Local Anesthetic Inhibition of m1 Muscarinic Acetylcholine Signaling

Background: Local anesthetics inhibit lipid mediator signaling (lysophosphatidate, thromboxane) by acting on intracellular domains of the receptor or on the G protein. On receptors for polar agonists, the ligand-binding pocket could form an additional site of interaction, possibly resulting in superadditive inhibition. The authors therefore investigated the effects of local anesthetics on m1 muscarinic receptor functioning.

Methods: The authors expressed receptors in isolation using Xenopus oocytes. Using a two-electrode voltage clamp, the authors measured the effects of lidocaine, QX314 (permanently charged), and benzocaine (permanently uncharged) on Ca2+-activated Cl- currents elicited by methylcholine. The authors also characterized the interaction of lidocaine with [3H] quinuclydinyl benzylate ([3H]QNB) binding to m1 receptors.

Results: Lidocaine inhibited muscarinic signaling with a half-maximal inhibitory concentration (IC50 18 nm) 140-fold less than that of extracellularly administered QX314 (IC50 2.4 [mu]m). Intracellularly injected QX314 (IC50 0.96 mm) and extracellularly applied benzocaine (IC50 1.2 mm) inhibited at high concentrations only. Inhibition of muscarinic signaling by extracellularly applied QX314 and lidocaine was the result of noncompetitive antagonism. Intracellularly injected QX314 and benzocaine inhibited muscarinic and lysophosphatidate signaling at similar concentrations, suggesting an action on the common G-protein pathway. Combined administration of intracellularly injected (IC50 19 [mu]m) and extracellularly applied QX314 (IC50 49 nm) exerted superadditive inhibition. Lidocaine did not displace specific [3H]QNB binding to m1 receptors.     

Inhibition of Mammalian Gq Protein Function by Local Anesthetics

Background: Local anesthetics have been shown to selectively inhibit functioning of Xenopus laevis Gq proteins. It is not known whether a similar interaction exists with mammalian G proteins. The goal of this study was to determine whether mammalian Gq protein is inhibited by local anesthetics.

Methods: In Xenopus oocytes, the authors replaced endogenous Gq protein with mouse Gq (expressed in Sf9 cells using baculovirus vectors). Cells endogenously expressing lysophosphatidic acid or recombinantly expressing muscarinic m3 receptors were injected with phosphorothioate DNA antisense (or sense as control) oligonucleotides against Xenopus Gq. Forty-eight hours later, oocytes were injected with purified mouse Gq (5 x 10-8 m) or solvent as control. Two hours later, the authors injected either lidocaine, its permanently charged analog QX314 (at IC50, 50 nl), or solvent (KCl 150 mm) as control and measured Ca-activated Cl currents in response to lysophosphatidic acid or methylcholine (one tenth of EC50).

Results: Injection of anti-Gq reduced the mean response size elicited by lysophosphatidic acid to 33 +/- 7% of the corresponding control response. In contrast, responses were unchanged (131 +/- 29% of control) in cells in addition injected with mouse Gq protein. Injection of mouse Gq protein "rescued" the inhibitory effect of intracellularly injected QX314: whereas QX314 was without effect on Gq-depleted oocytes, responses to lysophosphatidic acid after QX314 injection were inhibited to 44 +/- 10% of control response in cells in addition injected with mouse Gq protein (5 x 10-8 m). Similar results were obtained for m3 signaling and intracellularly injected lidocaine.     

Inhibition of mammalian Gq protein function by local anesthetics

BACKGROUND: Local anesthetics have been shown to selectively inhibit functioning of Xenopus laevis Gq proteins. It is not known whether a similar interaction exists with mammalian G proteins. The goal of this study was to determine whether mammalian Gq protein is inhibited by local anesthetics. METHODS: In Xenopus oocytes, the authors replaced endogenous Gq protein with mouse Gq (expressed in Sf9 cells using baculovirus vectors). Cells endogenously expressing lysophosphatidic acid or recombinantly expressing muscarinic m3 receptors were injected with phosphorothioate DNA antisense (or sense as control) oligonucleotides against Xenopus Gq. Forty-eight hours later, oocytes were injected with purified mouse Gq (5 x 10(-8) M) or solvent as control. Two hours later, the authors injected either lidocaine, its permanently charged analog QX314 (at IC50, 50 nl), or solvent (KCl 150 mM) as control and measured Ca-activated Cl currents in response to lysophosphatidic acid or methylcholine (one tenth of EC50). RESULTS: Injection of anti-Gq reduced the mean response size elicited by lysophosphatidic acid to 33 +/- 7% of the corresponding control response. In contrast, responses were unchanged (131 +/- 29% of control) in cells in addition injected with mouse Gq protein. Injection of mouse Gq protein "rescued" the inhibitory effect of intracellularly injected QX314: whereas QX314 was without effect on Gq-depleted oocytes, responses to lysophosphatidic acid after QX314 injection were inhibited to 44 +/- 10% of control response in cells in addition injected with mouse Gq protein (5 x 10(-8) M). Similar results were obtained for m3 signaling and intracellularly injected lidocaine. CONCLUSION: Inhibition of Gq function by local anesthetics is not restricted to Xenopus G proteins. Therefore, Gq should be considered as one additional intracellular target site for local anesthetics, especially relevant for those effects not explainable by sodium channel blockade (e.g., antiinflammatory effects).     

Synergistic inhibition of lysophosphatidic acid signaling by charged and uncharged local anesthetics.

We investigated the mechanism of benzocaine (permanently uncharged) and QX314 (permanently charged) inhibition of lysophosphatidic acid (LPA) signaling. To determine their site of action, we studied effects of these drugs, alone and in combination, on LPA-induced Ca2+-dependent Cl currents (I(Cl(Ca))) in Xenopus oocytes. After 10 min exposure to benzocaine, QX314 (10(-6)-10(-2) M), or both, we measured effects on I(Cl(Ca)) induced by LPA (with and without protein kinase [PKC] activation/inhibition) and on I(Cl(Ca)) induced by the intracellular injection of IP3 and GTPgammaS. LPA application to oocytes resulted in I(Cl(Ca)) (50% effective concentration approximately 10(-8) M). Both anesthetics inhibited LPA signaling concentration-dependently (50% inhibitory concentration [IC50] benzocaine 0.9 mM, QX314 0.66 mM). The combination acted synergistically (IC50 benzocaine 0.097 mM/QX314 0.048 mM). Intracellular signaling pathways were not affected. This study shows that benzocaine and QX314 inhibit LPA signaling and act synergistically, which is most easily explained by the existence of two different binding sites. Lack of inhibition of IP3 or GTPgammaS-induced I(Cl(Ca)) identifies the receptor as a target. Activation of PKC can be excluded as a potential mechanism. IMPLICATIONS: Lysophosphatidic acid may play a role in wound healing, and its signaling is inhibited by local anesthetics. We identified the membrane receptor as the local anesthetic site of action and showed that charged (QX314) and uncharged (benzocaine) local anesthetics inhibit lysophosphatidic acid signaling synergistically, which can be explained by the presence of different binding sites.     

Inhibition of Lysophosphatidate Signaling by Lidocaine and Bupivacaine

Background: Lidocaine and bupivacaine impair wound healing, but the mechanism of this side effect has not been determined. The phospholipid messenger lysophosphatidate is released from activated platelets and induces fibroblast and smooth muscle proliferation. Because it may play a role in wound healing, the authors studied the effects of local anesthetics on lysophosphatidate signaling in Xenopus oocytes.

Methods: Defolliculated Xenopus oocytes expressing endogenous G protein-coupled lysophosphatidate receptors were voltage clamped and studied in the presence or absence of lidocaine or bupivacaine. Lysophosphatidate-induced Ca2+ -activated Cl sup - currents (ICl(Ca)) were measured. To determine the site of action of the local anesthetics on the signaling pathway, the authors studied 1) the effects of local anesthetics on signaling induced by intracellular injection of the second messenger inositoltrisphosphate, and 2) the effects of local anesthetics on functioning of recombinantly expressed angiotensin II receptor signaling through the same pathways as the lysophosphatidate receptor.

Results: Lysophosphatidate signaling was inhibited in the presence of local anesthetics. The half maximal inhibitory concentration (IC50 s) for lidocaine and bupivacaine were 29.6 mM and 4.7 mM, respectively. Neither responses induced by inositoltrisphosphate injection nor angiotensin signaling were influenced by local anesthetics.     

Inhibition of brain cell excitability by lidocaine,QX314, and tetrodotoxin: a mechanism for analgesia from infused local anesthetics?

Local anesthetic infusions have been used to provide analgesia in a variety of painful conditions. The mechanism for this drug effect remains unknown. To better define the electrical effects of lidocaine concentrations comparable to those obtained during analgesic infusions, lidocaine (0.05-3 mmol l^-1), QX314 (an obligatorily charged, quaternary lidocaine derivative applied within the cells), and tetrodotoxin (10 nmoll^-1) were applied to rat hippocampal pyramidal cells. The three drugs, which inhibit Na⁺ currents by varying mechanisms, produced tonic increases in (firing) current threshold, and decreases in the amplitude of action potentials measured using an intracellular microelectrode technique. Lidocaine inhibited action potential spikes and increased current threshold in a concentration-dependent fashion. Lidocaine 50 and 100 μmol 1^-1 did not inhibit action potentials, but increased firing threshold by nearly 100%. Lidocaine 1–3 mmol l^-1 significantly inhibited action potential amplitude and increased threshold by as much as 800%. Similarly, QX314 and tetrodotoxin produced greater increases in current threshold than in action potential amplitude. QX314 produced phasic (or frequency-dependent) block during trains of stimuli at 1 Hz, even when almost no tonic block was present. Lidocaine produced less phasic block than QX314, and required both greater tonic block and more frequent stimulation to produce the phenomenon. Tetrodotoxin demonstrated no phasic block. Increases in current threshold occurred in lidocaine concentrations associated with analgesia and toxicity; inhibition of action potentials occurred scarcely at all at these concentrations. Thus, tonic increases in current threshold may underlie analgesia and supplementation of general anesthesia by intravenous lidocaine.     

Time-dependent Inhibition of G Protein-coupled Receptor Signaling by Local Anesthetics

Background: Several 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.

Methods: Measurement 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[alpha]q protein and inhibition of various proteins within the signaling pathway served for defining mechanisms and sites of action more specifically.

Results: LAs 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[alpha]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.     

Local anesthetics inhibit thromboxane A2 signaling in Xenopus oocytes and human k562 cells

Thromboxane A(2) (TXA(2)) has been proposed as a mediator of perioperative myocardial ischemia, vasoconstriction, and thrombosis. As these adverse events are minimized with epidural anesthesia, rather than general anesthesia, we hypothesized that local anesthetics would inhibit TXA(2)-receptor signaling. We used fluorometric determination of intracellular [Ca(2+)] in human K562 cells and 2-electrode voltage clamp measurements in Xenopus laevis oocytes expressing TXA(2) receptors. After 10-min incubation, lidocaine (IC(50): 1.02 +/- 0.2 x 10(-3) M), ropivacaine (IC(50): ropivacaine 6.3 +/- 0.9 x 10(-5) M), or bupivacaine (IC(50): 1.42 +/- 0.08 x 10(-7) M) inhibited TXA(2)-induced [Ca(2+)](i) in K562 cells. These data were confirmed in Xenopus oocytes recombinantly expressing TXA(2) receptors, with IC(50)s of bupivacaine 1.2 +/- 0.2 x 10(-5) M, R(+) ropivacaine 4.9 +/- 1.7 x 10(-4) M, S(-) ropivacaine 5.3 +/- 0.9 x 10(-5) M, and lidocaine 6.4 +/- 2.8 x 10(-4) M. Intracellular pathways activated by IP(3) and GTPgammaS were not significantly affected by the local anesthetics tested. QX314, a positively charged lidocaine analog, inhibited only if injected intracellularly (IC(50): 5.3 +/- 1.7 x 10(-4) M), indicating one local anesthetic target is most likely inside the cell. Benzocaine (largely uncharged) inhibited with an IC(50) of 8.7 +/- 1.8 x 10(-4) M. This suggests that some of the beneficial effects of regional anesthesia techniques might be due to direct interaction of local anesthetics with the functioning of membrane proteins.     

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

BACKGROUND: Several 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. METHODS: Measurement 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 alpha q protein and inhibition of various proteins within the signaling pathway served for defining mechanisms and sites of action more specifically. RESULTS: LAs 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 alpha 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. CONCLUSION: Inhibition of G protein-coupled receptor signaling by LAs was found to be time dependent and reversible. Critically requiring G alpha 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.     

The interaction of neuromuscular relaxants with rabbit lung muscarinic receptors

The interaction of muscle relaxants with airway muscarinic receptors of rabbit lung was investigated in vitro by the [3H]QNB binding technique. Pancuronium, vecuronium, alcuronium and succinyl choline chloride (SCC) inhibited the binding of [3H]QNB to rabbit lung muscarinic receptors in a dose-dependent manner. The values of IC50 (the concentration giving 50% inhibition) of pancuronium, vecuronium, alcuronium and SCC were 1.54 x 10(-5), 2.52 x 10(-5), 8.40 x 10(-5), and 4.00 x 10(-3) mol/l respectively. As the values of Kd increased with minimal change in the value of Bmax, while not influencing the number of receptors, these muscle relaxants had an inhibitory action on the affinity of muscarinic receptors to [3H]QNB in the order: pancuronium greater than or equal to vecuronium greater than alcuronium greater than SCC. Applying IC50 values to human conditions, clinical doses of these muscarinic relaxants are unlikely to exhibit any significant vagolytic action in lung tissue.     

Ropivacaine attenuates pulmonary vasoconstriction induced by thromboxane A2 analogue in the isolated perfused rat lung

BACKGROUND AND OBJECTIVES: Thromboxane A2 (TXA2) activation is involved in several pathophysiological states in producing pulmonary hypertension. Local anesthetics (LA) inhibit signaling of TXA2 receptors expressed in cell models. Therefore, we hypothesized that LA may inhibit pulmonary vasoconstriction induced by the TXA2 analogue U 46619 in an isolated lung model. METHODS: Isolated rat lungs were perfused with physiological saline solution and autologous blood with or without the LA lidocaine, bupivacaine, ropivacaine, or the permanently charged lidocaine analogue QX 314 (all 1 microg/mL) as a pretreatment. Subsequently, pulmonary vasoconstriction was induced by 3 concentrations of U 46619 (25, 50, and 100 ng/mL) and the change in pulmonary artery pressure (Pa) was compared with each LA. In a second experiment, Pa responses to angiotensin II (0.1 microg), hypoxic pulmonary vasoconstriction (HPV, 3% O2 for 10 minutes), or phenylephrine (0.1 microg) were assessed to determine the specificity of ropivacaine effects on TXA2 receptors. Finally, reversibility of pulmonary vasoconstriction was determined by adding ropivacaine to the perfusate after pulmonary vasoconstriction was established with U 46619. RESULTS: Ropivacaine, but not bupivacaine, lidocaine, or QX 314 significantly attenuated pulmonary vasoconstriction induced by 50 ng/mL U 46619 (35.9%, P<.003) or 100 ng/mL U 46619 (45.2%, P<.001). This effect of ropivacaine was likely to be specific for the thromboxane receptor because pulmonary vasoconstriction induced by angiotensin II, HPV, or phenylephrine was not altered. Ropivacaine did not reverse vasoconstriction when it was administered after U 46619. CONCLUSIONS: Ropivacaine, but not lidocaine, bupivacaine, or QX 314 at 1 microg/mL, attenuates U 46619-induced pulmonary vasoconstriction in an isolated perfused rat lung model. These results support evidence that the clinically used enantiomer S(-)-ropivacaine may inhibit TXA2 signaling.     

The interaction of pancuronium and vecuronium with cardiac muscarinic receptors

The interaction of vecuronium, a monoquaternary analogue of pancuronium, with the cardiac muscarinic receptors in canine hearts was investigated in vitro by [3H] QNB binding assay and compared with that of pancuronium. Both pancuronium and vecuronium, which are nicotinic antagonists of competitive types, inhibited the binding of [3H] QNB to cardiac muscarinic receptors with values of IC50 of 5.41 X 10(-7) mol/l and 3.97 X 10(-6) mol/l respectively. According to the Kd and Bmax values on the Scatchard plot, pancuronium, while not influencing the number of receptors, had a 3-fold greater inhibitory effect than vecuronium on the affinity of the muscarinic receptors. The KI values of vecuronium and pancuronium showed that pancuronium had a 7.3-fold greater affinity with the receptors than vecuronium. We concluded that vecuronium had a direct inhibitory action on the binding of [3H] QNB to the canine heart muscarinic receptors, but this action was much weaker than pancuronium.     

Lidocaine Induces a Reversible Decrease in Alveolar Epithelial Fluid Clearance in Rats

Background: Lidocaine is widely used in patients with acute cardiac disorders and has also been recently implicated as a possible cause of pulmonary edema after liposuction. The objective of this study was to assess the effect of lidocaine on alveolar fluid clearance, the primary mechanism responsible for the resolution of alveolar edema.

Methods: Alveolar fluid clearance was measured in 29 ventilated rats using our well-validated method over 1 h using a 5% albumin solution instilled into the distal air spaces of the lung. Lidocaine was added to the instilled albumin solution (10-5 m) or administered intravenously at a dose estimated to achieve a clinically relevant plasma concentration of 10-5 m. Standard agonists and antagonists were used to determine the effect of lidocaine on alveolar fluid clearance. To determine whether lidocaine acted predominantly on the apical or basal surface, we also used QX314, lidocaine n-ethyl bromide quaternary salt, an analog of lidocaine, which is unable to cross the alveolar epithelium. The effect of lidocaine on the apical epithelial sodium channel transfected in Xenopus oocytes was also studied.

Results: Alveolar or intravenous lidocaine decreased alveolar fluid clearance by 50%, an effect that was reversible with the [beta]2 agonist, terbutaline. Lidocaine acted predominantly on the basal surface of the epithelium because n-ethyl bromide quaternary salt decreased alveolar fluid clearance only when it was given intravenously and because lidocaine did not inhibit the apical epithelial sodium channel when expressed in oocytes.     

Magnesium enhances local anesthetic nerve block of frog sciatic nerve

The present study was designed to examine the effects of increased magnesium concentration on the nerve block produced by lidocaine, benzocaine, and QX 572 (a quaternary derivative of lidocaine). Desheathed whole sciatic nerves from northern Rana pipiens frogs were placed in a sucrose gap chamber and stimulated at various frequencies at supramaximal intensity for the activation of the compound action potential. After control studies, the nerve was bathed by a calcium-free frog Ringer's solution containing magnesium concentrations of 3.0, 10.0, or 20.0 mM with or without 0.5 mM lidocaine, 0.5 mM benzocaine, or 0.75 mM QX 572. Compound action potentials were measured, and nonfrequency and frequency dependent blocks were compared in each solution. Increased magnesium ion concentration, in the absence of local anesthetics, enhanced the nonfrequency dependent block but did not change the frequency dependent block. All three local anesthetics enhanced both types of block. Increased magnesium concentrations enhanced only the nonfrequency dependent benzocaine block. In contrast, increased magnesium enhanced both types of block produced by QX 572 and lidocaine. These results suggest a potentially important interaction between high magnesium concentrations and local anesthetic nerve blocks.     

The effects of lidocaine on the activity of glutamate transporter EAAT3: the role of protein kinase C and phosphatidylinositol 3-kinase

Using two electrode voltage clamps, we investigated the effects of lidocaine on one type of glutamate transporter, EAAT3, and the role of protein kinase C (PKC) and phosphatidylinositol 3-kinase (PI3K) in mediating the lidocaine effects. EAAT3 was expressed in Xenopus oocytes, and membrane currents were recorded after the application of L-glutamate (30 microM). Lidocaine increased glutamate-induced inward currents significantly at 2 concentrations (100 microM and 1 mM), but not at other concentrations. Lidocaine (100 microM) significantly increased the V(max), but not the K(m), of EAAT3 for glutamate compared with control. The action sites of lidocaine on EAAT3 seem to be intracellular, because only intracellularly injected QX314 (permanently charged lidocaine analog) increased the response. The combination of phorbol-12-myrisate-13-acetate, an activator of PKC, and lidocaine did not further increase the responses compared with phorbol-12-myrisate-13-acetate or lidocaine alone, although each of these three groups showed significantly bigger responses than controls. Three PKC inhibitors (staurosporine, calphostin C, and chelerythrine) did not affect the basal EAAT3 activity but abolished lidocaine-enhanced EAAT3 activity. Wortmannin (a specific PI3K inhibitor) inhibited EAAT3 basal activity and lidocaine-enhanced EAAT3 activity. Our results suggest that lidocaine enhances EAAT3 activity at certain concentrations and that PKC and PI3K may mediate these lidocaine effects. IMPLICATIONS: By using the Xenopus oocyte expression system, we investigated the effects of lidocaine on a glutamate transporter (EAAT3). Our findings suggest that lidocaine enhances EAAT3 activity at certain concentrations and that protein kinase C and phosphatidylinositol 3-kinase may mediate these lidocaine effects.     

Lidocaine induces a reversible decrease in alveolar epithelial fluid clearance in rats

BACKGROUND: Lidocaine is widely used in patients with acute cardiac disorders and has also been recently implicated as a possible cause of pulmonary edema after liposuction. The objective of this study was to assess the effect of lidocaine on alveolar fluid clearance, the primary mechanism responsible for the resolution of alveolar edema. METHODS: Alveolar fluid clearance was measured in 29 ventilated rats using our well-validated method over 1 h using a 5% albumin solution instilled into the distal air spaces of the lung. Lidocaine was added to the instilled albumin solution (10(-5) M) or administered intravenously at a dose estimated to achieve a clinically relevant plasma concentration of 10(-5) M. Standard agonists and antagonists were used to determine the effect of lidocaine on alveolar fluid clearance. To determine whether lidocaine acted predominantly on the apical or basal surface, we also used QX314, lidocaine n-ethyl bromide quaternary salt, an analog of lidocaine, which is unable to cross the alveolar epithelium. The effect of lidocaine on the apical epithelial sodium channel transfected in Xenopus oocytes was also studied. RESULTS: Alveolar or intravenous lidocaine decreased alveolar fluid clearance by 50%, an effect that was reversible with the beta2 agonist, terbutaline. Lidocaine acted predominantly on the basal surface of the epithelium because n-ethyl bromide quaternary salt decreased alveolar fluid clearance only when it was given intravenously and because lidocaine did not inhibit the apical epithelial sodium channel when expressed in oocytes. CONCLUSIONS: Lidocaine decreased alveolar fluid clearance by 50%, an effect that may have major clinical implications in the care of patients with cardiac disease or during the perioperative period in some patients. Importantly, the effect of lidocaine was completely reversible with beta2-adrenergic therapy.     

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 activationcontributes to postoperative hyperalgesia. Studies in volunteershave shown that intravenous local anaesthetics (LAs) preventthe development of hyperalgesic pain states. One potential explanationfor this beneficial effect is the inhibition of NMDA receptoractivation. Therefore, we studied the effects of LA on NMDAreceptor function. Methods. The human NR1A/NR2A NMDA receptor was expressed recombinantlyin Xenopus laevis oocytes. Peak currents were measured by voltageclamp in Mg- and Ca²⁺-free, Ba²⁺-containing Tyrode's solution.Holding potential was –70 mV. Oocytes were stimulatedwith glutamate/glycine (at EC₅₀) with or without 10 min priorincubation in bupivacaine, levobupivacaine, S-(–)-ropivacaine,or lidocaine (all at 10^–9–10^–4 M), procaine(10^–4 M), R-(+)-ropivacaine (10^–4 M), QX314 (permanentlycharged, 5x10^–4 M) extracellularly or intracellularlyor benzocaine (permanently uncharged, 5x10^–3 M). We alsodetermined the effect of the protein kinase C (PKC) inhibitorschelerythrine (5x10^–5 M), calphostin C (3x10^–6 M)and Ro 31-8220 (10^–7 M), and the effect of PKC activationwith phorbolester (10^–6 M). Results. Non-injected oocytes were unresponsive to agonist application,but oocytes expressing NMDA receptors responded with inwardcurrents (1.1±0.08 µA). All LA concentration-dependentlyinhibited agonist responses. The inhibition was reversible andstereoselective. Intracellular QX314 reduced responses to 59%of control, but extracellular QX314 was without effect. Benzocainereduced responses to 33% of control. PKC inhibitors had no additionalinhibitory effect beyond that of bupivacaine. The effect ofPKC activation was abolished in the presence of bupivacaine. Conclusion. All LA tested inhibited the activation of humanNMDA receptors in a concentration dependent fashion. This effectmay contribute to reduced hyperalgesia and opiate toleranceobserved after systemic administration of LA. The effect isindependent of the charge of LA; site of action is intracellular.The mechanism of action may be mediated by inhibition of PKC.     

The effects of the tramadol metabolite O-desmethyl tramadol on muscarinic receptor-induced responses in Xenopus oocytes expressing cloned M1 or M3 receptors

O-desmethyl tramadol is one of the main metabolites of tramadol. It has been widely used clinically and has analgesic activity. Muscarinic receptors are involved in neuronal functions in the brain and autonomic nervous system, and much attention has been paid to these receptors as targets for analgesic drugs in the central nervous system. We have reported that tramadol inhibits the function of type-1 muscarinic (M(1)) receptors and type-3 muscarinic (M(3)) receptors, suggesting that muscarinic receptors are sites of action of tramadol. However, the effects of O-desmethyl tramadol on muscarinic receptor functions have not been studied in detail. In this study, we investigated the effects of O-desmethyl tramadol on M(1) and M(3) receptors, using the Xenopus oocyte expression system. O-desmethyl tramadol (0.1-100 microM) inhibited acetylcholine (ACh)-induced currents in oocytes expressing the M(1) receptors (half-maximal inhibitory concentration [IC(50)] = 2 +/- 0.6 microM), whereas it did not suppress ACh-induced currents in oocytes expressing the M(3) receptor. Although GF109203X, a protein kinase C inhibitor, increased the ACh-induced current, it had little effect on the inhibition of ACh-induced currents by O-desmethyl tramadol in oocytes expressing M(1) receptors. The inhibitory effect of O-desmethyl tramadol on M(1) receptor was overcome when the concentration of ACh was increased (K(D) with O-desmethyl tramadol = 0.3 microM). O-desmethyl tramadol inhibited the specific binding of [(3)H]quinuclidinyl benzilate ([(3)H]QNB) to the oocytes expressed M(1) receptors (IC(50) = 10.1 +/- 0.1 microM), whereas it did not suppress the specific binding of [(3)H]QNB to the oocytes expressed M(3) receptors. Based on these results, O-desmethyl tramadol inhibits functions of M(1) receptors but has little effect on those of M(3) receptors. This study demonstrates the molecular action of O-desmethyl tramadol on the receptors and may help to explain its neural function.     

Propofol-induced Depression of Cultured Rat Ventricular Myocytes Is Related to the M2-acetylcholine Receptor-NO-cGMP Signaling Pathway

Background: It is well-known that propofol sometimes causes bradycardia or asystole during anesthesia; however, the direct effect of propofol on the myocardium remains unclear. Previous reports showed the contribution of muscarinic acetylcholine receptors to propofol-induced bradycardia. Conversely, it was suggested recently that nitric oxide (NO) plays an important role in mediating the effect of vagal stimulation in the autonomic regulation of the heart. Therefore, the authors investigated the effects of propofol on spontaneous contraction and NO production in cultured rat ventricular myocytes.

Methods: The authors measured chronotropic responses of cultured rat ventricular myocytes induced by propofol stimulation with a sensor, a fiber-optic displacement measurement instrument. The authors also quantitatively analyzed NO metabolite production in cultured myocytes by measuring the levels of nitrite and nitrate in a high-performance liquid chromatography reaction system. The influence of propofol on muscarinic acetylcholine receptors of myocyte membranes was also measured with a competitive binding assay using [3H]quinuclidinyl benzilate ([3H]QNB).

Results: Propofol caused negative chronotropy in a dose-dependent manner. Propofol (IC50) also caused the enhancement of nitrite production in cultured myocytes. Eighty percent of the enhancement of nitrite production induced by propofol (IC50) stimulation was abolished by pretreatment with atropine, methoctramine, or NG-monomethyl-L-arginine acetate (L-NMMA). The negative chronotropy induced by propofol (IC50) stimulation was reduced to 40-50% by pretreatment with atropine, methoctramine, L-NMMA, or 1 H[1,2,4]oxadiazolo[4,3-[alpha]]quanoxalin-1-one, a selective inhibitor of guanylyl cyclase. Propofol displaced [3H]QNB binding to the cell membrane of myocytes in a concentration-dependent manner.     

The quaternary lidocaine derivative, QX-314, produces long-lasting local anesthesia in animal models in vivo

BACKGROUND: QX-314 is a quaternary lidocaine derivative considered to be devoid of clinically useful local anesthetic activity. However, several reports document that extracellular QX-314 application affects action potentials. Hence, the authors tested the hypothesis that QX-314 could produce local anesthesia in animal models in vivo. METHODS: The authors tested QX-314 (10, 30, and 70 mm) in three standard in vivo local anesthetic animal models, using a randomized, blinded experimental design with negative (placebo) and positive (70 mm lidocaine) controls. The guinea pig intradermal wheal assay (n = 29) was used to test for peripheral inhibition of the cutaneous trunci muscle reflex, the mouse tail-flick test (n = 30) was used to test for sensory blockade, and the mouse sciatic nerve blockade model (n = 45) was used to test for motor blockade. RESULTS: In all three animal models, QX-314 concentration-dependently and reversibly produced local anesthesia of long duration, at concentrations equivalent to those clinically relevant for lidocaine. In the guinea pig intradermal wheal assay, QX-314 produced peripheral nociceptive blockade up to 6 times longer than lidocaine (650 +/- 171 vs. 100 +/- 24 min [mean +/- SD]; n = 6 per group; P < 0.0001). In the mouse tail-flick test, QX-314 produced sensory blockade up to 10 times longer than lidocaine (540 +/- 134 vs. 50 +/- 11 min; n = 6 per group; P < 0.0001). Finally, in the mouse sciatic nerve model, QX-314 produced motor blockade up to 12 times longer compared with lidocaine (282 +/- 113 vs. 23 +/- 10 min; n = 9 or 10 per group; P < 0.0001). The onset of QX-314-mediated blockade was consistently slower compared with lidocaine. Animals injected with saline exhibited no local anesthetic effects in any of the three models. CONCLUSION: In a randomized, controlled laboratory study, the quaternary lidocaine derivative, QX-314, concentration-dependently and reversibly produced long-lasting local anesthesia with a slow onset in animal models in vivo. The authors' results raise the possibility that quaternary ammonium compounds may produce clinically useful local anesthesia of long duration in humans and challenge the conventional notion that these agents are ineffective when applied extracellularly.