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Anesth Analg 2004;99:930-937
© 2004 International Anesthesia Research Society
doi: 10.1213/01.ANE.0000130351.76523.3F

REGIONAL ANESTHESIA

Local Anesthetics Inhibit Thromboxane A2 Signaling in Xenopus Oocytes and Human K562 Cells

Christian W. Hönemann, MD{dagger}, Klaus Hahnenkamp, MD*, Tobias Podranski*, Danja Strumper, MD*, Markus W. Hollmann, MD PhD{ddagger}, and Marcel E. Durieux, MD PhD§

*Klinik und Poliklinik für Anästhesiologie und operative Intensivmedizin, University Hospital, Münster; {dagger}Department of Anesthesiology, St. Marienhospital, Vechta; {ddagger}Department of Anesthesiology, University of Heidelberg, Germany; and §Department of Anesthesiology, University of Virginia, Charlottesville

Address correspondence to Marcel E. Durieux, MD, PhD, Department of Anesthesiology, University of Virginia, PO Box 800710, Charlottesville, VA 22908-0710. Address e-mail to durieux{at}virginia.edu


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 References
 
Thromboxane A2 (TXA2) 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 TXA2-receptor signaling. We used fluorometric determination of intracellular [Ca2+] in human K562 cells and 2-electrode voltage clamp measurements in Xenopus laevis oocytes expressing TXA2 receptors. After 10-min incubation, lidocaine (IC50: 1.02 ± 0.2 x 10–3 M), ropivacaine (IC50: ropivacaine 6.3 ± 0.9 x 10–5 M), or bupivacaine (IC50: 1.42 ± 0.08 x 10–7 M) inhibited TXA2-induced [Ca2+]i in K562 cells. These data were confirmed in Xenopus oocytes recombinantly expressing TXA2 receptors, with IC50s 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 IP3 and GTP{gamma}S were not significantly affected by the local anesthetics tested. QX314, a positively charged lidocaine analog, inhibited only if injected intracellularly (IC50: 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 IC50 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.

IMPLICATIONS: We demonstrated, using two different models, that thromboxane receptor functioning is inhibited by commonly used local anesthetics. One site of action seems to be inside the cell. 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.


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 References
 
Perioperative thromboembolic complications are common and seem to result from a combination of hypercoagulation and hypoperfusion (the latter often induced by vasoconstriction). After major orthopedic or urologic surgery, deep venous thrombosis occurs in 30%–80% of patients (1) unless preventive measures are taken. Myocardial ischemia induced by microthrombosis or coronary vasoconstriction is also common (2).

These complications seem to be reduced if epidural anesthesia is used (3–5). Unfortunately, epidural anesthesia is not always an option (because of patient preference, surgical procedure, or coexisting disease), and it is therefore important to know if these beneficial effects can be obtained by other means. Findings that local anesthetics (LA), when infused IV, decrease the incidence of thrombotic complications (1) suggest that some of the beneficial effects of epidural anesthesia can be explained by interactions between inflammatory mediator signaling and LA present in blood. In vitro, LA dilate blood vessels (6) and decrease platelet aggregation (7,8). The mechanism of action of these effects has not been described.

Thromboxane A2 (TXA2) is a potent platelet aggregator and vasoconstrictor. It is one of the major mediators released during orthopedic, cardiac (9), abdominal, vascular, and obstetric surgery. TXA2 has been proposed to act as a mediator of myocardial ischemia, coronary vasoconstriction, and thrombosis. Thus, interactions between LA and TXA2 signaling might play a role in the protective effects of LA. We hypothesized that the TXA2 signaling pathway might be a site of LA action.

To test this hypothesis, we investigated the effects of commonly used LA (lidocaine, bupivacaine, and ropivacaine) on TXA2 signaling. Two models were studied: (A) To determine if clinically used LA inhibit TXA2 signaling in a relevant human cell model, we studied their effects on TXA2 receptor-mediated intracellular Ca release in the hematopoietic cell line K562, a cell type with platelet-like properties. (B) To determine the molecular site of action of LA, we studied their effects on TXA2 receptors expressed recombinantly in Xenopus oocytes.

In either model, we investigated the effects on TXA2-induced activation of the inositoltrisphosphate(IP3)-Ca pathway, because this is the primary pathway involved in the signaling of these receptors.


   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 References
 
K562 cells were cultured in 500-mL flasks at 37°C in modified Eagle’s medium (4.0 mM of L-glutamine, 2.5 mM of pyridoxine HCl, 25 mM of sodium pyruvate, 10% fetal bovine serum, and 1% penicillin/streptomycin, 300 mOsm). Under sterile conditions, an aliquot of cells was removed from the flask and placed into a 50-mL conical tube. Cells were counted in a hemocytometer; 1–3 x 105 cells were used for each experiment. The media was removed, and phosphate buffered saline solution (120 mM of NaCl, 2.7 mM of KCl, and 10 mM of phosphate buffer salts, pH 7.6, 295 mOsm) was added.

To measure intracellular Ca2+ concentrations ([Ca2+]i), K562 cells were loaded with fura-2 acetoxymethylester (fura-2/AM; Molecular Probes, Junction City, OR; final concentration 2 µM) by incubation of cell suspension (1 x 106 cells/mL) in HKRB (Krebs-Ringers buffer with HEPES [N-(2-hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid) hemisodium]: 105 mM of NaCl, 5 mM of KCl, 0.1 mM of KH2PO4, 28 mM of NaHCO3, and 2 mM of HEPES) for 30 min at room temperature. Any fura-2/AM not internalized was removed by washing; 1.75 mL of 1 x HKRB, with or without LA, and 0.25-mL cell suspension (2.5 x 105 cells) were placed in a glass tube and incubated at 37°C for 10 min. The solution was then placed inside a fluorometer (Photon Technology International Inc, Felix analysis software, South Brunswick, NJ) in a 5-mL plastic cuvette. The solution was stirred throughout, and the temperature was controlled at 37°C. The fluorometer was calibrated and the stable thromboxane agonist U-46619 (Cayman Chemical, Ann Arbor, MI), diluted in 0.1% fatty acid free bovine serum albumin in 1 x HKRB, was added to the cuvette. Fura-2 fluorescence emission at 510 nm was then measured after excitation at 340 nm and 380 nm. Data were collected until fluorescence returned to baseline. Ionomycin (3 µL, 10–4 M) and EGTA (60 µL, 10–4 M) were used to determine maximum and minimum [Ca2+]i, respectively. Fluorescence data were converted to [Ca2+]i using the method of Grynkiewicz et al. (10). The LA studied (lidocaine, bupivacaine, and ropivacaine; Sigma Chemicals Inc, St Louis, MO) were prepared using 1 x HKRB adjusted to pH 7.4.

After approval of the protocol by the institutional Animal Care and Use Committee, oocytes were obtained from Xenopus laevis frogs. Surgical removal and defolliculation of oocytes for injection, as well as preparation and injection of rat TXA2-receptor mRNA and electrophysiologic recording techniques, were performed, as previously described (11). Oocytes were incubated for 10 min in bupivacaine, lidocaine, or ropivacaine isomers (AstraZeneca, Wayne, PA) diluted in modified Barth solution (MBS: 88 mM of NaCl, 2.4 mM of NaHCO3, 0.41 mM of CaCl2, 0.82 mM of MgSO4, 0.3 mM of Ca2NO3, 0.1 mM of gentamycin, and 15 mM of HEPES, pH adjusted to 7.4) to various concentrations or in MBS alone. Each oocyte was then transferred to a measurement chamber containing 500 µL of Tyrode solution (in mM: NaCl 150, KCl 5, CaCl2 2, MgSO4 1, dextrose 10, and HEPES 10, pH adjusted to 7.4, 300 mOsm) and the LA under investigation and was voltage-clamped and tested alternately with a control cell incubated in MBS only. Inward currents induced by TXA2-receptor stimulation are reported as µC.

A third micropipette was inserted into the voltage-clamped oocyte to study IP3- or GTP{gamma}S-induced ICl(Ca) or to test inhibition by the nonpermeable LA QX314 (AstraZeneca). The micropipette was connected to an automated microinjector (Nanoject; Drummond Scientific, Broomall, PA). Under voltage clamp, 30 nL of 2 mM of IP3 or 100 mM of GTP{gamma}S was injected, thereby activating the signaling pathway at the IP3 receptor or the G protein, respectively. We assumed an average Xenopus oocyte volume of approximately 500 nL. Since the injected volume is approximately 5% of the estimated oocyte volume, the estimated final concentrations were IP3 100 µM or GTP{gamma}S 5 mM (a "real" final concentration cannot be established, as distribution within the cell is not homogeneous). These concentrations were chosen to result in currents similar in size to those induced by the TXA2 agonist U-46619 at its EC50. Induced currents were recorded 5 s before and 55 s after intracellular injection and analyzed, as previously described (11,12). QX314, which does not penetrate the cell membrane because of its permanent charge, was diluted in 50 nL of KCl (150 mM) and injected into the oocyte or applied outside the cell to identify an intra- or extracellular site of action, respectively. Control cells were injected with 50 nL of KCl (150 mM). Each cell was then voltage clamped and tested 10 min after injection. Intracellular concentrations at the site of action were estimated using the assumptions listed above for IP3 or GTP{gamma}S.

For G protein {alpha} subunit knockdown experiments, phosphorothioate oligonucleotides were synthesized by the University of Virginia Research Facility. The antisense sequences are complementary to specific 20-base segments with <50% homology with other types of Xenopus laevis G{alpha} proteins (13). Sense oligonucleotides were used as control. Oocytes injected 24 h previously with cRNA encoding the TXA2 receptor were injected with 50 nL of sterile water containing 50 ng/cell of antisense or sense oligonucleotides. Control cells were injected with the same amount of sterile water. Twenty-four and 48 h after oligonucleotide injection, the cells were studied as described above.

Results are reported as mean ± SEM. Since receptor expression varies between batches of oocytes, responses from experiments with expressed receptors were normalized to same-day controls for each batch. Differences between treatment groups were analyzed using analysis of variance and Student’s unpaired t-test, appropriately corrected for multiple comparisons (Bonferroni). P < 0.05 was considered significant. Concentration response curves were constructed, and pooled data were fit to the following logistic function, derived from the Hill equation: equation


{50MM1}

where ymax and ymin are the maximum and minimum response obtained, n is the Hill coefficient, and X50 is the half-maximal effect concentration (EC50 for agonist) or the half-maximal inhibitory effect concentration (IC50 for antagonists).


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 References
 
We determined LA effects on TXA2 signaling by measuring [Ca2+]i in K562 cells, a hematopoietic cell line with platelet properties that expresses endogenous TXA2 receptors. The resting level of [Ca2+]i in K562 cells was 8–11 x 10–8 M. Application of U-46619, a selective TXA2 agonist (10–6 M), increased [Ca2+]i by 153 ± 45 nM within 90 s (Fig. 1A). [Ca2+]i returned to baseline levels within 5 min of U-46619 application. We calculated an EC50 for U-46619 of 6.4 ± 2.3 x 10–7 M (Fig. 1B) and therefore used 10–6 M of U-46619 for further experiments in this model.



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  		Figure 1. U-46619 increases intracellular [Ca2+] in human myeloid K562 cells via endogenous thromboxane A2 (TXA2) receptors in a concentration-dependent manner. Local anesthetics (LA) inhibit TXA2 signaling in the human myeloid K562 cell line concentration-dependently. (A) Example traces of U-46619 induced an increase in the intracellular calcium concentration ([Ca2+]i) in human myeloid K562 cells. Example traces show changes of [Ca2+]i of 48 nM (10–7 M of U-46619), 153 nM (10–6 M of U-46619), and 272 nM (10–5 M of U-46619). (B) Concentration-response curve of U-46619 in human myeloid K562 cells. Application of the TXA2 agonist increased the intracellular calcium concentration ([Ca2+]i) in a concentration-dependent manner (EC50: 6.4 x 10–7 M; fitting values in Table 1 [control]). (C) Concentration-inhibition curve of lidocaine on [Ca2+]i induced by 10–6 M of U-46619 in K562 cells (IC50: 1.02 x 10–3 M; fitting values in Table 1). (D) Concentration-inhibition curve of bupivacaine on [Ca2+]i induced by 10–6 M of U-46619 in K562 cells (IC50: 1.42 x 10–7 M; fitting values in Table 1). (E) Concentration-inhibition curve of S(-) ropivacaine on [Ca2+]i induced by 10–6 M of U-46619 in K562 cells (IC50: 6.42 x 10–5 M; fitting values in Table 1).

 

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  		Table 1. Fitting Values Using the Hill Equation for U46619-Induced Increase in Cytosolic [Ca2+] (control) and for Local Anesthetic Inhibition of U-46619-Induced Increase in Cytosolic [Ca2+]i in K562 Cells
 
Each of the 3 LA tested (bupivacaine, ropivacaine, and lidocaine) was without effect on baseline [Ca2+]i levels (data not shown) but inhibited U-46619–induced [Ca2+]i concentration-dependently. IC50 values, obtained from triplicate determinations, were as follows: lidocaine 1.1 x 10–3 M (Fig. 1C), bupivacaine 1.4 x 10–7 M (Fig. 1D), and S(-) ropivacaine 6.3 x 10–5 M (Fig. 1E; Table 1).

To investigate in more detail the mechanism of LA action on the TXA2 signaling pathway, we studied TXA2 receptors expressed recombinantly in Xenopus oocytes. Previously, we have shown that oocytes injected with TXA2 receptor mRNA respond to the application of U-46619 with inward currents (11). The EC50 was 3.2 x 10–7 M. We therefore chose U-46619 at a concentration of 10–6 M for subsequent experiments.

We have shown previously that TXA2-receptor signaling in oocytes is mediated by IP3-induced increases in intracellular Ca2+ (11). To ascertain that these effects are, in fact, mediated by G proteins, and to determine the G protein subtypes involved, we studied the effect of selective G{alpha} subunit depletion on TXA2-receptor signaling. We used antisense oligonucleotides for this purpose (Fig. 2A). To exclude the possibility that an injection of oligonucleotides per se affected responses to U-46619, we first studied the effect of sense-oligonucleotide injection. Neither 24 h nor 48 h (data not shown) after injection did the sense-oligonucleotide–injected oocytes show responses different from those obtained in control oocytes injected with sterile water. Thus, sense oligonucleotides are without effect on U-46619-induced effects. Injection, 48 h prior to testing, of antisense oligonucleotides directed against G{alpha}11 or G{alpha}q decreased responses induced by U-46619 to 62% ± 8% and 45% ± 7% of control, respectively (P < 0.05; Fig. 2B). In contrast, antisense sequences directed against G{alpha}14 or G{alpha}o were without significant effect compared with control oocytes (Fig. 2B). These findings indicate that recombinant TXA2 receptors signal primarily through G{alpha}11 and G{alpha}q, which, in agreement with our previous findings, couple to the IP3-Ca2+ pathway.



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  		Figure 2. In Xenopus oocytes, recombinant thromboxane A2 (TXA2) receptors signal via Gq proteins. Selective G{alpha}-subunits knockdown using antisense constructs. (A) Antisense oligonucleotide sequence. (B) Mean ± SEM of currents of TXA2 responses induced by U-46619 (10–6 M) in control oocytes (black bar, 2.6 ± 0.2 µC) and cells injected with the corresponding DNA antisense oligonucleotides (50 ng/oocyte) 48 h previoisly. Anti-G{alpha}11 inhibited responses to 62.6% ± 8.2% and anti-G{alpha}q to 44.7% ± 7.4%, as compared with control. In contrast, injection of anti-G{alpha}o (91.8% ± 8.9% of control response) or anti-G{alpha}14 (100% ± 11.7% of control response) was without significant effect compared with control oocytes.

 
We then studied the effects of lidocaine or bupivacaine on the functioning of recombinant TXA2 receptors in Xenopus oocytes. Either LA inhibited TXA2 signaling concentration-dependently (Fig. 3A–D; Table 2). The IC50 for lidocaine was 6.4 x 10–4 M; bupivacaine inhibited TXA2 signaling with a calculated IC50 of 1.2 x 10–5 M. Effects of both compounds were reversible (data not shown).



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  		Figure 3. Local anesthetics (LA) inhibit thromboxane A2 (TXA2)-receptor functioning recombinantly expressed in Xenopus oocytes. (A) Example traces of ICl(Ca) induced by U-46619 (10–6 M) in presence and absence of lidocaine in Xenopus oocytes. Charge movement in response to U-46619 was 8.2 µC (control), 4.4 µC (lidocaine 5 x 10–5 M), and 1.7 µC (lidocaine 5 x 10–2 M), respectively. (B) Lidocaine inhibited TXA2 receptor functioning in a concentration-dependent manner. IC50 is 0.64 x 10–4 M (fitting values in Table 2). (C) Example traces of ICl(Ca) induced by U-46619 (10–6 M) in the presence and absence of bupivacaine Xenopus oocytes. Charge movement in response to U-46619 was 7.9 µC (control), 3.9 µC (bupivacaine 1.5 x 10–6 M), and 0.9 µC (bupivacaine 1.5 x 10–5 M), respectively. (D) Bupivacaine TXA2-receptor functioning in a concentration-dependent manner. IC50 is 1.22 x 10–5 M (fitting values in Table 2). R(+) and S(-) ropivacaine inhibit stereoselective TXA2-receptor signaling recombinantly expressed in Xenopus oocytes. (E) Examples of ICl(Ca) induced by U-46619 (10–6 M) in Xenopus oocytes expressing TXA2 receptors. Charge movement in response to U-46619 is 9.1 µC. S(-) ropivacaine (2 x 10–4 M and 2 x 10–3 M) inhibit signaling, resulting in charge movements of 3.2 and 0.8 µC, respectively (upper 3 traces). Charge movement in response to U-46619 is 8.6 µC. R(+) ropivacaine (2 x 10–4 M and 2 x 10–3 M), resulted in charge movements of 4.1 and 0.5 µC, respectively (lower 3 traces). (F) Ropivacaine inhibits U-46619 (10–6 M)-induced ICl(Ca) in a concentration-dependent and stereoselective manner. IC50 for R(+) ropivacaine was 4.9 x 10–4 M and for S(-) ropivacaine 4.3 x 10–5 M (fitting value variable in Table 2). Calculations for the tested data points resulted in a statistically significant difference for the 2 x 10–4 M concentrations between both isomers (*P < 0.05; t-test).

 

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  		Table 2. Fitting Values Using the Hill Equation for U46619-Induced ICI(Ca) (control) and for Local Anesthetic Tested on Xenopus Oocytes, Recombinantly Expressing Rat TXA2 Receptors
 
We then tested the effects of R(+) or S(-) ropivacaine on recombinant TXA2-receptor function in Xenopus oocytes. R(+) ropivacaine inhibited TXA2 signaling with a calculated IC50 of 4.9 x 10–4 M, whereas the clinically used S(-) ropivacaine showed an approximately 10-fold greater inhibitory potency: IC50 was 5.3 x 10–5 M (Fig. 3, E and F). Therefore, we conclude that recombinant TXA2-receptor function is inhibited by LA in a stereoselective manner, which is consistent with an interaction with proteins. The rank-order of potency of lidocaine, bupivacaine, and ropivacaine in oocytes was similar to that observed in K562 cells.

To determine if the site of action for LA within the signaling pathway is located intra- or extracellularly, we studied the effect of QX314, which is positively charged and does not cross cell membranes. QX314, when applied intracellularly, blocked TXA2 signaling in a concentration-dependent manner (IC50: 5.35 x 10–4 M). In contrast, extracellular QX314 (5 x 10–4 M) was without significant effect (Fig. 4A–C), indicating that the effect of intracellularly applied QX314 is not caused by it reaching an extracellular site of action (by leakage or otherwise).



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  		Figure 4. Intracellular applied QX314 (positively charged) inhibited thromboxane A2 (TXA2)-receptor signaling as well as benzocaine (uncharged). Intracellular signaling pathways were not affected by all tested local anesthetics (LA) after 10 min of incubation. (A) The first bar represents ICl(Ca) of 14 oocytes (injected with mRNA encoding the TXA2 receptor) intracellulary injected with 150 mM of KCl (11.5 ± 2.39 µC, intern). The second bar shows ICl(Ca) of 14 oocytes intracellularly injected with 150 mM of KCl containing 500 µM of QX314 (5.59 ± 1.39 µC, positively charged), and the third bar represents ICl(Ca) of 7 oocytes exposed to 0.5 mM of QX314 extracellular for 10 min before agonist application (13.91 ± 1.86, extern). (B) Example traces of ICl(Ca) induced by U-46619 (10–6 M) in oocytes expressing TXA2 receptors; charge movement in the control cell (injected with 50 nL of 150 mM of KCl) was 13.4 µC, and in the cell injected with 150 mM of KCl containing 500 µM of QX314, ICl(Ca) was 7.3 µC. (C) As the other tested LA, QX314 inhibited ICl(Ca) responses to TXA2 agonist U-46619 (10–6M) in a concentration-dependent manner. IC50 is 5.35 x 10–4 M (fitting values in Table 2). (D) Benzocaine inhibited ICl(Ca) to the TXA2 agonist U-46619 (10–6M) in a concentration-dependent manner. IC50 was 8.72 x 10–4 M (fitting values in Table 2). (E) None of the tested local anesthetics inhibited intracellular signaling pathways activated by activation by G protein activation (GTP{gamma}S injection, black bar) or IP3 receptor activation (IP3 injection, white bar; n > 7 for each data point).

 
The results when using QX314 indicate that charged LA inhibit TXA2 signaling. To determine if TXA2 signaling block is restricted to charged LA, we studied the effect of benzocaine, which, at physiologic pH, is almost completely uncharged. Benzocaine inhibited TXA2 signaling with an IC50 of 8.7 x 10–4 M (Fig. 4D), indicating that both charged and uncharged LA can inhibit TXA2 signaling.

Our findings using QX314 suggest an effect of the LA on the intracellular signaling pathway coupling to the TXA2 receptor. To narrow down the site of action for LA within the TXA2 signaling pathway, we activated segments of the intracellular pathway directly by intracellular microinjection of IP3 (which directly activates its receptor-channel complex on intracellular Ca2+ stores) and GTP{gamma}S (which irreversibly activates G proteins). We previously reported a lack of effect of lidocaine and bupivacaine on IP3-induced currents (12,14). For comparative purposes, we repeated these experiments, and in addition, investigated effects of benzocaine, QX314, and ropivacaine on GTP{gamma}S- or IP3-induced currents. All LA tested were without effect on currents induced by microinjection of IP3 or GTP{gamma}S after 10 min of incubation (Fig. 4E). Lack of LA effects on currents induced by these mediators suggests that the signaling pathway downstream from the site of action of these compounds (i.e., downstream of the IP3 receptor) is unaffected by LA and that instead, the pathway between receptor and the IP3 receptor is the target of LA action.


   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
References
 
The LA lidocaine, bupivacaine, and ropivacaine inhibit TXA2-receptor signaling, both in a human cell line and in a recombinant model. These effects were concentration-dependent, reversible, and stereoselective. An intracellular site for charged LA was identified, but the uncharged LA benzocaine also inhibited TXA2 signaling.

Several clinical studies suggest that LA may play a role in preventing postoperative thrombosis. Cooke et al. (1) demonstrated beneficial effects of IV lidocaine infusion in preventing deep venous thrombosis. Modig and Borg (4,6,15) reported a reduced incidence of pulmonary thromboembolism as well as decreased pulmonary artery pressures when lumbar epidural anesthesia was used. We observed that epidural anesthesia prevented the hypercoagulation induced by major orthopedic surgery (16). These findings are supported by a meta-analysis that demonstrated reductions in the incidence of thrombosis by 44% and of pulmonary embolism by 55% if neuraxial blockade was used during surgery (5). However, the mechanisms that induce these beneficial effects remain unclear. Odoom et al. (7,8,17) suggested that the possible preventive effect of epidural bupivacaine on thrombosis might result from an inhibitory effect on platelet aggregation, in addition to an increase in lower limb blood flow. Because TXA2 is an important perioperative mediator that stimulates platelet aggregation (18,19) and constricts vascular and respiratory smooth muscle, and that has been suggested to play a role in thrombosis, unstable angina, and myocardial infarction (as well as asthma), it may be of relevance in this setting. Our data suggest that inhibition of TXA2 signaling could play a role in the beneficial effects of LA. The use of cell models precludes extrapolation of observed concentration-response data to the clinical setting. We found bupivacaine to be approximately 500-fold more potent than ropivacaine and almost 4 orders of magnitude more potent than lidocaine in inhibiting TXA2 signaling. It is conceivable (although unlikely) that partitioning across the membrane was not complete after 10 min of incubation. If such were the case, inhibiting concentrations would be less than those calculated by us. Although concentrations larger than the EC50 values observed in this study can certainly be reached when LA are applied for regional anesthesia (e.g., spinal administration induces millimolar levels at the site of action), they do not in most parts of the body. However, this does not negate the potential clinical relevance of our data for several reasons (1). The oocyte model, being an artificial construct, may not follow the same concentration response relationships as obtained in the clinical setting. In fact, our smaller IC50 obtained in K562 cells suggest this (2). We only investigated a single pathway. In vivo, multiple pathways and cascades work in parallel to induce thrombosis and vasoconstriction. Even a modest effect of LA on different segments of such pathways may therefore induce a clinical effect, even though the action on any single segment would be insufficient to provide a full effect (3). It is not necessary to obtain 50% blockade for a clinically relevant action. A 10% decrease in platelet adhesion might already greatly reduce the incidence of postoperative thrombosis. In fact, in many cases, profound inhibition of a pathway is not desired at all.

Only bupivacaine was effective at blocking TXA2 signaling at concentrations obtained during epidural anesthesia (20). If LA indeed decrease thrombotic events by inhibiting TXA2 signaling, bupivacaine might have advantages over ropivacaine. Moreover, our findings with ropivacaine isomers suggest that the TXA2-inhibitory properties of racemic bupivacaine may largely reside in the clinically relevant stereoisomer levobupivacaine, suggesting that this compound would be more effective in reducing the risk of thrombosis than the racemic preparation. However, the rank-order of potency may be model-dependent. Borg and Modig (6) suggested that lidocaine is the most effective antiaggregating LA. In a hamster cheek pouch model, topical application of lidocaine (60 µg) was much more effective in preventing laser-induced thrombosis than bupivacaine (15 µg) (21). We ourselves found ropivacaine to be more potent than bupivacaine in its effects on U-46619-induced pulmonary hypertension in the isolated lung model in the rat (22) and observed only limited effects of lidocaine, bupivacaine, and ropivacaine on TXA2-induced platelet aggregation in vitro (23). This latter finding suggests that the effects of LA on thrombosis may not be primarily mediated by an effect on platelet aggregation but rather on vascular tone. However, the systems under evaluation are so complex that any laboratory model is necessarily a vast simplification and may not reflect the events in the clinical setting.

In the present study, we showed that TXA2 receptors expressed in Xenopus oocytes couple to G{alpha}q and G{alpha}11. Together with the lack of LA effect on the downstream signaling pathway and results indicating that G{alpha}q proteins are a target for charged LA (24), this suggests that the inhibitory effect of intracellular QX314 may be mediated by interference with G{alpha}q signaling. In support of this hypothesis, IC50 for intracellular QX314 was similar to that reported for its effect on muscarinic and lysophosphatidate signaling (12,25). The stereoselectivity observed with ropivacaine is also compatible with this protein site of action. However, it was not possible to conclude on a target site with certainty, because we could not exclude that the effect of QX314 was indirect, i.e., that it affected an intracellular molecule that, in turn, modulated the TXA2-signaling pathway. In addition, data obtained with QX314 cannot necessarily be extrapolated to other compounds, and various LA might not all act at the same target. QX314 blocked TXA2 signaling only when applied intracellularly. Thus, TXA2 receptors seem to lack an extracellular binding site for charged LA, which is similar to findings obtained using lysophosphatidate and muscarinic m3 receptors but in contrast to muscarinic m1 receptors (25,26). Inhibition by the membrane permeable LA benzocaine may suggest an additional site of action for uncharged LA, either extracellularly or intracellularly.

In summary, our findings indicate that clinically used LA inhibit signaling through TXA2 receptors. This suggests that such an interaction might help explain, in part, their beneficial effects on various perioperative TXA2-mediated events.


   Acknowledgments
 
Dr. Hönemann was supported by "Ben Covino Award 1998" of the International Anesthesia Research Society sponsored by AstraZeneca Pain Control, Inc, and by a "Innovative Medizinische Forschung" grants (IMF I-6-II/96–8, IMF I-6-II/97–27), Münster, Germany, as well as by the German Research Society (DFG HO 1931/3–1), Bonn, Germany. Dr. Hollmann was supported by the German Research Society (DFG HO-2199/1–1), Bonn, Germany, and by "Ben Covino Award 2000" of the International Anesthesia Research Society sponsored by AstraZeneca Pain Control, Inc., Sweden. Supported by the Department of Anesthesiology, University of Virginia, Charlottesville, VA, and in part by grant GMS 52387 from the National Institutes of Health, Bethesda, Maryland.

We express our sincere thanks to Carl Lynch III (Professor and Chair of the Department of Anesthesiology, University of Virginia) and Cosmo DiFazio (Professor of Anesthesiology, University of Virginia). We thank Thomas Heyse, MS, and Sascha Berning, MS, for their assistance and Hugo Van Aken, MD, PhD, (University of Muenster, Department of Anesthesiology and Intensive Care, Germany) for his support.


   Footnotes
 
Dr. Hönemann was recipient of the second-place award in the 1998 American Society of Anesthesiologists Residents Research Essay Contest, for part of the work presented in this article.


   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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Accepted for publication April 6, 2004.





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Lippincott, Williams & Wilkins Anesthesia & Analgesia® is published for the International Anesthesia Research Society® by Lippincott Williams & Wilkins with the assistance of Stanford University Libraries' HighWire Press®. Copyright 2006 by the International Anesthesia Research Society. Online ISSN: 1526-7598   Print ISSN: 0003-2999 HighWire Press