Characterization of aldosterone binding sites in circulating human mononuclear leukocytes

1985 ◽  
Vol 248 (3) ◽  
pp. E388-E390 ◽  
Author(s):  
D. Armanini ◽  
T. Strasser ◽  
P. C. Weber
Keyword(s):  
Binding Sites ◽  
Physiological Significance ◽  
Mononuclear Leukocytes ◽  
Mineralocorticoid Receptors ◽  
Percoll Gradient ◽  
Single Class ◽  
Human Mononuclear Leukocytes

Aldosterone binding sites in human mononuclear leukocytes were characterized after separation of cells from blood by a Percoll gradient. After washing and resuspension in RPMI-1640 medium, cells were incubated at 37 degrees C for 1 h with different concentrations of [3H]aldosterone plus a 100-fold concentration of RU-26988 (11 alpha, 17 alpha-dihydroxy-17 beta-propynylandrost-1,4,6-trien-3-one), with or without an excess of unlabeled aldosterone. Aldosterone binds to a single class of receptors with an affinity of 2.7 +/- 0.5 nM (means +/- SD, n = 14) and a capacity of 290 +/- 108 sites/cell (n = 14). The specificity data show a hierarchy of affinity of desoxycorticosterone = corticosterone = aldosterone greater than hydrocortisone greater than dexamethasone. The results indicate that mononuclear leukocytes could be useful for studying the physiological significance of these mineralocorticoid receptors and their regulation in humans.

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.).

Characterization of gonadotropin-releasing hormone (GnRH) receptors in the ovary of common carp (Cyprinus carpio)

1992 ◽  
Vol 70 (2) ◽  
pp. 268-274 ◽  
Author(s):  
Debananda Pati ◽  
Hamid R. Habibi
Keyword(s):  
Common Carp ◽  
Cyprinus Carpio ◽  
Binding Sites ◽  
Tissue Concentration ◽  
Gonadotropin Releasing Hormone ◽  
Scatchard Analysis ◽  
Single Class ◽  
Releasing Hormone ◽  
Carp Cyprinus Carpio

Gonadotropin-releasing hormone (GnRH) binding sites have been characterized in the fully mature common carp ovary, using an analog of salmon GnRH ([D-Arg6,Trp7,Leu8,Pro9-NEt]-GnRH; sGnRH-A) as a labeled ligand. Binding of sGnRH-A to carp follicular membrane preparation was found to be time-, temperature-, and pH-dependent. Optimal binding was achieved after 40 min of incubation at 4 °C at pH 7.6; binding was found to be unstable at room temperature. Binding of radioligand was a function of tissue concentration, with a linear correlation over the range of 8.0–40.0 μg membrane protein per tube. Incubation of membrane preparations with increasing levels of [125I]sGnRH-A revealed saturable binding at radioligand concentrations greater than 400 nM. The binding of [125I]sGnRH-A to the carp ovary was also found to be reversible; addition of unlabeled sGnRH-A (10−6 M) after reaching equilibrium resulted in complete dissociation of [125I]sGnRH-A within 30 min, and the log dissociation plot indicated the existence of a single class of binding sites. Addition of unlabeled sGnRH-A displaced the bound [125I]sGnRH-A in a dose-related manner. Hill plot as well as Scatchard analysis suggested the presence of one class of high affinity GnRH binding sites. Bound [125I]sGnRH-A was also found to be displaceable by other GnRH peptides, including sGnRH ([Trp7,Leu8]-GnRH), cGnRH-II ([His5,Trp7,Tyr8]-GnRH) and a GnRH antagonist ([D-pGlu1,D-Phe2,D-Trp3,6]-GnRH; GnRH-ANT) in a parallel fashion, indicating that these peptides bind to the same class of binding sites. sGnRH-A and cGnRH-II were found to bind with greater affinities than sGnRH and GnRH-ANT to the carp ovarian binding sites. These results provide for the first time characterization of GnRH binding sites in the ovary of a teleost species, Cyprinus carpio.Key words: gonadotropin-releasing hormone, luteinizing hormone releasing hormone, receptor, ovary, carp, Cyprinus carpio.

Extrarenal receptor-effector-mechanisms for aldosterone: The sequence of effects on the cellular electrolyte transport in human lymphocytes and their implications for disorders of the water and electrolyte balances

1990 ◽  
Vol 123 (4) ◽  
pp. 385-394 ◽  
Author(s):  
Martin Wehling ◽  
Karl Theisen
Keyword(s):  
Cell Volume ◽  
Human Lymphocytes ◽  
Membrane Receptors ◽  
Mononuclear Leukocytes ◽  
Electrolyte Balance ◽  
Renal Tubules ◽  
Mineralocorticoid Receptors ◽  
Effector Mechanism ◽  
Vitro Effect ◽  
Human Mononuclear Leukocytes

Abstract. High affinity aldosterone binding sites have not only been described in the classic target tissues such as the renal tubules, but also in non-classic target tissues such as the hippocampus, mammary gland, endothelial cells and, recently, human mononuclear leukocytes. An in vitro effect of aldosterone on intracellular sodium, potassium and calcium concentrations and cell volume was shown in human mononuclear leukocytes. In the absence of aldosterone, the intracellular Na+, K+ and Ca2+ concentrations and the cell volume decreased significantly, but remained constant when aldosterone (1.4 nmol/l) was added to the incubation medium. These effects of aldosterone were blocked by the aldosterone antagonist canrenone (140 nmol/l). The sodium/proton exchanger of the cell membrane could be identified as the primary target of the aldosterone action, possibly non-genomically mediated through membrane receptors. The clinical significance of this model was underlined by the demonstration of absent or a decreased number of mineralocorticoid receptors and the lack of electrolyte response to aldosterone in human mononuclear leukocytes of patients with pseudohypoaldosteronism and aldosteronism. Additionally, an abnormal effector mechanism could be demonstrated in human mononuclear leukocytes from essential hypertensives. These studies are the first to demonstrate the significance of extrarenal, nonepithelial mineralocorticoid receptors and the related effector mechanism in different disorders of the water and electrolyte balance in man.

scholarly journals Characterization of high-affinity ryanodine-binding sites of rat liver endoplasmic reticulum. Differences between liver and skeletal muscle

1991 ◽  
Vol 276 (1) ◽  
pp. 41-46 ◽  
Author(s):  
V Shoshan-Barmatz ◽  
T A Pressley ◽  
S Higham ◽  
N Kraus-Friedmann
Keyword(s):  
Skeletal Muscle ◽  
Endoplasmic Reticulum ◽  
Binding Sites ◽  
Specific Binding ◽  
Dna Amplification ◽  
Single Class ◽  
High Affinity ◽  
Sodium Dantrolene

In this study, the binding of [3H]ryanodine to liver microsomal subfractions was investigated. The specific binding of [3H]ryanodine, as determined both by vacuum filtration and by ultracentrifugation, is to a single class of high-affinity binding sites with a Kd of 10 +/- 2.5 nM and density of 500 +/- 100 and 1200 +/- 200 fmol/mg of protein by the filtration and centrifugation methods respectively. [3H]Ryanodine binding reached equilibrium in about 1 min and 2 min at 36 degrees C and 24 degrees C respectively, and the half-time of dissociation at 37 degrees C was approx. 15 s. The binding of [3H]ryanodine is Ca(2+)-independent: it is slightly stimulated by NaCl, Mg2+, ATP and InsP3 but strongly inhibited by caffeine, diltiazem and sodium dantrolene. Thus the binding of ryanodine to endoplasmic reticulum membranes shares some of the characteristics of its binding to the sarcoplasmic reticulum but also differs from it in several important properties, such as its Ca(2+)-independence, its rapid association and dissociation, and its inhibition by caffeine. The structural similarities between the skeletal muscle and liver binding sites were further explored by employing in vitro DNA amplification techniques, using the known sequence of the skeletal muscle receptor as reference point. The data obtained with this method indicate that the liver does not process mRNA for the skeletal muscle ryanodine receptor.

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