. Saxitoxin binding correlated strongly with diprenorphine binding, but 5'-nucleotidase correlated poorly with diprenorphine or saxitoxin binding in these fractions. Opiate binding sites in membranes of myenteric and deep muscular plexus were of high affinity (Kd = 0.12 and 0.18 nM, respectively) with maximum binding capacity of 400 and 500 fmol/mg protein, respectively. Competition experiments using subtype-selective opiate ligands indicated that all three subtypes of opiate receptors were present in the same ratio of 40-45% mu-subtypes, 40-45% delta-subtypes, and 10-15% kappa-subtypes on both plexuses. Opiate receptors of canine small intestine, therefore, are located primarily or exclusively on nerves with similar distributions in nerve membranes containing only axonal varicosities (deep muscular plexus) as in those containing neurons, dendrites, and varicosities (myenteric plexus).

α2-Adrenergic receptors on the circular smooth muscle of canine small intestine

1991 ◽  
Vol 69 (6) ◽  
pp. 837-840 ◽  
Author(s):  
Sultan Ahmad ◽  
Hans D. Allescher ◽  
Elliott Jang ◽  
Jarmila Rausa ◽  
Edwin E. Daniel
Keyword(s):  
Smooth Muscle ◽  
Small Intestine ◽  
Binding Sites ◽  
Adrenergic Receptors ◽  
Plasma Membranes ◽  
Circular Muscle ◽  
Enteric Neurons ◽  
Hill Coefficient ◽  
Adrenergic Agents ◽  
Circular Smooth Muscle

We have studied the distribution and properties of α2-adrenergic receptors in the circular muscle layer (containing deep muscular plexus) of canine small intestine. Using radioactively labelled rauwolscine, we located the binding sites to the neuronal membranes supporting the prejunctional action of α2-adrenergic agents in the gut. Moreover, although the functional data to suggest the existence of postjunctional α2-adrenergic receptors coupled to contraction are not available so far, we measured a substantial number of rauwolscine binding sites on the smooth muscle plasma membranes. Scatchard and Hill analyses of the saturation data were indicative of the presence of a single high affinity site (Hill coefficient 0.996) with a KD value of 8.8 nM and the maximum number of binding sites (Bmax) of 313 fmol/mg of protein. Competition studies suggested the presence of multiple subtypes of α2-adrenoceptors.Key words: [3H]rauwolscine, prejunctional, postjunctional, smooth muscle, enteric neurons.

Agonist-induced internalisation of the angiotensin II-binding sites from plasma membranes of isolated rat hepatocytes

1997 ◽  
Vol 152 (3) ◽  
pp. 407-412 ◽  
Author(s):  
M Montiel ◽  
M C Caro ◽  
E Jiménez
Keyword(s):  
Plasma Membrane ◽  
Angiotensin Ii ◽  
Binding Sites ◽  
Plasma Membranes ◽  
Binding Capacity ◽  
Rat Hepatocytes ◽  
Receptor Complex ◽  
Ang Ii ◽  
Isolated Rat Hepatocytes ◽  
Isolated Rat

Angiotensin II (Ang II) provokes rapid internalisation of its receptor from plasma membranes in isolated rat hepatocytes. After 10 min stimulation with Ang II, plasma membrane lost about 60% of its 125I-Ang II-binding capacity. Internalisation was blocked by phenylarsine oxide (PhAsO), whereas okadaic acid, which markedly reduced the sustained phase of calcium mobilization, did not have a preventive effect on Ang II–receptor complex sequestration. These data suggest that Ang II receptor internalisation is probably independent of a phosphorylation/dephosphorylation cycle of critical serine/threonine residues in the receptor molecule. To establish a relationship between sequestration of the Ang II receptor and the physical properties of the Ang II-binding sites, 125I-Ang II–receptor complex profiles were analysed by isoelectric focusing. In plasma membrane preparations two predominant Ang II-binding sites, migrating to pI 6·8 and 6·5 were found. After exposure to Ang II, cells lost 125I-Ang II-binding capacity to the Ang II–receptor complex migrating at pI 6·8 which was prevented in PhAsO-treated cells. Pretreatment of hepatocytes with okadaic acid did not modify Ang II–receptor complex profiles, indicating that the binding sites corresponding to pI 6·5 and pI 6·8 do not represent a phosphorylated and/or non-phosphorylated form of the Ang II receptor. The results show that the Ang II–receptor complex isoform at pI 6·8 represents a functional form of the type-1 Ang II receptor. Further studies are necessary to identify the Ang II-related nature of the binding sites corresponding to pI 6·5. Journal of Endocrinology (1997) 152, 407–412

Characterization of galanin receptor in canine small intestinal circular muscle synaptosomes

1994 ◽  
Vol 266 (1) ◽  
pp. G106-G112 ◽  
Author(s):  
C. K. Chen ◽  
T. J. McDonald ◽  
E. E. Daniel
Keyword(s):  
Small Intestine ◽  
Binding Site ◽  
G Protein ◽  
Binding Capacity ◽  
Specific Binding ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Circular Muscle ◽  
Galanin Receptor ◽  
High Affinity

We used 125I-galanin (porcine) as ligand to study the galanin receptors in circular muscle and deep muscular plexus from canine small intestine. Specific binding sites were found in both nerve and muscle membranes. On synaptosomal membranes, the equilibrium binding study showed a high-affinity (dissociation constant, Kd = 1.1 +/- 0.13 nM; maximum binding capacity, Bmax = 244 +/- 2.1 fmol/mg) binding site. The specific binding of 125I-galanin to nerve membrane was inhibited by galanin or NH2-terminal galanin fragments but not by the COOH-terminal fragment. Computer analysis suggested a two-site model (inhibitor constants, Ki1 = 0.02 +/- 0.005 nM and Ki2 = 1.05 +/- 0.3 nM) for competition by galanin-(1-29). Kinetic and competition studies using guanosine 5'-O-(3-thiotriphosphate) or pertussis toxin (PTX) suggested that the high-affinity binding site involved a PTX-sensitive G protein which acted to slow dissociation of bound galanin from the receptor. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the galanin receptor complex revealed a radioactive band at 50 kDa. We conclude that, in canine small intestine, galanin may act as an inhibitory neuromodulator by a PTX-sensitive G protein-coupled interaction of galanin and its specific receptor on enteric nerve synaptosomes

Uptake of 3,5,3′-l-tri-iodothyronine in human erythrocytes

1989 ◽  
Vol 121 (3) ◽  
pp. 585-591 ◽  
Author(s):  
K. Yamauchi ◽  
R. Horiuchi ◽  
H. Takikawa
Keyword(s):  
Binding Sites ◽  
Binding Capacity ◽  
Human Erythrocytes ◽  
The Other ◽  
Transport Pathway ◽  
High Affinity ◽  
High Affinity Site ◽  
Maximum Binding Capacity ◽  
Energy Dependent ◽  
Dependent Pathway

ABSTRACT The mechanisms of 3,5,3′-l-tri-iodothyronine (T3) uptake into human erythrocytes were examined. Purified membranes of human erythrocytes were shown to have two classes of T3-binding sites with one being a high-affinity site (dissociation constant, 59·2±17·8 nmol/l; maximum binding capacity, 344·3 ± 95·5 fmol/μg protein). Furthermore, it was shown that there were two pathways for T3 uptake in human erythrocytes; one was saturable, stereospecific (T3»thyroxine > 3,5,3′-d-tri-iodothyronine), energydependent and dominant at 15 °C; the other was not displaced by unlabelled T3 and was energyindependent but did not occur by passive diffusion. The former pathway which, it is suggested, is a receptor-mediated transport pathway, was inhibited by monodansylcadaverine, phloretin or oligomycin at 15 or 37 °C, but the latter pathway was not inhibited by these inhibitors. Our results strongly suggest that uptake of T3 by the energy-independent pathway became predominant over the energy-dependent pathway at 37 °C and accounted for 83% of total T3 uptake of human erythrocytes. Journal of Endocrinology (1989) 121, 585–591

Analysis of the lipoprotein binding site of rat liver membranes

1994 ◽  
Vol 72 (3-4) ◽  
pp. 132-142 ◽  
Author(s):  
Lynda Adam ◽  
Louise Brissette
Keyword(s):  
Membrane Proteins ◽  
Rat Liver ◽  
Binding Sites ◽  
Plasma Membranes ◽  
Binding Capacity ◽  
New Information ◽  
Liver Membrane ◽  
Liver Membranes ◽  
Bona Fide ◽  
Lipoprotein Binding

Intermediate density lipoproteins (IDL) were shown to bind to high- and low-affinity binding sites on rat liver membranes. The low-affinity sites were named lipoprotein binding sites (LBS), since they bind all classes of lipoproteins. This study was undertaken to further characterize the interaction of 125I-labelled IDL with the LBS of rat liver membranes to determine the chemical nature of the LBS. We found that the binding of IDL to the LBS is insensitive to EDTA and sensitive to heparin and that it is present on plasma membranes. Also, membranes were pretreated with various enzymes that have an effect on the membrane constituents, and the activity of the LBS on these treated membranes was determined. Our results reveal that the LBS of rat liver membranes is insensitive to heparinase I, chondroitinase ABC, and phospholipase C, while it is partially sensitive to phospholipase A2 and sensitive to proteases and heat. Rat liver membrane proteins were solubilized with Triton X-100, reconstituted in liposomes, and analyzed for their ability to bind lipoproteins. 125I-labelled IDL were shown to bind to high- and low-affinity sites that are similar, in affinity and specificity, to the ones observed with intact rat liver membranes, indicating that a LBS activity is detectable on these liposomes. We found that the binding capacity of low-affinity sites in liposomes containing either no protein or containing proteins solubilized from Escherichia coli membranes is five times weaker than low-affinity sites in liposomes containing liver membrane proteins. Thus, a protein solubilized from rat liver membranes has LBS activity when reconstituted in liposomes. Taken altogether our results provide new information on the binding of IDL to the LBS and indicate that the LBS activity is in part mediated by a protein. Thus, the LBS appears to be a bona fide receptor.Key words: lipoprotein, receptor, binding, metabolism.

Interference with thyrotropin receptor antibody determination by a spuriously occurring anti-bovine TSH antibody

1986 ◽  
Vol 112 (2) ◽  
pp. 197-203 ◽  
Author(s):  
Makoto Iitaka ◽  
Toshinori Tanikawa ◽  
Yoshiki Sakatsume ◽  
Morifumi Yanagisawa ◽  
Yoshihito Hara ◽  
...  
Keyword(s):  
Binding Sites ◽  
Binding Capacity ◽  
Binding Activity ◽  
Tsh Receptor ◽  
Thyrotropin Receptor ◽  
Pepsin Digestion ◽  
Thyrotropin Receptor Antibody ◽  
Maximum Binding Capacity ◽  
Antibody Determination ◽  
Bovine Tsh

Abstract. Abnormally negative values of thyrotropin binding inhibitor immunoglobulin (TBII) were found in the sera from a patient with Graves' disease. This was due to the presence of potent bovine TSH (bTSH) binding activity in the sera. This activity was demonstrated to be in immunoglobulin G (IgG) with a λ light chain isotype, which was shown to have an affinity for bTSH with a Ka value of 3.5 × 1010 m−1 and a maximum binding capacity of 1.1 × 10−14m/mg IgG. F(ab')2 fragments obtained through pepsin digestion from the patient's IgG retained bTSH binding activity. [125I] bTSH binding to this IgG was inhibited by the TSH receptor. The inhibition was not completely competitive, suggesting the presence of different binding sites for this IgG and the TSH receptor on the TSH molecule. This IgG, however, could not bind labelled human TSH (hTSH). Since neither TSH nor other pituitary derivatives had ever been given to the patient, this bTSH binding activity was considered to be due to a spuriously occurring anti-bTSH antibody.

Characterization of binding sites for VIP-related peptides and activation of adenylate cyclase in developing pancreas

1991 ◽  
Vol 260 (2) ◽  
pp. G265-G274 ◽  
Author(s):  
V. Le Meuth ◽  
N. Farjaudon ◽  
W. Bawab ◽  
E. Chastre ◽  
G. Rosselin ◽  
...  
Keyword(s):  
Adenylate Cyclase ◽  
Postnatal Development ◽  
Binding Sites ◽  
Plasma Membranes ◽  
Early Stage ◽  
Binding Capacity ◽  
Human Growth ◽  
Inhibitory Peptide ◽  
Camp Generation ◽  
Glucagon Like Peptide 1

HPLC-purified 125I-labeled vasoactive intestinal peptide (VIP) bound in a specific, saturable, and reversible manner to pancreatic plasma membranes isolated from newborn calves, from milk-fed calves at 28 and 119 days, and from weaned calves at 119 days. A series of VIP analogues, including pituitary adenylate cyclase-activating polypeptide (PACAP), displaced 125I-VIP binding and activated adenylate cyclase in the same order of relative potency: PACAP-38 greater than helodermin greater than VIP, PACAP-27 greater than PHM (human peptide with NH2-terminal histidine and COOH-terminal methionine amide). At maximally effective concentrations, these five peptides produced the same two- to threefold increase of adenylate cyclase activity in pancreatic membranes from newborn and 28-day-old calves, and fourfold in ruminant or preruminant animals at 119 days. The activation constant for PACAP-38 ranged from 0.1 to 0.34 nM throughout the postnatal development. Helospectin I and II were three times less potent than VIP in inhibiting 125I-VIP binding. At concentrations up to 0.1 microM, secretin, rat and human growth hormone-releasing factors, glucagon, oxyntomodulin, the truncated form of glucagon-like peptide-1 lacking the 6 NH2-terminal amino acid sequence (TGLP-1), GLP-2, gastric inhibitory peptide, gastrin, CCK, and insulin had no effect on binding. Scatchard plots from 28- and 119-day-old calves were compatible with the presence of two classes of 125I-VIP binding sites: one with a high affinity for VIP and a low binding capacity (Kd = 0.11-0.4 nM, Bmax = 66-174 fmol/mg protein) and the other with a low affinity and high binding capacity. At birth, only one class of binding sites was observed (Kd = 0.4 nM, Bmax = 858 fmol/mg protein). The covalently cross-linked PACAP-preferring 125I-VIP binding site is a glycoprotein of 55 kDa with higher sensitivity to PACAP vs. helodermin and VIP. Our results suggest that calf pancreatic functions might be regulated at an early stage of postnatal development by PACAP receptors linked to cAMP generation.

Peptide YY receptor in submucosal and myenteric plexus synaptosomes of canine small intestine

1996 ◽  
Vol 271 (1) ◽  
pp. G36-G41 ◽  
Author(s):  
Y. K. Mao ◽  
Y. F. Wang ◽  
G. Ward ◽  
S. Cipris ◽  
E. E. Daniel ◽  
...  
Keyword(s):  
Small Intestine ◽  
Binding Sites ◽  
Peptide Yy ◽  
Binding Capacity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Receptor Complex ◽  
Submucosal Plexus ◽  
Small Intestinal ◽  
Dissociation Constant Kd

PYY receptors were characterized and their loci determined in canine small intestine. The density of 125I-labeled peptide tyrosine tyrosine (PYY) binding was highest in myenteric (MY) and submucosal (SUB) plexus fractions enriched in synaptosomes. Two binding sites [high affinity (H) and low affinity (L)] were found in the submucosal synaptosome-enriched membrane: dissociation constant (Kd)H = 7.6 pM, maximal binding capacity (Bmax)H = 28 fmol/mg; KdL = 0.18 nM, BmaxL = 120 fmol/mg protein. The binding of 125I-PYY reached a maximum within 30 min; dissociation was incomplete in the presence of unlabeled PYY. The rate of dissociation was enhanced after exposure of synaptosomes to guanosine 5'-O-(3-thiotriphosphate). Binding of 125I-PYY was completely inhibited by neuropeptide Y (NPY)-(13-36) (in SUB and MY) and by [Leu31,Pro34]NPY (in MY) but only partially by [Leu31,Pro34]NPY in SUB, suggesting the presence of Y2 receptor in SUB and the presence of Y1 and Y2 receptors in MY. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the PYY receptor complex revealed a radioactive band at 70 kDa. The PYY receptors in the canine small intestinal myenteric and submucosal plexus correspond in location to that of PYY in synaptosomes and are coupled with G proteins. Different subtypes are present in different loci.

Human syncytiotrophoblast NPY receptors are located on BBM and activate PLC-to-PKC axis

1998 ◽  
Vol 274 (3) ◽  
pp. E502-E509 ◽  
Author(s):  
Jacques Robidoux ◽  
Lucie Simoneau ◽  
Serge St-Pierre ◽  
Hafid Ech-Chadli ◽  
Julie Lafond
Keyword(s):  
Binding Sites ◽  
Plasma Membranes ◽  
Peptide Yy ◽  
Binding Capacity ◽  
Rank Order ◽  
Receptor Activation ◽  
Response Curves ◽  
Pi3k Activation ◽  
Basal Plasma ◽  
The Right

Neuropeptide Y (NPY) is abundant in plasma and amniotic fluid of women throughout pregnancy, during which its involvement in placental hormonogenesis has been proposed. In accordance with its putative role, the aim of this study was to characterize the human placental syncytiotrophoblast receptivity to NPY. Thus we performed this study on brush-border membranes (BBM) and basal plasma membranes (BPM). Specific125I-labeled NPY (125I-NPY) binding to BBM was rapid (20 min), saturable, with a maximum binding capacity of 604 ± 100 fmol/mg protein, and of high affinity, with a dissociation constant of 11 ± 3 nM. No saturable binding could be shown in BPM. The rank order of affinity of NPY and related peptides to compete for 125I-NPY binding sites was peptide YY (PYY) > NPY = [Leu31,Pro34]NPY > 13–36NPY >> pancreatic polypeptide (PP). It is noteworthy that PYY displaced only 45% of the binding sites. In BBM, both NPY and PYY were potent phospholipase C (PLC) stimulators, leading to a four- to fivefold increase of control phosphodiesterase activity. The latter effect could be prevented by preincubation of membranes with 5 μM U-73122, a known inhibitor of G protein-linked receptor activation of PLC-β. Furthermore, 5 μM BIBP-3226, a Y1-receptor antagonist, shifted both dose-response curves to the right in a similar fashion for both peptides. In accordance with the PLC stimulation, both peptides also induced stimulation of protein kinase C (PKC) activity, which could be partially but additively prevented by U-73122 and LY-294002, a selective inhibitor of phosphatidylinositol-3 kinase (PI3K). Taken together, these data suggest that placental and blood-derived NPY binds to a mixed population of receptors composed of Y1 and Y3 subtypes on the maternal side of the syncytiotrophoblast, where it can mediate its physiological purposes via PLC-β and PI3K activation, both of which lead to PKC activation. However, because BIBP-3226 antagonized both effects, the physiological relevance of the apparent Y3 fraction is still unsolved.

scholarly journals Contribution of gangliosides to thyroxin binding with plasma membranes

1995 ◽  
Vol 41 (2) ◽  
pp. 28-30
Author(s):  
T. S. Saatov ◽  
F. Ya. Gulyamova ◽  
G. U. Usmanova
Keyword(s):  
Binding Sites ◽  
Plasma Membranes ◽  
Binding Capacity ◽  
Specific Binding ◽  
Brain Cell ◽  
Cell Recognition ◽  
High Affinity ◽  
Specific Binding Sites

Besides intracellular receptors of thyroid hormones, specific binding sites for T3 and T4 were detected on plasma membranes (PM) of some cells and a relationship between membrane reception .and lipid composition of membranes shown. The parameters of 125I-T4 binding to highly purified PM of hepatic and cerebral cells of rats were studied. The hepatic and cerebral cellular membranes were found to contain two sites of hormone binding each, one of these sites being characterized by a high affinity and low capacity, and the other by low affinity and a higher binding capacity. The association constant of highly affine site of hepatocyte membranes was found to be higher than that of brain cell membranes. T4 membranous receptors may be significant in the process of cell “recognition" by the hormone. In vivo and in vitro experiments with 125I-T4 and 14C-labeled thyroxin in ganglioside fractions showed appreciable binding of the hormone to Gm3 fraction, this evidently pointing to participation of this, ganglioside in T4 interaction with membrane receptor. It is possible that gangliosides situated on membranous surface are components of or function as receptors.

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