Spectrum of the μ-, δ- and κ-binding sites in homogenates of rat brain

1 In homogenates of rat brain, the binding characteristics of tritiated opiates and opioid peptides were examined and the relative capacities of μ-, δ- and κ-binding sites of the opiate receptor determined by saturation analysis.

2 In competition experiments, binding of the selective μ-ligand [³H]-[D-Ala²,MePhe⁴,Gly-ol⁵]enkephalin at the μ-site was displaced by [D-Ala²,D-Leu⁵]enkephalin with rather low affinity (K_I = 12.6 nM) and more readily by the ketazocine-like compounds (-)-ethylketazocine (K_I = 3.1 nM) and (-)-bremazocine (K_I = 0.32 nM), which also displaced the binding of [³H]-[D-Ala²,D-Leu⁵]enkephalin from the δ-site. In contrast, the binding to the κ-site was easily displaced by ethylketazocine (1.0 nM) and bremazocine (0.37 nM) but not by the μ-ligand [D-Ala²,MePhe⁴,Gly-ol⁵]enkephalin (K_I = 2000-3000 nM) or the δ-ligand [D-Ala²,D-Leu⁵]enkephalin (K_I > 20,000 nM).

3 The dissociation equilibrium constant (K_D) and the binding capacity (pmol/g) of the μ-binding site were determined with the selective μ-ligand [³H]-[D-Ala²,MePhe⁴,Gly-ol⁵]enkephalin. For the δ-site, [³H]-[D-Ala²,D-Leu⁵]enkephalin was used in the presence of unlabelled [D-Ala²,MePhe⁴,Gly-ol⁵]enkephalin in order to suppress cross-reactivity to the μ-binding site. For the estimation of κ-binding, [³H]-(±)-ethylketazocine or [³H]-(-)-bremazocine were used in the presence of unlabelled μ- and δ-ligands for the suppression of cross-reactivities to the μ- and δ-binding sites.

4 In rat brain the capacity of the μ-binding site was 7.3 pmol/g brain, that of the δ-binding site 6.7 pmol/g brain and that of the κ-binding site 2.0 pmol/g brain. Thus, the κ-binding site had the lowest value whereas in the guinea-pig brain the capacity of the μ-binding site was lower than that of the δ- or κ-binding site.

     

Metabolism of the hallucinogen N,N-dimethyltryptamine in rat brain homogenates

The metabolism of the hallucinogen N,N-dimethyltryptamine (DMT) in whole rat brain homogenate is reported. Studies were conducted using tritiated DMT and DMT-N-oxide (DMT-NO), and metabolites were identified and quantified using thin-layer chromatography and liquid scintillation counting techniques. Metabolite confirmation was obtained by incubation of α,α,β,β-tetradeutero-DMT (DDMT) with whole brain homogenate followed by combined gas chromatographic/mass spectrometric analyses. The metabolites of DMT were identified as indoleacetic acid (IAA), DMT-NO, N-methyltryptamine (NMT), 2-methyl-1,2,3,4-tetrahydro-β-carboline (2-MTHBC), tryptamine (TA) and 1,2,3,4- tetrahydro-β-carboline (THBC). DMT-NO was metabolized to give DMT, NMT, IAA and 2-MTHBC. Formation of these metabolites from DMT-NO was stimulated by anaerobic incubation. Mechanisms for the formation of β-carbolines from DMT and DMT-NO are discussed. The effects of the monamine oxidase inhibitor iproniazid phosphate on DMT metabolism were also studied. Iproniazid inhibited the formation of IAA from DMT by 83 per cent. However, the formation of NMT and DMT-NO was inhibited by 90 per cent under these conditions. Thus, the reported extension of half-life and potentiation of DMT behavioral effects by iproniazid may be due to inhibition of NMT and DMT-NO formation rather than inhibition of monoamine oxidase. A cyclic pathway for the synthesis and metabolism of DMT in brain tissue is proposed.     

5-Hydroxytryptamine metabolism in rat brain and liver homogenates

1. The metabolism of 5-hydroxyindoleacetaldehyde derived from 5-hydroxytryptamine incubated with tissue homogenates was studied as an indicator of aldehyde dehydrogenase and alcohol dehydrogenase activities.2. In liver and brain from rats, there were indications of the presence of one or more aldehyde dehydrogenases which were stimulated by NAD(+) to a greater extent than by NADP(+).3. In liver from rats, there were indications of the presence of one or more alcohol dehydrogenases, which were stimulated by NADH to a greater extent than by NADPH.4. In brain from rats, there were indications of the presence of one or more alcohol dehydrogenases which were stimulated by NADPH to a greater extent than by NADH.     

Antioxidant effects of calcium antagonists in rat brain homogenates

We studied the antioxidant activities of calcium antagonists against autoxidation in rat brain homogenates. The homogenates were incubated for 30 min at 37 degrees C with or without a calcium antagonist and subsequently assayed for lipid peroxide content. Percent inhibition of the lipid peroxidation was used as an index of the antioxidant effect. Dihydropyridine calcium antagonists exhibited concentration-dependent (3-300 micromol/l) inhibitory effects against lipid peroxidation. The relative order of antioxidant potency and associated IC50 values (micromol/l) of the calcium antagonists for inhibition of the lipid peroxidation were as follows: nifedipine (51.5)>barnidipine (58.6)>benidipine (71.2)>nicardipine (129.3)>amlodipine (135.5)>nilvadipine (167.3)>nitrendipine (252.1)> diltiazem (>300)=verapamil (>300). These results suggest that some dihydropyridine calcium antagonists show antioxidant properties. The antioxidant effects of the calcium antagonists may contribute to their pharmacological actions.     

Ontogenetic studies on mu, delta and kappa opioid receptors in rat brain

The ontogenetic pattern of multiple opioid binding sites in rat brain from birth until weaning has been investigated. [3H]-dihydromorphine ([3H]-DHM)3 [3H]-D-Ala2-D-Leu5-enkephalin ([3H]-DADLE) and [3H]-dynorphin A (1-8) ([3H]-DYN) as markers of mu (mu), delta (delta) and Kappa (kappa) sites were utilized respectively. The analysis of the kinetic parameters of [3H]-DHM binding shows that, at birth, mu sites possess an affinity similar to that of adult animals, and a density of 50%, which reaches 80% of the adult value at day 4. On the contrary, [3H]-DADLE binding in the first post-natal days shows low affinity and low density and delta-sites do not reach values comparable to the adult ones until the second week of life. The kinetic parameters of [3H]-DYN binding are almost undetectable during the preweanling period, due to the very low density of kappa sites at this stage of life. Displacement studies with mu-, delta- and kappa-selective ligands show that the Ki values on [3H]-DHM binding sites were similar in 4 day old and adult animals for all the tested compounds, whereas Ki values on [3H]-DADLE and [3H]-DYN binding sites reflected an immaturity of delta and kappa receptors. In conclusion, our data suggest that multiple opioid receptors follow different ontogenetic patterns. In the first stages of life only mu receptors are almost mature and possibly mediate endogenous opioid actions and exogenous opiate pharmaco-toxicological effects.     

Regional variations in binding capacities at mu-, delta- and kappa-opioid sites in membrane suspensions from rabbit brain

The highest maximum binding capacities at the mu-sites of rabbit brain are in the striatum, with intermediate levels in the diencephalon, mesencephalon, cerebellum and cortex and low levels in the pons-medulla and hippocampus. For the delta-site the highest maximum binding capacity is also in the striatum; there are almost equally low levels in the other brain regions. At the kappa-sites the maximum binding capacities are highest in the diencephalon; there are intermediate levels in the cortex and striatum, and low levels in the mesencephalon, cerebellum, hippocampus and pons-medulla. The KD values lack reproducibility; there are no regional variations at the kappa-site as estimated with [3H](-)-bremazocine, but the possibility cannot be excluded that there are regional variations in the KD values for [3H][D-Ala2,MePhe4,Gly-ol5]enkephalin at the mu-site or for [3H][D-Ala2,D-Leu5]enkephalin at the delta-site. It may be important to use saturation analysis in future investigations of the distributions of the binding sites.     

Comparative binding of mu and delta selective ligands in whole brain and pons/medulla homogenates from rat: affinity profiles of fentanyl derivatives

The comparative binding characteristics of the mu opioid receptor selective ligand [3H]-[D-Ala2-MePhe4-glyol5]enkephalin [( 3H]-DAGO) and of the delta receptor ligand [3H]-[D-Pen2, D-Pen5]enkephalin[( 3H]-DPDPE) have been studied in homogenates of both whole brain and of pons/medulla regions from the rat. The receptor affinities of five 4-anilinopiperidine drugs (fentanyl derivatives) and of morphine have been determined by inhibition studies, using [3H]-DAGO and [3H]-DPDPE as markers of the mu and delta opioid binding sites, respectively. The concentration of delta opioid sites in pons/medulla was found to be approximately one third that of mu sites. The concentrations of both mu and delta sites in whole brain were similar to that of mu sites in pons/medulla. The rank order of affinities of the unlabelled drugs was dissimilar at the mu and delta sites. The most potent fentanyl derivatives exhibited negligible preference for the mu or delta sites, in contrast to the least potent compound, alfentanil which showed an extremely high mu-site selectivity.     

A microiontophoretic study of the actions of mu-, delta- and kappa-opiate receptor agonists in the rat brain.

The actions of mu-, delta- and kappa-opiate receptor agonists have been compared on the activity of single neurones in the brain stem, caudate nucleus and hippocampus of the rat, using the technique of microiontophoresis. In the brain stem and caudate nucleus the predominant effect of all the opiate agonists tested was depression of neuronal activity which was antagonized by naloxone. The selectivity of naloxone as an opiate receptor antagonist was indicated by its lack of effect on gamma-aminobutyric acid (GABA)-induced responses. In the hippocampus both mu- and delta-agonists mainly caused an increase in neuronal firing rates, though some neurones were depressed. In contrast, all the kappa-agonists, including the proposed endogenous ligand for the kappa-receptor, dynorphin, caused depression of neuronal activity. All of these effects were antagonized by naloxone. There was a clear distinction in the areas within the hippocampus in which the mu- and delta-agonists produced different effects. Neurones in the pyramidal cell layer were always excited by these drugs, whereas neurones in the granule cell layer of the dentate gyrus were always depressed by the same drug.     

Distribution,localization and characteristics of neurotensin binding sites in the rat brain

Neurotensin binds reversibly to brain membranes (0.44 pmol/mg) with high affinity (K_D = 8 nM). Stereospecific membrane binding sites for ¹²⁵I-neurotensin are localized in the thalamus, hypothalamus and cerebrum of rat brain: the hypothalamus and cerebrum contained 38% and 32% of the number of binding sites, respectively, compared to thalamus. The values of ¹²⁵I-neurotensin binding in the medulla and pons, cerebellum and pituitary were only 13, 9 and 2% of that found for the thalamus. Fractionation of brain tissue homogenates on discontinuous sucrose gradients resulted in an enrichment of binding sites in the membrane fraction banding at the interphase of 0.4–0.6 m sucrose. Maximum binding occurred at pH 7.0 in 25 mm buffer in the absence of mono- and divalent cations which are inhibitory. The degree of binding of neurotensin and neurotensin analogs to membranes correlates well with their biological activity as measured by their production of hyperglycemia and hyperglucogonemia in rats.     

Actions of enkephalin, mu and partial agonist analgesics on acetylcholine turnover in rat brain

The actions of μ agonists, partial agonists and enkephalin analogues on the turnover rate of acetylcholine (TR_Ach in several brain areas have been studied. The μ agonists and enkephalin analogues all suppressed TR_Ach in the parietal cortex and hippocampus but not in the striatum or frontal cortex. The partial agonists (κ agonists, μ antagonists), however, were unique in that they did not alter hippocampal TR_ACh, suggesting that the septal hippocampal cholinergic neurons do not possess κ receptors and/or are not regulated by neurons bearing κ receptors. These data along with those of Yaksh and Rudy (Pain4: 299–359, 1978) indicate that κ receptor mediated analgesia may involve different neural substrates from those of μ agonists and supports the concept of multiple opiate receptors within the CNS.     

Characterization of spironolactone binding sites distinct from aldosterone receptors in rat kidney homogenates

The binding of [3H]spironolactone to kidney homogenates from adrenalectomized rats was studied by dextran-charcoal absorption methods. [3H]Spironolactone binds with high affinity and low capacity (KD = 12.9 +/- 0.6 nM; Bmax = 93.4 +/- 3.8 fmoles/mg protein) at low temperatures (0 degrees-2 degrees). Its hormone specificity, as measured by relative binding affinity (RBA) is spironolactone greater than prorenone greater than methyltrienolone greater than testosterone greater than progesterone greater than aldosterone greater than dexamethasone. In the same tissue preparation, specific spironolactone binding sites and classical mineralocorticoid receptor sites labelled with [3H]aldosterone differ in their thermal stability, binding parameters and hormone specificities, whereas their tissue distributions are similar. In conclusion, [3H]spironolactone binds specifically to kidney homogenates from adrenalectomized rats and these binding sites, apparently, are different from the classical mineralocorticoid receptors. The theoretical and practical aspects of this finding are discussed.     

Age-related changes in reactive oxygen species production in rat brain homogenates

The generation of reactive oxygen species (ROS) and resultant oxidative stress have been implicated in the mechanism of brain dysfunction due to age-related neurodegenerative diseases or exposure to environmental chemicals. We have investigated intrinsic age-related differences in the ability of the various brain regions to generate ROS in the absence and presence of Fe(2)+. ROS production in crude brain homogenates from adult rats was linear with respect to time and tissue concentration, and was stimulated to a greater extent by Fe(2)+ than was TBARS production. ROS production was then determined in homogenates from cerebral cortex, striatum, hippocampus, and cerebellum of 7-day-old, 14-day-old, 21-day-old, adult (3-6-month old), and aged (24-month-old) rats using the fluorescent probe 2',7'-dichlorodihydrofluorescin (DCFH). Basal levels of ROS production were similar in 7-, 14-, and 21-day olds, increased in adults, and highest in aged rats, and did not differ between brain regions. ROS production was stimulated by Fe(2)+ (0. 3-30 microM) in a concentration-dependent manner in all brain regions. However, the stimulation of ROS production by Fe(2)+ varied with age. ROS production was greater in 14- and 21-day-old rats compared with adult and aged animals. ROS production in 7-day-old rats was decreased at low Fe(2)+ concentrations and increased at high Fe(2)+ concentrations compared to adult and aged rats. These data show that brain homogenates from neonatal rats respond differently to Fe(2)+, and suggest that developing animals may be more sensitive to oxidative stress in the brain after exposure to toxicants. Published by Elsevier Science Inc.