[Chromatographic determination of radiochemical purity - replacement of ITLC SG]. / Chromatographische Bestimmung der radiochemischen Reinheit von Radiopharmaka - Alternativen zu ITLC SG.

UNLABELLED: Thin layer chromatography is well established for quality control of radiopharmaceuticals. A convenient and widely used stationary phase are ITLC SG strips. However, the Pall Corporation stopped manufacturing of the silica gel impregnated glass fibre strips (ITLC SG). Material, Methode: As a replacement we tested silicic acid impregnated glass fibre strips from Varian (ITLC SA) and sufficient mobile phases. RESULTS: The chromatography with these strips takes two to three times longer than with ITLC SG, but it is in an acceptable range. Only three mobile phases are necessary to test most of the common in-house made radiopharmaceuticals. CONCLUSION: The proposed method is suitable for routinely measuring the radiochemical purity of radiophamaceuticals.

Anticonvulsant activity of Mimosa pudica decoction.

The decoction of Mimosa pudica leaves given intraperitoneally at dose of 1000-4000 mg/kg protected mice against pentylentetrazol and strychnine-induced seizures. M. pudica had no effect against picrotoxin-induced seizures It also antagonized N-methyl-D-aspartate- induced turning behavior. These properties could explain its use in African traditional medicine.

Effects of Cyperus articulatus compared to effects of anticonvulsant compounds on the cortical wedge.

Cyperus articulatus L. (Cyperaceae) is a plant commonly used in traditional medicine in Africa and Latin America to treat many diseases. The water extract from rhizomes of Cyperus articulatus concentration-dependently reduced spontaneous epileptiform discharges and NMDA-induced depolarisations in the rat cortical wedge preparation at concentrations at which AMPA-induced depolarisations are not affected. The two antiepileptic compounds, valproate and ethosuximide, possessed effect neither on epileptiform discharges nor on AMPA- and NMDA-induced depolarisations. Phenobarbital, pentobarbital and phenythoin inhibited both AMPA- and NMDA-induced depolarisations and spontaneous epileptiform discharges. The effects of Cyperus articulatus were very close to the effect of D-CPPene. D-CPPene also inhibited spontaneous epileptiform discharges and antagonised NMDA- but not AMPA-induced depolarisations. The extract of Cyperus articulatus could contain components acting as NMDA antagonists.

Anticonvulsant properties of the methanolic extract of Cyperus articulatus (Cyperaceae).

The methanolic extract of rhizomes of Cyperus articulatus, a plant used in traditional medicine in Africa and Latin America for many diseases, possesses anticonvulsant activity in mice. This extract protected mice against maximal electroshock (MES)- and pentylenetetrazol (PTZ)-induced seizures. It also delayed the onset of seizures induced by isonicotinic acid hydrazide and strongly antagonized N-methyl-D-aspartate-induced turning behavior. The ED(50) for protection against seizures was 306 (154-541) mg/kg intraperitoneally (i.p.) for the PTZ test and 1005 (797-1200) mg/kg i.p. for the MES test. The ED(50) of methanolic extract against N-methyl-D-aspartate-induced turning behavior was 875 (623-1123) mg/kg i.p. C. articulatus L. methanolic extract protected 54% of mice from seizures induced by strychnine at the dose of 1000 mg/kg i.p. but had no or a moderate effect only against picrotoxin- or bicuculline-induced seizures. With these effects, the rhizome of C. articulatus L. possesses anticonvulsant properties in animals that might explain its use as a traditional medicine for epilepsy in Africa.

Progress and potential for gene-based medicines.

During the past decade researchers have explored the potential of gene-based medicines to extend current treatments employing chemical entities and proteins. However, progress has been slower than was originally predicted due to our limited knowledge of the genetic components of major diseases, the complexity of developing active biological agents as therapies, and the stringent and time-consuming tests necessary to ensure safety prior to introduction of these novel modalities in the clinic. In spite of the present technology challenges and clinical setbacks in gene therapy it is anticipated that gene-based medicines will find their niche in disease prevention and management strategies in the coming decade, extending the repertoire of medicines available to satisfy key unmet medical needs. Additionally, progress in xenotransplantation research is creating the opportunity to use gene-modified porcine organs for human transplantation. This innovative approach aims to address the current insufficiency of human donor organs for clinical transplantation.

Impact of clinical pathways for total hip replacement: a community-based analysis.

The implementation of clinical pathways for total hip replacement was carried out by five hospitals in the metropolitan area of Syracuse, New York. This process occurred under the leadership of clinical nurse specialists and nurse managers. It was supported by preadmission patient education programs and active physician involvement. The participating hospitals shared utilization quality assurance data and benchmarked with respect to the experience of Sacramento, California, and each others' progress. The effort produced substantial reductions in hospital stays without adverse impacts on quality of care.

Extracts from rhizomes of Cyperus articulatus (Cyperaceae) displace [3H]CGP39653 and [3H]glycine binding from cortical membranes and selectively inhibit NMDA receptor-mediated neurotransmission.

The marshland plant Cyperus articulatus (Cyperaceae) is commonly used in traditional medicine in Africa and Latin America to treat a wide variety of human diseases ranging from headache to epilepsy. We tested the hypothesis that the purported anti-epileptic effect of this plant might be due to a functional inhibition of excitatory amino acid receptors. One or several component(s) contained in the extracts inhibited the binding of [3H]CGP39653 to the NMDA recognition site and of [3H]glycine to the strychnine-insensitive glycine site of the NMDA receptor complex from rat neocortex. Water extracts from rhizomes of Cyperus articulatus dose-dependently reduced spontaneous epileptiform discharges and NMDA-induced depolarizations in the rat cortical wedge preparation at concentrations at which AMPA-induced depolarizations were not affected. We conclude that the purported beneficial effects of Cyperus articulatus might at least partially be due to inhibition of NMDA-mediated neurotransmission.

Thalamocortical and corticocortical excitatory postsynaptic potentials mediated by excitatory amino acid receptors in the cat motor cortex in vivo.

Intracellular recordings were made from neurons in the motor cortex of an anaesthetized cat, together with iontophoretic application of excitatory amino acid receptor agonists and antagonists, in order to evaluate the role of such receptors in excitatory postsynaptic potentials evoked from stimulation of afferent and recurrent pathways in vivo. Excitatory postsynaptic potentials which were evoked by stimulation of the ventrolateral thalamus were found to be largely insensitive to antagonism by N-methyl-D-aspartate receptor antagonists, although they were susceptible to blockade by the non-N-methyl-D-aspartate receptor antagonist, 6-cyano-7-nitroquinoxaline-2,3-dione. Increasing the ventrolateral thalamus stimulation frequency from 0.5 or 1 to 5 Hz caused an increase of evoked excitatory postsynaptic potential amplitudes and number of action potentials. These augmented excitatory postsynaptic potentials remained insensitive to application of N-methyl-D-aspartate antagonists. In contrast, recurrent excitatory postsynaptic potentials evoked by stimulation of the pyramidal tract were found to be sensitive to N-methyl-D-aspartate receptor antagonists and/or non-N-methyl-D-aspartate receptor antagonists in some neurons. These results demonstrate the involvement of both N-methyl-D-aspartate- and non-N-methyl-D-aspartate receptors in synaptic responses of cat motor cortex neurons in vivo, and that the synaptic pharmacology of the thalamic input may differ from that of the local recurrent pathways.

N-methyl-D-aspartate induces regular firing patterns in the cat lateral habenula in vivo.

The present study was undertaken to elucidate the action of excitatory amino acids in the dorsal diencephalic pathway. Single neurons in the lateral habenula of halothane-anesthetized cats were recorded extracellularly, and excitatory amino acid receptor agonists and antagonists were applied by iontophoresis. Most neurons in the lateral habenula were spontaneously active. This spontaneous firing could be inhibited by kynurenic acid, a broad spectrum antagonist of excitatory amino acid receptors, but not by the selective N-methyl-D-aspartate receptor antagonist 2-amino-7-phosphono-heptanoic acid. Iontophoretic application of alpha-amino-3-hydroxy-5-methyl-5-isoxazolepropionate, quisqualate and kainate mostly elicited a non-burst, regular firing pattern which was sensitive to kynurenic acid. Surprisingly, 116 (96%) out of 121 neurons in the lateral habenula responded to iontophoretic application of N-methyl-D-aspartate with a regular non-burst firing pattern, in contrast to previously published observations from other brain regions where N-methyl-D-aspartate predominantly elicited phasic firing patterns. When cells were recorded with electrode assemblies where one iontophoretic barrel contained MgCl2 or MgSO4, only 10 (43%) out of 23 cells responded with regular firing upon application of N-methyl-D-aspartate, while 13 (57%) now displayed a phasic firing pattern. In these cells iontophoretically applied alpha-amino-3-hydroxy-5-methyl-5-isoxazolepropionate or quisqualate still evoked only regular firing. In a few cases, an initially regular N-methyl-D-aspartate-induced firing pattern could be changed to a phasic pattern following active ejection of Mg2+ ions.(ABSTRACT TRUNCATED AT 250 WORDS)

Synaptic physiology of excitatory amino acids.

This paper gives a short overview of the localization of the main excitatory amino acid pathways in the mammalian brain and lists a few of the most important agonists and antagonists of the 5 excitatory acid receptors described to-date: the N-methyl-D-aspartate-(NMDA), a-amino-3-hydroxy-5-methylisoxazole-4-propionic acid-(AMPA), kainic acid-, metabotropic- and presynaptic receptors. The main part summarizes work of this laboratory on excitatory amino acids involved in the synaptic physiology of the cortico-caudate, thalamo-cortical and a local cortical pathway studied in the cat brain in vivo. It concludes by presenting two hypotheses which describe how AMPA- and NMDA-receptors could interact in the brain to modulate synaptic function, the AMPA receptors being responsible for fast synaptic transmission while activation of NMDA-receptors enhance their gain either within a single synapse or from independent synapses or extrasynaptic sites.

Differential effects of (D)- and (L)-homocysteic acid on the membrane potential of cat caudate neurons in situ.

The enantiomers of homocysteic acid have been applied by microiontophoresis to neurons of the cat caudate nucleus in situ. The (L)-enantiomer elicited a bursty firing pattern similar to the one caused by N-methyl-D-aspartate, but differing from the N-methyl-D-aspartate pattern inasmuch as (L)-homocysteate induced depolarization shifts were shorter and had a smaller amplitude. (L)-Homocysteate induced excitations could be strongly inhibited by the selective N-methyl-D-aspartate antagonist 2-amino-7-phosphonoheptanoic acid but they were less sensitive to this antagonist than N-methyl-D-aspartate itself. (D)-Homocysteate elicited a more regular firing pattern similar to the one caused by non-N-methyl-D-aspartate excitatory amino acids such as quisqualate. These excitations were only rarely inhibited by 2-amino-7-phosphonoheptanoic acid. Our results suggest that (L)-homocysteate, a transmitter candidate at central mammalian synapses, is a mixed excitatory amino acid agonist with a strong preference for N-methyl-D-aspartate receptors in the cat caudate nucleus, while (D)-homocysteate has a predominant action at non-N-methyl-D-aspartate excitatory amino acid receptors.

6,7-Dichloro-3-hydroxy-2-quinoxalinecarboxylic acid is a relatively potent antagonist at NMDA and kainate receptors.

The 6,7-dichloro derivative of 3-hydroxy-2-quinoxalinecarboxylic acid (diCl-HQC) is a relatively potent antagonist at the two excitatory amino acid receptor subtypes activated by N-methyl-D-aspartate (NMDA) and kainic acid. It antagonizes NMDA-induced excitation in the frog spinal cord (pA2 5.8), i.e. with a potency similar to D-2-amino-7-phosphonoheptanoate (D-AP-7). It also antagonizes NMDA-induced sodium efflux from rat brain slices (pA2 5.6). The compound inhibits kainic acid-induced sodium efflux from rat brain slices with a pA2 of 5.4 and it inhibits [3H]kainic acid binding to rat brain membranes with a pKi of 5.4. DiCl-HQC is only weakly active at the quisqualate receptor. This spectrum of activities may make this compound a useful tool to investigate the pharmacology of excitatory amino acid receptors.

Excitatory amino acids in synaptic excitation of rat striatal neurones in vitro.

1. Intracellular recordings were made from rat striatal neurones in vitro. The cells had resting membrane potentials greater than -60 mV and action potentials greater than 70 mV with spike overshoot of 10-30 mV. 2. In the presence of bicuculline intrastriatal stimulation evoked an excitatory postsynaptic potential (EPSP). The relationship between EPSP amplitude and membrane potential was not linear. The EPSP decreased in amplitude and duration for values of membrane potential more negative than -80 mV and increased in amplitude and duration for values of membrane potential more positive than -50 mV. 3. The mean reversal potential for the EPSP recorded with electrodes filled with potassium methyl-sulphate was -9.2 +/- 1.7 mV (mean +/- S.E.M.) in presence of bicuculline (30 microM). A similar reversal potential was obtained with CsCl-filled electrodes. 4. The endogenous broad-spectrum excitatory amino acid antagonist, kynurenic acid (100-500 microM), reduced the EPSP in a dose-dependent way, maximally by 80% at 500 microM, but a residual depolarization remained even at high antagonist concentrations. This effect was associated sometimes with a membrane depolarization and an increase in input resistance. 5. In normal artificial cerebro-spinal fluid solution and at resting membrane potential the specific N-methyl-D-aspartate (NMDA) antagonist, (D,L)-2-amino-7-phosphonoheptanoic acid (([D,L)-AP7), did not affect the EPSP amplitude. However, this antagonist partially reduced the EPSP amplitude when the membrane was depolarized beyond -50 mV by intracellular current injection. 6. The nicotinic cholinergic antagonist mecamylamine (10 microM) caused a partial (24 +/- 3%) reduction of EPSP amplitude at resting potential in normal medium. However, in the cells where a reduction of EPSP amplitude was observed it was always accompanied by membrane depolarization (7.1 +/- 2.1 mV). (+)-Tubocurarine and hexamethonium were without effect at 10 microM. 7. When Mg2+ was removed from the bathing solution, the EPSP increased in amplitude (89 +/- 9.5%) and duration. In Mg2+-free medium at resting membrane potential (D,L)-AP7 (30 microM) partially reduced EPSP amplitudes (59 +/- 2.5%). 8. It is proposed that a major component of the EPSP evoked by intrastriatal stimulation is mediated by excitatory amino acids. At resting membrane potential and in normal medium only non-NMDA receptors seem to contribute to the synaptic depolarization, but at depolarized potentials and in Mg2+-free medium an NMDA receptor-mediated component of the EPSP can be demonstrated.

Release of neuroactive substances: homocysteic acid as an endogenous agonist of the NMDA receptor.

Sulfur containing amino acids such as homocysteic acid (HCA), cysteinsulfinic acid, homocysteinsulfinic acid are released by depolarization of slices from various rat brain regions in a Ca++-dependent manner. L-HCA excites caudate neurons through their N-methyl-D-aspartic acid (NMDA) receptor and potentiates their cortically evoked excitatory postsynaptic potentials. 35S-methionine can label the releasable pool of HCA, and thus appears as a precursor of HCA. Thus HCA is a transmitter candidate which acts predominantly on the NMDA receptor.

Effects of L-cysteine-sulphinate and L-aspartate, mixed excitatory amino acid agonists, on the membrane potential of cat caudate neurons.

Responses evoked by L-cysteine-sulphinate (L-CSA) and L-aspartate (L-Asp) were recorded with intracellular electrodes from caudate neurons in halothane anesthetized cats. L-CSA and L-Asp were applied microiontophoretically to caudate cells and their effects on membrane and action potentials, as well as on cortically evoked synaptic potentials were evaluated. L-CSA and L-Asp induced depolarizations accompanied by regular firing resembling kainate (KA)- or quisqualate (QUIS)-induced excitation patterns (type 1) in 82% and 72% of the recorded neurons, respectively, and a mixed pattern consisting of a N-methyl-D-aspartate (NMDA)-like excitation (type 2) followed by a regular type 1 pattern in the remaining cells. In about a quarter of the cells the effects of L-CSA and L-Asp, but not those of KA or QUIS, were partially antagonized by 2-amino-7-phosphonoheptanoate (AP-7), a specific NMDA receptor antagonist. Kynurenate, a broad spectrum excitatory amino acid antagonist, blocked responses elicited by either L-CSA or QUIS. The actions of L-CSA and L-Asp on the firing pattern and membrane potential of cat caudate neurons in situ provide evidence in favor of their mixed agonist nature with respect to NMDA and non-NMDA excitatory amino acid receptors.

Muscle relaxant and anticonvulsant activity of 3-((+/-)-2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid, a novel N-methyl-D-aspartate antagonist, in rodents.

A novel 4-substituted derivative of piperazine-2-carboxylic acid, 3-((+/-)-2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid (CPP), with potent N-methyl-D-aspartate (NMDA) antagonist activity was evaluated as a muscle relaxant in genetically spastic rats. CPP, 0.02-0.1 mmol/kg, given intraperitoneally reduced the tonic activity in the electromyogram recorded from the gastrocnemius muscle of genetically spastic rats in a dose- and time-dependent manner. Muscle relaxation was also seen following intrathecal application of CPP, 0.0002-0.002 mumol, in genetically spastic rats. CPP, 0.1 mmol/kg, while not affecting Hoffman (H-) reflexes, depressed flexor reflexes in anesthetized rats following intravenous administration. In mice, CPP, 0.001 mumol, given intracerebroventricularly preferentially antagonized myoclonic seizures induced by NMDA and quinolinate, and had no effect on convulsions elicited by kainate, quisqualate and L-glutamate. These observations identify CPP as the most potent preferential NMDA antagonist so far tested with muscle relaxant and anticonvulsant activity resembling the profile of action of 2-amino-7-phosphonoheptanoate.