Time-dependent modulation of muscarinic m1/m3 receptor signalling by local anaesthetics.

BACKGROUND: Signalling of several G-protein-coupled receptors of the Gq/11 family is time-dependently inhibited by local anaesthetics (LAs). Since LA-induced modulation of muscarinic m1 and m3 receptor function may explain their beneficial effects in clinical practice, such as decreased postoperative cognitive dysfunction or less bronchoconstriction, we studied how prolonged exposure affects muscarinic signalling (Wang D, Wu X, Li J, Xiao F, Liu X, Meng M. The effect of lidocaine on early postoperative cognitive dysfunction after coronary artery bypass surgery. Anesth Analg 2002; 95: 1134-41; Groeben H, Silvanus MT, Beste M, Peters J. Combined lidocaine and salbutamol inhalation for airway anesthesia markedly protects against reflex bronchoconstriction. Chest 2000; 118: 509-15). METHODS: A two-electrode voltage clamp was used to assess the effects of lidocaine or its permanently charged analogue QX314 on recombinantly expressed m1 and m3 receptors in Xenopus oocytes. Antisense knock-down of functional Gαq-protein and inhibition of protein kinase C (PKC) served to define mechanisms and sites of action. RESULTS: Lidocaine affected muscarinic signalling in a biphasic way: an initial decrease in methylcholine bromide-elicited m1 and m3 responses after 30 min, followed by a significant increase in muscarinic responses after 8 h. Intracellularly injected QX314 time-dependently inhibited muscarinic signalling, but had no effect in Gαq-depleted oocytes. PKC-antagonism enhanced m1 and m3 signalling, but completely abolished the LA-induced increase in muscarinic responses, unmasking an underlying time-dependent inhibition of m1 and m3 responses after 8 h. CONCLUSIONS: Lidocaine modulates muscarinic m1 and m3 receptors in a time- and Gαq-dependent manner, but this is masked by enhanced PKC activity. The biphasic time course may be due to interactions of LAs with an extracellular receptor domain, modulated by PKC activity. Prolonged exposure to LAs may not benefit pulmonary function, but may positively affect postoperative cognitive function.

Ketamine induces apoptosis via the mitochondrial pathway in human lymphocytes and neuronal cells.

BACKGROUND: Ketamine has been shown to have neurotoxic properties, when administered neuraxially. The mechanism of this local toxicity is still unknown. Therefore, we investigated the mechanism of cytotoxicity in different human cell lines in vitro. METHODS: We incubated the following cell types for 24 h with increasing concentrations of S(+)-ketamine and racemic ketamine: (i) human Jurkat T-lymphoma cells overexpressing the antiapoptotic B-cell lymphoma 2 protein, (ii) cells deficient of caspase-9, caspase-8, or Fas-associated protein with death domain and parental cells, and (iii) neuroblastoma cells (SHEP). N-Methyl-d-aspartate (NMDA) receptors and caspase-3 cleavage were identified by immunoblotting. Cell viability and apoptotic cell death were evaluated flowcytometrically by Annexin V and 7-aminoactinomycin D double staining. Mitochondrial metabolic activity and caspase-3 activation were measured. RESULTS: Ketamine, in a concentration-dependent manner, induced apoptosis in lymphocytes and neuroblastoma cell lines. Cell lines with alterations of the mitochondrial pathway of apoptosis were protected against ketamine-induced apoptosis, whereas alterations of the death receptor pathway did not reduce apoptosis. S(+)-Ketamine and racemic ketamine induced the same percentage of cell death in Jurkat cells, whereas in neuroblastoma cells, S(+)-ketamine was slightly less toxic. CONCLUSIONS: Ketamine at millimolar concentrations induces apoptosis via the mitochondrial pathway, independent of death receptor signalling. At higher concentrations necrosis is the predominant mechanism. Less toxicity of S(+)-ketamine was observed in neuroblastoma cells, but this difference was minor and therefore unlikely to be mediated via the NMDA receptor.

Monocyte phagocytosis of viable Staphylococcus aureus is impaired by barbiturates, but not by propofol.

BACKGROUND: Barbiturates and propofol are used for deep sedation of patients with elevated intracranial pressure refractory to standard therapeutic regimens. Such patients often suffer from bacterial infections, which are most commonly caused by Staphylococcus aureus. Various interactions of anesthetics with components of the host defense have been documented, but very little is known about the influence on monocytes, which are a first-line defense against bacterial invasion. Therefore, we studied the effects of thiopental, methohexital, and propofol on monocyte phagocytosis using an in vitro whole blood model of viable S. aureus. MATERIALS AND METHODS: Whole blood samples were preincubated with different concentrations of thiopental, methohexital, and propofol. Phagocytosis was stopped at different time points after addition of viable S. aureus. Monocytes then were stained with monoclonal antibodies for flow cytometric analysis of monocyte recruitment (ratio of ingesting monocytes). Furthermore, the fluorescence intensity of ingested bacteria served as semiquantitative measurement of phagocytosis activity. RESULTS: Both barbiturates inhibited monocyte recruitment and phagocytosis activity concentration-dependently, whereas propofol did not affect any of the investigated parameters. At concentrations of 7.6 x10(-3) M thiopental or 1.1 x 10(-3) M methohexital and greater, monocyte recruitment and phagocytosis activity were significantly inhibited. The calculated half-maximum inhibitory concentration (IC50) of thiopental was 8.4 x 10(-3) M for monocyte recruitment and 8.6 x 10(-3) M for phagocytosis activity. The corresponding values for methohexital were 4.1 x 10(-3) M and 1.1 x 10(-3) M, respectively. CONCLUSION: The two barbiturates induce concentration-dependent inhibition of monocyte phagocytosis, whereas propofol is without effect. In combination with previously described effects on granulocyte function, these findings suggest that defense against bacterial infection might be reduced by barbiturates.

Prevention and control of postoperative nausea and vomiting in post-craniotomy patients.

Postoperative nausea and vomiting (PONV) are the most frequent side-effects in the postoperative period, impairing subjective well-being and having economic impact due to delayed discharge. However, emetic symptoms can also cause major medical complications, and post-craniotomy patients may be at an increased risk. A review and critical appraisal of the existing literature on PONV in post-craniotomy patients, and a comparison of these findings with the current knowledge on PONV in the general surgical population, leads to the following conclusions: (1) Despite the lack of a documented case of harm caused by retching or vomiting in a post-craniotomy patient, the potential risk caused by arterial hypertension and high intra-abdominal/intra-thoracic pressure leading to high intracranial pressure, forces to avoid PONV in these patients. (2) There is unclarity about a specifically increased (or decreased) risk for PONV in post-craniotomy patients compared with other surgical procedures. (3) The decision whether or not to administer an antiemetic should not be based primarily on risk scores for PONV but on the likelihood for potential catastrophic consequences of PONV. If such a risk cannot be ruled out, a multimodal antiemetic approach should be considered regardless of the individual risk. (4) Randomized controlled trials with antiemetics in post-craniotomy patients are limited with respect to sample size and methodological quality. This also impacts upon the meaning of meta-analyses performed with trials that showed marked heterogeneity and inconclusive results. (5) No studies on the treatment of established PONV are available. This highlights the need to transfer knowledge about PONV treatment from other surgical procedures. (6) Despite the possibility that PONV in post-craniotomy patients can be triggered by specific conditions (e.g. surgery near the area postrema at the floor of the fourth ventricle with the vomiting centre located nearby), recommendations based on trials in post-craniotomy patients may be flawed. Thus, general knowledge on prevention and treatment of PONV must adopted for craniotomy settings.

Local anaesthetics inhibit signalling of human NMDA receptors recombinantly expressed in Xenopus laevis oocytes: role of protein kinase C.

BACKGROUND: N-methyl-D-aspartate (NMDA)-receptor activation contributes to postoperative hyperalgesia. Studies in volunteers have shown that intravenous local anaesthetics (LAs) prevent the development of hyperalgesic pain states. One potential explanation for this beneficial effect is the inhibition of NMDA receptor activation. Therefore, we studied the effects of LA on NMDA receptor function. METHODS: The human NR1A/NR2A NMDA receptor was expressed recombinantly in Xenopus laevis oocytes. Peak currents were measured by voltage clamp in Mg- and Ca2+-free, Ba2+-containing Tyrode's solution. Holding potential was -70 mV. Oocytes were stimulated with glutamate/glycine (at EC50) with or without 10 min prior incubation in bupivacaine, levobupivacaine, S-(-)-ropivacaine, or lidocaine (all at 10(-9)-10(-4) M), procaine (10(-4) M), R-(+)-ropivacaine (10(-4) M), QX314 (permanently charged, 5 x 10(-4) M) extracellularly or intracellularly or benzocaine (permanently uncharged, 5 x 10(-3) M). We also determined the effect of the protein kinase C (PKC) inhibitors chelerythrine (5 x 10(-5) M), calphostin C (3 x 10(-6) M) and Ro 31-8220 (10(-7) M), and the effect of PKC activation with phorbolester (10(-6) M). RESULTS: Non-injected oocytes were unresponsive to agonist application, but oocytes expressing NMDA receptors responded with inward currents (1.1+/-0.08 microA). All LA concentration-dependently inhibited agonist responses. The inhibition was reversible and stereoselective. Intracellular QX314 reduced responses to 59% of control, but extracellular QX314 was without effect. Benzocaine reduced responses to 33% of control. PKC inhibitors had no additional inhibitory effect beyond that of bupivacaine. The effect of PKC activation was abolished in the presence of bupivacaine. CONCLUSION: All LA tested inhibited the activation of human NMDA receptors in a concentration dependent fashion. This effect may contribute to reduced hyperalgesia and opiate tolerance observed after systemic administration of LA. The effect is independent of the charge of LA; site of action is intracellular. The mechanism of action may be mediated by inhibition of PKC.

Effects of cafedrine/theodrenaline, etilefrine and ephedrine on uterine blood flow during epidural-induced hypotension in pregnant sheep.

INTRODUCTION: Maternal hypotension is a major concern in obstetric anesthesia, and concerns have been raised about standard vasopressor therapy with ephedrine. Therefore, we evaluated the maternal and fetal hemodynamic effects of two potential alternatives to ephedrine. METHODS: Hypotension was induced by epidural administration of lidocaine in 6 chronically instrumented pregnant ewes (at 118-122 days of gestation, term 145 days). Three treatments were studied: 25 mg ephedrine, 5 mg etilefrine and 100 mg cafedrine/5 mg theodrenaline (C/T) intravenously. Mean fetal and maternal blood pressure and heart rate, uterine blood flow, as well as fetal and maternal arterial blood gases were recorded for 60 min. RESULTS: All three vasopressors increased maternal blood pressure, accompanied by a significant increase in uterine blood flow. C/T caused marked maternal tachycardia, whereas ephedrine decreased maternal heart rate. Maternal and fetal blood gases did not change during any of the three treatment regimens. CONCLUSION: All three vasopressors restored maternal blood pressure and uterine blood flow after epidurally induced maternal hypotension. However, restoration of uterine perfusion was delayed and less pronounced with C/T.

Epidural local anesthetics: a novel treatment for fetal growth retardation?

BACKGROUND: Chronically compromised uterine perfusion may lead to placental insufficiency and subsequent intrauterine growth restriction (IUGR). Various therapeutic approaches (e.g. vasodilators, low-dose aspirin, intravenous glucose infusion, and hemodilution) are often of limited efficacy. Local anesthetics have been shown to improve placental blood flow in pre-eclamptic women. We hypothesized that epidural administration of local anesthetics might improve outcome in IUGR independent of the underlying cause. In preparation for a clinical trial to test this hypothesis, we performed a pilot study in 10 patients. METHODS: After approval of the study protocol, 10 pregnant women presenting with oligohydramnios and IUGR were included in the study. In addition to our standard protocol (magnesium, glucose, betamethasone), each patient received an epidural catheter (T10/T12) with continuous infusion of bupivacaine 0.175% at a rate of 5 ml/h. Uteroplacental circulation was monitored by Doppler sonography and the amount of amniotic fluid was estimated daily. RESULTS: Epidural insertion and infusion was performed without complications. Four patients continued to deteriorate rapidly, amniotic fluid volume did not change and uterine artery pulsatility index (PI) tended to increase. In the remaining 6 patients the clinical status stabilized, amniotic fluid volume tended to increase and uterine artery PI tended to decrease during treatment. This improvement was associated with a prolonged interval to cesarean section and increased infant birth weight. CONCLUSION: Our data suggest that, even if the underlying cause of IUGR is not pre-eclampsia, epidural local anesthetic administration might improve placental blood flow and be beneficial in a subgroup of patients. A clinical trial to test this hypothesis appears warranted.

Clinical efficacy of postoperative autologous transfusion of filtered shed blood in hip and knee arthroplasty.

BACKGROUND: Total knee arthroplasty (TKA) or total hip arthroplasty (THA) regularly results in postoperative requirement of blood transfusion. Because of the disadvantages of allogeneic blood transfusion (ABT) such as the risk of transfusion-associated infections, incompatibility-related transfusion fatalities, or immunomodulatory effects, a continuing effort to reduce allogeneic blood transfusion is important. For this purpose, the effect of reinfusion of drain blood, via a postoperative wound drainage and reinfusion system, on the need for allogeneic blood transfusion was evaluated. STUDY DESIGN AND METHODS: Using a prospective observational quality assessment design, we compared 135 patients scheduled for TKA or THA with a historic group of 96 patients. In the study group the Bellovac ABT autotransfusion system was used. The shed blood was returned either when 500 mL were collected or at most 6 hours after surgery. Compared were the preoperative, postoperative, and discharge hemoglobin, as well as the number of allogeneic blood transfusions. RESULTS: There were no statistical differences between preoperative, postoperative, and discharge hemoglobin levels. Autologous transfusion reduced the number of patients receiving ABT overall from 35 percent (control) to 22 percent (study). The decrease of allogeneic transfusion requirement was most significant after TKA: from 18 percent to 6 percent (p < 0.001). CONCLUSION: We conclude that the Bellovac ABT device reduces allogeneic blood transfusions in TKA and THA.

COX 2 selectivity of non-steroidal anti-inflammatory drugs and perioperative blood loss in hip surgery. A randomized comparison of indomethacin and meloxicam.

BACKGROUND: In this prospective randomized study we tested the hypothesis that use of more cyclo-oxygenase 2 (COX 2)-selective non-steroidal anti-inflammatory drugs (NSAIDs) can reduce perioperative blood loss compared with non-selective NSAIDs. METHODS: Data from 200 patients who underwent total hip replacement were studied. Two NSAIDs were compared: indomethacin 50 mg (n = 82) and meloxicam 15 mg (n = 86). Both NSAIDs were given orally 1 h before surgery. RESULTS: The two groups were not different with respect to age, gender, ASA class or duration of surgery. When indomethacin was used preoperatively, intraoperative blood loss was 623 +/- 243 mL (mean +/- SD) and postoperative blood loss 410 +/- 340 mL. After meloxicam, these values were 524 +/- 304 mL and 358 +/- 272 mL, respectively. Total perioperative blood loss after meloxicam was 17% (P < 0.05) less than that observed after indomethacin. CONCLUSION: Perioperative blood loss after meloxicam is less than after indomethacin. These in vivo findings are consistent with in vitro results using selective COX 2 NSAIDs.

Effects of calcium and magnesium pretreatment on hyperkalaemic cardiac arrest in rats.

BACKGROUND AND OBJECTIVE: Administration of calcium safely and effectively reverses many of the electrophysiological actions of hyperkalaemia, but it has not been studied for pretreatment. Based on cellular studies, magnesium also has been suggested to prevent the effects of potassium on the heart. As their mechanisms of action differ, a combination of these drugs might have a synergistic protective action. Both compounds are inexpensive and can be administered safely in modest doses. We investigated whether magnesium, calcium or their combination could protect against hyperkalaemic cardiac arrest. METHODS: Twenty-four adult rats were anaesthetized with halothane and randomly pretreated with CaCl2 15 mg kg(-1), MgSO4 30 mg kg(-1), CaCl2 7.5 mg kg(-1) + MgSO4 15 mg kg(-1) or physiological saline. Potassium (0.01 mmol kg(-1) h(-1)) was infused. The times to the first dysrhythmia, mean arterial pressure decrease to <40% of baseline and cardiovascular collapse were measured. RESULTS: Serum potassium concentrations increased to similar values in all groups (to 12.0 +/- 0.2 mmol L(-1) at the time of collapse). No differences in survival times were observed between groups. There was a trend for respiratory values to be better in the group receiving magnesium. CONCLUSIONS: Pretreatment with magnesium, calcium or a combination of both did not influence the time to cardiovascular collapse, and is therefore--at least in our model--not of any benefit in preventing hyperkalaemic cardiac arrest.

Toxicity of local anaesthetics.

The complications of failure, neural injury and local anaesthetic toxicity are common to all regional anaesthetic techniques, and individual techniques are associated with specific complications. All potential candidates for regional anaesthesia should be thoroughly evaluated and informed of potential complications. Central neural blockades still account for more than 70% of regional anaesthesia procedures. Permanent neurological injury is 0.02-0.07%. Pain on injection and paraesthesias while performing regional anaesthesia are danger signals of potential injury and must not be ignored. The incidence of systemic toxicity to local anaesthetics has significantly decreased in the past 30 years, from 0.2 to 0.01%. Peripheral nerve blocks are associated with the highest incidence of systemic toxicity (7.5 per 10,000) and the lowest incidence of serious neural injury (1.9 per 10,000).

Local anesthetic actions on thromboxane-induced platelet aggregation.

UNLABELLED: Some local anesthetics (LA), in concentrations present in blood during IV or epidural infusion, inhibit thrombus formation in the postoperative period. Studies on thromboxane A2 (TXA2) signaling in a recombinant model suggest that interference with TXA2-induced platelet aggregation may explain, in part, the antithrombotic actions of epidural analgesia and IV LA infusion. In this study we investigated the effects of clinically used LAs (lidocaine, ropivacaine, and bupivacaine) on TXA2-induced early platelet aggregation (1-5 s) by using quenched-flow and optical aggregometry. Our findings demonstrate that the LAs tested seem to have only a limited ability to inhibit TXA2-induced platelet aggregation assessed at early times (1-5 s). Therefore, the clinical effects of LAs on thrombi formation are unlikely to be explained by this manner alone. At large LA concentrations, moderate effects were obtained. Prolonged incubation with LA did not significantly increase effectiveness, and the lack of an effect could not be explained by generation of secondary mediators. The results were independent of the anesthetic studied. Local anesthetic effects on TXA2-induced early platelet aggregation (1-5 s) are unlikely to play a major role in the clinically observed antithrombotic effects of local anesthetics. IMPLICATIONS: Local anesthetic effects on thromboxane A2-induced early platelet aggregation (1-5 s) are unlikely to play a major role in the clinically observed antithrombotic effects of local anesthetics. Thus, other potential targets need to be explored.

The effects of isoflurane on native and chimeric muscarinic acetylcholine receptors: the role of protein kinase C.

UNLABELLED: By using two electrode voltage clamps, we investigated the effects of isoflurane on m3 and chimeric m1/m3 muscarinic receptors and the role of protein kinase C (PKC) in the effects. Muscarinic receptors were expressed by injection of mRNA into Xenopus oocytes, and Ca(2+)-activated Cl(-) currents were measured after the application of acetyl-beta-methylcholine. We constructed chimeric m1/m3 receptor DNA encoding the third intracellular loop of m1 and the remainder from the m3 receptor. Chimeric and m3 receptors were inhibited by isoflurane, but the m1 receptor was not. PKC activation with phorbol-12-myrisate-13-acetate (50 nM) decreased signaling of both chimeric and m3 receptors significantly. Chelerythrine (20 microM, PKC inhibitor) abolished the effect of isoflurane on chimeric and m3 signaling. Whereas isoflurane inhibition of chimeric and m3 receptors was completely reversible after washout with Tyrode's solution for 3 min, treatment with okadaic acid (500 nM, protein phosphatase inhibitor) rendered the inhibition irreversible. Taken together, our results suggest that isoflurane inhibits m3 and chimeric m1/m3 muscarinic signaling by enhancing PKC activity and that the site of action is located outside of the third intracellular loop. IMPLICATIONS: By use of the Xenopus oocyte expression system, we investigated the effects of isoflurane on muscarinic signaling and the role of protein kinase C in these effects. Our findings suggest that isoflurane inhibits muscarinic receptors through activation of protein kinase C and that the relevant phosphorylation sites are located outside the third intracellular loop.

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. CONCLUSIONS: Local anesthetics in clinically relevant concentrations inhibit superoxide anion production of PAF-primed hPMNs. Effects on priming by these compounds might explain, at least in part, the previously unexplained difference between concentrations of LAs required for their antiinflammatory action in vitro and in vivo. This study suggests a target site for LAs within a Gq-coupled signaling pathway.