Christian W. Hoenemann

Modulation of NMDA receptor function by ketamine and magnesium: Part I.

N- methyl-d-aspartate (NMDA) receptors are important components of pain processing. Ketamine and Mg2+ block NMDA receptors and might therefore be useful analgesics, and combinations of Mg2+ and ketamine provide more effective analgesia. We investigated their interactions at NMDA receptors. Xenopus oocytes, expressing NR1/NR2A or NR1/NR2B glutamate receptors, were studied. The effects of Mg2+, racemic ketamine and its isomers, and the combination of Mg2+ and S(+)-ketamine on NMDA signaling were determined. Mg2+ and ketamine alone inhibited NMDA receptors noncompetitively (half-maximal inhibitory effect concentration: Mg2+ 4.2 ± 1.2 × 10−4 M at NR1/NR2A and 6.3 ± 2.4 × 10−4 M at NR1/NR2B; racemic ketamine 13.6 ± 8.5 × 10−6 M at NR1/NR2A and 17.6 ± 7.2 × 10−6 M at NR1/NR2B; S(+)-ketamine 4.1 ± 2.5 × 10−6 at NR1/NR2A and 3.0 ± 0.3 at NR1/NR2B; R(−)-ketamine 24.4 ± 4.1 × 10−6 M at NR1/NR2A and 26.0 ± 2.4 × 10−6 M at NR1/NR2B). The combined application of Mg2+ and ketamine decreased the half-maximal inhibitory effect concentration >90% at both receptors. Isobolographic analysis demonstrated super-additive interactions. Ketamine and Mg2+ inhibit responses of recombinantly expressed NR1/NR2A and NR1/NR2B glutamate receptors, and combinations of the compounds act in a super-additive manner. These findings may explain, in part, why combinations of ketamine and Mg2+ are more effective analgesics than either compound alone.

Modulation of NMDA receptor function by ketamine and magnesium. Part II: interactions with volatile anesthetics.

Mg2+ and ketamine interact superadditively at N- methyl-d-aspartate (NMDA) receptors, which may explain the clinical efficacy of the combination. Because patients are usually exposed concomitantly to volatile anesthetics, we tested the hypothesis that volatile anesthetics interact with ketamine and/or Mg2+ at recombinantly expressed NMDA receptors. NR1/NR2A or NR1/NR2B receptors were expressed in Xenopus oocytes. We determined the effects of isoflurane, sevoflurane, and desflurane on NMDA receptor signaling, alone and in combination with S(+)-ketamine (4.1 &mgr;M on NR1/NR2A, 3.0 &mgr;M on NR2/NR2B) and/or Mg2+ (416 &mgr;M on NR1/NR2A, 629 &mgr;M on NR1/NR2B). Volatile anesthetics inhibited NR1/NR2A and NR1/NR2B glutamate receptor function in a reversible, concentration-dependent, voltage-insensitive and noncompetitive manner (half-maximal inhibitory concentration at NR1/NR2A receptors: 1.30 ± 0.02 minimum alveolar anesthetic concentration [MAC] for isoflurane, 1.18 ± 0.03 MAC for desflurane, 1.24 ± 0.06 MAC for sevoflurane; at NR1/NR2B receptors: 1.33 ± 0.12 MAC for isoflurane, 1.22 ± 0.08 MAC for desflurane, and 1.28 ± 0.08 MAC for sevoflurane). On both NR1/NR2A and NR1/NR2B receptors, 50% inhibitory concentration for volatile anesthetics was reduced approximately 20% by Mg2+, approximately 30% by S(+)-ketamine, and approximately 50% by the compounds in combination. Volatile anesthetic effects on NMDA receptors can be potentiated significantly by Mg2+, S(+)-ketamine, or—most profoundly—both. Therefore, the analgesic effects of ketamine and Mg2+ are likely to be enhanced in the presence of volatile anesthetics.

The effects of local anesthetics on perioperative coagulation, inflammation, and microcirculation.

M ajor surgery is associated with a hypercoagulable and proinflammatory state that persists into the postoperative period (1,2). Perioperative inflammatory responses to trauma can trigger hypercoagulability, especially in patients undergoing vascular surgery, and are associated with vasoocclusive and thromboembolic events—major causes of postoperative morbidity and mortality (3–5). The mechanisms for these effects are poorly understood, but hypercoagulability seems to originate from what is known as the “stress response” to major surgery (4,6). Postoperative changes occur in all aspects of the coagulation system, including increased plasma levels of coagulation factors (1), decreased levels and more rapid inactivation of endogenous coagulation inhibitors (7), enhanced platelet reactivity (8), and impaired fibrinolysis (9). In addition, increasing attention has been given to the close link between hemostasis and inflammation (10), which is minutely influenced by general anesthesia with parenteral opioids (3). However, recent evidence suggests that regional anesthesia has a protective effect against the perioperative stress response. The beneficial effects of the epidural administration of local anesthetic (LA) have been attributed to the changes in physiology induced by neuraxial anesthesia and better pain management (5,11). However, as discussed below, there are hints that the pharmacodynamic effects of LA itself may contribute to these effects. This review is arranged according to the different effects of LAs—first on the hypercoagulable and inflammatory responses to surgery, and second on microcirculation. Stress Response

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

Background: Clinical studies suggest that intraoperative administration of the clinical remifentanil formulation Ultiva® (GlaxoWellcome GmbH & Co, Bad Oldesloe, Germany) increases postoperative pain and postoperative analgesic requirements, but mechanisms remain unclear. N-methyl-d-aspartate (NMDA) receptors are thought to play a major role in development of postoperative pain and opiate tolerance. The authors hypothesized that Ultiva® directly stimulates human NMDA receptors. Methods: To test this hypothesis, the authors expressed human NR1A/NR2A and NR1A/NR2B NMDA receptors in Xenopus laevis oocytes by injection of messenger RNA prepared in vitro. After protein expression, they used a two-electrode voltage clamp to measure currents induced by NMDA receptor agonists and opioids. Results: Noninjected cells were unresponsive to all compounds tested. Glutamate/glycine (1 nm–1 mm each) or Ultiva® (0.01 pm–0.1 mm) stimulated NMDA receptors concentration dependently. NR1A/2A EC50 values were 8.0 μM/12 μM for glutamate/glycine and 3.5 nM for Ultiva®, and NR1A/2B EC50 values were 3.9 μM/1.9 μM for glutamate/glycine and 0.82 μM for Ultiva®. Glycine in combination with Ultiva® showed no additive effect compared with Ultiva® alone. Ultiva®-induced currents were inhibited by MK-801 (pore blocker) but not by 7-CK (glycine antagonist), D-AP5 (glutamate antagonist), or naloxone. Fentanyl (10 μM) did not stimulate NMDA receptors. Conclusion: These data indicate that Ultiva® but not fentanyl stimulates NMDA receptors of different subunit combinations (NR1A/2A, NR1A/2B). The mechanism seems to be allosteric activation of the NMDA receptor.

Time-dependent inhibition of G protein-coupled receptor signaling by local anesthetics.

BackgroundSeveral beneficial effects of local anesthetics (LAs) were shown to be due to inhibition of G protein–coupled receptor signaling. Differences in exposure time might explain discrepancies in concentrations of LAs required to achieve these protective effects in vivo and in vitro (approximately 100-fold higher). Using Xenopus oocytes and human neutrophils, the authors studied time-dependent effects of LAs on G protein–coupled receptor signaling and characterized possible mechanisms and sites of action. MethodsMeasurement of agonist-induced Ca2+-activated Cl− currents, using a two-electrode voltage clamp technique, and determination of superoxide anion production by cytochrome c assay were used to assess the effects of LAs on G protein–coupled receptor signaling in oocytes and primed and activated human neutrophils, respectively. Antisense knockdown of G&agr;q protein and inhibition of various proteins within the signaling pathway served for defining mechanisms and sites of action more specifically. ResultsLAs attenuated G protein–coupled receptor signaling in both models in a time-dependent and reversible manner (lidocaine reduced lysophosphatidic acid signaling to 19 ± 3% after 48 h and 25 ± 2% after 6 h of control response in oocytes and human neutrophils, respectively). Whereas no effect was observed after extracellularly applied or intracellularly injected QX314, a lidocaine analog, using G&agr;q-depleted oocytes, time-dependent inhibition also occurred after intracellular injection of QX314 into undepleted oocytes. Inhibition of phosphatases or protein kinases and agonist-independent G-protein stimulation, using guanosine 5′-O-3-thiotriphosphate or aluminum fluoride, did not affect time-dependent inhibition by LAs. ConclusionInhibition of G protein–coupled receptor signaling by LAs was found to be time dependent and reversible. Critically requiring G&agr;q-protein function, this effect is located downstream of guanosine diphosphate–guanosine triphosphate exchange and is not dependent on increased guanosine triphosphatase activity, phosphatases, or protein kinases.

Bupivacaine inhibits whole blood coagulation in vitro.

BACKGROUND AND OBJECTIVES Epidural anesthesia decreases the risk of postoperative deep venous thrombosis in selected patients. Intravascular local anesthetic levels resulting from epidural anesthesia may contribute to this effect by impacting coagulation. We studied the effects of bupivacaine (1-10 micromol/L) on whole blood coagulation measured by thrombelastography (TEG) and activated clotting time (ACT). METHODS We incubated whole blood with bupivacaine (1, 2, and 10 micromol/L) or Tyrodes solution (control) for 60 minutes and measured TEG and ACT clotting parameters. RESULTS Bupivacaine (1 or 10 micromol/L) prolonged ACT when compared with control. The thromboxane A2 (TX) receptor antagonist SQ29548 also prolonged ACT significantly. The combination of SQ29548 and bupivacaine was equally effective as bupivacaine alone, compatible with the hypothesis that bupivacaine at these concentrations blocks TX signaling. Because SQ29548 + bupivacaine prolonged ACT more than did SQ29548 alone, bupivacaine likely inhibits processes in addition to TX signaling. This was evaluated further using TEG. After incubation with 2 microm bupivacaine, TEG reaction time and clot growth time increased significantly, and maximal amplitude decreased. CONCLUSIONS Bupivacaine in clinically relevant concentrations influences whole blood clotting characteristics as measured by TEG and ACT. Thromboxane receptor antagonism increases ACT, confirming a role for TX in coagulation. Bupivacaine may also inhibit TX signaling, but seems to block additional factors as well. These findings might partly explain the beneficial effects of epidural anesthesia on postoperative thrombotic events.

Delayed Onset of Malignant Hyperthermia in Desflurane Anesthesia

IMPLICATIONS Animal-experimental studies demonstrate desfluranes trigger effect for malignant hyperthermia (MH). In contrast to other anesthetics, the time interval from exposure to the occurrence of symptoms is much longer with desflurane. This case report focuses on MH induced by desflurane alone.

Bupivacaine inhibits thromboxane A2-Induced vasoconstriction in rat thoracic aorta

Plasma levels of thromboxane A2 (TXA2), an inflammatory mediator inducing platelet aggregation, bronchoconstriction, and vasoconstriction, are increased in the peri-operative period. A major role in the pathogenesis of perioperative thromboembolic and ischemic syndromes is attributed to this prostanoid. Local anesthetics (LA) inhibitsignalingofTXA2 receptorsexpressedincellmodels. Therefore, we hypothesized that LA may inhibit vasoconstriction induced by the TXA2 analog U46619 in rat thoracic aorta. Rings (3-mm length) of the rat thoracic aorta were mounted in organ baths and isometric contractile force was measured. Rings, with or without endothelium, were incubated for 60 min in bupivacaine (10−6 or 10−5 M)orKrebs-Henseleitsolution(controlgroup)andsubsequently exposed to cumulative concentrations of U46619 (10−10 to 10−6 M). The reversibility of the TXA2-induced vasoconstriction by bupivacaine was also studied. Pre-treatment of rings with bupivacaine concentration-dependently diminished TXA2-induced contraction in rat aortic rings. We found no significant differences in relaxing effect of bupivacaine between rings with and without endothelium. Contraction in rings established with U46619 could not be reversed by cumulative concentrations of bupivacaine. Bupivacaine inhibited carbachol-induced vascular relaxation. This study provides experimental evidence that bupivacaine is an endothelium-independent inhibitor of TXA2-induced vasoconstriction of rat thoracic aorta.