Design and development of 1,3,4-oxadiazole derivatives as potential inhibitors of acetylcholinesterase to ameliorate scopolamine-induced cognitive dysfunctions

The novel hybrids bearing 4-aminopyridine (4-AP) tethered with substituted 1,3,4-oxadiazole nucleus were designed, synthesized, and evaluated for their potential AChE inhibitory property along with significant antioxidant potential. The inhibitory potential (IC₅₀) of synthesized analogs was evaluated against human cholinesterases (hAChE and hBChE) using Ellman’s method. Among all the compounds, 9 with 4-hydroxyl substituent showed maximum hAChE inhibition with the non-competitive type of enzyme inhibition (IC₅₀ = 1.098 µM; Ki = 0.960 µM). Further, parallel artificial membrane permeation assay (PAMPA-BBB) showed significant BBB permeability in most of the synthesized compounds. Meanwhile, compound 9 also inhibited AChE-induced Aβ aggregation (38.2–65.9%) by thioflavin T assay. The in vivo behavioral studies showed dose-dependent improvement in learning and memory by compound 9. The ex vivo studies also affirmed the significant AChE inhibition and antioxidant potential of compound 9 in brain homogenates.     

Novel 2-aminopyridine liganded Pd(II) N-heterocyclic carbene complexes: Synthesis,characterization, crystal structure and bioactivity properties

In this work, the synthesis, crystal structure, characterization, and enzyme inhibition effects of the novel a series of 2-aminopyridine liganded Pd(II) N-heterocyclic carbene (NHC) complexes were examined. These complexes of the Pd-based were synthesized from PEPPSI complexes and 2-aminopyridine. The novel complexes were characterized by using ¹³C NMR, ¹H NMR, elemental analysis, and FTIR spectroscopy techniques. Also, crystal structures of the two compounds were recorded by using single-crystal X-ray diffraction assay. Also, these complexes were tested toward some metabolic enzymes like α-glycosidase, aldose reductase, butyrylcholinesterase, acetylcholinesterase enzymes, and carbonic anhydrase I, and II isoforms. The novel 2-aminopyridine liganded (NHC)PdI₂(2-aminopyridine) complexes (1a-i) showed Ki values of in range of 5.78 ± 0.33–22.51 ± 8.59 nM against hCA I, 13.77 ± 2.21–30.81 ± 4.87 nM against hCA II, 0.44 ± 0.08–1.87 ± 0.11 nM against AChE and 3.25 ± 0.34–12.89 ± 4.77 nM against BChE. Additionally, we studied the inhibition effect of these derivatives on aldose reductase and α-glycosidase enzymes. For these compounds, compound 1d showed maximum inhibition effect against AR with a Ki value of 360.37 ± 55.82 nM. Finally, all compounds were tested for the inhibition of α-glycosidase enzyme, which recorded efficient inhibition profiles with Ki values in the range of 4.44 ± 0.65–12.67 ± 2.50 nM against α-glycosidase.     

Cobalt(II) coordination compounds with acetate and 2-aminopyridine ligands: Synthesis, characterization, structures and magnetic properties of two polymorphic forms

The synthesis and characterization of two polymorphic modifications of new cobalt coordination compound with 2-aminopyridine are reported. The modifications were prepared by the reaction of a solution of cobalt acetate tetrahydrate and 2-aminopyridine. The crystal structures of both polymorphic modifications have been determined by single-crystal X-ray diffraction analysis. The structures of both modifications are quite similar. In both of them the Co²⁺ is six coordinated by four O atoms from two bidentate chelate acetate ligands and by two N atoms from two 2-aminopyridine molecules. Acetate and 2-aminopyridine ligands are lying cis about the metal centre. The most important difference is in asymmetry of acetate ligand chelate bonding and in H-bonding network. Compounds exhibit an extensive system of intra and intermolecular hydrogen bonding. Magnetic properties of both modifications were studied between 2 K and 300 K giving the result μ_eff = 4.6 BM for modification I and μ_eff = 4.7 BM for modification II in paramagnetic region.     

Delayed-rectifying potassium channels in mouse peritoneal macrophages: Pharmacological analysis

The effects of 4-aminopyridine, verapamil, and 4-bromophenacylbromide (4-BPB) on the kinetics of delayed outward-rectifying potassium currents (I _K) were investigated in cultured mouse peritoneal macrophages using a classical whole-cell patch-clamp technique. The outwardI _K was completely blocked by 4-aminopyridine at 1.0 mM concentration. Verapamil at the same concentration also blockedI _K completely. Lower concentration (50 µM) of verapamil demonstrated only partial blocking action, which was almost fully reversible, and markedly increased the rate ofI _K inactivation. The main effect of 4-BPB on the outwardI _K was a significant acceleration ofI _K activation and inactivation kinetics. It is suggested that this modulation results from a direct effect of 4-BPB on potassium channels or relates to the arachidonic acid cascade.Neirofiziologiya/Neurophysiology, Vol. 26, No. 1, pp. 49–53, January–February, 1994.     

Inhibition of corticosteroid 11 beta hydroxylation in bovine zona fasciculata cells by the potassium entry blocker 4-aminopyridine

The potassium entry blocker, 4-aminopyridine has been used to evaluate the importance of this cation in several tissues including the adrenal cortex. Since it inhibited corticosteroidogenesis, an important role for potassium entry in the control of this process has been implied. In the present in vitro study, 4-aminopyridine inhibited cortisol and corticosterone production and, at high concentrations, that of 11-deoxycortisol and 11-deoxycorticosterone. However, at lower concentrations, 11-deoxycorticosteroid production increased suggesting that 4-aminopyridine acts as an inhibitor of 11 beta hydroxylation. Its slight resemblance to metyrapone may suggest that it acts, at least in part, by competing for cytochrome P450. However, its use as a potassium inhibitor in studies of the adrenal cortex may give ambiguous results.     

A novel diazonium-sulfhydryl reaction in the inactivation of yeast alcohol dehydrogenase by diazotized 3-aminopyridine adenine dinucleotide.

Diazotized 3-aminopyridine adenine dinucleotide has been found to modify four sulfhydryl groups per molecule of enzyme during the complete inactivation of yeast alcohol dehydrogenase. The reaction of sulfhydryl groups was indicated by titration studies with 5,5-dithiobis(2-nitrobenzoic acid) as well as isolation and quantitation of the cysteinyl derivative released by acid hydrolysis of the modified enzyme. The cysteinyl derivative was identified as S-(3-pyridyl)cysteine. Authentic S-(3-pyridyl)cystein was synthesized and structurally characterized for these studies. Diazonium-sulfhydryl reactions were demonstrated for a number of diazonium derivatives with cysteine, homocysteine, glutathione, and mercaptoethanol at 0-4 degrees and neutral pH. Second order rate constants were determined in reactions of these sulfhydryl compounds with diazotized 1-methyl-3-aminopyridinium chloride, diazotized 3-aminopyridine adenine dinucleotide, and diazotized 3-aminopyridine adenine dinucleotide phosphate.     

Studies of yeast alcohol dehydrogenase with 3-aminopyridine mononucleotide

Summary 3-Aminopyridine mononucleotide, a nicotinamide mononucleotide analog, was prepared by enzymatic cleavage of 3-aminopyridine adenine dinucleotide by a snake venom phosphodiesterase and isolated by means of ion exchange chromatography. The spectrophotometric and fluorometric properties of this analog were studied. Several anions were shown to quench the fluorescence intensity of this analog. pH was shown to have a pronounced effect on the fluorescence intensity. 3-Aminopyridine mononucleotide was shown to be a coenzyme-competitive inhibitor of yeast alcohol dehydrogenase. The 3-aminopyridine mononucleotide was diazotized with the use of nitrous acid. A time dependent irreversible inactivation of yeast alcohol dehydrogenase resulted from incubation with the diazotized 3-aminopyridine mononucleotide at pH 7.0. Incubation of the enzyme with NAD prior to the addition of the diazotized 3-aminopyridine mononucleotide protected the enzyme against inactivation.Recently, 3-aminopyridine adenine dinucleotide (AAD) and 3-aminopyridine adenine dinucleotide phosphate (AADP), NAD and NADP analogs respectively, were synthesized by either chemical or enzymatic processes. The chemical, spectrophotometric properties of these dinucleotides have also been reported. It was demonstrated that these nucleotides serve as coenzyme-competitive inhibitors of dehydrogenases but did not function as coenzymes for oxidation-reduction reactions catalyzed by these enzymes. The pyridine amino group of AAD was diazotized and the diazotized derivative was shown to inactive yeast alcohol dehydrogenase irreversibly. Isolation of modified cysteine residue from the modified yeast alcohol dehydrogenase resulting from inactivation by diazotized AAD has been reported. Thus, diazotized AAD proved to be a site specific label for the coenzyme binding site of yeast alcohol dehydrogenase. It was of interest to prepared and determine the properties of a NMN analog, 3-aminopyridine mononucleotide (APMN). The preparation of APMN was accomplished by enzymatic cleavage of AAD with snake venom phosphodiesterase according to a method previously reported. This report deals with the preparation, properties and studies of APMN with yeast alcohol dehydrogenase.This work was supported in part by Research Grant GR-IX from Old Dominion University Research Foundation.     

Simultaneous determination of ritonavir and saquinavir, two human immunodeficiency virus protease inhibitors, in human serum by high-performance liquid chromatography

The aim of this study was to describe an high-performance liquid chromatographic assay for the simultaneous determination of two HIV protease inhibitors, saquinavir and ritonavir, in human serum. The method involved extraction of ritonavir and saquinavir from serum with the aid of solid-phase extraction C₁₈ cartridges followed by high-performance liquid chromatography with a C₈ column and ultraviolet detection set at a wavelength of 240 nm. The assay was linear and has been validated over the concentrations range of 0.5–32 μg/ml for ritonavir and 0.075–4.8 μg/ml for saquinavir, from 600 μl serum extracted. In future, the assay will be used to support human population pharmacokinetic studies, and therapeutic drug monitoring for ritonavir and saquinavir.     

A study on the inhibition of adenosine deaminase

Adenosine deaminase (ADA, EC 3.5.4.4) catalyses the irreversible deamination of adenosine and 2′-deoxyadenosine to inosine and 2′-deoxyinosine, respectively. In this study the inhibition of ADA from bovine spleen by several molecules with structure related to that of the substrate or product has been quantified. The inhibitors adenine, purine, inosine, 2-aminopurine, 4-aminopyrimidine, 4-aminopyridine, 4-hydroxypyridine and phenylhydrazine are shown to be competitive inhibitors with K_I (mM) values of 0.17, 1.1, 0.35, 0.33, 1.3, 1.8, 1.4 and 0.25, respectively. Synergistic inhibition by various combinations of molecules that imitate the structure of the substrate has never been observed. Some general conclusions are: i) the enzyme ADA from bovine spleen we have used is appropriate for kinetic studies of inhibition and mechanistic studies; it can be a reference catalytic system for the homogeneous comparison of various inhibitors; ii) this enzyme presents very rigid requirements for binding the substrate: variations in the structure of adenosine imply the loss of important interactions.     

Studies of NAD kinase and NMN:ATP adenylyltransferase in Haemophilus influenzae

Previous studies of Haemophilus influenzae documented the importance of several pyridine nucleotide-dependent enzymes in processing extracellular NAD and NMN to satisfy the V-factor growth requirement of the organism. The substrate specificities of two of these enzymes. NMN:ATP adenylyltransferase and NAD kinase, were investigated following partial purification. The ability of the transferase to utilize 3-acetylpyridine mononucleotide and 3-aminopyridine mononucleotide as substrates for the synthesis of the corresponding dinucleotides was demonstrated. The NAD kinase was observed to accept 3-acetylpyridine adenine dinucleotide as a substrate but failed to utilize 3-aminopyridine adenine dinucleotide. The mononucleotides of 3-acetylpyridine and 3-aminopyridine were shown to be as effective as the corresponding dinucleotides in the support of growth and inhibition of growth of H. influenzae, respectively. Inhibition of growth of H. influenzae by submicromolar 3-aminopyridine adenine dinucleotide was shown to occur because 3-aminopyridine mononucleotide was produced from it in reactions catalysed by the H. influenzae periplasmic nucleotide pyrophosphatase. The presence of an additional important pyridine nucleotide-dependent enzyme, NMN glycohydrolase, is also reported.     

A comparison of two methods for determining titanium dioxide marker content in broiler digestibility studies

The use of inert markers in broiler diets eliminates the need to quantitatively evaluate feed intake and excreta output to determine diet digestibility, and enables nutrient uptake at specific points along the gastrointestinal tract to be examined. Titanium dioxide (TiO₂) is commonly used for this purpose and measured using a UV-spectrophotometric assay. Two experiments were conducted to observe whether an inductively coupled plasma optical emission spectrophotometer (ICP-OES) assay is able to replace the UV-spectroscopy assay for rapid analysis of TiO₂ in broiler feed and ileal digesta samples. In the first experiment, TiO₂ was added at 5 g/kg to 19 broiler diets. Ross 308 male broilers (n=452) fed these diets were involved in a series of digestion studies to determine ileal digesta recovery of TiO₂. In the second experiment, defined amounts of TiO₂ were added to ileal digesta samples from Ross 308 male broilers (n=176) and TiO₂ recoveries were determined. The feed and ileal samples from both experiments were analysed by both UV-spectroscopy and ICP-OES, and relatedness of the findings from the two assays was determined. Overall relatedness of the two assays was strong for determination of TiO₂ concentration in both the broiler diets and ileal digesta samples (r=0.908 and r=0.884, respectively). Overall recovery of supplemented TiO₂ was 97.62% by the UV-spectroscopy assay and 98.77% by the ICP-OES assay. The ICP-OES assay in this study was as accurate as spectrophotometric determination for the quantification of TiO₂ content. The ICP-OES method can also be used to analyse several elements within one assay, with a single preparation step, and thus the measurement of TiO₂ may be incorporated into the analysis of other minerals. Time and resources dedicated to determining diet digestibility in broilers could be minimised by using the ICP-OES assay to replace the UV-spectroscopy assay when measuring TiO₂ concentration.     

Molecular docking study of the binding of aminopyridines within the K<Superscript>+</Superscript> channel

We present a molecular docking study aimed to identify the binding site of protonated aminopyridines for the blocking of voltage dependent K⁺ channels. Several active aminopyridines are considered: 2-aminopyridine, 3-aminopyridine, 4-aminopyridine, 3,4-diaminopyridine, and 4-aminoquinoleine. We apply the AutoDock force field with a lamarckian genetic algorithm, using atomic charges for the ligands derived from the electrostatic potential obtained at the B3LYP/cc-pVDZ level. We find a zone in the α-subunit of the K⁺ channel bearing common binding sites. This zone corresponds to five amino acids comprised between residuals Thr107 and Ala111, in the KcsA K⁺ channel (1J95 pdb structure). The 2-aminopyridine, 3-aminopyridine, 4-aminopyridine, and 3,4-diaminopyridine bind to the carboxylic oxygens of Thr107 and Ala111. In all cases aminopyridines are perpendicular to the axis of the pore. 4-aminoquinoleine binds to the carboxylic oxygen of Ala111. Due to its large size, the molecular plane is parallel to the axis of the pore. The charge distributions and the structures of the binding complexes suggest that the interaction is driven by formation of several hydrogen bonds. We find 2-aminopyridine, 3-aminopyridine, 4-aminopyridine, and 3,4-diaminopyridine with similar binding energy. Considering the standard error of the estimate of the AutoDock force field, this energy should lie, as a rough estimation, in the interval 3–7 kcal mol⁻¹. On the other hand, 4-aminoquinoleine seems to have a smaller binding energy. Figure Three-dimensional structure of the complex between 4-AQH⁺ and the binding sites of the K⁺ pore. Only the amino acid sequence from Thr107 to Ala111 is considered. Two different representations are included. In the left, the Thr107 position is marked. The right representation shows the CO oxygens of the peptide bond as spacefilled structures.     

Tetraethylammonium and 4-aminopyridine block calcium-dependent chloride current in rat cerebellum Purkinje cells

Using patch-clamp method (whole cell configuration), it was shown that tetraethylammonium (TEA) and 4-aminopyridine (4-AP) block calcium-dependent chloride currents in the membrane of freshly isolated cerebellar Purkinje cells of rats (12–15 days). In the concentration range studied (50 μM–10 mM TEA and 100 μM–1 mM 4-AP), both compounds blocked the chloride current at IC₅₀ 130 μM for TEA and 110 μM for 4-AP. TEA blockade was reversible after washing. The effect of 4-AP at concentrations greater than 100 μM was irreversible: both outward and inward chloride currents were blocked even after the removal of 4-AP from the incubation medium.     

Spectral characterization of a fluorescent nicotinamide adenine dinucleotide analog: 3-Aminopyridine adenine dinucleotide

Both absorption and fluorescence properties of 3-amino pyridine adenine dinucleotide (AAD) were examined. The shape of the AAD fluorescence emission spectrum, maximal at 425 nm, remains unchanged over the pH range 2 to 11, indicating that there is only one detectable emitting species. AAD fluorescence increases as the pH decreases, with an apparent pK_a of about 3.5. The absorption-pH profile indicates a pK_a of about 3.3 for the ground state of AAD. Effects of organic solvents on AAD fluorescence are somewhat diverse. The low fluorescence quantum yield of 0.022 corresponds well with the short lifetime of 1.15 ns at 23 °C in neutral aqueous solution. The steady-state polarization of AAD in water and that at infinite viscosity were determined at 23 °C to be 0.037 and 0.083, respectively. Since a smaller value of polarization for either donor or acceptor leads to a better estimate of the orientation factor for dipole-dipole interaction, AAD appears to be particularly suitable for energy transfer studies. Similar to NADH, AAD also assumes a folded conformation in aqueous solution. This is evident from effects of temperature and hydrolysis by phosphodiesterase on absorption and/or fluorescence properties of AAD. Energy transfer from the adenine group to the 3-aminopyridine ring has been detected to occur in aqueous solution at 23 °C with an efficiency of about 0.12, corresponding to a distance of 7.5 Å between these two ring moieties.     

4-Aminopyridine based amide derivatives as dual inhibitors of tissue non-specific alkaline phosphatase and ecto-5′-nucleotidase with potential anticancer activity

Ecto-nucleotidase members i.e., ecto-5′-nucleotidase and alkaline phosphatase, hydrolyze extracellular nucleotides and play an important role in purinergic signaling. Their overexpression are implicated in a variety of pathological states, including immunological diseases, bone mineralization, vascular calcification and cancer, and thus they represent an emerging drug targets. In order to design potent and selective inhibitors, new derivatives of 4-aminopyridine have been synthesized (10a-10m) and their structures were established on the basis of spectral data. The effect of nature and position of substituent was interestingly observed and justified on the basis of their detailed structure activity relationships (SARs) against both families of ecto-nucleotidase. Compound 10a displayed significant inhibition (IC₅₀ ± SEM = 0.25 ± 0.05 µM) that was found ≈168 fold more potent as compared to previously reported inhibitor suramin (IC₅₀ ± SEM = 42.1 ± 7.8 µM). This compound exhibited 6 times more selectivity towards h-TNAP over h-e5′NT. The anticancer potential and mechanism were also established using cell viability assay, flow cytometric analysis and nuclear staining. Molecular docking studies were also carried out to gain insight into the binding interaction of potent compounds within the respective enzyme pockets and herring-sperm DNA.     

The role of various calcium and potassium channels in the regulation of somatodendritic serotonin release

We prepared slices from midbrain containing the raphe nuclei and from hippocampus of rats. The brain slices were loaded with [³H]serotonin and superfused in order to measure the release of radioactivity at rest and in response to electrical stimulation. No difference was observed in the resting and stimulated fractional release of tritium in the somatodendritic and axon terminal parts of serotonergic neurons. The selective 5-HT_1A receptor agonist 8-OH-DPAT decreased the electrically induced tritium effux from raphe nuclei slices preloaded with [³H]serotonin, and this inhibition was reversed by 5-HT_1A receptor antagonist (+)WAY-100135. The 5-HT_1B receptor agonist CGS-12066B but not 8-OH-DPAT, inhibited the stimulation-evoked tritium efflux from hippocampal slices after labeling with [³H]serotonin. The electrical stimulation-evoked tritium efflux in raphe nuclei slices incubate with [³H]serotonin was completely external Ca²⁺-dependent, and omega-conotoxin GVIA and Cd²⁺, but not diltiazem, inhibited the tritium overflow. In raphe nuclei slices 4-aminopyridine enhanced the electrical stimulation-induced trititum release in a concentration-dependent manner. The inhibition of tritium efflux by 8-OH-DPAT was abolished with 4-aminopyridine. Glibenclamide or tolbutamide proved to be ineffective. These data indicate that (1) different 5-HT receptor subtypes (5-HT_1A and 5-HT_1B) regulate dendritic and axon terminal 5-HT release; (2) serotonin release from the dendrites may be regulated by the voltage-sensitive N-type Ca²⁺ channels; (3) the 5-HT_1A receptor-mediated inhibition of serotonin release may be due to opening of voltage-sensitive K⁺ channels.     

Scintillation proximity assay (SPA) as a new approach to determine a ligand’s kinetic profile. A case in point for the adenosine A<Subscript>1</Subscript> receptor

Scintillation proximity assay (SPA) is a radio-isotopic technology format used to measure a wide range of biological interactions, including drug-target binding affinity studies. The assay is homogeneous in nature, as it relies on a “mix and measure” format. It does not involve a filtration step to separate bound from free ligand as is the case in a traditional receptor-binding assay. For G protein-coupled receptors (GPCRs), it has been shown that optimal binding kinetics, next to a high affinity of a ligand, can result in more desirable pharmacological profiles. However, traditional techniques to assess kinetic parameters tend to be cumbersome and laborious. We thus aimed to evaluate whether SPA can be an alternative platform for real-time receptor-binding kinetic measurements on GPCRs. To do so, we first validated the SPA technology for equilibrium binding studies on a prototypic class A GPCR, the human adenosine A₁ receptor (hA₁R). Differently to classic kinetic studies, the SPA technology allowed us to study binding kinetic processes almost real time, which is impossible in the filtration assay. To demonstrate the reliability of this technology for kinetic purposes, we performed the so-called competition association experiments. The association and dissociation rate constants (k _on and k _off) of unlabeled hA₁R ligands were reliably and quickly determined and agreed very well with the same parameters from a traditional filtration assay performed simultaneously. In conclusion, SPA is a very promising technique to determine the kinetic profile of the drug-target interaction. Its robustness and potential for high-throughput may render this technology a preferred choice for further kinetic studies.     

Microtubule-associated protein 2-positive cells derived from microglia possess properties of functional neurons

Microglia are believed to play an important role in the regulation of phagocytosis, neuronal survival, neuronal cell death, and inflammation. Recent studies have demonstrated that microglia are multipotential stem cells that give rise to neurons, astrocytes, and oligodendrocytes. However, the functional properties of neurons derived from microglia are poorly understood. In this study, we investigated the possibility that microglia differentiate into functional neurons. Immunocytochemical study demonstrated that microtubule-associated protein 2 (MAP2)-positive cells were derived from microglia under differentiation conditions. Intracellular Ca²⁺ imaging study demonstrated that KCl caused no significant changes in [Ca²⁺]_i in microglia, whereas it caused a remarkable increase in [Ca²⁺]_i in microglia-derived cells. Furthermore, electrophysiological study demonstrated that the spike waveform, firing rate, and tetrodotoxin sensitivity of extracellular action potentials evoked by 4-aminopyridine from microglia-derived MAP2-positive cells were nearly identical to those from cultured cortical neurons. These results suggest that microglia-derived MAP2-positive cells possess properties of functional neurons.     

An insight into the ribonucleolytic and antiangiogenic activity of buffalo lactoferrin

Lactoferrin (LF) has several biological effects ranging from ribonuclease activity to antiangiogenic activity. It thus serves as a potential target protein for studies related to ribonucleolytic activity in association with its antiangiogenic activity. We have isolated buffalo LF and checked the ribonucleolytic activity via an agarose gel-based assay and precipitation assay. The ribonucleolytic activity of LF is lower compared to RNase A and the pH profile is a bell-shaped curve, with a pK₁ value of 5.43 and pK₂ of 7.65. The ribonuclease inhibitor that inhibits many ribonuclease-type proteins by forming a tight complex is unable to inhibit the ribonucleolytic property of LF. Fe(III) behaves as a noncompetitive inhibitor for the ribonucleolytic activity of protein. The superoxide-scavenging activity of the protein has also been measured. Histidine modification by diethylpyrocarbonate was monitored by UV–Vis spectroscopy at pH 7 and pH 8 and the effect towards the ribonucleolytic activity was determined. The antiangiogenic property of LF was investigated by the chorioallantoic membrane assay. Finally, the possible active site was analyzed via docking studies and correlated with the experimental study.     

Simultaneous Measurements of Estrogen Nuclear (E2Rn) and Progestin Cytosol (PRc) Receptors in the Hypothalamus and Pituitary Gland of Rats

Abstract

The present methods used to measure estrogen nuclear (E₂Rn) and progestin cytosol (PRc) receptors in the hypothalamus and pituitary gland require that separate assays be performed to determine the concentrations of each receptor. In the present studies we describe a method which simultaneously measures both E₂Rn and PRc in hypothalamic and pituitary tissue. Tissue samples were homogenized in tris-EDTA-glycerol-dithiothreitol buffer and centrifuged at 1500 × g for 5 min. The supernatant was purified for the PRc assay while the nuclear pellet was extracted for the E₂Rn exchange assay.

For the PRc assay, the supernatant was centrifuged at 106,500 × g for 30 min and aliquots from the resultant supernatant then were incubated with ³H-R5020. For the E₂Rn exchange assay, the original pellet was purified further by successively resuspending and centrifuging it through several sucrose solutions. Estrogen-receptor complexes were extracted from the chromatin pellet with a 0.4M KC1 solution and aliquots of the final supernatant were incubated with ³H-estradiol.

In both assays, the samples were placed onto Sephadex LH20 columns and the receptor bound ³H-steroid was eluted directly into scintillation vials. Scatchard analyses revealed that these assays measure a single class of binding sites for E₂Rn and PRc with dissociation constants (K_D) and maximal number of binding sites (Bmax) similar to those previously reported using a separate assay for each receptor.