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© 1999 International Anesthesia Research Society
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Abstract |
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 Top Abstract IntroductionMethodsResultsDiscussionAppendix 1References |
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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 (ICl(Ca)) in Xenopus oocytes. After 10 min exposure to benzocaine, QX314 (10-6–10-2 M), or both, we measured effects on ICl(Ca) induced by LPA (with and without protein kinase [PKC] activation/inhibition) and on ICl(Ca) induced by the intracellular injection of IP3 and GTP
S. LPA application to oocytes resulted in ICl(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 GTPS-induced ICl(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.
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Introduction |
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TopAbstractIntroductionMethodsResultsDiscussionAppendix 1References |
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Although best known for their ability to block Na channels, local anesthetics also interact with other cellular systems. The interactions of local anesthetics with ion channels other than Na channels have been studied in some detail (1); examples are effects on Ca channels (2–4) and nicotinic acetylcholine receptors (3, 5). In contrast, interactions with second messenger-linked systems, such as G-protein–coupled receptors, have not been investigated in depth.
One G-protein–coupled receptor that is inhibited in its function by local anesthetics is the lysophosphatidate (LPA) receptor (6). LPA is released by activated platelets and fibroblasts (7), suggesting a role in wound repair. Indeed, it has been reported to be generated after corneal injury (8), and we have shown it to be released after acute skin injury (unpublished data). Local anesthetics are frequently injected in high concentrations around surgical wounds and may delay wound healing (9–11); this could result from interference with LPA signaling. In a previous study, Nietgen et al. (6) demonstrated that, at concentrations such as those present when injected for postoperative analgesia, lidocaine and bupivacaine inhibit LPA signaling in Xenopus oocytes, but the mechanism of action was not investigated in detail.
In the present study, we continued this investigation. First, we determined in detail the site of local anesthetic action on the signaling cascade, including potential effects on protein kinase C (PKC) functioning. Second, we determined whether a charged group in the local anesthetic molecule is necessary for the inhibitory effect. The local anesthetics used in our previous study (6), lidocaine and bupivacaine, are both partially charged, depending on pH. Therefore, no conclusions could be drawn about the active species involved in LPA signaling blockade. To resolve this issue, in the present study, we investigated the effects on LPA signaling of two other local anesthetics: QX314 (N-(2,6)dimethylphenylcarbamoylmethyl triethylammonium bromide, a quaternary local anesthetic, 99.99% permanently charged) and benzocaine (permanently uncharged).
Our data indicate that LPA signaling inhibition by local anesthetics takes place at the membrane receptor. Both charged and uncharged local anesthetics inhibit LPA signaling, and they act synergistically when combined, which suggests the presence of two local anesthetic binding sites on the LPA receptor molecule, one of them intracellularly, and each of them able to inhibit LPA signaling when occupied by a local anesthetic molecule.
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Methods |
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TopAbstractIntroductionMethodsResultsDiscussionAppendix 1References |
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The study protocol was approved by the Animal Research Committee at the University of Virginia. Xenopus oocytes were removed as described previously (12). Oocytes were incubated for 10 min in benzocaine and diluted in modified Barth's solution (MBS) to four concentrations (0.01, 0.1, 1, and 10 mM). The pH of the MBS containing 10 mM benzocaine was adjusted to pH 5 to achieve solubility. Each oocyte was then voltage-clamped and tested alternately with a control cell incubated in MBS only. The control cells for the 10 mM benzocaine group were incubated in MBS at a pH of 5. Agonist application and voltage clamp techniques were performed as described previously (12). Intracellular Ca release in response to LPA was assessed by measuring Ca-activated Cl currents (ICl(Ca)) (Fig. 1A). Responses were quantified by integrating the current trace.
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7 for each data point.QX314, which does not penetrate the cell membrane because of its permanent charge, was injected into the oocyte or applied outside the cell to identify an intra- or extracellular site of action. For injection, QX314 was diluted in 50 nL of KCl (150 mM); control cells were injected with 50 nL of KCl (150 mM). Each cell was then voltage-clamped and tested 10 min after injection. Control oocytes, which were injected with 50 nL of KCl (150 mM) (Fig. 2A), showed currents approximately twice as large as those observed without intracellular injection (Fig. 1A), presumably as a result of increased intracellular Cl- concentration. Induced ICl(Ca) was recorded 5 s before and 55 s after intracellular injection and analyzed as described previously (12).
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A third micropipette was inserted into the voltage-clamped oocyte to study IP3- and GTP
S–induced ICl(Ca). Tips of intracellular micropipettes were beveled with a microgrinder (Narishige EG-6 Glass Electrode microgrinder; Narishige Laboratories, Tokyo, Japan). The micropipette was connected to an automated microinjector (Nanoject; Drummond Scientific, Broomall, PA). Under the voltage clamp, 30 nL of 2 mM IP3 or 100 mM GTPS was injected, thereby activating the signaling pathway at the IP3 receptor or the G protein, respectively. Because the volume of an average Xenopus oocyte is approximately 500 nL, the injected volume is approximately 5% of the oocyte volume. Therefore, the estimated final concentration was IP3 100 µM or GTPS 5 mM. These concentrations were chosen to result in an ICl(Ca) similar in size to those induced by LPA agonist at its 50% effective dose (EC50). PKC was activated by 5 min incubation in phorbol ester (50, 500, 1000 nM) before measuring LPA-induced ICl(Ca). PKC was inhibited by microinjecting 50 nL of 500 µM chelerythrine diluted in 150 mM KCl or, as a control, 150 mM KCl, into oocytes 2 h before measuring LPA-induced ICl(Ca).
Results are reported as mean ± SEM. Because variability among batches of oocytes is common, responses were, at times, normalized to same-day controls for each batch. Differences between treatment groups were analyzed by 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 fit to the following logistic function, derived from the Hill equation (13): y = ymin + (ymax –ymin) (1 - xn/[x50n - xn]) where ymax and ymin are the maximal and minimal responses obtained, n is the Hill coefficient, and x50 is the EC50 for agonist or the half-maximal inhibitory effect concentration (IC50 for antagonists). These were used for both algebraic and isobolographic analysis of drug interaction (14) (Appendix 1). Statistical analysis of the isobologram was used to establish the superadditive, subadditive, or simple additive effects of benzocaine and QX314 in combination. The drugs were tested in a 2:1 ratio based on their relative IC50 values (equiinhibitory concentrations). Concentration-effect data were plotted with 95% confidence intervals. Statistical analysis was then performed to determine the significance of the difference between the line of additivity and the intersection of the combination IC50 values.
LPA was obtained from Avanti Polar Lipids (Alabaster, AL) and was diluted in 0.1% fatty acid-free bovine serum albumin (ICN Pharmaceuticals, Costa Mesa, CA) in Tyrode's solution to the appropriate concentration. QX314 was a gift from Astra Inc. (Wayne, PA). All other chemicals were from Sigma Chemical Company (St. Louis, MO).
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Results |
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TopAbstractIntroductionMethodsResultsDiscussionAppendix 1References |
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In agreement with earlier studies (6, 15), we found that LPA induced transient inward currents in Xenopus oocytes (Fig. 1A). In contrast, the vehicle (BSA) was without effect (data not shown). We have previously shown that the oocyte response to LPA is a Ca-activated Cl current (ICl(Ca)) (16). It is induced via IP3-mediated Ca release, as we have shown the IP3 receptor antagonist heparin to be able to abolish responsiveness to LPA (17). Expanding our previous findings (6), we determined the concentration-response relationship for LPA. We found the oocyte response to LPA to be concentration-dependent, with an EC50 of 5.1 ± 0.3 x 10–9 M and a Hill coefficient of 0.6 ± 0.1 (Fig. 1B, Table 1). Maximal ICl(Ca) (8.9 ± 1.0 µC) was induced by 10-6 M LPA, and this concentration was used for the remainder of the study.
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We determined whether the site of local anesthetic block is intra- or extracellular using the permanently charged (and, therefore, virtually membrane-impermeant) lidocaine analog QX314.
When applied extracellularly, QX314 was unable to inhibit LPA-induced ICl(Ca) in oocytes, even when the local anesthetic was applied at high concentrations (50 mM) (Fig. 2, A and B). In contrast, when the compound was microinjected intracellularly, it inhibited LPA signaling effectively (Fig. 2, A and B). LPA signaling inhibition was concentration-dependent (Fig. 2C). Curve-fitting using the Hill equation revealed an IC50 of 660 ± 154 µM with a Hill coefficient of 0.4 ± 0.05 (Table 1). Maximal inhibition (using 5 mM QX314 intracellular concentration) was 28% of control.
These findings clarify two issues. First, they indicate that the site of QX314 block of LPA signaling is located intracellularly. Second, they indicate that a charged local anesthetic molecule is able to interact with LPA signaling.
Our finding that QX314 inhibits LPA signaling does not prove that only the charged local anesthetic molecule can exert inhibitory effects on LPA signaling. To determine whether an uncharged local anesthetic also interacts with LPA signaling, we studied the ability of benzocaine, a permanently uncharged (and, therefore, highly membrane-permeant) local anesthetic, to inhibit LPA signaling in oocytes.
LPA signaling was inhibited in a concentration-dependent manner when oocytes were exposed to benzocaine (Fig. 2D). Curve-fitting using the Hill equation revealed an IC50 of 909 ± 231 µM and a Hill coefficient of 0.7 ± 0.1 (Fig. 2E, Table 1). Thus, the compound was approximately half as potent as QX314. Maximal inhibition (using 10 mM benzocaine) was 20% of control. These findings show that charged and uncharged local anesthetics can inhibit LPA signaling. Two hypotheses can account for this finding: 1) the charged and uncharged compounds act at different sites, with different degrees of polarity; or 2) the compounds act at a single site, in which case the hydrophobic portion of the local anesthetic is likely to be the active moiety.
To differentiate between these two possibilities, we studied the inhibitory action of a combination of benzocaine (applied extracellularly) and QX314 (microinjected intracellularly). If the compounds acted on a single site, the combination would be anticipated to act in an additive manner. In contrast, if two sites in the signaling pathway were involved, the combination could act synergistically.
When applied in combination, benzocaine and QX314 (2:1 molar ratio, in view of their respective IC50 values) inhibited LPA signaling in a concentration-dependent manner (Fig. 3A). Curve-fitting using the Hill equation revealed an IC50 of 97 ± 12 µM benzocaine and 48 ± 6 µM QX314. The Hill coefficient was 0.5 ± 0.03 (Fig. 3B, Table 1). Thus, when applied in combination, the IC50 values for both local anesthetics are less than those obtained when the drugs were studied in isolation, which suggests a synergistic action. We therefore performed both algebraic and isobolographic analysis of our data to determine whether synergism was indeed present (Table 2). Algebraic analysis yielded an R of 5.56 (P = 0.035); isobolographic analysis (Fig. 3C, Appendix 1) similarly revealed evidence of superadditivity or synergism.
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= concentration response-curve of benzocaine in combination with intracellular applied QX314, 
= QX314 concentration response curve in combination with benzocaine. Curve-fitting using the Hill equation revealed a half-maximal effect concentration of benzocaine 0.097 ± 0.01 mM and QX314 0.048 ± 0.006 mM and a Hill coefficient of 0.48 ± 0.03. n
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Thus, when administered together, both charged and uncharged local anesthetics synergistically inhibit LPA signaling. This suggests that two sequential sites in the signaling pathway are blocked by these drugs. We therefore proceeded to determine the location of benzocaine and QX314 action within the signaling pathway.
To study the effect of local anesthetics on segments of the signaling pathway, we activated various segments of the intracellular signaling pathway by an intracellular microinjection of IP3 or GTPS.
We first tested the effects of the anesthetics on currents induced by IP3. As we (6,12,17) have demonstrated previously, IP3 induces ICl(Ca) indistinguishable from that induced by LPA application to oocytes. However, these previous studies were performed with a single concentration of IP3. We now sought to determine whether the effect was concentration-dependent. As shown in Figure 4A, the oocyte response to microinjected IP3 is concentration-dependent; 2 mM IP3 induces currents with an average charge movement of 11.3 ± 1.5 µC.
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We then investigated the effect of benzocaine (1 mM) and QX314 (0.5 mM) on currents induced by IP3. Benzocaine and QX314 did not affect the action of IP3 (Fig. 4B), which suggests that the LPA signaling pathway downstream of phospholipase C (including the IP3 receptor, the Ca release mechanism, and the Ca-activated Cl- channel) is unaffected by both local anesthetics.
Because the distal signaling pathway was not affected by local anesthetics, we determined their effects on the more proximal pathway. GTPS is a nonhydrolyzable GTP analog that irreversibly activates G proteins. We have previously shown that this compound induces ICl(Ca) in oocytes (12). We now demonstrate that these responses are concentration-dependent (Fig. 4C). GTPS (10 mM) induced currents with an average charge movement of 7.1 ± 0.5 µC.
Similar to the situation with IP3 signaling, neither benzocaine (1 mM) nor QX314 (0.5 mM injected intracellularly) affected the currents induced by 10 mM GTPS (Fig. 4D). This suggests that the LPA signaling pathway downstream from the receptor is not affected by the local anesthetics. Hence, their sites of action are most likely located on the LPA receptor molecule itself.
However, benzocaine and QX314 could exert their inhibitory effects on LPA receptors through a different mechanism. Instead of direct modulation of receptor configuration by interaction with the protein, they could act indirectly by activating PKC, which could then catalyze phosphorylation of LPA receptors and decrease receptor functioning. To determine whether PKC can indeed modulate LPA signaling, we studied the effect of PKC inhibition and activation.
Xenopus oocytes were pretreated for 2 h with the PKC inhibitor chelerythrine. Chelerythrine produced a 30% enhancement of the currents activated by 1 µM LPA (Fig. 5A) and 70% enhancement of the currents activated by 100 nM LPA (Fig. 5A). Conversely, the PKC activator PMA (50, 500, and 1000 nM for 5 min) inhibited currents evoked by 10-6 M LPA concentration-dependently to 56%, 40%, and 10% of the initial currents, respectively (Fig. 5B). Thus, LPA receptor functioning can be modulated by PKC.
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To determine whether local anesthetics could inhibit LPA signaling through PKC activation, we investigated the effects of chelerythrine on the inhibition of LPA signaling by QX314 and benzocaine. As shown in Figure 5C,l the inhibitory effects of QX314 and benzocaine (10 min incubation) were unchanged in oocytes treated with PKC inhibitor for 2 h. This suggests that local anesthetics most likely inhibit LPA receptor responses by direct interaction with the receptor protein, not by an indirect mechanism through phosphorylation processes induced by PKC activation.
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Discussion |
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TopAbstractIntroductionMethodsResultsDiscussionAppendix 1References |
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Our results demonstrate that benzocaine and QX314 inhibit LPA receptor function. Both a completely charged (QX314) and a completely uncharged local anesthetic (benzocaine) can inhibit LPA signaling, with a relatively similar IC50 (ratio 2:1). For either local anesthetic, the site of action is localized to the LPA receptor; neither the intracellular signaling pathway nor PKC-mediated modulation of LPA signaling are involved. For the charged species, the site of action is intracellular. This rules out the possibility that local anesthetic inhibition of LPA signaling could be due to direct interactions between the local anesthetic and the LPA molecule or to competition between LPA and local anesthetic for binding to albumin. The charged and uncharged local anesthetic interact synergistically, which indicates separate binding sites on the LPA receptor molecule.
These findings are in complete agreement with data obtained in previous studies (6,18), in which other local anesthetics were found to inhibit LPA signaling in a reversible manner but were without effect on angiotensin II signaling, which shares the same signaling pathway. Our present study expands these findings by investigating different local anesthetics, localizing the site of action in more detail, and elucidating the role of charge in the local anesthetic molecule. Binding studies and mutational analysis of the LPA receptor could clarify these issues further but are not possible until a reproducible binding assay is established and a clone has been definitely identified.
One item that we did find to be different between the studies are the IC50 values of the compounds. Whereas our previous study reported IC50 values of 30 ± 8 mM and 5 ± 1 mM (6) for lidocaine and bupivacaine, respectively, we found IC50 values of 0.66 ± 0.16 mM and 0.91 ± 0.23 mM for QX314 and benzocaine, respectively, in our present investigation. This difference may be due to pharmacological characteristics of the compounds or to pH (our previous study was performed at pH 6.4). Alternatively, small changes in experimental technique (continuous flow chamber, collagenase rather than manual defolliculation) may have yielded greater apparent potency.
As in our previous study, we chose the Xenopus oocyte model. In addition to providing consistency with the other investigation, the oocyte provides great advantages for studies of this kind. In particular, its size allows easy intracellular access, allowing study of intracellular pathways and site of action by microinjection of different compounds. However, potential disadvantages should also be kept in mind. Most relevant to the present study is the fact that the LPA receptor studied is amphibian, and its behavior may therefore diverge from its mammalian ortholog. However, LPA-induced Ca signaling in oocytes and in mammalian cells has been shown to be similar (7,17,19). Nonetheless, confirmatory studies of local anesthetic effects in other models, e.g., LPA-induced platelet aggregation or fibroblast proliferation, would be of interest.
As benzocaine is freely membrane permeable, we could not determine whether its site of action is intra- or extracellular. It might interact with the lipophilic agonist binding pocket on the LPA receptor. Unfortunately, lack of an established binding assay makes it impossible to test this hypothesis directly. In any case, the synergistic interaction between the two compounds makes it likely that two separate binding sites are involved: one for the charged and one for the uncharged species. Lack of effect on GTPS and IP3 signaling and lack of effect of PKC inhibition locates these two sites to the LPA receptor molecule itself. Our study does not rule out an effect of local anesthetics on PKC; there are many isoforms of this enzyme (20), and our results only apply to the (as yet not characterized) isoforms modulating LPA signaling. One alternative hypothesis to be considered is the presence of several subtypes of LPA receptor, one of which would be inhibited by charged, the other by uncharged, local anesthetics. Although there some evidence suggests the presence of multiple receptor subtypes in oocytes (21), definitive determination awaits molecular cloning of the receptor(s). Although some potential candidate clones have been reported (22), they have not yet been generally accepted by the scientific community. Nonetheless, it seems very unlikely that this could explain our findings, as these receptors would form parallel, rather than sequential, signaling pathways, and additive, rather than synergistic, interactions would therefore be expected.
In conclusion, the site of local anesthetic action on LPA signaling seems to be the membrane receptor. Intracellular signaling pathways and the PKC isoform modulating LPA signaling are not inhibited by local anesthetics. LPA receptors most likely contain multiple binding sites for charged and uncharged local anesthetics. Therefore, clinically used compounds, such as lidocaine (67% charged at physiological pH) and bupivacaine (88% charged at physiological pH), may influence this receptor by several means. As a result, they can be expected to modulate the effects of LPA after its release in the wound bed, as well as other LPA-mediated physiologic responses.
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Appendix 1 |
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TopAbstractIntroductionMethodsResultsDiscussionAppendix 1References |
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Isobolographic Analysis
An isobologram is a cartesian plot of pairs of doses that, in combination, yield a specified level of effect. It is a convenient way of graphically displaying results of a drug combination because paired values of experimental points that fall below or above the line connecting the axial points (usually the EC50 values) denote a supra- or subadditive combination, respectively. During our isobolographic and statistical analysis of the isobologram, we followed the suggestions of Tallarida et al. (14).
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We defined our variables: If the individual potencies of benzocaine and QX314 are denoted by their EC50 values, the classification of the result of a combination of both drugs as additive or nonadditive is based on the definition involving amounts of both in a mixture that produces the specified level of effect. This definition is expressed in the following equation:
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If C is <1, our tested drug combination is superadditive; if it =1, then our combination has a simple additive effect; if it is >1, we have a subadditive effect of the drug combination. In our study, C = 0.1796; therefore, the combination of benzocaine and QX314 had superadditive effects.
Statistical confidence limits must be expressed on the isobologram if this diagram is to establish superadditive effects. For two different drugs whose regression lines have slopes that do not differ significantly, a common slope ß is determined as a weighted mean of the individual slopes. Thus, benzocaine and QX314 have their respective regression lines calculated using the equations below. We used this method to calculate the variance and SEM of the fictive simple additive EC50 of the drug combination (point on the line of additivity) and analyzed the statistical significance of our findings using Student's t-tests. We used the following equations:
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We used the calculated values and solved the following equation. The result of this equation is the calculated variance of the fictive simple additive point on the line of additivity, which can be used to calculate the SEM.
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We then compared this mean ± SEM with the measured point (mean ± SEM) using Student's t-tests.
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Acknowledgments |
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This work was supported in part by National Institutes of Health Grant GMS 52387 to MED. CWH is supported by the Innovative Medizinische Forschung fund, Münster, Germany.
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References |
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TopAbstractIntroductionMethodsResultsDiscussionAppendix 1References |
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