Synaptic transmission at nicotinic acetylcholine receptors in rat hippocampal organotypic cultures and slices.
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1. Whole-cell clamp recordings of the compound synaptic current elicited by afferent stimulation of Schaffer collaterals showed that blockade of the NMDA, AMPA and GABAA receptor-mediated components by 6-nitro-7-sulphamoyl- benzo(f)quinoxaline-2,3-dione (NBQX), 3-((R)-2-carboxypiperazine-4-yl)propyl-1-phosphonate (R-CPP) and picrotoxin, respectively, left a small residual current in 39 out of 41 CA1 pyramidal neurones in organotypic cultures and 9 out of 16 CA1 cells in acutely prepared slices. 2. This current represented 2. 9 +/- 0.4 % of the compound evoked synaptic response in organoypic cultures and 1.4 +/- 0.5 % in slices. It was characterized by a slightly rectifying I-V curve and a reversal potential of 3.4 +/- 5. 1 mV. 3. This residual current was insensitive to blockers of GABAB, purinergic, muscarinic and 5-HT3 receptors, but it was essentially blocked by the nicotinic receptor antagonist d-tubocurarine (91 +/- 4 % blockade; 20 microM), and partly blocked by alpha-bungarotoxin (200 nM) and methyllycaconitine (10 nM), two antagonists with a higher selectivity for alpha7 subunit-containing nicotinic receptors (48 +/- 3 % and 55 +/- 11 % blockade, respectively). 4. The residual current was of synaptic origin, since it occurred after a small delay; its amplitude depended upon the stimulation intensity and it was calcium dependent and blocked by the sodium channel antagonist tetrodotoxin. 5. We conclude that afferent stimulation applied in the stratum radiatum evokes in some hippocampal neurones a small synaptic current mediated by activation of neuronal nicotinic receptors. (+info)
Cholinergic and GABAergic regulation of nitric oxide synthesis in the guinea pig ileum.
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Nitric oxide (NO) synthesis was examined in intact longitudinal muscle-myenteric plexus preparations of the guinea pig ileum by determining the formation of [3H]citrulline during incubation with [3H]arginine. Spontaneous [3H]citrulline production after 30 min was 80-90 dpm/mg, which constituted approximately 1% of the tissue radioactivity. Electrical stimulation (10 Hz) led to a threefold increase in [3H]citrulline formation. Removal of calcium from the medium or addition of NG-nitro-L-arginine strongly inhibited both spontaneous and electrically induced production of [3H]citrulline. TTX reduced the electrically induced but not spontaneous [3H]citrulline formation. The electrically induced formation of [3H]citrulline was diminished by (+)-tubocurarine and mecamylamine and enhanced by scopolamine, which suggests that endogenous ACh inhibits, via muscarinic receptors, and stimulates, via nicotinic receptors, the NO synthesis in the myenteric plexus. The GABAA receptor agonist muscimol and GABA also reduced the electrically evoked formation of [3H]citrulline, whereas baclofen was without effect. Bicuculline antagonized the inhibitory effect of GABA. It is concluded that nitrergic myenteric neurons are equipped with GABAA receptors, which mediate inhibition of NO synthesis. (+info)
Properties and expression of Ca2+-activated K+ channels in H9c2 cells derived from rat ventricle.
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H9c2 is a clonal myogenic cell line derived from embryonic rat ventricle that can serve as a surrogate for cardiac or skeletal muscle in vitro. Using whole cell clamp with H9c2 myotubes, we observed that depolarizing pulses activated slow outward K+ currents and then slow tail currents. The K+ currents were abolished in a Ca2+-free external solution, indicating that they were Ca2+-activated K+ currents. They were blocked by apamin, a small-conductance Ca2+-activated K+ (SK) channel antagonist (IC50 = 6.2 nM), and by d-tubocurarine (IC50 = 49.4 microM). Activation of SK channels exhibited a bell-shaped voltage dependence that paralleled the current-voltage relation for L-type Ca2+ currents (ICa,L). ICa,L exhibited a slow time course similar to skeletal ICa, L, were unaffected by apamin, and were only slightly depressed by d-tubocurarine. RT-PCR analysis of the mRNAs revealed that rSK3, but not rSK1 or rSK2, was expressed in H9c2 myotubes but not in myoblasts. These results suggest that rSK3 channels are expressed in H9c2 myotubes and are primarily activated by ICa,L directly or indirectly via Ca2+-induced Ca2+ release from sarcoplasmic reticulum. (+info)
An autoradiographic analysis of [3H]alpha-bungarotoxin distribution in the rat brain after intraventricular injection.
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Purified alpha-bungarotoxin was isolated by chromatography and made radioactive with tritium ([3H]acetamidino-alpha-bungarotoxin). Infusions of [3H]alpha-bungarotoxin alone or preceded by tubocurarine or atropine were given into the third ventricle. 2. 12, or 24 h after injection the brains were prepared for autoradiography. Injections of alpha-bungarotoxin (radioinert) in buffer, or of [3H]parathyroid hormone in buffer, served as controls. The various patterns of labeling suggest the presence of nicotinic-cholinergic neurons within the arcuate and basolateral regions of the hypothalamus including the supraoptic and suprachiasmatic nuclei and, in addition, the central nucleus of the amygdala. (+info)
Activation and Ca2+ permeation of stably transfected alpha3/beta4 neuronal nicotinic acetylcholine receptor.
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The alpha3/beta4 rat neuronal nicotinic acetylcholine receptor, stably transfected in human embryonic kidney cells, was examined using the whole-cell-clamp technique and 2-dimensional confocal imaging. Application of agonists (nicotine, cytisine, epibatidine) activated a large (100-200 pA/pF) inwardly rectifying monovalent current, with little current at voltages between 0 and +40 mV. Rapid application of nicotine and cytisine indicated EC50 values of congruent with22 and congruent with64 microM, respectively, and suggested second order binding kinetics (Hill coefficient approximately 2). The time constant of desensitization (decay) of nicotine-activated current was concentration-dependent (typically approximately 10 s at 30 microM versus approximately 1.0 s at 100-1000 microM), but not voltage-dependent and was significantly smaller than the approximately 200 s reported for the alpha3/beta4 receptor expressed in Xenopus oocytes. Nicotine-activated current was rapidly and reversibly blocked by coapplication of mecamylamine and d-tubocurarine. At -80 mV holding potentials, the current was also suppressed by approximately 25% either upon complete removal or elevation of Ca2+ to 10 mM. Total replacement of Na+ by Ca2+ also completely blocked the current. On the other hand, evidence for permeation of Ca2+ was indicated by increased inward current at -40 mV upon elevation of Ca2+ from 2 to 10 mM, as well as a rise in the cytosolic Ca2+ proportional to the current carried by the receptor. These findings are consistent with the idea that Ca2+, in addition to its channel-permeating properties, may also regulate the receptor from an extracellular site. Our results suggest that the alpha3/beta4 neuronal nicotinic acetylcholine receptor, when stably expressed in human embryonic kidney 293 cells, has desensitization kinetics and Ca2+ regulatory mechanisms somewhat different from those described for the receptor expressed in Xenopus oocytes. (+info)
Molecular determinants of (+)-tubocurarine binding at recombinant 5-hydroxytryptamine3A receptor subunits.
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The 5-hydroxytryptamine type 3 (5-HT3) receptor is a transmitter-gated ion channel mediating neuronal excitation. The receptor native to neurons, or as a homopentameric assembly of 5-HT3A receptor subunits, displays a species-dependent pharmacology exemplified by a 1800-fold difference in the potency of (+)-tubocurarine [(+)-Tc] as an antagonist of the current response mediated by mouse and human receptor orthologs. Here, we attempt to identify amino acid residues involved in binding (+)-Tc by use of chimeric and mutant 5-HT3A subunits of mouse and human expressed in Xenopus laevis oocytes. Replacement of the entire extracellular N-terminal domain of the mouse 5-HT3A (m5-HT3A) subunit by that of the human ortholog and vice versa exchanged the differential potency of (+)-Tc, demonstrating the ligand binding site to be contained wholly within this region. Mutagenesis of multiple amino acid residues within a putative binding domain that exchanged nonconserved residues between mouse and human receptors shifted the apparent affinity of (+)-Tc in a reciprocal manner. The magnitude of the shift increased with the number of residues (3, 5, or 7) exchanged, with septuple mutations of m5-HT3A and human 5-HT3A subunits producing a 161-fold decrease and 53-fold increase in the apparent affinity of (+)-Tc, respectively. The effect of point mutations was generally modest, the exception being m5-HT3A D206E, which produced a 9-fold decrease in apparent affinity. We conclude that multiple amino acids within a binding loop of human and mouse 5-HT3A subunits influence the potency of (+)-Tc. (+info)
Target-dependent regulation of acetylcholine secretion at developing motoneurons in Xenopus cell cultures.
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1. Myocyte-dependent regulation of acetylcholine (ACh) quantal secretion from developing motoneurons was studied in day-3 Xenopus nerve-muscle co-cultures. Spontaneous synaptic currents (SSCs) were measured in manipulated synapses by using whole-cell voltage-clamped myocytes. Changes in SSC amplitude were assumed to reflect changes in the ACh content of secreted quantal packets. Compared with natural synapses, motoneurons without any contact with a myocyte (naive neurons) released ACh in smaller quantal packets. 2. Bipolar cultured motoneurons, which were in contact with a myocyte with one axon branch (contact-end) but remained free at another axon branch (free-end), were further used to examine quantal ACh secretion. The ACh quantal size recorded at free-end terminals was similar to that of naive neurons and was smaller than that at the contact-end, indicating that myocyte contact exerts differential regulation on quantal secretion in the same neuron. 3. Some of the neurons that formed a natural synapse with a myocyte continued to grow forward and ACh quantal secretion from the free growth cone was examined. The ACh quantal size recorded at free growth cones was inversely proportional to the distance to the natural synapse, implying localized regulation of quantal secretion by the myocyte. 4. Chronic treatment of day-1 cultures with veratridine and d-tubocurarine, respectively, increased and decreased the neurotrophic action of myocytes when assayed on day 3. 5. Taken together, these findings suggest that the myocyte is an important postsynaptic target in the regulation of quantal secretion and that the trophic action is spatially restricted to the neighbourhood of the neuromuscular junction. (+info)
Deep sedation and mechanical ventilation without paralysis for 3 weeks in normal beagles: exaggerated resistance to metocurine in gastrocnemius muscle.
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BACKGROUND: Patients in the intensive care unit may have muscle weakness in the recovery phase, and disuse atrophy may play a role in this weakness. To assess this problem, the authors measured changes in the potency of the nondepolarizing neuromuscular blocking agent metocurine in a canine model that involved 3 weeks of intensive care, nonparalyzing anesthesia with pentobarbital, and positive-pressure ventilation. METHODS: Six dogs were anesthetized with pentobarbital to a sufficient depth that spontaneous and reflex muscle movements were absent. Their tracheas were intubated, their lungs were mechanically ventilated, and they received round-the-clock intensive medical and nursing care for 3 weeks. Transduced gastrocnemius muscle responses to metocurine were determined weekly. A 4- to 15-min infusion of 148-4,300 microg/min (longer durations and greater concentrations on progressive weeks) yielded more than 80% paralysis. Serial metocurine plasma concentrations during the onset of the block and recovery provided data to determine pharmacokinetics using NONMEM. Metocurine plasma concentrations and the degree of paralysis were used to model the effect compartment equilibration constant, and the Hill equation was used to yield the slope factor and potency within the effect compartment. RESULTS: The metocurine effect compartment concentration associated with a 50% diminution of twitch height after 3 weeks was 1,716+/-1,208 ng/ml (mean +/- SD), which was significantly different from 257+/-34 ng/ml, the value on day 0. There were no pharmacokinetic differences. CONCLUSION: The absence of muscle tone and reflex responsiveness for 3 weeks was associated with exaggerated resistance to the neuromuscular blocker metocurine. (+info)