Evidence for functional thromboxane A2-prostaglandin H2 receptors in human placenta

1989 ◽  
Vol 256 (2) ◽  
pp. E256-E263
Author(s):  
A. Hedberg ◽  
P. F. Mento ◽  
E. C. Liu ◽  
A. M. Hollander ◽  
B. M. Wilkes
Keyword(s):  
Human Placenta ◽  
Radioligand Binding ◽  
Specific Binding ◽  
Scatchard Analysis ◽  
Binding Studies ◽  
Single Class ◽  
Receptor Sites ◽  
H2 Receptors ◽  
Placental Membranes

The aim of this investigation was to study the role of thromboxane (TX) A2 in the modulation of human fetoplacental vascular resistance. By use of the isolated perfused fetoplacental cotyledon, TX generation (measured by direct radioimmunoassay of TXB2) was demonstrated on the fetal side of the placental circulation. The stable TX mimetic U-46619 caused a dose-dependent increase in perfusion pressure that was inhibited by the TX receptor antagonist SQ 29548. To further characterize the putative TXA2-prostaglandin H2 receptors, binding studies were performed in placental membranes using [3H]SQ 29548. Kinetic analysis revealed rapid and reversible specific binding of [3H]SQ 29548. Saturation binding and Scatchard analysis indicated radioligand binding to a single class of receptors (dissociation constant, 9.11 +/- 0.60 nM; receptor density, 103 +/- 8 fmol/mg protein, n = 4). Prostaglandins D2, E1, E2, F2a, and I2 did not inhibit the specific binding of [3H]SQ 29548 at concentrations less than or equal to 10 microM. This study demonstrates that the human placenta produces and releases TXA2, which can act locally via specific receptor sites to constrict the fetoplacental vasculature.

Endothelin receptors in human placenta: relationship to vascular resistance and thromboxane release

1990 ◽  
Vol 258 (5) ◽  
pp. E864-E870
Author(s):  
B. M. Wilkes ◽  
P. F. Mento ◽  
A. M. Hollander ◽  
M. E. Maita ◽  
S. Sung ◽  
...  
Keyword(s):  
Vascular Resistance ◽  
Human Placenta ◽  
Competitive Inhibition ◽  
Scatchard Analysis ◽  
Binding Studies ◽  
Single Class ◽  
Endothelin 1 ◽  
Binding Data ◽  
Endothelin 3

This investigation was performed to study the potential role of endothelin in the modulation of fetoplacental vascular resistance in the human placenta. Full-term placentas from uncomplicated pregnancies were studied within 30 min of delivery. The umbilical artery and vein to a single placental cotyledon were cannulated and the artery perfused with RPMI media (0.82 ml/min). Endothelin 1 caused a sustained dose-dependent increase in perfusion pressure. Infused endothelin 1 (50 nM) stimulated thromboxane release 2.3-fold compared with basal values. Thromboxane release persisted for 15 min after discontinuation of endothelin. Properties of human placental endothelin 1 receptors were defined in binding studies performed on a crude membrane fraction of placental cotyledons. Binding was saturable, reached steady state by 3 h at 25 degrees C, and was linear with protein concentration. Scatchard analysis of binding data indicated a single class of high-affinity binding sites with a Kd of 36.1 +/- 9.7 pM and a density of 185.4 +/- 9.6 fmol/mg protein (n = 5). The potency order for competitive inhibition of the binding of 125I-labeled endothelin 1 was endothelin 1 greater than endothelin 2 = endothelin 3 = sarafotoxin S6b greater than big endothelin (human) = big endothelin (porcine). Phenylephrine, bradykinin, norepinephrine, atrial natriuretic factor, diltiazem, U46619, and angiotensin II did not displace 125I-endothelin 1 from its receptors. These experiments demonstrate that endothelin 1 is a potent pressor substance in the human fetoplacental cotyledon. Pressor effects of endothelin may be mediated by a combination of direct effects and stimulation of vasoconstrictor prostanoids.

Characterization of glomerular thromboxane receptor sites in the rat

1989 ◽  
Vol 256 (6) ◽  
pp. F1111-F1116 ◽  
Author(s):  
B. M. Wilkes ◽  
J. Solomon ◽  
M. Maita ◽  
P. F. Mento
Keyword(s):  
Specific Binding ◽  
Binding Studies ◽  
Single Class ◽  
Thromboxane Receptor ◽  
Renal Glomeruli ◽  
Receptor Sites ◽  
High Affinity Receptor ◽  
Pg E2 ◽  
Txa2 Receptor Antagonist

The aim of this study was to identify and characterize thromboxane (Tx) receptor sites in renal glomeruli. Binding studies were performed on freshly isolated glomeruli using the stable TxA2 receptor antagonist, [3H]SQ 29548. Specific binding was saturable, reversible, and varied with glomerular protein. Scatchard plots revealed a single class of high-affinity receptor sites (Kd = 14.3 +/- 2.4 nM, Bmax = 361 +/- 22 fmol/mg; n = 5). Specific binding was inhibited by Tx agonists (U-46619 and U-44069) and antagonist (SQ 29548) and was highly specific for Tx, since prostaglandin (PG)E2 and PGF2 alpha were 1,000-fold less potent in inhibiting binding. In vivo, U-46619 (1.75 micrograms.kg-1.min-1) was without effect on mean arterial pressure, but reduced renal blood flow by 71% (P less than 0.01) and glomerular filtration rate by 67% (P less than 0.01) and increased filtration fraction by 24% (P less than 0.05). SQ 29548 (10 micrograms.kg-1.min-1) completely blocked the renal effects of U-46619. These studies demonstrate the presence of specific receptor sites for Tx on renal glomeruli that are linked to modulation of renal hemodynamics.

Endothelin-1 conversion and receptor characterization in human placental arteries

1994 ◽  
Vol 267 (2) ◽  
pp. E242-E249
Author(s):  
B. M. Wilkes ◽  
C. M. Macica ◽  
P. F. Mento
Keyword(s):  
Blood Vessels ◽  
Competitive Inhibition ◽  
Polyacrylamide Gel Electrophoresis ◽  
Receptor Subtype ◽  
Scatchard Analysis ◽  
Binding Studies ◽  
Single Class ◽  
Endothelin 1 ◽  
Active Peptide ◽  
Receptor Sites

Endothelin-1-(1-21), a potent pressor peptide, is transcribed as big endothelin-(1-38) and converted to active peptide by endothelin-converting enzyme. The current investigation tested the hypothesis that human fetoplacental blood vessels convert big endothelin-1 to active peptide and that fetoplacental blood vessels respond to endothelin-1 by binding of the peptide to specific receptor sites. In the isolated perfused placental cotyledon the addition of big endothelin-1 to the perfusate caused a time-dependent increase in perfusion pressure that corresponded to the appearance of endothelin-1 in the perfusate. The properties of human placental endothelin-1 receptors were defined in binding studies performed on a plasma membrane fraction of small arteries (<1.0 mm) dissected from the placenta. Binding was saturable, reached steady state by 3 h at 25 degrees C, and was linear with protein concentration. Scatchard analysis of binding data indicated a single class of high-affinity binding sites with a dissociation constant of 27.6 +/- 2.3 pM and a density of 856 +/- 119 fmol/mg protein (n = 5). The potency order for competitive inhibition of the binding of 125I-labeled endothelin-1 [endothelin-1 = endothelin-2 > endothelin-3 = sarafotoxin S6b >> big endothelin-1 (human) = big endothelin-1 (porcine)] is most consistent with a type A endothelin receptor subtype. Phenylephrine, bradykinin, norepinephrine, atrial natriuretic factor, diltiazem, U-46619, and angiotensin II did not displace 125I-endothelin-1 binding. Endothelin receptors were shown to have an approximate molecular weight of 36,600 by polyacrylamide gel electrophoresis.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals Overexpression of the insulin-like growth factor I receptor in human pheochromocytomas

2006 ◽  
Vol 36 (2) ◽  
pp. 279-287 ◽  
Author(s):  
Christian Fottner ◽  
Timo Minnemann ◽  
Sarah Kalmbach ◽  
Matthias M Weber
Keyword(s):  
Scatchard Analysis ◽  
Mrna Levels ◽  
Therapeutic Approaches ◽  
Binding Studies ◽  
Single Class ◽  
Normal Tissues ◽  
Igf System ◽  
Igf I ◽  
Polymerase Chain

In order to determine the role of the IGF-I receptor (IGF-IR) in human pheochromocytomas we have compared the expression of the IGF-IR in normal tissues and in pheochromocytomas with regard to the IGF-IR mRNA levels and ligand binding. By semiquantitative reverse transcription polymerase chain reaction (RT-PCR), the mRNA of the IGF-IR could be detected in all samples of normal adrenomedullary cells (n=13) and pheochromocytomas (n=16). However, pheochromocytomas exhibited 2.8-fold higher mean IGF-IR mRNA levels than normal adrenomedullary cells (2.8±0.5×105 molecules/μg RNA vs 7.8±1.2×105 molecules/μg RNA; P < 0.001). This overexpression of the IGF-IR in pheochromocytomas could be confirmed at the protein level by binding studies. Radioligand assays and Scatchard analysis revealed a single class of high affinity IGF-IR binding sites with a similar dissociation constant (Kd: 0.32±0.1 nmol/l vs 0.22±0.08 nmol/l) for both normal adrenomedullary cells and pheochromocytomas. However, specific 125I-labeled IGF-I binding and the calculated receptor concentration were significantly elevated in pheochromocytomas as compared with normal adrenomedullary cells (58.3±5 vs 24.3±12 nmol/kg protein; P < 0.05). In summary, our results demonstrate significant overexpression of the IGF-IR in human pheochromocytomas. This suggests a possible role of the IGF system in the pathogenesis of adrenal neoplasia and thus IGF-IR may be a target for future therapeutic approaches.

Identification and transmembrane signaling of leukotriene D4 receptors in human mesangial cells

1988 ◽  
Vol 255 (6) ◽  
pp. C771-C780 ◽  
Author(s):  
M. S. Simonson ◽  
P. Mene ◽  
G. R. Dubyak ◽  
M. J. Dunn
Keyword(s):  
Binding Sites ◽  
Radioligand Binding ◽  
Mesangial Cells ◽  
Specific Binding ◽  
Transmembrane Signaling ◽  
Binding Studies ◽  
Single Class ◽  
Leukotriene D4 ◽  
Human Mesangial Cells ◽  
Leukotriene Receptors

Although peptidoleukotriene (LTC4, LTD4) receptors have been characterized by radioligand binding studies, pathways of transmembrane signaling by activated leukotriene receptors remain obscure. We employed [3H]LTD4 binding studies and fluorescent measurements of intracellular Ca2+ concentration ([Ca2+]) and pH to identify LTD4 receptors and mechanisms of transmembrane signaling in cultured human mesangial cells. Mesangial cells expressed a single class of saturable, specific binding sites for [3H]LTD4. Kinetic, competition, and saturation analyses gave an average KD of approximately 12.0 nM with a Bmax of 987 fmol/mg protein. LTC4 competed with high affinity for [3H]LTD4 binding sites, as did LTB4 but with much lower affinity. [3H]LTD4 binding was blocked by a specific LTD4 receptor antagonist, SKF 102922. LTD4 and LTC4 also evoked a rapid (2-3 s), transient increase in intracellular [Ca2+], followed by a second, sustained increase. The transient phase was independent of extracellular Ca2+, whereas the sustained phase was dependent on extracellular Ca2+. Intracellular [Ca2+] was unaffected by LTB4. The LTD4-stimulated Ca2+ transients were dose dependent (1 nM-1 microM) and, similar to [3H]LTD4 binding, Ca2+ transients were inhibited by LTD4 receptor antagonists. We also report evidence that LTD4 affects intracellular pH and activates Na+-H+ exchange. Specifically, LTD4 induced an initial acidification within 1-2 min, followed by net alkalinization at 5 min. Alkalinization was due to activation of an amiloride-inhibitable Na+-H+ exchanger. LTD4 receptors were apparently not coupled to adenylate cyclase or phospholipase A2 as we detected no changes of adenosine 3',5'-cyclic monophosphate (cAMP) or prostanoids. Thus we conclude that [3H]LTD4 binding sites on human mesangial cells are coupled to a Ca2+-signaling system and Na+-H+ exchange. Moreover LTD4, a potent inflammatory mediator, failed to stimulate cAMP or prostaglandin E2/prostaglandin I2, two counterregulatory autacoids that preserve normal mesangial function.

scholarly journals Specific binding of cardiac glycoside drugs and endogenous digitalis-like substances to particulate membrane fractions from human placenta.

1989 ◽  
Vol 35 (10) ◽  
pp. 2093-2097 ◽  
Author(s):  
A Paci ◽  
F Cocci ◽  
F Piras ◽  
G Ciarimboli ◽  
A Clerico
Keyword(s):  
Human Placenta ◽  
Cardiac Glycoside ◽  
Cardiac Glycosides ◽  
Specific Binding ◽  
Placental Tissue ◽  
Scatchard Analysis ◽  
Pseudo First Order Reaction ◽  
Single Class ◽  
First Order ◽  
Endogenous Substances

Abstract We studied the characteristics of binding of cardiac glycosides to particulate membrane fractions from human placenta, to demonstrate that placental tissue is a suitable source of receptors for digitalis drugs. Moreover, we performed preliminary experiments with 125I-labeled digoxin and placental particulates to develop a radioreceptor assay for measurement of endogenous substances with activity similar to cardiac glycoside drugs (EDLS). Placental membrane fractions were incubated with [3H]ouabain (10 nmol/L) or 125I-labeled digoxin (50 pmol/L). With both ligands, binding followed a pseudo-first-order reaction kinetics and was saturable. Scatchard analysis revealed a single class of sites [for ouabain, KD = 20.2 +/- 5.8 nmol/L (mean +/- SEM), Bmax = 3.1 +/- 0.9 nmol per gram of protein; for digoxin, KD = 29.7 +/- 1.9 nmol/L, Bmax = 24.3 +/- 1.1 nmol per gram of protein]. As expected, digoxin was less potent than ouabain in displacing both tracers from digitalis drugs receptors; progesterone, cortisone, digitoxose, furosemide, bumetanide, and propranolol had no or little effect. Specific 125I-labeled digoxin binding was competitively inhibited by plasma and (or) urine extracts from newborns, adults, pregnant women, and patients with renal insufficiency. Inhibition of binding and volume of plasma and urine assayed were linearly related. These findings support the hypothesis that cardiac glycosides and EDLS can interact with the human placenta and suggest placental tissue to be a suitable source of receptors for cardiac glycosides.

The identification and characterization of 125I-labelled iodomelatonin-binding sites in the duck kidney

1992 ◽  
Vol 135 (2) ◽  
pp. 353-359 ◽  
Author(s):  
Y. Song ◽  
E. A. Ayre ◽  
S. F. Pang
Keyword(s):  
Acetic Acid ◽  
Binding Sites ◽  
Radioligand Binding ◽  
Specific Binding ◽  
Hill Coefficient ◽  
Scatchard Analysis ◽  
Equilibrium Studies ◽  
Single Class ◽  
Urine Production ◽  
Daily Urine

ABSTRACT The melatonin-binding sites in membrane preparations of duck kidney were demonstrated by utilizing 125I-labelled iodomelatonin as a radioligand. Binding at these sites was found to be reversible, saturable, specific and of high affinity. Scatchard analysis of the specific binding revealed an equilibrium binding constant (Kd) of 44·6±4·4 pmol/l (n= 6) and a total number of binding sites (Bmax) of 6·43 ±0·60 fmol/mg protein (n= 6) at the mid-point of the light period (mid-light). The Hill coefficient approached 1·0, suggesting a single class of 125I-labelled iodomelatoninbinding site in the duck kidney. Diurnal variation in 125I-labelled iodomelatonin binding showed that the Bmax was 53·4% higher at mid-light than at mid-point of the dark period (P<0·05), with no significant variation in Kd. The Kd value determined from kinetic analysis was 22·5 pmol/l in birds at mid-light, which was comparable with values determined from equilibrium studies. The order of pharmacological affinity for 125I-labelled iodomelatonin-binding sites in the duck kidney membrane preparations was: 2-iodomelatonin > melatonin > 6-chloromelatonin > 6-hydroxymelatonin > N-acetylserotonin » 5-methoxytryptamine, 5-methoxytryptophol, 5-hydroxytryptamine, tryptamine, 1-acetylindole-3-carboxaldehyde, 5-hydroxyindole-3-acetic acid, l-tryptophan, 5-methoxyindole-3-acetic acid, 3-acetylindole, acetylcholine, epinephrine, norepinephrine and harmaline. The pharmacological characteristics indicated that 125I-labelled iodomelatonin-binding sites are highly specific for melatonin. Our finding of 125I-labelled iodomelatonin-binding sites in the kidney suggests that melatonin may regulate kidney function. The possibility of a direct effect of melatonin on renin secretion and daily urine production is hypothesized. Journal of Endocrinology (1992) 135, 353–359

scholarly journals Solubilization of functional calcitonin receptors

1988 ◽  
Vol 253 (2) ◽  
pp. 505-510 ◽  
Author(s):  
G C Nicholson ◽  
C S D'Santos ◽  
T Evans ◽  
J M Moseley ◽  
B E Kemp ◽  
...  
Keyword(s):  
Binding Sites ◽  
Human Cancer ◽  
Rank Order ◽  
Biologically Active ◽  
Target Cells ◽  
Scatchard Analysis ◽  
Binding Studies ◽  
Single Class ◽  
Binding Characteristics ◽  
Placental Membranes

Calcitonin (CT) binding activity has been extracted from a membrane fraction of human placenta using the zwitterionic detergent, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulphonic acid (Chaps). Approximately two-thirds of the available binding sites were extracted using 5 mM-Chaps. The binding characteristics of 125I-labelled salmon CT(125I-sCT) to the solubilized extract were similar to those obtained previously with placental membranes and other targets such as osteoclasts, renal cells and certain human cancer cell lines. 125I-sCT binding was saturable (Bmax. 75 +/- 6 fmol/mg of protein, n = 3) and Scatchard analysis revealed a single class of high-affinity binding sites (Kd 165 +/- 28 pM, n = 3). In competitive-binding studies, various species-specific CTs and CT analogues showed the same rank order of potencies as seen in CT bioassays and several unrelated peptides did not compete at high doses. A biologically active CT analogue, [Arg11,18, Lys14]sCT, derivatized with the photoreactive phenylazide cross-linking agent, N-hydroxysuccinimidyl-4-azidobenzoate, was used to identify receptor components of Mr approximately 88,000 and approximately 71,000 in both particulate placental membranes and the solubilized extract. Receptor components of Mr 85-90,000 have been identified in other CT target cells previously using chemical- and photoaffinity-labelling techniques. These results demonstrate the first successful solubilization of the CT receptor in a form which purification.

Characterization of Thromboxane A2/Prostaglandin H2 Receptors in Porcine Coronary Artery -The Inhibitory Effect of a Novel Dibenzoxepin Derivative, KW-3635

1992 ◽  
Vol 67 (05) ◽  
pp. 582-584 ◽  
Author(s):  
Ichiro Miki ◽  
Akio Ishii
Keyword(s):  
Coronary Artery ◽  
Binding Sites ◽  
Thromboxane A2 ◽  
Inhibitory Effect ◽  
Scatchard Analysis ◽  
Single Class ◽  
Porcine Coronary Artery ◽  
Equilibrium Binding ◽  
H2 Receptors

SummaryWe characterized the thromboxane A2/prostaglandin H2 receptors in porcine coronary artery. The binding of [3H]SQ 29,548, a thromboxane A2 antagonist, to coronary arterial membranes was saturable and displaceable. Scatchard analysis of equilibrium binding showed a single class of high affinity binding sites with a dissociation constant of 18.5 ±1.0 nM and the maximum binding of 80.7 ± 5.2 fmol/mg protein. [3H]SQ 29,548 binding was concentration-dependently inhibited by thromboxane A2 antagonists such as SQ 29,548, BM13505 and BM13177 or the thromboxane A2 agonists such as U46619 and U44069. KW-3635, a novel dibenzoxepin derivative, concentration-dependently inhibited the [3H]SQ 29,548 binding to thromboxane A2/prosta-glandin H2 receptors in coronary artery with an inhibition constant of 6.0 ± 0.69 nM (mean ± S.E.M.).

Expression of insulin-like growth factor receptors in primary human thyroid neoplasms

1989 ◽  
Vol 121 (1) ◽  
pp. 112-120 ◽  
Author(s):  
Tohru Yashiro ◽  
Yoshito Ohba ◽  
Hitomi Murakami ◽  
Takao Obara ◽  
Toshio Tsushima ◽  
...  
Keyword(s):  
Thyroid Cancer ◽  
Binding Capacity ◽  
Specific Binding ◽  
Human Thyroid ◽  
Scatchard Analysis ◽  
Type I ◽  
Single Class ◽  
Normal Tissues ◽  
Igf I ◽  
Cancer Tissues

Abstract. The presence of IGF-I receptors was demonstrated in normal and neoplastic tissues of human thyroid. Binding of [125I]IGF-I to thyroid membranes was dependent on time and temperature of incubation, and maximal binding was achieved at 4°C and 18 h of incubation. [125I] IGF-I binding was dose-dependently displaced by unlabelled IGF-I; half-maximal inhibition occurred at concentrations of 10–20 μg/l. IGF-II and insulin had relative potencies of 5 and 1% compared with IGF-I. Scatchard analysis of binding data revealed a single class of IGF-I receptors with high affinity (Ka: 1.2–8.6 × 109 1/mol) in normal thyroid tissues. Affinity cross-linking and autoradiography demonstrated the type I IGF receptors. Specific binding of [125I] IGF-I in thyroid cancer tissues (9.69 ± 2.07% per 200 μg protein; mean ± sem, N = 8) was significantly (p <0.05) higher than that in the surrounding normal tissues (3.03 ± 0.35%, N = 8). In contrast, there was no difference in the binding between adenoma tissues (4.19 ± 0.53%, N = 5) and the adjacent normal tissues (2.94 ± 0.24%, N = 5). The higher IGF-I binding in cancer tissues was due to an increase in the binding capacity without any change in the affinity. The presence of IGF-I receptors suggests a possible role of IGF-I and its receptors in the growth of thyroid cancer cells.

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