Zhiming Tan

Increased Activity of Small GTP-binding Protein-dependent Phospholipase D during Differentiation in Human Promyelocytic Leukemic HL60 Cells

In response to dibutyryl cyclic AMP (dbcAMP) and all-trans retinoic acid, human promyelocytic leukemic HL60 cells differentiate into granulocyte-like cells. In cell lysate and in vitro reconstitution system, phospholipase D (PLD) activity in response to guanosine 5′-O-(3-thiotriphosphate) (GTPγS) was up-regulated by dbcAMP or all-trans retinoic acid treatment. In the present study, the mechanism(s) for increased PLD activity during differentiation was examined. Western blot analysis revealed that the contents of ADP-ribosylation factor, Rac2, and Cdc42Hs but not RhoA and Rac1 in the cytosolic fraction were elevated during differentiation. However, the cytosolic fraction from undifferentiated cells was almost equally potent as the cytosolic fraction from differentiated cells in the ability to stimulate membrane PLD activity. It was shown that the GTPγS-dependent PLD activity in membranes from differentiated cells was much higher than that in membranes from undifferentiated cells, suggesting that the increased PLD activity during differentiation was due to alterations in some membrane component(s). Clostridium botulinum ADP-ribosyltransferase C3 and C. difficile toxin B, which are known as inhibitors of RhoA and Rho family proteins, respectively, effectively suppressed PLD activity in membranes from differentiated cells. In fact, the amount of membrane-associated RhoA was increased during differentiation. Furthermore, the extent of GTPγS-dependent PLD activity partially purified from membranes from differentiated cells was greater than that from membranes from undifferentiated cells in the presence of recombinant ADP-ribosylation factor 1. The PLD (hPLD1) mRNA level was observed to be up-regulated during differentiation, as inferred by reverse transcription-polymerase chain reaction. Our results suggest the possibility that the increased Rho proteins in membranes and the changed level of PLD itself may be, at least in part, responsible for the increase in GTPγS-dependent PLD activity during granulocytic differentiation of HL60 cells.

Involvement of the mitogen-activated protein kinase family in tetracaine-induced PC12 cell death

Background To explore whether cytotoxicity of local anesthetics is related to apoptosis, the authors examined how local anesthetics affect mitogen-activated protein kinase (MAPK) family members, extracellular signal-regulated kinases (ERKs), c-Jun N-terminal kinases (JNKs)–stress-activated protein kinases, and p38 kinase, which are known to play important roles in apoptosis. Methods Cell death was evaluated using PC12 cells. Morphologic changes of cells, cellular membrane, and nuclei were observed. DNA fragmentation was electrophoretically assayed. Western blot analysis was performed to analyze phosphorylation of the MAPK family, cleavage of caspase-3 and poly(adenosine diphosphate–ribose) polymerase. Intracellular Ca2+ concentration was measured using a calcium indicator dye. Results Tetracaine-induced cell death was shown in a time- and concentration-dependent manner and characterized by nuclear condensation or fragmentation, membrane blebbing, and internucleosomal DNA fragmentation. Casepase-3 activation and phosphorylation of ERK, JNK, and p38 occurred in the cell death. PD98059, an inhibitor of ERK, enhanced tetracaine-induced cell death and JNK phosphorylation, whereas ERK phosphorylation was inhibited. Curcumin, an inhibitor of JNK pathway, attenuated the cell death. Increase of intracellular Ca2+ concentration was detected. In addition to the increase of ERK phosphorylation and the decrease of JNK phosphorylation, two Ca2+ chelators protected cells from death. Neither cell death nor phosphorylation of the MAPK family was caused by tetrodotoxin. Nifedipine did not affect tetracaine-induced apoptosis. Conclusions Tetracaine induces apoptosis of PC12 cells via the MAPK family. ERK activation protects cells from death, but JNK plays the opposite role. Toxic Ca2+ influx caused by tetracaine seems to be responsible for the cell death, but blocking of Na+ channels or L-type Ca2+ channels is unlikely involved in the tetracaines action for apoptosis.

Local Anesthetics Inhibit Muscarinic Receptor-mediated Activation of Extracellular Signal-regulated Kinases in Rat Pheochromocytoma PC12 Cells

BACKGROUND Because protein phosphorylation is a key mechanism for controlling cellular functions and extracellular signal-regulated kinase (ERK) plays a role in cellular signal transduction, the authors wanted to determine whether local anesthetics interfere with biochemical signaling molecules. METHODS Protein tyrosine phosphorylation and ERK activation induced by carbachol, an agonist for muscarinic acetylcholine receptors, were examined in rat pheochromocytoma PC12 cells, a model for investigating signal transduction. Carbachol-induced tyrosine-phosphorylated proteins of 44 and 42 kd were determined by Western blot analysis and identified as activated ERK1 and ERK2 using anti-ERK antibody. The ERK activation was blocked by preincubation with atropine or an M3 muscarinic acetylcholine receptor antagonist 4-diphenyacetooxy-1, 1-dimethylpiperidinium, indicating that is was mediated by M3 muscarinic acetylcholine receptor activation. Then, in the presence of local anesthetic, the carbachol-induced tyrosine phosphorylation and ERK activation were evaluated. The effects of three Na+ current-modifying reagents on carbachol-induced ERK activation were also evaluated. RESULTS Procaine (10(-4) to 10(-3) M) inhibited carbachol-induced tyrosine phosphorylation and ERK activation in a concentration-dependent manner. Although tetracaine, lidocaine, and bupivacaine similarly suppressed carbachol-induced tyrosine phosphorylation and ERK activation, neither tetrodotoxin, veratridine, nor ouabain affected the carbachol-induced ERKs activation. Both ERKs were also activated by 4beta-phorbol 12-myristate 13-acetate, an activator of protein kinase C, and fluoroaluminate (AlF4-), respectively, but procaine did not affect ERK activation induced by these two substances. The inhibition of carbachol-induced ERK activation by procaine was not modified by a phosphatase inhibitor, calyculin A. CONCLUSIONS The current results indicate that local anesthetics inhibit the activity of the signal-transducing molecule(s) leading to M3 muscarinic acetylcholine receptor-mediated ERK activation in PC12 cells. Such action is unlikely to be a result of the drugs action on Na+ channels or on the electrochemical gradients of the neuronal cell membrane.

Involvement of EphB1 receptor/ephrinB1 ligand in bone cancer pain.

In prior studies, Eph/ephrin system was demonstrated to be involved in inflammatory and neuropathic pain modulation. The present study was to investigate whether the spinal Eph/ephrin signaling was involved in modulation of spinal inflammatory cytokines in bone cancer pain (BCP) of rats. BCP was induced by intra-tibial inoculation of Walker 256 mammary gland carcinoma cells. The expressions of EphB1/ephrinB1 in spinal cord (SC) and dorsal root ganglia (DRG) were determined. At 16 days post inoculation, the pain relieving effect and the mRNA levels of inflammatory cytokines were detected after intrathecal administration of EphB1-Fc (blocker of EphB1 receptor, 10μg). The results showed that the EphB1/ephrinB1 expression was significantly increased in SC, but ephrinB1 was decreased in DRG after Walker 256 inoculation. The mechanical allodynia induced by bone cancer was significantly alleviated by intrathecal administration of EphB1-Fc. Furthermore, the RT-PCR analysis showed that the mRNA levels of IL-1β, IL-6 and TNF-α were significantly increased at 16 days post Walker 256 inoculation and were significantly suppressed by intrathecal administration of EphB1-Fc in SC. We concluded that Eph/ephrin might be involved in the maintenance of mechanical allodynia, via modulating the expression of spinal inflammatory cytokines, in the present rat model of BCP. This study suggested that Eph/ephrin signaling would be a potential target for the treatment of BCP.

Intravenous flurbiprofen axetil accelerates restoration of bowel function after colorectal surgery

Purpose: Flurbiprofen axetil (FA) is a potent non-steroidal antiinflammatory drug (NSAID). We examined the effects that perioperative intravenous administration of FA, combined with thoracic epidural anesthesia and postoperative patient-controlled epidural analgesia (PCEA), have on bowel function, postoperative pain, and cytokine release, after open colorectal surgery.Methods: This was a prospective, randomized, double-blind, placebo-controlled study. Forty patients were randomly assigned to one of two groups (n=20 in each group). The FA group patients received FA 1 mg·kg−1iv, 30 min before and six hours after skin incision; whereas the control group patients received an equal volume of intralipid. Blood cytokine levels were measured before FA administration, at the end of surgery, and six hours and 24 hr postoperatively. All patients received postoperative PCEA for pain control. Analgesic efficacy was evaluated for 72 hr postoperatively using visual analogue scale (VAS) pain scores both at rest and during coughing. Gastrointestinal motility was recorded. Temperature and leukocyte count were measured preoperatively, and 24 hr postoperatively.Results: The times to first bowel movement (87±23vs 105±19 hr,P=0.008) and first flatus (63±16vs 75±11 hr,P=0.01) were earlier in the FA group compared to the control group. For the first 24 hr, the pain scores in the FA group were also lower during coughing (P<0.001 compared to control). The plasma concentrations of interleukin (IL)-6 and IL-8 in the FA group were lower, postoperatively (P<0.01 andP<0.05, respectively, compared to control). In contrast, the IL-10 levels were significantly increased at six hours, postoperatively, in the FA group (P=0.009). The total leukocyte count and the incidence of pyrexia were also lower in patients of the FA group (P=0.001 0.001 andP=0.006, respectively, compared to control).Conclusion: Flurbiprofen axetil may have an anti-inflammatory effect in major abdominal surgery. The combination of perioperative intravenous FA, intraoperative thoracic epidural anesthesia, and postoperative PCEA facilitated recovery of bowel function, enhanced analgesia, and attenuated the cytokine response.RésuméObjectif: Le flurbiprofène axétil (FA) est un agent antiinflammatoire non stéroïdien (AINS) puissant. Nous avons évalué les effets d’une administration intraveineuse périopératoire de FA, en présence d’une anesthésie péridurale thoracique et d’une analgésie péridurale contrôlée par le patient (APCP) postopératoire, sur la fonction intestinale, la douleur postopératoire et la libération de cytokines à la suite d’une chirurgie colorectale ouverte.Méthode: Nous avons mené une étude prospective, randomisée, à double insu et contrôlée par placebo. Quarante patients ont été randomisés dans deux groupes (n=20 dans chaque groupe). Les patients du groupe FA ont reçu du FA 1 mg·kg-1 iv 30 min avant et six heures après l’incision de la peau ; les patients du groupe témoin ont, quant à eux, reçu un volume équivalent d’Intralipide. Les niveaux de cytokines plasmatiques ont été mesurés avant l’administration de FA, à la fin de la chirurgie, et six et 24 h après l’opération. Tous les patients ont reçu une APCP pour le contrôle de la douleur. L’efficacité analgésique a été évaluée à 72 h après l’opération sur la base des scores de douleur sur une échelle visuelle analogue (EVA) au repos et à la toux. La motilité gastro-intestinale a été enregistrée. La température et la numération leucocytaire ont été mesurées avant l’opération et 24 h après l’opération.Résultats: Les temps jusqu’à la première défécation (87±23 vs 105±19 h, P=0,008) et jusqu’aux premières flatuosités (63±16 vs 75±11 h, P=0,01) étaient plus courts dans le groupe FA que dans le groupe témoin. Durant les 24 premières heures, les scores de douleur à la toux étaient également moindres dans le groupe FA (P<0,001 par rapport au groupe témoin). Les concentrations plasmatiques d’interleukine (IL)-6 et IL-8 étaient plus basses dans le groupe FA après l’opération (P<0,01 et P<0,05, respectivement, par rapport au groupe témoin). En revanche, les niveaux IL-10 étaient significativement plus élevés à six heures après l’opération dans le groupe FA (P=0,009). La numération leucocytaire totale et l’incidence de pyrexie étaient également plus bas dans le groupe FA (P=0,001 et P=0,006, respectivement, par rapport au groupe témoin).Conclusion: Le flurbiprofène axétil pourrait avoir un effet antiinflammatoire lors de chirurgies abdominales majeures. La combinaison de FA intraveineux périopératoire, d’anesthésie péridurale thoracique peropératoire et d’APCP postopératoire a facilité le rétablissement de la fonction intestinale, amélioré l’analgésie, et atténué la sécrétion des cytokines.

Propofol protects against high glucose-induced endothelial dysfunction in human umbilical vein endothelial cells.

BACKGROUND: Hyperglycemia, via peroxynitrite-mediated endothelial nitric oxide synthase (eNOS) enzymatic uncoupling, induced endothelial dysfunction. Propofol has been reported to improve high glucose–induced endothelial dysfunction. However, its mechanisms of action remain unclear. We hypothesized that propofol could improve hyperglycemia-induced endothelial dysfunction by decreasing the peroxynitrite level and thus restoring eNOS coupling. METHODS: At the end of 3 days of incubation in medium with 30 mM glucose, human umbilical vein endothelial cells were treated with different concentrations (0.2, 1, 5, and 25 &mgr;M) of propofol for different times (0.5, 1, 2, and 4 hours). In parallel experiments, cells were cultured in 5 mM glucose for 3 days as a control. Nitric oxide (NO) production was measured with a nitrate reductase assay. Superoxide anion (O2·−) accumulation was measured with the reduction of ferricytochrome c and dihydroethidine fluorescence assay. The treatment that had maximal effect on 30 mM glucose–induced NO production and O2·− accumulation was applied in the following studies to examine the underlying signaling pathways. eNOS total protein, eNOS dimer and monomer expression, eNOS phosphorylation at Ser1177, inducible NO synthase total protein, inducible NO synthase dimer and monomer expression, peroxynitrite, and guanosine triphosphate cyclohydrolase I expression were measured by Western blot. Tetrahydrobiopterin (BH4) level was measured with liquid chromatography–mass spectrometry. RESULTS: Compared with 5 mM glucose treatment, 30 mM glucose significantly decreased NO production by 60% (P < 0.001) and increased O2·− accumulation by 175% (P = 0.0026), which were both attenuated by propofol in a concentration- and time-dependent manner. Compared with 5 mM glucose treatment, total eNOS protein expression was increased by 30 mM glucose (P < 0.001), whereas the ratio of eNOS dimer/monomer (P = 0.0001) and eNOS phosphorylation (P < 0.001) were decreased by 30 mM glucose. Propofol did not affect 30 mM glucose–induced total eNOS protein expression, but restored the ratio of eNOS dimer/monomer (P = 0.0005) and increased eNOS phosphorylation (P < 0.001). 30 mM glucose–induced O2·− accumulation was inhibited by the eNOS inhibitor hydrochloride. Furthermore, compared with 5 mM glucose treatment, 30 mM glucose decreased the BH4 level (P = 0.0001) and guanosine triphosphate cyclohydrolase I expression (P < 0.001), whereas it increased peroxynitrite level (P = 0.0003), which could all be reversed by propofol (P = 0.0045, P < 0.001, P = 0.0001 vs 30 mM glucose treatment, respectively). CONCLUSIONS: Propofol has beneficial effects on 30 mM glucose–induced NO reduction and O2·− accumulation in human umbilical vein endothelial cells. This may be mediated through inhibiting peroxynitrite-mediated BH4 reduction, and restoring eNOS coupling.

A smaller endotracheal tube combined with intravenous lidocaine decreases post-operative sore throat – a randomized controlled trial

Post‐operative sore throat (POST) has increasingly been a common clinical complication particularly in thyroid surgery. We conducted a trial to evaluate the effect of non‐pharmacological [smaller‐sized endotracheal tube (ETT)] combined with pharmacological intervention [lidocaine intravenous (i.v.)] on POST in women undergoing thyroid surgery.

Propofol attenuates angiotensin II-induced apoptosis in human coronary artery endothelial cells

BACKGROUND Angiotensin II (Ang II) induces oxidative stress and apoptosis in vascular endothelial cells. We hypothesized that propofol may attenuate Ang II-induced apoptosis in human coronary artery endothelial cells (HCAECs) and aimed to identify the underlying mechanisms. METHODS Endothelial cells were pre-treated with propofol and then stimulated with Ang II. Apoptosis was examined by TUNEL, DNA laddering, and caspase-3 activity assays. The effect of propofol on Ang II-modulated NADPH oxidase expression and activity, nitric oxide synthase III (NOSIII) expression and phosphorylation and activity, lipid peroxidation, superoxide anion generation, nitric oxide production, caspase activity, and protein expression of cytochrome c, Bcl-2, and C-IAP-1 were measured. RESULTS Ang II induced apoptosis, which was attenuated by 50 µM propofol (P<0.05). Propofol ameliorated Ang II-induced NADPH oxidase expression and activation (P<0.01), lipid peroxidation (P<0.05), and superoxide anion generation (P<0.05), whereas restoring NOSIII phosphorylation and activity (P<0.01) were down-regulated by Ang II. Propofol attenuated Ang II-modulated cytochrome c release, and the expression of Bcl-2 and C-IAP-1. In addition, propofol inhibited Ang II-induced caspase-9 (P<0.01) and caspase-3 activity (P<0.01). CONCLUSIONS Propofol protected HCAECs from Ang II-induced apoptosis by interfering with the generation of oxidative stress and redox-sensitive apoptotic pathways.

Effect of Thoracic Epidural Anesthesia with Different Concentrations of Ropivacaine on Arterial Oxygenation during One-lung Ventilation

Background:Thoracic epidural anesthesia can contribute to facilitate the fast-track approach in lung surgery. However, data regarding the effects of thoracic epidural anesthesia on oxygenation during one-lung ventilation (OLV) are scarce and contradictory. Therefore, the authors conducted a prospective, randomized, double-blinded trial in patients undergoing lung surgery under spectral entropy-guided intravenous anesthesia to evaluate the effects of thoracic epidural anesthesia with different concentrations of ropivacaine on oxygenation, shunt fraction (Qs/Qt) during OLV, and maintenance doses of propofol. Methods:One hundred twenty patients scheduled for lung surgery were randomly divided into four groups to epidurally receive saline (Group S), 0.25% (Group R0.25), 0.50% (Group R0.50), and 0.75% (Group R0.75) ropivacaine. Ropivacaine was administered intraoperatively (6–8 ml of first bolus + 5 ml/h infusion). Arterial oxygen tension (Pao2) and Qs/Qt were measured before, during, and after OLV. Results:Pao2 was significantly lower in Group R0.75 compared with that in Group S and Group R0.25 10 min (170 ± 61 vs. 229 ± 68 mmHg, P = 0.01; 170 ± 61 vs. 223 ± 70 mmHg, P = 0.03) and 20 min after OLV (146 ± 52 vs. 199 ± 68 mmHg, P = 0.009; 146 ± 52 vs. 192 ± 67 mmHg, P = 0.03). During OLV, Qs/Qt was significantly higher in Group R0.75 compared with that in Group S and Group R0.25 (P < 0.05). Maintenance doses of propofol were significantly lower in Group R0.75. Vasopressor requirements were higher in Group R0.75. Conclusion:A decrease in oxygenation during OLV occurred only at the highest dose of epidural local anesthetic and not at lower doses. Higher doses of epidural medication required less propofol and more vasopressors.

The inhibitory effect of local anesthetics on bradykinin-induced phospholipase D activation in rat pheochromocytoma PC12 cells.

Bradykinin induces activation of phospholipase D (PLD) via B2 receptors in neuronal cells. To demonstrate molecular mechanism(s) of local anesthetics, we examined whether and how local anesthetics affect bradykinin-induced PLD activation in PC12 cells. Using [3H]Palmitic acid-labeled PC12 cells stimulated with bradykinin, formation of [3H]phosphatidylbutanol was measured as a variable of PLD activity. Bradykinin-stimulated PLD activity seemed to peak at 2 min. Procaine, lidocaine, ropivacaine, bupivacaine, and tetracaine suppressed the bradykinin-induced PLD activation. We chose tetracaine, the most potent drug among the local anesthetics tested, to examine how local anesthetics affect phospholipase C, protein tyrosine kinase, and extracellular signal-regulated kinase, which are the molecules upstream of PLD. Tetra- caine at clinically relevant concentrations (1∼10 × 10−4 M) inhibited the bradykinin-induced PLD activation in a dose- and time-dependent manner, but neither tetrodotoxin nor nifedipine affected the PLD activation. Tetracaine (5 × 10−4 M) slightly potentiated brady-kinin-induced phospholipase C activation. Bradykinin-stimulated protein tyrosine-phosphorylation and extracellular signal-regulated kinase activation were not affected by tetracaine. Tetracaine significantly decreased PLD activity of membrane fraction in PC12 cells. These results indicate that local anesthetics depress bradykinin-induced lipid signaling pathway(s) and may provide some clues to understanding the molecular mechanisms of these drugs for anesthesia or analgesia.