Binding of botulinum neurotoxin to pure cholinergic nerve terminals isolated from the electric organ of Torpedo

Summary Torpedo electric organ has been used to study the binding of botulinum neurotoxin type A to pure cholinergic synaptosomes and presynaptic plasma membrane.¹²⁵I-labeled botulinum neurotoxin type A exhibits specific binding to cholinergic fractions. Two binding sites have been determined according to data analysis: a high affinity binding site (synaptosomes: K_d=0.11±0.03 nM, B_max=50±10 fmol · mg prot^–1; presynaptic plasma membrane: K_d=0.2±0.05 nM, B_max=150±15 fmol · mg prot^–1) and a low affinity binding site (synaptosomes: K_d 26 nM, B_max 7.5 pmol · mg prot^–1; presynaptic plasma membrane: K_d 30 nM, B_max 52 pmol · mg prot^–1). The binding of¹²⁵I-botulinum neurotoxin type A is decreased by previous treatment of synaptosomes by neuraminidase and trypsin, and by a preincubation with bovine brain gangliosides or antiserum raised against Torpedo presynaptic plasma membrane. When presynaptic plasma membranes are blotted to nitrocellulose sheet, either¹²⁵I-botulinum neurotoxin or botulinum toxin-gold complexes bind to a M_r 140,000 protein. Botulinum toxin-gold complexes have also been used to study the toxin internalization process into Torpedo synaptosomes. The images fit the three step sequence model in the pathway of botulinum neurotoxin poisoning.     

Cytoplasmic and axoplasmic density variations in relation to hyperactivity

Summary The density of the cytoplasm and axoplasm of the anterior horn cell in rats was determined by X-ray microradiography. The average density of the cytoplasm of more than 400 cells from control rats was 0.31 g/³, while that of over 600 cells from rats fed IDPN (- iminodipropionitrile) was 0.43 g/³.Hyperactivity developed during the first 5 weeks and was associated with a gradual increase in cytoplasmic density to 0.51 g/³.At 6 weeks there was a drop in density to 0.36 g/³ which coincided with the appearance of axonal balloons having a density of 0.17 g/³.During the 7–12th week on the diet, the cytoplasmic density showed a gradual increase to 0.59 g/³ and the balloons to 0.29 g/³.The volume of the nerve cells remained fairly constant. The density increases were discussed in relation to hypertrophy, dystrophy, and hyperactivity.

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Zusammenfassung Die Dichte des Cytoplasmas und Axoplasmas der Vorderhornzellen von Ratten wurde durch Röntgenmikroradiographie bestimmt. Die mittlere Dichte des Cytoplasmas von mehr als 400 Zellen der Kontrollratten war 0,31 g/³, während die mittlere Dichte von mehr als 600 Zellen der Ratten, die mit IDPN (- iminodipropionitrile) gefüttert waren, 0,43 g/³ war.Hyperaktivität entwickelte sich während der ersten 5 Wochen und war mit einer progressiven Zunahme der Cytoplasmadichte bis auf 0,51 g/³ verbunden.Nach 6 Wochen sank die Dichte auf 0,36 g/³. Diese Tatsache traf mit dem Auftreten der Axonauftreibungen zusammen, die eine Dichte von 0,17 g/³ hatten.Nach 7–12 Wochen zeigte die Cytoplasmadichte eine progressive Zunahme auf 0,59 g/³ und die der Auftreibungen eine Zunahme auf 0,29 g/³.Das Volumen der Nervenzellen blieb ziemlich konstant.Die möglichen Zusammenhänge zwischen Zunahme der Dichte, Hypertrophie, Dystrophie und Hyperaktivität werden dargestellt.

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Supported by U. S. Public Health Grant NB 1305.     

Synthetic peptides corresponding to the sequence of noxiustoxin indicate that the active site of this K+ channel blocker is located on its amino-terminal portion

Summary A nonapeptide Thr-Ile-Ile-Asn-Val-Lys-Cys-Thr-Ser (NTX_1–9) and a decapeptide Met-Asn-Gly-Lys-Cys-Lys-Cys-Tyr-Asn-Asn (NTX_30–39) corresponding to the N-terminal and C-terminal sequences respectively of Noxiustoxin (NTX) were synthesized by the solid phase method of Merrifield (1963). The first synthetic peptide (NTX_1–9) was shown to be toxic to mice independently of the route of administration: intraperitoneally, subcutaneously or intraventricularly (100–200 g/20 g mouse weight). The second (NTX_30–39) was not toxic even at higher dose (400 g/20 g mouse). When the effects of the peptide NTX_1–9 and of the authentic toxin (Noxiustoxin) were studied on the liberation of [³H] 4-aminobutyric acid (³H-GABA) from mouse synaptosomes, both gave essentially the same results, except that peptide NTX_1–9 was needed at higher concentration. Synthetic peptide NTX_30–39 had no effect in the same preparation at even higher doses. The GABA release produced by toxic peptide NTX_1–9 was not affected by tetrodotoxin but was completely abolished by the presence of the K⁺ ionophore valinomycin, mimicking the effect of native NTX in the same system (Sitges et al., 1986). These results indicate that the toxic active site of Noxiustoxin is possibly located in or near the N-terminal amino acid portion of the molecule.Abbreviations used BOC amino acids ter-butyloxycarbonyl-amino acids - BOC amino acid-PAM-resin ter-butyloxycarbonyl-aminoacyl-4-(oxymethyl)-phenacetamidomethyl-resin - GABA 4 aminobutyric acid - HPLC high performance liquid chromatography - MSA mouse serum albumin - NTX Noxiustoxin - NTX _(numbers) synthetic peptides with amino acid sequences of NTX at position start (first number) to position end (second number) of the sequence according to Fig. 1 - TTX tetrodotoxin Supported in part by the Mexican Council of Science and Technology (CONACyT), grants PVT/QF/NAL/84/2182, PVT/AI/NAL/85/3029 to L.D.P.; PCSACNA 022640 to A.B. A patent request claiming rights on the use of synthetic NTX and related peptides was presented in Washington, DC (U.S.A.), Serial number 07/132,169, filing date December 14, 1987.     

Effect of 1-methyl-4-phenylpyridinium (MPP+) on mitochondrial membrane potential in cerebellar neurons: Interaction with the NMDA receptor

Summary The effect of MPP⁺, a dopaminergic neurotoxin, in mitochondrial membrane potential was investigated in dissociated cerebellar granule cells using rhodamine 123 and flow cytometry. MPP⁺ (1 mM) decreased the mitochondrial membrane potential by 30%. Antagonists of the NMDA receptor complex, such as MK-801 (IC₅₀ value of 20.92 ± 0.02 nM), 5,7-dichlorokynurenic acid (IC₅₀ value of 6.46 ± 1.06 M) and D-AP5 (IC₅₀ value of 8.29 ± 0.63 M), inhibited the action of MPP⁺. Neither NBQX, nor riluzole, nor desipramine modified the action of MPP⁺. Dibucaine restored the basal values of mitochondrial membrane potential altered by MPP⁺. Since, in the presence of NMDA, MPP⁺ antagonized the effect of this total agonist, it can be concluded that, in this preparation, MPP⁺ interacts with the NMDA receptor complex as a partial agonist. This interaction could be the result of an allosteric modulation of the NMDA receptor complex by MPP⁺. The decrease of mitochondrial membrane potential induced by MPP⁺ is antagonized by dibucaine, suggesting that this effect is mediated by an activation of phospholipase A₂.     

Effects of dopamine D-1 and D-2 antagonists on decision making by rats: No reversal of neuroleptic-induced attenuation by scopolamine

Summary The presynaptic actions of the potassium channel blocker Dendrotoxin (DTX) on the Ca⁺²-dependent release of endogenous glutamate (GLU) and aspartate (ASP) have been tested in synaptosome-enriched preparations from rat striatum.24 hours after the intrastriatal administration of DTX the K⁺-evoked release of GLU and ASP from the striatal synaptosomes was decreased by 40–45%. No changes in the total synaptosomal content of the amino acids were observed. Superfusion of immobilized synaptosomes with DTX or 4-aminopyridine resulted in a dose-dependent increase in the basal outflow of GLU and ASP. The release of GLU stimulated by DTX was Ca⁺²-dependent and was not abolished by supervising the synaptosomes with 50 M D-ASP. Moreover, continous superfusion of DTX (7 M) to synaptosomes almost completely dumped the subsequent release of GLU and ASP stimulated by 20mM K⁺. It is concluded that blockade of presyanptic K⁺ channels by DTX leads to a massive release of the transmitter pool of GLU (and possible also ASP) from isolated nerve terminals and to a depletion of the amino acid releasable pool.     

Presynaptic effects of gamma-aminobutyric acid on norepinephrine release and uptake in rat pineal gland

Summary The effect of -aminobutyric acid (GABA) on pineal norepinephrine (NE) release was examined in vitro in the rat pineal gland. Exposure of pineal expiants previously loaded with³H-NE to 1–100 M GABA caused a dosedependent decrease of³H-NE release triggered by 60 mM K⁺, with a threshold GABA concentration of 1 M and IC₅₀ of about 10 M. The inhibitory effect of GABA was mimicked by the type B GABA agonist baclofen, displaying a similar dose-response relationship as GABA. The type A GABA agonist muscimol increased depolarization-induced³H-NE release, while the co-incubation with GABA and the type A receptor antagonist bicuculline augmented significantly GABA's depressive effect on³H-NE release. Bicuculline alone brought about a significant decrease of³H-NE release. Neither GABA, nor baclofen, muscimol or bicuculline, modified the spontaneous pineal³H-NE efflux. Assessment of³H-NE uptake at a low NE concentration (0.5 M) indicated that GABA decreased it in a dose-dependent manner (IC₅₀=100 M) through an effect blocked by bicuculline and mimicked by muscimol but not by baclofen; at a 5 M-³H-NE concentration a bicuculline-sensitive GABA augmentation of uptake was found. A kinetic analysis study of the pineal NE uptake process indicated that GABA augmented both Vmax and Km of transmitter uptake. These results indicate that GABA may be a significant regulatory signal for rat pineal sympathetic synapses.     

Succinoxidase activity in reticular neurons of hyperactive rats

Summary The succinoxidase activity of single neurons from the reticular formation was measured with the Cartesian diver technique. The average activity of nerve cell bodies from the nucleus gigantocellularis or pontis caudalis from control rats was 4.5 l O₂×10^–4 per hour at 37°C. After rats had received --iminodipropionitrile, the succinoxidase activity of the same type neuron increased to an average of 8.0 l O₂×10^–4. It was suggested that this increase was of etiological significance for the symptoms of hyperactivity which developed later. Also when --iminodipropionitrile had been added to the reticular neurons from normal rats in vitro, the succinoxidase activity increased to 8.4 l O₂×10^–4 per hour.Supported by USPH Grant NB 1305.     

Reduced serotonin transporter–availability in obsessive–compulsive disorder (OCD)

Abstract. We investigated the availability of brain serotonin transporters in 10 drug–free patients with obsessive–compulsive disorder (OCD) and age–matched healthy controls in vivo using single–photon emission computed tomography (SPECT) and the radioligand [¹²³I]–2–carbomethoxy–3–(4–idiophenyl)-tropane ([¹²³I]–CIT). For quantification of regional serotonin transporter a ratio of specific to non–specific [¹²³I]–CIT–binding was used. The availability of serotonin transporter was calculated using regions of interests (ROI) for thalamus/hypothalamus, midbrain, brainstem (highest density of serotonin transporter) and cerebellum as a reference. The mean specific to non–specific [¹²³I]–CIT binding ratios in the thalamic/hypothalamic ROI were 4.95 ± 0.57 (OCD patients), and 5.48 ± 0.87 (control group). The mean ratios in the midbrain ROI were 3.51 ± 0.45 (OCD patients) and 4.89 ± 1.23 (controls) and in the brainstem ROI the ratios were 2.38 ± 0.76 (OCD patients) and 3.53 ± 1.01 (controls). This in vivo finding of significant reduced serotonin transporter availability in midbrain/brainstem using [¹²³I] –CIT SPECT further supports the serotonin deficit hypothesis of OCD.     

β-Adrenoceptors in the extraorbital lacrimal gland of the Syrian hamster characterization with [125I]-iodopindolol and evidence of sexual dimorphism

Summary Saturation and competition experiments with the radiolabeled -adrenergic antagonist (–)-[¹²⁵I]-iodopindolol were used to characterize -adrenoceptor density (B_max) and receptor affinity in the extraorbital lacrimal gland of male and female Syrian hamsters. Specific binding to the receptor was saturable. Scatchard analysis of saturation isotherms revealed a single population of receptor sites. Male glands had a significantly higher B_max (38.9±5.0 vs. 23.3±2.1 fmole/mg protein, ¯x±SEM, p< 0.02) and receptor affinity (expressed in a lower dissociation constant K_d: 0.065±0.013 vs. 0.120±0.015 nM, p<0.02) than female glands. Binding of the radiolabeled ligand in competition with various adrenergic antagonists showed the receptor to be stereospecific and of the ₂-subtype.     

Intracerebroventricular galanin and N-terminal galanin fragment enhance the morphine-induced analgesia in the rat

Summary Behavioral effect of galanin and its fragments, galanin_1–15 and galanin_16–29 (200 ng, 1 and 5 g), after intracerebroventricular (i.c.v.) administration was studied in rats. The number of crossings and pippings and the time of locomotion (an open field test) showed a similar sedative action of galanin and galanin_16–29, with no significant effect of galanin_1–15. Galanin and its fragments, injected in doses of 200 ng, 1 and 5 g, did not affect nociception, as measured by a tail-flick and paw pressure test. Galanin and galanin_1–15, but not galanin_16–29 (5 g i.c.v.), injected together with morphine (2.5 g i.c.v.), significantly potentiated the analgetic effect of morphine assessed by a paw pressure test; a similar tendency was also observed in a tail-flick test. Galanin and its two fragments injected in doses of 200 ng, 1 and 5 g, did not change the effect of morphine given in a dose of 1 g. These data suggest that galanin, having no effect when given alone, potentiate the analgetic effect of morphine. The fact that the N-terminal fragment of galanin acts like a natural peptide suggests a receptor mediated action.In conclusion, the analgesic effect of morphine was potentiated by galanin and its N-terminal fragment galanin_1–15. On the other hand, behavioral study showed a similar sedative action of galanin and C-terminal fragment galanin_16–29. This suggests that the N- and C-terminal fragments of galanin are differentially involved in behavioral effects of the peptide.     

Cell membrane structure of human giant-celled glioblastoma

Summary A giant-cell glioblastoma was examined by electron microscopy and by the freeze-fracture technique. The cell membranes bordering the extensive extracellular space often showed complicated undulations and peripheral vacuoles as well as occasional microvilli or filopodia. The undulations were mainly composed of plasmalemmal vesicles as well as of large (400–800 nm in diameter) and small (30–50 nm in diameter) localized protrusions and invaginations of the cell membrane. Deep invaginations of the cell membrane apparently resulted in two separate cytoplasmic portions. Locking of protruded cytoplasmic tongues and adherens junctions were sometimes seen in closely approximated cell membranes. The average number of membrane particles per m² was 630±130 on the P face and 180±30 on the E face. The membrane particles were occasionally aggregated to form clusters about 30 to 150 m in diameter. Gap junctions were occasionally found, but there were no tight junctions. Large particles about 30 nm in diameter were found in places.     

In vitro studies on the possible effects of 1-aminoadamantanes on the serotonergic system in M. Parkinson

Summary Synaptosomes, synaptic vesicles, and membranes were isolated from rat brain homogenates by differential and density gradient centrifugation in sucrose. Synaptosomes incorporated serotonin (5-HT) with two different uptake mechanisms, high affinity: K_t1=47 nM and low affinity: K_t2=660 nM. Both uptake mechanisms are non-competitively inhibited by the potential antiparkinson drugs 1-aminoadamantane (amantadine, D 1: K_i1=57M, K_i2=96M) and 1-amino-3.5-dimethyladamantane (memantine, D 145: K_i1=97M, K_i2=150M). The incorporated 5-HT is released from Synaptosomes on incubation with high concentrations (0.5–5 mM) of the drugs or on electrical stimulation with rectangular pulses of alternating polarity. Subthreshold concentrations of these drugs (5–50M) which are too low to liberate 5-HT increase the electrically stimulated release of 5-HT.—The effect of D 1, D 145, and electrical stimulation on DA release parallels the results observed with 5-HT.The uptake of 5-HT into isolated synaptic vesicles and the binding to isolated nerve ending membranes is non-competitively inhibited by 1-aminoadamantanes. D 145 inhibits the binding of 5-HT to membranes more effectively (K_i=0.95 mM) than its uptake into vesicles (K_i=1.2mM) contrasting with D 1 which is a weaker inhibitor affecting vesicular uptake (K_i=2.5 mM) slightly more than membrane binding (K_i=3.1 mM).The results obtained suggest that, in addition to other mechanisms like receptor stimulation, 1-aminoadamantanes may act in parkinsonian patients by enriching the transmitter content in the synaptic cleft.     

Concentrations of remoxipride and its phenolic metabolites in rat brain and plasma. Relationship to extrapyramidal side effects and atypical antipsychotic profile

Summary The cataleptic effect of remoxipride was examined in the horizontal bar test after i.v.,i.p. and s.c. administration to male rats. Remoxipride induced immediate catalepsy after high i.v. doses (ED₅₀=49 mol/kg) while peak effects were seen 60–90 min after i.p. administration (ED₅₀=38 mol/kg). Following s.c. administration remoxipride failed to produce a statistically significant catalepsy in the 20–100 mol/kg dose range (ED₅₀ > 100 mol/kg). In contrast, haloperidol was found to be more effective in inducing catalepsy after i.v. (ED₅₀=0.4 mol/kg) than after i.p. or s.c. administration (ED₅₀=0.9 mol/ kg). The atypical antipsychotic profile of remoxipride was more pronounced when the compound was given i.v. or s.c. as compared with the i.p. route. Plasma and brain (striatum and nucleus accumbens) concentrations of remoxipride and its active phenolic metabolites FLA 797(–) and FLA 908(–) were measured by high performance liquid chromatography. The 40 mol/kg dose of remoxipride resulted in plasma and brain concentrations of remoxipride which were 300–1000-fold higher (depending on the route of administration) than the most potent of the phenolic metabolites, e.g., FLA 797(–). The plasma and brain concentrations of remoxipride and its phenolic metabolites were related to DA D₂ receptor blocking potency and to the temporal course and effectiveness to induce catalepsy. This analysis suggested that the unbound concentrations of the phenolic metabolites were too low to play a major role in the DA blocking action of remoxipride. However, FLA 797(–) may contribute marginally to the cataleptic effects following high (i.p.) doses of remoxipride.     

Effect of noradrenaline and vanadium on Na+K+-activated ATPase in rat cerebral cortex synaptosomal preparation

Summary The effect of l-noradrenaline and vanadate on the activity of Na⁺K⁺-activated ATPase was studied on synaptosomal brain cortex preparation. Using neutron activation analysis it was found that the rat cerebral cortex synaptosomal preparation contains 0.16M. vanadium. The concentration of vanadium needed to reduce enzyme activity by 50% proved to be 2×10^–6 M. Evidence has been provided that the increase by noradrenaline of enzyme activity in synaptosomal preparation depends on the presence of an inhibitory contaminant in commercial ATP preparations. In homogenate, however, noradrenaline was able to enhance enzyme activity even when vanadium-free ATP was used. This fact indicates that noradrenaline removes the inhibitory effect of cytoplasmic factor thereby stimulating enzyme activity.     

Different spinal effects of opioid agonists on spinal and spino-bulbo-spinal reflexes in rats

Summary The effects of morphine-HCl (MOR), methionine-enkephalin (ME) and dynorphin_1–13 (DYN) on spinal and spino-bulbo-spinal (SBS) reflexes were studied. Although spinal intrathecal administration of MOR (15g) did not produce any apparent effect on these reflexes, systemically administered MOR (3mg/kg i.v.) reduced the electrical toe stimulation-induced SBS reflex. Furthermore, MOR (3mg/kg i.v.) increased the polysynaptic reflex induced by electrical stimulation of low-threshold dorsal root afferents in intact (non-spinal) rats, but not in spinal rats. Intrathecally administred DYN (0.5 and 5 g) reduced both the electrical toe stimulation-induced spinal and SBS reflexes, while ME (15g) only reduced the SBS reflex. These results indicate the physiological multiplicity of spinal opioid receptors. MOR may affect supraspinal nuclei but not the spinal pathway which possesses MOR-sensitive opioid receptors, whereas ME and DYN affect spinal opioid peptide receptors and modulate the reflex activities in which they participate.     

Effect of parasympathetic blockade on the signalaveraged electrocardiogram

Low parasympathetic activity is associated with late potentials detected at a noise level of 0.4 V in a signal-averaged electrocardiogram (SAECG) following myocardial infarction. In contrast, at a noise level of 0.2 V, lowering parasympathetic activity influences late potential parameters in the opposite direction in healthy subjects. The aim of this study was to estimate the relationship between parasympathetic activity and the SAECG obtained at noise levels of 0.4 and 0.2 V in healthy subjects. Two SAECG recordings in 10 healthy subjects were obtained at noise levels of 0.2 and 0.4 V before and after parasympathetic blockade using atropine (1 mg). Signal-averaged QRS duration (SA-QRS), late potential duration (LPD) defined as duration of terminal signals below 40 V, and root mean square voltage of the terminal 40 ms of the averaged QRS (RMS₄₀) were measured. At a noise level of 0.2 V SA-QRS reduced from 124±14 to 114±17 ms (P=0.008), LPD from 37±10 to 28±14 ms (P=0.01), and RMS₄₀ increased from 26±22 to 41±25 V (P=0.006) during parasympathetic blockade compared to baseline values. At a noise level of 0.4 V the SA-QRS (115±15 ms) and LPD (29±11 ms) were lower and the RMS₄₀ (37±23 V) was higher compared to the noise level 0.2 V, and no systematic alterations of the three variables were found during parasympathetic blockade. The parasympathetic nervous system may induce a very low-amplitude late potential in the SAECG. The data suggest that parasympathetic activity and a low noise level may lead to a false late potential-positive SAECG in low arrhythmia risk subjects. Therefore, we recommend the use of a noise level of 0.4 V or identification of high arrhythmia risk patients by late potentialand low parasympathetic activity.     

Determination of 2-phenylethylamine in rat brain after MAO inhibitors,and in human CSF and urine by capillary GC and chemical ionization MS

Summary A highly specific and sensitive method for the determination of-phenylethylamine (PEA) in biological material is presented. It involves prepurification of the extracts on Sep-Pak C₁₈ cartridges, derivatization with heptafluorobutyric acid anhydride, gas chromatography on 50 m capillary columns and quantification by chemical ionization mass spectrometry.Using this method, levels of PEA in the rat brain and the effects of various monoamine oxidase (MAO) inhibitors thereon have been determined. PEA control levels were found to vary considerably: the lowest and highest values found were 0.4 and 12.5 ng/g tissue (n=25). Within one and the same control group, the variation was somewhat smaller. The preferential or specific inhibitors of MAO A, amiflamine, cimoxatone, CGP11305 A, moclobemide and toloxatone did not alter rat brain PEA even at high doses. In contrast, the preferential inhibitors of MAO B, deprenil, pargyline and MD 780236, as well as the nonselective agent tranylcypromine, caused strong (up to about 60-fold) increases. The threshold doses corresponded to those which caused about an 80 % inhibition of MAO B as measuredex vivo.The method was also used to determine the concentration of PEA in human CSF (mean 17.3±3.3 ng/ml, range 3–45 ng/ml, n=15) and urine (males: mean 35±5g/g creatinine, range 3.8–219g/g, 78 control days of a total of 12 subjects; females: mean 35±6g/g creat., range 2.7–266g/g, 55 control days of a total of 8 subjects).     

Intestinal neurohormones in protein-deficient rats

Summary The levels of noradrenaline, adrenaline, and serotonin in the jejunum and ileum of weanling rats fed protein-free (deficient) and high-protein (control) diets were analyzed. The concentration of noradrenaline of the deficient rats was markedly increased, both in the jejunum (0.430±0.039g/g vs. 0.188±0.019g/g in the control animals, +228%, P<0.001) and in the ileum (0.492±0.041g/g vs. 0.212±0.014g/g in the control rats, +232%, P<0.001). However, the levels of adrenaline and serotonin were unaltered in deficient rats as compared to controls, both in the jejunum (0.049±0.009g/g of adrenaline and 1.233±0.178g/g of serotonin vs. 0.047±0.006g/g of adrenaline and 1.364±0.131g/g of serotonin in the controls) and in the ileum (0.027±0.005g/g of adrenaline and 0.902±0.150g/g of serotonin vs. 0.038±0.006g/g of adrenaline and 1.118±0.192g/g of serotonin in the controls). In view of these results, it can be speculated that the abdominal distension and the reduced intestinal motility usually seen in the states of protein malnutrition could be caused, at least in part, by the accumulation of noradrenaline in the intestine.     

Alpha-1 and alpha-2 adrenoceptor binding in cerebral cortex: Competition studies with [3H]prazosin and [3H]idazoxan

Summary The tritiated adrenergic antagonists prazosin ([³H]PRZ) and idazoxan ([³H]IDA, or RX-781094) bind specifically and with high affinity in membrane preparations from cerebral cortex to alpha-1- and alpha-2-adrenoceptors respectively. Saturation experiments, performed to determine the density of receptors (B_max; maximum binding capacity) and the dissociation constant (K_d 25 °C), were analyzed by the methods of Eadie and Hofstee, iterative modelling, and the procedure of Hill. The pharmacologic properties and specificity of the labelling was verified by displacement experiments using alpha-adrenergic antagonists and agonists. The antagonist drugs showed the following order of potency to displace [³H]prazosin: prazosin phentolamine corynanthine > pyrextramine yohimbine piperoxan > benextramine > idazoxan; for the agonists: clonidine (–)-noradrenaline (–)-adrenaline phenylephrine, while other drugs, such as (–)-propranolol, dopamine, (–)-isoproterenol and serotonin only competed with the alpha-1-ligand at concentrations above 20 M. The alpha₂-sites labelled by [³H]idazoxan were characterized by the antagonist displacement sequence idazoxan phentolamine > yohimbine = > piperoxan pyrextramine benextramine prazosin corynanthine. The agonists order of potency to compete with [³H]idazoxan was clonidine phenylephrine = > (–)-adrenaline > (–)-noradrenaline, and for other related drugs it was (–)-propranolol dopamine serotonin > (–)-isoproterenol. These competition experiments clearly showed two pharmacologically distinct sites, but question the relative specificity of some of the adrenergic drugs.Abreviations [³H]PRZ [³H]prazosin - [³H]IDA [³H]idazoxan - B_max maximum binding capacity - K_d dissociation constant - IC 50 inhibitory concentration that reduces binding by 50% - K_i inhibition-dissociation constant - nH Hill coefficient - CMC coefficient of multiple correlation - fmol/mg p fentomoles per mg of protein - nM nanomolar Recipient of a F.R.S.Q. Studentship.     

Effect of dantrolene on KCl- or NMDA-induced intracellular Ca2+ changes and spontaneous Ca2+ oscillation in cultured rat frontal cortical neurons

Summary Dantrolene has been known to affect intracellular Ca²⁺ concentration ([Ca²⁺]_i) by inhibiting Ca²⁺ release from intracellular stores in cultured neurons. We were interested in examining this property of dantrolene in influencing the [Ca²⁺]_i affected by the NMDA receptor ligands, KCl, L-type Ca²⁺ channel blocker nifedipine, and two other intracellular Ca²⁺-mobilizing agents caffeine and bradykinin. Effect of dantrolene on the spontaneous oscillation of [Ca²⁺]_i was also examined. Dantrolene in M concentrations dose-dependently inhibited the increase in [Ca²⁺]_i elicited by NMDA and KCl. AP-5, MK-801 (NMDA antagonists), and nifedipine respectively reduced the NMDA and KCl-induced increase in [Ca²⁺]_i. Dantrolene, added to the buffer solution together with the antagonists or nifedipine, caused a further reduction in [Ca²⁺]_i to a degree similar to that seen with dantrolene alone inhibiting the increase in [Ca₂₊]_i caused by NMDA or KCl. At 30 M, dantrolene partially inhibited caffeine-induced increase in [Ca²⁺]_i whereas it has no effect on the bradykinin-induced change in [Ca²⁺]_i. The spontaneous oscillation of [Ca²⁺]_i in frontal cortical neurons was reduced both in amplitude and in base line concentration in the presence of 10 M dantrolene. Our results indicate that dantrolene's mobilizing effects on intracellular Ca²⁺ stores operate independently from the influxed Ca²⁺ and that a component of the apparent increase in [Ca²⁺]_i elicited by NMDA or KCl represents a dantrolene-sensitive Ca₂₊ release from intracellular stores. Results also suggest that dantrolene does not affect the IP₃-gated release of intracellular Ca²⁺ and that the spontaneous Ca²⁺ oscillation is, at least partially, under the control of Ca²⁺ mobilization from internal stores.Abbreviations AP-5 (±)-2-amino-5-phosphonopentanoic acid - AMPA amino-3-hydroxy-5-methyl-isoxazole-4-propionate - BSS balanced salt solution - CNS central nervous system - CICR Ca²⁺-induced Ca²⁺ release - DCKA 5,7-dichlorokynurenate - DNasel deoxyribonuclease I - DMEM Dulbecco's Modified Eagle's Medium - EGTA ethylene glycol-bis(-aminoethyl ether)N,N,N,N,-tetraacetic acid - FCS fetal calf serum - fura-2-AM 1-(2-(5-carboxyoxazol-2-yl)-6-aminobenzofuran-5-oxy-2-ethane-N,N,N,N-te-traacetic acid, pentaacetoxymethyl ester - HEPES N-[2-hydroxyethyl] piperazine-N-[2-ethanesulfonic acid] - [Ca ²⁺] _i intracellular free Ca²⁺ concentration - LTP long-term potantiation - MK-801 (5R, 10S)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,b]-cyclohepten-5,10-imine hydrogen maleate - NMDA N-methyl-D-aspartate