Supplemental treatment to atropine improves the efficacy to reverse nerve agent induced bronchoconstriction.

Exposure to highly toxic organophosphorus compounds causes inhibition of the enzyme acetylcholinesterase resulting in a cholinergic toxidrome and innervation of receptors in the neuromuscular junction may cause life-threatening respiratory effects. The involvement of several receptor systems was therefore examined for their impact on bronchoconstriction using an ex vivo rat precision-cut lung slice (PCLS) model. The ability to recover airways with therapeutics following nerve agent exposure was determined by quantitative analyses of muscle contraction. PCLS exposed to nicotine resulted in a dose-dependent bronchoconstriction. The neuromuscular nicotinic antagonist tubocurarine counteracted the nicotine-induced bronchoconstriction but not the ganglion blocker mecamylamine or the common muscarinic antagonist atropine. Correspondingly, atropine demonstrated a significant airway relaxation following ACh-exposure while tubocurarine did not. Atropine, the M3 muscarinic receptor antagonist 4-DAMP, tubocurarine, the ß2-adrenergic receptor agonist formoterol, the Na+-channel blocker tetrodotoxin and the K+ATP-channel opener cromakalim all significantly decreased airway contractions induced by electric field stimulation. Following VX-exposure, treatment with atropine and the Ca2+-channel blocker magnesium sulfate resulted in significant airway relaxation. Formoterol, cromakalim and magnesium sulfate administered in combinations with atropine demonstrated an additive effect. In conclusion, the present study demonstrated improved airway function following nerve agent exposure by adjunct treatment to the standard therapy of atropine.

Curare alkaloids from Matis Dart Poison: Comparison with d-tubocurarine in interactions with nicotinic, 5-HT3 serotonin and GABAA receptors.

Several novel bisbenzylisoquinoline alkaloids (BBIQAs) have recently been isolated from a Matis tribe arrow poison and shown by two-electrode voltage-clamp to inhibit mouse muscle nicotinic acetylcholine receptors (nAChR). Here, using radioligand assay with Aplysia californica AChBP and radioiodinated α-bungarotoxin ([125I]-αBgt), we show that BBIQA1, BBIQA2, and d-tubocurarine (d-TC) have similar affinities to nAChR orthosteric site. However, a competition with [125I]-αBgt for binding to the Torpedo californica muscle-type nAChR revealed that BBIQAs1, 2, and 3 are less potent (IC50s = 26.3, 8.75, and 17.0 µM) than d-TC (IC50 = 0.39 µM), while with α7 nAChR in GH4C1 cells, BBIQA1 was less potent that d-TC (IC50s = 162 µM and 7.77 µM, respectively), but BBIQA2 was similar (IC50 = 5.52 µM). In inhibiting the Ca2+ responses induced by acetylcholine in Neuro2a cells expressing the mouse adult α1ß1εδ nAChR or human α7 nAChR, BBIQAs1 and 2 had similar potencies to d-TC (IC50s in the range 0.75-3.08 µM). Our data suggest that BBIQA1 and BBIQA2 can inhibit adult muscle α1ß1εδ nAChR by both competitive and noncompetitive mechanisms. Further experiments on neuronal α3ß2, α4ß2, and α9α10 nAChRs, expressed in Xenopus laevis oocytes, showed that similar potencies for BBIQAs1, 2, and d-TC. With α3ß2γ2 GABAAR currents were almost completely inhibited by d-TC at a high (100 µM) concentration, but BBIQAs1 and 2 were less potent (only 40-50% inhibition), whereas in competition with Alexa Fluor 546-α-cobratoxin for binding to α1ß3γ2 GABAAR in Neuro2a cells, d-TC and these analogs had comparable affinities. Especially interesting effects of BBIQAs1 and 2 in comparison with d-TC were observed for 5-HT3AR: BBIQA1 and BBIQA2 were 5- and 87-fold less potent than d-TC (IC50 = 22.63 nM). Thus, our results reveal that these BBIQAs differ from d-TC in their potencies towards certain Cys-loop receptors, and we suggest that understanding the reasons behind this might be useful for future drug design.

Curare Alkaloids: Constituents of a Matis Dart Poison.

A phytochemical study of dart and arrow poison from the Matis tribe led to the identification of D-(-)-quinic acid, L-malic acid, ethyldimethylamine, magnoflorine, and five new bisbenzyltetrahydroisoquinoline alkaloids (BBIQAs), 1-5. D-Tubocurarine could not be identified among these products. BBIQA (3) contains a unique linkage at C-8 and C-11'. All structures were characterized by a combination of NMR and HRESIMS data. The effects of Matis poison and individual BBIQAs (1-3) on rat muscle nAChR expressed in Xenopus oocytes have been investigated using the two-electrode voltage clamp technique.

Where fMRI and electrophysiology agree to disagree: corticothalamic and striatal activity patterns in the WAG/Rij rat.

The relationship between neuronal activity and hemodynamic changes plays a central role in functional neuroimaging. Under normal conditions and in neurological disorders such as epilepsy, it is commonly assumed that increased functional magnetic resonance imaging (fMRI) signals reflect increased neuronal activity and that fMRI decreases represent neuronal activity decreases. Recent work suggests that these assumptions usually hold true in the cerebral cortex. However, less is known about the basis of fMRI signals from subcortical structures such as the thalamus and basal ganglia. We used WAG/Rij rats (Wistar albino Glaxo rats of Rijswijk), an established animal model of human absence epilepsy, to perform fMRI studies with blood oxygen level-dependent and cerebral blood volume (CBV) contrasts at 9.4 tesla, as well as laser Doppler cerebral blood flow (CBF), local field potential (LFP), and multiunit activity (MUA) recordings. We found that, during spike-wave discharges, the somatosensory cortex and thalamus showed increased fMRI, CBV, CBF, LFP, and MUA signals. However, the caudate-putamen showed fMRI, CBV, and CBF decreases despite increases in LFP and MUA signals. Similarly, during normal whisker stimulation, the cortex and thalamus showed increases in CBF and MUA, whereas the caudate-putamen showed decreased CBF with increased MUA. These findings suggest that neuroimaging-related signals and electrophysiology tend to agree in the cortex and thalamus but disagree in the caudate-putamen. These opposite changes in vascular and electrical activity indicate that caution should be applied when interpreting fMRI signals in both health and disease from the caudate-putamen, as well as possibly from other subcortical structures.

Pharmacological effects of Catharanthus roseus root alkaloids in acetylcholinesterase inhibition and cholinergic neurotransmission.

The leaves of Catharanthus roseus constitute the only source of the well known indolomonoterpenic alkaloids vincristine and vinblastine. In this work we studied the biological potential of the roots, which are used in several countries as decocts or hot water extracts for the treatment of a number of conditions. The aqueous extract strongly inhibited acetylcholinesterase (AchE) in an in vitro microassay, an effect ascribable mainly to serpentine (IC(50) = 0.775 microM vs physostigmine IC(50) = 6.45 microM) as assessed with the pure compound. Pure alkaloids were tested for muscarinic and nicotinic antagonism using rat ex-vivo preparations, namely, ileum and diaphragm/phrenic-nerve, respectively. Serpentine competitively blocked muscarinic receptors with a pA(2) of 5.2, whereas the precursor ajmalicine up to 80 microM was undistinguishable from control, and catharanthine exhibited an unsurmountable muscarinic antagonism at greater than 10 microM concentrations. Nicotinic receptor mediated diaphragm contractions were fully inhibited by catharanthine (IC(50) = 59.6 microM) and ajmalicine (IC(50) = 72.3 microM), in a reversible but non-competitive manner, unlike the more potent nicotinic antagonist tubocurarine (IC(50) = 0.35 microM) whose competitive blockade was overcome by a physostigmine-induced increase in acetylcholine. Serpentine up to 100 microM did not change diaphragm contractions suggesting reduced affinity for neuromuscular nicotinic receptors. Despite strong in vitro AchE inhibition, serpentine failed to restore diaphragm contractions upon submaximal tubocurarine blockade, suggesting that poor tissue penetration may prevent serpentine from inhibiting AchE in deep neuromuscular synapses in the ex-vivo preparation. To our knowledge, the present study is the first to assess the effect of C. roseus root extracts, as well as of serpentine, ajmalicine and catharanthine on AchE. The results described herein suggest that the currently overlooked C. roseus roots may constitute a promising source of compounds with pharmaceutical interest. Moreover, given serpentine's potent in vitro AchE inhibitory activity and low cholinergic receptor affinity, it is conceivable that minor structural modifications may yield a potent and selective AchE inhibitor, potentially useful for the pharmacological management of conditions such as Alzheimer's disease and/or myasthenia gravis.

Site selectivity of competitive antagonists for the mouse adult muscle nicotinic acetylcholine receptor.

The muscle-type nicotinic acetylcholine receptor has two nonidentical binding sites for ligands. The selectivity of acetylcholine and the competitive antagonists (+)-tubocurarine and metocurine for adult mouse receptors is known. Here, we examine the site selectivity for four other competitive antagonists: cisatracurium, pancuronium, vecuronium, and rocuronium. We rapidly applied acetylcholine to outside-out patches from transfected BOSC23 cells and measured macroscopic currents. We have reported the IC(50) of the antagonists individually in prior publications. Here, we determined inhibition by pairs of competitive antagonists. At least one antagonist was present at a concentration producing > or =67% receptor inhibition. Metocurine shifted the apparent IC(50) of (+)-tubocurarine in quantitative agreement with complete competitive antagonism. The same was observed for pancuronium competing with vecuronium. However, pancuronium and vecuronium each shifted the apparent IC(50) of (+)-tubocurarine less than expected for complete competition but more than expected for independent binding. The situation was similar for cisatracurium and (+)-tubocurarine or metocurine. Cisatracurium did not shift the apparent IC(50) of pancuronium or vecuronium, indicating independent binding of these two pairs. The data were fit to a two-site, two-antagonist model to determine the antagonist binding constants for each site, L(alphaepsilon) and L(alphadelta). We found L(alphaepsilon)/L(alphadelta) = 0.22 (range, 0.14-0.34), 20 (9-29), 21 (4-36), and 1.5 (0.3-2.9) for cisatracurium, pancuronium, vecuronium, and rocuronium, respectively. The wide range of L(alphaepsilon)/L(alphadelta) for some antagonists may reflect experimental uncertainties in the low affinity site, relatively poor selectivity (rocuronium), or possibly that the binding of an antagonist at one site affects the affinity of the second site.

[Neuromuscular monitoring: methods and machines]. / Uberwachung der neuromuskulären Blockade - Methoden und Geräte.

Muscle relaxing agents are clinically in use for general anaesthesia to optimize the conditions to the endotracheal intubation as well as the surgical conditions. Therefore different musclerelaxants with specific pharmacological characteristics are available. Many factors that depend on the condition of the patient and the used musclerelaxant agent influence the duration of the neuromuscular blockade. Rapid reversal of their effects, particularly in cases of profound blockades, proved to be difficult. In cases of postoperative residual paralysis hypoxic complications because of failure of the ventilation increase the morbidity and mortality of the perioperative period. To avoid these complications in cause of postoperative residual neuromuscular blockade it seems to be necessary to evaluate the status of the muscle function. For the tactile or visual assessment or the objective measurement of stimulation the train-of-four (TOF), double-burst (DBS) or tetanus-stimulation of peripheral nerves like the ulnar nerve may be used. Established methods for the objective monitoring of neuromuscular function is the mechanomyography (MMG), the acceleromyography (AMG), the electromyography (EMG), the kinemyography (KMG) and the phonomyography (PMG). A sufficient recovery of the neuromuscular transmission is reached to a TOF-ratio of 0,9 and should be aimed before the extubation at the end of surgery. No subjective evaluation of the neuromuscular recovery is able to identify residual paralysis above a TOF-ratio of 0,5. Recent studies suggest that objective methods should be used to monitor neuromuscular function to avoid postoperative residual blockades.

Roles of amino acids and subunits in determining the inhibition of nicotinic acetylcholine receptors by competitive antagonists.

BACKGROUND: Binding sites for agonists and competitive antagonists (nondepolarizing neuromuscular blocking agents) are located at the alpha-delta and alpha-epsilon subunit interfaces of adult nicotinic acetylcholine receptors. Most information about the amino acids that participate in antagonist binding comes from binding studies with (+)-tubocurarine and metocurine. These bind selectively to the alpha-epsilon interface but are differentially sensitive to mutations. To test the generality of this observation, the authors measured current inhibition by five competitive antagonists on wild-type and mutant acetylcholine receptors. METHODS: HEK293 cells were transfected with wild-type or mutant (alphaY198F, epsilonD59A, epsilonD59N, epsilonD173A, epsilonD173N, deltaD180K) mouse muscle acetylcholine receptor complementary DNA. Outside-out patches were excised and perfused with acetylcholine in the absence and presence of antagonist. Concentration-response curves were constructed to determine antagonist IC50. An antagonist-removal protocol was used to determine dissociation and association rates. RESULTS: Effects of mutations were antagonist specific. alphaY198F decreased the IC50 of (+)-tubocurarine 10-fold, increased the IC50 of vecuronium 5-fold, and had smaller effects on other antagonists. (+)-Tubocurarine was the most sensitive antagonist to epsilonD173 mutations. epsilonD59 mutations had large effects on metocurine and cisatracurium. deltaD180K decreased inhibition by pancuronium, vecuronium, and cisatracurium. Inhibition by these antagonists was increased for receptors containing two delta subunits but no epsilon subunit. Differences in IC50 arose from differences in both dissociation and association rates. CONCLUSION: Competitive antagonists exhibited different patterns of sensitivity to mutations. Except for pancuronium, the antagonists were sensitive to mutations at the alpha-epsilon interface. Pancuronium, vecuronium, and cisatracurium were selective for the alpha-delta interface. This suggests the possibility of synergistic inhibition by pairs of antagonists.

Enhanced nicotinic acetylcholine receptor-mediated [3H]norepinephrine release from neonatal rat hypothalamus.

Nicotinic acetylcholine receptor (nAChR)-evoked release of norepinephrine (NE) has been demonstrated in a number of brain regions that receive sole noradrenergic innervation from the locus coeruleus (LC). Many of these structures display enhanced nicotine-stimulated NE release in the neonate. We have examined the hypothalamus in order to determine if this region, which receives NE projections from both the LC and medullary catecholaminergic nuclei, also demonstrates maturational changes in nAChR-mediated NE release. Quantification of radiolabeled-NE release from rat hypothalamus slices by a maximally effective dose of nicotine revealed a peak response during the first postnatal week. This was followed by a decrease at postnatal day (P) 14, and a second peak at P21. Thereafter, release was equivalent to that observed at P14. Comparison of the pharmacological properties of nAChRs mediating NE release in neonatal (P7) and mature hypothalamus suggested involvement of different nAChR subtypes at the two ages. Using the selective toxin, DSP-4, nAChR-mediated NE release in the neonatal hypothalamus was shown to be from LC terminals. Our findings demonstrate an early sensitivity of hypothalamic LC terminals to nAChR regulation that may be associated with development of systems controlling critical homeostatic functions such as stress, feeding and cardiovascular regulation.

Cholinergic sensory inputs to command neurons in edible snail.

We studied cholinergic component of visceral sensory input to defensive behavior command neurons in edible snail. Nicotinic receptor antagonist tubocurarine and muscarinic receptor antagonist atropine reversibly decreased the amplitude of the total excitatory postsynaptic potential induced by electrostimulation of the peripheral region in the mechanosensory receptor field of command neurons on the surface of internal organs. Our results indicate that acetylcholine is involved in sensory signal transduction from the visceral sac to command neurons of snail parietal ganglia. The subsynaptic membrane of visceral synaptic input contains nicotinic and muscarinic receptors.

The theaflavin fraction is responsible for the facilitatory effect of black tea at the skeletal myoneural junction.

The effect of various fractions of black tea [(Camellia Sinensis) (L) O. Kuntze (Theaceae)] on the function of mammalian skeletomotor apparatus was studied. The theaflavin fraction (Tfs) produced a concentration- dependent facilitation of indirect twitch responses of the rat phrenic nerve diaphragm preparation and the facilitation was dependent on the amount of calcium present in the bathing fluid. Nifedipine reduced the facilitatory effect of Tfs as a function of its concentration. Tfs failed to produce facilitation when the twitch height was reduced to about 50% of the control value in presence of magnesium chloride. Tfs completely antagonized the submaximal paralytic effect of d- tubocurarine and decamethonium bromide. Tfs did not have any effect on direct twitch responses or on acetylcholine (Ach) and potassium chloride (KCl) induced contractures of denervated diaphragm. The results revealed that the site of action of Tfs is on the contractile mechanism of the voluntary muscle and point to a critical role of calcium in the mechanism of action of Tfs. N omega-nitro-L-arginine-methyl ester (L-NAME), a nitric oxide synthase (NOS) inhibitor, antagonized both the facilitatory and inhibitory effects on indirect twitch responses of rat diaphragm induced by L-arginine and Tfs when the phrenic nerve was stimulated at 5 Hz and 50 Hz respectively. The thearubigin (Trs) fraction of black tea and the aqueous part which is completely devoid of Tfs, did not potentiate the twitch responses. The findings suggest that Tfs have a potentiating effect on the contractile mechanism of skeletal muscle and that calcium and nitric oxide may modulate this action of Tfs.

Sepsis stage dependently and differentially attenuates the effects of nondepolarizing neuromuscular blockers on the rat diaphragm in vitro.

We investigated the effects of early and late sepsis on the actions of nondepolarizing neuromuscular blockers by using a rat sepsis model induced by cecal ligation and puncture. Isometric twitch tensions of nerve-hemidiaphragm preparations elicited by indirect (phrenic nerve) supramaximal stimulation at 0.1 Hz were evaluated. Rocuronium, pancuronium, and d-tubocurarine dose-dependently decreased the twitch tensions of the nonseptic, early septic, and late septic diaphragms (P < 0.01 each by analysis of variance [ANOVA]). Late sepsis shifted the concentration-twitch tension curves rightward from those of nonsepsis to larger degrees than did early sepsis, as indicated by increases in 50% inhibitory concentration (IC(50)) values (P < 0.01 each by ANOVA and P < 0.01 or 0.05 by the Scheffe F test). The standardized rightward shifts in early and late sepsis were largest for pancuronium, second largest for rocuronium, and smallest for d-tubocurarine (5.741, 2.979, and 1.660 times in late sepsis, respectively; P < 0.01 each by ANOVA and the Scheffe F test). Sepsis-induced increases in IC(50) values did not accompany the decreases in slopes. The results indicate that sepsis induces hyposensitivities to nondepolarizing neuromuscular blockers, the degree of which depends on the stage of sepsis and on the kind of neuromuscular blocker.

Acute effect of an extract of Ambrosia paniculata (Willd.) O. E. Schultz (mugwort) in several models of experimental epilepsy.

The acute effect of Ambrosia paniculata was studied in several animal models of epilepsy. Intraperitoneal injections (0.01 mL/g body wt) of a decoction of the dry leaves significantly enhanced the latency to the first convulsion and survival time in mice injected with picrotoxin (7 mg/kg) or isoniazid (210 mg/kg). Epileptic spikes were induced by topical application of penicillin through a glass electrode filled with a penicillin-agar-saline mixture and recorded in sensorimotor and occipital cortices, in rats immobilized with d-tubocurarine. The plant decoction reduced significantly the spike amplitude in both sites. The mentioned effects were elicited at doses that also reduced general motor activity (Irwin test) and exploratory behavior. The decoctions were not effective against electroshock-induced convulsions in mice. The convulsions induced by isoniazid, picrotoxin, and penicillin differed from those induced by electroshock implicating selective disruption of GABAergic neurotransmission. The results suggest that A. paniculata, like several conventional antiepileptic drugs, might act by enhancing GABAergic neurotransmission, a hypothesis that requires further demonstration. These results explain and justify the traditional use of the plant in epilepsy.

Agrin signaling in cortical neurons is mediated by a tyrosine kinase-dependent increase in intracellular Ca2+ that engages both CaMKII and MAPK signal pathways.

Agrin has been implicated in multiple aspects of central nervous system (CNS) neuron differentiation and function including neurite formation, synaptogenesis, and synaptic transmission. However, little is known about the signaling mechanisms whereby agrin exerts its effects. We have recently identified a neuronal receptor for agrin, whose activation induces expression of c-fos, and provided evidence that agrin binding to this receptor is associated with a rise in intracellular Ca2+, a ubiquitous second messenger capable of mediating a wide range of effects. To gain further insight into agrin's role in brain, we used Ca2+ imaging to explore agrin signal transduction in cultured cortical neurons. Bath application of either z+ or z-agrin isoforms resulted in marked changes in intracellular Ca2+ concentration specifically in neurons. Propagation of the Ca2+ response was a two-step process characterized by an initial increase in intracellular Ca2+ mediated by ryanodine receptor (RyR) release from intracellular stores, supplemented by influx through voltage-gated calcium channels (VGCCs). Agrin-induced increases in intracellular Ca2+ were blocked by genistein and herbimycin, suggesting that the agrin receptor is a tyrosine kinase. Ca2+ release from intracellular stores activates both calcium/calmodulin-dependent kinase II (CaMKII) and mitogen activated protein kinase (MAPK). Activation of CaMKII is required for propagation of the Ca2+ wave itself, whereas both MAPK and CaMKII play a role in mediating long latency responses such as induction of c-fos. These results suggest that an agrin-dependent tyrosine kinase could play a critical role in modulating levels of intracellular Ca2+ and activity of MAPK and CaMKII in CNS neurons.

Soluble, oligomeric, and ligand-binding extracellular domain of the human alpha7 acetylcholine receptor expressed in yeast: replacement of the hydrophobic cysteine loop by the hydrophilic loop of the ACh-binding protein enhances protein solubility.

The N-terminal extracellular domain (ECD; amino acids 1-208) of the neuronal nicotinic acetylcholine receptor (AChR) alpha7 subunit, the only human AChR subunit known to assemble as a homopentamer, was expressed as a glycosylated form in the yeast Pichia pastoris in order to obtain a native-like model of the extracellular part of an intact pentameric nicotinic AChR. This molecule, alpha7-ECD, although able to bind the specific ligand alpha-bungarotoxin, existed mainly in the form of microaggregates. Substitution of Cys-116 in the alpha7-ECD with serine led to a decrease in microaggregate size. A second mutant form, alpha7-ECD(C116S,Cys-loop), was generated in which, in addition to the C116S mutation, the hydrophobic Cys-loop (Cys(128)-Cys(142)) was replaced by the corresponding hydrophilic Cys-loop from the snail glial cell acetylcholine-binding protein. This second mutant protein was water-soluble, expressed at a moderate level (0.5 +/- 0.1 mg/liter), and had a size corresponding approximately to a pentamer as judged by gel filtration and electron microscopy studies. It also bound (125)I-alpha-bungarotoxin with relatively high affinity (K(d) = 57 nm), the binding being inhibited by unlabeled alpha-bungarotoxin, d-tubocurarine, or nicotine (K(i) = 0.8 x 10(-7) m, K(i) = 1 x 10(-5) m, and K(i) = 0.9 x 10(-2) m, respectively). All three constructs were expressed as glycosylated forms, but in vitro deglycosylation reduced the heterogeneity without affecting their ligand binding properties. These results show that alpha7-ECD(C116S,Cys-loop) was expressed in P. pastoris as an oligomer (probably a pentamer) with a near native conformation and that its deglycosylated form seems to be suitable starting material for structural studies on the ligand-binding domain of a neurotransmitter receptor.

Óxido Nítrico produzido durante a sepse intensifica a redução nos valores da razão de "train-of-four" (TOF) induzida por d-Tubocurarina e pancurônio / Produced Nitric oxide during sepse intensifies the reduction in the values of the reason of “train-of-four” (TOF) induced for d-Tubocurarine and pancuronium

Durante a sepse, grandes quantidades de óxido nítrico (NO) são sintetizadas pela enzima NO sintase tipo II. Em preparações neuromusculares, o NO produz ou acentua a fadiga de transmissão. 'Train-of-four'(TOF) são pulsos elétricos de 2 Hz aplicados ao nervo motor durante 2 segundos (4 contrações musculares) e o quociente entre as tensões musculares produzidas pela quarta (T4) e primeira (T1) contrações musculares (RTOF = T4/T1) é utilizado para avaliação do bloqueio da transmissão neuromuscular. A redução de RTOF é determinada por bloqueio dos receptores nicotínicos (Nn) facilitatórios e por estimulação dos receptores muscarínicos (M2) inibitórios do terminal nervoso motor. Estudos utilizando preparações neuromusculares de animais sépticos tratadas com bloqueadores neuromusculares em doses que reduzem os valores de RTOF sem promover alterações nas amplitudes das contrações musculares a 0,2 Hz não foram executados. Assim, utilizando preparações nervo ciático-músculo tibial anterior de ratos, verificamos que as menores doses de d-tubocurarina (d-TC), pancurônio (PANC) e galamina (GAL) capazes de produzir 30 por cento de redução em RTOF nos animais sham foram, respectivamente: 54,29 ± 2,86 mg/kg (n=7), 69,02 ± 6,13 mg/kg (n=5), 2,94 ± 0,33 mg/kg (n=4). A dose de d-TC foi menor em animais sépticos (34,53 ± 2,76 mg/kg, n=4), que em animais sham, porém os efeitos foram mais intensos e prolongados na sepse. d-TC (26,50 ± 0,68 mg/kg (n=5), promoveu 30 por cento de redução em RTOF nos animais sham submetidos à infusão de nitroprussiato de sódio (10 mg/kg/min). O tratamento com S-metilisotiouréia (SMT, 13 mg/kg) antagonizou a redução de RTOF em animais sépticos. As menores doses de PANC e GAL que reduziram RTOF em 30 por cento foram semelhantes em todos os grupos experimentais estudados e GAL promoveu efeitos similares em todas as condições experimentais. A redução de RTOF produzida por PANC foi menor que a induzida por d-TC e foi antagonizada pela administração de SMT....

[Sensitive nonradioactive screening method for compounds interacting with alpha7-cholinoreceptor]. / Chuvstvitel'nyi neradioaktivnyi metod skrininga soedinenii, vzaimodeistvuiushchikh s neironnym alpha7-kholinoretseptorom.

A sensitive nonradioactive method for detection of substances interacting with the neuronal nicotinic acetylcholine alpha 7-type receptor (AChR) was proposed. The method uses biotinylated alpha-cobratoxin (Bt-CTX) and is based on the ability of the N-terminal ligand-binding extracellular domain (LBED) of AChR to interact with alpha-cobratoxin (CTX) as does the whole receptor. LBED was produced by heterologic expression of a gene fragment of the alpha 7 subunit of AChR from the rat brain in Escherichia coli cells sorbed on wells of a 96-well plate and incubated with Bt-CTX. The specifically bound Bt-CTX was determined by staining with streptavidin-peroxidase complex. The ability of other compounds to interact with alpha 7-AChR was checked according to the degree with which they inhibit Bt-CTX binding to LBED. Nicotine, carbamylcholine, d-tubocurarin, anabaseine, conotoxin ImI, and neurotoxin II were used as model compounds. The sensitivity of this method was comparable with that of the radioligand method (up to 10 pmol).

Óxido Nítrico produzido durante a sepse intensifica a redução nos valores da razão de "train-of-four" (TOF) induzida por d-Tubocurarina e pancurônio / Produced Nitric oxide during sepse intensifies the reduction in the values of the reason of “train-of-four” (TOF) induced for d-Tubocurarine and pancuronium

Durante a sepse, grandes quantidades de óxido nítrico (NO) são sintetizadas pela enzima NO sintase tipo II. Em preparações neuromusculares, o NO produz ou acentua a fadiga de transmissão. 'Train-of-four'(TOF) são pulsos elétricos de 2 Hz aplicados ao nervo motor durante 2 segundos (4 contrações musculares) e o quociente entre as tensões musculares produzidas pela quarta (T4) e primeira (T1) contrações musculares (RTOF = T4/T1) é utilizado para avaliação do bloqueio da transmissão neuromuscular. A redução de RTOF é determinada por bloqueio dos receptores nicotínicos (Nn) facilitatórios e por estimulação dos receptores muscarínicos (M2) inibitórios do terminal nervoso motor. Estudos utilizando preparações neuromusculares de animais sépticos tratadas com bloqueadores neuromusculares em doses que reduzem os valores de RTOF sem promover alterações nas amplitudes das contrações musculares a 0,2 Hz não foram executados. Assim, utilizando preparações nervo ciático-músculo tibial anterior de ratos, verificamos que as menores doses de d-tubocurarina (d-TC), pancurônio (PANC) e galamina (GAL) capazes de produzir 30 por cento de redução em RTOF nos animais sham foram, respectivamente: 54,29 ± 2,86 mg/kg (n=7), 69,02 ± 6,13 mg/kg (n=5), 2,94 ± 0,33 mg/kg (n=4). A dose de d-TC foi menor em animais sépticos (34,53 ± 2,76 mg/kg, n=4), que em animais sham, porém os efeitos foram mais intensos e prolongados na sepse. d-TC (26,50 ± 0,68 mg/kg (n=5), promoveu 30 por cento de redução em RTOF nos animais sham submetidos à infusão de nitroprussiato de sódio (10 mg/kg/min). O tratamento com S-metilisotiouréia (SMT, 13 mg/kg) antagonizou a redução de RTOF em animais sépticos. As menores doses de PANC e GAL que reduziram RTOF em 30 por cento foram semelhantes em todos os grupos experimentais estudados e GAL promoveu efeitos similares em todas as condições experimentais. A redução de RTOF produzida por PANC foi menor que a induzida por d-TC e foi antagonizada pela administração de SMT....