Remifentanil Directly Activates Human N-methyl-d-aspartate Receptors Expressed in Xenopus laevis Oocytes

Background: Clinical studies suggest that intraoperative administration of the clinical remifentanil formulation Ultiva(R) (GlaxoWellcome GmbH & Co, Bad Oldesloe, Germany) increases postoperative pain and postoperative analgesic requirements, but mechanisms remain unclear. N-methyl-d-aspartate (NMDA) receptors are thought to play a major role in development of postoperative pain and opiate tolerance. The authors hypothesized that Ultiva(R) directly stimulates human NMDA receptors.

Methods: To test this hypothesis, the authors expressed human NR1A/NR2A and NR1A/NR2B NMDA receptors in Xenopus laevis oocytes by injection of messenger RNA prepared in vitro. After protein expression, they used a two-electrode voltage clamp to measure currents induced by NMDA receptor agonists and opioids.

Results: Noninjected cells were unresponsive to all compounds tested. Glutamate/glycine (1 nm-1 mm each) or Ultiva(R) (0.01 pm-0.1 mm) stimulated NMDA receptors concentration dependently. NR1A/2A EC50 values were 8.0 [mu]M/12 [mu]M for glutamate/glycine and 3.5 nM for Ultiva(R), and NR1A/2B EC50 values were 3.9 [mu]M/1.9 [mu]M for glutamate/glycine and 0.82 [mu]M for Ultiva(R). Glycine in combination with Ultiva(R) showed no additive effect compared with Ultiva(R) alone. Ultiva(R)-induced currents were inhibited by MK-801 (pore blocker) but not by 7-CK (glycine antagonist), D-AP5 (glutamate antagonist), or naloxone. Fentanyl (10 [mu]M) did not stimulate NMDA receptors.     

Local Anesthetic Inhibition of m1 Muscarinic Acetylcholine Signaling

Background: Local anesthetics inhibit lipid mediator signaling (lysophosphatidate, thromboxane) by acting on intracellular domains of the receptor or on the G protein. On receptors for polar agonists, the ligand-binding pocket could form an additional site of interaction, possibly resulting in superadditive inhibition. The authors therefore investigated the effects of local anesthetics on m1 muscarinic receptor functioning.

Methods: The authors expressed receptors in isolation using Xenopus oocytes. Using a two-electrode voltage clamp, the authors measured the effects of lidocaine, QX314 (permanently charged), and benzocaine (permanently uncharged) on Ca2+-activated Cl- currents elicited by methylcholine. The authors also characterized the interaction of lidocaine with [3H] quinuclydinyl benzylate ([3H]QNB) binding to m1 receptors.

Results: Lidocaine inhibited muscarinic signaling with a half-maximal inhibitory concentration (IC50 18 nm) 140-fold less than that of extracellularly administered QX314 (IC50 2.4 [mu]m). Intracellularly injected QX314 (IC50 0.96 mm) and extracellularly applied benzocaine (IC50 1.2 mm) inhibited at high concentrations only. Inhibition of muscarinic signaling by extracellularly applied QX314 and lidocaine was the result of noncompetitive antagonism. Intracellularly injected QX314 and benzocaine inhibited muscarinic and lysophosphatidate signaling at similar concentrations, suggesting an action on the common G-protein pathway. Combined administration of intracellularly injected (IC50 19 [mu]m) and extracellularly applied QX314 (IC50 49 nm) exerted superadditive inhibition. Lidocaine did not displace specific [3H]QNB binding to m1 receptors.     

Different Effects of Estrogen and Progesterone on K+ Currents Expressed in Xenopus Oocytes

Estrogen has received considerable attention recently as a potential therapeutic agent in vascular pathophysiological states such as stroke. The mechanisms by which estrogen influences cerebral arteries are incompletely understood. The present study was to examine the effect of ovariectomy and chronic estrogen or tamoxifen treatment on vascular reactivity in rat posterior communicating cerebral arteries with intact endothelium. Changes in vascular tension were measured by microvessel myograph. Ovariectomy significantly enhanced the constricting responses to endothelin I, but not to phenylephrine. Chronic treatment with estrogen or tamoxifen partially reversed or abolished the effect of ovariectomy. The contraction induced by high K⁺ solution was also enhanced in the ovariectomized rats and this enhancement was abolished by estrogen or tamoxifen treatment. Ovariectomy potentiated the relaxant response to nicardipine but not NS 1619. Estrogen but not tamoxifen reversed the effect of ovariectomy. The present results indicate that chronic tamoxifen may not act as an antagonist of estrogen, instead, chronic treatment with estrogen and tamoxifen has similar effect in inhibiting the increased vascular tension induced by ovariectomy. This study suggests the influence of physiological level of estrogen on vascular contractility. It is at present unknown what may have caused increased relaxant effect of nicardipine, a L-type Ca²⁺ channel blocker. More experiments are needed to show the role of endothelium in the altered vascular contractility in the ovariectomized and estrogen-treated rats. (supported by UPGC Direct Grant).     

Bupivacaine Inhibits Activation of Neuronal Spinal Extracellular Receptor-activated Kinase through Selective Effects on Ionotropic Receptors

Background: Central terminals of primary nociceptors release neurotransmitters glutamate and substance P, which bind to ionotropic or metabotropic receptors on spinal neurons to induce cellular responses. Extracellular signal-regulated kinases are activated by these receptors and are important modulators of pain at the dorsal horn. The authors investigated these pathways as potential targets for antinociceptive actions of local anesthetics.

Methods: The effects of bupivacaine on the activation of extracellular receptor-activated kinase (phosphorylation to pERK) in rat spinal cord slices, induced by presynaptic release (capsaicin), by presynaptic or postsynaptic ionotropic or metabotropic receptor activation, or by activation of intracellular protein kinase C or protein kinase A and also by a receptor-independent Ca2+ ionophore, were quantitated by immunohistochemistry, counting pERK-positive neurons in the superficial dorsal horn.

Results: Capsaicin (3 [mu]m, 10 min)-stimulated pERK was reduced by bupivacaine (IC50 approximately 2 mm, approximately 0.05%), which similarly suppressed pERK induced by the ionotropic glutamate receptors for N-methyl-d-aspartate and (S)-[alpha]-amino-3-hydroxy-5-methyle-4-isoxazole propionic acid but not that induced by the metabotropic receptors for glutamate, bradykinin, or substance P. Extracellular receptor-activated kinase activation by the Ca+2 ionophore ionomycin was also sensitive to bupivacaine, but direct activation by protein kinase A or protein kinase C was not.     

Ketamine Inhibits Monoamine Transporters Expressed in Human Embryonic Kidney 293 Cells

Background: Ketamine has been characterized as having psychotomimetic and sympathomimetic effects. These symptoms have raised the possibility that ketamine affects monoaminergic neurotransmission. To elucidate the relation between ketamine and monoamine transporters, the authors constructed three cell lines that stably express the norepinephrine, dopamine, and serotonin transporters and investigated the effects of ketamine on these transporters.

Methods: Human embryonic kidney cells were transfected using the Chen-Okayama method with the human norepinephrine rat dopamine, and rat serotonin transporter cDNA subcloned into the eukaryotic expression vector. Using cells stably expressing these transporters, the authors investigated the effects of ketamine on the uptake of these compounds and compared them with those of pentobarbital.

Results: Inhibition analysis showed that ketamine significantly inhibited the uptake of all three monoamine transporters in a dose-dependent manner. The Ki (inhibition constant) values of ketamine on the norepinephrine, dopamine, and serotonin transporters were 66.8 micro Meter, 62.9 micro Meter, and 162 micro Meter, respectively. Pentobarbital, a typical general anesthetic agent with no psychotic symptoms, did not affect the uptake of monoamines, however. Further, neither the glycine transporter 1 nor the glutamate/aspartate transporter was affected by ketamine, indicating that ketamine preferentially inhibits monoamine transporters.     

The Inhibitory Role of Acetylcholine and Muscarinic Receptors in Bladder Afferent Activity

Background

The main treatment for overactive bladder (OAB) is the use of anticholinergic drugs initially believed to inhibit the effect of parasympathetic acetylcholine (ACh) on the detrusor; however, there is now evidence to suggest that anticholinergic drugs could interact with sensory pathways.

Objective

Investigate the role of muscarinic receptors and ACh in modulating bladder afferent sensitivity in the mouse.

Design, setting, and participants

Bladder and surrounding tissue were removed from wild-type male mice, placed in a recording chamber, and continually perfused with fresh oxygenated Krebs solution at 35 °C. Bladders were cannulated to allow infusion and intravesical pressure monitoring, and afferent nerve fibres innervating the bladder were dissected and put into a suction electrode for recording.

Measurements

Multiunit afferent activity and intravesical pressure were recorded at baseline and during bladder distension. Experiments were conducted in the presence of muscarinic agonists and antagonist or in the presence of the cholinesterase inhibitor physostigmine.

Results and limitations

Blocking muscarinic receptors using atropine (1 μM) had no effect on spontaneous afferent discharge, the afferent response to bladder distension, or on bladder compliance. However, stimulation of muscarinic receptors directly using bethanechol (100 μM) and carbachol (100 μM) or indirectly using physostigmine (10 μM) significantly inhibited the afferent response to bladder distension and concurrently reduced bladder compliance. Furthermore, prior application of nifedipine prevented the changes in bladder tone but did not prevent the attenuation of afferent responses by bethanechol or physostigmine.

Conclusions

These data indicate that stimulation of muscarinic receptor pathways can depress sensory transduction by a mechanism independent of changes in bladder tone, suggesting that muscarinic receptor pathways and ACh could contribute to normal or pathologic bladder sensation.     

Human Neuronal Nicotinic Acetylcholine Receptors Expressed in Xenopus Oocytes Predict Efficacy of Halogenated Compounds That Disobey the Meyer-Overton Rule

Background: According to the Meyer-Overton rule, anesthetic potency of a substance can be predicted by its lipid solubility, but a group of halogenated volatile compounds predicted to induce anesthesia does not obey this rule. Thus, these compounds are useful tools for studies of molecular targets of anesthetics. Human neuronal nicotinic acetylcholine receptor (hnAChR) subunits have been recently cloned, which allowed the authors to assess whether these receptors could differentiate among volatile anesthetic and nonimmobilizer compounds. This study provides the first data regarding anesthetic sensitivity of hnAChRs.

Methods: [alpha]2[beta]4, [alpha]3[beta]4, and [alpha]4[beta]2 hnAChRs were expressed in Xenopus oocytes, and effects of volatile anesthetics isoflurane and F3 (1-chloro-1,2,2-triflurocyclobutane, 1A) and nonimmobilizers F6 (1,2-dichlorohexafluorocyclobutane, 2N) and F8 (2,3-dichlorooctafluorobutane) on the peak acetylcholine-gated currents were studied using the two-electrode voltage-clamp technique.

Results: Isoflurane and F3 inhibited all the hnAChRs tested in a concentration-dependent manner. Isoflurane at a concentration corresponding to 1 minimum alveolar concentration (MAC) inhibited 83, 69, and 71% of ACh-induced currents in [alpha]2[beta]4, [alpha]3[beta]4, and [alpha]4[beta]2 hnAChRs, respectively, and 1 MAC of F3 inhibited 64, 44, and 61% of currents gated in those receptors. F6 (8-34[mu]M) did not cause any changes in currents gated by any of the receptors tested. F8 (4-18[mu]M) did not alter the currents gated in either [alpha]3[beta]4 or [alpha]4[beta]2 receptors, but caused a small potentiation of [alpha]2[beta]4 hnAChRs without a concentration-response relation.     

Halothane Attenuation of Calcium Sensitivity in Airway Smooth Muscle: Mechanisms of Action during Muscarinic Receptor Stimulation

Background: In airway smooth muscle, muscarinic receptor stimulation is thought to increase calcium (Ca2+) sensitivity via a guanosine 5'-triphosphate (GTP)-binding protein/protein kinase C (PKC)-mediated mechanism. This study tested the hypothesis that halothane reduces Ca2+ sensitivity during muscarinic receptor stimulation by inhibiting these second messenger pathways.

Methods: A beta-escin permeabilized canine tracheal smooth muscle preparation was used in which the cytosolic Ca2+ concentration ([Ca sup 2+]i) is controlled and the GTP-binding protein/PKC pathways remain intact and can be activated. The muscarinic receptor was activated with acetylcholine plus GTP; the GTP-binding proteins were directly activated with a nonhydrolyzable form of GTP, guanosine 5'-O-(3-thiotriphosphate; GTP gamma S); and PKC was directly activated with the PKC agonist phorbol 12,13-dibutyrate (PDBu).

Results: Free Ca2+ caused a concentration-dependent increase in force. Acetylcholine plus GTP significantly decreased the median effective concentration for free Ca2+ from 0.52 +/- 0.06 micro Meter to 0.21 +/- 0.02 micro Meter, demonstrating an increase in Ca2+ sensitivity. Halothane (0.99 +/- 0.04 mM, equivalent to approximately 4 minimum alveolar concentration in dogs) significantly attenuated this increase in Ca2+ sensitivity induced by acetylcholine plus GTP, increasing the median effective concentration for free Ca2+ from 0.21 +/- 0.02 micro Meter to 0.31 +/- 0.03 micro Meter. However, halothane did not affect the increases in Ca2+ sensitivity induced by GTP gamma S or PDBu.     

Acetylcholine-Induced Contractions in the Porcine Internal Mammary Artery: Possible Role of Muscarinic Receptors

The effect of acetylcholine on the isolated, non-precontracted, porcine internal mammary artery (IMA) was investigated. Acetylcholine induced concentration-dependent contractions of non-precontracted IMA rings with denuded endothelium (pEC₅₀ = 5.80 ± 0.04) and was without effect on arterial segments with intact endothelium. The muscarinic receptor antagonists atropine, pirenzepine, methoctramine and p-fluoro-hexahydro-sila-diphenidol (pFHHSiD) antagonized the response to acetylcholine. The constrained pA₂ values were 10.14, 7.74, 7.34 and 10.5, respectively. It is concluded that acetylcholine induces concentration-dependent contractions of porcine internal mammary artery rings on basal tone and that this contractile effect is probably due to direct cholinergic stimulation of smooth muscle cells, maybe including activation of muscarinic M₁ receptors.     

Isoflurane Modulation of Neuronal Nicotinic Acetylcholine Receptors Expressed in Human Embryonic Kidney Cells

Background: It is well established that neuronal nicotinic acetylcholine receptors (nAChRs) are sensitive to inhalational anesthetics. The authors previously reported that halothane potently blocked [alpha]4[beta]2-type nAChRs of rat cortical neurons. However, the effect of isoflurane, which is widely used clinically, on nAChRs largely remains to be seen. The authors studied the effects of isoflurane as compared with sevoflurane and halothane on the human [alpha]4[beta]2 nAChRs expressed in human embryonic kidney cells.

Methods: The whole-cell and single-channel patch clamp techniques were used to record currents induced by acetylcholine.

Results: Isoflurane, sevoflurane, and halothane suppressed the acetylcholine-induced currents in a concentration-dependent manner with 50% inhibitory concentrations of 67.1, 183.3, and 39.8 [mu]m, respectively, which correspond to 0.5 minimum alveolar concentration or less. When anesthetics were coapplied with acetylcholine, isoflurane and sevoflurane decreased the apparent affinity of receptor for acetylcholine, but halothane, in addition, decreased the maximum acetylcholine current. When isoflurane was preapplied and coapplied, its inhibitory action was independent of acetylcholine concentration. Isoflurane blocked the nAChR in both resting and activated states. Single-channel analyses revealed that isoflurane at 84 [mu]m decreased the mean open time and burst duration without inducing "flickering" during channel openings. Isoflurane increased the mean closed time. As a result, the open probability of single channels was greatly reduced by isoflurane.     

Intrathecal Clonidine Alleviates Allodynia in Neuropathic Rats: Interaction with Spinal Muscarinic and Nicotinic Receptors

Background: Intrathecally administered clonidine increases release of spinal acetylcholine, which may be related to its analgesic action in neuropathic pain. The current study determined the role of spinal muscarinic and nicotinic receptors in the antiallodynic effect of intrathecally administered clonidine in spinal nerve-ligated rats.

Methods: Allodynia was produced in rats by ligation of the left L5-L6 spinal nerves. Mechanical allodynia was determined by application of von Frey filaments to the left hindpaw. The effect of intrathecal injection of saline, two muscarinic receptor antagonists (atropine and scopolamine), and two nicotinic receptor antagonists (mecamylamine and hexamethonium) on the antiallodynic action produced by intrathecal administration of 20 [micro sign]g clonidine was assessed in six groups of animals. Each group consisted of six to eight rats.

Results: Intrathecal injection of saline or muscarinic or nicotinic receptor antagonists did not alter the withdrawal thresholds. The antiallodynic effect produced by intrathecally administered clonidine was attenuated in a dose-dependent manner by intrathecal treatment with muscarinic and nicotinic antagonists. Although nicotinic receptor antagonists only partially attenuated the effect of clonidine, blockade of spinal muscarinic receptors almost abolished the antiallodynic effect of clonidine.     

TNF Receptors Differentially Signal and Are Differentially Expressed and Regulated in the Human Heart

Tumor necrosis factor (TNF) utilizes two receptors, TNFR1 and 2, to initiate target cell responses. We assessed expression of TNF, TNFRs and downstream kinases in cardiac allografts, and compared TNF responses in heart organ cultures from wild‐type (^WTC57BL/6), TNFR1‐knockout (^KO), TNFR2^KO, TNFR1/2^KO mice. In nonrejecting human heart TNFR1 was strongly expressed coincidentally with inactive apoptosis signal‐regulating kinase‐1 (ASK1) in cardiomyocytes (CM) and vascular endothelial cells (VEC). TNFR2 was expressed only in VEC. Low levels of TNF localized to microvessels. Rejecting cardiac allografts showed increased TNF in microvessels, diminished TNFR1, activation of ASK1, upregulated TNFR2 co‐expressed with activated endothelial/epithelial tyrosine kinase (Etk), increased apoptosis and cell cycle entry in CM. Neither TNFR was expressed significantly by cardiac fibroblasts. In ^WTC57BL/6 myocardium, TNF activated both ASK1 and Etk, and increased both apoptosis and cell cycle entry. TNF‐treated TNFR1^KO myocardium showed little ASK1 activation and apoptosis but increased Etk activation and cell cycle entry, while TNFR2^KO myocardium showed little Etk activation and cell cycle entry but increased ASK1 activation and apoptosis. These observations demonstrate independent regulation and differential functions of TNFRs in myocardium, consistent with TNFR1‐mediated cell death and TNFR2‐mediated repair.     

Suppressor of Cytokine Signaling 1 Inhibits Apoptosis of Islet Grafts Through Caspase 3 and Apoptosis-Inducing Factor Pathways in Rats

A significant portion of pancreatic islet grafts can be destroyed by apoptosis, failing to engraft in the early period after transplantation. Recently, we observed that overexpression of suppressor of cytokine signaling 1 (SOCS1) in islet grafts achieved an antiapoptotic effect, prolonging graft survival in a rat transplant model. Caspase 3 is the central executioner caspase that is activated by upstream cascades in a caspase-dependent apoptosis pathway. Apoptosis inducing factor (AIF) is a key protein that can be released from mitochondria, translocating to the nucleus in the caspase-independent apoptosis pathway. In this study, we investigated whether these two pathways were involved in cytoprotection afforded by SOCS1 on islet grafts. We used a chimeric adenovirus vector (Ad5F35-SOCS1) to enhance SOCS1 expression in isolated Sprague-Dawley rat islets, which were transplanted into recipients experiencing streptozotocin-induced diabetes. We analyzed the expressions of active (cleaved) caspase 3 and AIF on islets. The Ad5F35-SOCS1-infected islets with higher SOCS1 expression showed decreased levels of active caspase 3 and intranuclear AIF after treatment with tumor necrosis factor-α and cycloheximide in vitro. The diabetic recipients transplanted with Ad5F35-SOCS1-infected islets showed longer periods of normoglycemia versus recipients transplanted with mock-infected islets (P < .05) due to prolonged graft survival. A histological analysis indicated that the Ad5F35-SOCS1-infected islet grafts displayed decreased caspase 3 activation and AIF translocation (to nucleus) in the early posttransplant period. These results demonstrated that the expression of SOCS1 in islet grafts protected them from apoptosis through caspase 3 dependent and AIF caspase-independent-pathways.     

Toll样受体信号传导通路及其在器官移植中作用的研究进展

目的了解Toll样受体(TLRs)信号传导通路及其在器官移植中的作用的研究进展。方法通过文献检索并总结TLRs及其配体结构、功能的特点,就近年来TLRs信号传导通路在动物实验和临床器官移植中的作用的研究进展进行综述。结果 TLRs在器官移植中发挥着重要作用,TLRs的活化可激活特异性免疫系统,使移植物发生缺血再灌注损伤、急性排斥反应和慢性排斥反应,同时也可通过TLRs诱导免疫耐受。早期治疗措施的干预可减少器官移植中移植物因缺血再灌注损伤所致的TLRs激活,从而提高移植物存活率;同时,针对TLRs及其介导的信号传导通路的相关免疫抑制靶点研发出的高效免疫抑制药物可减轻器官移植后缺血再灌注损伤和免疫排斥反应。结论 TLRs信号传导通路在缺血再灌注损伤、免疫排斥及免疫调节中起着重要作用。     

糖尿病大鼠膀胱重构中M3受体改变的实验研究

目的:研究病程24周的2型糖尿病大鼠膀胱重构时,逼尿肌收缩功能的改变和M3受体含量及其基因转录水平的改变情况,并探讨二者之间的相关性。方法:2d龄雌性Wistar大鼠随机分成实验组和正常对照组,应用链脲佐菌素腹腔注射并结合高糖高脂饮食进行2型糖尿病大鼠动物模型制备。于糖尿病病程24周时进行下列实验:应用离体膀胱灌注方法观察逼尿肌收缩功能的变化;应用RT-PCR和Western blotting方法观察逼尿肌M3受体mRNA和蛋白表达的变化。结果:2型糖尿病组大鼠逼尿肌收缩功能低于正常对照组,为(16.52±2.97)cmH2O/100mgVS(25.66±3.56)cmH2O/100mg;2型糖尿病组大鼠逼尿肌M3受体mRNA和蛋白的表达均高于正常对照组,分别为(65.27±4.61)%VS(37.53±4.02)%和(45.19±2.37)%VS(23.67±2.85)%。结论:本研究证实了2型糖尿病大鼠在病程24周时膀胱逼尿肌的收缩力降低,但M3受体的生物合成却上调,这种不平行现象可能是病变进展的表现,为深入研究糖尿病膀胱病的发病机制提供了有价值