Endothelin ETA and ETB receptors in postnatal intestine

2001 ◽  
Vol 280 (4) ◽  
pp. G555-G562 ◽  
Author(s):  
Craig A. Nankervis ◽  
David J. Dunaway ◽  
Charles E. Miller

We aimed to characterize endothelin (ET) receptors in the swine intestinal vasculature and to determine ischemia-reperfusion (I/R) effects on these receptors. Saturation and competitive binding assays were performed on mesenteric artery protein membranes from 1- and 40-day-old animals, both control and those subjected to 1 h of partial ischemia followed by 6 h of reperfusion in vivo. Scatchard analysis of saturation binding with 125I-labeled ET-1 in membranes from endothelium-denuded (E−) vessels revealed that the maximum number of binding sites was greater in younger animals. Competitive125I-ET-1 binding was significant for a one-site model with ET-1, ET-3, and sarafotoxin S6c (S6c) in membranes from endothelium-intact (E+) and E− vessels in both age groups. The maximum number of ET-1 binding sites was significantly greater in younger animals. In the presence of the ETAreceptor antagonist BQ-123, competitive 125I-ET-1 binding was significant for a one-site model with ET-1 and S6c in membranes from E+ vessels in both age groups. The maximum number of ET-1 binding sites was significantly greater in younger animals. After I/R, the maximum number of ET-1 binding sites was unchanged. In the presence of BQ-123, specific binding by ET-1 and S6c was eliminated in both age groups after I/R. These results suggest that both ET receptor populations are expressed to a greater degree in younger animals and I/R significantly affects the ETB receptor.

1981 ◽  
Author(s):  
P Silber ◽  
T H Finlay

The effect of ristocetin on the binding of 125I-porcine von Willebrand factor to human platelets was studied. Previously, we had shown that 125I-porcine von Willebrand factor binds to human platelets in the absence of ristocetin. The present work demonstrates that binding is stimulated by ristocetin and this stimulation is maximal at a ristocetin concentration of 2 mg/ml. At a ristocetin concentration of 0.5 mg/ml, Scatchard analysis indicates a binding constant of 5.18 × 10-9M and the presence of 105,000 binding sites. This compares with our previous finding, in the absence of ristocetin, of a binding constant of 2.92 × 10-7M and 4760 binding sites. These binding data assume the porcine von Willebrand factor to be a tetramer with a molecular weight of 9 × 105. This study indicates that ristocetin causes tighter binding and increases the number of binding sites on human platelets for porcine von Willebrand factor. Unlabelled porcine von Willebrand factor competitively inhibits the specific binding of the labelled protein and gives a binding constant of 0.17 × 10-9M. Similar results were obtained using human von Willebrand factor.


2003 ◽  
Vol 284 (3) ◽  
pp. R689-R697 ◽  
Author(s):  
Michel B. Lortie ◽  
Thomas W. Moon

The presence and functionality of β-adrenoceptors (β-ARs) were examined in red (RM) and white muscle (WM) membranes isolated from the rainbow trout Oncorhynchus mykiss. Specific binding assays revealed the presence of a single class of binding sites with similar affinities in both muscle types ( K d in nM: 0.14 ± 0.03 and 0.18 ± 0.03 for RM and WM, respectively) but with a significantly higher number of binding sites in RM compared with WM (Bmax in fmol/mg protein: 3.22 ± 0.11 and 2.60 ± 0.13, respectively). Selective and nonselective β-adrenergic agonists (β-AAs) and antagonists indicated an atypical β-AR pharmacology. This result may represent a nonmammalian β-AR classification or, more likely, the presence of more than one β-AR subtype in trout muscles with similar affinities that could not be kinetically resolved. Adenylyl cyclase (ACase) assays showed a dose-dependent increase in cAMP production as concentrations of β2-AAs increased in both muscle membranes with significantly higher basal cAMP production in RM compared with WM (cAMP production in pmol cAMP · mg protein−1 · 10 min−1: 24.67 ± 3.06 and 9.64 ± 3.45, respectively). The agonist-induced increase in cAMP production was blocked by the β-adrenergic antagonist propranolol, while the ACase activator forskolin increased cAMP production by 7- to 14-fold above basal and ∼3-fold above all β-AAs tested. This study demonstrated the presence of atypical β2-ARs on RM and WM membranes of trout, suggesting that β2-AAs may be a tool to enhance protein accretion through this signaling pathway.


1985 ◽  
Vol 110 (1) ◽  
pp. 50-55 ◽  
Author(s):  
Stephen LaFranchi ◽  
Cheryl E. Hanna ◽  
Toni Torresani ◽  
Eugen Schoenle ◽  
Ruth Illig

Abstract. We undertook a comparison of human growth hormone (hGH) binding and metabolic responses in rat adipocytes of epididymal, subcutaneous, and retroperitoneal origin to determine whether the site of fat depot biopsy might affect the response to hGH stimulation. The results showed highest specific binding in epididymal (3.6%), followed by subcutaneous (2.3%) and retroperitoneal adipocytes (1.5%); half-maximal binding was achieved at 14–18 ng/ml hGH for the three sites. Scatchard analysis of the binding data from each site was linear; there was no significant difference in binding affinities (2.1 to 3.3 × 109, m−1), but the number of binding sites was statiticially higher in epididymal (9.8 × 103) as compared to subcutaneous (7.5 × 103, P < 0.05) and retroperitoneal cells (3.3 × 103, P < 0.01). Stimulation with 5 to 2500 ng pituitary hGH produced a dose-related increase in glucose incorporation, with the largest increase in epididymal fat cells (31%, P <0.05) followed by subcutaneous cells (18%, P < 0.05); no significant increase was seen with retroperitoneal cells. Biosynthetic hGH produced a similar pattern of glucose incorporation in the three sites. Addition of hGH antibodies blocked the glucose incorporation in epididymal adipocytes using both pituitary-derived and biosynthetic hGH. It seems clear that this insulin-like effect is caused by hGH, not an insulin-like impurity. We conclude that the number of binding sites, perhaps related to adipose cell size, differs in adipose tissue from different locations and this influences the metabolic response to hGH stimulation.


1990 ◽  
Vol 122 (6) ◽  
pp. 740-744 ◽  
Author(s):  
J. Eurin ◽  
A. Carayon ◽  
M. A. Zongazo ◽  
F. Masson ◽  
C. Barthelemy ◽  
...  

Abstract. The presence of functional receptors for human atrial natriuretic hormone in human pheochromocytomas was recently reported. The present study reports the binding of hANH as measured by Scatchard analysis in 4 human adrenal glands and in 5 human pheochromocytomas. Binding assays using [3H]ANH revealed a single class of high-affinity binding sites for hANH in both tissues. Human pheochromocytomas present a lower number of binding sites than normal human adrenal gland (Bmax of 7.1±2.1 vs 33.6±6.9 fmol/mg protein, respectively). However, the decreased number of ANH receptors was not paralleled by modifications of tissular cyclic GMP (cGMP). Moreover, plasma hANH concentrations in 7 patients with pheochromocytomas (20.2±2.7 pmol/l) were statistically higher than those obtained in 25 normal control humans (8.1±0.6 pmol/l, p<0.001). We also demonstrated the presence of immunoreactive ANH in the tumour itself.


1984 ◽  
Vol 219 (3) ◽  
pp. 1001-1007 ◽  
Author(s):  
Y A Lefebvre ◽  
J T Venkatraman

Nuclear envelopes and nuclear matrices were isolated from the male-rat liver. Incubation of 125I-labelled 3,3′,5-tri-iodothyronine (T3) with the nuclear-envelope fraction resulted in specific binding of T3 to the membranes. Maximum specific binding occurred at 30 degrees C after 2h incubation. Storage for 1 week at -80 degrees C resulted in no loss of binding. Scatchard analysis revealed a class of binding sites with KD 86 nM. 3,3′,5′-Tri-iodothyronine was as effective a competitor of [125I]T3 binding to nuclear envelopes as was L-T3 itself, and tri-iodothyroacetic acid was 70% as potent as T3. L- and D-thyronine did not compete for [125I]T3 binding. Incubation of nuclear envelopes with 0.6 M-NaCl before addition of T3 resulted in the complete loss of specific binding sites, whereas exposure of the membranes to 2.0 M-NaCl after incubation with T3 did not extract binding sites. Nuclear matrices, after incubation with [125I]T3 under the same conditions, were shown to possess a class of binding sites with a similar KD but with approx. 30% of the maximum binding capacity. Nuclear envelopes from hypothyroid animals may possess slightly lower numbers of binding sites compared with nuclear envelopes from the intact animal, whereas nuclear matrices from hypothyroid animals have the same number of binding sites as do nuclear envelopes from the intact animal. In conclusion, nuclear envelopes and nuclear matrices have a class of binding sites with relatively high affinity for T3. It is distinct from nuclear and cytosolic binding sites.


1981 ◽  
Vol 88 (3) ◽  
pp. 401-408 ◽  
Author(s):  
RYOSUKE NAKANO ◽  
MAREO YAMOTO ◽  
MASAFUMI IWASAKI

The binding of 125I-labelled human LH (hLH) to the 2000 g subcellular fraction of human corpora lutea of the menstrual cycle was examined. Displacement studies demonstrated that 125I-labelled hLH was specifically bound in the 2000 g fraction of human luteal tissue. Specific binding of 125I-labelled hLH was demonstrated in all the corpora lutea examined except for two aged corpora lutea at an early proliferative phase of the cycle. The number of binding sites for hLH increased between the early to mid-luteal phase and decreased towards the late luteal phase. However, the apparent dissociation constant (Kd) in each corpus luteum did not vary throughout the menstrual cycle. In addition, the effects of treatment with diethylstilboestrol diphosphate (DES) and prostaglandin F2α (PGF2α) on the binding of 125I-labelled hLH to the 2000 g fraction of luteal tissue were investigated and the changes in hLH receptors were estimated by Scatchard analysis. The number of binding sites were 1·59 × 10−14 mol/mg protein in control tissue, 0·86 × 10 −14 mol/mg protein in DES-treated luteal tissue and 2·95 × 10−14 mol/mg protein in PGF2α-treated luteal tissue. Thus, the binding sites for hLH decreased as a result of treatment with DES and increased by treatment with PGF2α. In contrast, the apparent Kd in each luteal tissue revealed almost the same value (4·24 × 10−10 to 6·07 × 10−10 mol/l) after treatment with DES or PGF2α. The results of the present study suggest that oestrogen and prostaglandin might have an important role in modulating hLH receptor in human corpora lutea.


1993 ◽  
Vol 138 (1) ◽  
pp. 51-57 ◽  
Author(s):  
Z. M. Liu ◽  
S. F. Pang

ABSTRACT The melatonin-binding sites in membrane preparations of the bursa of Fabricius of birds were studied using [125I]iodomelatonin as a radioligand. The binding sites were stable, saturable, reversible and of high affinity. Scatchard analysis of specific binding revealed equilibrium binding constants (Kd) of (means±s.e.m.) 43·1 ±5·9 73·3±7·6 and 35·3±4·8 pmol/l respectively at the mid-point of the light period (mid-light) in chickens, pigeons and quail, with a total number of binding sites (Bmax) of 1·23 ±0·15, 1·33±0·18 and 0·94 ±0·07 fmol/mg protein. The diurnal variation in [125I]iodomelatonin binding showed that the Bmax was 45, 115 and 70% higher at mid-light than at mid-dark in the bursae of chickens, pigeons and quail respectively. The Kd value determined from kinetic analysis was 49·0 ±6·4 pmol/l at mid-light in the chicken bursa. The [125I]iodomelatonin-binding sites of chicken bursal membranes had the following order of pharmacological affinities: 2-iodomelatonin > melatonin > 6-chloromelatonin > 6-hydroxymelatonin > N-acetylserotonin > 5-hydroxytryptamine, tryptamine, 5-methoxytryptophol, 1-acetylindole-3-carboxaldehyde, 3-acetylindole, l-tryptophan, 5-hydroxyindole3-acetic acid, 5-hydroxytryptophan suggesting that the [125I]iodomelatonin-binding sites were highly specific for melatonin. The subcellular distribution of binding sites in the chicken bursa was in the following descending order: nuclear > mitochondrial > microsomal > cytosolic fraction. There was an agerelated decrease in [125I]iodomelatonin-binding in chicken bursal membranes, with higher densities in the neonate. Our studies of [125I]iodomelatonin-binding sites in the bird bursa indicate that this primary lymphoid tissue is a target organ for melatonin, and that melatonin has a direct effect on the immune system. Journal of Endocrinology (1993) 138, 51–57


1988 ◽  
Vol 118 (2) ◽  
pp. 227-232 ◽  
Author(s):  
L. G. Guijarro ◽  
E. Arilla

ABSTRACT Atrophy of the exocrine pancreas was induced in rabbits by pancreatic duct ligation. Somatostatin concentration and binding in cytosol from rabbit duodenal mucosa were studied after 6 and 14 weeks of pancreatic duct ligation. Somatostatin-like immunoreactivity was significantly increased in the duodenal mucosa in both periods. Scatchard analysis showed a parallel increase in the number of binding sites rather than a change in their affinity. The physiological significance of these findings remains to be clarified. J. Endocr. (1988) 118, 227–232


1996 ◽  
Vol 313 (2) ◽  
pp. 537-541 ◽  
Author(s):  
Denis REYNAUD ◽  
Peter DEMIN ◽  
Cecil R. PACE-ASCIAK

Hepoxilins have been shown to release calcium from intracellular stores in human neutrophils [Dho, Grinstein, Corey, Su and Pace-Asciak (1990) Biochem. J. 266, 63-68; Laneuville, Reynaud, Grinstein, Nigam and Pace-Asciak (1993) Biochem. J. 295, 393-397]. In this paper we report that tritium-labelled hepoxilin A3 (8S) binds to broken neutrophil membranes in a time-, substrate- and temperature-dependent fashion. Specific binding was displaced with unlabelled hepoxilin A3. Specific binding was greatest at 37 °C. Competitive binding was best observed with unlabelled hepoxilin A3 (8S); the glutathione conjugate, HxA3-C (8S or 8R), or 12(S)-hydroxyeicosatetraenoic acid was less active. Similarly inactive in displacing the bound radiolabelled hepoxilin A3 was leukotriene B4 as well as a variety of prostaglandins and thromboxane B2. Formylmethionyl-leucylphenylalanine was similarly inactive in competing for the hepoxilin binding sites. Specific binding was inhibited by pretreatment of the broken membranes during 30 min at 37 °C with proteinase K, while specific binding of the intact cells was unaffected. Scatchard analysis of binding data revealed a single population of binding sites with apparent KD and Bmax. of 79.3±9.1 nM and 8.86±1.4 pmol/ml per 2×106 cells (±S.E.M.) respectively reflecting approx. 2.67×106 sites/cell. These results demonstrate for the first time that neutrophils contain specific binding sites to hepoxilin A3.


1979 ◽  
Author(s):  
H.R. Gralnick ◽  
D.K. Morisato

We have investigated the binding of radiolabelled factor VIII/von Willebrand factor (f. VIII/vWf) protein to human platelets (P) in the presence of ristocetin (R). In these atudies we have delineated the importance of the carbohydrate (CHO) moiety(s) in both the binding to the P and in cauaing agglutination of P. Binding of the f.VIII/vWf protein to human P was time and temperature dependent and dependent on the concentration of R. Binding was specific in that it could not be blocked by human fibrinogen but was inhibited by unlabelled f.VIII/vWf protein. In studies utilizing varying amounts of the f.VIII/vWf protein or by varying the number of P in the assay, the number of binding sites for the f. VIII/vWf protein were estimated at 9,500-9,800 per platelet. Scatchard analysis revealed 11,000 binding sites with 3,600 of high affinity and 7,400 of low affinity. Removal of the sialic acid of the f.VIII/vWf protein resulted in no significa nt change in its ability to bind to the P surface or cause agglutination in the presence, IR. Removal of the galactose by 6-galactosijase resulted in a 75% reduction of binding of the f.VIII/vWf protein and a 91% decrease in the agglutination of human P. Similar studies with galactose oxidase showed that oxidation of the penultimate galactose residue s results in a decrease in agglutination comparable to that seen with 6-galactosidase treatment. These studies indicate that the CHO moiety of the f.VIII/vWf protein is important in both binding to the P surface as well as causing agglutination of human P.


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