High affinity binding of [3H] cocaine to rat liver microsomes

[3H]Cocaine bound reversibly, with high affinity (KD 2.3 +/- 1.1 nM) and stereospecificity to rat liver microsomes. Little binding was detected in the lysosomal, mitochondrial and nuclear fractions. The binding kinetics were slow (T1/2 for association, 6 min and for dissociation 17 min), and the kinetically calculated KD was 2 nM. Induction of mixed function oxidases by phenobarbital did not produce significant change in [3H]cocaine binding. On the other hand, chronic administration of cocaine reduced [3H]cocaine binding drastically. Neither treatment affected the affinity of the liver binding protein for cocaine. Microsomes from mouse and human livers had less cocaine-binding protein and lower affinity for cocaine than those from rat liver. Binding of [3H]cocaine to rat liver microsomes was insensitive to monovalent cations and greater than 10 fold less sensitive to biogenic amines than the cocaine receptor in rat striatum. However, the liver protein had higher affinity for cocaine and metabolites except for norcocaine. Amine uptake inhibitors displaced [3H]cocaine binding to liver with a different rank order of potency than their displacement of [3H]cocaine binding to striatum. This high affinity [3H]cocaine binding protein in liver is not likely to be a monooxygenase, but may have a role in cocaine-induced hepatotoxicity.     

High affinity binding of [H]neurotensin to rat uterus

[³H]Neurotensin (NT) was found to bind specifically and with high affinity to crude membranes prepared from rat uterus. Scatchard analysis of saturation binding studies indicated that [³H]NT apparently binds to two sites (high affinity Kd 0.5 nM; low affinity Kd 9 nM) with the density of high affinity sites (41 fmoles/mg prot.) being about one-third that of the low affinity sites (100 fmoles/mg prot.). In competition studies, NT and various fragments inhibited [³H]NT binding with the following potencies (IC₅₀): NT 8–13 (0.4 nM), NT 1–13 (4 nM), NT 9–13 (130 nM), NT 1–11, NT 1–8 (>100 μM). Quantitatively similar results were obtained using brain tissue. These findings raise the possibility of a role for NT in uterine function.     

Proctolin antagonists bind to [(3)H]proctolin binding sites in the locust hindgut

Proctolin caused dose-dependent (1-200 nM) contraction of the isolated hindgut of S. gregaria which was abolished by [alpha-methyl-L-tyrosine(2)]-proctolin (1 microM). In comparison, cycloproctolin (5 microM) reduced the proctolin maximum response by 41%. Hindgut homogenates contained [(3)H]proctolin binding sites with a K(d) value of 660 nM, a B(max) value of 23.8 pmol/mg protein and a Hill coefficient of 0.934. Cycloproctolin (IC(50,) 220 nM; K(i), 204 nM), unlabeled proctolin (IC(50) 680 nM) and [alpha-methyl-L-tryosine(2)]-proctolin (IC(50) 3.1 microM, K(i), 2.9 microM) but not SchistoFLRFamide (1 nM-10 microM) were capable of displacing bound [(3)H]proctolin.     

Characterization of [(3)H]ryanodine binding sites in mammalian lung

The ryanodine-sensitive calcium channels, also called ryanodine receptors, are intracellular Ca(2+)-release channels that have been shown to bind the neutral plant alkaloid ryanodine with nanomolar affinity. The activity of the skeletal muscle (RyR1), cardiac muscle (RyR2), and brain (RyR3) ryanodine receptor isoforms have been shown to be highly regulated by physiological factors including pH, temperature, and ionic strength; endogenous compounds including Ca(2+), Mg(2+), and adenosine triphosphate (ATP); and pharmacological agents including caffeine, ruthenium red, and neomycin. RyR3 is reportedly expressed in diverse tissues including lung; however, specific [(3)H]ryanodine binding sites in mammalian lung tissue have not been characterized. In this study, hamster lung ryanodine binding proteins were shown to specifically bind [(3)H]ryanodine with an affinity similar to that of RyR isoforms found in other tissues and this binding was shown to be sensitive to Ca(2+) concentration, stimulation by caffeine and spermine, and inhibition by Mg(2+), ruthenium red, and neomycin. The solubilized, intact ryanodine binding protein from hamster lung demonstrated approximately the same 30S sedimentation coefficient as RyR1 and RyR2, but a putative ryanodine receptor subunit from hamster lung was not found to cross-react with antibodies specific for the three known isoforms. We conclude that the hamster lung ryanodine binding protein demonstrates sedimentation and binding characteristics that are similar to those of the known RyR isoforms, but may exhibit antigenic dissimilarity from the typical RyR isoforms found in muscle and brain.     

High affinity stereospecific binding of [3H] cocaine in striatum and its relationship to the dopamine transporter

A high affinity (KD 35 nM) binding site for [3H]cocaine is detected in rat brain striatum present at 2-3 pmol/mg protein of synaptic membranes. This binding is displaced by cocaine analogues with the same rank order as their inhibition of [3H]dopamine ([3H]DA) uptake into striatal synaptosomes (r = 0.99), paralleling the order of their central stimulant activity. The potent DA uptake inhibitors nomifensine, mazindol, and benztropine are more potent inhibitors of this high affinity [3H]cocaine binding than desipramine and imipramine. Cathinone and amphetamine, which are more potent central stimulants than cocaine, displace the high affinity [3H]cocaine binding stereospecifically, but with lower potency (IC50 approximately equal to 1 microM) than does cocaine. It is suggested that the DA transporter in striatum is the putative "cocaine receptor." Binding of [3H]cocaine, measured in 10 mM Na2HPO4-0.32 M sucrose, pH 7.4 buffer, is inhibited by physiologic concentrations of Na+ and K+ and by biogenic amines. DA and Na+ reduce the affinity of the putative "cocaine receptor" for [3H]cocaine without changing the Bmax, suggesting that inhibition may be competitive. However, TRIS reduces [3H]cocaine binding noncompetitively while Na+ potentiates it in TRIS buffer. Binding of [3H]mazindol is inhibited competitively by cocaine. In phosphate-sucrose buffer, cocaine and mazindol are equally potent in inhibiting [3H]mazindol binding, but in TRIS-NaCl buffer cocaine has 10 times lower potency. It is suggested that the cocaine receptor in the striatum may be an allosteric protein with mazindol and cocaine binding to overlapping sites, while Na+ and DA are allosteric modulators, which stabilize a lower affinity state for cocaine.     

The effect of melanotropic peptides on binding of [(3)H]dihydroalprenolol-hydrochloride to hypothalamic membranes

In the present work we studied the interaction of alpha-melanocyte-stimulating hormone (alpha-MSH) and ACTH-(1-24) with beta-adrenergic receptors in hypothalamic membranes from rat brain. Saturation curves for [(3)H]dihydroalprenolol-hydrochloride ([(3)H]DHA) binding in the presence of the peptides revealed a decreased binding capacity (Bmax). The dissociation constant (Kd) was, however, not affected by alpha-MSH or ACTH-(1-24). These data indicate a non competitive interaction between these melanocortin peptides and [(3)H]DHA on beta-adrenergic receptors in hypothalamic membranes.     

Kinetics of binding of [(3)H]glycine to transport proteins in channel catfish brain

[(3)H]Glycine was observed to bind to channel catfish brain particles in a manner displaying saturation kinetics. The dissociation constant was calculated to be 7.38 +/- 2.11 microM. Though some binding occurred in the absence of Na(+) ions, the presence of such ions stimulated binding in a concentration-dependent manner. Similarly, chloride ions had a stimulatory effect on [(3)H]glycine binding. Several inhibitors of binding were identified, the most effective being beta-alanine, pipecolic acid, 2,3-pyrazine dicarboxylic acid and 3,5-pyrazole dicarboxylic acid. Each is a structural analogue of glycine. Harmaline, a known inhibitor of Na(+) binding, also inhibited glycine binding. A previous study had shown the presence of a sodium-dependent, active uptake system for glycine in synaptosomes derived from catfish brain. The present results suggest that the binding of [(3)H]glycine was to a glycine transporter and that the amino acid functions as a neurotransmitter in this species.     

Photoaffinity labeling of insect nicotinic acetylcholine receptors with a novel [(3)H]azidoneonicotinoid

The nicotinic acetylcholine receptor (nAChR) is a ligand-gated ion channel in the insect CNS and a target for major insecticides. Here we use photoaffinity labeling to approach the functional architecture of insect nAChRs. Two candidate 5-azido-6-chloropyridin-3-yl photoaffinity probes are evaluated for their receptor potencies: azidoneonicotinoid (AzNN) with an acyclic nitroguanidine moiety; azidodehydrothiacloprid. Compared to their non-azido parents, both probes are of decreased potencies at Drosophila (fruit fly) and Musca (housefly) receptors but AzNN retains full potency at the Myzus (aphid) receptor. [(3)H]AzNN was therefore radiosynthesized at high specific activity (84 Ci/mmol) as a novel photoaffinity probe. [(3)H]AzNN binds to a single high-affinity site in Myzus that is competitively inhibited by imidacloprid and nicotine and further characterized as to its pharmacological profile with various nicotinic ligands. [(3)H]AzNN photoaffinity labeling of Myzus and Homalodisca (leafhopper) detects a single radiolabeled peak in each case displaceable with imidacloprid and nicotine and with molecular masses corresponding to approximately 45 and approximately 56 kDa, respectively. The photoaffinity-labeled receptor in both Drosophila and Musca has imidacloprid- and nicotine-sensitive profiles and migrates at approximately 66 kDa. These photoaffinity-labeled polypeptides are considered to be the insecticide-binding subunits of native insect nAChRs.     

High affinity (3H)tryptamine binding sites in various organs of the rat

(3H)Tryptamine binds with high affinity (KD values around 4 nmol/l) to membranes from different organs of the rat. The number of binding sites was high in the liver, intermediate in the heart and cerebral cortex and low in other organs including ileum, kidney, spleen, stomach and lung. Binding to kidney membranes was investigated in more detail. It was rapid and reversible (t1/2 7 min.). The structure-activity profile was characterized by high affinity of some beta-carbolines (IC50 = 20 - 200 nmol/l), medium affinity of serotonin (IC50 = 858 nmol/l) and low of methysergide (IC50 greater than 20 mumol/l). The findings correlate very well with binding characteristics to cerebral cortex membranes. Therefore, rat brain cortex and other organs contain the same type of (3H)tryptamine binding site and may be equally appropriate for further analysis.     

High affinity inhibition of [3H]-flunitrazepam binding to brain benzodiazepine receptors by CGS 9896, a novel pyrazoloquinoline

We had previously reported the extensive purification of snRNPs (ribonucleoprotein complexes containing small nuclear RNAs) from HeLa hnRNP (ribonucleoprotein complexes containing the heterogenous nuclear RNA) by taking advantage of their ability to withstand centrifugation in cesium chloride containing 0.5% sarkosyl (C. BRUNEL et al 1981, Nucleic Acids Research, 9, 815–830). Such purified snRNPs contained the five nucleoplasmic snRNAs (U₁, U₂, U₄, U₅ and U₆) and a very simple protein complement of 4–5 polypeptides between 10 and 14,000 daltons. The possible involvement of U₁ in messenger RNA splicing makes it crucial to obtain in pure form the individual U₁ snRNP, if an in vitro splicing system is to be reconstructed. The present work reports the purification of U₁ snRNP and shows that it contains exactly the same polypeptides as the mixture of all five snRNPs suggesting that the specific function, if any, depends on the RNA moiety.     

Identification of nuclear type II [(3)H]estradiol binding sites as histone H4

[3H]Luteolin binds covalently to uterine nuclear type II sites [B. Markaverich, K. Shoulars, M.A. Alejandro, T. Brown, Steroids 66 (2001) 707] and was used to identify this protein(s). SDS-PAGE analyses of [3H]luteolin-labeled type II site preparations revealed specific binding to 11- and 35-kDa proteins. The 11-kDa protein was identified as histone H4 by amino acid sequencing. Western blotting confirmed that the 11- and 35-kDa proteins were acetylated forms of histone H4. Anti-histone H4 antibodies (but not H2A, H2B, or H3 antibodies) quantitatively immunoadsorbed type II binding sites from nuclear extracts. Binding analyses by [3H]estradiol exchange, using luteolin as a competitor, detected specific type II binding activity to histone H4 (but not histones H2A, H2B, or H3) generated in a rabbit reticulocyte lysate translation system and confirmed that histone H4 is the type II site.     

3H]epinephrine and [3H]norepinephrine binding to alpha-noradrenergic.

(±)-[³H]Epinephrine and (?)-[³H]norepinephrine bind saturably to calf cerebral cortex membranes under appropriate incubation conditions in a fashion indicating that they label α-noradrenergic receptors. Binding of the two [³H]catecholamines is saturable with dissociation constants of 20–30 nM. Binding is stereoselective with (?)-norepinephrine displaying about twenty times greater affinity than (+)-norepinephrine. The relative potencies of catecholamines in competing for these binding sites parallels their relative pharmacologic effects at α-noradrenergic receptors in numerous tissues. Thus, (?)-epinephrine is 2–3 times more potent than (?)-norepinephrine and 500 times more potent than (?)-isoproterenol. Binding is inhibited by low concentrations of the α-antagonists phentolamine and phenoxybenzamine but not by the β-antagonist propranolol.     

Uptake of [(3)H]glycine by synaptosomes of channel

It is known that channel catfish erythrocytes can take up glycine by several distinct transport systems. Further, glycine is an inhibitory neurotransmitter in mammalian brain and spinal cord. Consequently, the uptake of [(3)H]glycine by catfish brain was investigated and found to be a saturable process, dependent on the presence of Na(++) and Cl(--) and sensitive to temperature. A kinetic analysis of transport was performed at 22C. This showed that a high-affinity system existed which exhibited a K(m) of 5.1 (+/- 2. 1) microM. Several structural analogues of glycine were capable of inhibiting uptake in a competitive manner. The most effective inhibitor was sarcosine (IC(50) 5 36 microM). Uptake was also able to be inhibited by harmaline, a drug known to interfere with Na(+)-dependent transport processes. It is concluded that glycine transport by channel catfish brain has much in common with transport by mammalian nervous tissue which is carried out by the membrane carriers GLYT1 and GLYT2. On the other hand, synaptosomal transport differs somewhat from glycine transport by channel catfish erythrocytes.     

Neurotensin decreases high affinity [3H]-ouabain binding to cerebral cortex membranes

Previous work from this laboratory showed the ability of neurotensin to inhibit synaptosomal membrane Na(+), K(+)-ATPase activity, the effect being blocked by SR 48692, a non-peptidic antagonist for high affinity neurotensin receptor (NTS1) [López Ordieres and Rodríguez de Lores Arnaiz 2000; 2001]. To further study neurotensin interaction with Na(+), K(+)-ATPase, peptide effect on high affinity [(3)H]-ouabain binding was studied in cerebral cortex membranes. It was observed that neurotensin modified binding in a dose-dependent manner, leading to 80% decrease with 1 × 10(-4)M concentration. On the other hand, the single addition of 1 × 10(-6)M, 1 × 10(-5)M and 1 × 10(-4)M SR 48692 (Sanofi-Aventis, U.S., Inc.) decreased [(3)H]-ouabain binding (in %) to 87 ± 16; 74 ± 16 and 34 ± 17, respectively. Simultaneous addition of neurotensin and SR 48692 led to additive or synergic effects. Partial NTS2 agonist levocabastine inhibited [(3)H]-ouabain binding likewise. Saturation assays followed by Scatchard analyses showed that neurotensin increased K(d) value whereas failed to modify B(max) value, indicating a competitive type interaction of the peptide at Na(+), K(+)-ATPase ouabain site. At variance, SR 48692 decreased B(max) value whereas it did not modify K(d) value. [(3)H]-ouabain binding was also studied in cerebral cortex membranes obtained from rats injected i. p. 30 min earlier with 100 μg and 250 μg/kg SR 48692. It was observed that the 250 μg/kg SR 48692 dose led to 19% decrease in basal [(3)H]-ouabain binding. After SR 48692 treatments, addition of 1 × 10(-6)M led to additive or synergic effect. Results suggested that [(3)H]-ouabain binding inhibition by neurotensin hardly involves NTS1 receptor.     

High affinity [3H]imipramine binding and serotonin uptake to platelets of adolescent females suffering from anorexia nervosa

High affinity [3H]imipramine binding and [3H]serotonin uptake to platelets were investigated in 17 anorexic females aged 15-18 years as compared to 15 healthy females of similar ages. A significant decrease in the density of [3H]imipramine binding sites was observed in anorexics as compared to controls (368 +/- 40 vs 517 +/- 38 fmoles/mg protein, p less than 0.01). No alteration in Kd values or in the kinetic parameters of serotonin uptake (Vmax, Km) were noted. The fact that the decrease in imipramine binding is not accompanied by a parallel reduction in serotonin uptake might indicate that anorexia nervosa is not ultimately related to major depression and that the imipramine binding site is not identical to the serotonin uptake site.     

Inhibition of [(3)H]ponasterone a binding by ecdysone agonists in the intact Sf-9 cell line

Ecdysone agonists, including dibenzoylhydrazines, significantly inhibited the binding of [(3)H]ponasterone A ([(3)H]PoA) in intact Sf-9 cells (Spodoptera frugiperda). The amount of [(3)H]PoA binding varied in a concentration-dependent manner. According to the IC(50), concentration at which there is 50% inhibition, the order of potency of typical ecdysone agonists is tebufenozide (RH-5992) > methoxyfenozide (RH-2485) > PoA > 20-hydroxyecdysone > cyasterone > RH-5849, makisterone A > or = inokosterone > ecdysone. The ranking is consistent with that obtained from a cultured integument system of the rice stem borer Chilo suppressalis except for methoxyfenozide. Other compounds whose modes of action are different from that of ecdysteroids, for example respiration inhibitors, plant steroid hormones, and chitin synthesis inhibitors, did not inhibit the binding of [(3)H]PoA significantly. The mammalian hormones estradiol and diethylstilbestrol, and a secondary bile acid, lithocholic acid, significantly inhibited the binding of [(3)H]PoA at 25 microM. However, their binding activity in terms of pIC(50) was either very low or not evaluated.     

Purification of L-[3H]nicotine eliminates low affinity binding

Some studies of L-[3H]nicotine binding to rodent and human brain tissue have detected two binding sites as evidenced by nonlinear Scatchard plots. Evidence presented here indicates that the low affinity binding site is not stereospecific, is not inhibited by low concentrations of cholinergic agonists and is probably due to breakdown products of nicotine since purification of the L-[3H]nicotine eliminates the low affinity site.     

Imperatoxin A (IpTx(a)) from Pandinus imperator stimulates [(3)H]ryanodine binding to RyR3 channels.

The effect of imperatoxin A (IpTx(a)) on the ryanodine receptor type 3 (RyR3) was studied. IpTx(a) stimulates [(3)H]ryanodine binding to RyR3-containing microsomes, but this effect requires toxin concentrations higher than those required to stimulate RyR1 channels. The effect of IpTx(a) on RyR3 channels was observed at calcium concentrations in the range 0.1 microM to 10 mM. By contrast, RyR2 channels were not significantly affected by IpTx(a) in the same calcium ranges. Single channel current measurements indicated that IpTx(a) induced subconductance state in RyR3 channels that was similar to those observed with RyR1 and RyR2 channels. These results indicate that IpTx(a) is capable of inducing similar subconductance states in all three RyR isoforms, while stimulation of [(3)H]ryanodine binding by this toxin results in isoform-specific responses, with RyR1 being the most sensitive channel, RyR3 displaying an intermediate response and RyR2 the least responsive ones.