Specific binding of 125I-LHRH agonist to hippocampal membranes: fluctuations during the estrous cycle.

Specific binding of 125I-[D-Ala6-CH3-Leu7-Pro9,NHET]LHRH, a LHRH agonist, to hippocampal membranes prepared from ovariectomized female rates was examined. One high affinity binding site was observed with a Kd of 0.12 +/- 0.01 nM and an apparent Bmax of 13.0 +/- 3.8 fmol/mg. Luteinizing hormone-releasing hormone and the behaviorally active Ac-LHRH(5-10) were able to compete for the agonist binding site. Native LHRH had an apparent Ki of 1.73 nM, while AC-LHRH(5-10) was 30 times less potent. Competition studies examined over the rat estrous cycle revealed an eighteenfold decrease in apparent affinity during diestrus I and estrus compared with ovariectomized animals. Tissue from animals in proestrus had a Ki of 5.0 nM. Specific binding studies indicate that receptor concentration is highest in proestrus (6.11 +/- 0.90 fmol/mg) and significantly lower during estrus (2.4 +/- 0.29 fmol/mg). These data suggest that at least one fragment of native LHRH can interact with neuronal LHRH receptors and that these receptors, like those in the pituitary, can be modulated by circulating steroid hormones.     

QSAR of adenosine A3 receptor antagonist 1,2,4-triazolo[4,3-a]quinoxalin-1-one derivatives using chemometric tools

Considering the potential of selective adenosine A3 receptor subtype ligands in the development of prospective therapeutic agents, an attempt has been made to explore physicochemical requirements of 1,2,4-triazolo[4,3-a]quinoxalin-1-one derivatives for A3 receptor binding. In this study, lipophilicity (logP), physicochemical substituent constants (pi, MR, sigma p) of phenyl ring substituents, and Wang-Ford charges of common atoms of the quinoxaline nucleus (calculated from molecular electrostatic potential surface of energy-minimized geometry using AM1 technique) were used as independent variables along with suitable dummy parameters. The best multiple linear regression (MLR) equation obtained from factor analysis (FA-MLR) as the preprocessing step could explain and predict 72.6% and 65.3%, respectively, of the variance of the binding affinity. The same equation also emerged as the best equation in the population of 100 equations obtained from genetic function approximation (GFA-MLR). The results suggested that presence of an electron-withdrawing group at the para position of the phenyl ring would be favorable for the binding affinity. Again, the presence of a nitro group at position R1 increases the binding affinity. When factor scores were used as predictor variables in the principal component regression analysis, the resultant model showed 78.6% explained variance and 63.1% predicted variance. The best equation derived from G/PLS could explain and predict 74.4% and 64.8%, respectively. The results have suggested the importance of Wang-Ford charges of atoms C15 and C19, apart from positive contributions of electron-withdrawing para substituents of the variance of the phenyl ring and nitro group at the R1 position.     

On the role of luteinizing hormone-releasing hormone in the in vitro synthesis of bioactive human chorionic gonadotropin in human pregnancies

The dynamics of luteinizing hormone-releasing hormone (LHRH) induced human chorionic gonadotropin (hCG) production were studied in isolated placental cells from normal and anencephalic midterm and term gestations. A spontaneous release of immunoreactive hCG was first detected after 24-36 h of preparation in term control cells. The addition of LHRH at a concentration ranging from 10(-9) to 10(-6) M induced a threefold increase in this output of hCG. Placental cell responsiveness to LHRH varied according to the number of days of cell cultures, with maximal response on days 1 and 6. Placental cells from normal pregnancies incubated with 1 X 10(-6) M LHRH showed a release of both immuno- and bio-assayable hCG, which was four- to six-fold higher at midgestation than at term (p less than 0.001). In contrast, placental cells from pregnancies with anencephalic fetuses showed, at both stages of gestation, an hCG production that was comparable to that observed with normal term placental cells. We conclude that LHRH at a concentration appropriate for its placental receptor binding affinity induces a production of bioactive hCG in humans. Furthermore, our data suggest that anencephaly changes the placental response of hCG to LHRH stimulation.     

Preparation of a monoclonal antibody to common amino acid sequence of LHRH and its application

In order to prepare an antibody directed at the common amino acid sequence of mammalian, avian, and fish luteinizing hormone-releasing hormones (LHRHs), C-terminal free LHRH was conjugated with bovine thyroglobulin, and was used as the antigen. A monoclonal antibody (LRH13) was obtained as an ascitic fluid by fusing the spleen cells of a BALB/c donor mouse immunized with the antigen to X63.Ag8.653 mouse myeloma cells followed by limiting dilution cloning and transplanting a positive clone to BALB/c mice. This monoclonal antibody seems to belong to IgG2b as it was eluted from protein A-Sepharose CL-4B with citrate buffer pH 3.5. competitive binding experiment using fragment peptides of LHRH indicated the binding site of LRH13 was a region around serine and tyrosine, and modification of mammalian LHRH by radioiodination caused a marked decrease in the binding activity. LRH13 has an affinity constant of 0.134 X 10(9) M-1 to native mammalian LHRH, and binds C-terminal free LHRH with a similar affinity (1.6X), however, it binds with higher affinities to N- and C-terminal free LHRH (12.9X), N-terminal free LHRH (10.4X), salmon LHRH (8.3X) and chicken LHRH-I (6.0X). Chicken LHRH-II, where tyrosine is replaced for histidine, has a lower affinity (0.3X) than that of mammalian LHRH. From its high affinity to N-, C-terminal free LHRH, LRH13 is also expected to bind possible precursor peptides of LHRH. Immunohistochemical staining of the brain sections obtained from rats, mice, chickens, Japanese quail, and rainbow trout successfully visualized cell bodies and fibers distributed from the olfactory bulb to the median eminence, indicating high LHRH specificity and wide crossreactivity in animal classes of this monoclonal antibody. With this antibody, LHRH-like immunoreactive substance in the pineal gland was also stained with fixation at neutral pH.     

Locating an antagonist in the 5-HT3 receptor binding site using modeling and radioligand binding

We have used a homology model of the extracellular domain of the 5-HT(3) receptor to dock granisetron, a 5-HT(3) receptor antagonist, into the binding site using AUTODOCK. This yielded 13 alternative energetically favorable models. The models fell into 3 groups. In model type A the aromatic rings of granisetron were between Trp-90 and Phe-226 and its azabicyclic ring was between Trp-183 and Tyr-234, in model type B this orientation was reversed, and in model type C the aromatic rings were between Asp-229 and Ser-200 and the azabicyclic ring was between Phe-226 and Asn-128. Residues located no more than 5 A from the docked granisetron were identified for each model; of 26 residues identified, 8 were found to be common to all models, with 18 others being represented in only a subset of the models. To identify which of the docking models best represents the ligand-receptor complex, we substituted each of these 26 residues with alanine and a residue with similar chemical properties. The mutant receptors were expressed in human embryonic kidney (HEK)293 cells and the affinity of granisetron determined using radioligand binding. Mutation of 2 residues (Trp-183 and Glu-129) ablated binding, whereas mutation of 14 other residues caused changes in the [(3)H]granisetron binding affinity in one or both mutant receptors. The data showed that residues both in and close to the binding pocket can affect antagonist binding and overall were found to best support model B.     

Antigen-binding receptors on T cells from long-term MLR. Evidence of binding sites for allogeneic and self-MHC products

Antibody inhibition of radiolabelled stimulator membrane vesicle binding by T blasts activated in the mixed lymphocyte reaction (MLR) was used to identify responder-cell determinants involved in the binding phenomenon. Antisera or monoclonal antibodies against Thy-1, Lyt-1, Lyt-2 and Ly-6 antigens were not inhibitory. However, antibodies against heavy-chain V region (V_H) determinants strongly inhibited vesicle binding by both primary and longterm MLR blasts. Anti-Ia (both alloantisera and monoclonal reagents) caused inhibition of antigen binding by primary MLR blasts only. T blasts from long-term MLR lines were neither Ia-positive, nor susceptible to blocking of antigen binding with anti-Ia. However, these cells were capable of specifically absorbing soluble syngeneic Ia material, with the concomitant appearance of vesiclebinding inhibition with anti-Ia sera. Acquisition of syngeneic Ia by T blasts was effectively blocked with the anti-V_H reagent. Passively bound self-Ia did not interfere with vesicle binding in the absence of anti-Ia. These results strongly suggest the existance of specific self-Ia acceptor sites closely linked to the receptors for stimulator alloantigens on T cells proliferating in MLR. A receptor model based on these findings is briefly discussed.Abbreviations used in this paper B10 C57BL/10 - Con A concanavalin A - FcR Fc receptor - FCS fetal calf serum - H heavy chain - Ia I-region associated antigen - Ig immunoglobulin - LPS lipopolysaccharide - Lyt T-lymphocyte differentiation antigen - MHC major histocompatibility complex - MLR mixed lymphocyte reaction - PM plasma membrane - T thymus derived - Tcr T-cell receptor - V variable region of Ig     

Variations in the number of pituitary LHRH receptors correlated with altered responsiveness to LHRH

Isolated pituitary cells from metestrous, ovariectomized (OVX), and ovariectomized-estradiol treated (OVX-EB) rats were employed to study the gonadotropin response to luteinizing hormone-releasing hormone (LHRH) challenge and to quantitate LHRH receptors, using a labeled LHRH analog. Ovariectomy (3–4 weeks post castration) resulted in a reduction of LHRH receptor concentration from 34.4 ± 2.1 in metestrous females to 14.3 ± 0.9 fmoles/10⁶ cells. Concomitantly, the luteinizing hormone (LH) response to a near-maximal dose of LHRH (5 ng/ml) decreased from a 3-fold stimulation in intact females to 1.13-fold stimulation in cells from OVX rats. Replacement therapy with EB (50 ug/rat for 2 days) to OVX rats restored LH response and LHRH binding sites (a 2.5-fold stimulation in LH secretion and 32.0 ± 2.1 fmoles/10⁶ cells, respectively). The LH response to LHRH stimulation was not altered after one day of EB treatment although the number of LHRH binding sites was increased. The changes in the number of LHRH binding sites were not accompanied by any alterations in the affinity of the LHRH analog ($\text{K}{}_{\mathrm{d}}\text{? 0.5 × 10}{}^{\mathrm{?9}}\text{M}$). It is concluded that variations in LHRH receptor number reflect the degree of pituitary sensitivity to LHRH and it may suggest that LHRH and estradiol modulation of gonadotropin release is mediated by these receptors.     

Effects of aging on pituitary and testicular luteinizing hormone-releasing hormone receptors in the rat

Aging exerts profound influences on the function of the hypothalamic-pituitary-testicular-axis. This work has been performed in order to verify whether, in male rats, the decreased secretion of LH and testosterone (T) occurring in old animals is reflected by modifications of luteinizing hormone-releasing hormone (LHRH) receptors at the level of the anterior pituitary and of the testes. To this purpose, the affinity constant (Ka) and the maximal binding capacity (Bmax) for the LHRH analog [D-Ser(tBu)6]des-Gly10-LHRH-N-ethylamide were evaluated, by means of a receptor binding assay, in membrane preparations derived from the anterior pituitary and testicular Leydig cells of male rats of 3 and 19 months of age. Serum levels of LH and T were measured by specific RIAs. The results obtained show that, in aged male rats, the concentration of pituitary LHRH receptors is significantly lower than that found in young animals. On the other hand, the concentration of LHRH binding sites is significantly increased on the membranes of Leydig cells of old rats. In no instance the Ka for the LHRH analog is significantly affected. Serum levels of LH and T are significantly lower in old than in young male rats. In conclusion, these results suggest that the reduced secretion of LH in old male rats may be linked, at least partially, to a decrease of the number of pituitary LHRH receptors. The impaired production of testosterone occurring in aged rats is accompanied by a significant increase of the number of testicular LHRH receptors, indicating that also the intratesticular mechanisms controlling testosterone release undergo significant alterations with aging.     

Effect of receptor-ligand affinity on the strength of endothelial cell adhesion.

The objective of this study was to determine the effect of receptor-ligand affinity on the strength of endothelial cell adhesion. Linear and cyclic forms of the fibronectin (Fn) cell-binding domain peptide Arg-Gly-Asp (RGD) were covalently immobilized to glass, and Fn was adsorbed onto glass slides. Bovine aortic endothelial cells attached to the surfaces for 15 min. The critical wall shear stress at which 50% of the cells detached increased nonlinearly with ligand density and was greater with immobilized cyclic RGD than with immobilized linear RGD or adsorbed Fn. To directly compare results for the different ligand densities, the receptor-ligand dissociation constant and force per bond were estimated from data for the critical shear stress and contact area. Total internal reflection fluorescence microscopy was used to measure the contact area as a function of separation distance. Contact area increased with increasing ligand density. Contact areas were similar for the immobilized peptides but were greater on surfaces with adsorbed Fn. The dissociation constant was determined by nonlinear regression of the net force on the cells to models that assumed that bonds were either uniformly stressed or that only bonds on the periphery of the contact region were stressed (peeling model). Both models provided equally good fits for cells attached to immobilized peptides whereas the peeling model produced a better fit of data for cells attached to adsorbed Fn. Cyclic RGD and linear RGD both bind to the integrin alpha v beta 3, but immobilized cyclic RGD exhibited a greater affinity than did linear RGD. Receptor affinities of Fn adsorbed to glycophase glass and Fn adsorbed to glass were similar. The number of bonds was calculated assuming binding equilibrium. The peeling model produced good linear fits between bond force and number of bonds. Results of this study indicate that 1) bovine aortic endothelial cells are more adherent on immobilized cyclic RGD peptide than linear RGD or adsorbed Fn, 2) increased adhesion is due to a greater affinity between cyclic RGD and its receptor, and 3) the affinity of RGD peptides and adsorbed Fn for their receptors is increased after immobilization.     

An <Emphasis Type="Italic">Insilico</Emphasis> approach to High Altitude Pulmonary Edema - Molecular modeling of human β<Subscript>2</Subscript> adrenergic receptor and its interaction with Salmeterol &; Nifedipine

Knowledge of the three-dimensional structures of protein targets from genomic data has the potential to accelerate researches pertaining to drug discovery. Human β₂ adrenergic receptor is a G-protein-coupled receptor with seven transmembrane helices, and is important in pharmaceutical targeting on pulmonary and cardiovascular diseases. The human β₂ adrenergic receptor has been found to play a very important role in the pathogenesis of high altitude pulmonary edema (HAPE). In the present study, a high quality of protein 3D structure has been predicted for the human β₂ adrenergic receptor sequence with primary accession number P07550. Homologous template protein sequence with known 3D structure was identified and the template-query protein sequence validation was done by multiple sequence alignment method. The homology model was performed through Modeller and depended on the quality of the sequence alignment by BLAST, template structure and the consolidated result performed by Gene silico meta-server. The statistical verification of the generated model was evaluated by PROCHECK which revealed that the structure modeled through Modeller to be of good quality with 84.1% of residues in the most favored region. Docking studies were carried out after modeling with two well known ligands namely Salmeterol and Nifedipine, and the fitness score revealed that Salmeterol has a higher fitness score than Nifedipine. Estimation of binding affinity by X-Score revealed that Salmeterol had −10.40 binding affinity while Nifedipine showed −9.62 binding affinity. From the present study, it can be concluded that the generated model of human β₂ adrenergic receptor can be used for further studies related to this receptor and Salmeterol was found to have a high binding affinity with human β₂ adrenergic receptor.