[H]lysergic acid diethylamide (LSD): differential agonist and antagonist binding properties at 5-HT receptor subtypes in rat brain

[³H]Lysergic acid diethylamide (LSD) in the presence of 40 nM ketanserin labeled the 5-HT_1A receptor subtype in rat hippocampal membranes. In the presence of guanosine triphosphate (GTP), the B_max and affinity of [³H]LSD binding to the 5-HT_1A binding site were significantly decreased. [³H]LSD in the presence of 40 nM WB4101 labeled the 5-HT₂ receptor subtype in homogenates of rat frontal cortex. In contrast to the effect on [³H]LSD binding to the 5-HT_1A binding site, GTP produced no significant effect on either the B_max or the K_D of [³H]LSD binding to the 5-HT₂ binding site. Competition of 5-HT for [³H]LSD binding to the 5-HT₂ binding site was best described by a computer-derived model assuming two binding sites. In the presence of GTP, the 5-HT competition curve was shifted significantly to the right with an approx. 3-fold increase in the IC₅₀. These binding characteristics are consistent with [³H]LSD acting as an antagonist at the 5-HT₂ receptor which has multiple affinity states for agonists and is coupled to a guanine nucleotide regulatory subunit. Thus, [³H]LSD has binding characteristics consistent with it acting as an agonist at the 5-HT_1A receptor subtype but as an antagonist at the 5-HT₂ receptor subtype in rat brain.     

Influence of lipid peroxidation on [H]ketanserin binding to 5-HT2 prefrontal cortex receptors

The influence of lipid peroxidation on 5-HT₂ receptor binding was examined in prefrontal cortex membranes from sheep brain. Lipid peroxidation was induced with ascorbic acid and ferrous sulphate and measured by the thiobarbituric acid method. In lipid-peroxidized membranes, [³H]ketanserin specific binding was inhibited. The B_max values decreased by 80%, from 50.1±3.5 fmol/mg protein in control membranes to 10.1±2.0 fmol/mg protein in peroxidized membranes, indicating a decrease in the number of 5-HT₂ binding sites. However, the K_D values for the [³H]ketanserin specific binding did not significantly change. In order to further characterize [³H]ketanserin binding, the inhibition potency (IC₅₀ values) of antagonists or agonists of serotonin and dopamine receptors for [³H]ketanserin specific binding was determined. In control membranes, the order of the inhibition potency of the drugs tested was the following: ketanserin (−log [IC₅₀] = 8.56±0.70) ritanserin (−log [IC₅₀] = 8.13±0.30) methysergide (−log [IC₅₀] = 7.42±0.50) spiperone (−log [IC₅₀] = 7.23±0.18) serotonin (−log [IC₅₀] = 6.99±0.65) haloperidol (−log [IC₅₀] = 6.95±0.65) dopamine (−log [IC₅₀] = 5.82±0.76). After membrane lipid peroxidation, the IC₅₀ value for ritanserin was significantly increased, suggesting a decreased capacity for displacing [³H]ketanserin specific binding. Other antagonists of 5-HT₂ receptors showed apparent increases in IC₅₀ values upon peroxidation, whereas spiperone was shown to be the most potent drug (−log [IC₅₀] = 7.19±1.06) in inhibiting [³H]ketanserin specific binding. A decrease in polyunsaturated fatty acids, namely docosahexaenoic acid (22:6) was also observed in peroxidized membranes. These results indicate a modulating role of the surrounding lipids and of the physical properties of the membranes on the binding activity of 5-HT₂ receptors upon the lipid peroxidation process, which can be involved in the tissue impairment that occurs during the aging process and in post-ischemic situations.     

Interaction of the nicotinic agonist (R,S)-3-pyridyl-1-methyl-2-(3-pyridyl)-azetidine (MPA) with nicotinic acetylcholine receptor subtypes expressed in cell lines and rat cortex

The interaction of the nicotinic agonist (R,S)-3-pyridyl-1-methyl-2-(3-pyridyl)-azetidine (MPA) with different nicotinic acetylcholine receptor (nAChR) subtypes was studied in cell lines and rat cortex. MPA showed an affinity (Ki = 1.21 nM) which was higher than anatoxin-a > (−)-nicotine > (+)-[R]nornicotine > (−)-[S]nornicotine > and (+)-nicotine, but lower than cytisine (Ki = 0.46 nM) in competing for (−)-[³H]nicotine binding in M10 cells, which stably express the recombinant ₄β₂ nAChR subtype. A one-binding site model was observed in all competing experiments between (−)-[³H]nicotine binding and each of the agonists studied in M10 cells. MPA showed a 13-fold higher affinity for (−)-[³H]nicotine binding sites compared to the [³H]epibatidine binding sites in rat cortical membranes. In human neuroblastoma SH-SY5Y cells, which predominantly express the ₃ nAChR subunit mRNA, MPA displaced [³H]epibatidine binding from a single population of the binding sites with an affinity in the same nM range as that observed MPA in displacing [³H]epibatidine binding in rat cortical membranes. Chronic treatment of M10 cells with MPA significantly up-regulated the number of (−)-[³H]nicotine binding sites in a concentration dependent manner. Thus MPA appears to have higher affinity to 4-subunit containing receptor subtype than ₃-subunit containing receptor subtype of nAChRs. Furthermore MPA binds to ₄β₂ receptor subtype with higher affinity than (−)-nicotine and behaves, opposite to cytisine, as a full agonist in up-regulating the number of nAChRs. © 1998 Elsevier Science Ltd. All rights reserved.     

A differential interaction of putative μ-selective agonists with opiate binding sites in rat brain

The availability of tritium-labelled sufentanil ([³H]SUF) allowed for a further radioligand analysis of opiate binding sites in rat brain. A comparison of the binding characteristics of [³H]SUF and [³H]dihydromorphine ([³H]DHM) revealed a very similar potency in their mutual displacement by unlabelled analogues. Furthermore, a series of putative μ-opiate agonists displayed equal potencies in displacing either [³H]SUF and [³H]DHM, the only striking exception being the highly μ-selective opioid peptide morphiceptin which was 33 times less potent in inhibiting [³H]SUF as compared to [³H]DHM binding. Additional experiments revealed further pronounced differences in [³H]SUF and [³H]DHM binding characteristics: the total amount of binding sites for [³H]SUF was 4 times higher than that for [³H]DHM and the regional distribution within particular brain areas displayed considerable differences. Furthermore, the binding of [³H]SUF was differentially modulated by sodium and GTP as compared to [³H]DHM binding. These data suggest that in rat brain, [³H]SUF interacts both with μ-opiate sites recognizing [³H]DHM and another type of opiate site, which cannot be equated with any of the, as yet, described δ- or κ-binding sites, and rather, represents a subclass of μ-opiate receptor sites. These experiments, thus, support the notion of subclasses (isoreceptors) for different types of opiate receptors.     

3-Mercaptopropionic acid administration increases the affinity of [H]Quinuclidinyl benzilate binding to membranes of the striatum and cerebellum

It is known that quinuclidinyl benzilate (QNB) binds specifically and with high affinity to the cholinergic muscarinic receptor and that behaves as a potent antagonist of this receptor.

We have analysed -[³H]QNB binding to rat CNS membranes after the administration of the convulsant 3-mercaptopropionic acid (MP) (150 mg·kg⁻¹, i.p.). The studies were done in rats killed at two stages: during and after seizures. No changes in [³H]QNB binding to hippocampus and cerebral cortex membranes were found. [³H]QNB binding increased about 40 and 80% in striatum and cerebellum membranes, respectively. The changes were observed both in seizure and postseizures states. The study was extended to the assay of [³H]QNB binding kinetic constants in the anatomical areas modified by the convulsant. The analysis of the saturation curves indicated an increase in the binding affinity but no change in the number of binding sites. Hill number values were near the unit suggesting a non-cooperative interaction between the ligand and the receptor, and the labelling of a homogeneous population of receptor sites.

The results suggest the participation of some cholinergic pathways in the development and maintenance of MP-induced seizures.     

Differentiation of [H]phencyclidine and ( + )-[H]SKF-10,047 binding sites in rat cerebral cortex

The potency of a series of opioid and non-opioid psychotomimetic drugs to inhibit the specific binding of [³H]PCP and ( + )-[³H]SKF-10,047 to rat cerebral cortical membranes was examined. ( + )-PCMP, the 3-methylpiperidino analog of PCP, was a potent inhibitor of the specific binding of both ligands. All of the other 12 compounds examined, however, displayed a 3-277-fold selectivity for either [³H]PCP or (+)-[³H]SKF-10,047 binding. These results suggest that although these opioid and non-opioid psychotomimetics bind to both sites, most have significantly different affinities. The binding sites for [³H]PCP appear to be distinct from the ‘sigma’ binding sites labeled with (+)-[³H]SKF-10,047.

SKF-10,047 Sigma receptor Phencyclidine Phencyclidine receptor Psychotomimetic activity     

Muscarinic cholinergic components in the carp brain

The activity of the muscarinic cholinergic system (acetylcholine, ACh; acetylcholinesterase, AChE; choline acetyltransferase, ChAT; muscarinic acetylcholine receptors) was studied in the carp brain. The ACh content (13.9 ± 1.1 nmol/g wet tissue) was estimated by gas chromatography after microwave irradiation focused to the head. The AChE and ChAT activities were 153 ± 13 nmol/min/mg protein and 817 ± 50 pmol/min/mg protein, respectively. The characteristics of [³H](−)quinuclidinyl benzilate ([³H](−)QNB) and [³H]pirenzepine ([³H]PZ) binding were also studied in brain membranes. Their specific binding was linearly dependent on the protein content and they appeared to bind with high affinity to a single, saturable binding site. A dissociation constant (K_d) of 47 ± 6.3 pM and a maximum number of binding sites (B_max) of 627 ± 65 fmol/mg protein were obtained for [³H](−)QNB, with a K_d value of 3.85 ± 0.67 nM and a B_max value of 95.3 ± 6.25 fmol/mg protein for [³H]PZ binding. The [³H]PZ binding amounted to only 15% of the [³H](−)QNB-labeled sites, as estimated from the ratio of the B_max values of [³H](−)QNB and [³H]PZ, suggesting a low density of M₁ subtype. Atropine sulfate, atropine methylnitrate and PZ inhibited the binding of both radioligands with Hill slopes (n_H) close to unity. The n_H value of AF-DX 116 was close to 1 against [³H](−)QNB binding, while it was 0.75 against [³H]PZ binding. The displacement curves of oxotremorine and carbachol were shallow for the binding of both radioligands. The rank order of potency of muscarinic ligands against [³H](−)QNB binding (K_i nM) was atropine sulfate (0.55) > atropine methylnitrate (1.61) > PZ (61.19) > oxotremorine (156.3) > AF-DX 116 (307) > carbachol (1301), while in the case of [³H]PZ binding it was atropine sulfate (0.24) > atropine methylnitrate (0.34) > PZ (10.38) > AF-DX 116 (55.87) > oxotremorine (62.79) > carbachol (1696). The results indicate the presence of a well-developed muscarinic cholinergic system with predominantly M₂ receptors in the carp brain.     

Direct interactions of androgenic/anabolic steroids with the peripheral benzodiazepine receptor in rat brain: Implications for the psychological and physiological manifestations of androgenic/anabolic steroid abuse

The peripheral benzodiazepine receptor (PBR) is a mitochondrial protein involved in regulating steroid synthesis and transport. We report here the effects of androgenic/anabolic steroids (AAS) on the binding of the PBR-specific ligand [³H] PK11195 to male rat brain cortical synaptoneurosomes. Two synthetic AAS, stanozolol and 17β-testosterone cypionate (17β-cyp), significantly inhibited 1 nM [³H] PK11195 binding at concentrations greater than 5 and 25 μM, respectively. Stanozolol was the most effective inhibitor, reducing [³H] PK11195 binding by up to 75%, compared to only 40% inhibition by 17β-cyp, at 50 μM AAS concentration. Two other AAS, 17-methyltestosterone and nortestosterone decanoate, were incapable of inhibiting [³H] PK11195 binding at concentrations up to 50 μM. On the basis of Scatchard/Rosenthal analysis, [³H] PK11195 binds to two classes of binding sites, and the inhibition of [³H] PK11195 binding by stanozolol appears to be allosteric, primarily reducing binding to the higher affinity [³H] PK11195 binding site. These results, in combination with earlier studies indicating the direct effects of AAS on the function of additional central nervous system receptor complexes, suggest that the behavioral and psychological effects of AAS result from the interactions of AAS with multiple regulatory systems in the brain.     

[H]WAY–100635 for 5–HT1A receptor autoradiography in human brain: a comparison with [H]8–OH–DPAT and demonstration of increased binding in the frontal cortex in schizophrenia

WAY–100635 is the first selective, silent 5–HT_1A (5-hydroxytryptamine_1A, serotonin-1A) receptor antagonist. We have investigated the use of [³H]WAY–100635 as a quantitative autoradiographic ligand in post-mortem human hippocampus, raphe and four cortical regions, and compared it with the 5–HT_1A receptor agonist, [³H]8–OH–DPAT. Saturation studies showed an average Kd for [³H]WAY–100635 binding in hippocampus of 1.1 nM. The regional and laminar distributions of [³H]WAY–100635 binding and [³H]8–OH–DPAT binding were similar. The density of [³H]WAY–100635 binding sites was 60–70% more than that of [³H]8–OH–DPAT in all areas examined except the cingulate gyrus where it was 165% higher. [³H]WAY–100635 binding was robust and was not affected by the post-mortem interval, freezer storage time or brain pH (agonal state). Using [³H]WAY–100635, we confirmed an increase of 5–HT_1A receptor binding sites in the frontal cortex in schizophrenia, previously demonstrated with [³H]8–OH–DPAT. Compared to [³H]8–OH–DPAT, [³H]WAY–100635 has two advantages: it has a higher selectivity and affinity for the 5–HT_1A receptor, and it recognizes 5–HT_1A receptors whether or not they are coupled to a G-protein, whereas [³H]8–OH–DPAT primarily detects coupled receptors. Given these considerations, the [³H]WAY–100635 binding data in schizophrenia clarify two points. First, they indicate that the elevated [³H]8–OH–DPAT binding seen in the same cases is attributable to an increase of 5–HT_1A receptors rather than any other binding site. Second, the enhanced [³H]8–OH–DPAT binding in schizophrenia reflects an increased density of 5–HT_1A receptors, not an increased percentage of 5–HT_1A receptors which are G-protein-coupled. We conclude that [³H]WAY–100635 is a valuable autoradiographic ligand for the qualitative and quantitative study of 5–HT_1A receptors in the human brain.     

Effects of detergents on binding of 5-hydroxytryptamine3 receptor antagonist [H]GR65630 to rat cortical membranes

In the absence of detergent, specific binding of [³H]GR65630, a 5-hydroxytryptamine₃ (5-HT₃) antagonist, determined in the presence of 5-HT₃ receptor antagonist ICS205-930, was at most 30% of the total binding. To decrease the level of nonspecific binding, the effects of detergents on [³H]GR65630 binding to rat cortical membranes were investigated. The use of a detergent (0.1% Lubrol PX or Triton X-100) decreased nonspecific binding, increasing the proportion of specific binding to 70% of total binding. In the presence of 0.1% Triton X-100, binding of [³H]GR65630 was rapid, reversible and saturable at 25°C. The rank order of 5-HT₃ receptor active drugs in inhibiting [³H]GR65630 binding was quipazine > ICS205-930 > 2-methyl-5-HT = 5-HT > metoclopramide, which confirmed that [³H]GR65630 efficiently labeled 5-HT₃ receptors in the presence of Triton X-100. Triton X-100 improved 5-HT₃ receptor binding with rat brain membranes.     

Selective changes of neurotransmitter receptors in middle-aged gerbil brain

Age-related alterations in major neurotransmitter receptors and voltage dependent calcium channels were analyzed by receptor autoradiography in the gerbil brain. [³H]Quinuclidinyl benzilate (QNB). [³H]cyclohexyladenosine (CHA), [³H]muscimol, [³H]MK-801, [³H]SCH 23390, [³H]naloxone, and [³H]PN200-110 were used to label muscarinic acetylcholine receptors, adenosine A₁ receptors, γ-aminobutyric acid_A (GABA_A) receptors, (NMDA) receptors, dopamine D₁ receptors, opioid receptors, and voltage dependent calcium channels, respectively. In middle-aged gerbils (16 months old), the hippocampus exhibited a significant elevation in [³H]QNB, [³H]MK-801, [³H]SCH 23390, [³H]naloxone, and [³H]PN200-110 binding, whereas [³H]CHA and [³H]muscimol binding showed a significant reduction in this area, compared with that of young animals (1 month). On the other hand, the cerebellum showed a significant alteration in [³H]QNB, [³H]CHA, and [³H]naloxone binding and the striatum also exhibited a significant alteration in [³H]SCH 23390 and [³H]CHA binding in middle-aged gerbils. The neocortex showed a significant elevation only in [³H]CHA binding in middle-aged animals. The nucleus accumbens and thalamus also showed a significant alteration only in [³H]muscimol binding. However, the hypothalamus and substantia nigra exhibited no significant alteration in these bindings in middle-aged gerbils. These results demonstrate the age-related alterations of various neurotransmitter receptors and voltage dependent calcium channels in most brain regions. Furthermore, they suggest that the hippocampus is most susceptible to aging processes and is altered at an early stage of senescence.     

Rat brain guanosine binding site. Biological studies and pseudo-receptor construction

Rat brain guanosine binding sites were studied by (i) a pharmacological approach to confirm the hypothesis of the existence of specific G-coupled receptors for guanosine (1) and, for the first time, delineate a structure–activity relationship for a series of guanosine derivatives; (ii) a molecular modelling approach to design a pseudo-receptor construction. GTP and its non-hydrolysable analogue Gpp[NH]p decreased [³H]-guanosine binding to rat brain membranes. Gpp[NH]p 30 and 100 μM induced a dose-dependent decrease in [³H]-guanosine affinity and PTX pretreatment of rat brain membranes caused a 50% reduction in binding. In slices from rat brain cortex, guanosine induced a dose-dependent increase in intracellular cAMP. This increase is specific for guanosine, since neither the pretreatment with adenosine deaminase nor the A₁ and A₂ adenosine receptor antagonists were able to modify the guanosine-induced cAMP accumulation. The structure–activity relationship showed that the potency order of the best substances able to displace 50 nM [³H]-guanosine was guanosine (1)=6-thioguanosine (3)>8-bromoguanosine (4)>inosine (10)>7-methylguanosine (6)=3′-deoxyguanosine (9)>2′-deoxyguanosine (8)=guanine (11)=6-thioguanine (12)>>N²-methylguanosine (5). The competition studies confirmed that [³H]-guanosine site was distinct from the well characterized ATP and adenosine binding sites. The present results are rationalized in terms of a putative pseudo-receptor construct which includes all the relevant physicochemical interaction between guanosine analogues and their putative binding sites. This construct will be useful for the in silico screening of compound libraries in search for new potent and structurally diverse pharmacological tools.     

G-protein-coupled A1 adenosine receptors in coated vesicles of mammalian brain: Characterization by radioligand binding and photoaffinity labelling

A₁ adenosine receptors in coated vesicles have been characterized by radioligand binding and photoaffinity labelling. Saturation experiments with the antagonist 8-cyclopentyl-1,3-[³H]dipropyl-xanthine ([³H]DPCPX) gave a K_d value of 0.7 nM and a B_max value of 82 ± 13 fmol/mg protein. For the highly A₁-selective agonist 2-chloro-N⁶-[³H]cyclopentyladenosine ([³H]CCPA) a K_d value of 1.7 nM and a B_max value of 72 ± 29 fmol/mg protein was estimated. Competition of agonists for [³H]DPCPX binding gave a pharmacological profile with R-N⁶-phenylisopropyladenosine (R-PIA) > CCPA > S-PIA > 5′-N-ethylcarboxamido-adenosine (NECA), which is identical to brain membranes. The competition curves were best fitted according to a two-site model, suggesting the existence of two affinity states. GTP shifted the competition curve for CCPA to the right and only one affinity state similar to the low affinity state in the absence of GTP was detected. The photoreactive agonist 2-azido-N⁶-¹²⁵I-p-hydroxyphenylisopropyladenosine ([¹²⁵I]AHPIA) specifically labelled a single protein with an apparent molecular weight of 35,000 in coated vesicles, which is identical to A₁ receptors labelled in brain membranes. Therefore, coated vesicles contain A₁ adenosine receptors with similar binding characteristics as membrane-bound receptors, including GTP-sensitive high-affinity agonist binding. Photoaffinity labelling data suggest that A₁ receptors in these vesicles are not a processed receptor form. These results confirm that A₁ receptors in coated vesicles are coupled to a G-protein, and it appears that the A₁ receptor systems in coated vesicles and in plasma membranes are identical.     

Solubilization of binding sites for IAPP and CGRP from rat lung

Functional binding sites for [¹²⁵I]IAPP and [¹²⁵I]CGRP were solubilized from rat lung membranes with CHAPSO (10 mM). Rat IAPP had a higher affinity (K_i = 22.9 nM) for [¹²⁵I]IAPP binding and rat CGRP (K_i = 0.904 nM) had a higher affinity for [¹²⁵I]CGRP binding over related peptides. [¹²⁵I]IAPP binding was unaffected by GTPγS, but [¹²⁵I]CGRP binding was 50% inhibited, indicating solubilization of a G-protein-receptor complex for CGRP but not IAPP binding. Wheat germ agglutinin affinity columns gave a 25-fold purification of IAPP binding sites, but no CGRP binding sites were eluted from the column, indicating different patterns of glycosylation of the two sites.     

Kinetic evidence for isomerization of the dopamine receptor-raclopride complex

The binding kinetics of the specific dopamine D₂ antagonist [³H]raclopride to dopamine D₂ receptors in rat neostriatum were studied. The pseudo-first-order rate constants of [³H]raclopride binding with these membranes revealed a hyperbolic dependence upon the antagonist concentration, indicating that the reaction had at least two consecutive and kinetically distinguished steps. The first step was fast binding equilibrium, characterized by the dissociation constant K_A = 12 ± 2 nM. The following step corresponded to a slow isomerization of the receptor-antagonist complex, characterized by the isomerization equilibrium constant K_i = 0.11. The dissociation constant K_d = 1.3 nM, calculated from these kinetic data, was similar to K_d = 2.4 nM, determined from equilibrium binding isotherm for the radioligand. Implications of the complex reaction mechanism on dopamine D₂ receptor assay by [³H]raclopride were discussed.     

Tris-azaaromatic quaternary ammonium salts: Novel templates as antagonists at nicotinic receptors mediating nicotine-evoked dopamine release

A series of tris-azaaromatic quaternary ammonium salts has been synthesized and evaluated for their ability to inhibit neuronal nicotinic acetylcholine receptors (nAChRs) mediating nicotine-evoked [³H]dopamine release from superfused rat striatal slices and for inhibition of [³H]nicotine and [³H]methyllycaconitine binding to whole rat brain membranes. The 3-picolinium compound 1,3,5-tri-{5-[1-(3-picolinium)]-pent-1-ynyl}benzene tribromide (tPy3PiB), 3b, exhibited high potency and selectivity for nAChR subtypes mediating nicotine-evoked [³H]dopamine release with an IC₅₀ of 0.2 nM and I_max of 67%.     

Identification of a novel 5-HT1 binding site in rat spinal cord

High affinity, specific [³H]5-hydroxytryptamine (5-HT) binding to spinal cord synaptosomes was examined to identify the 5-HT receptor subtypes present. Computer nonlinear regression analysis of competition studies employing 8-OH-DPAT indicated that this 5-HT_1A selective agonist demonstrated high affinity competition (K_{i = 1.3 nM}) for 24.6 ± 0.7% of the total [³H]5-HT binding sites. Competition studies employing the 5-HT_1B selective agonist RU24969, in the presence of 100 nM 8-OH-DPAT, indicated that RU24969 demonstrated high affinity (K_{i = 1.1 nM}) competitive inhibition for 26.2 ± 1.4% of all [³H]5-HT binding sites. Neither 5-HT_1C, 5-HT_1D, 5-HT₂ nor 5-HT₃ selective compounds demonstrated any high affinity competition for the residual 49% of specific [³H]5-HT binding. Therefore, three major classes of [³H]5-HT binding sites could be demonstrated in spinal cord synaptosomes: 5-HT_1A, 5-HT_1B and a novel [³H]5-HT binding site which respectively represented 25, 26 and 49% of spinal cord synaptosomal [³H]5-HT binding. Further studies focusing on the function of the latter binding site are needed to determine if the presently identified novel binding site is the major 5-HT₁ receptor subtype present in spinal cord.     

Temperature dependent binding of mebendazole to tubulin in benzimidazole-susceptible and -resistant strains of Trichostrongylus colubriformis and Caenorhabditis elegans.

The binding of [³H]mebendazole ([³H]MBZ) to tubulin in benzimidazole-susceptible (BZ-S) and benzimidazole-resistant (BZ-R) strains of Trichostrongylus colubriformis and Caenorhabditis elegans was examined in order to investigate the biochemical changes to tubulin that result in BZ resistance in parasitic and free-living nematodes. In both species the extent of [³H]MBZ binding to tubulin was significantly reduced in the BZ-R strain compared with the BZ-S strain. The decrease in [³H]MBZ binding in the BZ-R strain of each species was the result of a significant reduction in the amount of charcoal stable [³H]MBZ-tubulin complexes and was not related to a change in the association constant of the [³H]MBZ-tubulin interaction. [³H]MBZ binding to tubulin was temperature dependent, reaching maximum levels at 37°C in BZ-S T. colubriformis and 10°C in BZ-R T. colubriformis. Both the BZ-S and BZ-R strains of C. elegans displayed maximum [³H]MBZ binding at 4°C. Resistance ratios derived from the amount of [³H]MBZ binding in the BZ-S and BZ-R strains and in vitro development assays demonstrated that the temperature dependence and extent of drug binding was indicative of BZ resistance status and was species specific in the BZ-S isolates. These results indicate that biochemical differences exist in the binding of benzimidazole carbamates to tubulin in nematode species, and suggest that the susceptibility of the parasitic nematodes to the benzimidazole anthelmintics is the result of a unique high affinity and/or high capacity interaction ofbenzimidazole carbamates with tubulin.     

Biochemical and autoradiographic investigations of the retrograde axonal transport of labeled material following [H]norepinephrine injection in the olfactory bulb

Biochemical and autoradiographic methods were used to investigate the retrograde transport of labeled material after injection of [³H]norepinephrine ([³H]NE) in the olfactory bulb (OB) of rat. Mechanical obstruction of the ventricular recess prevented intraventricular diffusion. At different time intervals following bilateral [³H]NE injections, total radioactivity was measured in the OB, locus caeruleus (LC), raphe dorsalis and periaqueductal gray. Preferential accumulation occurred in the LC, and an approximate rate of retrograde transport of 1–6 mm/h could be calculated. Previous administration of 6-hydroxydopamine in the OB reduced this accumulation by 60%. Sixteen hours after [³H]NE injection, the radioactivity in LC was equally distributed between an ethanol-soluble and -insoluble fraction. A small proportion of the soluble material was recovered as [³H]NE and/or [³H]normetanephrine. Following unilateral injections of [³H]NE, light microscopic autoradiograms demonstrated nerve cell body labeling mainly in the ipsilateral LC and of greater intensity after 16 than 4 and 8 h. These data lead to the conclusion that the movement of radioactive material was indeed representative of retrograde axonal transport rather than of other mechanisms such as diffusion. The observation of a preferential labeling of noradrenergic perikarya in LC supports the hypothesis of a process mediated by specific binding and/or uptake of [³H]NE into noradrenergic axon terminals.     

Selective labeling of κ2 opioid receptors in rat brain by [I]IOXY: Interaction of opioid peptides and other drugs with multiple κ2a binding sites

Recent studies from our laboratory resolved two subtypes of the κ₂ binding site, termed κ_2a and κ_2b, using guinea pig, rat, and human brain membranes depleted of μ and δ receptors by pretreatment with the site-directed acylating agents BIT (μ-selective) and FIT (δ-selective). 6β-Iodo-3,14-dihydroxy-17-cyclopropylmethyl-4,5-epoxymorphinan (IOXY), an opioid antagonist that has high affinity for κ₂ sites, was radioiodinated to maximum specific activity (2200 Ci/mmol) and purified by high pressure liquid chromotography and used to characterize multiple κ₂ binding sites. The results indicated that [¹²⁵I]IOXY, like [³H]bremazocine, selectively labels κ₂ binding sites in rat brain membranes pretreated with BIT and FIT. Using 100 nM [ -Ala²-MePhe⁴,Gly-ol⁵]enkephalin to block [¹²⁵I]IOXY binding to the κ_2b site, two subtypes of the κ_2a binding site were resolved, both in the absence and presence of 50 μM 5′-guanylyimidodiphosphate. Viewed collectively, these results provide further evidence for heterogeneity of the κ opioid receptor, which may provide new targets for drug design, synthesis, and therapeutics.