Effect of Mechanogated Membrane Ion Channel Blockers on Experimental Traumatic Brain Oedema

Summary Traumatic head injury leads to marked swelling of endothelial cells, both in human patients and in Marmarou's rat model. We used this model to test the hypothesis of mechanogated ion channels being involved in the formation of traumatic brain oedema. All mechanogated channel blockers tested (gadolinium, amiloride, gentamicin) significantly reduced traumatic brain oedema evaluated by Evans blue extraction ratio, either when given 15 minutes before or 30 minutes after induction of trauma (evaluation 2 hours after trauma). These results clearly support our hypothesis, opening a new way for the investigation of the treatment of a clinical situation endowed with high morbidity and mortality.     

Anesthetic Effects on Mitochondrial ATP-sensitive K Channel

Background : Volatile anesthetics show an ischemic preconditioning-like cardioprotective effect, whereas intravenous anesthetics have cardioprotective effects for ischemic-reperfusion injury. Although recent evidence suggests that mitochondrial adenosine triphosphate-regulated potassium (mitoKATP) channels are important in cardiac preconditioning, the effect of anesthetics on mitoKATP is unexplored. Therefore, the authors tested the hypothesis that anesthetics act on the mitoKATP channel and mitochondrial flavoprotein oxidation.

Methods : Myocardial cells were isolated from adult guinea pigs. Endogenous mitochondrial flavoprotein fluorescence, an indicator of mitochondrial flavoprotein oxidation, was monitored with fluorescence microscopy while myocytes were exposed individually for 15 min to isoflurane, sevoflurane, propofol, and pentobarbital. The authors further investigated the effect of 5-hydroxydeanoate, a specific mitoKATP channel antagonist, on isoflurane- and sevoflurane-induced flavoprotein oxidation. Additionally, the effects of propofol and pentobarbital on isoflurane-induced flavoprotein oxidation were measured.

Results : Isoflurane and sevoflurane induced dose-dependent increases in flavoprotein oxidation (isoflurane: R2 = 0.71, n = 50; sevoflurane: R2 = 0.86, n = 20). The fluorescence increase produced by both isoflurane and sevoflurane was eliminated by 5-hydroxydeanoate. Although propofol and pentobarbital showed no significant effects on flavoprotein oxidation, they both dose-dependently inhibited isoflurane-induced flavoprotein oxidation.     

Volatile Anesthetic Actions on Contractile Proteins in Membrane-permeabilized Small Mesenteric Arteries

Background: Volatile anesthetics have been shown to have vasodilating or vasoconstricting actions in vitro that may contribute to their cardiovascular effects in vivo. However, the precise mechanisms of these actions in vitro have not been fully elucidated. Moreover, there are no data regarding the mechanisms of volatile anesthetic action on small resistance arteries, which play a critical role in the regulation of blood pressure and blood flow.

Methods: With the use of isometric tension recording methods, volatile anesthetic actions were studied in intact and beta-escin-membrane-permeabilized smooth muscle strips from rat small mesenteric arteries. In experiments with intact muscle, the effects of halothane (0.25-5.0%), isoflurane (0.25-5.0%), and enflurane (0.25-5.0%) were investigated on high Potassium sup + -induced contractions at 22 degrees Celsius and 35 degrees Celsius. All experiments were performed on endothelium-denuded strips in the presence of 3 micro Meter guanethidine and 0.3 micro Meter tetrodotoxin to minimize the influence of nerve terminal activities. In experiments with membrane-permeabilized muscle, the effects of halothane (0.5-4.0%), isoflurane (0.5-4.0%), and enflurane (0.5-4.0%) on the half-maximal and maximal Calcium2+ -activated contractions were examined at 22 degrees Celsius in the presence of 0.3 micro Meter ionomycin to eliminate intracellular Calcium sup 2+ stores.

Results: In the high Potassium sup + -stimulated intact muscle, all three anesthetics generated transient contractions, which were followed by sustained vasorelaxation. The IC50 values for this vasorelaxing action of halothane, isoflurane, and enflurane were 0.47 vol% (0.27 mM), 0.66 vol% (0.32 mM), and 0.53 vol% (0.27 mM), respectively, at 22 degrees Celsius and were 3.36 vol% (0.99 mM), 3.07 vol% (0.69 mM), and 3.19 vol% (0.95 mM), respectively, at 35 degrees Celsius. Ryanodine (10 micro Meter) eliminated the anesthetic-induced contractions but had no significant effect on the anesthetic-induced vasorelaxation in the presence of high Potassium sup +. In addition, no significant differences were observed in the dose dependence of the direct vasodilating action among these anesthetics with or without ryanodine at either the low or the high temperature. However, significant differences were observed in the vasoconstricting actions among the anesthetics, and the order of potency was halothane > enflurane > isoflurane. The Calcium sup 2+ -tension relation in the membrane-permeabilized muscle yielded a half-maximal effective Calcium2+ concentration (EC50) of 2.02 micro Meter. Halothane modestly but significantly inhibited 3 micro Meter (approximately the EC50) and 30 micro Meter (maximal) Calcium sup 2+ -induced contractions. Enflurane slightly but significantly inhibited 3 micro Meter but not 30 micro Meter Calcium2+ contractions. Isoflurane did not significantly inhibit either 3 micro Meter or 30 micro Meter Calcium2+ contractions.     

TOK1 Is a Volatile Anesthetic Stimulated K sup + Channel

Background: Volatile anesthetic agents can activate the S channel, a baseline potassium (K sup +) channel, of the marine mollusk Aplysia. To investigate whether cloned ion channels with electrophysiologic properties similar to the S channel (potassium selectivity, outward rectification, and activation independent of voltage) also are modulated by volatile anesthetic agents, the authors expressed the cloned yeast ion channel TOK1 (tandem pore domain, outwardly rectifying K sup + channel) in Xenopus oocytes and studied its sensitivity to volatile agents.

Methods: Standard two-electrode voltage and patch clamp recording methods were used to study TOK1 channels expressed in Xenopus oocytes.

Results: Studies with two-electrode voltage clamp at room temperature showed that halothane, isoflurane, and desflurane increased TOK1 outward currents by 48-65% in barium Frog Ringer's perfusate. The concentrations at which 50% potentiation occurred (EC50 values) were in the range of 768-814 micro meter (0.016-0.044 atm) and had a rank order of potency in atm in which halothane > isoflurane > desflurane. The potentiation of TOK1 by volatile anesthetic agents was rapid and reversible (onset and offset, 1-20 s). In contrast, the non-anesthetic 1,2-dichlorohexafluorocyclobutane did not potentiate TOK1 currents in concentrations up to five times the MAC value predicted by the Meyer-Overton hypothesis based on oil/gas partition coefficients. Single TOK1 channel currents were recorded from excised outside-out patches. The single channel open probability increased as much as twofold in the presence of isoflurane and rapidly returned to the baseline values on washout. Volatile anesthetic agents did not alter the TOK1 single channel current-voltage (I-V) relationship, however, suggesting that the site of action does not affect the permeation pathway of the channel.     

The Lipid Composition of Human Epididymis

In the present study the lipid composition of human epididymidis is reported. The total lipids represented 1.25 per cent of the total wet weight of the tissue. The phospholipids formed 64 per cent, cholesterol 12 per cent and glycerides 24 per cent of the total lipids. In patients with obstruction of efferent ducts, the total lipids were increased due to the significant increases observed in glycerides and cholesterol of the epididymidis. There was a marked fall in epididymal phospholipids due to a fall in phosphatidyl choline and phosphatidyl ethanolamine.     

脑片技术在麻醉药理学研究中的应用

脑片技术是一项兼具在体和离体实验优点的技术方法 ,随着电生理学研究在脑片中的深入和多种实验方法的整合 ,脑片技术越来越多地被麻醉药理学研究所采用 ,并在全麻原理、脑保护机制等领域发挥着重要的作用。     

钙通道拮抗剂作用机制及其对人类精子的影响

钙离子作为细胞内第二信使 ,广泛存在于体细胞内 ,因而钙通道拮抗剂 (CCB)被广泛应用于心脏、骨骼肌和神经元等研究及心血管疾病治疗。随着对人类精子细胞膜钙离子通道的研究深入 ,不仅阐明了CCB的作用机制 ,而且通过研究CCB对精子的形态、活力及顶体反应的影响 ,证明CCB对人类精子的功能有明显影响 ,并为男性药物避孕提供了一条新的途径。     

Neosaxitoxin as a Local Anesthetic: Preliminary Observations from a First Human Trial

Background: Neosaxitoxin is a phycotoxin that reversibly blocks the voltage-gated sodium channels at the neuronal level. Its activity results in blocking the axonal conduction, stopping the propagation of the nerve impulse. The objective of the present work was to evaluate neosaxitoxin as a local anesthetic in a human trial.

Methods: The authors conducted a randomized, double-blind, placebo-controlled trial with 10 healthy volunteers. Subcutaneous injections were made in the middle posterior skin of the calf: one leg received 50 [mu]g neosaxitoxin, and the contra-lateral leg received placebo. The anesthetic effect was evaluated using a standardized human sensory and pain model. TSA II Neurosensory Analyzer (Medoc Ltd, Minneapolis, MN) and von Frey technique were used to evaluate five parameters: sensory threshold for warm and cold, pain thresholds for heat and cold, and mechanical touch perception threshold. Measurements were made 0, 1, 3, 6, 9, 12, 16, 24, and 48 h after the injections.

Results: For all the patients, effective and complete blocking of the evaluated parameters was obtained. As the blocking began to revert gradually, heat pain was the first to return to normal values after 3 h. Cold pain was the longest sensation abolished, achieving 24 h of blockade. The toxin was undetected in blood and urine samples. No adverse reactions to neosaxitoxin were detected.     

Local Anesthetic Effects on Priming and Activation of Human Neutrophils

Background: Local anesthetics (LAs) have been shown to inhibit human polymorphonuclear neutrophil (hPMN) functions in vitro, but mechanisms are poorly understood. In this study the authors determined how LAs affect superoxide anion production of hPMNs primed with platelet-activating factor (PAF). The authors studied which pharmacologic properties of LAs are important for this action and assessed the LA site of action within the PAF signaling pathway.

Methods: Metabolic activity of primed and/or activated hPMNs were measured using the cytochrome-c assay. hPMNs were incubated with several LAs for 1 h to assess interference with PAF signaling. Using protein kinase C (PKC) inhibitors, the PKC activator phorbol myristate acetate (PMA), and the phospholipase C (PLC) antagonist U-73122, we studied involvement of PKC and PLC in the priming process. Pertussis toxin (PTX) was used to characterize the G proteins mediating this pathway. Combined administration of lidocaine with PMA or PTX was used to determine the LA site of action within the priming pathway.

Results: Platelet-activating factor effectively primed hPMNs. Ester LAs (tetracaine and benzocaine) exerted the most profound inhibitory effect on PAF-primed hPMNs, whereas inhibitory potency of amide LAs increased with decreased charged fraction. The major PAF-induced priming pathway is PLC- and PKC-dependent and mainly Gq-mediated. The main target site for LA in this pathway is located upstream of PKC.     

Nonhalogenated Alkane Anesthetics Fail to Potentiate Agonist Actions on Two Ligand-gated Ion Channels

Background: Although ether, alcohol, and halogenated alkane anesthetics potentiate agonist actions or increase the apparent agonist affinity of ligand-gated ion channels at clinically relevant concentrations, the effects of nonhalogenated alkane anesthetics on ligand-gated ion channels have not been studied. The current study assessed the abilities of two representative nonhalogenated alkane anesthetics (cyclopropane and butane) to potentiate agonist actions or increase the apparent agonist affinity of two representative ligand-gated ion channels: the nicotinic acetylcholine receptor and [gamma]-aminobutyric acid type A (GABAA) receptor.

Methods: Nicotinic acetylcholine receptors were obtained from the electroplax organ of Torpedo nobiliana, and human GABAA receptors ([alpha]1[beta]2[gamma]2L) were expressed in human embryonic kidney 293 cells. The Torpedo nicotinic acetylcholine receptors apparent agonist affinity in the presence and absence of anesthetic was assessed by measuring the apparent rates of desensitization induced by a range of acetylcholine concentrations. The GABAA receptor's apparent agonist affinity in the presence and absence of anesthetic was assessed by measuring the peak currents induced by a range of GABA concentrations.

Results: Neither cyclopropane nor butane potentiated agonist actions or increased the apparent agonist affinity (reduced the apparent agonist dissociation constant) of the Torpedo nicotinic acetylcholine receptor or GABAA receptor. At clinically relevant concentrations, cyclopropane and butane reduced the apparent rate of Torpedo nicotinic acetylcholine receptor desensitization induced by low concentrations of agonist.     

Nonhalogenated Alkanes Cyclopropane and Butane Affect Neurotransmitter-gated Ion Channel and G-protein-coupled Receptors: Differential Actions on GABAA and Glycine Receptors

Background: Anesthetic mechanisms of nonhalogenated alkanes cyclopropane and butane are not understood. This study was designed to look at which neurotransmitter receptors are possible targets for these anesthetics.

Methods: Effects of cyclopropane and butane on eight recombinant receptors expressed in Xenopus oocytes were examined electrophysiologically. To address molecular mechanisms of interaction with glycine and [gamma]-aminobutyric acid type A (GABAA) receptors, cyclopropane was further tested on [alpha]1(S267C) glycine receptor and [alpha]2(S270X)[beta]1 GABAA receptors that were mutated to amino acids with larger side chains.

Results: Cyclopropane (1, 2, and 5 minimum alveolar concentration [MAC]) potentiated glycine responses by 39, 62, and 161%, respectively, and butane (1 MAC) potentiated by 64% with an increase in apparent affinity for glycine, but yielded barely detectable potentiation of GABAA receptors. The efficacy of cyclopropane for glycine receptors was less than isoflurane and halothane. The potentiation by cyclopropane was eliminated for the [alpha]1(S267C) glycine receptor. Mutant GABAA receptors in which the corresponding amino acid was substituted with larger amino acids did not produce significant potentiation. Cyclopropane and butane inhibited nicotinic acetylcholine and N-methyl-d-aspartate receptors, potentiated G-protein-coupled inwardly rectifying potassium channels, and did not change 5-hydroxytryptamine3A or muscarinic1 receptor function. Only cyclopropane markedly inhibited [alpha]-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors.     

Effects of Fetal pH on Local Anesthetic Transfer across the Human Placenta

Background: Fetal acidemia increases umbilical venous bupivacaine concentrations in the in situ rabbit model. The authors studied the effects of decreasing fetal pH on the rate of maternal to fetal (M-->F) clearances of lidocaine, bupivacaine, 2-chloroprocaine, and antipyrine (a nonionic marker of placental transfer) across the isolated, dual perfused, human placental cotyledon.

Methods: Maternal to fetal clearances of bupivacaine, lidocaine, 2-chloroprocaine, and antipyrine were determined at fetal pH (7.4), during progressive fetal acidemia (pH 7.2-->7.0-->6.8), and after recovery to fetal pH 7.4 in experiments with both low protein state and in those with in vivo maternal and fetal protein-binding potentials.

Results: Placental transfer of all three agents increased linearly as the fetal pH decreased. Antipyrine transfer was unaffected. Clearance of lidocaine and bupivacaine, but not 2-chloroprocaine, returned to baseline when fetal pH was restored to 7.4. When maternal and fetal protein-binding potentials were increased, clearance at fetal pH 7.4 of bupivacaine, but not lidocaine, decreased significantly. During fetal acidemia, the transfer of both agents increased, but to a lesser extent than in the low protein concentration experiments.     

Effect of Local Anesthetic on Neuronal Cytoplasmic Calcium and Plasma Membrane Lysis (Necrosis) in a Cell Culture Model

Background: To investigate the mechanism by which rare cases of spinal local anesthetic (LA) neurotoxicity occur, we have tested the hypotheses that LAs elevate cytoplasmic calcium (Ca2+cyt), that this is associated with a neurotoxic effect, and that lidocaine and bupivacaine differ in their neurotoxicity.

Methods: Neurons of the ND7 cell culture line, derived from dorsal root ganglion, were loaded with fura-2 and analyzed by digitized video fluorescence microscopy during 60 min LA exposure, allowing determination of Ca2+cyt and time of necrotic cell death (plasma membrane lysis) at the single neuron level.

Results: Lidocaine 0.1% and bupivacaine 0.025% caused minimal changes in Ca2+cyt. Lidocaine 0.5-5% and bupivacaine 0.125-0.625% caused an early, small (less than threefold), concentration-dependent increase in Ca2+cyt that was transient and returned to near baseline within 10 min. Lidocaine 2.5% and 5% then caused a sustained, greater than ten-fold increase in Ca2+cyt and death in some neurons during the 60 min exposure period. Pretreatment with thapsigargin eliminated the initial transient increase in Ca2+cyt, consistent with endoplasmic reticulum (ER) as its source, and increased neuronal death with 5% lidocaine, suggesting that lidocaine neurotoxicity can be increased by failure of ER to take up elevated Ca2+cyt. The later sustained increase in Ca2+cyt seen with 2.5 and 5% lidocaine was prevented in Ca2+-free medium, and restored when Ca2+ was added back to the buffer in the presence of lidocaine, suggesting that higher concentrations of lidocaine increase influx of Ca2+ through the plasma membrane.     

The Young Adult Human Brain: An MRI-based Morphometric Analysis

Morphometric analysis was performed on three-dimensional MRIscans of 10 male and 10 female young adults with four principalobjectives: (1) to characterize in vivo volumes of whole brainand substructures, (2) to explore volumetric symmetry in bilateralstructures, (3) to consider the extent to which volumetric measuresare dimorphic in the male and female brain, and (4) to providea normal volumetric database for the young adult brain. Totalbrain volumes ranged between 1173 and 1626 cm³. All bilateralstructures were symmetric or nearly symmetric in volume, withthe exception of a slightly larger right neocortex and amygdala,and larger left lateral ventricle. Male brains were larger involume than female brains, a difference that reached significancefor cerebellar but not for cerebral hemisphere volume. In females,there was less cerebral white matter while caudate volume waslarger than in the male brains. The proportions of caudate andhippocampus relative to total cerebral volumes were larger infemales than in males. These four measures accurately predictedgender in 85% of the subjects by discriminant analysis. No genderdifferences were noted in the structural symmetry analysis.These results represent the first step in establishing a comprehensivedatabase of morphometric parameters, with unexpected findingsrelative to brain symmetry and sexual dimorphism.     

人精子质膜孕激素受体研究

目的 :观察人精子表面孕激素受体 (PR)的定位及阳性表达率。　方法 :精子体外获能后 ,应用异硫氰酸荧光素标记的牛血清白蛋白 孕酮复合物 (P BSA FITC)染色 ,荧光显微镜观察及流式细胞术 (FCM )定量分析法 ,分别观察与孕酮 (P)结合精子形态及标记精子所占比例。　结果 :P BSA FITC染色精子的形态主要表现为 2种类型 :整个顶体区域或仅赤道区呈绿色荧光 ,顶体后区及尾部不着色。与P结合精子的百分率为 (30 2± 2 4 ) %。　结论 :人精子顶体表面有PR表达 ,且这种表达呈选择性