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.     

No Orcadian Rhythms of Serotoninergic,Alpha-, Beta-Adrenergic and Imipramine Binding Sites in Rat Brain Regions

B_max values of the specific binding of [³H]-WB 4101, [³H]-dihydroalprenolol, [³H]-spiperone and [³H]-imipramine to various rat brain regions were determined at 4 hr intervals over 24 hr under circadian conditions. No significant circadian rhythm of binding sites number was found for any receptor investigated in cerebral cortex, hypothalamus or brain stem. Some methodological issues are discussed.     

A quantitative autoradiographic study of muscarinic cholinergic receptor subtypes in the brains of pyrithiamine-treated rats

Previous studies describe decreased acetylcholine synthesis in brain as well as neurobehavioural evidence for a central muscarinic cholinergic deficit in pyrithiamine-induced thiamine-deficient rats. In order to further evaluate this possibility, quantitative autoradiographic procedures using [³H]quinuclidinyl benzilate (for total muscarinic binding sites), [³H]pirenzepine (for muscarinic M₁ sites) and [³H]AF-DX 384 (for muscarinic M₂ sites) were performed at early (presymptomatic) and late (symptomatic) stages of thiamine deficiency induced in rats by administration of the central thiamine antagonist, pyrithiamine. No significant alterations in densities of M₁, M₂ or total muscarinic binding sites were observed in any brain structure evaluated at either early or late stages of thiamine deficiency. These findings do not support a major role for modifications of muscarinic cholinergic function in the pathogenesis of the neurological symptoms of thiamine deficiency.     

The role of light and polar auxin transport in root regeneration from hypocotyls of tomato seedling cuttings

A relationship between light conditions, auxin transport and adventitious root formation by hypocotyls of tomato seedling cuttings was demonstrated. Effective rooting of tomato seedling cuttings was observed under continuous white light (WL) irradiation. However, root formation was reduced in darkness or under red (RL) or blue light (BL). At least 3/4-day-long irradiation treatment with (WL) was necessary to increase the number of roots formed in comparison with control cuttings grown in darkness. Light was most effective if applied during the first half of the 13-day-long rooting period. The role of photoreceptor-dependent light perception in the light-regulation of rooting was tested using tomato photomorphogenic mutants: aurea (au) and high pigment (hp). When exposed to WL both mutants generated fewer roots then their isogenic wild type (WT). In darkness or under BL and RL less roots were formed on all plants and no difference was observed between mutants and WT plants. TIBA (2,3,5-triiodobenzoic acid) inhibited rooting in a dose-dependent manner both in darkness and under WL. However, although rooting was suppressed by 0.75 M TIBA in the dark, 8 M TIBA was necessary to block root formation in continuous WL. Inhibition of rooting by TIBA was most efficient when applied at the initial period of rooting, a 1-day-long treatment with TIBA being sufficient to suppress rooting if given during the first 2 days of culture. Later treatment had much less effect on the root formation.     

The role of the shoot apex in controlling rhizogenesis in vitro

This paper reports that rhizogenesis in woody plant species in vitro was mediated through the basipetal transport of auxin from the shoot apex. This can directly induce roots in easy-to-root species such as Betula pendula, but was dependent upon an interaction with exogenous auxin in more difficult-to-root species such as Daphne cneorum, and to a lesser extent in Quercus robur. Shoot apex removal reduced rhizogenesis in Quercus, and inhibited it in Daphne, even in the presence of exogenous auxin, whereas rooting in Betula was unaffected. That basipetally transported auxin modulates rhizogenesis was demonstrated by the inhibition of root induction in Betula shoots by the auxin transport inhibitor 2,3,5-triiodobenzoic acid (TIBA), and by the substitution of indole-3-acetic acid (IAA) for a bud in Betula internodal sections.Abbreviations IAA indole-3-acetic acid - IBA indole-3-butyric acid - TIBA 2,3,5-triiodobenzoic acid - MS Murashige and Skoog medium - WPM woody plant medium     

Guanidinoethane sulphonic acid interferes with the binding of [3H]dizocilpine and neurotoxic action of AF64A

Summary The binding of [³H]dizocilpine [[³H]MK-801] to the N-methylD-aspartate receptor complex of well washed rat cortical membranes was reduced by guanidinoethane sulphonic acid (GES). Micromolar concentrations of GES, which were high relative to those of dizocilpine, inhibited in a concentration dependent manner the binding of [3H]dizocilpine. The inhibitory effect of GES on [3H]dizocilpine binding was slightly influenced by concentration of glutamate. The glutamate antagonist DL-2-amino-5phosphonovaleric acid blocked the effect GES at concentrations higher relative to GES. The inhibitory effect of GES was still present during spermidine-induced stimulation of [³H]dizocilpine binding. GES reduced the binding of the glycine antagonist [³H]5,7-dichlorokynurenic acid with an IC₅₀ of 530 M.. Intraperitoneal injections of GES (0.2mmol/kg) protected against both amnesia and decrease in the choline acetyltransferase activity following local injections of the neurotoxin AF64A into the nucleus basalis magnocellularis. GES given to lesioned rats during the training period in the spatial learning task gradually improved the performance to the level of sham operated rats. It is concluded that GES interferes with the transmitter and the dizocilpine binding sites of the NMDA receptor complex and has the capacity to protect against neurotoxic brain damage.     

Affinities of muscarinic drugs for [3H]N-methylscopolamine (NMS) and [3H]oxotremorine (OXO) binding to a mixture of M1−M4 muscarinic receptors: Use of NMS/OXO-M ratios to group compounds into potential agonist,partial agonist,and antagonist classes

The relative affinities of various muscarinic drugs in the antagonist ([³H]N-methyl scopolamine ([³H]NMS)) and agonist ([³H]Oxotremorine-m ([³H]OXO-M)) binding assays using a mixture of tissues containing M₁–M₄ receptor subtypes have been determined. [³H]NMS bound with high affinity (K_d=25±5.9 pM; n=3) and to a high density (B_max=11.8±0.025 nmol/g wet weight) of muscarinic receptors. [³H]OXO-M appeared to bind to two binding sites with differing affinities (K_d1=2.5±0.1 nM; K_d2=9.0±4.9 M; n=4) and to a different population of binding sites (B_max1=5.0±0.26 nmol/g wet weight; B_max2=130±60 nmol/g wet weight). Well known antagonists exhibited high affinity for [³H]NMS binding but a lower affinity for [³H]OXO-M binding. The opposite was true for acetylcholine and other known agonists. However, pilocarpine and McN-A-343 had similar affinities for sites labeled by both radioligands. Using the ratios of antagonist-to-agonist binding affinities, it was possible to group compounds into apparently distinct full agonist (ratios of 180–665; e.g. carbachol, muscarine, OXO-M, OXO-S and arecoline), partial agonist (ratios of 14–132; e.g. McN-A-343, pilocarpine, aceclidine, bethanechol, OXA-22 and acetylcholine) and antagonist (ratios of 0.22–1.9; e.g. atropine, NMS, pirenzepine, methoctramine, 4-DAMP and p-fluorohexahydrosialo-difenidol) classes. These data suggest that the NMS/OXO-M affinity ratios using a mixture of M₁–M₄ muscarinic receptors may be a useful way to screen and group a large number of compounds into apparent agonist, partial agonist, and antagonist classes of cholinergic agents.     

Polymyxin B Enhances Formation of a New Pigment,a Peptide–Ferropyrimine Complex,in Serratia marcescens

We previously isolated a Serratia marcescens O5: HI Z-54 strain which produces a new reddish-violet pigment, a peptide- ferropyrimine complex. This study showed that polymyxin B enhances the formation of the pigment about threefold. This occurs because polymyxin B in the medium causes the formation of an iron-polymyxin B complex which imposes a low iron stress on the bacteria and, in turn, enhances pigment production. This shows that polymyxin B is both a membrane-disrupting and ionophoric antibiotic.     

Autoradiographic Analysis of GABAA Receptors in μ-Opioid Receptor Knockout Mice

Anatomical evidence indicates that γ-aminobutyric acid (GABA)-ergic and opioidergic systems are closely linked and act on the same neurons. However, the regulatory mechanisms between GABAergic and opioidergic system have not been well characterized. In the present study, we investigated whether there are changes in GABA_A receptors in mice lacking μ-opioid receptor gene. The GABA_A receptor binding was carried out by autoradiography using [³H]-muscimol (GABA_A), [³H]-flunitrazepam (FNZ, native type 1 benzodiazepine) and [³⁵S]-t-butylbicyclophosphorothionate (TBPS, binding to GABA_A-gated chloride channels) in brain slices of wild type and μ-opioid receptor knockout mice. The binding of [³H]-FNZ in μ-opioid receptor knockout mice was significantly higher than that of the wild type controls in most of the cortex and hippocampal CA1 and CA2 formations. μ-Opioid receptor knockout mice show significantly lower binding of [³⁵S]-TBPS than that of the wild type mice in few of the cortical areas including ectorhinal cortex layers I, III, and V, but not in the hippocampus. There was no significant difference in binding of [³H]-muscimol between μ-opioid receptor knockout and wild type mice in the cortex and hippocampus. These data indicate that there are specific regional changes in GABA_A receptor binding sites in μ-opioid receptor knockout mice. These data also suggest that there are compensatory up-regulation of benzodiazepine binding site of GABA_A receptors in the cortex and hippocampus and down-regulation of GABA-gated chloride channel binding site of GABA_A receptors in the cortex of the μ-opioid receptor knockout mice.     

Regional effects of neurotensin on the electrically stimulated release of [3H]dopamine and [14C]acetylcholine in the rat nucleus accumbens

This study has shown that neurotensin (NT) increases the electrically stimulated release of [³H]DA to a similar extent in all but the extreme caudolateral area of the rat nucleus accumbens and appears to modulate DA release equally in the medial and lateral zones of this brain area. The simultaneous release of ACh was not significantly affected by NT.     

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.     

The role of endogenous auxin in root initiation

This paper is the second part of a review which considers evidence for the involvement of auxin in root initiation. Part II examines the research being carried out with transformed plant tissues. Agrobacterium rhizogenes causes abundant root initiation at the site of inoculation. Ri plasmid T-DNA contains several genes which encode enzymes involved in the biosynthesis and metabolism of indole-3-acetic acid. Transfer of various fragments of the Ri plasmid has also been reported to confer increased sensitivity to auxin upon plant cells. Controlled expression of these genes in the plant genome potentially offer an insight for developmental plant physiologists into the role of plant growth substances in the process of root initiation. The importance of absolute levels of IAA in the stimulation of root initiation is discussed.     

Solubilization and characterization of [3H]Imipramine and [3H]Paroxetine binding sites from calf striatum

The serotonin (5-HT) transporter from calf striatum cerebral membranes was solubilized with digitonin and characterized by gel exclusion chromatography. [³H]Imipramine and [³H]paroxetine were utilized as markers for labeling it.³H-imipramine labels a high- and a low-affinity site on striaturn membranes, whereas it binds to a single high-affinity site on the solubilized fraction. [³H]Paroxetine binds with the same affinity to a single site on both membranes and solubilized preparations. After gel exclusion chromatography of the solubilizate both [³H]imipramine and [³H]paroxetine bind on an identical fraction of 205 kDa molecular weight, with a similar maximum number of binding sites (Bmax). Our results suggest that both³H-imipramine and [³H]paroxetine bind to a common site on the 5-HT transporter.     

The Fate of [3H]Ecdysone in Three Species of Annelids

Summary

The metabolism of [³H]ecdysone was examined in 3 species of annelids: the bloodworm, Tubifex vulgaris (a freshwater oligochaete), the earthworm, Lumbricus terrestris (a terrestrial oligochaete) and the ragworm, Nereis divtrsicolor (a marine polychaete). One of these species, N. diversicolor, metabolised injected [³H]ecdysone into compounds which co-chromatographed on both reversed-phase and adsorption HPLC with authentic 20-hydroxyecdysone, 26-hydroxyecdysone and 20,26-dihydroxyecdysone, thus demonstrating the occurrence of 20-hydroxylation and 26-hydroxylation capability in the Annelida. Furthermore, [³H]ecdysonoic acid was also formed and excreted by N. diversicolor, suggesting that 26-oic acid formation is involved in ecdysteroid inactivation in this species. Other, as yet unidentified, radioactive metabolites were also excreted by N. diversicolor. Several metabolites of [³H]ecdysone were also detected in the other 2 species examined, T. vulgaris and L. terrestris.     

Serotonergic activity of HP 184: Does spontaneous release have a role?

Examination of HP 184, [N-n-propyl)-N-(3-fluoro-4-pyridinyl)-1H-3-methylindodel-1-amine hydrochloride], in a variety of tests for serotonergic activity revealed some unique properties of this compound. We report here that 100 μM HP 184 enhanced spontaneous release of [³H]serotonin (5-HT) from rat hippocampal slices. This release was independent of the uptake carrier. In vivo assays confirmed that HP 184 (20 mg/kg, i.p.) lacked significant interactions at the norepinephrine (NE) or 5-HT uptake carrier itself. Notably, HP 184 (15 mg/kg, i.p.) reduced drinking behavior in schedule-induced polydipsic (SIP) rats. We previously reported that some selective 5-HT reuptake inhibitors decrease SIP 30–40% after a 14–21 day treatment. In the current study, HP 184 decreased SIP beginning with the first treatment, and this reduction (30%) was maintained for 28 days. We further investigated HP 184 and serotonin metabolite levels. One hour after i.p. administration of 30 mg/kg HP 184, the ratio of whole brain 5-hydroxyindolacetic acid (5-HIAA) to 5-HT was increased, suggesting serotonergic activation. Under these conditions, the brain: plasma ratio of HP 184 was approximately 2∶1, with brain concentrations of 1.6 μg/gram. We speculate that the spontaneous release effects of HP 184 may be responsible for the behavioral effects observed.     

Stem cells in microturbellarians

Summary A combination of microscopical, immunocytochemical, and autoradiographic techniques were employed to study stem cells and their fates during asexual reproduction and regeneration in two microturbellarians,Microstomum lineare (Macrostomida) andStenostomum leucops (Catenulida). Special attention was paid to the development of the immunoreactivity (IR) to FMRF/RF-amide and 5-HT in differentiating nerve cells.Asexual reproduction inM. lineare andS. leucops occurs by paratomy, i.e., fragmentation after completed differentiation of the new organs. Regeneration, on the other hand, involves a combination of morphallactic and epimorphic processes without the formation of a regeneration blastema. The only cells incorporating tritiated thymidine ([³H]T) were the mesenchymal and gastrodermal neoblasts, which proliferate continuously replenishing the population of stem cells available for growth, asexual reproduction and regeneration. These proliferative cells occurred in two ultrastructurally different forms, differing from each other only by the presence or absence of ciliar basal bodies in the cytoplasm. Few differentiated cells were labeled in the head piece after completed regeneration. A greater amount of labeled differentiated cells were, however, observed postpharyngeally in the first zooid as well as in zooids having developed during the same time (i.e., 20–45 h after the treatment with [³H]T). Furthermore, many labeled cells were still undifferentiated at that time or just in the beginning of the differentiation process. It can therefore be concluded that neoblasts function both as reserve cells and as functional stem cells for all differentiated cell types in these worms. IR to FMRF/RF-amide neuropeptides was not observed in nerve cells differentiating from neoblasts until the occurrence of dense-core vesicles in their cytoplasm. Due to methodological difficulties only weak or no IR to 5-HT could be traced in the nervous system of the asexual and regenerating worms.Abbreviations ICC Immunocytochemical - IR immunoreactivity - [³H]T tritiated thymidine     

NAD-299 ANTAGONISES 5-HT-STIMULATED AND SPIPERONE-INHIBITED [35S]GTPγS BINDING IN CLONED 5-HT1A RECEPTORS

ABSTRACT

In Chinese Hamster Ovary (CHO) cells expressing cloned human 5-hydroxy-tryptamine_1A (5-HT_1A) receptors, (R)-3-N,N-dicyclobutylamino-8-fluoro-[6-³H]-3,4-dihydro-2H-1-benzopyran-5-carboxamide ([³H]NAD-299) exhibited high affinity (K_d = 0.16?nM) and labeled 34% more receptors than 8-hydroxy-2-([2,3-³H]di-n-propylamino)tetralin ([³H]8-OH-DPAT). NAD-299 behaved as a silent antagonist in [³⁵S]GTPγS binding similar to N-tert-butyl-3-(4-(2-methoxyphenyl)-piperazin-1-yl)-2-phenylpropanamide (WAY-100635) and (S)-5-fluoro-8-hydroxy-2-(di-n-propylamino)tetralin ((S)UH-301). 5-HT and 5-carboxamidotryptamine (5-CT) stimulated [³⁵S]GTPγS binding 2.5-fold while spiperone and methiothepin inhibited [³⁵S]GTPγS binding 1.4-fold. Furthermore, NAD-299 antagonised both the 5-HT stimulated and the spiperone inhibited [³⁵S]GTPγS binding to basal levels. The K_iL/K_iH ratios for spiperone (0.66), methiothepin (0.39), WAY-100635 (0.32), (S)UH-301 (0.94), NAD-299 (1.29), NAN-190 (1.23), (S)pindolol (5.85), ipsapirone (13.1), buspirone (24.6), (±)8-OH-DPAT (47.3), flesinoxan (55.8), 5-HT (200) and 5-CT (389) correlated highly significantly with the intrinsic activity obtained with [³⁵S] GTPγS (r = 0.97).     

NMDA receptor function is enhanced in the hippocampus of aged rats

The density and functional activity of theN-methyl-D-aspartate (NMDA)-sensitive glutamate receptor was examined in various brain areas of 3-, 18- and 24-month-old rats. The total numbers of binding sites for the NMDA receptor antagonists [³H]CGP 39653 and [³H]MK 801 binding sites were decreased in the hippocampus, cerebral cortex and striatum of 18- and 24-month-old rats, relative to 3-month-old animals. In the hippocampus of 18-month-old rats, the reduced number of NMDA receptors was associated with an increased sensitivity of [³H]MK 801 binding to the stimulatory action of glycine and glutamate. Thus, 10 M glycine and 10 M glutamate increased [³H]MK 801 binding in the hippocampus of 18-month-old rats by 75 and 160%, respectively; in 3-month-old animals, the same concentration of these amino acids increased binding by 37 and 95%, respectively. The sensitivity of [³H]MK 801 binding to glycine and glutamate was not increased in the cerebral cortex and striatum of aged rats. Moreover, an increased efficacy of glycine and glutamate in stimulating the binding of [³H]MK 801 in the hippocampus was no longer apparent in the 24-month-old rats. The increased sensitivity of [³H]MK 801 binding to glycine and glutamate in the hippocampus of 18-month-old rats may reflect an increase in NMDA receptor activity to compensate for the decrease in receptor number.     

In vivo Effect of Antidepressants on [<Superscript>3</Superscript>H]paroxetine Binding to Serotonin Transporters in Rat Brain

Amine transporters are major target for development of various pharmacological agents to treat behavioral disorders. Serotonin transporters (SERT) have been implicated in the etiology of depression and drugs acting on SERT can be effective in treating depression. The aim of the present study was to study the in vivo effect of various antidepressants on [³H]paroxetine binding to SERT in regions of rat brain. Rats were treated with tricyclic antidepressant (TCAs) such as amitriptyline (AMI), serotonin/norepinephrine reuptake inhibitor (SNRIs) such as clomipramine (CMI), and selective serotonin reuptake inhibitors (SSRIs) such as fluoxetine (FLX) and citalopram (CIT) (10 mg/kg body wt.) for 30 days. Density of SERT was measured in cortex and hippocampus using [³H]paroxetine (0.03–1.0 nM) in presence and absence of 10 μM fluoxetine as displacer. It was observed that the density of cortical SERT was significantly decreased with CMI (68%, P < 0.0001), FLX (67%, P < 0.0001), CIT (54%, P < 0.0001), and AMI (52%, P < 0.0001) treatment, when compared to the density of 120.7 ± 4.0 fmol/mg protein in control rats, without altering the affinity (Kd) of [³H]paroxetine to the transporters. The density of SERT in hippocampus was also significantly decreased with FLX (65%, P < 0.0001), CMI (54%, P < 0.0001), CIT (52%, P < 0.0001) and AMI (46%, P < 0.0001) treatment, when compared to the density of 74.0 ± 2.6 fmol/mg protein in control rats, without altering the affinity of [³H]paroxetine to the transporters. Displacement study showed high affinity for CMI > CIT > FLX. The results suggest that chronic antidepressant treatment significantly down-regulates both cortical and hippocampal SERT in rat brain and SSRIs have high affinity for SERT than TCAs.     

Effects of osmotic stress on polar auxin transport in Avena mesocotyl sections

Segments of mesocotyls of Avena sativa L. transported [1-¹⁴C]indol-3yl-acetic acid (IAA) with strictly basipetal polarity. Treatment of the segments with solutions of sorbitol caused a striking increase in basipetal auxin transport, which was greatest at concentrations around 0.5 M. Similar effects were observed with mannitol or quebrachitol as osmotica, but with glucose or sucrose the increases were smaller. Polar transport was still detectable in segments treated with 1.2 M sorbitol. The effects of osmotic stress on the polar transport of auxin were reversible, but treatment with sorbital solutions more concentrated than 0.5 M reduced the subsequent ability of mesocotyl segments to grow in response to IAA. The increased transport of auxin in the osmotically stressed segments could not be explained in terms of an increased uptake from donor blocks. The velocity of transport declined with higher concentrations of osmoticum. The reasons for the enhancement of auxin transport by osmotic stress are not known.