Effect of phencyclidine on [3H]QNB binding

Intraperitoneal injection of phencyclidine before intravenous injection of [³H] Quinuclidinyl benzilate (QNE, 1.6 μg/kg) significantly increased the amount of radioactivity found in the brains of female C57BL/6J mice one hour after the ³H-QNB administration. This effect was found in hypothalamus, cortex, hippocampus and striatum and was decreased by pretreatment of the animal with atropine. The magnitude of the enhancement varied as a function of dose but did not change across the time span studied. These data are in contrast to our findings and those of others of inhibition of the specific binding of ³H-QNB to muscarinic cholinergic receptors by PCP $\text{in vitro}$. When atropine or PCP was administered $\text{in vivo}$ and the tissue later analyzed $\text{in vitro}$, no effects of the drugs were observed on ³H-QNB binding. The reasons for the differences remain a matter of speculation.     

Regional distribution of [3H] kainic acid after intraventricular injection

[³H] Kainic acid was administered intraventricularly to rats at a dose that selectively destroys the pyramidal cells of hippocampal area CA3. Only about one-third of the injected radioactivity was recovered in the brain 15 min later, but the residual radioactivity disappeared at a much slower rate. [³H]-Kainic acid distributed rather evenly throughout the brain; there was no correlation between accumulation of radioactivity and neurotoxicity. Almost 90% of the radioactivity in sucrose homogenates was recovered in the high-speed supernatant. No cerebral metabolism of [³H] kainic acid was detected by thin-layer chromatography. These data rule out the possibility that a lethal accumulation of the toxin by hippocampus accounts for the preferential vulnerability of hippocampal pyramidal cells.     

Intracellular disposition of [3H]-cocaine, [3H]-norcocaine, [3H]-benzoylecgonine and [3H]-benzoylnorecgonine in the brain of rats

[³H]-cocaine, [³H]-norcocaine, [³H]-benzoylecgonine and [³H]-benzoylnorecgonine were administered i.c. in equi-potent pharmacologic doses and the intracellular disposition and metabolism of each drug determined. Norcocaine and cocaine rapidly entered and egressed from the brain so that 4.8–6.1% of the radioactivity present in brain at one minute was observed at 30 minutes. The highest levels of subcellular radioactivity were generally found in the microsomal plus supernatant, followed by the nuclear and shocked mitochondrial fractions. No apparent localization of the radioactivity occured in synaptic membranes. The brain/plasma (B/P) ratio curves for cocaine and norcocaine were similar; however, the norcocaine values were considerably higher at each time interval. Benzoylecgonine and benzoylnorecgonine had higher comparative B/P ratios than cocaine or norcocaine and persisted in brain for a longer period of time so that 0.6–2.1% of the radioactivity present in brain at 1 hour was detected at 24 hours. Cocaine and norcocaine were extensively metabolized to the benzoylmetabolites. Benzoylecgonine was metabolized to benzoylnorecgonine and benzoylnorecgonine was unmetabolized. The brain disposition data and B/P ratios agreed quite well with the overall pharmacologic action of cocaine and its metabolites.     

Binding of [3H]cytochalasin B and [3H]colchicine to isolated liver plasma membranes

The binding to isolated hepatocyte plasma membranes of radioactively labelled inhibitors of microfilamentous and microtubular protein function ([³H]cytochalasin B and [³H]colchicine, respectively) was studied as one means of assessing the degree of association of these proteins with cell surface membranes. [³H]Cytochalasin B which behaved identically to the unlabelled compound with respect to binding to these membranes was prepared by reduction of cytochalasin A with NaB³H₄. The binding was rapid, readily reversible, proportional to the amount of membrane and relatively insentive to changes of pH or ionic strength. At 10^?6 M [³H]cytochalasin B, glucose or p-chloromercuribenzoate, an inhibitor of glucose transport inhibited binding by about 20%; treatment of membranes with 0.6 M KI which depolymerizes F actin to G actin caused about 60% inhibition of binding. These two types of inhibition were additive indicating two separate classes of binding sites, one associated with sugar transport and one with microfilaments. Filamentous structures with the diameter of microfilaments (50 Å) were seen in electron micrographs of thin sections of the membranes. At concentrations greater than 10^?5 M [³H]cytochalasin B, binding was proportional to drug concentration, characteristic of non-specific adsorption or partitioning. Intracellular membranes of the hepatocyte also bound [³H]cytochalasin B, those of the smooth endoplasmic reticulum to a greater extent than plasma membranes.[³H]Colchicine bound to plasma membranes in proportion to the amount of membrane and at a rate compatible with binding to tubulin. However, other properties of the binding including effects of temperature, drug concentration and antisera against tubulin were different from those of binding to tubulin. Hence, no evidence was obtained for association of microtubular elements with these membranes. Despite this there appeared to be an interdependence between microtubule and microfilament inhibitors: vinblastine sulfate stimulated [³H]cytochalasin B binding and cytochalasin B stimulated ³H colchicine binding. [³H]Colchicine also bound to intracellular membranes, especially smooth microsomes.     

The toxic effects in rats of some synthanecine carbamate and phosphate esters analogous to hepatotoxic pyrrolizidine alkaloids

Five synthetic compounds analogous to pyrrolizidine alkaloids have been tested for toxicity in rats. These were the bis-N-ethylcarbamate esters of synthanecines A, B, C and D (Compounds I–IV) and the bis-diethylphosphate ester (V) of synthanecine A. The amino alcohol moiety in each of these had a single 5-membered heterocyclic ring in place of the pyrrolizidine amino alcohol (necine) moiety of natural pyrrolizidine alkaloids.The toxicity of these compounds differed considerably. The synthanecine A carbamate (I) was the most toxic, male and female rats being similarly susceptible. Like many hepatotoxic pyrrolizidine alkaloids, a single dose of compound I caused acute centrilobular necrosis of the liver, chronic hepatotoxicity involving the development of persistent giant hepatocytes, and chronic lung injury. Compound III had similar actions but was less toxic. The synthanecine D carbamate (IV) caused acute liver necrosis but no chronic hepatotoxicity, whereas the synthanecine A phosphate (V) had the opposite effect, with only chronic hepatotoxicity.The different toxic effects were related to the structure and metabolism of the compounds. Doses of compounds I, III and IV associated with a similar degree of acute hepatotoxicity led to similar levels of pyrrolic metabolites in the liver. Compound II, which was not hepatotoxic, gave very little liver pyrrole. The liver level of pyrrolic metabolite from the phosphate ester (V) decreased more rapidly than that from (I), and was not associated with acute toxicity.Antimitotic activity, indicated by the appearance of bizarre giant cells, was shown by compounds capable of forming pyrrolic metabolites which were bifunctional alkylating agents, but not by compound IV, which could only form a monofunctional alkylating agent. Pretreatment with phenobarbitone lowered the susceptibility of rats to compound I and greatly increased the liver level of pyrrolic metabolites associated with acute hepatotoxicity. Some rats given compounds I and III had kidney lesions primarily involving the glomerulus. The results confirm that toxic effects characteristic of many natural pyrrolizidine alkaloids can be reproduced using simplified synthetic analogues, and that such toxicity is associated with pyrrolic metabolites.     

Modulation of [3H]-dopamine binding by cholecystokinin octapeptide (CCK-8)

Cholecystokinin-octapeptide (CCK-8) is a putative neurotransmitter which has been demonstrated previously to occur in midbrain dopamine neurones. We observe that CCK-8 causes changes in both the affinity and density of binding sites for [³H]-dopamine in rat striatal homogenates, in vitro, upon incubation with the peptide at a concentration of 1 micromolar. A dose-response study of the competetion of CCK-8 with [³H]-dopamine binding indicates an IC₅₀ for the peptide of 450 nM; desulfated CCK-8 and the related peptide caerulin are at least 4-fold less active than CCK-8. CCK-8 was also administered to rats in a separate study; the binding of [³H]-dopamine was evaluated to homogenates of striata and olfactory tubercles obtained from these animals, which had been treated with systemic injection at a dose of 20 micrograms/kg, daily, for four days. A decrease in the number of striatal binding sites for the radioligand was observed, with a concomitant increase in the number of binding sites in the olfactory tubercle. These data collectively suggest a possible regulatory role for CCK-8 in the ascending dopamine systems.     

The interaction of [3H]TPA with bovine lymph node lymphocytes in vitro

The potent tumor promoter, 12-O-tetradecanoyl-phorbol-13-acetate (TPA), is an effective modulator of DNA synthesis in bovine lymph node lymphocytes in culture. The effect of TPA on cellular proliferation depends to a great extent on the length of exposure and the mitogenic stimulus. Addition of TPA along with mitogenic lectins enhances DNA synthesis. Exposure to TPA for 2 days before addition of lectins depresses DNA synthesis. There is evidence that some inhbitory material other than TPA is formed during the longer incubation. Therefore, in this study, we used [³H]TPA to determine (i) the amount of material retained by the cells after preincubation and (ii) if TPA was metabolized during this culture period. We found that after incubation with 10^?7 M, [³H]TPA, less than 3% of the radioactivity was retained by the cells. This was released by incubation in fresh medium. All of the cell associated material appeared to be TPA. However, the TPA in the medium was degraded by about 30% during a 2-day incubation to 12-O-tetradecanoyl-phorbol (TP), phorbol-13-acetate (PA) and phorbol. The source of the hydrolytic activity appeared to be serum because the same effect was seen in the absence of cells but was not seen in the absence of serum. These metabolites when added directly to the lymphocytes had no effect on DNA synthesis. Moreover, the amount of TPA retained by the cells and released into the medium was too small to account for the inhibitory activity of medium from TPA-treated cells. Studies are in progress to determine the nature of the inhibitory material after exposure to TPA.     

Ascorbic acid decreases [3H]dopamine binding in striatum without inhibiting dopamine-sensitive adenylate cyclase

Ascorbic acid is found in very high concentrations in cells of neural crest origin such as the central nervous system and the adrenal gland. A variety of evidence has been marshalled to support a role for ascorbate as a chemical messenger. One of the first non-biosynthetic biochemical effects ascribed to ascorate in the CNS was its ability to inhibit dopamine-stimulated adenylate cyclase (DA-ACase) in homogenates from striata of Long Evans rats (J. Neurochem.28, 663, 1977). Using an adenylate cyclase assay based on preparative HPLC, we were unable to detect any inhibition of DA-ACase by ascorbate at concentrations as high as 1 mM. Moreover, this failure to find inhibitory effects of ascorbate on DA-ACase occurred not only when striatal homogenates from Long-Evans rats were used, but also when tissue from Sprague-Dawley rats of N.C. Board of Health mice was tested. Although ascorbate may play a neuromodulatory role, it does not appear that its effects are mediated through effects on cAMP biosynthesis. Despite our inability to detect effects of ascorbate on DA-ACase, we did confirm that ascorbate significantly altered the binding of [³H]dopamine to striatal membranes. Thus, it is clear that the sites binding [³H]dopamine that are affected by ascorbate are unlikely to be the same ones coupled to aenylate cyclase.     

Binding of [3H]estradiol- and [3H]H1285-receptor complexes to rabbit uterine chromatin

In the present study we investigated the binding characteristics of estrogen and antiestrogen-receptor complexes to rabbit uterine chromatin. Activated or nonactivated estrogen receptors were partially purified by DEAE-cellulose chromatography using low (1 mM) or high (10 mM) concentrations of sodium molybdate. Activated [³H]estradiol-receptor complexes showed enhanced binding to chromatin acceptor sites unmasked by 1 M, 4 M and 6 M guanidine hydrochloride. We also examined the chromatin-binding characteristics of the estrogen receptors when bound by the high-affinity triphenylethylene antiestrogen, H1285. The acceptor site activity for the [³H]H1285-receptor complexes was markedly decreased at sites unmasked by 4 M and 6 M guanidine hydrochloride. Further, the nonactivated receptor complexes showed very low binding to deproteinized chromatin. The estrogen-receptor chromatin-acceptor sites were tissue specific and saturable. These chromatin acceptor sites differ in their affinity and capacity (number of binding sites per cell) for the estrogen- and antiestrogen-receptor complexes. Thus, we suggest that the differences in the physiological and physicochemical properties of estrogens and antiestrogens may be related to their differential interaction with uterine chromatin subfractions.     

The metabolism of [3−3H]oleanolic acid-3-O-mono-[14C]glucoside in isolated cells from Calendula officinalis leaves

The radioactive precursor, [3?³H]oleanolic acid-3-O-mono-[¹⁴C]glucoside was administrated to isolated cells obtained from the leaves of Calendula officinalis. The radioactivity of the precursor was incorporated into fractions containing free oleanolic acid, individual glucosides, glucuronide F and other glucuronides. The ratio of ³H: ¹⁴C radioactivity in these fractions indicated that glucosides were formed in a process involving direct glycosylation of the precursor, whereas the glucuronides were formed from oleanolic acid released by hydrolysis of the precursor. Dynamics curves showed that glucoside II formed by direct glycosylation of the precursor was intensively transformed to other derivatives.     

The incorporation of [3H]glucosamine, [3H]fucose and [14C]mannose into protein in the rat anterior pituitary incubated in vitro

Rat anterior hemipituitaries incubated in vitro rapidly take up and incorporate into protein D-[6-³H]-glucosamine · HCl, D-[1-¹⁴C]mannose and L-[G-³H]fucose. The newly labeled protein was only slowly released into a Krebs-Ringer bicarbonate incubation medium. Glucosamine- or mannose-labeled protein was barely detectable in the medium after a 30–60 min incubation whereas about 4% of all fucose-labeled protein had already been released into the incubation medium by 30 min. Puromycin · 2HCl (1 mM) inhibited incorporation of glucosamine or mannose into protein to 40% or less of control values within 30 min; fucose incorporation was not significantly inhibited before 45 min. Acid hydrolysis followed by amino acid analysis of glucosamine-labeled protein yielded significant amounts of label in glucosamine, galactosamine and apparent glucosamine-degradation products but no significant amount of label in any amino acid.     

Mechanism of Cu2+-induced elevation of [3H]cimetidine binding to membranes in rat brain

The mechanism of increasing effect of CuCl₂ on specific [³H]cimetidine binding was examined in brain membranes of rats. CuCl₂-Induced elevation of [³H]cimetidine binding was high in Krebs-Ringer solution (pH 7.4) compared to those in 50 mM Na, K-phosphate buffer (pH 7.4) and in 50 mM Tris-HCl buffer (pH 7.4). CaCl₂ (5–50 mM) inhibited effect of CuCl₂, but NaCl (25–200 mM), KCl (5–100 mM) or MgCl₂ (5–50 mM) did not. CuCl₂ (50 μM) elevated 9.3- and 2.5-fold the binding in phosphate- and Tris—HCl buffer, respectively. EDTA-2Na decreased the binding elevated by 50 μM CuCl₂ in phosphate buffer to the similar level in Tris-HCl buffer, whereas it did not affect those in Tris-HCl buffer. The absorption spectra of cimetidine and CuCl₂ mixture showed a peak at 317 nm in phosphate buffer that was not observed in Tris-HCl buffer. It is suggested that cimetidine-Cu²⁺ chelate complex could be formed in phosphate buffer, resulting in higher amount of binding in phosphate buffer than in Tris-HCl buffer. PdCl₂ also caused a marked elevation in [³H]cimetidine binding, seeming to be due to formation of cimetidine-Pd²⁺ chelate complex. There were two types of [³H]cimetidine binding in the presence of 20 nM PdCl₂: high affinity binding with K_d = 0.7 ± 0.1 nM and low affinity binding with K_d = 44.3 ± 3.0 nM. It is suggested that cimetidine-Cu²⁺ complex binds to cimetidine binding sites in brain with higher affinity than cimetidine alone.     

Elevation of [3H]cimetidine binding by CuCl2 in brain membranes of rats

Influences of dithiothreitol (DTT), p-chloromercuriphenyl sulfonate (PCMPS) and ascorbate on CuCl₂-induced elevation of [³H]cimetidine binding were investigated in brain membranes of rats. CuCl₂ (10–500 μM) elevated specific [³H]cimetidine binding in a concentration-dependent manner. There were two types of [³H]cimetidine binding in the presence of 50 μM CuCl₂: high affinity binding with K_d = 1.97 nM and low affinity with K_d = 21.6 nM. PCMPS (10 and 100 μM) reduced the binding in both media with and without CuCl₂. DTT (1–30 μM) or ascorbate (0.1 and 1.0 mM) markedly elevated the binding in the presence of CuCl₂ but showed no effect and ascorbate rather inhibited the binding in the absence of CuCl₂. DTT (0.1 mM) diminished the binding in the presence and absence of CuCl₂. CuCl₂ (50 μM) significantly (P < 0.01) increased the IC₅₀ of histamine for [³H]cimetidine binding and the effect was greater than that from 100 μM GTP. It is suggested that sulfhydryl groups sensitive to PCMPS could interact with Cu²⁺ and thus be involved in an elevation of cimetidine binding. Cu²⁺ seems to regulate affinity of agonist binding for cimetidine binding sites presumably by acting on cimetidine binding sites and/or GTP binding regulatory proteins.     

Surface interaction of [3H]norepinephrine with cultured chick embryo myocardial cells

Cultured chick embryo cardiac myoblasts specifically bind [³H]nonrepinephrine. The binding is rapid and reversible. Bound [³H]nonrepinephrine, dissociated by 1 M HCl, can be rebound to fresh cells. β-Adrenergic catecholamines were most potent in displacing [³H]nonrepinephrine from the cellular bindign sites. The binding reaction did not show stereospecificity. α-Adrenergic amines were much less potent. Propranolol, but no phentolamine, competed for the sites. Approximately 2.5 · 10⁶ specific binding sites are present per myocardial cell. The sites appear to be present predominantly at the cell surface in that nonrepinephrine linked to agarose beads competes for th sites. Similarly, the sites were degraded by either trypsin or trypsin bound to agarose. Two different binding constants, K = 2 · 10⁶ and 1 · 10⁵, were observed. Proteolytic enzymes decreased binding whereas certain hospholipases led to an increase in specific binding. Divalent cations at concentrations > 1 mM diminished binding as did chelating agents.     

Subcellular distribution of [3H]-prostaglandin E2 binding sites in porcine gastric mucosa

The distribution of PGE₂ binding sites in four subcellular fractions (F1–F4) from porcine fundic mucosa obtained by gradient centrifugation was examined. Binding of HPGE₂ to fractions F2–F4 was specific, dissociable, saturable and pH dependent. A significant degree of specific binding was not evident in F1. The Scatchard analysis of binding to F2 and F3 revealed heterogenous populations of binding sites with similar dissociation constants but greater concentrations of binding sites than was evident in the initial 30,000 xg homogenate protein. A single class of low affinity binding sites was evident in F4. The ratio of total: nonspecific binding was approximately equal in F2 and F3. The ratio was considerably smaller in F4. The activity of 5' nucleotidase the marker enzyme for plasma membranes followed this ratio. There was no correlation between the binding ratio and marker enzyme activities for mitochondrial membranes and endoplasmic reticulum. These data suggest that high affinity PGE₂ binding sites occur predominantly on the plasma membrane from gastric mucosal tissue.     

Decreased [3H]serotonin and [3H]spiperone binding consequent to lipid peroxidation in rat cortical membranes

The effect of lipid peroxidation on two types of serotonin binding sites was investigated. Incubation of rat cortical membranes with an ascobate-dependent peroxidizing system resulted in the formation of malonyldialdehyde and significant decreases in the specific binding of [³H]serotonin and [³H]spiperone to the treated membranes. When ascorbate concentrations were varied from 0.025 to 6.0 mM, malonyldialdehyde production increased to a maximum at 0.5 mM ascorbate and then declined. Conversely, the specific binding of [³H]serotonin and [³H]spiperone decreased to a minimum at 0.5 mM ascorbate and then increased. Regression analysis of the data revealed that the decrease in the two binding sites was linearly correlated to lipid peroxidation.     

[3H]norepinephrine binding and lipolysis by isolated fat cells

[³H]norepinephrine was shown to bind to specific sites on isolated fat cells. A Scatchard plot of norepinephrine binding showed two apparent K_a of 1.9 · 10⁶ and 1.2 · 10⁵ LM^?. 1.4 · 10^?4 M Norepinephrine covalently-linked to agarose beads reduced [³H]norepinephrine binding by over 50%. Several structurally related drugs were compared as inhibitors of [³H]norepinephrine binding and as stimulators of lipolysis in preparations of similarly prepared cells. Dose-response curves for norepinephrine, epinephrine and isoproterenol showed the affinities for binding inhibition and for stimulation of lipolysis to be in the same range of 6 · 10^?7-2 · 10^?6 M. Dopamine and dopa were potent inhibitors of [³H]norepinephrine binding at 8.5 · 10^?7 M and 2.0 · 10^?6 M respectively, but did not stimulate lipolysis even at 10^?4 M. Propranolol, a β-adrenergic antagonist, had no effect on [³H]norepinephrine binding at 10^?4 M but completely inhibited catecholamine-stimulated lipolysis at 10^?5 M. Phentolamine, an α-adrenergic antagonist, did not inhibit binding or catecholamine-stimulated lipolysis at 10^?4 M. Ephedrine, metaraminol, phenylephrine and normetanephrine were also ineffective both as [³H]norepinephrine binding inhibitors and as stimulators of lipolysis. The results suggested the catechol ring of catecholamines is more important than the ethanolamine side chain as a requirement for binding, while both an intact catechol moiety and ethanolamine function appear necessary for physiological effect.     

Dopamine receptors in the rat brain: quantitative autoradiographic localization using [3H]sulpiride

In vitro labeling of tissue sections with [³H]sulpiride has been utilized in the present study to autoradiographically localize D₂-dopamine receptors in the rat brain. Preliminary biochemical studies, using slide-mounted tissue sections, were performed to define the optimal labeling conditions for this binding. Autoradiograms were generated by apposition of the labeled tissue sections to tritium-sensitive film. Specific binding sites for [³H]sulpiride were localized to the caudate-putamen, nucleus accumbens, olfactory tubercle, glomerular layer of the olfactory bulb, pituitary, laminae I and III of the entorhinal cortex, substantia nigra, lateral mammillary nucleus and the stratum-lacunosum moleculare of the hippocampus. The high selectivity of [³H]sulpiride for the D₂-dopamine receptor indicates that it is a valuable tool for the autoradiographic localization and quantitation of neuroleptic receptors.     

Binding Sites for [3H]Glutamate and [3H]Aspartate in Human Cerebellum

Abstract The binding of [³H]aspartate and [³H]glutamate to membranes prepared from frozen human cerebellar cortex was studied. The binding sites differed in their relative proportions, their inhibition by amino acids and analogues, and by the effects of cations. A proportion (about 30%) of [³H]glutamate binding was to sites similar to those labelled by [³H]aspartate. An additional component of [³H]gluta-mate binding (about 50%) was displaced by quisqualate and aL-amino-3-hydroxy-5-methylisoxazole-4-propionic acid, and may represent a “quisqualate-preferring” receptor. Neither N-methyl-d-aspartic acid-sensitive nor dl-2-amino-4-phosphonobutyric acid-sensitive [³H]glutamate binding was detected.