Inhibitory effect of glucocorticoids on human-cloned 5-hydroxytryptamine3A receptor expressed in xenopus oocytes

BACKGROUND: Methylprednisolone, dexamethasone, and other glucocorticoids have been found effective against nausea and vomiting induced by chemotherapy and surgery. Although the specific 5-hydroxytriptamine3 (5-HT3) receptor antagonists such as ondansetron and ramosetron are used as antiemetics, reports show that the use of 5-HT3 receptor antagonists with some glucocorticoids brings additional effects. Glucocorticoids are reported to be antiemetic. The effect of glucocorticoids on 5-HT3 receptor, however, has not been well characterized. This study was designed to examine whether dexamethasone and methylprednisolone had direct effects on human-cloned 5-HT3A receptor expressed in Xenopus oocytes. METHODS: Homomeric human-cloned 5-HT3A receptor was expressed in Xenopus oocytes. The authors used the two-electrode voltage-clamping technique to study the effect of methylprednisolone and dexamethasone on 5-HT-induced current. RESULTS: Both dexamethasone and methylprednisolone concentration-dependently attenuated 5-HT-induced current. Dexamethasone inhibited 2 microm 5-HT-induced current, which was equivalent to EC30 concentration for 5-HT3A receptor, with an inhibitory concentration 50% of 5.29 +/- 1.02 microm. Methylprednisolone inhibited 2 microm 5-HT-induced current with an inhibitory concentration 50% of 1.07 +/- 0.15 mm. The mode of inhibition with either dexamethasone or methylprednisolone was noncompetitive and voltage-independent. When administered together with the 5-HT3 receptor antagonists, ramosetron or metoclopramide, both glucocorticoids showed an additive effect on 5-HT3 receptor. CONCLUSION: The glucocorticoids had a direct inhibitory effect on 5-HT3 receptors. The combined effect of glucocorticoids and the 5-HT3 receptor antagonists seems additive.     

The Diverse Actions of Volatile and Gaseous Anesthetics on Human-cloned 5-Hydroxytryptamine3 Receptors Expressed in Xenopus Oocytes

Background: General anesthetics can modulate the 5-hydroxytryptamine type 3 (5-HT3) receptor, which may be involved in processes mediating nausea and vomiting, and peripheral nociception. The effects of the new volatile anesthetic sevoflurane and the gaseous anesthetics nitrous oxide (N2O) and xenon (Xe) on the 5-HT3 receptor have not been well-characterized.

Methods: Homomeric human-cloned 5-HT3A receptors were expressed in Xenopus oocytes. The effects of halothane, isoflurane, sevoflurane, N2O, and Xe on 5-HT-induced currents were studied using a two-electrode, voltage clamping technique.

Results: Halothane (1%) and isoflurane (1%) potentiated 1 [mu]m 5-HT-induced currents to 182 +/- 12 and 117 +/- 2%, respectively. In contrast, sevoflurane (1%), N2O (70%), and Xe (70%) inhibited 5-HT-induced currents to 76 +/- 1, 77 +/- 4, and 34 +/- 4%, respectively. The inhibitory effects were noncompetitive for sevoflurane and competitive for N2O and Xe. None of these inhibitory effects showed voltage dependency.     

The diverse actions of volatile and gaseous anesthetics on human-cloned 5-hydroxytryptamine3 receptors expressed in Xenopus oocytes

BACKGROUND: General anesthetics can modulate the 5-hydroxytryptamine type 3 (5-HT3) receptor, which may be involved in processes mediating nausea and vomiting, and peripheral nociception. The effects of the new volatile anesthetic sevoflurane and the gaseous anesthetics nitrous oxide (N2O) and xenon (Xe) on the 5-HT3 receptor have not been well-characterized. METHODS: Homomeric human-cloned 5-HT3A receptors were expressed in Xenopus oocytes. The effects of halothane, isoflurane, sevoflurane, N2O, and Xe on 5-HT-induced currents were studied using a two-electrode, voltage clamping technique. RESULTS: Halothane (1%) and isoflurane (1%) potentiated 1 mum 5-HT-induced currents to 182 +/- 12 and 117 +/- 2%, respectively. In contrast, sevoflurane (1%), N2O (70%), and Xe (70%) inhibited 5-HT-induced currents to 76 +/- 1, 77 +/- 4, and 34 +/- 4%, respectively. The inhibitory effects were noncompetitive for sevoflurane and competitive for N2O and Xe. None of these inhibitory effects showed voltage dependency. CONCLUSION: Inhalational general anesthetics produce diverse effects on the 5-HT3 receptor. Both halothane and isoflurane enhanced 5-HT3 receptor function in a concentration-dependent manner, which is consistent with previous studies. Sevoflurane inhibited the 5-HT3 receptor noncompetitively, whereas N2O and Xe inhibited the 5-HT3 receptor competitively, suggesting the inhibitory mechanism of sevoflurane might be different from those of N2O and Xe.     

In Vitro Electrophysiologic Effects of Morphine in Rabbit Ventricular Myocytes

Background: Morphine is widely used in patients undergoing surgical operations and is also reported to mediate cardioprotection of preconditioning. The current study determined effects of morphine at therapeutic to pharmacologic concentrations on cardiac action potential, L-type Ca2+ current (ICa.L), delayed rectifier K+ current (IK), and inward rectifier K+ current (IK1) in isolated rabbit ventricular myocytes.

Methods: Ventricular myocytes were enzymatically isolated from rabbit hearts. Action potential and membrane currents were recorded in current and voltage clamp modes.

Results: Morphine at concentrations from 0.01 to 1 [mu]m significantly prolonged cardiac action potential, and at 0.1 and 1 [mu]m slightly but significantly hyperpolarized the resting membrane potential. In addition, morphine at 0.1 [mu]m significantly augmented ICa.L (at +10 mV) from 5.9 +/- 1.9 to 7.3 +/- 1.7 pA/pF (by 23%; P < 0.05 vs. control) and increased IK1 (at -60 mV) from 2.8 +/- 1.0 to 3.5 +/- 0.9 pA/pF (by 27%; P < 0.05 vs. control). Five [mu]m naltrindole (a selective [delta]-opioid receptor antagonist) or 5 [mu]m norbinaltorphimine (a selective [kappa]-opioid receptor antagonist) prevented the increase in ICa.L induced by morphine, but 5 [mu]m CTOP (a selective [mu]-opioid receptor antagonist) did not. The three types of opioid antagonists did not affect the augmentation of IK1 by morphine. Morphine had no effect on IK.     

Droperidol Inhibits GABAA and Neuronal Nicotinic Receptor Activation

Background: Droperidol is used in neuroleptanesthesia and as an antiemetic. Although its antiemetic effect is thought to be caused by dopaminergic inhibition, the mechanism of droperidol's anesthetic action is unknown. Because [gamma]-aminobutyric acid type A (GABAA) and neuronal nicotinic acetylcholine receptors (nAChRs) have been implicated as putative targets of other general anesthetic drugs, the authors tested the ability of droperidol to modulate these receptors.

Methods: [gamma]-Aminobutyric acid type A [alpha]1[beta]1[gamma]2 receptor, [alpha]7 and [alpha]4[beta]2 nAChRs were expressed in Xenopus oocytes and studied with two-electrode voltage clamp recording. The authors tested the ability of droperidol at concentrations from 1 nm to 100 [mu]m to modulate activation of these receptors by their native agonists.

Results: Droperidol inhibited the GABA response by a maximum of 24.7 +/- 3.0%. The IC50 for inhibition was 12.6 +/- 0.47 nm droperidol. At high concentrations, droperidol (100 [mu]m) activates the GABAA receptor in the absence of GABA. Inhibition of the GABA response is significantly greater at hyperpolarized membrane potentials. The activation of the [alpha]7 nAChR is also inhibited by droperidol, with an IC50 of 5.8 +/- 0.53 [mu]m. The Hill coefficient is 0.95 +/- 0.1. Inhibition is noncompetitive, and membrane voltage dependence is insignificant.     

Morphine Preconditions Purkinje Cells against Cell Death under In Vitro Simulated Ischemia-Reperfusion Conditions

Background: Morphine pretreatment via activation of [delta]1-opioid receptors induces cardioprotection. In this study, the authors determined whether morphine preconditioning induces ischemic tolerance in neurons.

Methods: Cerebellar brain slices from adult Sprague-Dawley rats were incubated with morphine at 0.1-10 [mu]m in the presence or absence of various antagonists for 30 min. They were then kept in morphine- and antagonist-free buffer for 30 min before they were subjected to simulated ischemia (oxygen-glucose deprivation) for 20 min. After being recovered in oxygenated artificial cerebrospinal fluid for 5 h, they were fixed for morphologic examination to determine the percentage of undamaged Purkinje cells.

Results: The survival rate of Purkinje cells was significantly higher in slices preconditioned with morphine (>= 0.3 [mu]m) before the oxygen-glucose deprivation (57 +/- 4% at 0.3 [mu]m morphine) than that of the oxygen-glucose deprivation alone (39 +/- 3%, P < 0.05). This morphine preconditioning-induced neuroprotection was abolished by naloxone, a non-type-selective opioid receptor antagonist, by naltrindole, a selective [delta]-opioid receptor antagonist, or by 7-benzylidenenaltrexone, a selective [delta]1-opioid receptor antagonist. However, the effects were not blocked by the [mu]-, [kappa]-, or [delta]2-opioid receptor antagonists, [beta]-funaltrexamine, nor-binaltorphimine, or naltriben, respectively. Morphine preconditioning-induced neuroprotection was partially blocked by the selective mitochondrial adenosine triphosphate-sensitive potassium channel antagonist, 5-hydroxydecanoate, or the mitochondrial electron transport inhibitor, myxothiazol. None of the inhibitors used in this study alone affected the simulated ischemia-induced neuronal death.     

The inhibitory effects of tramadol on 5-hydroxytryptamine type 2C receptors expressed in Xenopus oocytes

Although tramadol is widely available as an analgesic, its mechanism of antinociception remains unresolved. Serotonin (5-hydroxytryptamine, 5-HT) is a monoaminergic neurotransmitter that modulates numerous sensory, motor, and behavioral processes. The 5-HT type 2C receptor (5-HT(2C)R) is one of the major 5-HT receptor subtypes and is implicated in many important effects of 5-HT, including pain, feeding, and locomotion. In this study, we used a whole-cell voltage clamp to examine the effects of tramadol on 5-HT-induced Ca(2+)-activated Cl(-) currents mediated by 5-HT(2C)R expressed in Xenopus oocytes. Tramadol inhibited 5-HT-induced Cl(-) currents at pharmacologically relevant concentrations. The protein kinase C (PKC) inhibitor, bisindolylmaleimide I (GF109203x), did not abolish the inhibitory effects of tramadol on the 5-HT(2C)R-mediated events. We also studied the effects of tramadol on [(3)H]5-HT binding to 5-HT(2C)R expressed in Xenopus oocytes, and found that it inhibited the specific binding of [(3)H]5-HT to 5-HT(2C)R. Scatchard analysis of [(3)H]5-HT binding revealed that tramadol altered the apparent dissociation constant for binding without changing maximal binding, indicating competitive inhibition. The results suggest that tramadol inhibits 5-HT(2C)R function, and the mechanism of this inhibitory effect seems to involve competitive displacement of the 5-HT binding to the 5-HT(2C)R, rather than via activation of the PKC pathway. IMPLICATIONS: We examined the effects of tramadol on 5-hydroxytryptamine type 2C receptor (5-HT(2C)R) expressed in Xenopus oocytes. Tramadol inhibited 5-HT(2C)R function and the specific binding of [(3)H]5-HT to 5-HT(2C)R in a competitive manner. From these data, the mechanism of the inhibitory effect on 5-HT(2C)R might involve the competitive displacement of 5-HT binding to the 5-HT(2C)R.     

Mechanisms of Desflurane-induced Preconditioning in Isolated Human Right Atria In Vitro

Background: The authors examined the role of adenosine triphosphate-sensitive potassium (KATP) channels, adenosine A1 receptor, and [alpha] and [beta] adrenoceptors in desflurane-induced preconditioning in human myocardium, in vitro.

Methods: The authors recorded isometric contraction of human right atrial trabeculae suspended in oxygenated Tyrode's solution (34[degrees]C; stimulation frequency, 1 Hz). Before a 30-min anoxic period, 3, 6, and 9% desflurane was administered during 15 min. Desflurane, 6%, was also administered in the presence of 10 [mu]m glibenclamide, a KATP channels antagonist; 10 [mu]m HMR 1098, a sarcolemmal KATP channel antagonist; 800 [mu]m 5-hydroxy-decanoate (5-HD), a mitochondrial KATP channel antagonist; 1 [mu]m phentolamine, an [alpha]-adrenoceptor antagonist; 1 [mu]m propranolol, a [beta]-adrenoceptor antagonist; and 100 nm 8-cyclopentyl-1,3-dipropylxanthine (DPX), the adenosine A1 receptor antagonist. Developed force at the end of a 60-min reoxygenation period was compared (mean +/- SD).

Results: Desflurane at 3% (95 +/- 13% of baseline), 6% (86 +/- 6% of baseline), and 9% (82 +/- 6% of baseline) enhanced the recovery of force after 60 min of reoxygenation as compared with the control group (50 +/- 11% of baseline). Glibenclamide (60 +/- 12% of baseline), 5-HD (57 +/- 21% of baseline), DPX (63 +/- 19% of baseline), phentolamine (56 +/- 20% of baseline), and propranolol (63 +/- 13% of baseline) abolished desflurane-induced preconditioning. In contrast, HMR 1098 (85 +/- 12% of baseline) did not modify desflurane-induced preconditioning.     

Structural Requirements of Human Ether-a-go-go-related Gene Channels for Block by Bupivacaine

Background: Local anesthetics interact with human ether-a-go-go-related gene (HERG) channels via the aromatic amino acids Y652 and F656 in the S6 region. This study aimed to establish whether the residues T623, S624, and V625 residing deeper within the pore are also involved in HERG channel block by bupivacaine. In addition, the study aimed to further define the role of the aromatic residues Y652 and F656 in bupivacaine inhibition by mutating these residues to threonine.

Methods: Alanine and threonine mutants were generated by site-directed mutagenesis. Electrophysiologic and pharmacologic properties of wild-type and mutant HERG channels were established using two-electrode voltage-clamp recordings of Xenopus laevis oocytes expressing HERG channels.

Results: Tail currents at -120 mV through HERG wild-type channels were inhibited with an IC50 value of 132 +/- 22 [mu]m (n = 33). Bupivacaine (300 [mu]m) inhibited wild-type tail currents by 62 +/- 12% (n = 7). Inhibition of HERG tail currents by bupivacaine (300 [mu]m) was reduced by all mutations (P < 0.001). The effect was largest for F656A (inhibition 5 +/- 2%, n = 6) in the lower S6 region and for T623A (inhibition 13 +/- 4%, n = 9) near the selectivity filter. Introducing threonine at positions 656 and 652 significantly reduced inhibition by bupivacaine compared with HERG wild type (P < 0.001).     

Meperidine Exerts Agonist Activity at the α2B-Adrenoceptor Subtype

Background: The opioid agonist meperidine has actions, such as antishivering, that are more pronounced than those of other opioid agonists and that are not blocked with nonselective opioid antagonists. Agonists at the [alpha]2 adrenoceptors, such as clonidine, are very effective antishivering drugs. Preliminary evidence also indicates that meperidine interacts with [alpha]2 adrenoceptors. The authors therefore studied the ability of meperidine to bind and activate each of the [alpha]2-adrenoceptor subtypes in a transfected cell system.

Methods: The ability of meperidine to bind to and inhibit forskolin-stimulated cyclic adenosine monophosphate formation as mediated by the three [alpha]2-adrenoceptor subtypes transiently transfected into COS-7 cells has been tested. The ability of the opioid antagonist naloxone and the [alpha]2-adrenoceptor antagonists yohimbine and RX821002 to block the analgesic action of meperidine in the hot-plate test was also assessed. The ability of meperidine to fit into the [alpha]2B adrenoceptor was assessed using molecular modeling techniques.

Results: Meperidine bound to all [alpha]2-adrenoceptor subtypes, with [alpha]2B having the highest affinity ([alpha]2B, 8.6 +/- 0.3 [mu]m; [alpha]2C, 13.6 +/- 1.5 [mu]m, P < 0.05; [alpha]2A, 38.6 +/- 0.7 [mu]m). Morphine was ineffective at binding to any of the receptor subtypes. Meperidine inhibited the production of forskolin-stimulated cyclic adenosine monophosphate mediated by all receptor subtypes but was most effective at the [alpha]2B adrenoceptor ([alpha]2B, 0.6 [mu]m; [alpha]2A, 1.3 mm; [alpha]2C, 0.3 mm), reaching the same level of inhibition (approximately 70%) as achieved with the [alpha]2-adrenoceptor agonist dexmedetomidine. The analgesic action of meperidine was blocked by naloxone but not by the [alpha]2-adrenoceptor antagonists yohimbine and RX821002. The modeling studies demonstrated that meperidine can fit into the [alpha]2B-adrenoceptor subtype.     

The γ Subunit Determines Whether Anesthetic-induced Leftward Shift Is Altered by a Mutation at α1S270 in α1β2γ2L GABAA Receptors

Background: A major action of volatile anesthetics is enhancement of [gamma]-aminobutyric acid receptor type A (GABAAR) currents. In recombinant GABAARs consisting of several subunit mixtures, mutating the [alpha]1 subunit serine at position 270 to isoleucine [[alpha]1(S270I)] was reported to eliminate anesthetic-induced enhancement at low GABA concentrations. In the absence of studies at high GABA concentrations, it remains unclear whether [alpha]1(S270I) affects enhancement versus inhibition by volatile anesthetics. Furthermore, the majority of GABAARs in mammalian brain are thought to consist of [alpha]1, [beta]2, and [gamma]2 subunits, and the [alpha]1(S270I) mutation has not been studied in the context of this combination.

Methods: Recombinant GABAARs composed of [alpha]1[beta]2 or [alpha]1[beta]2[gamma]2L subunit mixtures were studied electrophysiologically in whole Xenopus oocytes in the voltage clamp configuration. Currents elicited by GABA (0.03 [mu]m to 1 mm) were measured in the absence and presence of isoflurane or halothane. Anesthetic effects on GABA concentration responses were evaluated for individual oocytes.

Results: In wild-type [alpha]1[beta]2[gamma]2L GABAA, anesthetics at approximately 2 minimum alveolar concentration (MAC) shifted GABA concentration response curves to the left approximately threefold, decreased the Hill coefficient, and enhanced currents at all GABA concentrations. The [alpha]1(S270I) mutation itself rendered the GABAAR more sensitive to GABA and reduced the Hill coefficient. At low GABA concentrations (EC5), anesthetic enhancement of peak current was much smaller in [alpha]1(S270I)[beta]2[gamma]2Lversus wild-type channels. Paradoxically, the leftward shift of the whole GABA concentration-response relation by anesthetics was the same in both mutant and wild-type channels. At high GABA concentrations, volatile anesthetics reduced currents in [alpha]1(S270I)[beta]2[gamma]2L GABAARs. In parallel studies on [alpha]1[beta]2 ([gamma]-less) GABAARs, anesthetic-induced leftward shifts in wild-type receptors were more than eightfold at 2 MAC, and the [alpha]1(S270I) mutation nearly eliminated anesthetic-induced leftward shift.     

Protein Tyrosine Kinase-Dependent Modulation of Isoflurane Effects on Cardiac Sarcolemmal KATP Channel

Background: Cardiac adenosine triphosphate-sensitive potassium (KATP) channels and protein tyrosine kinases (PTKs) are mediators of ischemic preconditioning, but the interaction of both and a role in myocardial protection afforded by volatile anesthetics have not been defined.

Methods: Whole cell and single channel patch clamp techniques were used to investigate the effects of isoflurane and the PTK inhibitor genistein on the cardiac sarcolemmal KATP channel in acutely dissociated guinea pig ventricular myocytes.

Results: At 0.5 mm internal ATP, genistein (50 [mu]m) elicited whole cell KATP current (22.5 +/- 7.9 pA/pF). Genistein effects were concentration-dependent, with an EC50 of 32.3 +/- 1.4 [mu]m. Another PTK inhibitor, tyrphostin B42, had a similar effect. The inactive analog of genistein, daidzein (50 [mu]m), did not elicit KATP current. Isoflurane (0.5 mm) increased genistein (35 [mu]m)- activated whole cell KATP current from 14.5 +/- 3.1 to 32.5 +/- 6.6 pA/pF. Stimulation of receptor PTKs with epidermal growth factor, nerve growth factor, or insulin attenuated genistein and isoflurane effects, and the protein tyrosine phosphatase inhibitor orthovanadate (1 mm) prevented their actions on KATP current. In excised inside-out membrane patches, and at fixed 0.2 mm internal ATP, genistein (50 [mu]m) increased channel open probability from 0.053 +/- 0.016 to 0.183 +/- 0.039, but isoflurane failed to further increase open probability (0.162 +/- 0.051) of genistein-activated channels. However, applied in the presence of genistein and protein tyrosine phosphatase 1B (1 [mu]g/ml), isoflurane significantly increased open probability to 0.473 +/- 0.114.     

Effects of Antidepressants on G Protein-coupled Receptor Signaling and Viability in Xenopus laevis Oocytes

Background: Tricyclic antidepressants are structurally related to local anesthetics, suggesting that part of their analgesic action may result from properties shared with local anesthetics. Because local anesthetics block G protein-coupled receptor signaling (which explains, in part, their inflammatory modulating properties), the authors studied whether antidepressants have similar effects.

Methods: Peak Ca-activated Cl currents induced in Xenopus laevis oocytes by lysophosphatidic acid (10-4 m) were measured using a voltage clamp. The effects of a 30-, 120-, or 240-min incubation in amitriptyline, nortriptyline, imipramine, or fluoxetine were determined.

Results: After a 30-min incubation, low concentrations (10-7-10-5 m) of antidepressants had no effect on lysophosphatidic acid-induced currents. After prolonged incubation, only amitriptyline or nortriptyline inhibited lysophosphatidic acid signaling (each to 58% of the control response at 10-7 m after 240 min). At low concentrations, none of the compounds induced membrane damage (defined as a holding current of > 1 [mu]A, 2% in control cells). Imipramine at 10-3 m induced damage in 100% of oocytes, and fluoxetine at 10-4 m induced damage in 71% of oocytes (P < 0.05 vs. control). Amitriptyline and nortriptyline had no effect.     

Amitriptyline versus Bupivacaine in Rat Sciatic Nerve Blockade

Background: Amitriptyline, a tricyclic antidepressant, is frequently used orally for the management of chronic pain. To date there is no report of amitriptyline producing peripheral nerve blockade. The authors therefore investigated the local anesthetic properties of amitriptyline in rats and in vitro.

Methods: Sciatic nerve blockade was performed with 0.2 ml amitriptyline or bupivacaine at selected concentrations, and the motor, proprioceptive, and nociceptive blockade was evaluated. Cultured rat GH3 cells were externally perfused with amitriptyline or bupivacaine, and the drug affinity toward inactivated and resting Na+ channels was assessed under whole-cell voltage clamp conditions. In addition, use-dependent blockade of these drugs at 5 Hz was evaluated.

Results: Complete sciatic nerve blockade for nociception was obtained with amitriptyline for 217 +/- 19 min (5 mm, n = 8, mean +/- SEM) and for 454 +/- 38 min (10 mm, n = 7) versus bupivacaine for 90 +/- 13 min (15.4 mm, n = 6). The time to full recovery of nociception for amitriptyline was 353 +/- 12 min (5 mm) and 656 +/- 27 min (10 mm) versus 155 +/- 9 min for bupivacaine (15.4 mm). Amitriptyline was approximately 4.7-10.6 times more potent than bupivacaine in binding to the resting channels (50% inhibitory concentration [IC50] of 39.8 +/- 2.7 vs. 189.6 +/- 22.3 [mu]m) at -150 mV, and to the inactivated Na+ channels (IC50 of 0.9 +/- 0.1 vs. 9.6 +/- 0.9 [mu]m) at -60 mV. High-frequency stimulation at 3 [mu]m caused an additional approximately 14% blockade for bupivacaine, but approximately 50% for amitriptyline.     

Norepinephrine Release from Spinal Synaptosomes: Auto-α2-adrenergic Receptor Modulation

Background: Clonidine produces analgesia after spinal injection by activating [alpha]2-adrenergic receptors. Recently, clonidine has been demonstrated to increase spinal release of norepinephrine (NE) in vivo, in contrast to that anticipated by classic presynaptic autoinhibition. The purpose of the current study was to determine if clonidine could inhibit release of NE in a preparation of spinal cord tissue lacking synaptic circuits.

Methods: Crude synaptosomes were prepared from male Sprague-Dawley rat spinal cord, loaded with [3H]NE, and stimulated by potassium chloride to release [3H]NE. Samples were incubated with clonidine in the absence or presence of various inhibitors. To study the effect of [alpha]2a-adrenergic receptor subtypes, some animals were pretreated with an oligodeoxynucleotide (ODN) composed of a sense or antisense sequence to a portion of this receptor.

Results: Potassium chloride produced a concentration-dependent increase in [3H]NE release, and this release was inhibited by clonidine with a concentration producing 50% maximal inhibition (IC50) of 1.3 [mu]m. The effect of clonidine was inhibited by the [alpha]2-adrenergic antagonists, yohimbine and idazoxan, but not by [alpha]1-adrenergic, muscarinic, or opioid antagonists. Intrathecal pretreatment with antisense ODN to [alpha]2A-adrenergic receptors reduced [alpha]2A-adrenergic receptor protein expression compared with sense ODN control and also reduced clonidine-induced inhibition of [3H]NE release.     

Meperidine and Lidocaine Block of Recombinant Voltage-Dependent Na+ Channels: Evidence that Meperidine is a Local Anesthetic

Background: The opioid meperidine induces spinal anesthesia and blocks nerve action potentials, suggesting it is a local anesthetic. However, whether it produces effective clinical local anesthesia in peripheral nerves remains unclear. Classification as a local anesthetic requires clinical local anesthesia but also blockade of voltage-dependent Na+ channels with characteristic features (tonic and phasic blockade and a negative shift in the voltage-dependence of steady-state inactivation) involving an intrapore receptor. The authors tested for these molecular pharmacologic features to explore whether meperidine is a local anesthetic.

Methods: The authors studied rat skeletal muscle [mu]1 (RSkM1) voltage-dependent Na+ channels or a mutant form heterologously coexpressed with rat brain Na+ channel accessory [beta]1 subunit in Xenopus oocytes. Polymerase chain reaction was used for mutagenesis, and mutations were confirmed by sequencing. Na+ currents were measured using a two-microelectrode voltage clamp. Meperidine and the commonly used local anesthetic lidocaine were applied to oocytes in saline solution at room temperature.

Results: Meperidine and lidocaine produced tonic current inhibition with comparable concentration dependence. Meperidine caused phasic current inhibition in which the concentration-response relationship was shifted to fivefold greater concentration relative to lidocaine. Meperidine and lidocaine negatively shifted the voltage dependence of steady-state inactivation. Mutation of a putative local anesthetic receptor reduced phasic inhibition by meperidine and lidocaine and tonic inhibition by lidocaine, but not meperidine tonic inhibition.     

Single Amino Acid Residue in the Extracellular Portion of Transmembrane Segment 2 in the Nicotinic α7 Acetylcholine Receptor Modulates Sensitivity to Ketamine

Background: Ketamine inhibits the activation of both heteromeric and homomeric nicotinic acetylcholine receptors. The site of molecular interaction is unknown.

Methods: The inhibition of [alpha]7 nicotinic acetylcholine receptors by ketamine was compared to that of 5-hydroxytryptamine-3A (5HT3A)receptors that are resistant to ketamine inhibition in Xenopus laevis oocytes. To determine whether the region of transmembrane segments 2 and 3 is relevant for ketamine inhibition of nicotinic receptors, the authors identified single amino acid residues that differ in the sequence alignment of the two proteins. They created 22 mutant [alpha]7 nicotinic receptors that contain the single homologous amino acid residue in the 5HT3A sequence.

Results: Of the 22 mutant [alpha]7 nicotinic receptors tested, only one ([alpha]7 A258S) was significantly resistant to 20 [mu]m ketamine. The ketamine concentration response relationship for the [alpha]7 A258S mutant was shifted to the right with the IC50 for ketamine increased from 17 +/- 2 for wild type to 30 +/- 3 [mu]m in the mutant (P < 0.001). Agonist activation was unchanged by the mutation. The homologous amino acid residue in the 5HT3A receptor was mutated to the alanine that occurs in the wild-type nicotinic receptor. This mutation made the previously insensitive 5HT3A receptor sensitive to ketamine (P < 0.001).     

Enflurane Directly Depresses Glutamate AMPA and NMDA Currents in Mouse Spinal Cord Motor Neurons Independent of Actions on GABAA or Glycine Receptors

Background: The spinal cord is an important anatomic site at which volatile agents act to prevent movement in response to a noxious stimulus. This study was designed to test the hypothesis that enflurane acts directly on motor neurons to inhibit excitatory synaptic transmission at glutamate receptors.

Methods: Whole-cell recordings were made in visually identified motor neurons in spinal cord slices from 1- to 4-day-old mice. Excitatory postsynaptic currents (EPSCs) or potentials (EPSPs) were evoked by electrical stimulation of the dorsal root entry area or dorsal horn. The EPSCs were isolated pharmacologically into glutamate N-methyl-d-aspartate (NMDA) receptor- and non-NMDA receptor-mediated components by using selective antagonists. Currents also were evoked by brief pulse pressure ejection of glutamate under various conditions of pharmacologic blockade. Enflurane was made up as a saturated stock solution and diluted in the superfusate; concentrations were measured using gas chromatography.

Results: Excitatory postsynaptic currents and EPSPs recorded from motor neurons by stimulation in the dorsal horn were mediated by glutamate receptors of both non-NMDA and NMDA subtypes. Enflurane at a general anesthetic concentration (one minimum alveolar anesthetic concentration) reversibly depressed EPSCs and EPSPs. Enflurane also depressed glutamate-evoked currents in the presence of tetrodotoxin (300 nm), showing that its actions are postsynaptic. Block of inhibitory [gamma]-aminobutyric acid A and glycine receptors by bicuculline (20 [mu]m) or strychnine (2 [mu]m) or both did not significantly reduce the effects of enflurane on glutamate-evoked currents. Enflurane also depressed glutamate-evoked currents if the inhibitory receptors were blocked and if either D,L-2-amino-5-phosphonopentanoic acid (50 [mu]m) or 6-cyano-7-nitroquinoxaline-2,3-dione disodium (10 [mu]m) was applied to block NMDA or [alpha]-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid-kainate receptors respectively.     

Direct Inhibition of the N-methyl-D-aspartate Receptor Channel by High Concentrations of Opioids

Background: Electrophysiologic and receptor binding studies showed that some opioids have noncompetitive N-methyl-D-aspartate (NMDA) receptor antagonist properties.

Methods: The effects and mechanisms of action of various opioid compounds were examined on four kinds of heteromeric NMDA receptor channels, namely the [epsilon]1/[zeta]1, [epsilon]2/[zeta]1, [epsilon]3/[zeta]1, and [epsilon]4/[zeta]1 channels, expressed in Xenopus oocytes. Furthermore, the action sites of opioids on NMDA receptor channels were investigated by site-directed mutagenesis.

Results: Meperidine inhibited four kinds of channels to a similar extent with inhibitor concentrations for half-control response (IC50s) of 210-270 [mu]M. Morphine, fentanyl, codeine, and naloxone also inhibited NMDA receptor channels with affinities comparable to meperidine. Opioid inhibition exhibited voltage dependence and was quite effective at negative potentials. Opioids also shifted the inhibition curve of Mg2+ to the right. Furthermore, replacement of the conserved asparagine residue with glutamine in the channel-lining segment M2 of the [zeta]1 subunit, which constitutes the block sites of Mg2+ and ketamine, reduced the sensitivity to opioids, whereas that of the [epsilon]2 subunit barely affected the opioid sensitivity.     

Propofol Stimulates Ciliary Motility via the Nitric Oxide-Cyclic GMP Pathway in Cultured Rat Tracheal Epithelial Cells

Background: Airway ciliary motility is impaired by inhaled anesthetics. Recent reports show that nitric oxide (NO) induces upregulation in ciliary beat frequency (CBF), and others report that propofol, an intravenous anesthetic, stimulates NO release; this raises the possibility that propofol increases CBF by stimulating the NO-cyclic guanosine monophosphate (cGMP) signal pathway. In this study, the authors investigated the effects of propofol on CBF and its relation with the NO-cGMP pathway using the pharmacologic blockers NG-monomethyl-l-arginine (l-NMMA), an NO synthase inhibitor; 1 H-[1,2,4]oxidazole[4,3-a]quinoxalin-1-one (ODQ), a soluble guanylyl cyclase inhibitor; and KT5823, a cGMP-dependent protein kinase inhibitor, in cultured rat tracheal epithelial cells.

Methods: Rat tracheal tissues were explanted and cultured for 3-5 days. Images of ciliated cells were videotaped using a phase-contrast microscope. Baseline CBF and CBF 25 min after exposure to propofol or blocker were measured using video analysis.

Results: Vehicle (0.1% dimethyl sulfoxide; n = 11) increased CBF by 0.2 +/- 1.7% (mean +/- SD) from baseline. Propofol stimulated CBF significantly (P < 0.01) and dose dependently (1 [mu]m, 2.0 +/- 1.9%, n = 6; 10 [mu]m, 8.2 +/- 6.7%, n = 9; 100 [mu]m, 14.0 +/- 4.7%, n = 10). Intralipid (0.05%), the clinical vehicle of propofol, did not affect CBF (-0.2 +/- 2.2%; n = 5). The enhancement of CBF with use of 100 [mu]m propofol was abolished (P < 0.01) by coadministration of 10 m[mu]m l-NMMA (2.4 +/- 3.6%; n = 5), 100 [mu]m ODQ (-0.3 +/- 2.2%; n = 6) or 30 [mu]m KT5823 (-0.1 +/- 4.1%; n = 8). l-NMMA, ODQ, or KT5823 alone did not change CBF.