Effect of bupivacaine and levobupivacaine on exocytotic norepinephrine release from rat atria

BACKGROUND: The cardiotoxic mechanism of local anesthetics may include interruption of cardiac sympathetic reflexes. The authors undertook this investigation to determine if clinically relevant concentrations of bupivacaine and levobupivacaine interfere with exocytotic norepinephrine release from cardiac sympathetic nerve endings. METHODS: Rat atria were prepared for measurements of twitch contractile force and [H]-norepinephrine release. After nerve endings were loaded with [H]-norepinephrine, the tissue was electrically stimulated in 5-min episodes during 10 10-min sampling periods. After each period, a sample of bath fluid was analyzed for radioactivity and [H]-norepinephrine release was expressed as a fraction of tissue counts. Atria were exposed to buffer alone during sampling periods 1 and 2 (S1 and S2). Control atria received saline (100 microl each, n = 6 atria) in S3-S10. Experimental groups (n = 6 per group) received either bupivacaine or levobupivacaine at concentrations (in microM) of 5 (S3-S4), 10 (S5-S6), 30 (S7-S8), and 100 (S9-S10). RESULTS: Bupivacaine and levobupivacaine decreased stimulation-evoked fractional [H]-norepinephrine release with inhibitory concentration 50% values of 5.1 +/- 0.5 and 6.1 +/- 1.3 microM. The inhibitory effect of both local anesthetics (approximately 70%) approached that of tetrodotoxin. Local anesthetics abolished the twitch contractions of atria with inhibitory concentration 50% values of 12.6 +/- 5.0 microM (bupivacaine) and 15.7 +/- 3.9 microM (levobupivacaine). In separate experiments, tetrodotoxin inhibited twitch contractile force by only 30%. CONCLUSIONS: The results indicate that clinically relevant cardiotoxic concentrations of bupivacaine and levobupivacaine markedly depress cardiac sympathetic neurotransmission. A possible mechanism of local anesthetics in reducing evoked norepinephrine release from sympathetic endings is blockade of tetrodotoxin-sensitive fast sodium channels.     

Effects of Bupivacaine and Ropivacaine on High-voltage-activated Calcium Currents of the Dorsal Horn Neurons in Newborn Rats

Background: Local anesthetics, such as bupivacaine, have been reported to block calcium currents in primary sensory neurons and to interfere with the release of neurotransmitters in central nervous system neurons. However, it is unknown whether local anesthetics affect the calcium current activity of central nervous system neurons.

Methods: Using a traditional whole cell voltage clamp technique, effects of bupivacaine and ropivacaine on high-voltage-activated calcium currents (HVA-Ica) were investigated in enzymatically dissociated dorsal horn neurons of neonatal rats. Calcium currents were evoked by testing pulses from a holding potential of -90 to 0 mV.

Results: Bupivacaine significantly reduced HVA-Ica in a dose-dependent manner. The peak HVA-Ica decreased by 24.5 +/- 2.5, 32.0 +/- 6.8, 59.4 +/- 6.2, 88.3 +/- 1.5, and 91.6 +/- 1.1% in response to 10, 30, 50, 100 and 200 [mu]m bupivacaine, respectively. Unlike bupivacaine, ropivacaine markedly increased HVA-Ica at lower concentrations (< 50 [mu]m) but decreased HVA-Ica at higher concentrations (>= 50 [mu]m). The percent increases in peak HVA-Ica induced by 10 and 30 [mu]m ropivacaine were 95 +/- 19.1 and 41.6 +/- 8.3%, respectively. The percent decreases in response to 50, 100, and 200 [mu]m ropivacaine were 21.1 +/- 2.1, 63.2 +/- 6.0 and 79.1 +/- 7.6%, respectively. Results indicate that the inhibitory potency of ropivacaine on HVA-Ica was significantly lower than that of bupivacaine at the same concentrations.     

Effects of bupivacaine, levobupivacaine and ropivacaine on myocardial relaxation

PURPOSE: Ropivacaine and levobupivacaine were developed to reduce the risk of occasional toxicity reported with bupivacaine. While the effects of long-acting local anesthetics (LAAs) on myocardial contractility (inotropy) are well described, their effects on relaxation (lusitropy) remain largely unknown. The present study aimed to compare the effects of LAAs on rat myocardium. METHODS: Left ventricular papillary muscles of male Wistar rats were used to compare the inotropic and lusitropic responses of increasing concentrations of LAAs (10(-8) to 10(-3) M) under isometric and isotonic conditions. Data are mean % (SD) of baseline value. RESULTS: Long-acting local anesthetics induced a significant impairment of relaxation in isotonic and isometric conditions. As compared to ropivacaine, bupivacaine and levobupivacaine induced greater negative lusitropic effects in isotony [at 10(-3) M, maximum unloaded shortening velocity ((max)Vr) = 27 +/- 11 vs 13 +/- 6 and 8 +/- 5%] and isometry (at 10(-3) M, time-to-half-relaxation: 106 +/- 10 vs 127 +/- 17 and 133 +/- 17%). When the comparison was made with equipotent concentrations, the negative lusitropic effects induced with levobupivacaine were significantly greater than those of bupivacaine and ropivacaine in isometric and isotonic conditions (at 10(-3) M, (max)Vr = 7 +/- 4 vs 13 +/- 6 and 17 +/- 4 %). As previously described, LAAs also induced concentration-dependent negative inotropic effects that were greater for levobupivacaine compared to equivalent or equipotent concentrations of bupivacaine and ropivacaine. CONCLUSIONS: Long-acting local anesthetics induce marked negative inotropic and lusitropic effects. Among LAAs, levobupivacaine exerts the greater depressant effects. Impairment of calcium handling and sarcoplasmic reticulum could explain the differential responses to local anesthetics.     

Molecular Mechanisms of the Inhibitory Effects of Bupivacaine, Levobupivacaine, and Ropivacaine on Sarcolemmal Adenosine Triphosphate-sensitive Potassium Channels in the Cardiovascular System

Background: Sarcolemmal adenosine triphosphate-sensitive potassium (KATP) channels in the cardiovascular system may be involved in bupivacaine-induced cardiovascular toxicity. The authors investigated the effects of local anesthetics on the activity of reconstituted KATP channels encoded by inwardly rectifying potassium channel (Kir6.0) and sulfonylurea receptor (SUR) subunits.

Methods: The authors used an inside-out patch clamp configuration to investigate the effects of bupivacaine, levobupivacaine, and ropivacaine on the activity of reconstituted KATP channels expressed in COS-7 cells and containing wild-type, mutant, or chimeric SURs.

Results: Bupivacaine inhibited the activities of cardiac KATP channels (IC50 = 52 [mu]m) stereoselectively (levobupivacaine, IC50 = 168 [mu]m; ropivacaine, IC50 = 249 [mu]m). Local anesthetics also inhibited the activities of channels formed by the truncated isoform of Kir6.2 (Kir6.2[DELTA]C36) stereoselectively. Mutations in the cytosolic end of the second transmembrane domain of Kir6.2 markedly decreased both the local anesthetics' affinity and stereoselectivity. The local anesthetics blocked cardiac KATP channels with approximately eightfold higher potency than vascular KATP channels; the potency depended on the SUR subtype. The 42 amino acid residues at the C-terminal tail of SUR2A, but not SUR1 or SUR2B, enhanced the inhibitory effect of bupivacaine on the Kir6.0 subunit.     

Retigabine Stimulates Human KCNQ2/Q3 Channels in the Presence of Bupivacaine

Background: Inhibition of KCNQ2/Q3 channels may cause convulsion in humans. The interaction of bupivacaine with these channels is unknown. The anticonvulsant retigabine activates KCNQ2/Q3 channels and may reverse inhibitory actions of bupivacaine. Potassium channel stimulation may thus constitute a novel approach to treat local anesthetic-induced seizures. The aim of this study was to characterize bupivacaine effects on KCNQ2/Q3 channels and to investigate whether retigabine reverses the effects of the local anesthetic.

Methods: KCNQ2/Q3 channels were transiently expressed in Chinese hamster ovary cells. The effects of bupivacaine and retigabine were studied with the patch-clamp technique.

Results: Bupivacaine inhibited KCNQ2/Q3 channels in a concentration-dependent and reversible manner. The concentration-response curve was described by a Hill equation (IC50 = 173 +/- 7 [mu]m, Hill coefficient = 1.4 +/- 0.1, mean +/- SEM, n = 37). The inhibitory effect did not differ between bupivacaine and levobupivacaine (42 +/- 4%, n = 7, versus 42 +/- 5%, n = 10; P > 0.05). Ropivacaine was four times less potent than bupivacaine. The inhibition of KCNQ2/Q3 channels by bupivacaine resulted in a significant and reversible depolarization of the membrane potential. Retigabine (300 nm-10 [mu]m) reversed the inhibitory action of bupivacaine on KCNQ2/Q3 channels as well as the depolarization of the membrane potential.     

Efficacy, Safety, and Pharmacokinetics of Levobupivacaine with and without Fentanyl after Continuous Epidural Infusion in Children: A Multicenter Trial

Background: Levobupivacaine, the levo-enantiomer of bupivacaine, is as potent as bupivacaine but less toxic. Therefore, the authors investigated the efficacy, safety, and pharmacokinetics of perioperative epidural levobupivacaine with and without fentanyl in children.

Methods: After Research Ethics Board approval and informed written consent, 120 healthy children aged 6 months to 12 yr who were scheduled to undergo urologic or abdominal surgery were randomized in a double-blinded and concealed manner to receive one of four epidural solutions as a continuous infusion for 24 h: 0.125% levobupivacaine; 0.0625% levobupivacaine; 1 [mu]g/ml fentanyl; or the combination, 0.0625 levobupivacaine and 1 [mu]g/ml fentanyl. After induction of anesthesia and tracheal intubation, a lumbar epidural catheter was sited, a loading dose was administered (0.75 ml/kg levobupivacaine, 0.175%), and the epidural infusion was commenced. The primary endpoint was the need for rescue analgesia (morphine) in the first 10 h after surgery. Pain, motor strength, and side effects were recorded for 24 h. Venous blood was collected from 18 children to determine the plasma concentrations of levobupivacaine and/or fentanyl before and 2, 4, 8, 16, 24, and 26 or 30 h after the start of the epidural infusion.

Results: Of the 114 children who were analyzed for intention to treat, a similar number of children in each group reached the 10-h mark. The time to the first dose of morphine in the first 10 h was less in the plain fentanyl group (P < 0.044). All other effects were similar among the four groups. The plasma concentration of levobupivacaine increased during the infusion period, reaching a maximum of 0.76 +/- 0.11 [mu]g/ml in the 0.125% group and 0.48 +/- 0.12 [mu]g/ml in the 0.0625% group by 24 h. The plasma concentration of fentanyl also increased steadily, reaching a maximum concentration of 0.37 +/- 0.11 ng/ml by 24 h.     

Interaction of bupivacaine and tetracaine with the sarcoplasmic reticulum Ca2+ release channel of skeletal and cardiac muscles

BACKGROUND: Although various local anesthetics can cause histologic damage to skeletal muscle when injected intramuscularly, bupivacaine appears to have an exceptionally high rate of myotoxicity. Research has suggested that an effect of bupivacaine on sarcoplasmic reticulum Ca2+ release is involved in its myotoxicity, but direct evidence is lacking. Furthermore, it is not known whether the toxicity depends on the unique chemical characteristics of bupivacaine and whether the toxicity is found only in skeletal muscle. METHODS: The authors studied the effects of bupivacaine and the similarly lipid-soluble local anesthetic, tetracaine, on the Ca2+ release channel-ryanodine receptor of sarcoplasmic reticulum in swine skeletal and cardiac muscle. [3H]Ryanodine binding was used to measure the activity of the Ca2+ release channel-ryanodine receptors in microsomes of both muscles. RESULTS: Bupivacaine enhanced (by two times at 5 mM) and inhibited (66% inhibition at 10 mM) [3H]ryanodine binding to skeletal muscle microsomes. In contrast, only inhibitory effects were observed with cardiac microsomes (about 3 mM for half-maximal inhibition). Tetracaine, which inhibits [3H]ryanodine binding to skeletal muscle microsomes, also inhibited [3H]ryanodine binding to cardiac muscle microsomes (half-maximal inhibition at 99 microM). CONCLUSIONS: Bupivacaine's ability to enhance Ca2+ release channel-ryanodine receptor activity of skeletal muscle sarcoplasmic reticulum most likely contributes to the myotoxicity of this local anesthetic. Thus, the pronounced myotoxicity of bupivacaine may be the result of this specific effect on Ca2+ release channel-ryanodine receptor superimposed on a nonspecific action on lipid bilayers to increase the Ca2+ permeability of sarcoplasmic reticulum membranes, an effect shared by all local anesthetics. The specific action of tetracaine to inhibit Ca2+ release channel-ryanodine receptor activity may in part counterbalance the nonspecific action, resulting in moderate myotoxicity.     

Comparative Somatic and Visceral Antinociception and Neurotoxicity of Intrathecal Bupivacaine, Levobupivacaine, and Dextrobupivacaine in Rats

Background: The current study investigated whether racemic bupivacaine and its S(-)- and R(+)-enantiomers, levobupivacaine and dextrobupivacaine, differ in somatic and visceral antinociception and neurotoxicity when administered intrathecally in rats.

Methods: In experiment 1, rats intrathecally received 15 [mu]l saline or 0.125, 0.25, 0.5, or 1% bupivacaine, levobupivacaine, or dextrobupivacaine. The tail-flick and colorectal distension tests were performed to assess somatic and visceral antinociceptive effects, respectively, for 180 min after injection. In experiment 2, rats given 0.25% anesthetic solutions were evaluated with colorectal distension-induced response in blood pressure and heart rate. In experiment 3, four groups of rats received a 1-h infusion of saline or 2.5% bupivacaine, levobupivacaine, or dextrobupivacaine. Additional rats received either 1.25% bupivacaine or levobupivacaine for 1 h. Four days after infusion, animals were assessed for persistent sensory impairment using the tail-flick test. Spinal cords and nerve roots were obtained for histologic analysis.

Results: In experiment 1, the three drugs produced similar time course effects and dose-effect relations in tail-flick latency. Colorectal distension thresholds and motor paralysis were slightly lower and less apparent, respectively, at some concentrations in rats given levobupivacaine than in those given the other agents. In experiment 2, colorectal distension-induced response in heart rate was less depressed in rats given levobupivacaine than in those given the other anesthetics. In experiment 3, three groups of rats given 2.5% anesthetic solutions developed similar significant increases in tail-flick latency and incurred similar morphologic damage. Two groups of rats receiving 1.25% anesthetic solutions were similar in functional impairment and nerve injury scores.     

A comparison of epidural levobupivacaine 0.75% with racemic bupivacaine for lower abdominal surgery

Levobupivacaine, the S(-) isomer of bupivacaine, is less cardiotoxic than racemic bupivacaine. In this prospective, randomized, double-blinded study of epidural anesthesia, the onset, extent, and duration of sensory and motor block produced by 0.75% levobupivacaine (20 mL, 150 mg) was compared with that of 0.75% racemic bupivacaine in 56 patients undergoing elective lower abdominal surgery. The time to onset of adequate sensory block (T10 dermatome) was similar in both treatment groups (13.6 +/- 5.6 min for levobupivacaine and 14.0 +/- 9.9 min for bupivacaine), with an average peak block height of T5 reached at 24.3 +/- 9.4 and 26.5 +/- 13.2 min, respectively. Time to complete regression of sensory block was significantly longer with levobupivacaine (550.6 +/- 87.6 min) than bupivacaine (505.9 +/- 71.1 min) (P = 0.016). Abdominal muscle relaxation was adequate for the scheduled procedure in all patients, and there were no significant differences between the groups in rectus abdominis muscle scores (P = 0.386) and quality of muscle relaxation as determined by the surgeon and anesthesiologist (P = 0. 505 and 0.074, respectively). In conclusion, both 0.75% levobupivacaine and 0.75% bupivacaine produced effective epidural anesthesia and their effects were clinically indistinguishable. Implications: The results of this study indicate that the sensory and motor block produced by 0.75% levobupivacaine is equivalent to that of 0.75% racemic bupivacaine. Both local anesthetics are well tolerated and effective in producing epidural anesthesia for patients undergoing lower abdominal surgery.     

Comparative Effects of Bupivacaine and Ropivacaine on Intracellular Calcium Transients and Tension in Ferret Ventricular Muscle

Background: Recent evidence suggests that ropivacaine exerts markedly less cardiotoxicity compared with bupivacaine; however, the mechanisms are not fully understood at the molecular level.

Methods: Isolated ferret ventricular papillary muscles were microinjected with the Ca2+-binding photoprotein aequorin, and intracellular Ca2+ transients and tension were simultaneously measured during twitch in the absence and presence of bupivacaine or ropivacaine.

Results: Bupivacaine and ropivacaine (10, 30, and 100 [mu]m) reduced peak systolic [Ca2+]i and tension in a concentration-dependent manner. The effects were significantly greater for bupivacaine, particularly on tension (approximately twofold). The percentage reduction of tension was linearly correlated with that of [Ca2+]i for both anesthetics, with the slope of the relationship being [almost equal to]1.0 for ropivacaine and [almost equal to]1.3 for bupivacaine (slope difference, P < 0.05), suggesting that the cardiodepressant effect of ropivacaine results predominantly from inhibition of Ca2+ transients, whereas bupivacaine suppresses Ca2+ transients and the reaction beyond Ca2+ transients, i.e., myofibrillar activation, as well. BAY K 8644, a Ca2+ channel opener, abolished the inhibitory effects of ropivacaine on Ca2+ transients and tension, whereas BAY K 8644 only partially inhibited the effects of bupivacaine, particularly the effects on tension.     

Effects of Intravenous Anesthetic Agents on Glutamate Release: A Role for GABAA Receptor-Mediated Inhibition

Background: Many anesthetic agents are known to enhance the [alpha]1[beta]2[gamma]2S[gamma]-aminobutyric acid type A (GABAA) chloride current; however, they also depress excitatory neurotransmission. The authors evaluated two hypotheses: intravenous anesthetic agents inhibit glutamate release and any observed inhibition may be secondary to GABAA receptor activation.

Methods: Cerebrocortical slices were prepared from Wistar rats. After perfusion in oxygenated Krebs buffer for 60 min at 37[degrees]C, samples for glutamate assay were obtained at 2-min intervals. After 6 min, a 2-min pulse of 46 mM K+ was applied to the slices (S1); this was repeated after 30 min (S2). Bicuculline (1-100 [mu]M) was applied when the S1 response returned to basal level, and 10 min later, thiopental (1-300 [mu]M), propofol (10 [mu]M), or ketamine (30 [mu]M) were also applied until the end of S2. Perfusate glutamate concentrations were measured fluorometrically, and the area under the glutamate release curves was expressed as a ratio (S2/S1).

Results: Potassium (46 mM) evoked a monophasic release of glutamate during S1 and S2, with a mean control S2/S1 ratio of 1.07 +/- 0.33 (mean +/- SD, n = 96). Ketamine and thiopental produced a concentration-dependent inhibition of K+-evoked glutamate release with half-maximum inhibition of release values of 18.2 and 10.9 [mu]M, respectively. Release was also inhibited by propofol. Bicuculline produced a concentration dependent reversal of thiopental inhibition of glutamate release with a half-maximum reversal of the agonist effect of 10.3 [mu]M. Bicuculline also reversed the effects of propofol but not those of ketamine.     

Local Anesthetic Interaction with Human Ether-a-go-go-related Gene (HERG) Channels: Role of Aromatic Amino Acids Y652 and F656

Background: Human ether-a-go-go-related gene (HERG) potassium channels constitute a potential target involved in cardiotoxic side effects of amino-amide local anesthetics. The molecular interaction site of these low-affinity blockers with HERG channels is currently unknown. The aim of this study was to determine the effect of the mutations Y652A and F656A in the putative drug binding region of HERG on the inhibition by bupivacaine, ropivacaine, and mepivacaine.

Methods: The authors examined the inhibition of wild-type and mutant HERG channels, transiently expressed in Chinese hamster ovary cells by bupivacaine, ropivacaine, and mepivacaine. Whole cell patch clamp recordings were performed at room temperature.

Results: Inhibition of HERG wild-type and mutant channels by the different local anesthetics was concentration dependent, stereoselective, and reversible. The sensitivity decreased in the order bupivacaine > ropivacaine > mepivacaine for wild-type and mutant channels. The mutant channels were approximately 4-30 times less sensitive to the inhibitory action of the different local anesthetics than the wild-type channel. The concentration-response data were described by Hill functions (bupivacaine: wild-type IC50 = 22 +/- 2 [mu]m, n = 38; Y652A IC50 = 95 +/- 5 [mu]m, n = 31). The mutations resulted in a change of the stereoselectivity of HERG channel block by ropivacaine. The potency of the local anesthetics to inhibit wild-type and mutant channels correlated with the lipophilicity of the drug (r > 0.9).     

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).     

Determination of the Full Dose-Response Relation of Intrathecal Bupivacaine, Levobupivacaine, and Ropivacaine, Combined with Sufentanil, for Labor Analgesia

Background: Ropivacaine and levobupivacaine are local anesthetics that produce less motor block and greater sensory-motor separation when compared with equal milligram doses of bupivacaine. Although minimum local analgesic concentration studies suggested that they are less potent than bupivacaine, full dose-response studies have not been performed. The current trial describes the dose-response relation of levobupivacaine, ropivacaine, and bupivacaine, combined with sufentanil, when used for intrathecal labor analgesia.

Methods: Four hundred fifty term parturients in active labor were included in this double-blind, randomized trial. Combined spinal-epidural anesthesia was performed, and ropivacaine, levobupivacaine, or bupivacaine was intrathecally administered in a dose of 1.0, 1.5, 2.0, 2.5, 3.0, or 3.5 mg, always combined with 1.5 [mu]g sufentanil. Patients were considered responders to spinal analgesia if the visual analog scale score for pain was less than 25 mm within 15 min and the visual analog scale score remained less than 25 mm for 45 min. Patient demographics, obstetric data, maternal side effects, and fetal and neonatal well-being were noted. Group-specific dose-response curves were constructed using a probit regression model.

Results: The ED95 of bupivacaine was 3.3 mg (95% confidence interval, 2.9-4.1). The ED95s of ropivacaine and levobupivacaine were 4.8 mg (95% confidence interval, 4.0-6.7) and 5.0 mg (95% confidence interval, 4.1-7.0), respectively. Racemic bupivacaine was significantly more potent than ropivacaine (P = 0.0027) and levobupivacaine (P = 0.0006). Ropivacaine and levobupivacaine were of similar potency (P = 0.91).     

Effects of Intravenous General Anesthetics on [(3) H]GABA Release from Rat Cortical Synaptosomes

Background: Potentiation by general anesthetics of [Greek small letter gamma]-aminobutyric acid (GABA)-mediated inhibitory transmission in the central nervous system is attributed to GABAA receptor-medicated postsynaptic effects. However, the role of presynaptic mechanisms in general anesthetic action is not well characterized, and evidence for anesthetic effects on GABA release is controversial. The effects of several intravenous general anesthetics on [(3) H]GABA release from rat cerebrocortical synaptosomes (isolated nerve terminals) were investigated.

Methods: Purified synaptosomes were preloaded with [(3) H]GABA and superfused with buffer containing aminooxyacetic acid and nipecotic acid to inhibit GABA metabolism and reuptake, respectively. Spontaneous and elevated potassium chloride depolarization-evoked [(3) H]GABA release were evaluated in the superfusate in the absence or presence of various anesthetics, extracellular Ca2+, GABA receptor agonists and antagonists, and 2,4-diaminobutyric acid.

Results: Propofol, etomidate, pentobarbital, and alphaxalone, but not ketamine, potentiated potassium chloride-evoked [(3) H]GABA release (by 1.3 to 2.9 times) in a concentration-dependent manner, with median effective concentration values of 5.4 +/- 2.8 [micro sign]M (mean +/- SEM), 10.1 +/- 2.1 [micro sign]M, 18.8 +/- 5.8 [micro sign]M, and 4.4 +/- 2.0 [micro sign]M. Propofol also increased spontaneous [(3) H]GABA release by 1.7 times (median effective concentration = 7.1 +/- 3.4 [micro sign]M). Propofol facilitation of [(3) H]GABA release was Ca2+ dependent and inhibited by bicuculline and picrotoxin, but was insensitive to pretreatment with 2,4-diaminobutyric acid, which depletes cytoplasmic GABA pools.     

Interaction of Bupivacaine and Tetracaine with the Sarcoplasmic Reticulum Ca2+ Release Channel of Skeletal and Cardiac Muscles

Background: Although various local anesthetics can cause histologic damage to skeletal muscle when injected intramuscularly, bupivacaine appears to have an exceptionally high rate of myotoxicity. Research has suggested that an effect of bupivacaine on sarcoplasmic reticulum Ca2+ release is involved in its myotoxicity, but direct evidence is lacking. Furthermore, it is not known whether the toxicity depends on the unique chemical characteristics of bupivacaine and whether the toxicity is found only in skeletal muscle.

Methods: The authors studied the effects of bupivacaine and the similarly lipid-soluble local anesthetic, tetracaine, on the Ca2+ release channel-ryanodine receptor of sarcoplasmic reticulum in swine skeletal and cardiac muscle. [(3) H]Ryanodine binding was used to measure the activity of the Ca2+ release channel-ryanodine receptors in microsomes of both muscles.

Results: Bupivacaine enhanced (by two times at 5 mM) and inhibited (66% inhibition at 10 mM) [(3) H]ryanodine binding to skeletal muscle microsomes. In contrast, only inhibitory effects were observed with cardiac microsomes (about 3 mM for half-maximal inhibition). Tetracaine, which inhibits [(3) H]ryanodine binding to skeletal muscle microsomes, also inhibited [(3) H]ryanodine binding to cardiac muscle microsomes (half-maximal inhibition at 99 [micro sign]M).     

Comparative Ventricular Electrophysiologic Effect of Racemic Bupivacaine, Levobupivacaine, and Ropivacaine on the Isolated Rabbit Heart

Background: Numerous local anesthetics have an asymmetric tetrahedron carbon, which confers stereoselective differences between the isomers. The authors attempted to quantify the depressant effect of racemic bupivacaine, levobupivacaine, and ropivacaine on myocardial ventricular conduction and on myocardial contractility.

Methods: The authors studied the pharmacokinetics (outflow concentration) and pharmacodynamics (QRS widening) of the three drugs infused in an isolated rabbit heart preparation. All data were fitted simultaneously with use of mixed-effect modeling, thus allowing precise statistical comparison between the three drug parameters. The rate dependence of QRS widening was fitted separately.

Results: Racemic bupivacaine, levobupivacaine, and ropivacaine induced a calculated maximum increase in QRS duration in the ratio 1:0.4:0.3. Css50, the dose which caused half the maximum increase in QRS duration at steady state, was similar for all three drugs (22 [mu]m free concentration). A rate dependence of QRS widening was observed, which was in the ratio 1:0.5:0.25 for racemic bupivacaine, levobupivacaine, and ropivacaine, respectively.     

Effects of CNS Site-directed Carotid Arterial Infusions of Bupivacaine, Levobupivacaine, and Ropivacaine in Sheep

Background: Previous preclinical safety studies in ewes have found intravenous levobupivacaine and ropivacaine to be less potent toward causing central nervous system (CNS) and cardiac toxicity than bupivacaine. Analogous cardiotoxicity has been demonstrated directly in various cardiac preparations ex vivo. Moreover, drug-related arrhythmogenicity has been demonstrated from direct CNS injection of local anesthetic agents in vivo, suggesting CNS-related cardiotoxicity. This study investigated whether CNS site-directed blood-borne drug administration (with minimal systemic recirculation) would demonstrate drug-related cardiotoxicity.

Methods: Direct CNS effects and indirect cardiotoxic sequelae were determined after bilateral carotid arterial infusions of levobupivacaine, bupivacaine, or ropivacaine in ewes. After pilot studies to validate the procedures, equimolar doses (24-96 [mu]mol, [almost equal to]7.5-30 mg) were infused over 3 min using a crossover design. Behavioral CNS signs, quantitative electroencephalographic (EEG), cardiovascular, and electrocardiographic effects were recorded. Drug blood concentrations in superior sagittal sinus and aorta were measured serially.

Results: Blood drug concentrations in the superior sagittal sinus were 5-10 times those concurrently in the aorta, confirming highly selective CNS delivery with minimal systemic recirculation. Dose-dependent CNS excitatory behavior and EEG changes, with increased mean arterial blood pressure, heart rate, cardiac output, and myocardial contractility, were found, consistent with sympathetic nervous system stimulation. The overall rank order of potency for these effects was ropivacaine < levobupivacaine < bupivacaine. Nonfatal cardiac arrhythmias were observed, but the type or frequency did not differ between drugs.     

The inhibitory effects of bupivacaine,levobupivacaine, and ropivacaine on K2P (two-pore domain potassium) channel TREK-1

Purpose

Bupivacaine, levobupivacaine, and ropivacaine are amide local anesthetics. Levobupivacaine and ropivacaine are stereoisomers of bupivacaine and were developed to circumvent the bupivacaine’s severe toxicity. The recently characterized background potassium channel, K_2P TREK-1, is a well-known target for various local anesthetics. The purpose of study is to investigate the differences in inhibitory potency and stereoselectivity among bupivacaine, levobupivacaine, and ropivacaine on K_2P TREK-1 channels overexpressed in COS-7 cells.

Methods

We investigated the effects of bupivacaine, levobupivacaine, and ropivacaine (10, 50, 100, 200, and 400 μM) on TREK-1 channels expressed in COS-7 cells by using the whole cell patch clamp technique with a voltage ramp protocol ranging from ?100 to 100 mV for 200 ms from a holding potential of ?70 mV.

Results

Bupivacaine, levobupivacaine, and ropivacaine showed reversible inhibition of TREK-1 channels in a concentration-dependent manner. The half-maximal inhibitory concentrations (IC₅₀) of bupivacaine, levobupivacaine, and ropivacaine were 95.4 ± 14.6, 126.1 ± 24.5, and 402.7 ± 31.8 μM, respectively. IC₅₀ values indicated a rank order of potency (bupivacaine > levobupivacaine > ropivacaine) with stereoselectivity. Hill coefficients were 0.84, 0.93, and 0.89 for bupivacaine, levobupivacaine, and ropivacaine, respectively.

Conclusion

Inhibitory effects on TREK-1 channels by bupivacaine, levobupivacaine, and ropivacaine demonstrated stereoselectivity: bupivacaine was more potent than levobupivacaine and ropivacaine. Inhibition of TREK-1 channels and consecutive depolarization of the cell membrane by bupivacaine, levobupivacaine, and ropivacaine may contribute to the blockade of neuronal conduction and side effects.     

Propofol Modulates γ-Aminobutyric Acid-mediated Inhibitory Neurotransmission to Cardiac Vagal Neurons in the Nucleus Ambiguus

Background: Although it is well recognized that anesthetics modulate the central control of cardiorespiratory homeostasis, the cellular mechanisms by which anesthetics alter cardiac parasympathetic activity are poorly understood. One common site of action of anesthetics is inhibitory neurotransmission. This study investigates the effect of propofol on [gamma]-aminobutyric acid-mediated (GABAergic) and glycinergic neurotransmission to cardiac parasympathetic neurons.

Methods: Cardiac parasympathetic neurons were identified in vitro by the presence of a retrograde fluorescent tracer, and spontaneous GABAergic and glycinergic synaptic currents were examined using whole cell patch clamp techniques.

Results: Propofol at concentrations of 1.0 [mu]m and greater significantly (P < 0.05) increased the duration and decay time of spontaneous GABAergic inhibitory postsynaptic currents. To determine whether the action of propofol was at presynaptic or postsynaptic sites, tetrodotoxin was applied to isolate miniature inhibitory postsynaptic currents. Propofol at concentrations of 1.0 [mu]m and greater significantly (P < 0.05) prolonged the decay time and duration of miniature inhibitory postsynaptic currents, indicating that propofol directly alters GABAergic neurotransmission at a postsynaptic site. Propofol at high concentrations (>=50 [mu]m) also inhibited the frequency of both GABAergic inhibitory postsynaptic currents and miniature inhibitory postsynaptic currents. Propofol at concentrations up to 50 [mu]m had no effect on glycinergic neurotransmission.