To compare the difference in action sites between mecamylamine (MEC) and hexamethonium (HEX) on nicotinic receptors of sympathetic neurons, we investigated the effects of MEC and HEX on the nicotine-induced currents in cultured superior cervical ganglion neurons by whole-cell patch clamp technique. The IC(50) of MEC and HEX for antagonizing the effect of 0.08 mmol/L nicotine was 0.0012 and 0.0095 mmol/L, respectively. Both MEC and HEX accelerated the desensitization of nicotinic receptors. Furthermore, by comparing their effects at holding potentials 30, 70 and 110 mV, it was indicated that their suppressing effect on the nicotine-induced currents was voltage-dependent. However, different from that of HEX, the inhibitory effect of MEC increased with administering the mixture of MEC and nicotine at intervals of 3 min, indicating a use-dependent effect of MEC. It is concluded that the action site of MEC on nicotinic receptors of sympathetic neurons is different from that of HEX.
Animals ; Animals, Newborn ; Cells, Cultured ; Hexamethonium ; pharmacology ; Mecamylamine ; pharmacology ; Neurons ; drug effects ; physiology ; Nicotinic Antagonists ; pharmacology ; Patch-Clamp Techniques ; Rats ; Rats, Wistar ; Receptors, Nicotinic ; drug effects ; physiology ; Superior Cervical Ganglion ; cytology ; physiology

BACKGROUND: Intrathecal gabapentin is effective on nociceptive states evoked by tissue injury. In addition, gabapentin interacts synergistically with clonidine at the spinal level, suggesting that a mechanism of gabapentin may be related to spinal adrenoceptors. However, it has not been established whether this drug is associated with cholinergic receptors. The aim of this study was to examine the role of spinal adrenergic and cholinergic receptors on the antinociceptive action of intrathecal gabapentin. METHODS: Rats were implanted with lumbar intrathecal catheters. For a nociceptive test, 50nl of 5% formalin solution was injected into the hindpaw. The effect of intrathecal gabapentin, administered 10 min before the formalin injection, was assessed. Next, antagonistic effects of intrathecal prazosin, yohimbine, atropine and mecamylamine for the action of intrathecal gabapentin were evaluated. RESULTS: Formalin injection caused a biphasic incidence of flinching of the injected paw. Intrathecal gabapentin produced a dose-dependent suppression of only the phase 2 flinching response in the formalin test. Intrathecal atropine, but not prazosin, yohimbine nor mecamylamine, reversed the antinociception of intrathecal gabapentin. CONCLUSIONS: The antinociceptive effect of intrathecal gabapentin on facilitated states may be mediated through the muscarinic receptor but by neither the nicotinic receptor nor the adrenergic receptor at the spinal level.
Animals ; Atropine ; Catheters ; Cholinergic Antagonists* ; Clonidine ; Formaldehyde ; Incidence ; Mecamylamine ; Nociception ; Pain Measurement ; Prazosin ; Rats ; Receptors, Adrenergic ; Receptors, Cholinergic ; Receptors, Muscarinic ; Receptors, Nicotinic ; Spinal Cord ; Yohimbine

The signal pathways involved in the regulation of AP-1 DNA binding activity in long-term nicotine stimulated bovine adrenal medullary chromaffin (BAMC) cells have not been well characterized. To understand the involvement of second messengers in the regulation of AP-1 DNA binding activity, the present study was designed to define the time-course for inhibition of nicotine-induced responses by cholinergic antagonists, Ca2+ and calmodulin (CaM) antagonists, and calcium/calmodulin-dependent protein kinase (CaMK) II inhibitor using electrophoretic mobility shift assay. Nicotine (10microM) stimulation increased AP-1 DNA binding activity at 24 hr after treatment. Posttreatment with hexamethonium (1 mM) plus atropine (1microM) (HA), nimodipine (1microM), or calmidazolium (1microM) at 0.5, 3, and 6 hr after the nicotine treatment significantly inhibited the AP-1 DNA binding activity increased by long-term nicotine stimulation. However, posttreatment with HA, nimodipine, or calmidazolium at 9 or 12 hr after the nicotine treatment did not affect the nicotine-induced increase of AP-1 DNA binding activity. The pretreatment of BAMC cells with various concentrations of KN-62 inhibited the increase of AP-1 DNA binding activity induced by nicotine in a concentration-dependent manner. KN-62 (10microM) posttreatment beginning at 0.5, 3, or 6 hr after the nicotine treatment significantly inhibited the increase of AP-1 DNA binding activity. However, KN-62 posttreatment beginning at 9 or 12 hr after the nicotine treatment did not affect the increase of AP-1 DNA binding activity. This study suggested that stimulation (for at least 6 hr) of nicotinic receptors on BAMC cells was necessary for increase of AP-1 DNA binding activity, and activation of Ca2+, CaM, and CaMK II up to 6 hr at least seemed to be required for the increase of nicotine-induced AP-1 DNA binding activity.
Atropine ; Calmodulin ; Cholinergic Antagonists ; Chromaffin Cells* ; DNA* ; Electrophoretic Mobility Shift Assay ; Hexamethonium ; Nicotine* ; Nimodipine ; Protein Kinases ; Receptors, Nicotinic ; Second Messenger Systems* ; Signal Transduction ; Transcription Factor AP-1*

AIMTo investigate the roles of acetylcholine (ACh) receptors in the rapid effects of corticosterone (CORT) on the presympathetic neurons in the rostral ventrolateral medulla (RVLM) of rats, and study the non-genomic mechanism of glucocorticoid (GC) in the integration of sympathetic cardiovascular activity.

METHODSThe effects of microelectrophoresis of CORT on the discharge of the presympathetic neurons in the RVLM were observed by extracellular recording in urethane-anaesthetized rats. The responses of atropine (a blocker for M type of ACh receptor, ATR), d-tubocurarine (a blocker for N1 type of ACh receptor, d-TC) and hexamethonium (a blocker for N2 type of ACh receptor, C6) to the effects of CORT on the presympathetic neurons were investigated respectively.

RESULTSTotally 33 presympathetic neurons in the RVLM were recorded. Among them the firing rate of 25 (76%) presympathetic neurons was increased by microelectrophoresis of CORT. The effects of CORT were also positively correlated with the currents. In the other 8 presympathetic neurons, had was shown no effect after microelectrophoresis of CORT. In 10 presympathetic neurons, which discharge was increased by CORT, microelectrophoresis of ATR decreased the firing rate of these presympathetic neurons (P < 0.05), and did not fully block the excitatory effect induced by CORT. In both 7 and 6 presympathetic neurons, application of d-TC and C6 had no effect on these neurons respectively, and did not fully block the excitatory effect induced by CORT.

CONCLUSIONCORT had rapid excitatory effects on the presympathetic neurons in the RVLM, which effect might be independent on ACh receptors.

Animals ; Cholinergic Antagonists ; pharmacology ; Corticosterone ; pharmacology ; Electrophoresis, Microchip ; Male ; Medulla Oblongata ; drug effects ; physiology ; Neuromuscular Nondepolarizing Agents ; pharmacology ; Neurons ; drug effects ; physiology ; Nicotinic Antagonists ; pharmacology ; Rats ; Rats, Sprague-Dawley ; Receptors, Cholinergic ; physiology

Recent studies have reported that the "cholinergic anti-inflammatory pathway" regulates peripheral inflammatory responses via alpha7 nicotinic acetylcholine receptors (alpha7 nAChRs) and that acetylcholine and nicotine regulate the expression of proinflammatory mediators such as TNF-alpha and prostaglandin E2 in microglial cultures. In a previous study we showed that ATP released by beta-amyloid-stimulated microglia induced reactive oxygen species (ROS) production, in a process involving the P2X7 receptor (P2X7R), in an autocrine fashion. These observations led us to investigate whether stimulation by nicotine could regulate fibrillar beta amyloid peptide (1-42) (fA beta(1-42))-induced ROS production by modulating ATP efflux-mediated Ca2+ influx through P2X7R. Nicotine inhibited ROS generation in fA beta(1-42)-stimulated microglial cells, and this inhibition was blocked by mecamylamine, a non-selective nAChR antagonist, and a-bungarotoxin, a selective alpha7 nAChR antagonist. Nicotine inhibited NADPH oxidase activation and completely blocked Ca2+ influx in fA beta(1-42)-stimulated microglia. Moreover, ATP release from fA beta(1-42)-stimulated microglia was significantly suppressed by nicotine treatment. In contrast, nicotine did not inhibit 2',3'-O-(4-benzoyl)-benzoyl ATP (BzATP)-induced Ca2+ influx, but inhibited ROS generation in BzATP-stimulated microglia, indicating an inhibitory effect of nicotine on a signaling process downstream of P2X7R. Taken together, these results suggest that the inhibitory effect of nicotine on ROS production in fA beta(1-42)-stimulated microglia is mediated by indirect blockage of ATP release and by directly altering the signaling process downstream from P2X7R.
Adenosine Triphosphate/analogs & derivatives/metabolism/pharmacology ; Amyloid/*metabolism ; Amyloid beta-Protein/*pharmacology ; Animals ; Calcium/metabolism ; Enzyme Activation/drug effects ; Microglia/cytology/*drug effects/enzymology/*metabolism ; NADPH Oxidase/metabolism ; Nicotine/pharmacology ; Nicotinic Antagonists/pharmacology ; Peptide Fragments/*pharmacology ; Rats ; Rats, Sprague-Dawley ; Reactive Oxygen Species/*metabolism ; Receptors, Nicotinic/*metabolism ; Receptors, Purinergic P2/metabolism

BACKGROUND: The serotonin type 3 receptors are diffusely distributed in both the central and the peripheral nervous system. Physiological and pathophysiological processes thought to be mediated by this receptor include nausea and vomiting, peripheral nociception and central antinociception, conditioned aversion response to drugs, anxiety, and cognition. Because of the structural similarity between the nicotinic acetylcholine receptor and the 5HT3 receptor, we investigated the effects of clinically used neuromuscular blockers on the 5HT3 receptor function related with PONV. METHODS: A cDNA clone encoding the full length murine 5HT3a receptor was subcloned into an oocyte expression vector and 50 ng of cRNA transcribed in vitro injected per oocyte. After 24 72 h incubation, oocytes were placed into a recording chamber continuously perfused with frog Ringer's solution and electrophysiological recordings were obtained by the two electrode voltage clamp technique. Serotonin with or without the various drugs were bath applied by a computer controlled solenoid valve. Peak currents induced by the drug applications were measured and dose responses were obtained. RESULTS: The 5HT3 receptor expression in Xenopus oocyte was identified by the pharmacologic tools. Serotonin induced rapid inward currents, and thus was showed dose-dependent: KD = 2.5 micrometer, Hill coefficiency = 2.09. Inhibition by the neuromuscular blockers showed dose-dependence and their inhibitory potency on 5HT3 receptor (IC50) was in order of d-tubocurarine (0.046 micrometer) > vecuronium (16.32 micrometer) > gallamine (1,169 micrometer). CONCLUSIONS: There was a different inhibitory effect of nicotinic cholinergic antagonists, clinically used neuromuscular blockers, on the 5HT3 receptor and a judicious selection of them might contribute to reducing the incidence of PONV clinically.
Anxiety ; Baths ; Cholinergic Antagonists ; Clone Cells ; Cognition ; DNA, Complementary ; Electrodes ; Gallamine Triethiodide ; Incidence ; Nausea ; Neuromuscular Blockade* ; Neuromuscular Blocking Agents* ; Nociception ; Oocytes* ; Peripheral Nervous System ; Postoperative Nausea and Vomiting ; Receptors, Nicotinic ; RNA, Complementary ; Serotonin* ; Tubocurarine ; Vecuronium Bromide ; Vomiting ; Xenopus*

BACKGROUND: The serotonin type 3 receptors are diffusely distributed in both the central and the peripheral nervous system. Physiological and pathophysiological processes thought to be mediated by this receptor include nausea and vomiting, peripheral nociception and central antinociception, conditioned aversion response to drugs, anxiety, and cognition. Because of the structural similarity between the nicotinic acetylcholine receptor and the 5HT3 receptor, we investigated the effects of clinically used neuromuscular blockers on the 5HT3 receptor function related with PONV. METHODS: A cDNA clone encoding the full length murine 5HT3a receptor was subcloned into an oocyte expression vector and 50 ng of cRNA transcribed in vitro injected per oocyte. After 24 72 h incubation, oocytes were placed into a recording chamber continuously perfused with frog Ringer's solution and electrophysiological recordings were obtained by the two electrode voltage clamp technique. Serotonin with or without the various drugs were bath applied by a computer controlled solenoid valve. Peak currents induced by the drug applications were measured and dose responses were obtained. RESULTS: The 5HT3 receptor expression in Xenopus oocyte was identified by the pharmacologic tools. Serotonin induced rapid inward currents, and thus was showed dose-dependent: KD = 2.5 micrometer, Hill coefficiency = 2.09. Inhibition by the neuromuscular blockers showed dose-dependence and their inhibitory potency on 5HT3 receptor (IC50) was in order of d-tubocurarine (0.046 micrometer) > vecuronium (16.32 micrometer) > gallamine (1,169 micrometer). CONCLUSIONS: There was a different inhibitory effect of nicotinic cholinergic antagonists, clinically used neuromuscular blockers, on the 5HT3 receptor and a judicious selection of them might contribute to reducing the incidence of PONV clinically.
Anxiety ; Baths ; Cholinergic Antagonists ; Clone Cells ; Cognition ; DNA, Complementary ; Electrodes ; Gallamine Triethiodide ; Incidence ; Nausea ; Neuromuscular Blockade* ; Neuromuscular Blocking Agents* ; Nociception ; Oocytes* ; Peripheral Nervous System ; Postoperative Nausea and Vomiting ; Receptors, Nicotinic ; RNA, Complementary ; Serotonin* ; Tubocurarine ; Vecuronium Bromide ; Vomiting ; Xenopus*

Spinal gabapentin has been known to show the antinociceptive effect. Although several assumptions have been suggested, mechanisms of action of gabapentin have not been clearly established. The present study was undertaken to examine the action mechanisms of gabapentin at the spinal level. Male SD rats were prepared for intrathecal catheterization. The effect of gabapentin was assessed in the formalin test. After pretreatment with many classes of drugs, changes of effect of gabapentin were examined. General behaviors were also observed. Intrathecal gabapentin produced a suppression of the phase 2 flinching, but not phase 1 in the formalin test. The antinociceptive action of intrathecal gabapentin was reversed by intrathecal NMDA, AMPA, D-serine, CGS 15943, atropine, and naloxone. No antagonism was seen following administration of bicuculline, saclofen, prazosin, yohimbine, mecamylamine, L-leucine, dihydroergocristine, or thapsigargin. Taken together, intrathecal gabapentin attenuated only the facilitated state. At the spinal level, NMDA receptor, AMPA receptor, nonstrychnine site of NMDA receptor, adenosine receptor, muscarinic receptor, and opioid receptor may be involved in the antinociception of gabapentin, but GABA receptor, L-amino acid transporter, adrenergic receptor, nicotinic receptor, serotonin receptor, or calcium may not be involved.
Acetic Acids/administration & dosage ; Acetic Acids/metabolism ; Acetic Acids/pharmacology* ; Adrenergic Antagonists/metabolism ; Adrenergic alpha-Antagonists/metabolism ; Analgesics/administration & dosage ; Analgesics/metabolism ; Analgesics/pharmacology* ; Animals ; Atropine/metabolism ; Dihydroergocristine/metabolism ; Enzyme Inhibitors/metabolism ; Excitatory Amino Acid Agonists/metabolism ; GABA Antagonists/metabolism ; Injections, Spinal ; Leucine/metabolism ; Male ; Mecamylamine/metabolism ; Muscarinic Antagonists/metabolism ; N-Methylaspartate/metabolism ; Naloxone/metabolism ; Narcotic Antagonists/metabolism ; Nicotinic Antagonists/metabolism ; Pain Measurement ; Quinazolines/metabolism ; Rats ; Rats, Sprague-Dawley ; Serine/metabolism ; Spinal Cord/drug effects* ; Thapsigargin/metabolism ; Triazoles/metabolism ; alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid/metabolism

OBJECTIVESTo investigate the neuroprotective function of alpha 3 nicotinic acetylcholine receptor (nAChR) by inhibiting the gene expression in human neuroblastoma (SH-SY5Y) cells using small interference RNA (siRNA).

METHODSThe siRNA coding oligonucleotide sequences targeting alpha 3 nAChR were designed and synthesized. The annealed product was cloned into pSilencer 3.1-H1 neo vector. The recombinant alpha 3 nAChR pSilencer 3.1-H1 neo vector was transfected into the SH-SY5Y cells. The stable clones were screened by G418 medium, and the levels of alpha 3 nAChR mRNA and protein were monitored by using real-time PCR and Western blotting, respectively. After the SH-SY5Y cells with siRNA treatment were exposed to 1 micromol/L Abeta(1-42), MTT [3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide], SOD, GSH-px and the lipid peroxidation were measured by spectrophotometry.

RESULTSCompared with the controls, the expression levels of mRNA and protein in the stable SH-SY5Y clone cells transfected with the recombinant alpha 3 nAChR pSilencer 3.1-H1 neo vector were decreased with inhibitory efficiency of 98% and 66%, respectively, the MTT reduction decreased; the product of lipid peroxidation was increased and the activities of SOD and GSH-px were decreased. Biologically, the gene expression inhibition of alpha 3 nAChR enhanced the toxicity induced by Abeta in SH-SY5Y cells.

CONCLUSIONSThe expression inhibition of alpha 3 nAChR as a result of recombinant alpha 3 nAChR siRNA can induce oxidative stress and improve the toxicity of Abeta on SH-SY5Y cells, indicating that alpha 3 nAChR may play a significant neuroprotective role in the pathogenesis of Alzheimer disease.

Amyloid beta-Peptides ; pharmacology ; Cell Line, Tumor ; Cell Membrane ; drug effects ; Gene Expression Regulation ; drug effects ; Humans ; Neuroblastoma ; pathology ; Oxidation-Reduction ; drug effects ; Peptide Fragments ; pharmacology ; RNA Interference ; immunology ; RNA, Small Interfering ; pharmacology ; Receptors, Nicotinic ; drug effects ; genetics ; metabolism ; Superoxide Dismutase ; antagonists & inhibitors ; genetics ; metabolism

OBJECTIVETo investigate the efficacy of continuous propofol infusion via the common carotid artery for general anesthesia.

METHODSForty adult patients scheduled for abdominal surgery were randomly assigned into 2 groups to receive propopol via the common carotid artery (IC group, n=20) or via the median cubital vein (IV group, n=20). Anesthesia was induced with intravenous administration of drugs and maintained with continuous propofol infusion via the common carotid artery or the median cubital vein, with the CSI stabilized at 40-/+5 till the end of the operation. During the anesthesia, intravenous injection of fentanyl (3 microg.kg(-1).h(-1)) and vecuronium (50 microg.kg(-1).h(-1)) were given intermittently to maintain the analgesia and muscular relaxation. The dose of propofol used, hemodynamics and recovery of the patients were observed.

RESULTSThe dose of propofol used during the surgery to maintain a CSI of 40-/+5 was significantly lower in group IC and than in group IV (2.57-/+0.67 vs 5.72-/+1.37 mg.kg(-1).h(-1), P<0.01). In group IC, the blood pressure was elevated in more than half of the patients and in some cases, the elevation exceeded one third of baseline value and needed intervention with hypotensive drugs. In the IV group, the patients' blood pressure remained stable and varied within the amplitude of 15% of the baseline level. Recovery of spontaneous breathing and consciousness was more quickly in group IC than in group IV (P<0.05).

CONCLUSIONLoss of consciousness and nervous reflex can be achieved with propofol infusion via the common carotid artery, which reduces propofol dose by about 50% in comparison with intravenous infusion and allows more rapid recovery of spontaneous breath and consciousness.

Abdomen ; surgery ; Adult ; Aged ; Analgesics, Opioid ; administration & dosage ; Anesthesia, General ; methods ; Carotid Artery, Common ; Female ; Fentanyl ; administration & dosage ; Humans ; Hypnotics and Sedatives ; administration & dosage ; Infusions, Intra-Arterial ; Male ; Middle Aged ; Nicotinic Antagonists ; administration & dosage ; Propofol ; administration & dosage ; Treatment Outcome ; Vecuronium Bromide ; administration & dosage