Effects of Sulfhydryl Reagents on [3H] Inositol Trisphosphate Binding to Dog Cerebellar Membranes

Abstract

Homogenates from dog cerebellum were fractionated using sucrose gradient centrifugation. The [³H]inositol 1, 4, 5-trisphosphate binding and the glucose 6-phosphatase activities were found to co-purify. The binding was saturable and had high affinity (B_max=44 pmol/mg protein, K_d=116 nM). Selective chemical modification was used to examine amino acid residues of the microsomal receptor that might be critical for the binding of inositol trisphophate. Sulfhydryl reagents, p-chloromercuric-phenyl sulfonic acid, eosin 5-maleimide, N-ethyl maleimide and fluorescein 5-maleimide were found to be highly potent inhibitors of the binding with half-maximal inhibition occurring at about 20 µM, 70 µM, 1 mM, and 0.1 mM, respectively. The inhibition was specific since the presence of 10 µM of inositol trisphosphate during the reaction completely protected against the inhibition by these reagents. These results suggest that sulfhydryl group is essential for inositol trisphosphate binding to its receptor.     

Effects of Salt Extraction on the Quantitation of Nuclear Estrogen Receptors: Interference by Secondary Estrogen Binding Sites

Abstract

The effects of salt-extraction on type I and type II estrogen binding sites were examined in uterine nuclei. Injection (10 ug) of estradiol or estriol in adult ovariectomized rats induced maximum numbers (80–100%, ~ 1 pmole/uterus) of 0.4 M KCL resistant type I estrogen complexes at 1 hour. Only estradiol, which sustained these levels for long periods of time (4–24 hours) stimulated true uterine growth.

Likewise, a single injection of estradiol, but not estriol, also elevated nuclear type II sites throughout the entire uterine growth period (1–48 hours). However extraction of these nuclei from estradiol injected rats with 0.4 M KCL increased the numbers of type II sites from ~ 1 pmole/uterus (non-extracted nuclei) to ~ 8 pmoles/uterus (salt resistant plus salt-extractable fractions). Sixty percent of these sites were resistant to salt-extraction. Continuous exposure to either estradiol or estriol by beeswax implants stimulated nuclear type II sites which were highly resistant (80%) to KCL-extraction, and additional sites were not exposed by high salt. Thus chronic treatment with both estrogens “locked in” nuclear type II sites such that they were resistant to KCL-extraction. This resistance of type II sites to salt-extraction correlated with the ability of estradiol and estriol implants to stimulate true uterine growth. The procedures presented here for nuclear preparation and assay have reduced non-specific binding considerably in the uterine system, and may eliminate the need to perform exchange assays on salt-extracted nuclei in other systems.     

An Exchange Assay for the Measurement of Cell Nuclear Estrogen Receptors in Microdissected Brain Regions

An exchange assay is described for the measurement of nuclear estrogen receptors (ERn) in microdissected brain regions. The distribution of ERn in the hypothalamus and amygdala of the rat 1 h after an injection of estradiol (E) is presented. Combining the exchange assay with a previously described method for measurement of cytosol estrogen receptors (ERc) in microdissected brain samples, gonadectomized male and female rats were compared for ERc and ERn. While ERc concentrations tended to be higher in females than in males in all regions of the hypothalamus, with a significant sex difference in the arcuate-median eminence, no sex difference in ERn concentrations was observed after E injection. These results suggest that ERc measurements alone are not sufficient to establish the capacity of the E receptor system: ERn measurements are also necessary to establish the relationship between receptor levels and physiologic estrogen responsiveness.     

Presynaptic Nicotinic Cholinergic Receptors Labeled by [3H]Acetylcholine on Catecholamine and Serotonin Axons in Brain

Nicotinic cholinergic receptor binding sites labeled by [3H]acetylcholine were measured in the cerebral cortices, thalami, striata, and hypothalami of rats lesioned by intraventricular injection of either 6-hydroxydopamine or 5, 7-dihydroxytryptamine. In addition, [3H]acetylcholine binding sites were measured in the cerebral cortices of rats lesioned by injection of ibotenic acid into the nucleus basalis magnocellularis. [3H]Acetylcholine binding was significantly decreased in the striata and hypothalami of both 6-hydroxydopamine- and 5,7-dihydroxytryptamine-lesioned rats. There was no change in binding in the cortex or thalamus by either lesion. Ibotenic acid lesions of the nucleus basalis magnocellularis, which projects cholinergic axons to the cortex, did not alter [3H]acetylcholine binding. These results provide evidence for a presynaptic location of nicotinic cholinergic binding sites on catecholamine and serotonin axons in the striatum and hypothalamus.     

Effects of Valeriana Officinalis Extracts on [3H]Flunitrazepam Binding,Synaptosomal [3H]GABA Uptake,and Hippocampal [3H]GABA Release

Extracts of Valeriana officinalis have been used in folkloric medicine for its sedative, hypnotic, tranquilizer and anticonvulsant effects, and may interact with -aminobutyric acid (GABA) and/or benzodiazepine sites. At low concentrations, valerian extracts enhance [³H]flunitrazepam binding (EC₅₀ 4.13 × 10^–10 mg/ml). However, this increased [³H]flunitrazepam binding is replaced by an inhibition at higher concentrations (IC₅₀ of 4.82 × 10^–1 mg/ml). These results are consistent with the presence of at least two different biological activities interacting with [³H]flunitrazepam binding sites. Valerian extracts also potentiate K⁺ or veratridine-stimulated release of radioactivity from hippocampal slices preloaded with [³H]GABA. Finally, inhibition of synaptosomal [³H]GABA uptake by valerian extracts also displays a biphasic interaction with guvacine. The results confirm that valerian extracts have effects on GABA_A receptors, but can also interact at other presynaptic components of GABAergic neurons.     

Ethanol Extraction Requirement for Purification of Protein Labeled with [3H]Leucine in Aquatic Bacterial Production Studies

The trichloroacetic acid (TCA)-insoluble fraction of water column bacteria labeled with [³H]leucine contained an ethanol-soluble fraction accounting for up to 44% of the label. A component of the ethanol-soluble fraction is [³H]leucine. Labeled-protein purification requires an ethanol wash step. Cold TCA can replace hot TCA for precipitation of labeled proteins.     

Effects of chronic antidepressant treatment on dopamine-related [3H]SCH 23390 and [3H]Spiperone binding in the rat striatum

1. The effects of chronic administration of antidepressants on dopamine-related [3H]SCH 23390 and [3H]spiperone binding to rat striatal membranes were assessed. 2. The monoamine oxidase inhibitors phenelzine (5 or 10 mg kg-1/day) and tranylcypromine (1 mg kg-1/day) and the tricyclic desipramine (10 mg kg-1/day) were administered for 28 days by constant subcutaneous infusion using Alzet (2ML4) osmotic minipumps. 3. These treatments did not alter Kd estimates for either [3H]SCH 23390 or [3H]spiperone binding sites. The monoamine oxidase inhibitors induced a decrease in the Bmax values for both [3H]SCH 23990 and [3H]spiperone binding sites. Desipramine induced a decrease in the Bmax value for [3H]SCH 23390 binding but had no effect on the Bmax value for [3H]spiperone binding.     

Inhibition by theanine of binding of [3H]AMPA, [3H]kainate,and [3H]MDL 105,519 to glutamate receptors

In an investigation of the mechanisms of the neuroprotective effects of theanine (gamma-glutamylethylamide) in brain ischemia, inhibition by theanine of the binding of [3H](RS)-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA), [3H]kainate, and [3H](E)-3-(2-phenyl-2-carboxyethenyl)-4,6-dichloro-1-H-indole-2-carboxylic acid (MDL 105,519) to glutamate receptors was studied in terms of its possible inhibiting effects on the three receptor subtypes (AMPA, kainate, and NMDA glycine), with rat cortical neurons. Theanine bound the three receptors, but its IC50 of theanine was 80- to 30,000-fold less than that of L-glutamic acid.     

Alpha1 Adrenergic Receptors in the Porcine Pituitary Neurointermediate Lobe: Detection with [3H]Ketanserin

Abstract

[³H]Ketanserin, a serotonin receptor antagonist, labelled high affinity, saturable sites in homogenates of porcine neurointermediate lobe tissue. Cinanserin, a potent and selective serotonin receptor antagonist, inhibited the specific binding of 5 × 10^-10M [³H]ketanserin with a high affinity component representing 20% of the total binding. Prazosin, a potent and selective alpha₁ adrenergic antagonist, inhibited [³H]ketanserin binding with a high affinity component representing 60% of total binding. The prazosin-specific component was demonstrated to be distinct from the cinanserin-specific component. 10^-7M cinanserin was co-incubated with [³H]ketanserin to eliminate the serotonergic component of the binding and allow pharmacological characterization of the remaining prazosin-specific component. The prazosin-specific binding of [³H]ketanserin binding closely resembled the results of experiments using [³H]prazosin to label alpha₁ receptors in neurointermediate lobe tissue homogenates. Ketanserin was found to be sevenfold more potent in inhibiting [³H]prazosin binding to alpha₁ adrenergic receptors in the neurointermediate lobe tissue than in brain tissue. This observation explains why low concentrations of [³H]ketanserin can selectively label serotonin receptors in the brain but will label both adrenergic and serotonin receptors in the neurointermediate lobe.     

An improved method for labeling carboxyatractyloside by [3H]KBH4

Carboxyatractyloside was labeled with [3H]KBH4 after oxidation of the primary alcohol of the glucose disulfate moiety by dicyclohexylcarbodiimide and P2O5 under anhydrous conditions in a dimethylsulfoxide medium. The 3H-labeled product was purified by DE 52 column chromatography followed by Cellulofine GCL 25 column chromatography. The final 3H-labeled product gave a single spot on a thin-layer chromatogram, and its Rf value was the same as that of authentic carboxyatractyloside. The biological activities (such as inhibition of state 3 respiration and binding to the adenine nucleotide carrier) were also comparable with those of authentic carboxyatractyloside.     

Degradation of [3H]chondroitin 4-sulphate and re-utilization of the [3H]hexosamine component by the isolated perfused rat liver.

Radiolabelled chondroitin 4-sulphate was isolated after incubation of rat rib cartilage with N-acetyl-D-[6-3H]galactosamine. After proteolytic digestion of the tissue with either papain or trypsin the released [3H]chondroitin 4-sulphate was added to an isolated perfused rat liver system. Analysis of perfusate after several hours perfusion showed that radiolabelled amino sugars were secreted by the liver in a low-molecular-weight form and as components of glycoproteins.     

Changes of [3H]MK-801, [3H]muscimol and [3H]flunitrazepam Binding in Rat Brain by the Prolonged Ventricular Infusion of Transformed Ginsenosides

Ameliorating effects of ginseng were observed on neuronal cell death associated with ischemia or glutamate toxicity. Ginseng saponins are transformed by intestinal microflora and the transformants would be absorbed from intestine. In the present study, we have investigated the effects of transformed ginsenoside Rg3, Rh2 and compound K on the modulation of NMDA receptor and GABA_A receptor binding in rat brain. The NMDA receptor binding was analyzed by quantitative autoradiography using [³H]MK-801 binding, and GABA_A receptor bindings were analyzed by using [³H]muscimol and [³H]flunitrazepam binding in rat brain slices. Ginsenoside Rg3, Rh2 and compound K were infused (10 g/10 l/h) into rat brain lateral ventricle for 7 days, through pre-implanted cannula by osmotic minipumps (Alzet, model 2ML). The levels of [³H]MK-801 binding were highly decreased in almost all regions of frontal cortex and hippocampus by ginsenoside Rh2 and compound K. The levels of [³H]muscimol binding were elevated in part of frontal cortex and granule layer of cerebellum by the treatment of ginsenoside Rh2 and compound K. However, the [³H]flunitrazepam binding was not modulated by any tested ginsenosides. Ginsenoside Rh2 and compound K induced the downregulation of the [³H]MK-801 binding as well as upregulation of the and [³H]muscimol binding in a region-specific manner after prolonged infusion into lateral ventricle. However, ginsenoside Rg3 did not show the significant changes of ligand bindings. In addition, ginsenoside Rh2 decreased the expression of nNOS in the hippocampus although Rg3 decreased the expression in the cortex. These results suggest that biotransformed ginsenoside Rh2 and compound K could play an important role in the biological activities in the central nervous systems and neurodegenerative disease.     

Effects of reserpine and adenosine agonist on α2-adrenergic receptor of rat vas deferens examined with [3H]yohimbine and [3H]clonidine

The changes of [³H]yohimbine and [³H]clonidine binding sites in rat vas deferens on treatments with adenosine receptor agonists (2-chloroadenosine, adenosine) or reserpine were examined. Treatment with adenosine agonist in vitro increased [³H]clonidine binding sites but had no influence on affinity and number of binding sites of α₂-antagonist, [³H]yohimbine. Amount of [³H]yohimbine binding sites was found to be higher than that of [³H]clonidine with or without the treatment. Inhibition curves of α₂-agonists, clonidine and norepinephrine, on [³H]yohimbine binding were less than unity though α₂-antagonist inhibited with about 1.0 of n_H. The treatment with adenosine agonist reduced the IC₅₀ value of agonists on the [³H]yohimbine binding but had no influence on the inhibitory effect of antagonist. These effect of adenosine agonists was completely blocked by theophylline. Accordingly it was considered that activation of adenosine receptor caused configurational change in α₂-adrenergic receptor from low affinity state for agonist to the high affinity state, though both states had same affinity for antagonist.On the other hand, treatment with reserpine in vivo increased the affinity of clonidine for α₂-adrenergic receptors and also increased the amount of the α₂-receptors.     

Distinct Binding Patterns of [3H]Raclopride and [3H]Spiperone at Dopamine D2 Receptors in vivo in Rat Brain. Implications for Pet Studies

Abstract

Positron emission tomography studies (PET) on dopamine (DA) D₂ receptors of schizophrenics provided conflicting data, perhaps because the ligands generally used, raclopride (RAC) and spiperone (SPI), did not label the same sites. In this study, we found that the in vivo binding characteristics of [³H]RAC and of [³H]SPI in rat brain, differed in many ways. 1) [³H]RAC labeled twice as many sites in striatum and olfactory tubercle and [³H]SPI twice as many sites in pituitary. 2) The kinetic was much shorter with [³H]RAC than [³H]SPI in striatum. 3) RAC, unlike SPI, did not exhibit limbic selectivity. 4) The modulation of [³H]RAC and [³H]SPI binding by endogenous DA were diametrically opposite: D-amphetamine decreased, and reserpine + α-methyl-p-tyrosine increased [³H]RAC binding in striatum whereas the opposite occured with [³H]SPI. This distinct binding pattern of [³H]RAC and [³H]SPI suggests that these two radioligands do not label the same receptor sites.     

Dynamic Studies on Estrogen Response in Fetal Guinea Pig Uterus: Effect of Estradiol Administration on Estradiol Receptor,Progesterone Receptor and Uterine Growth

Abstract

The uterus of the guinea pig fetus has been shown to respond to estradiol treatment by an increase in uterine wet weight and a stimulation of the progesterone receptor protein. A study of the kinetics of these two parameters of estrogen response in the fetal uterus was undertaken in order to correlate these responses with changes in the estrogen receptor. Administration of estradiol to pregnant guinea pigs (1 mg/kg/body weight) leads to a rapid stimulation of the progesterone receptor by 6h after treatment which reaches maximal values by 15.5h, which are increased 7-fold in estradiol-primed guinea pigs above values in untreated animals. The estradiol receptor undergoes rapid translocation from the cytosol into the nucleus by 1h after hormone treatment and is retained in the nucleus for at least 6h. At the same time, there is a 50% decrease in the total occupied and available estradiol receptor concentration at 6h after treatment. Estradiol treatment also provokes an increase in wet weight of the fetal uterus which is significantly greater after 3 consecutive days of treatment (171% ± 24 (S.D.) above wet weights of untreated uteri which were considered as 100%) than after only 1 day (121% ± 25 (S.D.)). These estrogen responses were found to be of long duration since uterine wet weights and progesterone receptor concentrations remained well above control values even 5 days after a single treatment with estradiol. In conclusion, the fetal uterus responds to estradiol treatment by a slow increase in wet weight and a rapid stimulation of the progesterone receptor protein with a concomitant loss in estradiol receptor concentration.     

Effects of N-LAAM on [3H]etorphine binding in neuronal-enriched cell cultures

Stereospecific [3H]etorphine binding sites are present in neuronal-enriched cell cultures dissociated from 7-day-old chick embryonic brain. Moreover, binding was regulated by both ions and GTP in a manner similar to that of in vivo brain tissue. When cultures were exposed to N-LAAM (10(-6) M) from day 6 to day 7 or 8 and assayed for binding at day 8, Bmax was decreased and KD was increased. These findings support our view that primary neuronal cultures are a suitable model with which to study interactions of drugs with opiate receptors.