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).     

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 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.     

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 microm). 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 microm) and extracellularly applied QX314 (IC50 49 nm) exerted superadditive inhibition. Lidocaine did not displace specific [3H]QNB binding to m1 receptors. CONCLUSIONS: m1 Muscarinic signaling is inhibited by clinically relevant concentrations of lidocaine and by extracellularly administered QX314, suggesting that the major site of action is a extracellular domain of the muscarinic receptor. An additional less potent but superadditive inhibitory effect on the G-protein is suggested.     

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.     

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.     

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.     

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.     

Effects of Antidepressants on G Protein-coupled Receptor Signaling and Viability in Xenopus laevis Oocytes

Background: Tricyclic antidepressants are structurally related to local anesthetics, suggesting that part of their analgesic action may result from properties shared with local anesthetics. Because local anesthetics block G protein-coupled receptor signaling (which explains, in part, their inflammatory modulating properties), the authors studied whether antidepressants have similar effects.

Methods: Peak Ca-activated Cl currents induced in Xenopus laevis oocytes by lysophosphatidic acid (10-4 m) were measured using a voltage clamp. The effects of a 30-, 120-, or 240-min incubation in amitriptyline, nortriptyline, imipramine, or fluoxetine were determined.

Results: After a 30-min incubation, low concentrations (10-7-10-5 m) of antidepressants had no effect on lysophosphatidic acid-induced currents. After prolonged incubation, only amitriptyline or nortriptyline inhibited lysophosphatidic acid signaling (each to 58% of the control response at 10-7 m after 240 min). At low concentrations, none of the compounds induced membrane damage (defined as a holding current of > 1 [mu]A, 2% in control cells). Imipramine at 10-3 m induced damage in 100% of oocytes, and fluoxetine at 10-4 m induced damage in 71% of oocytes (P < 0.05 vs. control). Amitriptyline and nortriptyline had no effect.     

Effects of antidepressants on G protein-coupled receptor signaling and viability in Xenopus laevis oocytes

BACKGROUND: Tricyclic antidepressants are structurally related to local anesthetics, suggesting that part of their analgesic action may result from properties shared with local anesthetics. Because local anesthetics block G protein-coupled receptor signaling (which explains, in part, their inflammatory modulating properties), the authors studied whether antidepressants have similar effects. METHODS: Peak Ca-activated Cl currents induced in Xenopus laevis oocytes by lysophosphatidic acid (10(-4) m) were measured using a voltage clamp. The effects of a 30-, 120-, or 240-min incubation in amitriptyline, nortriptyline, imipramine, or fluoxetine were determined. RESULTS: After a 30-min incubation, low concentrations (10(-7)-10(-5) m) of antidepressants had no effect on lysophosphatidic acid-induced currents. After prolonged incubation, only amitriptyline or nortriptyline inhibited lysophosphatidic acid signaling (each to 58% of the control response at 10(-7) m after 240 min). At low concentrations, none of the compounds induced membrane damage (defined as a holding current of > 1 microA, 2% in control cells). Imipramine at 10(-3) m induced damage in 100% of oocytes, and fluoxetine at 10(-4) m induced damage in 71% of oocytes (P < 0.05 vs. control). Amitriptyline and nortriptyline had no effect. CONCLUSIONS: These findings are in part different from those obtained with local anesthetics and suggest that interference with G protein-coupled signaling might explain, in part, the analgesic properties of some antidepressants. However, use of antidepressants in high concentrations may be associated with cellular toxicity.     

Local Anesthetic Inhibition of Baseline Potassium Channels with Two Pore Domains in Tandem

Background: Recently, a new structural family of potassium channels characterized by two pore domains in tandem within their primary amino acid sequence was identified. These tandem pore domain potassium channels are not gated by voltage and appear to be involved in the control of baseline membrane conductances. The goal of this study was to identify mechanisms of local anesthetic action on these channels.

Methods: Oocytes of Xenopus laevis were injected with cRNA from five cloned tandem pore domain baseline potassium channels (TASK, TREK-1, TOK1, ORK1, and TWIK-1), and the effects of several local anesthetics on the heterologously expressed channels were assayed using two-electrode voltage-clamp and current-clamp techniques.

Results: Bupivacaine (1 mM) inhibited all studied tandem pore potassium channels, with TASK inhibited most potently. The potency of inhibition was directly correlated with the octanol: buffer distribution coefficient of the local anesthetic, with the exception of tetracaine, to which TASK is relatively insensitive. The approximate 50% inhibitory concentrations of TASK were 709 [micro sign]M mepivacaine, 222 [micro sign]M lidocaine, 51 [micro sign]M R(+)-ropivacaine, 53 [micro sign]M S(-)-ropivacaine, 668 [micro sign]M tetracaine, 41 [micro sign]M bupivacaine, and 39 [micro sign]M etidocaine. Local anesthetics (1 mM) significantly depolarized the resting membrane potential of TASK cRNA-injected oocytes compared with saline-injected control oocytes (tetracaine 22 +/- 6 mV vs. 7 +/- 1 mV, respectively, and bupivacaine 31 +/- 7 mV vs. 6 +/- 4 mV).     

Local anesthetic inhibition of baseline potassium channels with two pore domains in tandem

BACKGROUND: Recently, a new structural family of potassium channels characterized by two pore domains in tandem within their primary amino acid sequence was identified. These tandem pore domain potassium channels are not gated by voltage and appear to be involved in the control of baseline membrane conductances. The goal of this study was to identify mechanisms of local anesthetic action on these channels. METHODS: Oocytes of Xenopus laevis were injected with cRNA from five cloned tandem pore domain baseline potassium channels (TASK, TREK-1, TOK1, ORK1, and TWIK-1), and the effects of several local anesthetics on the heterologously expressed channels were assayed using two-electrode voltage-clamp and current-clamp techniques. RESULTS: Bupivacaine (1 mM) inhibited all studied tandem pore potassium channels, with TASK inhibited most potently. The potency of inhibition was directly correlated with the octanol: buffer distribution coefficient of the local anesthetic, with the exception of tetracaine, to which TASK is relatively insensitive. The approximate 50% inhibitory concentrations of TASK were 709 microM mepivacaine, 222 microM lidocaine, 51 microM R(+)-ropivacaine, 53 microM S(-)-ropivacaine, 668 microM tetracaine, 41 microM bupivacaine, and 39 microM etidocaine. Local anesthetics (1 mM) significantly depolarized the resting membrane potential of TASK cRNA-injected oocytes compared with saline-injected control oocytes (tetracaine 22+/-6 mV rs. 7+/-1 mV, respectively, and bupivacaine 31+/-7 mV vs. 6+/-4 mV). CONCLUSIONS: Local anesthetics inhibit tandem pore domain baseline potassium channels, and they could depolarize the resting membrane potential of cells expressing these channels. Whether inhibition of these channels contributes to conduction blockade or to the adverse effects of local anesthetics remains to be determined.     

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.     

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.     

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.     

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.     

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.     

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.     

Modulation of Xenopus laevis Ca-activated Cl currents by protein kinase C and protein phosphatases: implications for studies of anesthetic mechanisms

Ca-activated Cl currents (I(Cl(Ca))) are used frequently as reporters in functional studies of anesthetic effects on G protein-coupled receptors using Xenopus laevis oocytes. However, because anesthetics affect protein kinase C (PKC), they could indirectly affect I(Cl(Ca)) if this current is regulated by phosphorylation. We therefore studied the effect of modulation of either PKC or protein phosphatases PP1alpha and PP2A on I(Cl(Ca)) stimulated either by lysophosphatidate (LPA) signaling or by microinjection of Ca. X. laevis oocytes were studied under voltage clamp. Rat PP1alpha and PP2A were overexpressed in oocytes. PP, inositoltrisphosphate (IP(3)), the PP inhibitor okadaic acid (OA), the PKC inhibitor chelerythrine, or CaCl(2) were directly injected into the oocyte. Responses to agonists (LPA 10(-6) M, IP(3) 10(-4) M, CaCl(2) 0.5 M) were measured at a holding potential of -70 mV in the presence or absence of the PP inhibitors cantharidin or OA. PP1 alpha and PP2A inhibited I(Cl(Ca)) from 7.6 +/- 0.9 microC to 2.5 +/- 0.9 microC and 3.2 +/- 1.4 microC, respectively. PP inhibition enhanced I(Cl(Ca)) in control oocytes and reversed the inhibitory effect in oocytes expressing PP1 alpha or PP2A. PKC inhibition by chelerythrine enhanced both LPA- and CaCl(2)-induced I(Cl(Ca)). Our data indicate that the Xenopus I(Cl(Ca)) is modulated by phosphorylation. This may complicate design and interpretation of studies of G protein-coupled receptors using this model.     

Differential Sensitivities of Mammalian Neuronal and Muscle Nicotinic Acetylcholine Receptors to General Anesthetics

Background: Nicotinic acetylcholine receptors (nAChRs) are members of a superfamily of fast neurotransmitter-gated receptor channels that includes the gamma-aminobutyric acidA (GA-BAA), glycine and serotonin type 3 (5-HT3) receptors. Most previous work on the interactions of general anesthetics with nAChRs has involved the muscle-type receptor. The authors investigate the effects of general anesthetics on defined mammalian neuronal and muscle nAChRs expressed in Xenopus oocytes.

Methods: Complementary deoxyribonucleic acid (cDNA) or messenger ribonucleic acid (mRNA) encoding for various neuronal or muscle nAChR subunits was injected into Xenopus oocytes, and the resulting ACh-activated currents were studied using the two-electrode voltage-clamp technique. The effects of halothane, isoflurane, sevoflurane, and propofol on the peak acetylcholine-induced currents were investigated, and concentration-response curves were constructed.

Results: The neuronal nAChRs were found to be much more sensitive to general anesthetics than were the muscle nAChRs, with IC50 concentrations being 10- to 35-fold less for the neuronal receptors. For the inhalational general anesthetics, the IC50 concentrations were considerably less than the free aqueous concentrations that cause general anesthesia in mammals. In addition, qualitative (dependence on acetylcholine concentration) and quantitative (steepness of concentration-response curves) differences in the anesthetic interactions between the neuronal and muscle nAChRs suggest that different mechanisms of inhibition may be involved.